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Sample records for previously isolated human

  1. Molecular analysis of clinical isolates previously diagnosed as Mycobacterium intracellulare reveals incidental findings of "Mycobacterium indicus pranii" genotypes in human lung infection.

    Science.gov (United States)

    Kim, Su-Young; Park, Hye Yun; Jeong, Byeong-Ho; Jeon, Kyeongman; Huh, Hee Jae; Ki, Chang-Seok; Lee, Nam Yong; Han, Seung-Jung; Shin, Sung Jae; Koh, Won-Jung

    2015-09-30

    Mycobacterium intracellulare is a major cause of Mycobacterium avium complex lung disease in many countries. Molecular studies have revealed several new Mycobacteria species that are closely related to M. intracellulare. The aim of this study was to re-identify and characterize clinical isolates from patients previously diagnosed with M. intracellulare lung disease at the molecular level. Mycobacterial isolates from 77 patients, initially diagnosed with M. intracellulare lung disease were re-analyzed by multi-locus sequencing and pattern of insertion sequences. Among the 77 isolates, 74 (96 %) isolates were designated as M. intracellulare based on multigene sequence-based analysis. Interestingly, the three remaining strains (4 %) were re-identified as "Mycobacterium indicus pranii" according to distinct molecular phylogenetic positions in rpoB and hsp65 sequence-based typing. In hsp65 sequevar analysis, code 13 was found in the majority of cases and three unreported codes were identified. In 16S-23S rRNA internal transcribed spacer (ITS) sequevar analysis, all isolates of both species were classified within the Min-A ITS sequevar. Interestingly, four of the M. intracellulare isolates harbored IS1311, a M. avium-specific element. Two of three patients infected with "M. indicus pranii" had persistent positive sputum cultures after antibiotic therapy, indicating the clinical relevance of this study. This analysis highlights the importance of precise identification of clinical isolates genetically close to Mycobacterium species, and suggests that greater attention should be paid to nontuberculous mycobacteria lung disease caused by "M. indicus pranii".

  2. Effect of media composition, including gelling agents, on isolation of previously uncultured rumen bacteria.

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    Nyonyo, T; Shinkai, T; Tajima, A; Mitsumori, M

    2013-01-01

    The aim of this study was to develop novel anaerobic media using gellan gum for the isolation of previously uncultured rumen bacteria. Four anaerobic media, a basal liquid medium (BM) with agar (A-BM), a modified BM (MBM) with agar (A-MBM), an MBM with phytagel (P-MBM) and an MBM with gelrite (G-MBM) were used for the isolation of rumen bacteria and evaluated for the growth of previously uncultured rumen bacteria. Of the 214 isolates composed of 144 OTUs, 103 isolates (83 OTUs) were previously uncultured rumen bacteria. Most of the previously uncultured strains were obtained from A-MBM, G-MBM and P-MBM, but the predominant cultural members, isolated from each medium, differed. A-MBM and G-MBM showed significantly higher numbers of different OTUs derived from isolates than A-BM (P rumen bacteria were isolated from all media used, the ratio of previously uncultured bacteria to total isolates was increased in A-MBM, P-MBM and G-MBM. © 2012 The Society for Applied Microbiology.

  3. Cultivation-based multiplex phenotyping of human gut microbiota allows targeted recovery of previously uncultured bacteria

    DEFF Research Database (Denmark)

    Rettedal, Elizabeth; Gumpert, Heidi; Sommer, Morten

    2014-01-01

    The human gut microbiota is linked to a variety of human health issues and implicated in antibiotic resistance gene dissemination. Most of these associations rely on culture-independent methods, since it is commonly believed that gut microbiota cannot be easily or sufficiently cultured. Here, we...... microbiota. Based on the phenotypic mapping, we tailor antibiotic combinations to specifically select for previously uncultivated bacteria. Utilizing this method we cultivate and sequence the genomes of four isolates, one of which apparently belongs to the genus Oscillibacter; uncultivated Oscillibacter...

  4. Evaluation of an interdisciplinary re-isolation policy for patients with previous Clostridium difficile diarrhea.

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    Boone, N; Eagan, J A; Gillern, P; Armstrong, D; Sepkowitz, K A

    1998-12-01

    Diarrhea caused by Clostridium difficile is increasingly recognized as a nosocomial problem. The effectiveness and cost of a new program to decrease nosocomial spread by identifying patients scheduled for readmission who were previously positive for toxin was evaluated. The Memorial Sloan-Kettering Cancer Center is a 410-bed comprehensive cancer center in New York City. Many patients are readmitted during their course of cancer therapy. In 1995 as a result of concern about the nosocomial spread of C difficile, we implemented a policy that all patients who were positive for C difficile toxin in the previous 6 months with no subsequent toxin-negative stool as an outpatient would be placed into contact isolation on readmission pending evaluation of stool specimens. Patients who were previously positive for C difficile toxin were identified to infection control and admitting office databases via computer. Admitting personnel contacted infection control with all readmissions to determine whether a private room was required. Between July 1, 1995, and June 30, 1996, 47 patients who were previously positive for C difficile toxin were readmitted. Before their first scheduled readmission, the specimens for 15 (32%) of these patients were negative for C difficile toxin. They were subsequently cleared as outpatients and were readmitted without isolation. Workup of the remaining 32 patients revealed that the specimens for 7 patients were positive for C difficile toxin and 86 isolation days were used. An additional 25 patients used 107 isolation days and were either cleared after a negative specimen was obtained in-house or discharged without having an appropriate specimen sent. Four patients (9%) had reoccurring C difficile after having toxin-negative stools. We estimate (because outpatient specimens were not collected) the cost incurred at $48,500 annually, including the incremental cost of hospital isolation and equipment. Our policy to control the spread of nosocomial C

  5. Genetic differences between avian and human isolates of Candida dubliniensis.

    LENUS (Irish Health Repository)

    McManus, Brenda A

    2009-09-01

    When Candida dubliniensis isolates obtained from seabird excrement and from humans in Ireland were compared by using multilocus sequence typing, 13 of 14 avian isolates were genetically distinct from human isolates. The remaining avian isolate was indistinguishable from a human isolate, suggesting that transmission may occur between humans and birds.

  6. Total excitation of the isolated human heart

    NARCIS (Netherlands)

    Durrer, D.; Dam, R.Th. van; Freud, G.E.; Janse, M.J.; Meijler, F.L.; Arzbaecher, R.C.

    To obtain information conceming the time course and instantaneous distribution of the excitatory process of the normal human healt, studies were made on isolated human hearts from seven individuals who died from various cerebral conditions, but who had no history of cardiac disease. Measurements

  7. Isolation of previously uncultured rumen bacteria by dilution to extinction using a new liquid culture medium.

    Science.gov (United States)

    Kenters, Nikki; Henderson, Gemma; Jeyanathan, Jeyamalar; Kittelmann, Sandra; Janssen, Peter H

    2011-01-01

    A new anaerobic medium that mimics the salts composition of rumen fluid was used in conjunction with a dilution method of liquid culture to isolate fermentative bacteria from the rumen of a grass-fed sheep. The aim was to inoculate a large number of culture tubes each with a mean of 97% sequence identity to genes of uncultured bacteria detected in various gastrointestinal environments. This strategy has therefore allowed us to cultivate many novel rumen bacteria, opening the way to overcoming the lack of cultures of many of the groups detected using cultivation-independent methods. Copyright © 2010 Elsevier B.V. All rights reserved.

  8. Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:H- Isolates from Czech Patients with Novel Plasmid Composition Not Previously Seen in German Isolates.

    Science.gov (United States)

    Bauwens, Andreas; Marejková, Monika; Middendorf-Bauchart, Barbara; Prager, Rita; Kossow, Annelene; Zhang, Wenlan; Karch, Helge; Mellmann, Alexander; Bielaszewska, Martina

    2017-12-01

    -uremic syndrome in Europe. They account for 10 to 20% of sporadic cases of this disease and have caused several large outbreaks. The strains isolated throughout Europe share conserved chromosomal and plasmid characteristics. Here we identified novel sorbitol-fermenting enterohemorrhagic E. coli O157:H - patient isolates in the Czech Republic which differ from all such strains reported previously by their unique plasmid characteristics, including plasmid number, composition of plasmid-carried virulence genes, and plasmid origins. Our findings contribute substantially to understanding the evolution of E. coli O157 strains and their plasmids. In practical terms, they enable the identification of strains with these novel plasmid characteristics in patient stool samples and thus the investigation of their roles as human pathogens in other geographic areas. Copyright © 2017 American Society for Microbiology.

  9. Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:H− Isolates from Czech Patients with Novel Plasmid Composition Not Previously Seen in German Isolates

    Science.gov (United States)

    Bauwens, Andreas; Marejková, Monika; Middendorf-Bauchart, Barbara; Prager, Rita; Kossow, Annelene; Zhang, Wenlan; Karch, Helge

    2017-01-01

    -uremic syndrome in Europe. They account for 10 to 20% of sporadic cases of this disease and have caused several large outbreaks. The strains isolated throughout Europe share conserved chromosomal and plasmid characteristics. Here we identified novel sorbitol-fermenting enterohemorrhagic E. coli O157:H− patient isolates in the Czech Republic which differ from all such strains reported previously by their unique plasmid characteristics, including plasmid number, composition of plasmid-carried virulence genes, and plasmid origins. Our findings contribute substantially to understanding the evolution of E. coli O157 strains and their plasmids. In practical terms, they enable the identification of strains with these novel plasmid characteristics in patient stool samples and thus the investigation of their roles as human pathogens in other geographic areas. PMID:28970221

  10. Previous experience in manned space flight: A survey of human factors lessons learned

    Science.gov (United States)

    Chandlee, George O.; Woolford, Barbara

    1993-01-01

    Previous experience in manned space flight programs can be used to compile a data base of human factors lessons learned for the purpose of developing aids in the future design of inhabited spacecraft. The objectives are to gather information available from relevant sources, to develop a taxonomy of human factors data, and to produce a data base that can be used in the future for those people involved in the design of manned spacecraft operations. A study is currently underway at the Johnson Space Center with the objective of compiling, classifying, and summarizing relevant human factors data bearing on the lessons learned from previous manned space flights. The research reported defines sources of data, methods for collection, and proposes a classification for human factors data that may be a model for other human factors disciplines.

  11. Transepithelial Transport of PAMAM Dendrimers Across Isolated Human Intestinal Tissue.

    Science.gov (United States)

    Hubbard, Dallin; Enda, Michael; Bond, Tanner; Moghaddam, Seyyed Pouya Hadipour; Conarton, Josh; Scaife, Courtney; Volckmann, Eric; Ghandehari, Hamidreza

    2015-11-02

    Poly(amido amine) (PAMAM) dendrimers have shown transepithelial transport across intestinal epithelial barrier in rats and across Caco-2 cell monolayers. Caco-2 models innately lack mucous barriers, and rat isolated intestinal tissue has been shown to overestimate human permeability. This study is the first report of transport of PAMAM dendrimers across isolated human intestinal epithelium. It was observed that FITC labeled G4-NH2 and G3.5-COOH PAMAM dendrimers at 1 mM concentration do not have a statistically higher permeability compared to free FITC controls in isolated human jejunum and colonic tissues. Mannitol permeability was increased at 10 mM concentrations of G3.5-COOH and G4-NH2 dendrimers. Significant histological changes in human colonic and jejunal tissues were observed at G3.5-COOH and G4-NH2 concentrations of 10 mM implying that dose limiting toxicity may occur at similar concentrations in vivo. The permeability through human isolated intestinal tissue in this study was compared to previous rat and Caco-2 permeability data. This study implicates that PAMAM dendrimer oral drug delivery may be feasible, but it may be limited to highly potent drugs.

  12. Experimental infection of one-day-old chicks with Salmonella Serotypes Previously isolated from poultry facilities, wild birds, and swine

    Directory of Open Access Journals (Sweden)

    E de Sousa

    2013-12-01

    Full Text Available In order to maintain the high production and export rates achieved by the Brazilian poultry industry, it is necessary to prevent and control certain disease agents, such as Salmonella spp. Using bacterial cultures, the aim of the present study was to investigate the prevalence of Salmonella spp. in specimens collected from broiler facilities. Local wild birds were also sampled, as well as the feces of swine housed on the poultry farm. After sample collection, the isolated serotypes were subsequently inoculated into broiler chicks to determine their effects. Positive samples were collected from the following locations in the poultry facilities: poultry litter (S. serotype 4,5,12:R:-; S. Heidelberg; S. Infantis, broiler feces (S. Heidelberg; S. serotype 6,7:R:-; S. serotype 4,5,12:R:-; S. Tennessee, water (S. Glostrup; S. serotype 6,8:d:-;, and lesser mealworms (Alphitobius diaperinus found in the litter (S. Tennessee. Among the 36 wild birds captured, S. Heidelberg was isolated from one bird's organs and intestinal contents (Colaptes campestris, and S. Enteritidis was isolated from another bird's intestinal contents (Zenaida auriculata. Salmonella Panama and Salmonella Typhimurium were isolated from swine feces. One-day-old chicks (150 were divided into 10 groups of 15 animals each. Each group was orally inoculated with a previously isolated serotype of Salmonella. Soft stools were observed on the cage floor and around the birds' cloaca between 3 and 12 days post-infection (dpi. The different serotypes of Salmonella used to inoculate the chicks were re-isolated from the spleen, liver, and cecal content samples of the infected birds on 15 and 21 dpi.

  13. Isolation of Human Skin Dendritic Cell Subsets.

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    Gunawan, Merry; Jardine, Laura; Haniffa, Muzlifah

    2016-01-01

    Dendritic cells (DCs) are specialized leukocytes with antigen-processing and antigen-presenting functions. DCs can be divided into distinct subsets by anatomical location, phenotype and function. In human, the two most accessible tissues to study leukocytes are peripheral blood and skin. DCs are rare in human peripheral blood (skin covering an average total surface area of 1.8 m(2) has approximately tenfold more DCs than the average 5 L of total blood volume (Wang et al., J Invest Dermatol 134:965-974, 2014). DCs migrate spontaneously from skin explants cultured ex vivo, which provide an easy method of cell isolation (Larsen et al., J Exp Med 172:1483-1493, 1990; Lenz et al., J Clin Invest 92:2587-2596, 1993; Nestle et al., J Immunol 151:6535-6545, 1993). These factors led to the extensive use of skin DCs as the "prototype" migratory DCs in human studies. In this chapter, we detail the protocols to isolate DCs and resident macrophages from human skin. We also provide a multiparameter flow cytometry gating strategy to identify human skin DCs and to distinguish them from macrophages.

  14. Isolation of Human Innate Lymphoid Cells.

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    Krabbendam, Lisette; Nagasawa, Maho; Spits, Hergen; Bal, Suzanne M

    2018-06-29

    Innate lymphoid cells (ILCs) are innate immune cells of lymphoid origin that have important effector and regulatory functions in the first line of defense against pathogens, but also regulate tissue homeostasis, remodeling, and repair. Their function mirrors T helper cells and cytotoxic CD8 + T lymphocytes, but they lack expression of rearranged antigen-specific receptors. Distinct ILC subsets are classified in group 1 ILCs (ILC1s), group 2 ILCs (ILC2s), and group 3 ILCs (ILC3s and lymphoid tissue-inducer cells), based on the expression of transcription factors and the cytokines they produce. As the frequency of ILCs is low, their isolation requires extensive depletion of other cell types. The lack of unique cell surface antigens further complicates the identification of these cells. Here, methods for ILC isolation and characterization from human peripheral blood and different tissues are described. © 2018 by John Wiley & Sons, Inc. © 2018 John Wiley & Sons, Inc.

  15. Genetic divergence of influenza A NS1 gene in pandemic 2009 H1N1 isolates with respect to H1N1 and H3N2 isolates from previous seasonal epidemics

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    Campanini Giulia

    2010-09-01

    Full Text Available Abstract Background The Influenza A pandemic sustained by a new H1N1 variant (H1N1v started in Mexico and the USA at the end of April 2009 spreading worldwide in a few weeks. In this study we investigate the variability of the NS1 gene of the pandemic H1N1v strain with respect to previous seasonal strains circulating in humans and the potential selection of virus variants through isolation in cell culture. Methods During the period April 27th 2009-Jan 15th 2010, 1633 potential 2009 H1N1v cases have been screened at our center using the CDC detection and typing realtime RT-PCR assays. Virus isolation on MDCK cells was systematically performed in 1/10 positive cases. A subset of 51 H1N1v strains isolated in the period May-September 2009 was selected for NS1 gene sequencing. In addition, 15 H1N1 and 47 H3N2 virus isolates from three previous seasonal epidemics (2006-2009 were analyzed in parallel. Results A low variability in the NS1 amino acid (aa sequence among H1N1v isolates was shown (aa identity 99.5%. A slightly higher NS1 variability was observed among H1N1 and H3N2 strains from previous epidemics (aa identity 98.6% and 98.9%, respectively. The H1N1v strains were closely related (aa identity 92.1% to swine reference strain (A/swine/Oklahoma/042169/2008. In contrast, substantial divergence (aa identity 83.4% with respect to human reference strain A/Brevig Mission/1/1918 and previous epidemic strains H1N1 and H3N2 (aa identity 78.9% and 77.6%, respectively was shown. Specific sequence signatures of uncertain significance in the new virus variant were a C-terminus deletion and a T215P substitution. Conclusions The H1N1v NS1 gene was more conserved than that of previous epidemic strains. In addition, a closer genetic identity of H1N1v with the swine than the human reference strains was shown. Hot-spots were shown in the H1N1v NS1 aa sequence whose biologic relevance remains to be investigated.

  16. A diverse group of previously unrecognized human rhinoviruses are common causes of respiratory illnesses in infants.

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    Wai-Ming Lee

    2007-10-01

    Full Text Available Human rhinoviruses (HRVs are the most prevalent human pathogens, and consist of 101 serotypes that are classified into groups A and B according to sequence variations. HRV infections cause a wide spectrum of clinical outcomes ranging from asymptomatic infection to severe lower respiratory symptoms. Defining the role of specific strains in various HRV illnesses has been difficult because traditional serology, which requires viral culture and neutralization tests using 101 serotype-specific antisera, is insensitive and laborious.To directly type HRVs in nasal secretions of infants with frequent respiratory illnesses, we developed a sensitive molecular typing assay based on phylogenetic comparisons of a 260-bp variable sequence in the 5' noncoding region with homologous sequences of the 101 known serotypes. Nasal samples from 26 infants were first tested with a multiplex PCR assay for respiratory viruses, and HRV was the most common virus found (108 of 181 samples. Typing was completed for 101 samples and 103 HRVs were identified. Surprisingly, 54 (52.4% HRVs did not match any of the known serotypes and had 12-35% nucleotide divergence from the nearest reference HRVs. Of these novel viruses, 9 strains (17 HRVs segregated from HRVA, HRVB and human enterovirus into a distinct genetic group ("C". None of these new strains could be cultured in traditional cell lines.By molecular analysis, over 50% of HRV detected in sick infants were previously unrecognized strains, including 9 strains that may represent a new HRV group. These findings indicate that the number of HRV strains is considerably larger than the 101 serotypes identified with traditional diagnostic techniques, and provide evidence of a new HRV group.

  17. MLVA genotyping of human Brucella isolates from Peru

    NARCIS (Netherlands)

    Smits, Henk L.; Espinosa, Benjamin; Castillo, Rosa; Hall, Eric; Guillen, Alfredo; Zevaleta, Milagros; Gilman, Robert H.; Melendez, Paolo; Guerra, Carlos; Draeger, Angelika; Broglia, Alessandro; Nöckler, Karsten

    2009-01-01

    Recent human Brucella melitensis isolates from Peru were genotyped by multiple locus variable number repeat analysis. All 24 isolates originated from hospitalized patients living in the central part of Peru and consisted of six genomic groups comprising two to four isolates and nine unique

  18. Electron tomography study of isolated human centrioles.

    Science.gov (United States)

    Ibrahim, Rana; Messaoudi, Cédric; Chichon, Francisco Javier; Celati, Claude; Marco, Sergio

    2009-01-01

    Centrioles are components of the centrosome, which is present in most eukaryotic cells (from protozoa to mammals). They organize the microtubule skeleton during interphase and the mitotic spindle during cell division. In ciliate cells, centrioles form basal bodies that are involved in cellular motility. Despite their important roles in biology, the detailed structure of centrioles remains obscure. This work contributes to a more complete model of centriole structure. The authors used electron tomography of isolated centrosomes from the human lymphoblast KE37 to explore the details of subdistal appendages and centriole lumen organization in mother centrioles. Their results reveal that each of the nine subdistal appendages is composed of two halves (20 nm diameter each) fused in a 40 nm tip that extends 100 nm from where it anchors to microtubules. The centriole lumen is filled at the distal domain by a 45 nm periodic stack of rings. Each ring has a 30 nm diameter, is 15 nm thick, and appears to be tilted at 53 degrees perpendicular to the centriole axis. The rings are anchored to microtubules by arms. Based on their results, the authors propose a model of the mother centriole distal structure. Copyright 2008 Wiley-Liss, Inc.

  19. Genome sequence analysis of five Canadian isolates of strawberry mottle virus reveals extensive intra-species diversity and a longer RNA2 with increased coding capacity compared to a previously characterized European isolate.

    Science.gov (United States)

    Bhagwat, Basdeo; Dickison, Virginia; Ding, Xinlun; Walker, Melanie; Bernardy, Michael; Bouthillier, Michel; Creelman, Alexa; DeYoung, Robyn; Li, Yinzi; Nie, Xianzhou; Wang, Aiming; Xiang, Yu; Sanfaçon, Hélène

    2016-06-01

    In this study, we report the genome sequence of five isolates of strawberry mottle virus (family Secoviridae, order Picornavirales) from strawberry field samples with decline symptoms collected in Eastern Canada. The Canadian isolates differed from the previously characterized European isolate 1134 in that they had a longer RNA2, resulting in a 239-amino-acid extension of the C-terminal region of the polyprotein. Sequence analysis suggests that reassortment and recombination occurred among the isolates. Phylogenetic analysis revealed that the Canadian isolates are diverse, grouping in two separate branches along with isolates from Europe and the Americas.

  20. Effect of Previous Chemotherapy on the Quality of Cryopreserved Human Ovarian Tissue In Vitro.

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    Babak Asadi Azarbaijani

    Full Text Available Cryopreservation of ovarian tissue has been widely accepted as an option for fertility preservation among cancer patients. Some patients are exposed to chemotherapy prior to ovarian tissue cryopreservation. Consequently, assessment of the developmental capacity of human ovarian tissue after chemotherapy is of primary importance.In order to study the impact of previous chemotherapy on in vitro development and viability of ovarian follicles, quality control samples from 34 female cancer patients at median age of 15 years (range 1‒35, cryopreserved for fertility preservation before (n = 14 or after (n = 20 initiation of chemotherapy, were thawed and cultured for 7 days. The morphology and developmental stages of ovarian follicles were studied by light microscopy before and after culture. Possible associations between follicular densities, age and exposure to alkylating agents, expressed as cyclophosphamide equivalent dose (CED were tested.Exposure to chemotherapy significantly impaired the survival and development of ovarian follicles in culture. After seven days, significantly higher densities of intermediary, primary and secondary follicles and lower densities of atretic follicles was detected in the samples collected before chemotherapy. Increasing dose of alkylating agents was identified by multivariate linear regression analysis as an independent predictor of a higher density of atretic follicles, whereas increasing age of the patient predicted a better outcome with less follicle atresia and a higher density of maturing follicles.This study provides quantitative in vitro evidence of the impact of chemotherapy on developmental capacity of cryopreserved human ovarian tissue. The results indicate that fertility preservation should be carried out, if possible, before initiation of alkylating agents in order to guarantee better in vitro survival of ovarian follicles. In addition, ovarian samples from younger girls show lower viability and fewer

  1. Human Papilloma Virus Identification in Breast Cancer Patients with Previous Cervical Neoplasia.

    Science.gov (United States)

    Lawson, James S; Glenn, Wendy K; Salyakina, Daria; Clay, Rosemary; Delprado, Warick; Cheerala, Bharathi; Tran, Dinh D; Ngan, Christopher C; Miyauchi, Shingo; Karim, Martha; Antonsson, Annika; Whitaker, Noel J

    2015-01-01

    Women with human papilloma virus (HPV)-associated cervical neoplasia have a higher risk of developing breast cancer than the general female population. The purpose of this study was to (i) identify high-risk HPVs in cervical neoplasia and subsequent HPV positive breast cancers which developed in the same patients and (ii) determine if these HPVs were biologically active. A range of polymerase chain reaction and immunohistochemical techniques were used to conduct a retrospective cohort study of cervical precancers and subsequent breast cancers in the same patients. The same high-risk HPV types were identified in both the cervical and breast specimens in 13 (46%) of 28 patients. HPV type 18 was the most prevalent. HPVs appeared to be biologically active as demonstrated by the expression of HPV E7 proteins and the presence of HPV-associated koilocytes. The average age of these patients diagnosed with breast cancer following prior cervical precancer was 51 years, as compared to 60 years for all women with breast cancer (p for difference = 0.001). These findings indicate that high-risk HPVs can be associated with cervical neoplasia and subsequent young age breast cancer. However, these associations are unusual and are a very small proportion of breast cancers. These outcomes confirm and extend the observations of two similar previous studies and offer one explanation for the increased prevalence of serious invasive breast cancer among young women.

  2. Human papilloma virus identification in breast cancer patients with previous cervical neoplasia

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    James Sutherland Lawson

    2016-01-01

    Full Text Available Purpose: Women with human papilloma virus (HPV associated cervical neoplasia have a higher risk of developing breast cancer than the general female population. The purpose of this study was to (i identify high risk for cancer HPVs in cervical neoplasia and subsequent HPV positive breast cancers which developed in the same patients and (ii determine if these HPVs were biologically active.Methods: A range of polymerase chain reaction (PCR and immunohistochemical techniques were used to conduct a retrospective cohort study of cervical precancers and subsequent breast cancers in the same patients. Results: The same high risk HPV types were identified in both the cervical and breast specimens in 13 (46% of 28 patients. HPV type 18 was the most prevalent. HPVs appeared to be biologically active as demonstrated by the expression of HPV E7 proteins and the presence of HPV associated koilocytes. The average age of these patients diagnosed with breast cancer following prior cervical precancer was 51 years, as compared to 60 years for all women with breast cancer (p for difference = 0.001. Conclusions: These findings indicate that high risk HPVs can be associated with cervical neoplasia and subsequent young age breast cancer. However these associations are unusual and are a very small proportion of breast cancers. These outcomes confirm and extend the observations of 2 similar previous studies and offer one explanation for the increased prevalence of serious invasive breast cancer among young women.

  3. Reciprocity, culture and human cooperation: previous insights and a new cross-cultural experiment.

    Science.gov (United States)

    Gächter, Simon; Herrmann, Benedikt

    2009-03-27

    Understanding the proximate and ultimate sources of human cooperation is a fundamental issue in all behavioural sciences. In this paper, we review the experimental evidence on how people solve cooperation problems. Existing studies show without doubt that direct and indirect reciprocity are important determinants of successful cooperation. We also discuss the insights from a large literature on the role of peer punishment in sustaining cooperation. The experiments demonstrate that many people are 'strong reciprocators' who are willing to cooperate and punish others even if there are no gains from future cooperation or any other reputational gains. We document this in new one-shot experiments, which we conducted in four cities in Russia and Switzerland. Our cross-cultural approach allows us furthermore to investigate how the cultural background influences strong reciprocity. Our results show that culture has a strong influence on positive and in especially strong negative reciprocity. In particular, we find large cross-cultural differences in 'antisocial punishment' of pro-social cooperators. Further cross-cultural research and experiments involving different socio-demographic groups document that the antisocial punishment is much more widespread than previously assumed. Understanding antisocial punishment is an important task for future research because antisocial punishment is a strong inhibitor of cooperation.

  4. The effects of social isolation on steroid hormone levels are modulated by previous social status and context in a cichlid fish.

    Science.gov (United States)

    Galhardo, L; Oliveira, R F

    2014-01-01

    Social isolation is a major stressor which impacts the physiology, behaviour and health of individuals in gregarious species. However, depending on conditional and contextual factors, such as social status and group composition, social isolation may be perceived differently by different individuals or even by the same individuals at different times. Here we tested the effects of social status (territorial vs. non-territorial) and previous group composition (i.e. type of social group: mixed sex group with two territorial males, TT vs. mixed sex group with one territorial and one non-territorial male, TnT) on the hormonal response (androgens and cortisol) to social isolation in a cichlid fish (Oreochromis mossambicus). The different steroid hormones measured responded differentially to social isolation, and their response was modulated by social factors. Social isolation elicited a decrease of 11-keto formation only in territorial males, whereas non-territorial males present a non-significant trend for increasing KT levels. Testosterone did not respond to social isolation. Cortisol only increased in isolated individuals from TnT groups irrespective of social status (i.e. both in territorials and non-territorials). These results suggest that it is the perception of social isolation and not the objective structure of the situation that triggers the hormonal response to isolation. © 2013.

  5. Antigenic Relationships among Human Pathogenic Orientia tsutsugamushi Isolates from Thailand.

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    Sarah L James

    2016-06-01

    Full Text Available Scrub typhus is a common cause of undiagnosed febrile illness in certain tropical regions, but can be easily treated with antibiotics. The causative agent, Orientia tsutsugamushi, is antigenically variable which complicates diagnosis and efforts towards vaccine development.This study aimed to dissect the antigenic and genetic relatedness of O. tsutsugamushi strains and investigate sero-diagnostic reactivities by titrating individual patient sera against their O. tsutsugamushi isolates (whole-cell antigen preparation, in homologous and heterologous serum-isolate pairs from the same endemic region in NE Thailand. The indirect immunofluorescence assay was used to titrate Orientia tsutsugamushi isolates and human sera, and a mathematical technique, antigenic cartography, was applied to these data to visualise the antigenic differences and cross-reactivity between strains and sera. No functional or antigen-specific analyses were performed. The antigenic variation found in clinical isolates was much less pronounced than the genetic differences found in the 56kDa type-specific antigen genes. The Karp-like sera were more broadly reactive than the Gilliam-like sera.Antigenic cartography worked well with scrub typhus indirect immunofluorescence titres. The data from humoral responses suggest that a Karp-like strain would provide broader antibody cross-reactivity than a Gilliam-like strain. Although previous exposure to O. tsutsugamushi could not be ruled out, scrub typhus patient serum antibody responses were characterised by strong homologous, but weak heterologous antibody titres, with little evidence for cross-reactivity by Gilliam-like sera, but a broader response from some Karp-like sera. This work highlights the importance of antigenic variation in O. tsutsugamushi diagnosis and determination of new serotypes.

  6. Discovery of previously unidentified genomic disorders from the duplication architecture of the human genome

    NARCIS (Netherlands)

    Sharp, Andrew J.; Hansen, Sierra; Selzer, Rebecca R.; Cheng, Ze; Regan, Regina; Hurst, Jane A.; Stewart, Helen; Price, Sue M.; Blair, Edward; Hennekam, Raoul C.; Fitzpatrick, Carrie A.; Segraves, Rick; Richmond, Todd A.; Guiver, Cheryl; Albertson, Donna G.; Pinkel, Daniel; Eis, Peggy S.; Schwartz, Stuart; Knight, Samantha J. L.; Eichler, Evan E.

    2006-01-01

    Genomic disorders are characterized by the presence of flanking segmental duplications that predispose these regions to recurrent rearrangement. Based on the duplication architecture of the genome, we investigated 130 regions that we hypothesized as candidates for previously undescribed genomic

  7. Characterization of Candidate probionts isolated from human breast milk.

    Science.gov (United States)

    Khalkhali, S; Mojgani, N

    2017-05-20

    This study was designed to isolate and identify the potential probionts present in 32 healthy mothers' breast milk. Microbial culture media and 16SrRNA sequencing were used to isolate and identify the bacteria and all isolates were analyzed for their antagonistic potential, resistance to acidic pH, bile salts and survival under simulated gastric and intestinal conditions. The colonization potential was further assessed based on adherence to human enterocyte-like Caco-2 cell lines. The breast milk samples harbored significant numbers of Gram positive and catalase negative (85%) bacteria. Based on 16SrRNA sequencing, these isolates were identified as Lactobacillus casei, L.gasseri, L.fermentum, L.plantarum, Pediococcus acidilactici, and Enterococcus facieum. Among the isolates, P. acidilactici was the most frequent species (71%) present in these samples. Few Gram and catalase positive isolates, Staphylococcus aureus and S.hominiis were also observed. The isolates were viable and unviable in pH 3 and 1.5, respectively, while all isolates survived in 1.0% bile salt. As putative probionts, P.acidilactici 1C showed a significantly higher percentage of adhesion to Caco-2 cells (p< 0.05)than the other two isolates L.plantarum 7A and E.facieum 2C. Bacterial strains isolated from human breast milk were shown to have probiotic properties including anti-infective protection and may be considered as future therapeutics for infants.

  8. Draft Genome Sequence of Lactobacillus delbrueckii Strain #22 Isolated from a Patient with Short Bowel Syndrome and Previous d-Lactic Acidosis and Encephalopathy.

    Science.gov (United States)

    Domann, Eugen; Fischer, Florence; Glowatzki, Fabian; Fritzenwanker, Moritz; Hain, Torsten; Zechel-Gran, Silke; Giffhorn-Katz, Susanne; Neubauer, Bernd A

    2016-07-28

    d-Lactic acidosis with associated encephalopathy caused by overgrowth of intestinal lactic acid bacteria is a rarely diagnosed neurological complication of patients with short bowel syndrome. Here, we report the draft genome sequence of Lactobacillus delbrueckii strain #22 isolated from a patient with short bowel syndrome and previous d-lactic acidosis/encephalopathy. Copyright © 2016 Domann et al.

  9. Effect of previous exhaustive exercise on metabolism and fatigue development during intense exercise in humans

    DEFF Research Database (Denmark)

    Iaia, F. M.; Perez-Gomez, J.; Nordsborg, Nikolai

    2010-01-01

    The present study examined how metabolic response and work capacity are affected by previous exhaustive exercise. Seven subjects performed an exhaustive cycle exercise ( approximately 130%-max; EX2) after warm-up (CON) and 2 min after an exhaustive bout at a very high (VH; approximately 30 s), high...

  10. A simple method for human peripheral blood monocyte Isolation

    Directory of Open Access Journals (Sweden)

    Marcos C de Almeida

    2000-04-01

    Full Text Available We describe a simple method using percoll gradient for isolation of highly enriched human monocytes. High numbers of fully functional cells are obtained from whole blood or buffy coat cells. The use of simple laboratory equipment and a relatively cheap reagent makes the described method a convenient approach to obtaining human monocytes.

  11. Isolation and in vitro expansion of human colonic stem cells

    NARCIS (Netherlands)

    Jung, P.; Sato, T.; Merlos-Suarez, A.; Barriga, F.M.; Iglesias, M.; Rossell, D.; Auer, H.; Gallardo, M.; Blasco, M.A.; Sancho, E.; Clevers, H.; Batlle, E.

    2011-01-01

    Here we describe the isolation of stem cells of the human colonic epithelium. Differential cell surface abundance of ephrin type-B receptor 2 (EPHB2) allows the purification of different cell types from human colon mucosa biopsies. The highest EPHB2 surface levels correspond to epithelial colonic

  12. Sterols of Pneumocystis carinii hominis organisms isolated from human lungs

    DEFF Research Database (Denmark)

    Kaneshiro, E S; Amit, Z; Chandra, Jan Suresh

    1999-01-01

    in conjunction with analyses of chemically synthesized authentic standards. The sterol composition of isolated P. carinii hominis organisms has yet to be reported. If P. carinii from animal models is to be used for identifying potential drug targets and for developing chemotherapeutic approaches to clear human...... infections, it is important to determine whether the 24-alkylsterols of organisms found in rats are also present in organisms in humans. In the present study, sterol analyses of P. carinii hominis organisms isolated from cryopreserved human P. carinii-infected lungs and from bronchoalveolar lavage fluid were...

  13. Isolation & characterization of Brucella melitensis isolated from patients suspected for human brucellosis in India

    Directory of Open Access Journals (Sweden)

    Anita Barua

    2016-01-01

    Interpretation & conclusions: The present study showed an overall isolation rate of 17.64 per cent for B. melitensis. There is a need to establish facilities for isolation and characterization of Brucella species for effective clinical management of the disease among patients as well as surveillance and control of infection in domestic animals. Further studies are needed from different geographical areas of the country with different level of endemicity to plan and execute control strategies against human brucellosis.

  14. Discovery of previously unidentified genomic disorders from the duplication architecture of the human genome.

    Science.gov (United States)

    Sharp, Andrew J; Hansen, Sierra; Selzer, Rebecca R; Cheng, Ze; Regan, Regina; Hurst, Jane A; Stewart, Helen; Price, Sue M; Blair, Edward; Hennekam, Raoul C; Fitzpatrick, Carrie A; Segraves, Rick; Richmond, Todd A; Guiver, Cheryl; Albertson, Donna G; Pinkel, Daniel; Eis, Peggy S; Schwartz, Stuart; Knight, Samantha J L; Eichler, Evan E

    2006-09-01

    Genomic disorders are characterized by the presence of flanking segmental duplications that predispose these regions to recurrent rearrangement. Based on the duplication architecture of the genome, we investigated 130 regions that we hypothesized as candidates for previously undescribed genomic disorders. We tested 290 individuals with mental retardation by BAC array comparative genomic hybridization and identified 16 pathogenic rearrangements, including de novo microdeletions of 17q21.31 found in four individuals. Using oligonucleotide arrays, we refined the breakpoints of this microdeletion, defining a 478-kb critical region containing six genes that were deleted in all four individuals. We mapped the breakpoints of this deletion and of four other pathogenic rearrangements in 1q21.1, 15q13, 15q24 and 17q12 to flanking segmental duplications, suggesting that these are also sites of recurrent rearrangement. In common with the 17q21.31 deletion, these breakpoint regions are sites of copy number polymorphism in controls, indicating that these may be inherently unstable genomic regions.

  15. Contribution of Rare Copy Number Variants to Isolated Human Malformations

    Science.gov (United States)

    Serra-Juhé, Clara; Rodríguez-Santiago, Benjamín; Cuscó, Ivon; Vendrell, Teresa; Camats, Núria; Torán, Núria; Pérez-Jurado, Luis A.

    2012-01-01

    Background Congenital malformations are present in approximately 2–3% of liveborn babies and 20% of stillborn fetuses. The mechanisms underlying the majority of sporadic and isolated congenital malformations are poorly understood, although it is hypothesized that the accumulation of rare genetic, genomic and epigenetic variants converge to deregulate developmental networks. Methodology/Principal Findings We selected samples from 95 fetuses with congenital malformations not ascribed to a specific syndrome (68 with isolated malformations, 27 with multiple malformations). Karyotyping and Multiplex Ligation-dependent Probe Amplification (MLPA) discarded recurrent genomic and cytogenetic rearrangements. DNA extracted from the affected tissue (46%) or from lung or liver (54%) was analyzed by molecular karyotyping. Validations and inheritance were obtained by MLPA. We identified 22 rare copy number variants (CNV) [>100 kb, either absent (n = 7) or very uncommon (n = 15, malformations (21%), including 11 deletions and 11 duplications. One of the 9 tested rearrangements was de novo while the remaining were inherited from a healthy parent. The highest frequency was observed in fetuses with heart hypoplasia (8/17, 62.5%), with two events previously related with the phenotype. Double events hitting candidate genes were detected in two samples with brain malformations. Globally, the burden of deletions was significantly higher in fetuses with malformations compared to controls. Conclusions/Significance Our data reveal a significant contribution of rare deletion-type CNV, mostly inherited but also de novo, to human congenital malformations, especially heart hypoplasia, and reinforce the hypothesis of a multifactorial etiology in most cases. PMID:23056206

  16. Blastocystis phylogeny among various isolates from humans to insects.

    Science.gov (United States)

    Yoshikawa, Hisao; Koyama, Yukiko; Tsuchiya, Erika; Takami, Kazutoshi

    2016-12-01

    Blastocystis is a common unicellular eukaryotic parasite found not only in humans, but also in various kinds of animal species worldwide. Since Blastocystis isolates are morphologically indistinguishable, many molecular biological approaches have been applied to classify these isolates. The complete or partial sequences of the small subunit rRNA gene (SSU rDNA) are mainly used for comparisons and phylogenetic analyses among Blastocystis isolates. However, various lengths of the partial SSU rDNA sequence have been used for phylogenetic inference among genetically different isolates. Based on the complete SSU rDNA sequences, consensus terminology of nine subtypes (STs) of Blastocystis sp. that were supported by phylogenetically monophyletic nine clades was proposed in 2007. Thereafter, eight additional kinds of STs comprising non-human mammalian Blastocystis isolates have been reported based on the phylogeny of SSU rDNA sequences, while STs 11 and 12 were only proposed on the base of partial sequences. Although many sequence data from mammalian and avian Blastocystis are registered in GenBank, only limited data on SSU rDNA are available for poikilotherm-derived Blastocystis isolates. Therefore, the phylogenetic positions of the reptilian/amphibian Blastocystis clades are unstable. The phylogenetic inference of various STs comprising mammalian and/or avian Blastocystis isolates was verified herein based on comparisons between partial and complete SSU rDNA sequences, and the phylogenetic positions of reptilian and amphibian Blastocystis isolates were also investigated using 14 new Blastocystis isolates from reptiles with all known isolates from other reptilians, amphibians, and insects registered in GenBank. Copyright © 2016. Published by Elsevier Ireland Ltd.

  17. Molecular characterization of Cryptosporidium isolates from humans in Equatorial Guinea.

    Science.gov (United States)

    Blanco, María Alejandra; Iborra, Asunción; Vargas, Antonio; Nsie, Eugenia; Mbá, Luciano; Fuentes, Isabel

    2009-12-01

    The aim of the study was to perform a molecular characterization of clinical isolates of Cryptosporidium species from Equatorial Guinea. Standard laboratory methods were used to identify 35 cryptosporidiosis cases among 185 patients. PCR-RFLP successfully identified 34 Cryptosporidium species from these 35 cases, comprising C. parvum (52.9%), C. hominis (44.1%) and C. meleagridis (2.9%); over 90% of the species were isolated from HIV-positive patients. This is the first report of the molecular characterization of Cryptosporidium species isolated from humans in Equatorial Guinea and shows that zoonotic and anthroponotic transmission is present in this country.

  18. Isolation & characterization of Brucella melitensis isolated from patients suspected for human brucellosis in India

    Science.gov (United States)

    Barua, Anita; Kumar, Ashu; Thavaselvam, Duraipandian; Mangalgi, Smita; Prakash, Archana; Tiwari, Sapana; Arora, Sonia; Sathyaseelan, Kannusamy

    2016-01-01

    Background & objectives: Brucellosis is endemic in the southern part of India. A combination of biochemical, serological and molecular methods is required for identification and biotyping of Brucella. The present study describes the isolation and biochemical, molecular characterization of Brucella melitensis from patients suspected for human brucellosis. Methods: The blood samples were collected from febrile patients suspected to have brucellosis. A total of 18 isolates were obtained from 102 blood samples subjected to culture. The characterization of these 18 isolates was done by growth on Brucella specific medium, biochemical reactions, CO2 requirement, H2S production, agglutination with A and M mono-specific antiserum, dye sensitivity to basic fuchsin and thionin. Further, molecular characterization of the isolates was done by amplification of B. melitensis species specific IS711 repetitive DNA fragment and 16S (rRNA) sequence analysis. PCR-restriction fragment length polymorphism (RFLP) analysis of omp2 locus and IS711 gene was also done for molecular characterization. Results: All 102 suspected samples were subjected to bacteria isolation and of these, 18 isolates could be recovered on blood culture. The biochemical, PCR and PCR-RFLP and 16s rRNA sequencing revealed that all isolates were of B. melitensis and matched exactly with reference strain B. melitensis 16M. Interpretation & conclusions: The present study showed an overall isolation rate of 17.64 per cent for B. melitensis. There is a need to establish facilities for isolation and characterization of Brucella species for effective clinical management of the disease among patients as well as surveillance and control of infection in domestic animals. Further studies are needed from different geographical areas of the country with different level of endemicity to plan and execute control strategies against human brucellosis. PMID:27488010

  19. Antibiotic Resistance Escherichia coli isolated from Faecal of Healthy Human

    OpenAIRE

    , S. Budiarti

    2011-01-01

    The objective of this research was to examine antibiotic resistant of Escherechia coli as intestinal normal şora, isolated from healthy human. The samples were collected from faeces of new born children, children under 3 and 5years-old, and human adult. Bacteria were isolated at Eosin Methylen Blue solid media followed by biochemistry reaction for physiological E.coli identiŞcation. Antibiotic resistant test was carried out using Kirby-Bauer method. The result showed that 95 % bacterial strai...

  20. Isolation of human simple repeat loci by hybridization selection.

    Science.gov (United States)

    Armour, J A; Neumann, R; Gobert, S; Jeffreys, A J

    1994-04-01

    We have isolated short tandem repeat arrays from the human genome, using a rapid method involving filter hybridization to enrich for tri- or tetranucleotide tandem repeats. About 30% of clones from the enriched library cross-hybridize with probes containing trimeric or tetrameric tandem arrays, facilitating the rapid isolation of large numbers of clones. In an initial analysis of 54 clones, 46 different tandem arrays were identified. Analysis of these tandem repeat loci by PCR showed that 24 were polymorphic in length; substantially higher levels of polymorphism were displayed by the tetrameric repeat loci isolated than by the trimeric repeats. Primary mapping of these loci by linkage analysis showed that they derive from 17 chromosomes, including the X chromosome. We anticipate the use of this strategy for the efficient isolation of tandem repeats from other sources of genomic DNA, including DNA from flow-sorted chromosomes, and from other species.

  1. Subtyping of Salmonella enterica isolated from humans and food animals using Pulsed-Field Gel Electrophoresis

    Directory of Open Access Journals (Sweden)

    Golab, N.

    2016-07-01

    Full Text Available Salmonella infections are the second leading cause of zoonotic bacterial foodborne illness. Main source of infection in human is contaminated food products. The aim of this study was sub typing isolates of Salmonella enterica obtained during our previous study by Pulsed Field Gel Electrophoresis (PFGE technique. All 46 Salmonella isolates were serotyped and then subjected to PFGE. Total isolates were analyzed by means of the molecular technique XbaI PFGE. In this study, PFGE and serotyping were used to subtype 46 Salmonella isolates belonging to 27different serovars and derived from human and different food origins. Among these isolates, S. Typhimurium was found to be the most predominant serovar. 40 PFGE patterns out of 46 isolates were obtained. The Discrimination Index obtained by serotyping (DI = 0.93 was lower than PFGE (DI = 0.99. Subtyping of Salmonella enterica is very important and shows that animal origin can be one of a reservoir that potentially could be transferred to human through the food chain. In addition, results of this study also revealed that this procedure is a golden standard for genotyping of such salmonella serotypes.

  2. Teaching basic lung isolation skills on human anatomy simulator: attainment and retention of lung isolation skills.

    Science.gov (United States)

    Latif, Rana K; VanHorne, Edgar M; Kandadai, Sunitha Kanchi; Bautista, Alexander F; Neamtu, Aurel; Wadhwa, Anupama; Carter, Mary B; Ziegler, Craig H; Memon, Mohammed Faisal; Akça, Ozan

    2016-01-20

    Lung isolation skills, such as correct insertion of double lumen endobronchial tube and bronchial blocker, are essential in anesthesia training; however, how to teach novices these skills is underexplored. Our aims were to determine (1) if novices can be trained to a basic proficiency level of lung isolation skills, (2) whether video-didactic and simulation-based trainings are comparable in teaching lung isolation basic skills, and (3) whether novice learners' lung isolation skills decay over time without practice. First, five board certified anesthesiologist with experience of more than 100 successful lung isolations were tested on Human Airway Anatomy Simulator (HAAS) to establish Expert proficiency skill level. Thirty senior medical students, who were naive to bronchoscopy and lung isolation techniques (Novice) were randomized to video-didactic and simulation-based trainings to learn lung isolation skills. Before and after training, Novices' performances were scored for correct placement using pass/fail scoring and a 5-point Global Rating Scale (GRS); and time of insertion was recorded. Fourteen novices were retested 2 months later to assess skill decay. Experts' and novices' double lumen endobronchial tube and bronchial blocker passing rates showed similar success rates after training (P >0.99). There were no differences between the video-didactic and simulation-based methods. Novices' time of insertion decayed within 2 months without practice. Novices could be trained to basic skill proficiency level of lung isolation. Video-didactic and simulation-based methods we utilized were found equally successful in training novices for lung isolation skills. Acquired skills partially decayed without practice.

  3. Prevotella timonensis sp. nov., isolated from a human breast abscess.

    Science.gov (United States)

    Glazunova, Olga O; Launay, Thierry; Raoult, Didier; Roux, Véronique

    2007-04-01

    Gram-negative anaerobic rods were isolated from a human breast abscess. Based on genotypic and phenotypic characteristics, the novel strain belonged to the genus Prevotella. Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed that it was closely related to Prevotella buccalis (94 % 16S rRNA gene sequence similarity), Prevotella salivae (90 %) and Prevotella oris (89.1 %). The major cellular fatty acid was C(14 : 0) (19.5 %). The new isolate represents a novel species in the genus Prevotella, for which the name Prevotella timonensis sp. nov. is proposed. The type strain is strain 4401737(T) (=CIP 108522(T)=CCUG 50105(T)).

  4. Isolation and characterization of human apolipoprotein M-containing lipoproteins

    DEFF Research Database (Denmark)

    Christoffersen, Christina; Nielsen, Lars Bo; Axler, Olof

    2006-01-01

    Apolipoprotein M (apoM) is a novel apolipoprotein with unknown function. In this study, we established a method for isolating apoM-containing lipoproteins and studied their composition and the effect of apoM on HDL function. ApoM-containing lipoproteins were isolated from human plasma...... with immunoaffinity chromatography and compared with lipoproteins lacking apoM. The apoM-containing lipoproteins were predominantly of HDL size; approximately 5% of the total HDL population contained apoM. Mass spectrometry showed that the apoM-containing lipoproteins also contained apoJ, apoA-I, apoA-II, apoC-I, apo...

  5. Francisella tularensis subsp. novicida isolated from a human in Arizona

    Directory of Open Access Journals (Sweden)

    Birdsell Dawn N

    2009-11-01

    Full Text Available Abstract Background Francisella tularensis is the etiologic agent of tularemia and is classified as a select agent by the Centers for Disease Control and Prevention. Currently four known subspecies of F. tularensis that differ in virulence and geographical distribution are recognized:tularensis (type A, holarctica (type B, mediasiatica, and novicida. Because of the Select Agent status and differences in virulence and geographical location, the molecular analysis of any clinical case of tularemia is of particular interest. We analyzed an unusual Francisella clinical isolate from a human infection in Arizona using multiple DNA-based approaches. Findings We report that the isolate is F. tularensis subsp. novicida, a subspecies that is rarely isolated. Conclusion The rarity of this novicida subspecies in clinical settings makes each case study important for our understanding of its role in disease and its genetic relationship with other F. tularensis subspecies.

  6. The requirement for freshly isolated human colorectal cancer (CRC) cells in isolating CRC stem cells.

    Science.gov (United States)

    Fan, F; Bellister, S; Lu, J; Ye, X; Boulbes, D R; Tozzi, F; Sceusi, E; Kopetz, S; Tian, F; Xia, L; Zhou, Y; Bhattacharya, R; Ellis, L M

    2015-02-03

    Isolation of colorectal cancer (CRC) cell populations enriched for cancer stem cells (CSCs) may facilitate target identification. There is no consensus regarding the best methods for isolating CRC stem cells (CRC-SCs). We determined the suitability of various cellular models and various stem cell markers for the isolation of CRC-SCs. Established human CRC cell lines, established CRC cell lines passaged through mice, patient-derived xenograft (PDX)-derived cells, early passage/newly established cell lines, and cells directly from clinical specimens were studied. Cells were FAC-sorted for the CRC-SC markers CD44, CD133, and aldehyde dehydrogenase (ALDH). Sphere formation and in vivo tumorigenicity studies were used to validate CRC-SC enrichment. None of the markers studied in established cell lines, grown either in vitro or in vivo, consistently enriched for CRC-SCs. In the three other cellular models, CD44 and CD133 did not reliably enrich for stemness. In contrast, freshly isolated PDX-derived cells or early passage/newly established CRC cell lines with high ALDH activity formed spheres in vitro and enhanced tumorigenicity in vivo, whereas cells with low ALDH activity did not. PDX-derived cells, early passages/newly established CRC cell lines and cells from clinical specimen with high ALDH activity can be used to identify CRC-SC-enriched populations. Established CRC cell lines should not be used to isolate CSCs.

  7. Isolation and characterization of the human uracil DNA glycosylase gene

    International Nuclear Information System (INIS)

    Vollberg, T.M.; Siegler, K.M.; Cool, B.L.; Sirover, M.A.

    1989-01-01

    A series of anti-human placental uracil DNA glycosylase monoclonal antibodies was used to screen a human placental cDNA library in phage λgt11. Twenty-seven immunopositive plaques were detected and purified. One clone containing a 1.2-kilobase (kb) human cDNA insert was chosen for further study by insertion into pUC8. The resultant recombinant plasmid selected by hybridization a human placental mRNA that encoded a 37-kDa polypeptide. This protein was immunoprecipitated specifically by an anti-human placenta uracil DNA glycosylase monoclonal antibody. RNA blot-hybridization (Northern) analysis using placental poly(A) + RNA or total RNA from four different human fibroblast cell strains revealed a single 1.6-kb transcript. Genomic blots using DNA from each cell strain digested with either EcoRI or PstI revealed a complex pattern of cDNA-hydridizing restriction fragments. The genomic analysis for each enzyme was highly similar in all four human cell strains. In contrast, a single band was observed when genomic analysis was performed with the identical DNA digests with an actin gene probe. During cell proliferation there was an increase in the level of glycosylase mRNA that paralleled the increase in uracil DNA glycosylase enzyme activity. The isolation of the human uracil DNA glycosylase gene permits an examination of the structure, organization, and expression of a human DNA repair gene

  8. Protein biosynthesis in isolated human scalp hair follicles.

    Science.gov (United States)

    Vermorken, A J; Weterings, P J; Bloemendal, H

    1979-02-15

    The present study demonstrates that protein biosynthesis can be studied in single isolated human scalp hair follicles. The matrix and the sheath are the main regions where amino acids are built in. Incorporation is linear for at least five hours. The newly synthesized proteins can be separated into a water-soluble, a urea-soluble and a urea-insoluble fraction. Product analysis has been performed on the first two fractions, revealing different protein patterns.

  9. Color-Removal by Microorganisms Isolated from Human Hands

    Directory of Open Access Journals (Sweden)

    Tsukasa Ito

    2013-08-01

    Full Text Available Microorganisms are essential for human life. Microorganisms decompose the carbon compounds in dead animals and plants and convert them into carbon dioxide. Intestinal bacteria assist in food digestion. Some vitamins are produced by bacteria that live in the intestines. Sewage and industrial wastewater are treated by activated sludge composed of microbial communities. All of these are due to the ability of microbes to produce many enzymes that can degrade chemicals. How do teachers make students understand that microorganisms are always associated with humans, and that microorganisms have the ability to degrade chemicals? The presence of microorganisms on humans can be shown by incubating agar plates after they are touched by the hands of students. The ability of microorganisms to degrade chemicals can be shown by an analytical measurement of the degradation of chemicals. When the chemicals are dyes (colorants in water, microbial activity on degradation of dyes can be demonstrated by observing a decreasing degree of color as a result of the enzymatic activity (e.g., azoreductase. Dyes are widely used in the textile, food, and cosmetic industries. They are generally resistant to conventional biological wastewater treatment systems such as the activated sludge process (4. The discharge of wastewater containing dye pollutes surface water. The ability of microorganisms to decolorize and degrade dyes has been widely investigated to use for bioremediation purposes (5. The goal of this tip is to understand the presence of bacteria on human skin and the ability of bacteria to degrade colorant chemicals (decolorization. In this tip, students first cultivate and isolate bacteria on their hands, and then examine potential decolorization activity of each bacterium by observing the degree of color of the liquid in tubes in which bacteria isolated from students’ hands were inoculated. Decolorization activity of bacterial isolates from human skin has been

  10. Innate immune functions of microglia isolated from human glioma patients

    Directory of Open Access Journals (Sweden)

    Grimm Elizabeth

    2006-03-01

    Full Text Available Abstract Background Innate immunity is considered the first line of host defense and microglia presumably play a critical role in mediating potent innate immune responses to traumatic and infectious challenges in the human brain. Fundamental impairments of the adaptive immune system in glioma patients have been investigated; however, it is unknown whether microglia are capable of innate immunity and subsequent adaptive anti-tumor immune responses within the immunosuppressive tumor micro-environment of human glioma patients. We therefore undertook a novel characterization of the innate immune phenotype and function of freshly isolated human glioma-infiltrating microglia (GIM. Methods GIM were isolated by sequential Percoll purification from patient tumors immediately after surgical resection. Flow cytometry, phagocytosis and tumor cytotoxicity assays were used to analyze the phenotype and function of these cells. Results GIM expressed significant levels of Toll-like receptors (TLRs, however they do not secrete any of the cytokines (IL-1β, IL-6, TNF-α critical in developing effective innate immune responses. Similar to innate macrophage functions, GIM can mediate phagocytosis and non-MHC restricted cytotoxicity. However, they were statistically less able to mediate tumor cytotoxicity compared to microglia isolated from normal brain. In addition, the expression of Fas ligand (FasL was low to absent, indicating that apoptosis of the incoming lymphocyte population may not be a predominant mode of immunosuppression by microglia. Conclusion We show for the first time that despite the immunosuppressive environment of human gliomas, GIM are capable of innate immune responses such as phagocytosis, cytotoxicity and TLR expression but yet are not competent in secreting key cytokines. Further understanding of these innate immune functions could play a critical role in understanding and developing effective immunotherapies to malignant human gliomas.

  11. Epidemiological relationship of human and swine Streptococcus suis isolates.

    Science.gov (United States)

    Tarradas, C; Luque, I; de Andrés, D; Abdel-Aziz Shahein, Y E; Pons, P; González, F; Borge, C; Perea, A

    2001-06-01

    Two cases of meningitis due to Streptococcus suis in humans are reported here. A butcher and an abattoir worker were referred to a health centre in Castellón (Spain) with fever and symptoms of meningitis. After adequate treatment, a slight hipoacusia persisted as sequelae in both cases. Colonies of S. suis group R, serotype 2 and phenotype MRP+EF+ were isolated from cerebroespinal fluid. Epidemiological studies showed that both workers had in common the handling of pork meat of slaughtered healthy pigs from three closed farms. A study of the tonsils from apparently healthy, slaughtered pigs was carried out. A total of 234 tonsillar samples were obtained and 81 strains of S. suis were isolated from them. Serotype 2 appeared to be the most frequent (50.6%), and the analysis for phenotype showed a high percentage of tonsillar strains with the phenotype MRP+EF+ (35.9%). The humans and 28 tonsillar swine strains showed a similar profile (S. suis group R, serotype 2 and phenotype MRP+EF+). A total of 26 of the swine isolates were analysed by ribotyping using EcoRI. The human strains showed the same six-band hybridization pattern that shared five bands with the pattern most frequently shown by most of the tonsillar N. suis group R, serotype 2 and phenotype MRP+EF+ strains, differing only in the lightest, faintest band which was slightly less anodical in human (> or = 1.8 kb) than in swine (approximately 1.8 kb). From these results, both groups of strains, humans and porcine, showed differences; how can these differences in the pattern of ribotyping be explained if they should have the same origin? Is it possible that they have undergone an adaptation to the new host or perhaps the modification is due to other unknown causes? Further studies in this area are required in order to answer these questions.

  12. Isolation and characterization of human articular chondrocytes from surgical waste after total knee arthroplasty (TKA

    Directory of Open Access Journals (Sweden)

    Jakob Naranda

    2017-03-01

    Full Text Available Background Cartilage tissue engineering is a fast-evolving field of biomedical engineering, in which the chondrocytes represent the most commonly used cell type. Since research in tissue engineering always consumes a lot of cells, simple and cheap isolation methods could form a powerful basis to boost such studies and enable their faster progress to the clinics. Isolated chondrocytes can be used for autologous chondrocyte implantation in cartilage repair, and are the base for valuable models to investigate cartilage phenotype preservation, as well as enable studies of molecular features, nature and scales of cellular responses to alterations in the cartilage tissue. Methods Isolation and consequent cultivation of primary human adult articular chondrocytes from the surgical waste obtained during total knee arthroplasty (TKA was performed. To evaluate the chondrogenic potential of the isolated cells, gene expression of collagen type 2 (COL2, collagen 1 (COL1 and aggrecan (ACAN was evaluated. Immunocytochemical staining of all mentioned proteins was performed to evaluate chondrocyte specific production. Results Cartilage specific gene expression of COL2 and ACAN has been shown that the proposed protocol leads to isolation of cells with a high chondrogenic potential, possibly even specific phenotype preservation up to the second passage. COL1 expression has confirmed the tendency of the isolated cells dedifferentiation into a fibroblast-like phenotype already in the second passage, which confirms previous findings that higher passages should be used with care in cartilage tissue engineering. To evaluate the effectiveness of our approach, immunocytochemical staining of the evaluated chondrocyte specific products was performed as well. Discussion In this study, we developed a protocol for isolation and consequent cultivation of primary human adult articular chondrocytes with the desired phenotype from the surgical waste obtained during TKA. TKA is a

  13. Azoreductase activity of anaerobic bacteria isolated from human intestinal microflora.

    Science.gov (United States)

    Rafii, F; Franklin, W; Cerniglia, C E

    1990-01-01

    A plate assay was developed for the detection of anaerobic bacteria that produce azoreductases. With this plate assay, 10 strains of anaerobic bacteria capable of reducing azo dyes were isolated from human feces and identified as Eubacterium hadrum (2 strains), Eubacterium spp. (2 species), Clostridium clostridiiforme, a Butyrivibrio sp., a Bacteroides sp., Clostridium paraputrificum, Clostridium nexile, and a Clostridium sp. The average rate of reduction of Direct Blue 15 dye (a dimethoxybenzidine-based dye) in these strains ranged from 16 to 135 nmol of dye per min per mg of protein. The enzymes were inactivated by oxygen. In seven isolates, a flavin compound (riboflavin, flavin adenine dinucleotide, or flavin mononucleotide) was required for azoreductase activity. In the other three isolates and in Clostridium perfringens, no added flavin was required for activity. Nondenaturing polyacrylamide gel electrophoresis showed that each bacterium expressed only one azoreductase isozyme. At least three types of azoreductase enzyme were produced by the different isolates. All of the azoreductases were produced constitutively and released extracellularly. Images PMID:2202258

  14. Azoreductase activity of anaerobic bacteria isolated from human intestinal microflora.

    Science.gov (United States)

    Rafii, F; Franklin, W; Cerniglia, C E

    1990-07-01

    A plate assay was developed for the detection of anaerobic bacteria that produce azoreductases. With this plate assay, 10 strains of anaerobic bacteria capable of reducing azo dyes were isolated from human feces and identified as Eubacterium hadrum (2 strains), Eubacterium spp. (2 species), Clostridium clostridiiforme, a Butyrivibrio sp., a Bacteroides sp., Clostridium paraputrificum, Clostridium nexile, and a Clostridium sp. The average rate of reduction of Direct Blue 15 dye (a dimethoxybenzidine-based dye) in these strains ranged from 16 to 135 nmol of dye per min per mg of protein. The enzymes were inactivated by oxygen. In seven isolates, a flavin compound (riboflavin, flavin adenine dinucleotide, or flavin mononucleotide) was required for azoreductase activity. In the other three isolates and in Clostridium perfringens, no added flavin was required for activity. Nondenaturing polyacrylamide gel electrophoresis showed that each bacterium expressed only one azoreductase isozyme. At least three types of azoreductase enzyme were produced by the different isolates. All of the azoreductases were produced constitutively and released extracellularly.

  15. Acrolein generation stimulates hypercontraction in isolated human blood vessels

    International Nuclear Information System (INIS)

    Conklin, D.J.; Bhatnagar, A.; Cowley, H.R.; Johnson, G.H.; Wiechmann, R.J.; Sayre, L.M.; Trent, M.B.; Boor, P.J.

    2006-01-01

    Increased risk of vasospasm, a spontaneous hyperconstriction, is associated with atherosclerosis, cigarette smoking, and hypertension-all conditions involving oxidative stress, lipid peroxidation, and inflammation. To test the role of the lipid peroxidation- and inflammation-derived aldehyde, acrolein, in human vasospasm, we developed an ex vivo model using human coronary artery bypass graft (CABG) blood vessels and a demonstrated acrolein precursor, allylamine. Allylamine induces hypercontraction in isolated rat coronary artery in a semicarbazide-sensitive amine oxidase activity (SSAO) dependent manner. Isolated human CABG blood vessels (internal mammary artery, radial artery, saphenous vein) were used to determine: (1) vessel responses and sensitivity to acrolein, allylamine, and H 2 O 2 exposure (1 μM-1 mM), (2) SSAO dependence of allylamine-induced effects using SSAO inhibitors (semicarbazide, 1 mM; MDL 72274-E, active isomer; MDL 72274-Z, inactive isomer; 100 μM), (3) the vasoactive effects of two other SSAO amine substrates, benzylamine and methylamine, and (4) the contribution of extracellular Ca 2+ to hypercontraction. Acrolein or allylamine but not H 2 O 2 , benzylamine, or methylamine stimulated spontaneous and pharmacologically intractable hypercontraction in CABG blood vessels that was similar to clinical vasospasm. Allylamine-induced hypercontraction and blood vessel SSAO activity were abolished by pretreatment with semicarbazide or MDL 72274-E but not by MDL 72274-Z. Allylamine-induced hypercontraction also was significantly attenuated in Ca 2+ -free buffer. In isolated aorta of spontaneously hypertensive rat, allylamine-induced an SSAO-dependent contraction and enhanced norepinephrine sensitivity but not in Sprague-Dawley rat aorta. We conclude that acrolein generation in the blood vessel wall increases human susceptibility to vasospasm, an event that is enhanced in hypertension

  16. Wide-field spectral imaging of human ovary autofluorescence and oncologic diagnosis via previously collected probe data

    Science.gov (United States)

    Renkoski, Timothy E.; Hatch, Kenneth D.; Utzinger, Urs

    2012-03-01

    With no sufficient screening test for ovarian cancer, a method to evaluate the ovarian disease state quickly and nondestructively is needed. The authors have applied a wide-field spectral imager to freshly resected ovaries of 30 human patients in a study believed to be the first of its magnitude. Endogenous fluorescence was excited with 365-nm light and imaged in eight emission bands collectively covering the 400- to 640-nm range. Linear discriminant analysis was used to classify all image pixels and generate diagnostic maps of the ovaries. Training the classifier with previously collected single-point autofluorescence measurements of a spectroscopic probe enabled this novel classification. The process by which probe-collected spectra were transformed for comparison with imager spectra is described. Sensitivity of 100% and specificity of 51% were obtained in classifying normal and cancerous ovaries using autofluorescence data alone. Specificity increased to 69% when autofluorescence data were divided by green reflectance data to correct for spatial variation in tissue absorption properties. Benign neoplasm ovaries were also found to classify as nonmalignant using the same algorithm. Although applied ex vivo, the method described here appears useful for quick assessment of cancer presence in the human ovary.

  17. Isolation of Mesenchymal Stromal Cells (MSCs from Human Adenoid Tissue

    Directory of Open Access Journals (Sweden)

    Yoon Se Lee

    2013-04-01

    Full Text Available Background: Mesenchymal stromal cells (MSCs are multipotent progenitor cells that originally derived from bone marrow. Clinical use of bone marrow-derived MSC is difficult due to morbidity and low MSC abundance and isolation efficiency. Recently, MSCs have been isolated from various adult tissues. Here we report the isolation of adenoid tissue-derived MSCs (A-MSCs and their characteristics. Methods: We compared the surface markers, morphologies, and differentiation and proliferation capacities of previously established tonsil-derived MSCs (T-MSCs and bone marrow-derived MSCs (BM-MSCs with cells isolated from adenoid tissue. The immunophenotype of A-MSCs was investigated upon interferon (IFN-γ stimulation. Results: A-MSCs, T-MSCs, and BM-MSCs showed negative CD45, CD31 HLA-DR, CD34, CD14, CD19 and positive CD 90, CD44, CD73, CD105 expression. A-MSCs were fibroblast-like, spindle-shaped non-adherent cells, similar to T-MSCs and BM-MSCs. Adipogenesis was observed in A-MSCs by the formation of lipid droplets after Oil Red O staining. Osteogenesis was observed by the formation of the matrix mineralization in Alizarin Red staining. Chondrogenesis was observed by the accumulation of sulfated glycosaminoglycan-rich matrix in collagen type II staining. These data were similar to those of T-MSCs and BM-MSCs. Expression of marker genes (i.e., adipogenesis; lipoprotein lipase, proliferator-activator receptor-gamma, osteogenesis; osteocalcin, alkaline phasphatase, chondrogenesis; aggrecan, collagen type II α1 in A-MSCs were not different from those in T-MSCs and BM-MSCs. Conclusions: A-MSCs possess the characteristics of MSCs in terms of morphology, multipotent differentiation capacity, cell surface markers, and immunogeneity. Therefore, A-MSCs fulfill the definition of MSCs and represent an alternate source of MSCs.

  18. Staphylococcus massiliensis sp. nov., isolated from a human brain abscess.

    Science.gov (United States)

    Al Masalma, Mouhamad; Raoult, Didier; Roux, Véronique

    2010-05-01

    Gram-positive, catalase-positive, coagulase-negative, non-motile, non-fermentative and novobiocin-susceptible cocci were isolated from a human brain abscess sample (strain 5402776(T)). This novel strain was analysed by a polyphasic taxonomic approach. The respiratory quinones detected were MK-7 (93 %) and MK-6 (7 %) and the major fatty acids were C(15 : 0) iso (60.5 %), C(17 : 0) iso (8.96 %) C(15 : 0) anteiso (7.93 %) and C(19 : 0) iso (6.78 %). The peptidoglycan type was A3alpha l-Lys-Gly(2-3)-l-Ser-Gly. Based on cellular morphology and biochemical criteria, the new isolate was assigned to the genus Staphylococcus, although it did not correspond to any recognized species. The G+C content of the DNA was 36.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed that the new isolate was most closely related to Staphylococcus piscifermentans, Staphylococcus condimenti, Staphylococcus carnosus subsp. carnosus, S. carnosus subsp. utilis and Staphylococcus simulans (97.7 %, 97.6 %, 97.6 %, 97.6 % and 96.5 % sequence similarity, respectively). Comparison of tuf, hsp60, rpoB, dnaJ and sodA gene sequences was also performed. In phylogenetic analysis inferred from tuf, dnaJ and rpoB gene sequence comparisons, strain 5402776(T) clustered with Staphylococcus pettenkoferi (93.7 %, 82.5 % and 89 % sequence similarity, respectively) and on phylogenetic analysis inferred from sodA gene sequence comparisons, it clustered with Staphylococcus chromogenes (82.8 %). On the basis of phenotypic and genotypic data, this isolate represents a novel species for which the name Staphylococcus massiliensis sp. nov. is proposed (type strain 5402776(T)=CCUG 55927(T)=CSUR P23(T)).

  19. Characterization of Listeria monocytogenes isolated from Ganges water, human clinical and milk samples at Varanasi, India.

    Science.gov (United States)

    Soni, Dharmendra K; Singh, Rakesh K; Singh, Durg V; Dubey, Suresh K

    2013-03-01

    Listeria monocytogenes isolated from Ganges water, human clinical and milk samples were characterized by antibiotic susceptibility, serotype identification, detection of virulence genes and ERIC- and REP-PCR fingerprint analyses. All isolates were uniformly resistant to ampicillin, except two isolates, and showed variable resistance to gentamicin, cotrimoxazole, ofloxacin, rifampicin and tetracycline. Of the 20 isolates found positive for pathogens, seven (four human and three water isolates) belong to serogroups 4b, 4d and 4e; six (one human and five water isolates) belong to serogroups 1/2c and 3c; four milk isolates belong to serogroups 1/2b and 3b; and three milk isolates belong to serogroups 1/2a and 3a. Two water isolates, all human isolates, except one (Pb1) lacking inlJ gene, and three milk isolates possess inlA, inlC, plcA, prfA, actA, hlyA and iap genes. The remaining water and milk isolates showed variable presence of inlJ, plcA, prfA, and iap genes. ERIC- and REP-PCR based analyses collectively indicated that isolates of human clinical samples belong to identical or similar clone and isolates of water and milk samples belong to different clones. Overall study demonstrates the prevalence of pathogenic L. monocytogenes species in the environmental and clinical samples. Most of the isolates were resistant to commonly used antibiotics. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Similarities between Salmonella Enteritidis isolated from humans and captive wild animals in South Africa.

    Science.gov (United States)

    Smith, Anthony M; Ismail, Husna; Henton, Maryke M; Keddy, Karen H

    2014-12-15

    Salmonella is well recognized as an aetiological agent of gastrointestinal and diarrhoeal disease. Salmonella enterica serotype Enteritidis (Salmonella Enteritidis) is one of the commonest serotypes associated with foodborne illness. In South Africa, we compared Salmonella Enteritidis strains isolated from humans with gastroenteritis and strains isolated from captive wild animals, between June 2011 and July 2012. Bacteria were phenotypically characterized using standard microbiological techniques. Genotypic relatedness of isolates was investigated by pulsed-field gel electrophoresis (PFGE) analysis. a diversity of 27 PFGE patterns amongst 196 human non-invasive isolates was shown; two PFGE patterns predominated and accounted for 74% of all human isolates. Human isolates showed a 12% prevalence rate for nalidixic acid resistance. Animal isolates from 5 different sources were investigated. With the exception of an isolate from a ground hornbill, all animal isolates (jaguar, crocodile, lion and poultry) showed PFGE pattern matches to a human isolate. Animal isolates showed susceptibility to all antimicrobial agents tested, with the exception of nalidixic acid resistance in isolates from the lion and poultry source. Our data showed similarities between Salmonella Enteritidis strains isolated from humans and captive wild animals, suggesting a probable common source for strains from humans and animals.

  1. Genetic diversity of human isolates of Salmonella enterica serovar Enteritidis in Malaysia.

    Science.gov (United States)

    Bakeri, S A; Yasin, R M; Koh, Y T; Puthucheary, S D; Thong, K L

    2003-01-01

    The study was undertaken to determine clonal relationship and genetic diversity of the human strains of Salmonella enterica serovar Enteritidis isolated from 1995 to 2002 from different parts of Malaysia. Antimicrobial susceptibility test, plasmid profiling and pulsed-field gel electrophoresis were applied to analyse 65 human isolates of S. Enteritidis obtained over an eight year period from different parts of Malaysia. Four nonhuman isolates were included for comparison. A total of 14 distinct XbaI-pulsed-field profiles (PFPs) were observed, although a single PFP X1 was predominant and this particular clone was found to be endemic in Malaysia. The incidence of drug resistant S. Enteritidis remained relatively low with only 37% of the strains analysed being resistant to one or more antimicrobial agents. All except one resistant strain carried at least one plasmid ranging in size from 3.7 to 62 MDa giving nine plasmid profiles. The three isolates from raw milk and one from well-water had similar PFPs to that of the human isolates. Salmonella Enteritidis strains were more diverse than was previously thought. Fourteen subtypes were noted although one predominant clone persisted in Malaysia. The combination of pulsed-field gel electrophoresis, plasmid profiling and antibiograms provided additional discrimination to the highly clonal strains of S. Enteritidis. This is the first report to assess the genotypes of the predominant clinical S. Enteritidis in different parts of the country. As S. Enteritidis is highly endemic in Malaysia, the data generated would be useful for tracing the source during outbreaks of gastroenteritis in the study area.

  2. Data on isolating mesenchymal stromal cells from human adipose tissue using a collagenase-free method

    Directory of Open Access Journals (Sweden)

    Wassim Shebaby

    2016-03-01

    Full Text Available The present dataset describes a detailed protocol to isolate mesenchymal cells from human fat without the use of collagenase. Human fat specimen, surgically cleaned from non-fat tissues (e.g., blood vessels and reduced into smaller fat pieces of around 1–3 mm size, is incubated in complete culture media for five to seven days. Then, cells started to spread out from the fat explants and to grow in cultures according to an exponential pattern. Our data showed that primary mesenchymal cells presenting heterogeneous morphology start to acquire more homogenous fibroblastic-like shape when cultured for longer duration or when subcultured into new flasks. Cell isolation efficiency as well as cell doubling time were also calculated throughout the culturing experimentations and illustrated in a separate figure thereafter. This paper contains data previously considered as an alternative protocol to isolate adipose-derived mesenchymal stem cell published in “Proliferation and differentiation of human adipose-derived mesenchymal stem cells (ASCs into osteoblastic lineage are passage dependent” [1]. Keywords: Adipose tissue, mesenchymal stromal cell, cell culture, doubling time

  3. DETECTION AND ISOLATION OF CD59 FROM HUMAN SEMINAL PLASMA

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    A REZAIE

    2002-06-01

    Full Text Available Introduction. CD59 is one of the complement regulatory proteins (CRPs which exert inhibitory function by blocking the formation of C5b-9 complex or Membrane Attacks complex (MAC. Regarding the therapeutic role of CD59 in treatment of pathological effects in uncontrolled activation of complements system and its efficiency to overcome the hyper-acute rejection, CD59 was suggested for maintenance of transplanted organ. In this study We determined and isolated CD59 from seminal plasma. Methods. Six normospermic sample according to WHO standards were chosen. Plasma of samples was separated and to remove the postasomes, the seminal plama was ultra centrifuged. CD59 was detected by Dot-Blot using CD59 mAb (MEM43. The molecular weight and purity of protein was detected by SOS-PAGE method follwed by Westerm Blot. Results. Protein was present in the 6.5 ml and 15ml of gel fitration fractions. Molecular weight based on marker size in these two fractions was 65 and 21KD respectively. Discussion. CD59 had previously beem purified by lysis of erythrocyte cell membrane. Because of use of detergent and preservative agents, this method decreased physiologic effects of the protein. In this study the isolation was performed from prostasome granules" without using of any detergent and preservative agents.

  4. Isolation of human foetal myoblasts and its application for microencapsulation

    Science.gov (United States)

    Li, Anna Aihua; Bourgeois, Jacqueline; Potter, Murray; Chang, Patricia L

    2008-01-01

    Abstract Foetal cells secrete more growth factors, generate less immune response, grow and proliferate better than adult cells. These characteristics make them desirable for recombinant modification and use in microencapsulated cellular gene therapeutics. We have established a system in vitro to obtain a pure population of primary human foetal myoblasts under several rounds of selection with non-collagen coated plates and identified by desmin staining. These primary myoblasts presented good proliferation ability and better differentiation characteristics in monolayer and after microencapsulation compared to murine myoblast C2C12 cells based on creatine phosphokinase (CPK), major histocompatibility complex (MHC) and multi-nucleated myotubule determination. The lifespan of primary myoblasts was 70 population doublings before entering into senescent state, with a population time of 18–24 hrs. Hence, we have developed a protocol for isolating human foetal primary myoblasts with excellent differentiation potential and robust growth and longevity. They should be useful for cell-based therapy in human clinical applications with microencapsulation technology. PMID:18366454

  5. Isolation of Mesenchymal Stem Cells from Human Deciduous Teeth Pulp

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    Aileen I. Tsai

    2017-01-01

    Full Text Available This study aimed to identify predictors of success rate of mesenchymal stem cell (MSC isolation from human deciduous teeth pulp. A total of 161 deciduous teeth were extracted at the dental clinic of Chang Gung Memorial Hospital. The MSCs were isolated from dental pulps using a standard protocol. In total, 128 colonies of MSCs were obtained and the success rate was 79.5%. Compared to teeth not yielding MSCs successfully, those successfully yielding MSCs were found to have less severe dental caries (no/mild-to-moderate/severe: 63.3/24.2/12.5% versus 12.5/42.4/42.4%, P<0.001 and less frequent pulpitis (no/yes: 95.3/4.7% versus 51.5/48.5%, P<0.001. In a multivariate regression model, it was confirmed that the absence of dental caries (OR = 4.741, 95% CI = 1.564–14.371, P=0.006 and pulpitis (OR = 9.111, 95% CI = 2.921–28.420, P<0.001 was significant determinants of the successful procurement of MSCs. MSCs derived from pulps with pulpitis expressed longer colony doubling time than pulps without pulpitis. Furthermore, there were higher expressions of proinflammatory cytokines, interleukin- (IL- 6 and monocyte chemoattractant protein- (MCP- 1, P<0.01, and innate immune response [toll-like receptor 1 (TLR1 and TLR8, P<0.05; TLR2, TLR3, and TLR6, P<0.01] in the inflamed than noninflamed pulps. Therefore, a carious deciduous tooth or tooth with pulpitis was relatively unsuitable for MSC processing and isolation.

  6. Biofilm formation in Hafnia alvei HUMV-5920, a human isolate

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    Itziar Chapartegui-González

    2016-11-01

    Full Text Available Hafnia alvei is a Gram-negative, rodshaped, facultative anaerobic bacterium of the family Enterobacteriaceae that has been isolated from various mammals, fish, insects and birds. In humans, case reports of Hafnia-associated enteric infections have been chiefly reported in Spain. Although H. alvei shares some virulence mechanisms with other Gram-negative enteropathogens little is known about the factors that contribute to its pathogenesis or virulence factors and regulatory circuits that may enhance the establishment and survival of H. alvei in the environment. The goal of the present study was to analyze the capacity of a H. alvei clinical isolate (strain HUMV-5920 to form biofilms. Biofilm formation by this strain increases during growth at 28 °C compared to 37 °C. Investigation of multicellular behavior by confocal microscopy, crystal violet and calcofluor staining in this strain showed biofilm formation associated with the production of cellulose. Importantly, several genes related to cellulose production including bcsABZC and yhjQ are present in the H. alvei HUMV-5920 chromosome. The ability of H. alvei to adhere to abiotic surfaces and to form biofilms likely contributes to its persistence in the hospital environment or food processing environments, increasing the probability of causing infections. Therefore, a better understanding of the adherence properties of this species will provide greater insights into the diseases it causes.

  7. Candida nivariensis isolated from an Indonesian human immunodeficiency virus-infected patient suffering from oropharyngeal candidiasis

    NARCIS (Netherlands)

    Wahyuningsih, Retno; SahBandar, Ivo N.; Theelen, Bart; Hagen, Ferry; Poot, Ge; Meis, Jacques F.; Rozalyani, Anna; Sjam, Ridhawati; Widodo, Djoko; Djauzi, Samsuridjal; Boekhout, Teun

    Candida nivariensis was isolated from an Indonesian human immunodeficiency virus-infected patient who suffered from oropharyngeal candidiasis and was identified with molecular tools. Our isolate demonstrated low MICs to amphotericin B, flucytosine, posaconazole, caspofungin, and isavueonazole and

  8. Candida nivariensis isolated from an Indonesian human immunodeficiency virus-infected patient suffering from oropharyngeal candidiasis.

    NARCIS (Netherlands)

    Wahyuningsih, R.; SahBandar, IN; Theelen, B.; Hagen, F.; Poot, G.; Meis, J.F.; Rozalyani, A.; Sjam, R.; Widodo, D.; Djauzi, S.; Boekhout, T.

    2008-01-01

    Candida nivariensis was isolated from an Indonesian human immunodeficiency virus-infected patient who suffered from oropharyngeal candidiasis and was identified with molecular tools. Our isolate demonstrated low MICs to amphotericin B, flucytosine, posaconazole, caspofungin, and isavuconazole and

  9. Prevotella maculosa sp. nov., isolated from the human oral cavity.

    Science.gov (United States)

    Downes, Julia; Sutcliffe, Iain C; Booth, Veronica; Wade, William G

    2007-12-01

    Three strains of anaerobic Gram-negative bacilli isolated from human oral sites were subjected to a comprehensive range of phenotypic and genotypic tests and were found to comprise a homogeneous group. 16S rRNA gene sequence analysis revealed the strains to constitute a novel group within the genus Prevotella, most closely related to Prevotella oris and Prevotella salivae. A novel species, Prevotella maculosa sp. nov., is proposed to accommodate these strains. Prevotella maculosa is saccharolytic and produces acetic and succinic acids as end products of fermentation. The G+C content of the DNA of the type strain is 48 mol%. The type strain of Prevotella maculosa is W1609(T) (=DSM 19339(T) =CCUG 54766(T)).

  10. Isolation and Characterization of Human Lung Lymphatic Endothelial Cells

    Science.gov (United States)

    Lorusso, Bruno; Falco, Angela; Madeddu, Denise; Frati, Caterina; Cavalli, Stefano; Graiani, Gallia; Gervasi, Andrea; Rinaldi, Laura; Lagrasta, Costanza; Maselli, Davide; Gnetti, Letizia; Silini, Enrico M.; Quaini, Eugenio; Ampollini, Luca; Carbognani, Paolo; Quaini, Federico

    2015-01-01

    Characterization of lymphatic endothelial cells from the respiratory system may be crucial to investigate the role of the lymphatic system in the normal and diseased lung. We describe a simple and inexpensive method to harvest, isolate, and expand lymphatic endothelial cells from the human lung (HL-LECs). Fifty-five samples of healthy lung selected from patients undergoing lobectomy were studied. A two-step purification tool, based on paramagnetic sorting with monoclonal antibodies to CD31 and Podoplanin, was employed to select a pure population of HL-LECs. The purity of HL-LECs was assessed by morphologic criteria, immunocytochemistry, flow cytometry, and functional assays. Interestingly, these cells retain in vitro several receptor tyrosine kinases (RTKs) implicated in cell survival and proliferation. HL-LECs represent a clinically relevant cellular substrate to study lymphatic biology, lymphoangiogenesis, interaction with microbial agents, wound healing, and anticancer therapy. PMID:26137493

  11. Comparative genomics of pathogenic Leptospira interrogans serovar Canicola isolated from swine and human in Brazil

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    Luisa Z Moreno

    Full Text Available Leptospira interrogans serovar Canicola is one of the most important pathogenic serovars for the maintenance of urban leptospirosis. Even though it is considered highly adapted to dogs, serovar Canicola infection has already been described in other animals and even a few human cases. Here, we present the genomic characterisation of two Brazilian L. interrogans serovar Canicola strains isolated from slaughtered sows (L0-3 and L0-4 and their comparison with human strain Fiocruz LV133. It was observed that the porcine serovar Canicola strains present the genetic machinery to cause human infection and, therefore, represent a higher risk to public health. Both human and porcine serovar Canicola isolates also presented sequences with high identity to the Chinese serovar Canicola published plasmids pGui1 and pGui2. The plasmids identification in the Brazilian and Chinese serovar Canicola strains suggest that extra-chromosomal elements are one more feature of this serovar that was previously unnoticed.

  12. Isolation and characterization of human umbilical cord-derived endothelial colony-forming cells

    Science.gov (United States)

    Zhang, Hao; Tao, Yanling; Ren, Saisai; Liu, Haihui; Zhou, Hui; Hu, Jiangwei; Tang, Yongyong; Zhang, Bin; Chen, Hu

    2017-01-01

    Endothelial colony-forming cells (ECFCs) are a population of endothelial progenitor cells (EPCs) that display robust proliferative potential and vessel-forming capability. Previous studies have demonstrated that a limited number of ECFCs may be obtained from adult bone marrow, peripheral blood and umbilical cord (UC) blood. The present study describes an effective method for isolating ECFCs from human UC. The ECFCs derived from human UC displayed the full properties of EPCs. Analysis of the growth kinetics, cell cycle and colony-forming ability of the isolated human UC-ECFCs indicated that the cells demonstrated properties of stem cells, including relative stability and rapid proliferation in vitro. Gene expression of Fms related tyrosine kinase 1, kinase insert domain receptor, vascular endothelial cadherin, cluster of differentiation (CD)31, CD34, epidermal growth factor homology domains-2, von Willebrand factor and endothelial nitric oxide synthase was assessed by reverse transcription-polymerase chain reaction. The cells were positive for CD34, CD31, CD73, CD105 and vascular endothelial growth factor receptor-2, and negative for CD45, CD90 and human leukocyte antigen-antigen D related protein according to flow cytometry. 1,1′-dioctadecyl-3,3,3′,3′-tetra-methyl-indocarbocyanine perchlorate-labeled acetylated low-density lipoprotein and fluorescein isothiocyanate-Ulex europaeus-l were used to verify the identity of the UC-ECFCs. Matrigel was used to investigate tube formation capability. The results demonstrated that the reported technique is a valuable method for isolating human UC-ECFCs, which have potential for use in vascular regeneration. PMID:29067104

  13. Comparison of phenotypic and virulence genes characteristics in human and chicken isolates of Proteus mirabilis.

    Science.gov (United States)

    Barbour, Elie K; Hajj, Zahi G; Hamadeh, Shadi; Shaib, Houssam A; Farran, Mohamad T; Araj, George; Faroon, Obaid; Barbour, Kamil E; Jirjis, Faris; Azhar, Esam; Kumosani, Taha; Harakeh, Steve

    2012-10-01

    The objective of this work is to compare the phenotypic and virulence genes characteristics in human and chicken isolates of Proteus mirabilis. The bacterial examination of 50 livers of individual broilers, marketed by four major outlets, revealed a high recovery of P. mirabilis (66%), and a low recovery frequency of Salmonella spp. (4%), Serratia odorifera (2%), Citrobacter brakii (2%), and Providencia stuartii (2%). The phenotypic biochemical characterization of the recovered 33 chicken isolates of P. mirabilis were compared to 30 human isolates (23 urinary and six respiratory isolates). The comparison revealed significant differences in the presence of gelatinase enzyme (100% presence in chicken isolates versus 91.3 and 83.3% presence in human urinary and respiratory isolates, respectively, P,0.05). The H(2)S production occurred in 100% of chicken isolates versus 95.6 and 66.7% presence in human urinary and respiratory isolates, respectively, P,0.05). The other 17 biochemical characteristics did not differ significantly among the three groups of isolates (P.0.05). Two virulence genes, the mrpA and FliL, were having a typical 100% presence in randomly selected isolates of P. mirabilis recovered from chicken livers (N510) versus isolates recovered from urinary (N55) and respiratory specimens of humans (N55) (P.0.05). The average percentage similarity of mrpA gene nucleotide sequence of poultry isolates to human urinary and respiratory isolates was 93.2 and 97.5-%, respectively. The high similarity in phenotypic characteristics, associated with typical frequency of presence of two virulence genes, and high similarity in sequences of mrpA gene among poultry versus human P. mirabilis isolates justifies future investigations targeting the evaluation of adaptable pathogenicity of avian Proteus mirabilis isolates to mammalian hosts.

  14. Mycobacterium talmoniae sp. nov., a slowly growing mycobacterium isolated from human respiratory samples.

    Science.gov (United States)

    Davidson, Rebecca M; DeGroote, Mary Ann; Marola, Jamie L; Buss, Sarah; Jones, Victoria; McNeil, Michael R; Freifeld, Alison G; Elaine Epperson, L; Hasan, Nabeeh A; Jackson, Mary; Iwen, Peter C; Salfinger, Max; Strong, Michael

    2017-08-01

    A novel slowly growing, non-chromogenic species of the class Actinobacteria was isolated from a human respiratory sample in Nebraska, USA, in 2012. Analysis of the internal transcribed spacer sequence supported placement into the genus Mycobacterium with high sequence similarity to a previously undescribed strain isolated from a patient respiratory sample from Oregon, USA, held in a collection in Colorado, USA, in 2000. The two isolates were subjected to phenotypic testing and whole genome sequencing and found to be indistinguishable. The bacteria were acid-fast stain-positive, rod-shaped and exhibited growth after 7-10 days on solid media at temperatures ranging from 25 to 42°C. Colonies were non-pigmented, rough and slightly raised. Analyses of matrix-assisted laser desorption ionization time-of-flight profiles showed no matches against a reference library of 130 mycobacterial species. Full-length 16S rRNA gene sequences were identical for the two isolates, the average nucleotide identity (ANI) between their genomes was 99.7 % and phylogenetic comparisons classified the novel mycobacteria as the basal most species in the slowly growing Mycobacterium clade. Mycobacterium avium is the most closely related species based on rpoB gene sequence similarity (92 %), but the ANI between the genomes was 81.5 %, below the suggested cut-off for differentiating two species (95 %). Mycolic acid profiles were more similar to M. avium than to Mycobacterium simiae or Mycobacterium abscessus. The phenotypic and genomic data support the conclusion that the two related isolates represent a novel Mycobacterium species for which the name Mycobacterium talmoniae sp. nov. is proposed. The type strain is NE-TNMC-100812T (=ATCC BAA-2683T=DSM 46873T).

  15. Profile of Shiga toxin-producing Escherichia coli strains isolated from dogs and cats and genetic relationships with isolates from cattle, meat and humans.

    Science.gov (United States)

    Bentancor, A; Rumi, M V; Carbonari, C; Gerhardt, E; Larzábal, M; Vilte, D A; Pistone-Creydt, V; Chinen, I; Ibarra, C; Cataldi, A; Mercado, E C

    2012-05-04

    Pets can be reservoirs of Shiga toxin-producing Escherichia coli (STEC) strains. The aim of this study was to examine nine strains belonging to several serotypes (O91:H21, O91:H16, O178:H19, O8:H19, O22:H8, O22:HNT, ONT:H8), previously recovered from cats or dogs. To this end, we assessed a set of additional virulence genes (stx(2) subtype, subAB, ehxA, eae and saa), cytotoxic activity, and genetic relationships with strains isolated from cattle, meat and humans using pulsed-field gel electrophoresis (PFGE). Most of the isolates carried the stx(2) and/or stx(2vh-b) sequences, while only the O91:H21 isolate presented the mucus-activatable stx(2d) variant, as confirmed by sequencing the genes of subunits A and B. All the strains showed cytotoxic activity in cultured cells. One of the two O178:H19, selected for its high level of cytotoxicity in Vero cells, showed the ability to cause functional alterations in the human colon mucosa in vitro. None of the strains possessed the subAB, eae or saa genes and only the strains belonging to serotype O8:H19 carried the ehxA gene. The isolates shared 90-100% similarity by PFGE to epidemiologically unrelated strains of the corresponding serotypes recovered from cattle, meat or humans. Our results demonstrate that dogs and cats may have a role in the infection of humans by STEC, probably serving as a vehicle for bovine strains in the cycle of human infection, and thus emphasize the health risks for owners and their families. Copyright © 2011 Elsevier B.V. All rights reserved.

  16. Antimicrobial drug resistance of Salmonella isolates from meat and humans, Denmark

    DEFF Research Database (Denmark)

    Skov, Marianne Nielsine; Andersen, Jens Strodl; Aabo, Søren

    2007-01-01

    We compared 8,144 Salmonella isolates collected from meat imported to or produced in Denmark, as well as from Danish patients. Isolates from imported meat showed a higher rate of antimicrobial drug resistance, including multidrug resistance, than did isolates from domestic meat. Isolates from...... humans showed resistance rates lower than those found in imported meat but higher than in domestic meat. These findings indicate that programs for controlling resistant Salmonella spp. are a global issue...

  17. Antimicrobial drug resistance of Salmonella isolates from meat and humans, Denmark

    DEFF Research Database (Denmark)

    Skov, Marianne; Andersen, Jens Strodl; Aabo, Søren

    2007-01-01

    We compared 8,144 Salmonella isolates collected from meat imported to or produced in Denmark, as well as from Danish patients. Isolates from imported meat showed a higher rate of antimicrobial drug resistance, including multidrug resistance, than did isolates from domestic meat. Isolates from...... humans showed resistance rates lower than those found in imported meat but higher than in domestic meat. These findings indicate that programs for controlling resistant Salmonella spp. are a global issue....

  18. Occurrence of Putative Virulence Genes in Arcobacter Species Isolated from Humans and Animals

    Science.gov (United States)

    Douidah, Laid; de Zutter, Lieven; Baré, Julie; De Vos, Paul; Vandamme, Peter; Vandenberg, Olivier; Van den Abeele, Anne-Marie

    2012-01-01

    Interest in arcobacters in veterinary and human public health has increased since the first report of the isolation of arcobacters from food of animal origin. Since then, studies worldwide have reported the occurrence of arcobacters on food and in food production animals and have highlighted possible transmission, especially of Arcobacter butzleri, to the human population. In humans, arcobacters are associated with enteritis and septicemia. To assess their clinical relevance for humans and animals, evaluation of potential virulence factors is required. However, up to now, little has been known about the mechanisms of pathogenicity. Because of their close phylogenetic affiliation to the food-borne pathogen Campylobacter and their similar clinical manifestations, the presence of nine putative Campylobacter virulence genes (cadF, ciaB, cj1349, hecA, hecB, irgA, mviN, pldA, and tlyA) previously identified in the recent Arcobacter butzleri ATCC 49616 genome sequence was determined in a large set of human and animal Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii strains after the development of rapid and accurate PCR assays and confirmed by sequencing and dot blot hybridization. PMID:22170914

  19. Isolation of a new herpes virus from human CD4+ T cells

    International Nuclear Information System (INIS)

    Frenkel, N.; Schirmer, E.C.; Wyatt, L.S.; Katsafanas, G.; Roffman, E.; Danovich, R.M.; June, C.H.

    1990-01-01

    A new human herpes virus has been isolated from CD4 + T cells purified from peripheral blood mononuclear cells of a healthy individual (RK), following incubation of the cells under conditions promoting T-cell activation. The virus could not be recovered from nonactivated cells. Cultures of lymphocytes infected with the RK virus exhibited a cytopathic effect, and electron microscopic analyses revealed a characteristic herpes virus structure. RK virus DNA did not hybridize with large probes derived from herpes simplex virus, Epstein-Barr virus, varicella-zoster virus, and human cytomegalovirus. The genetic relatedness of the RK virus to the recently identified T-lymphotropic human herpes virus 6 (HHV-6) was investigated by restriction enzyme analyses using 21 different enzymes and by blot hydridization analyses using 11 probes derived from two strains of HHV-6 (Z29 and U1102). Whereas the two HHV-6 strains exhibited only limited restriction enzyme polymorphism, cleavage of the RK virus DNA yielded distinct patterns. Of the 11 HHV-6 DNA probes tested, only 6 cross-hybridized with DNA fragments derived from the RK virus. Taken together, the maximal homology amounted to 31 kilobases of the 75 kilobases tested. The authors conclude that the RK virus is distinct from previously characterized human herpesviruses. The authors propose to designate it as the prototype of a new herpes virus, the seventh human herpes virus identified to date

  20. Description of Campylobacter fetus subsp. testudinum subsp. nov., isolated from humans and reptiles

    Science.gov (United States)

    A polyphasic study was undertaken to determine the taxonomic position of 13 Campylobacter fetus-like isolates from humans (n=8) and reptiles (n=5). Phenotypic characterization, Genusgenus-specific and sap insertion-PCR initially identified all human isolates as type A Campylobacter fetus. Phylogenet...

  1. Comparison of virulence factors and capsular types of Streptococcus agalactiae isolated from human and bovine infections.

    Science.gov (United States)

    Emaneini, Mohammad; Khoramian, Babak; Jabalameli, Fereshteh; Abani, Samira; Dabiri, Hossein; Beigverdi, Reza

    2016-02-01

    Streptococcus agalactiae is a leading cause of human and bovine infections. A total of 194 S. agalactiae isolates, 55 isolates from bovines and 139 from humans, were analyzed for capsular types, virulence genes (scpB, hly, rib, bca and bac) and mobile genetic elements (IS1548 and GBSi1) using polymerase chain reaction (PCR) and multiplex PCR. Capsular type III was predominant (61%), followed by types V, II, Ib, and IV. The scpB, hly, bca and bac virulence genes were only found among human isolates. Twelve and 2 distinct virulence gene profiles were identified among human and bovine isolates respectively. The virulence gene profiles scpB- hly- IS1548- rib-bca (51%) and scpB- hly- IS1548- bca (19%) were only predominant among human isolates. The rib gene was the most common virulence gene in both human and bovine isolates. The study showed a high prevalence of virulence genes in S. agalactiae strains isolated from human infections, these result can support the idea that S. agalactiae isolated from humans and bovines are generally unrelated and probably belonged to separate populations. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Epidemiology, Clinical Characteristics, and Antimicrobial Susceptibility Profiles of Human Clinical Isolates of Staphylococcus intermedius Group.

    Science.gov (United States)

    Yarbrough, Melanie L; Lainhart, William; Burnham, C A

    2018-03-01

    The veterinary pathogens in the Staphylococcus intermedius group (SIG) are increasingly recognized as causes of human infection. Shared features between SIG and Staphylococcus aureus may result in the misidentification of SIG in human clinical cultures. This study examined the clinical and microbiological characteristics of isolates recovered at a tertiary-care academic medical center. From 2013 to 2015, 81 SIG isolates were recovered from 62 patients. Patients were commonly ≥50 years old, diabetic, and/or immunocompromised. Documentation of dog exposure in the electronic medical record was not common. Of the 81 SIG isolates, common sites of isolation included 37 (46%) isolates from wound cultures and 17 (21%) isolates from respiratory specimens. Although less common, 10 (12%) bloodstream infections were documented in 7 unique patients. The majority of SIG (65%) isolates were obtained from polymicrobial cultures. In comparison to S. aureus isolates from the same time period, significant differences were noted in proportion of SIG isolates that were susceptible to doxycycline (74% versus 97%, respectively; P SIG isolates. All MR isolates detected by an oxacillin disk diffusion test would have been misclassified as methicillin susceptible using a cefoxitin disk diffusion test. Thus, SIG is recovered from human clinical specimens, and distinction of SIG from S. aureus is critical for the accurate characterization of MR status in these isolates. Copyright © 2018 American Society for Microbiology.

  3. Porphyromonas pasteri sp. nov., isolated from human saliva.

    Science.gov (United States)

    Sakamoto, Mitsuo; Li, Dan; Shibata, Yukie; Takeshita, Toru; Yamashita, Yoshihisa; Ohkuma, Moriya

    2015-08-01

    A bacterial strain, designated KUFDS01T, isolated from human saliva was characterized using a polyphasic taxonomic approach that included analysis of physiological and biochemical features, cellular fatty acid profiles and phylogenetic position based on 16S rRNA gene sequence analysis. Cells of the strain were obligately anaerobic, non-pigmented, non-spore-forming, non-motile, Gram-stain-negative rods. Growth of the strain was inhibited on medium containing 20% bile. The 16S rRNA gene sequence analysis showed that the strain was a member of the genus Porphyromonas. Strain KUFDS01T was closely related to Porphyromonas catoniae JCM 13863T (96.6% sequence similarity). An hsp60 gene sequence analysis indicated that strain KUFDS01T was different from P. catoniae JCM 13863T, with a sequence similarity value of 87.8%. The major cellular fatty acids of strain KUFDS01T were C16 : 0, iso-C15 : 0, anteiso-C15 : 0, C18 : 2ω6, 9c and C18 : 1ω9c. The DNA G+C content of strain KUFDS01T was 57.7 ± 0.66 mol%. On the basis of these data, strain KUFDS01T represents a novel species of the genus Porphyromonas, for which the name Porphyromonas pasteri sp. nov. is proposed. The type strain of P. pasteri is KUFDS01T ( = JCM 30531T = CCUG 66735T).

  4. Tire tread wear particles in ambient air--a previously unknown source of human exposure to the biocide 2-mercaptobenzothiazole.

    Science.gov (United States)

    Avagyan, Rozanna; Sadiktsis, Ioannis; Bergvall, Christoffer; Westerholm, Roger

    2014-10-01

    Urban particulate matter (PM), asphalt, and tire samples were investigated for their content of benzothiazole and benzothiazole derivates. The purpose of this study was to examine whether wear particles, i.e., tire tread wear or road surface wear, could contribute to atmospheric concentrations of benzothiazole derivatives. Airborne particulate matter (PM10) sampled at a busy street in Stockholm, Sweden, contained on average 17 pg/m(3) benzothiazole and 64 pg/m(3) 2-mercaptobenzothiazole, and the total suspended particulate-associated benzothiazole and 2-mercaptobenzothiazole concentrations were 199 and 591 pg/m(3), respectively. This indicates that tire tread wear may be a major source of these benzothiazoles to urban air PM in Stockholm. Furthermore, 2-mercaptobenzothiazole was determined in urban air particulates for the first time in this study, and its presence in inhalable PM10 implies that the human exposure to this biocide is underestimated. This calls for a revision of the risk assessments of 2-mercaptobenzothiazole exposure to humans which currently is limited to occupational exposure.

  5. Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

    Science.gov (United States)

    Pfeiffer, Elisa; Zeilinger, Katrin; Seehofer, Daniel; Damm, Georg

    2016-01-01

    Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the "gold standard" for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique. Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation. Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models. PMID:27077489

  6. Biotin IgM Antibodies in Human Blood: A Previously Unknown Factor Eliciting False Results in Biotinylation-Based Immunoassays

    Science.gov (United States)

    Chen, Tingting; Hedman, Lea; Mattila, Petri S.; Jartti, Laura; Jartti, Tuomas; Ruuskanen, Olli; Söderlund-Venermo, Maria; Hedman, Klaus

    2012-01-01

    Biotin is an essential vitamin that binds streptavidin or avidin with high affinity and specificity. As biotin is a small molecule that can be linked to proteins without affecting their biological activity, biotinylation is applied widely in biochemical assays. In our laboratory, IgM enzyme immuno assays (EIAs) of µ-capture format have been set up against many viruses, using as antigen biotinylated virus like particles (VLPs) detected by horseradish peroxidase-conjugated streptavidin. We recently encountered one serum sample reacting with the biotinylated VLP but not with the unbiotinylated one, suggesting in human sera the occurrence of biotin-reactive antibodies. In the present study, we search the general population (612 serum samples from adults and 678 from children) for IgM antibodies reactive with biotin and develop an indirect EIA for quantification of their levels and assessment of their seroprevalence. These IgM antibodies were present in 3% adults regardless of age, but were rarely found in children. The adverse effects of the biotin IgM on biotinylation-based immunoassays were assessed, including four inhouse and one commercial virus IgM EIAs, showing that biotin IgM do cause false positivities. The biotin can not bind IgM and streptavidin or avidin simultaneously, suggesting that these biotin-interactive compounds compete for the common binding site. In competitive inhibition assays, the affinities of biotin IgM antibodies ranged from 2.1×10−3 to 1.7×10−4 mol/L. This is the first report on biotin antibodies found in humans, providing new information on biotinylation-based immunoassays as well as new insights into the biomedical effects of vitamins. PMID:22879954

  7. Hybrids of Shigatoxigenic and Enterotoxigenic Escherichia coli (STEC/ETEC) Among Human and Animal Isolates in Finland.

    Science.gov (United States)

    Nyholm, O; Heinikainen, S; Pelkonen, S; Hallanvuo, S; Haukka, K; Siitonen, A

    2015-11-01

    Diarrhoeagenic Escherichia coli (DEC) cause serious foodborne infections in humans. Total of 450 Shigatoxigenic E. coli (STEC) strains isolated from humans, animals and environment in Finland were examined by multiplex PCR targeting the virulence genes of various DEC pathogroups simultaneously. One per cent (3/291) of the human STEC and 14% (22/159) of the animal and environmental STEC had genes typically present in enterotoxigenic E. coli (ETEC). The strains possessed genes encoding both Shiga toxin 1 and/or 2 (stx1 and/or stx2 ) and ETEC-specific heat-stable (ST) enterotoxin Ia (estIa). The identified stx subtypes were stx1a, stx1c, stx2a, stx2d and stx2g. The three human STEC/ETEC strains were isolated from the patients with haemolytic uraemic syndrome and diarrhoea and from an asymptomatic carrier. The animal STEC/ETEC strains were isolated from cattle and moose. The human and animal STEC/ETEC strains belonged to 11 serotypes, of which O2:H27, O15:H16, O101:H-, O128:H8 and O141:H8 have previously been described to be associated with human disease. Identification of multiple virulence genes offers further information for assessing the virulence potential of STEC and other DEC. The emergence of novel hybrid pathogens should be taken into account in the patient care and epidemiological surveillance. © 2015 Blackwell Verlag GmbH.

  8. Molecular Characterization and Antimicrobial Susceptibility of Salmonella Isolates from Infections in Humans in Henan Province, China

    DEFF Research Database (Denmark)

    Xia, S.L.; Hendriksen, Rene S.; Xie, Z.Q.

    2009-01-01

    We characterized 208 human Salmonella isolates from 2006 to 2007 and 27 human Salmonella enterica serovar Typhimurium isolates from 1987 to 1993 from Henan Province, China, by serotyping, by antimicrobial susceptibility testing, and, for the most common serovars, by pulsed-field gel electrophoresis...... (PFGE). The most common serovars among the 2006-2007 isolates were S. enterica serovar Typhimurium (27%), S. enterica serovar Enteritidis (17%), S. enterica serovar Derby (10%), S. enterica serovar Indiana (6%), and S. enterica serovar Litchfield (6%). A high percentage of the isolates were multiple-drug...

  9. Antimicrobial Susceptibilities of Geographically Diverse Clinical Human Isolates of Leptospira▿

    OpenAIRE

    Ressner, Roseanne A.; Griffith, Matthew E.; Beckius, Miriam L.; Pimentel, Guillermo; Miller, R. Scott; Mende, Katrin; Fraser, Susan L.; Galloway, Renee L.; Hospenthal, Duane R.; Murray, Clinton K.

    2008-01-01

    Although antimicrobial therapy of leptospirosis has been studied in a few randomized controlled clinical studies, those studies were limited to specific regions of the world and few have characterized infecting strains. A broth microdilution technique for the assessment of antibiotic susceptibility has been developed at Brooke Army Medical Center. In the present study, we assessed the susceptibilities of 13 Leptospira isolates (including recent clinical isolates) from Egypt, Thailand, Nicarag...

  10. Streptococcus dysgalactiae subsp. dysgalactiae isolated from milk of the bovine udder as emerging pathogens: In vitro and in vivo infection of human cells and zebrafish as biological models.

    Science.gov (United States)

    Alves-Barroco, Cinthia; Roma-Rodrigues, Catarina; Raposo, Luís R; Brás, Catarina; Diniz, Mário; Caço, João; Costa, Pedro M; Santos-Sanches, Ilda; Fernandes, Alexandra R

    2018-03-25

    Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) is a major cause of bovine mastitis and has been regarded as an animal-restricted pathogen, although rare infections have been described in humans. Previous studies revealed the presence of virulence genes encoded by phages of the human pathogen Group A Streptococcus pyogenes (GAS) in SDSD isolated from the milk of bovine udder with mastitis. The isolates SDSD VSD5 and VSD13 could adhere and internalize human primary keratinocyte cells, suggesting a possible human infection potential of bovine isolates. In this work, the in vitro and in vivo potential of SDSD to internalize/adhere human cells of the respiratory track and zebrafish as biological models was evaluated. Our results showed that, in vitro, bovine SDSD strains could interact and internalize human respiratory cell lines and that this internalization was dependent on an active transport mechanism and that, in vivo, SDSD are able to cause invasive infections producing zebrafish morbidity and mortality. The infectious potential of these isolates showed to be isolate-specific and appeared to be independent of the presence or absence of GAS phage-encoded virulence genes. Although the infection ability of the bovine SDSD strains was not as strong as the human pathogenic S. pyogenes in the zebrafish model, results suggested that these SDSD isolates are able to interact with human cells and infect zebrafish, a vertebrate infectious model, emerging as pathogens with zoonotic capability. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  11. Effect of 30 mCi radioiodine on multinodular goiter previously treated with recombinant human thyroid-stimulating hormone

    Energy Technology Data Exchange (ETDEWEB)

    Paz-Filho, G.J.; Mesa-Junior, C.O.; Boguszewski, C.L.; Carvalho, G.A.; Graf, H. [Universidade Federal do Parana (UFPR), Curitiba, PR (Brazil). Hospital de Clinicas. Servico de Endocrinologia e Metabologia; Olandoski, M. [Pontificia Univ. Catolica do Parana, Curitiba, PR (Brazil). Nucleo de Bioestatistica; Woellner, L.C. [Centro de Medicina Nuclear, Curitiba, PR (Brazil); Goedert, C.A. [Centro de Tomografia Computadorizada, Curitiba, PR (Brazil)

    2007-12-15

    Recombinant human thyroid-stimulating hormone (rhTSH) enhances {sup 131}I uptake, permitting a decrease in radiation for the treatment of multinodular goiter (MNG). Our objective was to evaluate the safety and efficacy of a single 0.1-mg dose of rhTSH, followed by 30 mCi {sup 131}I, in patients with MNG. Seventeen patients (15 females, 59.0 {+-} 13.1 years), who had never been submitted to {sup 131}I therapy, received a single 0.1-mg injection of rhTSH followed by 30 mCi {sup 131}I on the next day. Mean basal thyroid volume measured by computed tomography was 106.1 {+-} 64.4 mL. {sup 131}I 24-h uptake, TSH, free-T4, T3, thyroglobulin, anti-thyroid antibodies, and thyroid volume were evaluated at regular intervals of 12 months. Mean {sup 131}I 24-h uptake increased from 18.1 {+-} 9.7 to 49.6 {+-} 13.4% (P < 0.001), a median 2.6-fold increase (1.2 to 9.2). Peak hormonal levels were 10.86 {+-} 5.44 mU/L for TSH (a median 15.5-fold increase), 1.80 {+-} 0.48 ng/dL for free-T4, 204.61 {+-} 58.37 ng/dL for T3, and a median of 557.0 ng/mL for thyroglobulin. The adverse effects observed were hyperthyroidism (17.6%), painful thyroiditis (29.4%) and hypothyroidism (52.9%). Thyroid volume was reduced by 34.3 {+-} 14.3% after 6 months (P < 0.001) and by 46.0 {+-} 14.6% after 1 year (P < 0.001). Treatment of MNG with a single 0.1-mg dose of rhTSH, followed by a fixed amount of radioactivity of {sup 131}I, leads to an efficacious decrease in thyroid volume for the majority of the patients, with a moderate incidence of non-serious and readily treatable adverse effects. (author)

  12. Effect of 30 mCi radioiodine on multinodular goiter previously treated with recombinant human thyroid-stimulating hormone

    Directory of Open Access Journals (Sweden)

    G.J. Paz-Filho

    2007-12-01

    Full Text Available Recombinant human thyroid-stimulating hormone (rhTSH enhances 131I uptake, permitting a decrease in radiation for the treatment of multinodular goiter (MNG. Our objective was to evaluate the safety and efficacy of a single 0.1-mg dose of rhTSH, followed by 30 mCi 131I, in patients with MNG. Seventeen patients (15 females, 59.0 ± 13.1 years, who had never been submitted to 131I therapy, received a single 0.1-mg injection of rhTSH followed by 30 mCi 131I on the next day. Mean basal thyroid volume measured by computed tomography was 106.1 ± 64.4 mL. 131I 24-h uptake, TSH, free-T4, T3, thyroglobulin, anti-thyroid antibodies, and thyroid volume were evaluated at regular intervals of 12 months. Mean 131I 24-h uptake increased from 18.1 ± 9.7 to 49.6 ± 13.4% (P < 0.001, a median 2.6-fold increase (1.2 to 9.2. Peak hormonal levels were 10.86 ± 5.44 mU/L for TSH (a median 15.5-fold increase, 1.80 ± 0.48 ng/dL for free-T4, 204.61 ± 58.37 ng/dL for T3, and a median of 557.0 ng/mL for thyroglobulin. The adverse effects observed were hyperthyroidism (17.6%, painful thyroiditis (29.4% and hypothyroidism (52.9%. Thyroid volume was reduced by 34.3 ± 14.3% after 6 months (P < 0.001 and by 46.0 ± 14.6% after 1 year (P < 0.001. Treatment of MNG with a single 0.1-mg dose of rhTSH, followed by a fixed amount of radioactivity of 131I, leads to an efficacious decrease in thyroid volume for the majority of the patients, with a moderate incidence of non-serious and readily treatable adverse effects.

  13. Assessment of Shiga toxin-producing Escherichia coli isolates from wildlife meat as potential pathogens for humans.

    Science.gov (United States)

    Miko, Angelika; Pries, Karin; Haby, Sabine; Steege, Katja; Albrecht, Nadine; Krause, Gladys; Beutin, Lothar

    2009-10-01

    A total of 140 Shiga toxin-producing Escherichia coli (STEC) strains from wildlife meat (deer, wild boar, and hare) isolated in Germany between 1998 and 2006 were characterized with respect to their serotypes and virulence markers associated with human pathogenicity. The strains grouped into 38 serotypes, but eight O groups (21, 146, 128, 113, 22, 88, 6, and 91) and four H types (21, 28, 2, and 8) accounted for 71.4% and 75.7% of all STEC strains from game, respectively. Eighteen of the serotypes, including enterohemorrhagic E. coli (EHEC) O26:[H11] and O103:H2, were previously found to be associated with human illness. Genes linked to high-level virulence for humans (stx(2), stx(2d), and eae) were present in 46 (32.8%) STEC strains from game. Fifty-four STEC isolates from game belonged to serotypes which are frequently found in human patients (O103:H2, O26:H11, O113:H21, O91:H21, O128:H2, O146:H21, and O146:H28). These 54 STEC isolates were compared with 101 STEC isolates belonging to the same serotypes isolated from farm animals, from their food products, and from human patients. Within a given serotype, most STEC strains were similar with respect to their stx genotypes and other virulence attributes, regardless of origin. The 155 STEC strains were analyzed for genetic similarity by XbaI pulsed-field gel electrophoresis. O103:H2, O26:H11, O113:H21, O128:H2, and O146:H28 STEC isolates from game were 85 to 100% similar to STEC isolates of the same strains from human patients. By multilocus sequence typing, game EHEC O103:H2 strains were attributed to a clonal lineage associated with hemorrhagic diseases in humans. The results from our study indicate that game animals represent a reservoir for and a potential source of human pathogenic STEC and EHEC strains.

  14. Whole genome sequencing of Mycobacterium bovis to obtain molecular fingerprints in human and cattle isolates from Baja California, Mexico.

    Science.gov (United States)

    Sandoval-Azuara, Sarai Estrella; Muñiz-Salazar, Raquel; Perea-Jacobo, Ricardo; Robbe-Austerman, Suelee; Perera-Ortiz, Alejandro; López-Valencia, Gilberto; Bravo, Doris M; Sanchez-Flores, Alejandro; Miranda-Guzmán, Daniela; Flores-López, Carlos Alberto; Zenteno-Cuevas, Roberto; Laniado-Laborín, Rafael; de la Cruz, Fabiola Lafarga; Stuber, Tod P

    2017-10-01

    To determine genetic diversity by comparing the whole genome sequences of cattle and human Mycobacterium bovis isolates from Baja California. A whole genome sequencing strategy was used to obtain the molecular fingerprints of 172 isolates of M. bovis obtained from Baja California, Mexico; 155 isolates were from cattle and 17 isolates were from humans. Spoligotypes were characterized in silico and single nucleotide polymorphism (SNP) differences between the isolates were evaluated. A total of 12 M. bovis spoligotype patterns were identified in cattle and humans. Two predominant spoligotypes patterns were seen in both cattle and humans: SB0145 and SB1040. The SB0145 spoligotype represented 59% of cattle isolates (n=91) and 65% of human isolates (n=11), while the SB1040 spoligotype represented 30% of cattle isolates (n=47) and 30% of human isolates (n=5). When evaluating SNP differences, the human isolates were intimately intertwined with the cattle isolates. All isolates from humans had spoligotype patterns that matched those observed in the cattle isolates, and all human isolates shared common ancestors with cattle in Baja California based on SNP analysis. This suggests that most human tuberculosis caused by M. bovis in Baja California is derived from M. bovis circulating in Baja California cattle. These results reinforce the importance of bovine tuberculosis surveillance and control in this region. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  15. Molecular characterization of Mycobacterium avium subspecies hominissuis isolated from humans, cattle and pigs in the Uganda cattle corridor using VNTR analysis.

    Science.gov (United States)

    Muwonge, Adrian; Oloya, James; Kankya, Clovice; Nielsen, Sigrun; Godfroid, Jacques; Skjerve, Eystein; Djønne, Berit; Johansen, Tone B

    2014-01-01

    Members of the Mycobacterium avium complex (MAC) cause disease in both human and animals. Their ubiquitous nature makes them both successful microbes and difficult to source track. The precise characterization of MAC species is a fundamental step in epidemiological studies and evaluating of possible reservoirs. This study aimed at identifying and characterizing Mycobacterium avium subsp. hominissuis isolated from human, slaughter cattle and pigs in various parts of the Uganda cattle corridor (UCC) at two temporal points using variable number of tandem repeat (VNTR) analysis. A total of 46 M. avium isolates; 31 from 997 pigs, 12 from 43 humans biopsies and three from 61 cattle lesions were identified to subspecies level using IS1245 and IS901 PCR, thereafter characterized using VNTR. Twelve loci from two previously described VNTR methods were used and molecular results were analyzed and interpreted using Bionumerics 6.1. 37 of the isolates were identified as M. avium subsp. hominissuis and four as M. avium subsp. avium, while five could not be differentiated, possibly due to mixed infection. There was distinct clustering that coincides with the temporal and spatial differences of the isolates. The isolates from humans and cattle in the North Eastern parts of the UCC shared identical VNTR genotypes. The panel of loci gave an overall discriminatory power of 0.88. Some loci were absent in several isolates, probably reflecting differences in isolates from Uganda/Africa compared to isolates previously analyzed by these methods in Europe and Asia. The findings indicate a molecular difference between M. avium subsp. hominissuis isolates from pigs in Mubende and cattle and human in the rest of the UCC. Although human and cattle shared VNTR genotypes in the North Eastern parts of the UCC, it is most likely a reflection of a shared environmental source. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Diversity and antimicrobial susceptibility profiling of staphylococci isolated from bovine mastitis cases and close human contacts.

    Science.gov (United States)

    Schmidt, T; Kock, M M; Ehlers, M M

    2015-09-01

    The objectives of this study were to examine the diversity of Staphylococcus spp. recovered from bovine intramammary infections and humans working in close contact with the animals and to evaluate the susceptibility of the staphylococcal isolates to different antimicrobials. A total of 3,387 milk samples and 79 human nasal swabs were collected from 13 sampling sites in the KwaZulu-Natal province of South Africa. In total, 146 Staph. aureus isolates and 102 coagulase-negative staphylococci (CNS) were recovered from clinical and subclinical milk samples. Staphylococcusaureus was isolated from 12 (15.2%) of the human nasal swabs and 95 representative CNS were recovered for further characterization. The CNS were identified using multiplex-PCR assays, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and tuf gene sequencing. Seven Staphylococcus spp. were identified among the CNS of bovine origin, with Staph.chromogenes (78.4%) predominating. The predominant CNS species recovered from the human nasal swabs was Staph.epidermidis (80%) followed by Staph.chromogenes (6.3%). The antimicrobial susceptibility of all staphylococcal isolates was evaluated using disk diffusion and was supplemented by screening for specific antimicrobial resistance genes. Ninety-eight (67.1%) Staph.aureus isolates of bovine origin were pansusceptible; 39 (26.7%) isolates were resistant to a single class, and 7 (4.8%) isolates were resistant to 2 classes of antimicrobials. Two Staph. aureus (1.4%) isolates were multidrug-resistant. Resistance to penicillin was common, with 28.8% of the bovine and 75% of the human Staph. aureus isolates exhibiting resistance. A similar observation was made with the CNS, where 37.3% of the bovine and 89.5% of the human isolates were resistant to penicillin. Multidrug-resistance was common among the human CNS, with 39% of the isolates exhibiting resistance to 3 or more classes of antimicrobials. The antimicrobial

  17. Protein phosphorylation in isolated human adipocytes - Adrenergic control of the phosphorylation of hormone-sensitive lipase

    International Nuclear Information System (INIS)

    Smiley, R.M.; Paul, S.; Browning, M.D.; Leibel, R.L.; Hirsch, J.

    1990-01-01

    The effect of adrenergic agents on protein phosphorylation in human adipocytes was examined. Freshly isolated human fat cells were incubated with 32 PO 4 in order to label intracellular ATP, then treated with a variety of adrenergic and other pharmacologic agents. Treatment with the β-adrenergic agonist isoproterenol led to a significant increase in phosphate content of at least five protein bands (M r 52, 53, 63, 67, 84 kDa). The increase in phosphorylation was partially inhibited by the α-2 agonist clonidine. Epinephrine, a combined α and β agonist, was less effective at increasing phosphate content of the proteins than was isoproterenol. Neither insulin nor the α-1 agonist phenylephrine had any discernible effect on the pattern of protein phosphorylation. The 84 kDa phosphorylated peptide band appears to contain hormone-sensitive lipase, a key enzyme in the lipolytic pathway which is activated by phosphorylation. These results are somewhat different than previously reported results for rat adipocytes, and represent the first report of overall pattern and adrenergic modulation of protein phosphorylation in human adipocytes

  18. Genotyping of Giardia lamblia isolates from humans in China and Korea using ribosomal DNA Sequences.

    Science.gov (United States)

    Yong, T S; Park, S J; Hwang, U W; Yang, H W; Lee, K W; Min, D Y; Rim, H J; Wang, Y; Zheng, F

    2000-08-01

    Genetic characterization of a total of 15 Giardia lamblia isolates, 8 from Anhui Province, China (all from purified cysts) and 7 from Seoul, Korea (2 from axenic cultures and 5 from purified cysts), was performed by polymerase chain reaction amplification and sequencing of a 295-bp region near the 5' end of the small subunit ribosomal DNA (eukaryotic 16S rDNA). Phylogenetic analyses were subsequently conducted using sequence data obtained in this study, as well as sequences published from other Giardia isolates. The maximum parsimony method revealed that G. lamblia isolates from humans in China and Korea are divided into 2 major lineages, assemblages A and B. All 7 Korean isolates were grouped into assemblage A, whereas 4 Chinese isolates were grouped into assemblage A and 4 into assemblage B. Two Giardia microti isolates and 2 dog-derived Giardia isolates also grouped into assemblage B, whereas Giardia ardeae and Giardia muris were unique.

  19. Complete genome sequence of Bifidobacterium breve CECT 7263, a strain isolated from human milk.

    Science.gov (United States)

    Jiménez, Esther; Villar-Tajadura, M Antonia; Marín, María; Fontecha, Javier; Requena, Teresa; Arroyo, Rebeca; Fernández, Leónides; Rodríguez, Juan M

    2012-07-01

    Bifidobacterium breve is an actinobacterium frequently isolated from colonic microbiota of breastfeeding babies. Here, we report the complete and annotated genome sequence of a B. breve strain isolated from human milk, B. breve CECT 7263. The genome sequence will provide new insights into the biology of this potential probiotic organism and will allow the characterization of genes related to beneficial properties.

  20. Isolation of tannin-degrading lactobacilli from humans and fermented foods.

    Science.gov (United States)

    Osawa, R; Kuroiso, K; Goto, S; Shimizu, A

    2000-07-01

    Lactobacilli with tannase activity were isolated from human feces and fermented foods. A PCR-based taxonomic assay revealed that the isolates belong to Lactobacillus plantarum, L. paraplantarum, and L. pentosus. Additional studies on a range of Lactobacillus species from established culture collections confirmed that this enzymatic activity is a phenotypic property common to these three species.

  1. Molecular typing of Brucella species isolates from Human and livestock bloods in Isfahan province

    Directory of Open Access Journals (Sweden)

    Ebtehaj Pishva

    2015-01-01

    Conclusion: Our findings confirm abundance of B. melitensis, particularly biovar 1 in human and sheep are identical but B. abortus biovar 3 as the etiological agent of cattle brucellosis most frequently isolated in the Isfahan area.

  2. Association between antimicrobial resistance in Escherichia coli isolates from food animals and blood stream isolates from humans in Europe: an ecological study.

    Science.gov (United States)

    Vieira, Antonio R; Collignon, Peter; Aarestrup, Frank M; McEwen, Scott A; Hendriksen, Rene S; Hald, Tine; Wegener, Henrik C

    2011-12-01

    In addition to medical antimicrobial usage, the use of antimicrobials in food animals contributes to the occurrence of resistance among some bacterial species isolated from infections in humans. Recently, several studies have indicated that a large proportion of Escherichia coli causing infections in humans, especially those resistant to antimicrobials, have an animal origin. We analyzed the correlation between the prevalence of antimicrobial resistance in E. coli isolates from blood stream infections in humans and in E. coli isolates from poultry, pigs, and cattle between 2005 and 2008 for 11 countries, using available surveillance data. We also assessed the correlation between human antimicrobial usage and the occurrence of resistance in E. coli isolates from blood stream infections. Strong and significant correlations between prevalences of resistance to ampicillin (r=0.94), aminoglycosides (r=0.72), third-generation cephalosporins (r=0.76), and fluoroquinolones (r=0.68) were observed for human and poultry E. coli isolates. Similar significant correlations were observed for ampicillin (r=0.91), aminoglycosides (r=0.73), and fluoroquinolone resistance (r=0.74) in pig and human isolates. In cattle isolates, only ampicillin resistance (r=0.72) was significantly correlated to human isolates. When usage of antimicrobials in humans was analyzed with antimicrobial resistance among human isolates, only correlations between fluoroquinolones (r=0.90) and third-generation cephalosporins (r=0.75) were significant. Resistance in E. coli isolates from food animals (especially poultry and pigs) was highly correlated with resistance in isolates from humans. This supports the hypothesis that a large proportion of resistant E. coli isolates causing blood stream infections in people may be derived from food sources.

  3. Human bocavirus isolated from children with acute respiratory tract infections in Korea, 2010-2011.

    Science.gov (United States)

    Ahn, Jong Gyun; Choi, Seong Yeol; Kim, Dong Soo; Kim, Ki Hwan

    2014-12-01

    Human bocavirus (HBoV) was first recognized in respiratory samples in 2005. The clinical importance of HBoV infection remains unclear. This report describes the clinical features and molecular phylogeny of HBoV isolates in children with acute respiratory infections. Nasopharyngeal aspirates were obtained from 1,528 children with acute respiratory infections between 2010 and 2011. Respiratory samples were screened for HBoV by multiplex PCR. A phylogenetic analysis of the HBoV VP1/VP2 gene was also undertaken. HBoV was detected in 187 (12.2%) of the 1,528 patients with a peak incidence of infection observed in patients aged 12-24 months. Coinfection with other respiratory viruses was observed in 107 (57.2%) of the HBoV-positive children. The peak of HBoV activity occurred during the month of June in both 2010 and 2011. A higher previous history of wheezing (P = 0.016), a higher frequency of chest retraction (P respiratory symptom score (P = 0.002), and a longer duration of hospital stay (P = 0.021) were observed in HBoV-positive children compared with the HBoV-negative group. Phylogenetic analysis showed all 187 HBoV-positive isolates were identified as HBoV 1, indicating minimal sequence variations among the isolates. A single lineage of HBoV 1 was found to have circulated in children with acute respiratory infections between 2010 and 2011 and was associated with several clinical characteristics including age, seasonality, and clinical severity with retraction, wheezing, and longer hospitalization. The clinical relevance of the minimal sequence variations of HBoV remains to be determined. © 2014 Wiley Periodicals, Inc.

  4. Comparison of Campylobacter jejuni isolates from human, food, veterinary and environmental sources in Iceland using PFGE, MLST and fla-SVR sequencing.

    Science.gov (United States)

    Magnússon, S H; Guðmundsdóttir, S; Reynisson, E; Rúnarsson, A R; Harðardóttir, H; Gunnarson, E; Georgsson, F; Reiersen, J; Marteinsson, V Th

    2011-10-01

    Campylobacter jejuni isolates from various sources in Iceland were genotyped with the aim of assessing the genetic diversity, population structure, source distribution and campylobacter transmission routes to humans. A collection of 584 Campylobacter isolates were collected from clinical cases, food, animals and environment in Iceland in 1999-2002, during a period of national Campylobacter epidemic in Iceland. All isolates were characterized by pulse field gel electrophoresis (PFGE), and selected subset of 52 isolates representing the diversity of the identified PFGE types was further genotyped using multilocus sequence typing (MLST) and fla-SVR sequencing to gain better insight into the population structure. The results show a substantial diversity within the Icelandic Campylobacter population. Majority of the human Campylobacter infections originated from domestic chicken and cattle isolates. MLST showed the isolates to be distributed among previously reported and common sequence type complexes in the MLST database. The genotyping of Campylobacter from various sources has not previously been reported from Iceland, and the results of the study gave a valuable insight into the population structure of Camp. jejuni in Iceland, source distribution and transmission routes to humans. The geographical isolation of Iceland in the north Atlantic provides new information on Campylobacter population dynamics on a global scale. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology No claim to Icelandic Government works.

  5. A locus for isolated cataract on human Xp.

    Science.gov (United States)

    Francis, P J; Berry, V; Hardcastle, A J; Maher, E R; Moore, A T; Bhattacharya, S S

    2002-02-01

    To genetically map the gene causing isolated X linked cataract in a large European pedigree. Using the patient registers at Birmingham Women's Hospital, UK, we identified and examined 23 members of a four generation family with nuclear cataract. Four of six affected males also had complex congenital heart disease. Pedigree data were collated and leucocyte DNA extracted from venous blood. Linkage analysis by PCR based microsatellite marker genotyping was used to identify the disease locus and mutations within candidate genes screened by direct sequencing. The disease locus was genetically refined to chromosome Xp22, within a 3 cM linkage interval flanked by markers DXS9902 and DXS999 (Zmax=3.64 at theta=0 for marker DXS8036). This is the first report of a locus for isolated inherited cataract on the X chromosome. The disease interval lies within the Nance-Horan locus suggesting allelic heterogeneity. The apparent association with congenital cardiac anomalies suggests a possible new oculocardiac syndrome.

  6. Molecular identification of Giardia lamblia isolates from adult human ...

    African Journals Online (AJOL)

    ARL

    2013-02-27

    Feb 27, 2013 ... Giardia lamblia is a flagellated protozoa that infects the intestinal tract of a wide range of mammalian hosts, including both wild and domestic animals as well as humans. Two genotypes A and B are commonly reported among humans. The purpose of this study was to investigate the genotypes of G.

  7. Pheno- and genotyping of Staphylococcus epidermidis isolated from bovine milk and human skin

    DEFF Research Database (Denmark)

    Thorberg, B. M.; Kuhn, I.; Aarestrup, Frank Møller

    2006-01-01

    showed one pattern, which was identical to the most common pattern found in the milk isolates. Isolates from herd 2 showed three to four patterns, two of these being identical to skin isolates from the milker. As dairy cows are not a natural host for S. epidermidis the results suggest a human source...... (PFGE) and 122 by ribotyping. PFGE showed single patterns in the human strains with one exception; one strain was categorised as the same clone as four of the milk strains. PFGE divided 73 of the milk strains into 62 different patterns. The PFGE method had high discriminatory power and shows that many...... different S. epidermidis types exist in milk samples. Antibiotic resistance patterns matched the SmaI profiles closely in the two herds, but poorly in the routinely collected milk samples. Isolates from herd I showed one to five patterns, depending on the typing method used. Isolates from the milker's skin...

  8. Characterization of Leptospira isolates from humans and the environment in Uruguay.

    Science.gov (United States)

    Meny, Paulina; Menéndez, Clara; Quintero, Jair; Hernández, Elba; Ríos, Cristina; Balassiano, Ilana Teruszkin; Trindade, Camilla Nunes Dos Reis; Vital-Brazil, Juliana Magalhães; Ramos, Tatiane Mendes Varela; Ashfield, Natalia; Feble, Camila; Avila, Esthefani; Schelotto, Felipe; Varela, Gustavo

    2017-12-21

    Laboratory diagnosis of human leptospirosis usually relies on indirect methods exploring specific immune response. Isolation and identification of the involved strains are cumbersome, but can provide biological resources for pathogenic studies and relevant information for guiding prevention and control measures. The aim of the research we are hereby reporting was the characterization of Leptospira isolates obtained from humans and the environment in Uruguay. Blood cultures were performed from early samples of 302 Uruguayan patients, mainly rural workers, and from 36 water samples taken from their living or working environments. Eight human isolates and seven environmental isolates were obtained and analyzed by end point Polymerase Chain Reaction (PCR), Multilocus Variable Number of Tandem Repeat Analysis (MLVA) and other molecular methods. Human isolates corresponded to several serogroups and serovars of Leptospira interrogans and Leptospira kirschneri species, probably reflecting the infection with similar involved Leptospira species and serovars of an extended animal reservoir in rural settings of the country, mostly dedicated to meat and dairy production. Culture-positive patients were older than usually affected workers, and presented signs and symptoms of severe illness. A high organic and circulating bacterial burden may explain an easier positive result from these workers' samples. Environmental isolates were mainly identified as Leptospira biflexa strains, with a single L. meyeri isolate of uncertain significance.

  9. Social isolation disrupts hippocampal neurogenesis in young non-human primates

    Directory of Open Access Journals (Sweden)

    Simone M Cinini

    2014-03-01

    Full Text Available Social relationships are crucial for the development and maintenance of normal behavior in non-human primates. Animals that are raised in isolation develop abnormal patterns of behavior that persist even when they are later reunited with their parents. In rodents, social isolation is a stressful event and is associated with a decrease in hippocampal neurogenesis but considerably less is known about the effects of social isolation in non-human primates during the transition from adolescence to adulthood. To investigate how social isolation affects young marmosets, these were isolated from other members of the colony for one or three weeks and evaluated for alterations in their behavior and hippocampal cell proliferation. We found that anxiety-related behaviors like scent-marking and locomotor activity increased after social isolation when compared to baseline levels. In agreement, grooming - an indicative of attenuation of tension - was reduced among isolated marmosets. These results were consistent with increased cortisol levels after one and three weeks of isolation. After social isolation (one or three weeks, reduced proliferation of neural cells in the subgranular zone of dentate granule cell layer was identified and a smaller proportion of BrdU-positive cells underwent neuronal fate (doublecortin labeling. Our data is consistent with the notion that social deprivation during the transition from adolescence to adulthood leads to stress and produces anxiety-like behaviors that in turn might affect neurogenesis and contribute to the deleterious consequences of prolonged stressful conditions.

  10. Isolation and characterization of human salivary gland cells for stem cell transplantation to reduce radiation-induced hyposalivation

    International Nuclear Information System (INIS)

    Feng Jielin; Zwaag, Marianne van der; Stokman, Monique A.; Os, Ronald van; Coppes, Robert P.

    2009-01-01

    Background: Recently, we showed that transplantation of 100-300 c-Kit + stem cells isolated from cultured salispheres ameliorates radiation-damage in murine salivary glands. The aim of this study is to optimize and translate these findings from mice to man. Methods: Mouse and human non-malignant parotid and submandibular salivary gland tissue was collected and enzymatically digested. The remaining cell suspension was cultured according to our salisphere culture method optimized for murine salispheres. Salisphere cells were tested using 3D matrix culturing for their in vitro stem cell characteristics such as the potential to differentiate into tissue specific cell types. Several potential mouse and human salivary gland stem cells were selected using FACS. Results: In human salivary gland, c-Kit + cells were only detected in excretory ducts as shown previously in mice. From both human parotid and submandibular gland cell suspensions salispheres could be grown, which when placed in 3D culture developed ductal structures and mucin-expressing acinar-like cells. Moreover, cells dispersed from primary salispheres were able to form secondary spheres in matrigel, a procedure that could be repeated for at least seven passages. Approximately 3000 c-Kit + cells could be isolated from primary human salispheres per biopsy. Conclusion: Human salivary glands contain a similar 'putative' stem cell population as rodents, expressing c-kit and capable of in vitro differentiation and self-renewal. In the future, these cells may have the potential to reduce radiotherapy-induced salivary gland dysfunction in patients.

  11. Long Chain N-acyl Homoserine Lactone Production by Enterobacter sp. Isolated from Human Tongue Surfaces

    Science.gov (United States)

    Yin, Wai-Fong; Purmal, Kathiravan; Chin, Shenyang; Chan, Xin-Yue; Chan, Kok-Gan

    2012-01-01

    We report the isolation of N-acyl homoserine lactone-producing Enterobacter sp. isolate T1-1 from the posterior dorsal surfaces of the tongue of a healthy individual. Spent supernatants extract from Enterobacter sp. isolate T1-1 activated the biosensor Agrobacterium tumefaciens NTL4(pZLR4), suggesting production of long chain AHLs by these isolates. High resolution mass spectrometry analysis of these extracts confirmed that Enterobacter sp. isolate T1-1 produced a long chain N-acyl homoserine lactone, namely N-dodecanoyl-homoserine lactone (C12-HSL). To the best of our knowledge, this is the first isolation of Enterobacter sp., strain T1-1 from the posterior dorsal surface of the human tongue and N-acyl homoserine lactones production by this bacterium. PMID:23202161

  12. Isolation of primary human hepatocytes from normal and diseased liver tissue: a one hundred liver experience.

    Directory of Open Access Journals (Sweden)

    Ricky H Bhogal

    2011-03-01

    Full Text Available Successful and consistent isolation of primary human hepatocytes remains a challenge for both cell-based therapeutics/transplantation and laboratory research. Several centres around the world have extensive experience in the isolation of human hepatocytes from non-diseased livers obtained from donor liver surplus to surgical requirement or at hepatic resection for tumours. These livers are an important but limited source of cells for therapy or research. The capacity to isolate cells from diseased liver tissue removed at transplantation would substantially increase availability of cells for research. However no studies comparing the outcome of human hepatocytes isolation from diseased and non-diseased livers presently exist. Here we report our experience isolating human hepatocytes from organ donors, non-diseased resected liver and cirrhotic tissue. We report the cell yields and functional qualities of cells isolated from the different types of liver and demonstrate that a single rigorous protocol allows the routine harvest of good quality primary hepatocytes from the most commonly accessible human liver tissue samples.

  13. A genetic comparison of human and wildlife isolates of Echinococcus granulosus in Queensland: public health implications.

    Science.gov (United States)

    Hope, M; Bowles, J; Prociv, P; McManus, D P

    1992-01-06

    To test the hypothesis that the hydatid parasite infecting macropods and dingoes in Queensland is a sylvatic strain of Echinococcus granulosus, distinct from the domestic strain which produces cysts in sheep and humans. Molecular biological techniques were used to compare DNA isolated from hydatid cysts from humans, local macropods and sheep from New South Wales and the United Kingdom, as well as from adult tapeworms in dingoes. The human cysts were surgically resected from two patients seen with hydatidosis in Brisbane teaching hospitals over a one-year period. Neither patient had had previous contact with sheep farms. Macropods and dingoes were shot randomly in the localities where the patients presumably acquired their infections. Sheep liver cysts were obtained from abattoirs. Studies comprised extraction of DNA from cysts, digestion by a series of restriction endonucleases, slab gel electrophoresis. Southern blotting and then hybridisation with defined DNA probes. Polymerase chain reaction, in combination with direct DNA sequencing, was used to compare DNA from cysts and adult worms from dingoes. The restriction fragment length polymorphism (RFLP) patterns of DNA from all cysts and a defined mitochondrial DNA sequence from all sources were indistinguishable. This finding is significant as both techniques can clearly distinguish between genetically distinct, well characterised strains of E. granulosus. Hydatid cysts are prevalent in some macropod populations and adult worms are common in dingoes. Since there are relatively few sheep-rearing areas in Queensland, contact with wild animals may be the main source of human hydatid infection in this State. The strain of E. granulosus in both patients was genetically indistinguishable from that found in macropods, dingoes and sheep from New South Wales and the United Kingdom. This strongly suggests that the domestic strain of E. granulosus, or a form very close genetically, freely infects Australian wildlife, and

  14. Isolating human DNA repair genes using rodent-cell mutants

    International Nuclear Information System (INIS)

    Thompson, L.H.; Weber, C.A.; Brookman, K.W.; Salazar, E.P.; Stewart, S.A.; Mitchell, D.L.

    1987-01-01

    The DNA repair systems of rodent and human cells appear to be at least as complex genetically as those in lower eukaryotes and bacteria. The use of mutant lines of rodent cells as a means of identifying human repair genes by functional complementation offers a new approach toward studying the role of repair in mutagenesis and carcinogenesis. In each of six cases examined using hybrid cells, specific human chromosomes have been identified that correct CHO cell mutations affecting repair of damage from uv or ionizing radiations. This finding suggests that both the repair genes and proteins may be virtually interchangeable between rodent and human cells. Using cosmid vectors, human repair genes that map to chromosome 19 have cloned as functional sequences: ERCC2 and XRCC1. ERCC1 was found to have homology with the yeast excision repair gene RAD10. Transformants of repair-deficient cell lines carrying the corresponding human gene show efficient correction of repair capacity by all criteria examined. 39 refs., 1 fig., 1 tab

  15. MLVA Typing of Streptococcus pneumoniae Isolates with Emphasis on Serotypes 14, 9N and 9V: Comparison of Previously Described Panels and Proposal of a Novel 7 VNTR Loci-Based Simplified Scheme.

    Science.gov (United States)

    Costa, Natália S; Pinto, Tatiana C A; Merquior, Vânia L C; Castro, Luciana F S; da Rocha, Filomena S P; Morais, Jaqueline M; Peralta, José M; Teixeira, Lúcia M

    2016-01-01

    Streptococcus pneumoniae remains as an important cause of community-acquired bacterial infections, and the nasopharynx of asymptomatic carriers is the major reservoir of this microorganism. Pneumococcal strains of serotype 14 and serogroup 9 are among the most frequently isolated from both asymptomatic carriers and patients with invasive disease living in Brazil. Internationally disseminated clones belonging to such serotypes have been associated with the emergence and spread of antimicrobial resistance in our setting, highlighting the need for epidemiological tracking of these isolates. In this scenario, Multiple Loci VNTR Analysis (MLVA) has emerged as an alternative tool for the molecular characterization of pneumococci, in addition to more traditional techniques such as Multi-Locus Sequence Typing (MLST) and Pulsed-Field Gel Electrophoresis (PFGE). In the present study, 18 VNTR loci, as well as other previously described reduced MLVA panels (7 VNTR loci), were evaluated as tools to characterize pneumococcal strains of serotypes 14, 9N and 9V belonging to international and regional clones isolated in Brazil. The 18 VNTR loci panel was highly congruent with MLST and PFGE, being also useful for indicating the genetic relationship with international clones and for discriminating among strains with indistinguishable STs and PFGE profiles. Analysis of the results also allowed deducing a novel shorter 7 VNTR loci panel, keeping a high discriminatory power for isolates of the serotypes investigated and a high congruence level with MLST and PFGE. The newly proposed simplified panel was then evaluated for typing pneumococcal strains of other commonly isolated serotypes. The results indicate that MLVA is a faster and easier to perform, reliable approach for the molecular characterization of S. pneumoniae isolates, with potential for cost-effective application, especially in resource-limited countries.

  16. Previously unknown species of Aspergillus.

    Science.gov (United States)

    Gautier, M; Normand, A-C; Ranque, S

    2016-08-01

    The use of multi-locus DNA sequence analysis has led to the description of previously unknown 'cryptic' Aspergillus species, whereas classical morphology-based identification of Aspergillus remains limited to the section or species-complex level. The current literature highlights two main features concerning these 'cryptic' Aspergillus species. First, the prevalence of such species in clinical samples is relatively high compared with emergent filamentous fungal taxa such as Mucorales, Scedosporium or Fusarium. Second, it is clearly important to identify these species in the clinical laboratory because of the high frequency of antifungal drug-resistant isolates of such Aspergillus species. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been shown to enable the identification of filamentous fungi with an accuracy similar to that of DNA sequence-based methods. As MALDI-TOF MS is well suited to the routine clinical laboratory workflow, it facilitates the identification of these 'cryptic' Aspergillus species at the routine mycology bench. The rapid establishment of enhanced filamentous fungi identification facilities will lead to a better understanding of the epidemiology and clinical importance of these emerging Aspergillus species. Based on routine MALDI-TOF MS-based identification results, we provide original insights into the key interpretation issues of a positive Aspergillus culture from a clinical sample. Which ubiquitous species that are frequently isolated from air samples are rarely involved in human invasive disease? Can both the species and the type of biological sample indicate Aspergillus carriage, colonization or infection in a patient? Highly accurate routine filamentous fungi identification is central to enhance the understanding of these previously unknown Aspergillus species, with a vital impact on further improved patient care. Copyright © 2016 European Society of Clinical Microbiology and

  17. Human intestinal mucus proteins isolated by transanal irrigation and proctosigmoidoscopy

    Directory of Open Access Journals (Sweden)

    Paola Andrea Gómez Buitrago

    2014-10-01

    Full Text Available Human intestinal mucus essentially consists of a network of Mucin2 glycoproteins embedded in many lower molecular weight proteins. This paper contributes to the proteomic study of human intestinal mucus by comparing two sample collection methods (transanal irrigation and brush cytology during proctosigmoidoscopy and analysis techniques (electrophoresis and digestion in solution. The entire sample collection and treatment process is explained, including protein extraction, digestion and desalination and peptide characterisation using a nanoAcquity UPLC chromatograph coupled to an HDMS spectrometer equipped with a nanoESI source. Collecting mucus via transanal irrigation provided a larger sample volume and protein concentration from a single patient. The proctosigmoidoscopy sample could be analysed via digestion in solution after depleting albumin. The analysis indicates that a simple mucus lysis method can evaluate the electrophoresis and digestion in solution techniques. Studying human intestinal mucus complexes is important because they perform two essential survival functions for humans as the first biochemical and physical defences for the gastrointestinal tract and a habitat for intestinal microbiota, which are primarily hosted in the colon and exceeds the human genetic information and cell number 100- and 10-fold (1.

  18. Comparative Analysis of Human and Canine Campylobacter upsaliensis Isolates by Amplified Fragment Length Polymorphism

    DEFF Research Database (Denmark)

    Damborg, Peter; Guardabassi, Luca; Pedersen, Karl

    2008-01-01

    Human (n = 33) and canine (n = 53) Campylobacter upsaliensis isolates from seven countries were genotyped by a new amplified fragment length polymorphism method. We observed 100% typeability and high overall diversity. The majority of human strains (23/33) clustered separately from canine strains...

  19. Supravital dithizone staining in the isolation of human and rat pancreatic islets

    DEFF Research Database (Denmark)

    Hansen, W A; Christie, M R; Kahn, R

    1989-01-01

    Dithizone, a zinc chelating agent, is known to selectively stain the islets of Langerhans in the pancreas. In the present study, we have used this stain to aid the identification of islets in material obtained by collagenase digestion of human pancreas. Islets were shown to rapidly and reversibly...... techniques for the large scale isolation of functionally intact human islets....

  20. Relaxant mechanisms of 3, 5, 7, 30, 40-pentamethoxyflavone on isolated human cavernosum

    DEFF Research Database (Denmark)

    Jansakul, Chaweewan; Tachanaparuksa, Kuldej; Mulvany, Michael J.

    2012-01-01

    We have investigated effects and mechanisms responsible for the activity of 3, 5, 7, 30, 40-pentamethoxyflavone (PMF) on isolated human cavernosum. PMF is the major flavone isolated from Kaempferia parviflora claimed to act as an aphrodisiac. PMF caused relaxation of phenylephrine precontracted...... Krebs solution with nifedipine (blocker of L-type Ca2þ channels), or in Ca2þ-free Krebs solution, PMF caused a further inhibition of human cavernosum contracted with phenylephrine. In human cavernosum treated with thapsigargin (inhibitor of sarcoplasmic reticulum Ca2þ-ATPase) in Ca2þ-free medium, PMF...

  1. Translational neuropharmacology: the use of human isolated gastrointestinal tissues.

    Science.gov (United States)

    Sanger, G J; Broad, J; Kung, V; Knowles, C H

    2013-01-01

    Translational sciences increasingly emphasize the measurement of functions in native human tissues. However, such studies must confront variations in patient age, gender, genetic background and disease. Here, these are discussed with reference to neuromuscular and neurosecretory functions of the human gastrointestinal (GI) tract. Tissues are obtained after informed consent, in collaboration with surgeons (surgical techniques help minimize variables) and pathologists. Given the difficulties of directly recording from human myenteric neurones (embedded between muscle layers), enteric motor nerve functions are studied by measuring muscle contractions/relaxations evoked by electrical stimulation of intrinsic nerves; responses are regionally dependent, often involving cholinergic and nitrergic phenotypes. Enteric sensory functions can be studied by evoking the peristaltic reflex, involving enteric sensory and motor nerves, but this has rarely been achieved. As submucosal neurones are more accessible (after removing the mucosa), direct neuronal recordings are possible. Neurosecretory functions are studied by measuring changes in short-circuit current across the mucosa. For all experiments, basic questions must be addressed. Because tissues are from patients, what are the controls and the influence of disease? How long does it take before function fully recovers? What is the impact of age- and gender-related differences? What is the optimal sample size? Addressing these and other questions minimizes variability and raises the scientific credibility of human tissue research. Such studies also reduce animal use. Further, the many differences between animal and human GI functions also means that human tissue research must question the ethical validity of using strains of animals with unproved translational significance. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

  2. Identifying Effective Enzyme Activity Targets for Recombinant Class I and Class II Collagenase for Successful Human Islet Isolation

    OpenAIRE

    Balamurugan, Appakalai N.; Green, Michael L.; Breite, Andrew G.; Loganathan, Gopalakrishnan; Wilhelm, Joshua J.; Tweed, Benjamin; Vargova, Lenka; Lockridge, Amber; Kuriti, Manikya; Hughes, Michael G.; Williams, Stuart K.; Hering, Bernhard J.; Dwulet, Francis E.; McCarthy, Robert C.

    2015-01-01

    Isolation following a good manufacturing practice-compliant, human islet product requires development of a robust islet isolation procedure where effective limits of key reagents are known. The enzymes used for islet isolation are critical but little is known about the doses of class I and class II collagenase required for successful islet isolation.

  3. Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography.

    Science.gov (United States)

    Blans, Kristine; Hansen, Maria S; Sørensen, Laila V; Hvam, Michael L; Howard, Kenneth A; Möller, Arne; Wiking, Lars; Larsen, Lotte B; Rasmussen, Jan T

    2017-01-01

    Studies have suggested that nanoscale extracellular vesicles (EV) in human and bovine milk carry immune modulatory properties which could provide beneficial health effects to infants. In order to assess the possible health effects of milk EV, it is essential to use isolates of high purity from other more abundant milk structures with well-documented bioactive properties. Furthermore, gentle isolation procedures are important for reducing the risk of generating vesicle artefacts, particularly when EV subpopulations are investigated. In this study, we present two isolation approaches accomplished in three steps based on size-exclusion chromatography (SEC) resulting in effective and reproducible EV isolation from raw milk. The approaches do not require any EV pelleting and can be applied to both human and bovine milk. We show that SEC effectively separates phospholipid membrane vesicles from the primary casein and whey protein components in two differently obtained casein reduced milk fractions, with one of the fractions obtained without the use of ultracentrifugation. Milk EV isolates were enriched in lactadherin, CD9, CD63 and CD81 compared to minimal levels of the EV-marker proteins in other relevant milk fractions such as milk fat globules. Nanoparticle tracking analysis and electron microscopy reveals the presence of heterogeneous sized vesicle structures in milk EV isolates. Lipid analysis by thin layer chromatography shows that EV isolates are devoid of triacylglycerides and presents a phospholipid profile differing from milk fat globules surrounded by epithelial cell plasma membrane. Moreover, the milk EV fractions are enriched in RNA with distinct and diverging profiles from milk fat globules. Collectively, our data supports that successful milk EV isolation can be accomplished in few steps without the use of ultracentrifugation, as the presented isolation approaches based on SEC effectively isolates EV in both human and bovine milk.

  4. Genetic relatedness of Legionella longbeachae isolates from human and environmental sources in Australia

    International Nuclear Information System (INIS)

    Lanser, J.A.; Doyle, R.; Sangster, N.; Steele, T.W.; Adams, M.

    1990-01-01

    The genetic relatedness of Legionella longbeachae isolated in Australia since 1987 was investigated by restriction fragment length polymorphism (RFLP) analysis and allozyme electrophoresis. Three radiolabeled probes were used in Southern hybridizations for the RFLP studies. They were Escherichia coli 16S and 23S rRNA and cloned fragments of L. longbeachae selected empirically from genomal banks in lambda and a cosmid. The legionellae included in the study comprised 11 Legionella longbeachae serogroup 1 organisms isolated form humans, 28 L. longbeachae serogroup 1 isolates from environmental sources, 3 L. longbeachae serogroup 2 environmental isolates. These were compared with the American Type Culture Collection reference strains of both serogroups and some other related Legionella species. Results of allozyme and RFLP analysis showed that all the isolates from humans and all but three of the environmental L. longbeachae serogroup 1 isolates were closely related. They were also closely related to L. longbeachae serogroup 1 ATCC 33462. There was wider variation among the three L. longbeachae serogroup 2 environmental isolates. One of these was closely related to L. longbeachae serogroup 2 ATCC 33484. RFLP studies with the rRNA probe provided the most discrimination among isolates but did not distinguish between the two serogroups

  5. CRISPR Diversity in E. coli Isolates from Australian Animals, Humans and Environmental Waters.

    Directory of Open Access Journals (Sweden)

    Maxim S Sheludchenko

    Full Text Available Seventy four SNP genotypes and 54 E. coli genomes from kangaroo, Tasmanian devil, reptile, cattle, dog, horse, duck, bird, fish, rodent, human and environmental water sources were screened for the presence of the CRISPR 2.1 loci flanked by cas2 and iap genes. CRISPR 2.1 regions were found in 49% of the strains analysed. The majority of human E. coli isolates lacked the CRISPR 2.1 locus. We described 76 CRISPR 2.1 positive isolates originating from Australian animals and humans, which contained a total of 764 spacer sequences. CRISPR arrays demonstrated a long history of phage attacks especially in isolates from birds (up to 40 spacers. The most prevalent spacer (1.6% was an ancient spacer found mainly in human, horse, duck, rodent, reptile and environmental water sources. The sequence of this spacer matched the intestinal P7 phage and the pO111 plasmid of E. coli.

  6. High resolution of heterogeneity among human neutrophil granules: physical, biochemical, and ultrastructural properties of isolated fractions.

    Science.gov (United States)

    Rice, W G; Kinkade, J M; Parmley, R T

    1986-08-01

    Previous studies on the fractionation of human neutrophil granules have identified two major populations: myeloperoxidase (MPO)-containing azurophil, or primary, granules and MPO-deficient specific, or secondary, granules. Peripheral blood neutrophils from individual donors were lysed in sucrose-free media by either hypotonic shock or nitrogen cavitation. Using a novel two-gradient Percoll density centrifugation system, the granule-rich postnuclear supernatant was rapidly (ten minutes) and reproducibly resolved into 13 granule fractions (L1 through L8 and H1 through H5). Granule flotation and recentrifugation experiments on both continuous, self-generated and multiple-step gradients using individual and mixed isolated fractions demonstrated that the banding patterns were isopycnic and nonartifactual. Isolated granules were intact based on the findings that biochemical latency of several granule enzymes was greater than 95%, and thin-sectioned electron micrographs demonstrated intact granule profiles. Biochemical analyses of the granule marker proteins MPO, beta-glucuronidase, lysozyme, and lactoferrin indicated that a number of the fractions were related to the major azurophil and specific granule populations. Lactoferrin was found in ten of 13 fractions (L1 through L8, H1 to H2), whereas MPO was found in every fraction. Consistent with these biochemical data, all fractions exhibited varying degrees of heterogeneity based on ultrastructural morphology and cytochemistry, including diaminobenzidine (DAB) reactivity for peroxidase and periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining for complex glycoconjugates. A variable but significant percentage (23% to 70%) of the granules in fractions L1 through L8 and H1 and H2 showed DAB reactivity, while about 90% of the granules in fractions H3 through H5 were peroxidase positive. These results demonstrated that DAB-reactive granules spanned the entire range of granule size and density. Ultrastructural PA

  7. Contribution of Avian Salmonella enterica Isolates to Human Salmonellosis Cases in Constantine (Algeria

    Directory of Open Access Journals (Sweden)

    Rachid Elgroud

    2015-01-01

    Full Text Available An epidemiological investigation was carried out on one hundred Salmonella isolates from broiler farms, slaughterhouses, and human patients in the Constantine region of Algeria, in order to explore the contribution of avian strains to human salmonellosis cases in this region over the same period of time. The isolates were characterized by phenotypic as well as genotypic methods. A large variety of antimicrobial resistance profiles was found among human isolates, while only seven profiles were found among avian isolates. Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR, Insertion Sequence 200-PCR (IS200-PCR, and Pulsed Field Gel Electrophoresis (PFGE resulted in the allocation of the isolates to 16, 20, and 34 different profiles, respectively. The 3 genotyping methods led to complementary results by underlining the clonality of some serovars with the diffusion and persistence of a single clone in the Constantine area as well as stressing the polymorphism present in isolates belonging to other serovars, indicating the diversity of potential reservoirs of nontyphoidal Salmonella. Altogether, our results seem to indicate that nontyphoidal avian Salmonella may play an important role in human salmonellosis in the Constantine region.

  8. Transcriptional profiling of human breast cancer cells cultured under microgravity conditions revealed the key role of genetic gravity sensors previously detected in Drosophila melanogaster

    Science.gov (United States)

    Valdivia-Silva, Julio E.; Lavan, David; Diego Orihuela-Tacuri, M.; Sanabria, Gabriela

    2016-07-01

    Currently, studies in Drosophila melanogaster has shown emerging evidence that microgravity stimuli can be detected at the genetic level. Analysis of the transcriptome in the pupal stage of the fruit flies under microgravity conditions versus ground controls has suggested the presence of a few candidate genes as "gravity sensors" which are experimentally validated. Additionally, several studies have shown that microgravity causes inhibitory effects in different types of cancer cells, although the genes involved and responsible for these effects are still unknown. Here, we demonstrate that the genes suggested as the sensors of gravitational waves in Drosophila melanogaster and their human counterpart (orthologous genes) are highly involved in carcinogenesis, proliferation, anti-apoptotic signals, invasiveness, and metastatic potential of breast cancer cell tumors. The transcriptome analyses suggested that the observed inhibitory effect in cancer cells could be due to changes in the genetic expression of these candidates. These results encourage the possibility of new therapeutic targets managed together and not in isolation.

  9. Salmonella Brandenburg in the pork chain in Italy: Genetic comparison with the human isolates.

    Science.gov (United States)

    Bonardi, Silvia; Morganti, Marina; Pupillo, Giovanni; Brindani, Franco

    2018-03-31

    Salmonella Brandenburg ranked 16 th among the serovars responsible for human infections in EU in 2015 and it was found to be associated with swine. In Emilia- Romagna and Lombardy regions of northern Italy, S. Brandenburg was isolated from mesenteric lymph nodes, fecal matter, carcasses and conveyor belts at pig slaughterhouses in 2014 and 2015. In the same area, S. Brandenburg was detected in pork salami in 2015. In the present study, 12 isolates of S. Brandenburg recovered from the pork food-chain were typed by Xba I PFGE and their three profiles were compared to all human S . Brandenburg isolates processed by the Surveillance System of Emilia- Romagna region from 2012 to 2017 (105 isolates). The most frequent pulsotype of porcine origin (6/12) was the second most frequent in humans (16/105). Of the other two pulsotypes of porcine origine (3/12 each), one was the most frequent in humans (41/105), the other was undetected among human isolates.

  10. Effects of vasopressin on the isolated perfused human collecting tubule.

    Science.gov (United States)

    Yanagawa, N; Trizna, W; Bar-Khayim, Y; Fine, L G

    1981-05-01

    Cortical collecting tubules (CCT) were dissected from the surviving normal tissue of human kidneys removed at operation for either carcinoma or calculus. These CCT's were perfused in vitro shortly after the nephrectomy was performed. Transtubular potential differences in different tubules varied from +3.2 to -2.0 mV and were reduced towards zero by lowering the temperature or by adding ouabain to the bath. In the absence of vasopressin, tubules were essentially impermeable to water with extremely low net water fluxes even in the presence of a transtubular osmotic gradient. Addition of vasopressin to the bath caused the transtubular osmotic water permeability coefficient to increase to values of 125, 175, and 155 X 10(-4) cm/sec in three tubules thus studied. These results demonstrate close similarities between the human CCT and the more extensively studied rabbit CCT.

  11. Draft genome sequences of two opportunistic pathogenic strains of Staphylococcus cohnii isolated from human patients.

    Science.gov (United States)

    Mendoza-Olazarán, Soraya; Garcia-Mazcorro, José F; Morfín-Otero, Rayo; Villarreal-Treviño, Licet; Camacho-Ortiz, Adrián; Rodríguez-Noriega, Eduardo; Bocanegra-Ibarias, Paola; Maldonado-Garza, Héctor J; Dowd, Scot E; Garza-González, Elvira

    2017-01-01

    Herein, we report the draft-genome sequences and annotation of two opportunistic pathogenic strains of Staphylococcus cohnii isolated from humans. One strain (SC-57) was isolated from blood from a male patient in May 2006 and the other (SC-532) from a catheter from a male patient in June 2006. Similar to other genomes of Staphylococcus species, most genes (42%) of both strains are involved in metabolism of amino acids and derivatives, carbohydrates and proteins. Eighty (4%) genes are involved in virulence, disease, and defense and both species show phenotypic low biofilm production and evidence of increased antibiotic resistance associated to biofilm production. From both isolates, a new Staphylococcal Cassette Chromosome mec was detected: mec class A, ccr type 1. This is the first report of whole genome sequences of opportunistic S. cohnii isolated from human patients.

  12. [Corynebacterium imitans isolated from blood culture in a patient with suspected bacteremia - the first isolation from human clinical material in the Czech Republic].

    Science.gov (United States)

    JeŽek, Petr; Zavadilová, Jana; Kolínská, Renáta; Švec, Pavel; Guttwirth, Jiří; Petráš, Petr

    2014-09-01

    The current view of the clinical importance of nondiphtherial corynebacteria recovered from human clinical material has changed considerably in recent decades; in many cases, a direct etiological role is assumed or has already been demonstrated. Presented is a case of suspected bacteremia in a hospitalized elderly woman with isolation of the very rare species Corynebacterium imitans from blood culture. However, the etiological significance of the isolated microorganism remains unclear. The aim was not to demonstrate the etiological significance of the isolated C. imitans strain but to report the occurrence of this very rare species which is considered to be the first isolation from humans in the Czech Republic.

  13. Mycobacterium bovis in Burkina Faso: epidemiologic and genetic links between human and cattle isolates.

    Directory of Open Access Journals (Sweden)

    Adama Sanou

    2014-10-01

    Full Text Available In sub-Saharan Africa, bovine tuberculosis (bTB is a potential hazard for animals and humans health. The goal of this study was to improve our understanding of bTB epidemiology in Burkina Faso and especially Mycobacterium bovis transmission within and between the bovine and human populations.Twenty six M. bovis strains were isolated from 101 cattle carcasses with suspected bTB lesions during routine meat inspections at the Bobo Dioulasso and Ouagadougou slaughterhouses. In addition, 7 M. bovis strains were isolated from 576 patients with pulmonary tuberculosis. Spoligotyping, RDAf1 deletion and MIRU-VNTR typing were used for strains genotyping. The isolation of M. bovis strains was confirmed by spoligotyping and 12 spoligotype signatures were detected. Together, the spoligotyping and MIRU-VNTR data allowed grouping the 33 M. bovis isolates in seven clusters including isolates exclusively from cattle (5 or humans (1 or from both (1. Moreover, these data (genetic analyses and phenetic tree showed that the M. bovis isolates belonged to the African 1 (Af1 clonal complex (81.8% and the putative African 5 (Af5 clonal complex (18.2%, in agreement with the results of RDAf1 deletion typing.This is the first detailed molecular characterization of M. bovis strains from humans and cattle in Burkina Faso. The distribution of the two Af1 and putative Af5 clonal complexes is comparable to what has been reported in neighbouring countries. Furthermore, the strain genetic profiles suggest that M. bovis circulates across the borders and that the Burkina Faso strains originate from different countries, but have a country-specific evolution. The genetic characterization suggests that, currently, M. bovis transmission occurs mainly between cattle, occasionally between cattle and humans and potentially between humans. This study emphasizes the bTB risk in cattle but also in humans and the difficulty to set up proper disease control strategies in Burkina Faso.

  14. Mycobacterium bovis in Burkina Faso: epidemiologic and genetic links between human and cattle isolates.

    Science.gov (United States)

    Sanou, Adama; Tarnagda, Zekiba; Kanyala, Estelle; Zingué, Dezemon; Nouctara, Moumini; Ganamé, Zakaria; Combary, Adjima; Hien, Hervé; Dembele, Mathurin; Kabore, Antoinette; Meda, Nicolas; Van de Perre, Philippe; Neveu, Dorine; Bañuls, Anne Laure; Godreuil, Sylvain

    2014-10-01

    In sub-Saharan Africa, bovine tuberculosis (bTB) is a potential hazard for animals and humans health. The goal of this study was to improve our understanding of bTB epidemiology in Burkina Faso and especially Mycobacterium bovis transmission within and between the bovine and human populations. Twenty six M. bovis strains were isolated from 101 cattle carcasses with suspected bTB lesions during routine meat inspections at the Bobo Dioulasso and Ouagadougou slaughterhouses. In addition, 7 M. bovis strains were isolated from 576 patients with pulmonary tuberculosis. Spoligotyping, RDAf1 deletion and MIRU-VNTR typing were used for strains genotyping. The isolation of M. bovis strains was confirmed by spoligotyping and 12 spoligotype signatures were detected. Together, the spoligotyping and MIRU-VNTR data allowed grouping the 33 M. bovis isolates in seven clusters including isolates exclusively from cattle (5) or humans (1) or from both (1). Moreover, these data (genetic analyses and phenetic tree) showed that the M. bovis isolates belonged to the African 1 (Af1) clonal complex (81.8%) and the putative African 5 (Af5) clonal complex (18.2%), in agreement with the results of RDAf1 deletion typing. This is the first detailed molecular characterization of M. bovis strains from humans and cattle in Burkina Faso. The distribution of the two Af1 and putative Af5 clonal complexes is comparable to what has been reported in neighbouring countries. Furthermore, the strain genetic profiles suggest that M. bovis circulates across the borders and that the Burkina Faso strains originate from different countries, but have a country-specific evolution. The genetic characterization suggests that, currently, M. bovis transmission occurs mainly between cattle, occasionally between cattle and humans and potentially between humans. This study emphasizes the bTB risk in cattle but also in humans and the difficulty to set up proper disease control strategies in Burkina Faso.

  15. Complete genome sequence of Bifidobacterium breve CECT 7263, a strain isolated from human milk

    OpenAIRE

    Jiménez, Esther; Villar-Tajadura, M. Antonia; Marín, María; Fontecha, F. Javier; Requena, Teresa; Arroyo, Rebeca; Fernández, Leónides; Rodríguez, Juan M.

    2012-01-01

    Bifidobacterium breve is an actinobacterium frequently isolated from colonic microbiota of breastfeeding babies. Here, we report the complete and annotated genome sequence of a B. breve strain isolated from human milk, B. breve CECT 7263. The genome sequence will provide new insights into the biology of this potential probiotic organism and will allow the characterization of genes related to beneficial properties. © 2012, American Society for Microbiology.

  16. Draft genome sequences of two opportunistic pathogenic strains of Staphylococcus cohnii isolated from human patients

    OpenAIRE

    Mendoza-Olazar?n, Soraya; Garcia-Mazcorro, Jos? F.; Morf?n-Otero, Rayo; Villarreal-Trevi?o, Licet; Camacho-Ortiz, Adri?n; Rodr?guez-Noriega, Eduardo; Bocanegra-Ibarias, Paola; Maldonado-Garza, H?ctor J.; Dowd, Scot E.; Garza-Gonz?lez, Elvira

    2017-01-01

    Herein, we report the draft-genome sequences and annotation of two opportunistic pathogenic strains of Staphylococcus cohnii isolated from humans. One strain (SC-57) was isolated from blood from a male patient in May 2006 and the other (SC-532) from a catheter from a male patient in June 2006. Similar to other genomes of Staphylococcus species, most genes (42%) of both strains are involved in metabolism of amino acids and derivatives, carbohydrates and proteins. Eighty (4%) genes are involved...

  17. Microbiological and molecular characterization of human clinical isolates of Staphylococcus cohnii, Staphylococcus hominis, and Staphylococcus sciuri.

    Science.gov (United States)

    Garza-González, Elvira; Morfin-Otero, Rayo; Martínez-Vázquez, Manuel A; Gonzalez-Diaz, Esteban; González-Santiago, Omar; Rodríguez-Noriega, Eduardo

    2011-12-01

    The incidence of coagulase-negative staphylococci reported as causative agents of nosocomial infections has risen in the last decade. The aim of this study was to characterize biofilm formation, antibiotic resistance, SCCmec type, and genetic relatedness in clinical isolates of Staphylococcus cohnii, Staphylococcus hominis, and Staphylococcus sciuri recovered from humans. Clinically relevant isolates of S. cohnii (n = 15), S. hominis (n = 9), and S. sciuri (n = 6), were collected from patients. Biofilm formation was evaluated using crystal violet staining, drug susceptibility was assessed using the broth microdilution method, and methicillin resistance was measured using the cefoxitin disk test. SCCmec was typed using 2 different methodologies, and genetic relatedness was determined by pulsed-field gel electrophoresis (PFGE). Sixty percent (9/15) of S. cohnii, 33% (3/9) of S. hominis, and 50% (3/6) of S. sciuri isolates were categorized as weak producers of biofilm. None of the isolates were resistant to vancomycin or linezolid. All 3 species showed a high resistance (> 66%) to ampicillin, levofloxacin, erythromycin, and ceftriaxone, and the majority of the isolates were methicillin-resistant. PFGE revealed that the S. cohnii isolates comprised 1 dominant clone. The S. cohnii, S. hominis, and S. sciuri isolates analyzed in this study showed a high methicillin resistance and resistance to other antimicrobials. The results of this study strongly suggest that coagulase-negative staphylococci harbour new SCCmec elements. We report the first case of a clone of S. cohnii associated with human disease.

  18. Human cultured cells are capable to incorporate isolated plant mitochondria loaded with exogenous DNA

    Directory of Open Access Journals (Sweden)

    Laktionov P. P.

    2012-07-01

    Full Text Available Aim. To investigate the possibility of human cultured cells to incorporate isolated mitochondria together with exogenous DNA introduced into organelles. Methods. Two approaches were used for this purpose, fluorescent labelling of mitochondria and/or DNA with subsequent analysis of the cells subjected to incubation by microscopy or by quantitative PCR. Results. We have shown that human cultured cells lines, HeLa and HUVEC, are capable to uptake isolated plant mitochondria and that this process depends on the incubation time and concentration of organelles present in medium. The incorporated mitochondria can serve as vehicles to deliver exogenous DNA into human cells, this DNA is then distributed in different cell compartments. Conclusions. These results are preliminary and need further investigations, including testing the possibility of human cells to incorporate the mitochondria of human or animal origin and creating genetic construction which could provide certain selectivity or stability of the transferred exogenous DNA upon cell uptake of the mitochondria as vectors.

  19. Affinity-purified human interleukin I is cytotoxic to isolated islets of Langerhans

    DEFF Research Database (Denmark)

    Mandrup-Poulsen, T; Bendtzen, K; Nerup, J

    1986-01-01

    Addition of highly purified human Interleukin-1 to the culture medium of isolated rat islets of Langerhans for 6 days led to 88% inhibition of glucose-induced insulin-release, reduction of islet contents of insulin and glucagon to 31% and 8% respectively, and disintegration of the islets. These e......Addition of highly purified human Interleukin-1 to the culture medium of isolated rat islets of Langerhans for 6 days led to 88% inhibition of glucose-induced insulin-release, reduction of islet contents of insulin and glucagon to 31% and 8% respectively, and disintegration of the islets...

  20. RNA isolation for transcriptomics of human and mouse small skin biopsies

    Directory of Open Access Journals (Sweden)

    Breit Timo M

    2011-10-01

    Full Text Available Abstract Background Isolation of RNA from skin biopsies presents a challenge, due to the tough nature of skin tissue and a high presence of RNases. As we lacked the dedicated equipment, i.e. homogenizer or bead-beater, needed for the available RNA from skin isolation methods, we adapted and tested our zebrafish single-embryo RNA-isolation protocol for RNA isolation from skin punch biopsies. Findings We tested our new RNA-isolation protocol in two experiments: a large-scale study with 97 human skin samples, and a small study with 16 mouse skin samples. Human skin was sampled with 4.0 mm biopsy punches and for the mouse skin different punch diameter sizes were tested; 1.0, 1.5, 2.0, and 2.5 mm. The average RNA yield in human samples was 1.5 μg with an average RNA quality RIN value of 8.1. For the mouse biopsies, the average RNA yield was 2.4 μg with an average RIN value of 7.5. For 96% of the human biopsies and 100% of the mouse biopsies we obtained enough high-quality RNA. The RNA samples were successfully tested in a transcriptomics analysis using the Affymetrix and Roche NimbleGen platforms. Conclusions Using our new RNA-isolation protocol, we were able to consistently isolate high-quality RNA, which is apt for further transcriptomics analysis. Furthermore, this method is already useable on biopsy material obtained with a punch diameter as small as 1.5 mm.

  1. [Microbiological characterisation of Listeria monocytogenes isolates from human cases in Andalusia].

    Science.gov (United States)

    Lepe, José A; Torres, María José; Liró, Julia; Luque, Rafael; Aznar, Javier

    2012-12-01

    The aim of this study was to perform a retrospective study by genotyping 154 isolates from human listeriosis cases occurred in the region of Andalusia (southern Spain) in the period 2005-2009. Serotyping was performed for 1 and 4 somatic antigens using commercial Listeria antisera, and by multiplex-PCR serogrouping according to the method described by Doumith et al. (2004). The antimicrobial susceptibility was performed by Epsilon test and interpreted by CLSI criteria. PFGE was performed according to the PulseNet protocol with the ApaI enzyme. The similarity of PFGE profiles was evaluated using the Bionumerics software. The multiplex PCR protocol described by Chen and Knabel (2007) was used for the identification of isolates belonging to L. monocytogenes ECI, ECII, and ECIII epidemic clones. The 154 isolates were grouped into four serotypes: 4b [94 (61%)] strains, 1/2b [30 (19%)] strains, 1/2a [27 (18%)] strains, and 1/2c [3 (2%)] strains, with 100% of susceptibility to ampicillin and cotrimoxazole. A further sixty-two ApaI distinct pulsotypes were recognized. Thirty-seven isolates (24%) showed unique ApaI pulsotypes, and the remaining 117 strains (76%) were assigned to 25 ApaI clusters (60% in clusters of more than two isolates). The EC markers were found in 62 (40.3%) of the L. monocytogenes isolates tested. The ECI marker was present in 43 (46.2%) 4b serotype isolates, ECII in 10 (10.7%) 4b serotype isolates, and ECIII in 9 (33,3%) 1/2a serotype isolates. A large proportion of the human listeriosis cases under investigation could be grouped into molecular subtype clusters, and our cases could be related to international food-borne outbreaks. Copyright © 2011 Elsevier España, S.L. All rights reserved.

  2. Whole genome sequencing of Mycobacterium bovis to obtain molecular fingerprints in human and cattle isolates from Baja California, Mexico

    Directory of Open Access Journals (Sweden)

    Sarai Estrella Sandoval-Azuara

    2017-10-01

    Conclusions: All isolates from humans had spoligotype patterns that matched those observed in the cattle isolates, and all human isolates shared common ancestors with cattle in Baja California based on SNP analysis. This suggests that most human tuberculosis caused by M. bovis in Baja California is derived from M. bovis circulating in Baja California cattle. These results reinforce the importance of bovine tuberculosis surveillance and control in this region.

  3. Characterization of RNA isolated from eighteen different human tissues: results from a rapid human autopsy program.

    Science.gov (United States)

    Walker, Douglas G; Whetzel, Alexis M; Serrano, Geidy; Sue, Lucia I; Lue, Lih-Fen; Beach, Thomas G

    2016-09-01

    Many factors affect the integrity of messenger RNA from human autopsy tissues including postmortem interval (PMI) between death and tissue preservation and the pre-mortem agonal and disease states. In this communication, we describe RNA isolation and characterization of 389 samples from 18 different tissues from elderly donors who were participants in a rapid whole-body autopsy program located in Sun City, Arizona ( www.brainandbodydonationprogram.org ). Most tissues were collected within a PMI of 2-6 h (median 3.15 h; N = 455), but for this study, tissue from cases with longer PMIs (1.25-29.25 h) were included. RNA quality was assessed by RNA integrity number (RIN) and total yield (ng RNA/mg tissue). RIN correlated with PMI for heart (r = -0.531, p = 0.009) and liver (r = -558, p = 0.0017), while RNA yield correlated with PMI for colon (r = -485, p = 0.016) and skin (r = -0.460, p = 0.031). RNAs with the lowest integrity were from skin and cervix where 22.7 and 31.4 % of samples respectively failed to produce intact RNA; by contrast all samples from esophagus, lymph node, jejunum, lung, stomach, submandibular gland and kidney produced RNA with measurable RINs. Expression levels in heart RNA of 4 common housekeeping normalization genes showed significant correlations of Ct values with RIN, but only one gene, glyceraldehyde-3 phosphate dehydrogenase, showed a correlation of Ct with PMI. There were no correlations between RIN values obtained for liver, adrenal, cervix, esophagus and lymph node and those obtained from corresponding brain samples. We show that high quality RNA can be produced from most human autopsy tissues, though with significant differences between tissues and donors. The RNA stability and yield did not depend solely on PMI; other undetermined factors are involved, but these do not include the age of the donor.

  4. Isolate-dependent growth, virulence, and cell wall composition in the human pathogen Aspergillus fumigatus.

    Directory of Open Access Journals (Sweden)

    Nansalmaa Amarsaikhan

    Full Text Available The ubiquitous fungal pathogen Aspergillus fumigatus is a mediator of allergic sensitization and invasive disease in susceptible individuals. The significant genetic and phenotypic variability between and among clinical and environmental isolates are important considerations in host-pathogen studies of A. fumigatus-mediated disease. We observed decreased radial growth, rate of germination, and ability to establish colony growth in a single environmental isolate of A. fumigatus, Af5517, when compared to other clinical and environmental isolates. Af5517 also exhibited increased hyphal diameter and cell wall β-glucan and chitin content, with chitin most significantly increased. Morbidity, mortality, lung fungal burden, and tissue pathology were decreased in neutropenic Af5517-infected mice when compared to the clinical isolate Af293. Our results support previous findings that suggest a correlation between in vitro growth rates and in vivo virulence, and we propose that changes in cell wall composition may contribute to this phenotype.

  5. CRISPR Typing and Antibiotic Resistance Correlates with Polyphyletic Distribution in Human Isolates of Salmonella Kentucky.

    Science.gov (United States)

    Vosik, Dorothy; Tewari, Deepanker; Dettinger, Lisa; M'ikanatha, Nkuchia M; Shariat, Nikki W

    2018-02-01

    Although infrequently associated with reported salmonellosis in humans, Salmonella enterica, subsp. enterica serovar Kentucky (ser. Kentucky) is the most common nonclinical, nonhuman serovar reported in the United States. The goal of this study was to use Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-multi-virulence-locus sequence typing (MVLST) to subtype a collection of human clinical isolates of ser. Kentucky submitted to the Pennsylvania Department of Health and to determine the extent of antibiotic resistance in these strains. This analysis highlighted the polyphyletic nature of ser. Kentucky, and separated our isolates into two groups, Group I and Group II, which were equally represented in our collection. Furthermore, antimicrobial susceptibility testing on all isolates using a National Antimicrobial Resistance Monitoring System (NARMS) panel of antibiotics demonstrated that resistance profiles could be divided into two groups. Group I isolates were resistant to cephems and penicillins, whereas Group II isolates were resistant to quinolones, gentamicin, and sulfisoxazole. Collectively, 50% of isolates were resistant to three or more classes of antibiotics and 30% were resistant to five or more classes. The correlation of antibiotic resistance with the two different lineages may reflect adaptation within two distinct reservoirs of ser. Kentucky, with differential exposure to antimicrobials.

  6. Association Between Antimicrobial Resistance in Escherichia coli Isolates from Food Animals and Blood Stream Isolates from Humans in Europe: An Ecological Study

    DEFF Research Database (Denmark)

    Vieira, Antonio; Collignon, Peter; Aarestrup, Frank Møller

    2011-01-01

    Background: In addition to medical antimicrobial usage, the use of antimicrobials in food animals contributes to the occurrence of resistance among some bacterial species isolated from infections in humans. Recently, several studies have indicated that a large proportion of Escherichia coli causing...... infections in humans, especially those resistant to antimicrobials, have an animal origin.Methods: We analyzed the correlation between the prevalence of antimicrobial resistance in E. coli isolates from blood stream infections in humans and in E. coli isolates from poultry, pigs, and cattle between 2005...... and 2008 for 11 countries, using available surveillance data. We also assessed the correlation between human antimicrobial usage and the occurrence of resistance in E. coli isolates from blood stream infections.Results: Strong and significant correlations between prevalences of resistance to ampicillin (r...

  7. Virulence-associated gene pattern of porcine and human Yersinia enterocolitica biotype 4 isolates.

    Science.gov (United States)

    Schneeberger, M; Brodard, I; Overesch, G

    2015-04-02

    Yersinia enterocolitica 4/O:3 is the most important human pathogenic bioserotype in Europe and the predominant pathogenic bioserotype in slaughter pigs. Although many studies on the virulence of Y. enterocolitica strains have showed a broad spectrum of detectable factors in pigs and humans, an analysis based on a strict comparative approach and serving to verify the virulence capability of porcine Y. enterocolitica as a source for human yersiniosis is lacking. Therefore, in the present study, strains of biotype (BT) 4 isolated from Swiss slaughter pig tonsils and feces and isolates from human clinical cases were compared in terms of their spectrum of virulence-associated genes (yadA, virF, ail, inv, rovA, ymoA, ystA, ystB and myfA). An analysis of the associated antimicrobial susceptibility pattern completed the characterization. All analyzed BT 4 strains showed a nearly similar pattern, comprising the known fundamental virulence-associated genes yadA, virF, ail, inv, rovA, ymoA, ystA and myfA. Only ystB was not detectable among all analyzed isolates. Importantly, neither the source of the isolates (porcine tonsils and feces, humans) nor the serotype (ST) had any influence on the gene pattern. From these findings, it can be concluded that the presence of the full complement of virulence genes necessary for human infection is common among porcine BT 4 strains. Swiss porcine BT 4 strains not only showed antimicrobial susceptibility to chloramphenicol, cefotaxime, ceftazidime, ciprofloxacin, colistin, florfenicol, gentamicin, kanamycin, nalidixic acid, sulfamethoxazole, streptomycin, tetracycline and trimethoprim but also showed 100% antibiotic resistance to ampicillin. The human BT 4 strains revealed comparable results. However, in addition to 100% antibiotic resistance to ampicillin, 2 strains were resistant to chloramphenicol and nalidixic acid. Additionally, 1 of these strains was resistant to sulfamethoxazole. The results demonstrated that Y. enterocolitica BT 4

  8. Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography

    DEFF Research Database (Denmark)

    Blans, Kristine Ingrid Marie; Hansen, Maria Stenum; Sørensen, Laila V.

    2017-01-01

    -marker proteins in other relevant milk fractions such as milk fat globules. Nanoparticle tracking analysis and electron microscopy reveals the presence of heterogeneous sized vesicle structures in milk EV isolates. Lipid analysis by thin layer chromatography shows that EV isolates are devoid of triacylglycerides...... accomplished in three steps based on size-exclusion chromatography (SEC) resulting in effective and reproducible EV isolation from raw milk. The approaches do not require any EV pelleting and can be applied to both human and bovine milk. We show that SEC effectively separates phospholipid membrane vesicles...... from the primary casein and whey protein components in two differently obtained casein reduced milk fractions, with one of the fractions obtained without the use of ultracentrifugation. Milk EV isolates were enriched in lactadherin, CD9, CD63 and CD81 compared to minimal levels of the EV...

  9. Identification of species and genetic variation in Taenia isolates from human and swine of North India.

    Science.gov (United States)

    Singh, Satyendra K; Prasad, Kashi N; Singh, Aloukick K; Gupta, Kamlesh K; Chauhan, Ranjeet S; Singh, Amrita; Singh, Avinash; Rai, Ravi P; Pati, Binod K

    2016-10-01

    Taenia solium is the major cause of taeniasis and cysticercosis/neurocysticercosis (NCC) in the developing countries including India, but the existence of other Taenia species and genetic variation have not been studied in India. So, we studied the existence of different Taenia species, and sequence variation in Taenia isolates from human (proglottids and cysticerci) and swine (cysticerci) in North India. Amplification of cytochrome c oxidase subunit 1 gene (cox1) was done by polymerase chain reaction (PCR) followed by sequencing and phylogenetic analysis. We identified two species of Taenia i.e. T. solium and Taenia asiatica in our isolates. T. solium isolates showed similarity with Asian genotype and nucleotide variations from 0.25 to 1.01 %, whereas T. asiatica displayed nucleotide variations ranged from 0.25 to 0.5 %. These findings displayed the minimal genetic variations in North Indian isolates of T. solium and T. asiatica.

  10. Successful isolation, in vitro expansion and characterization of stem cells from Human Dental Pulp

    OpenAIRE

    Preethy SP; Srinivasan T; Tholcopiyan L; Thamaraikannan P; Srinivasan V; Murugan P; Manjunath S; Kannan TA; Shalini R; Sunil PM; Manikandhan R; Muthu MS; Abraham S

    2010-01-01

    BACKGROUND: Recent studies have shown that mesenchymal stem cells isolated from post natal human dental pulp, (Dental pulp stem cells-DPSCs) which is from permanent teeth and SHED (stem cells from human exfoliated deciduous teeth),the Periodontal ligament stem cells (PDLSC) and Stem cells from root Apical papilla(SCAP)have the potential to differentiate into cells of a variety of tissues including heart, muscle, cartilage, bone, nerve, salivary glands, teeth etc(1,2,3,4).This multipotential a...

  11. Molecular Characterisation of Salmonella enterica Serovar Typhi Isolated from Typhoidial Humans

    Directory of Open Access Journals (Sweden)

    Arunava Das

    2012-09-01

    Full Text Available Aims: Salmonella enterica serovar Typhi is the major causative agent for typhoidial fever around the globe among human population reported till date. Present research work was carried out for detection and molecular characterisation of Salmonella enterica serovar Typhi isolated from humans with Typhoidial fever by biochemical, phenotypical and virulence gene based polymerase chain reaction (PCR techniques. The isolated strains were also investigated for antibiotic susceptibility patterns as a control measure. Methodology and Results: A total of 16 clinical samples were collected from the same numbers of patients (7 males and 9 females from Coimbatore, Erode and Salem districts of Tamil Nadu and were processed via broth enrichment methods for isolation and identification of the causative agent S. enterica serovar Typhi. Microbiological and biochemical investigations revealed the presence of S. Typhi from 16 samples. The biotyping of the isolates showed that all the isolates belonged to biotype IV. The PCR analysis confirmed the presence of invA (Invasion gene, 244bp, tyv (Tyveloseepimerase gene, 615 bp, fliC-d (Phage-1 flagellin gene for d-antigen, 750 bp and viaB (Vi antigen gene, 439bp in all 16 clinical samples. The antibiotic susceptibility test that was carried out among the isolates against 12 antimicrobial agents, showed 100 % resistance to only ampicillin and 100 % sensitivity to carbenicillin, chloramphenicol, clindamycin, gentamycin, kanamycin and tetracycline.Conclusion, significance and impact of study: This study confirmed the association of virulent strains of S. enterica serovar Typhi from Typhoidial fever among human population and suggested that PCR based diagnostic could be very useful for the rapid detection of S. Typhi isolates. Present study emphasized the use of antibiotic like chloramphenicol or in combination with other antibiotics for the effective control of S. Typhi.

  12. Probiotic attributes of Lactobacillus strains isolated from food and of human origin.

    Science.gov (United States)

    Gaudana, Sandeep B; Dhanani, Akhilesh S; Bagchi, Tamishraha

    2010-06-01

    Lactobacilli isolated from various sources were identified on the basis of 16S-23S rRNA gene intergenic region amplification and subsequent sequencing of the smaller intergenic region. An in vitro analysis of probiotic properties including binding, ability to tolerate different concentrations of bile, survival in acidic buffer and antimicrobial activity of four different isolates and two standard strains (Lactobacillus plantarum American Type Culture Collection (ATCC) 8014 and L. rhamnosus GG (LGG)) was carried out. The ability of each isolate to stimulate Caco-2 cells, human peripheral blood mononuclear cells (PBMC) and THP-1 cells resulting in immunomodulation of these cells was analysed. Isolates L. rhamnosus CS25 and L. delbrueckii M and standard strain ATCC 8014 showed broad antimicrobial activity, and isolates CS25 (percentage of survival 6.9 % at pH 2.5, 5.1 % at pH 2.0) and L. plantarum CS23 (5.7 % at pH 2.5, 4.9 % at pH 2.0) have shown good tolerance to acidic pH. Isolate CS23 showed a good survival (14 %) after 2 h incubation in de Man, Rogosa and Sharpe (MRS) medium containing 3 % bile salts. Isolates CS23, CS25 and L. fermentum ASt1 could stimulate Caco-2 cells, human PBMC and THP-1 cells for a strong and varied immunomodulatory response in these cells. Though LGG showed poor antimicrobial activity as well as bile and acid tolerance, it was found to be the best binding strain tested. Child faecal isolate CS23 from the present study showed high binding ability (seventeen bacteria/Caco-2), high tolerance to acidic pH and bile salts and significant immunomodulation; therefore it is a good potential probiotic candidate.

  13. Isolation and identification of the human homolog of a new p53-binding protein, Mdmx

    NARCIS (Netherlands)

    Shvarts, A.; Bazuine, M.; Dekker, P.; Ramos, Y. F.; Steegenga, W. T.; Merckx, G.; van Ham, R. C.; van der Houven van Oordt, W.; van der Eb, A. J.; Jochemsen, A. G.

    1997-01-01

    We recently reported the identification of a mouse cDNA encoding a new p53-associating protein that we called Mdmx because of its structural similarity to Mdm2, a well-known p53-binding protein. Here we report the isolation of a cDNA encoding the human homolog of Mdmx. The ORF of the cDNA encodes a

  14. The Psychology of Isolated and Confined Environments: Understanding Human Behavior in Antarctica.

    Science.gov (United States)

    Palinkas, Lawrence A.

    2003-01-01

    Reviews lessons learned from research in Antarctica with relevance to understanding human behavior in other isolated and confined environments. Outlines four distinct characteristics of psychosocial adaptation to such environments and discusses some of the benefits for individuals seeking challenging experiences. (Contains references.) (SLD)

  15. Body temperature predicts the direction of internal desynchronization in humans isolated from time cues

    NARCIS (Netherlands)

    Daan, Serge; Honma, Sato; Honma, Ken-ichi

    2013-01-01

    This publication presents a new analysis of experiments that were carried out in human subjects in isolation from time cues, under supervision of Jurgen Aschoff and Rutger Wever at the Max Planck Institute for Behavioural Physiology (Erling-Andechs, Germany, 1964-1974). Mean rectal temperatures

  16. MICs of Oxazolidinones for Rhodococcus equi Strains Isolated from Humans and Animals

    OpenAIRE

    Bowersock, Terry L.; Salmon, Sarah A.; Portis, Ellen S.; Prescott, John F.; Robison, Denise A.; Ford, Charles W.; Watts, Jeffrey L.

    2000-01-01

    Eperezolid and linezolid are representatives of a new class of orally active, synthetic antimicrobial agents. The in vitro activity values (MICs) of linezolid, eperezolid, and comparator antibiotics against 102 strains of Rhodococcus equi isolated from humans and animals were determined. Linezolid was more active than eperezolid against the strains tested; premafloxacin was the most active comparator antibiotic.

  17. MLVA and LPS Characteristics of Brucella canis Isolated from Humans and Dogs in Zhejiang, China

    Directory of Open Access Journals (Sweden)

    Dongri Piao

    2017-12-01

    Full Text Available BackgroundBrucella canis is a pathogenic bacterium that causes brucellosis in dogs, and its zoonotic potential has been increasing in recent years. B. canis is a rare source of human brucellosis in China, where Brucella melitensis has been the major pathogen associated with human brucellosis outbreaks. In late 2011, a case of a B. canis infection was detected in a human patient in Zhejiang Province, China. To compare the genotypes between strains of B. canis isolated from the patient and from dogs, a multiple-locus variable-number tandem-repeat analysis (MLVA-16 was performed. In addition, the lipopolysaccharide-synthesis-related genes were analyzed with the B. canis reference strain RM6/66.Results32 B. canis strains were divided into 26 genotypes using MLVA-16 [Hunter-Gaston Diversity Index (HGDI = 0.976]. The HGDI indexes for various loci ranged between 0.000 and 0.865. All four Hangzhou isolates were indistinguishable using panel 1 (genotype 3 and panel 2A (genotype 28. However, these strains were distinctly different from other isolates from Beijing, Jiangsu, Liaoning, and Inner Mongolia at Bruce 09. The emergence of a human B. canis infection was limited to an area. Comparative analysis indicated B. canis from canines and humans have no differences in lipopolysaccharide-synthesis locus.ConclusionThe comprehensive approaches have been used to analyze human and canine B. canis isolates, including molecular epidemiological and LPS genetic characteristics. Further detailed analysis of the whole genomic sequencing will contribute to understanding of the pathogenicity of B. canis in humans.

  18. Simultaneous isolation of vascular endothelial cells and mesenchymal stem cells from the human umbilical cord.

    Science.gov (United States)

    Kadam, Sachin S; Tiwari, Shubha; Bhonde, Ramesh R

    2009-01-01

    The umbilical cord represents the link between mother and fetus during pregnancy. This cord is usually discarded as a biological waste after the child's birth; however, its importance as a "store house" of stem cells has been explored recently. We developed a method of simultaneous isolation of endothelial cells (ECs) from the vein and mesenchymal stem cells from umbilical cord Wharton's jelly of the same cord. The isolation protocol has been simplified, modified, and improvised with respect to choice of enzyme and enzyme mixture, digestion time, cell yield, cell growth, and culture medium. Isolated human umbilical vascular ECs (hUVECs) were positive for von-Willibrand factor, a classical endothelial marker, and could form capillary-like structures when seeded on Matrigel, thus proving their functionality. The isolated human umbilical cord mesenchymal stem cells (hUCMSCs) were found positive for CD44, CD90, CD 73, and CD117 and were found negative for CD33, CD34, CD45, and CD105 surface markers; they were also positive for cytoskeleton markers of smooth muscle actin and vimentin. The hUCMSCs showed multilineage differentiation potential and differentiated into adipogenic, chondrogenic, osteogenic, and neuronal lineages under influence of lineage specific differentiation medium. Thus, isolating endothelial cells as well as mesenchymal cells from the same umbilical cord could lead to complete utilization of the available tissue for the tissue engineering and cell therapy.

  19. Should we isolate human preantral follicles before or after cryopreservation of ovarian tissue?

    Science.gov (United States)

    Vanacker, Julie; Luyckx, Valérie; Amorim, Christiani; Dolmans, Marie-Madeleine; Van Langendonckt, Anne; Donnez, Jacques; Camboni, Alessandra

    2013-04-01

    To evaluate the survival and growth potential of human preantral follicles isolated before and after cryopreservation. Pilot study. Gynecology research unit in a university hospital. Six women aged 27 to 32 years. Six ovarian biopsy samples were cut into two equal parts, half subjected to slow-freezing followed by follicle isolation (cryo-iso group) and alginate-matrigel embedding, and half immediately processed for follicle isolation and alginate-matrigel embedding followed by slow-freezing (iso-cryo group) or used as fresh controls (fresh group). Follicle number, viability, diameter, and morphology. After 1,134 preantral follicles had been isolated from fresh biopsy samples and 1,132 from frozen specimens, the three groups were compared before and after 7 days of in vitro culture (IVC) in alginate-matrigel beads. No statistically significant differences in viability were found between the three groups before or after IVC, but follicle diameter increased in all three groups after IVC. Morphology analysis revealed well-preserved follicles in both the iso-cryo and cryo-iso groups after IVC. Human preantral follicles can be successfully cryopreserved before or after isolation without impairing their ability to survive and grow in vitro. This could lead to development of new protocols for follicle cryopreservation, IVC, and grafting in clinical and research settings for fertility preservation. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  20. Prevalence of plant beneficial and human pathogenic bacteria isolated from salad vegetables in India.

    Science.gov (United States)

    Nithya, Angamuthu; Babu, Subramanian

    2017-03-14

    The study aimed at enumerating, identifying and categorizing the endophytic cultivable bacterial community in selected salad vegetables (carrot, cucumber, tomato and onion). Vegetable samples were collected from markets of two vegetable hot spot growing areas, during two different crop harvest seasons. Crude and diluted vegetable extracts were plated and the population of endophytic bacteria was assessed based on morphologically distinguishable colonies. The bacterial isolates were identified by growth in selective media, biochemical tests and 16S rRNA gene sequencing. The endophytic population was found to be comparably higher in cucumber and tomato in both of the sampling locations, whereas lower in carrot and onion. Bacterial isolates belonged to 5 classes covering 46 distinct species belonging to 19 genera. Human opportunistic pathogens were predominant in carrot and onion, whereas plant beneficial bacteria dominated in cucumber and tomato. Out of the 104 isolates, 16.25% are human pathogens and 26.5% are human opportunistic pathogens. Existence of a high population of plant beneficial bacteria was found to have suppressed the population of plant and human pathogens. There is a greater potential to study the native endophytic plant beneficial bacteria for developing them as biocontrol agents against human pathogens that are harboured by plants.

  1. Genetic diversity and antimicrobial resistance of Yersinia enterocolitica isolated from pigs and humans in Lithuania.

    Science.gov (United States)

    Novoslavskij, Aleksandr; Kudirkienė, Eglė; Marcinkutė, Audronė; Bajoriūnienė, Almina; Korkeala, Hannu; Malakauskas, Mindaugas

    2013-06-01

    Yersiniosis is one of the three leading foodborne zoonoses in Lithuania, and the incidence of 12.86 per 100,000 population was the highest among EU member states in 2010. Contaminated pig carcasses and subsequently undercooked pig meat are considered to be the primary transmission vehicle of enteropathogenic Y. enterocolitica to consumers. With the aim of evaluating pigs as a possible source of human yersiniosis in Lithuania, this study investigated the genetic diversity of Y. enterocolitica isolated from pigs and human cases of yersiniosis. In addition, the antimicrobial resistance of selected isolates from both sources was compared. In total, 83 Y. enterocolitica strains were characterised using pulsed field gel electrophoresis. Overall, 68% of Y. enterocolitica 4/O:3 pulsotypes found in human clinical samples were identical to 81% of pulsotypes found in the pig production chain. Yersinia enterocolitica pulsotype II was confirmed as the dominant pulsotype in the pig production chain and was identical to nine of 19 Y. enterocolitica strains found in humans. All tested Y. enterocolitica 4/O:3 strains were resistant to ampicillin and erythromycin and sensitive to ciprofloxacin. Of the strains studied, 5% were resistant to tetracycline and streptomycin. This study showed that pigs may be the main source of human yersiniosis in Lithuania. In addition, Y. enterocolitica 4/O:3 strains isolated from the pig production chain and from yersiniosis patients shared similar resistance to different antimicrobials. © 2012 Society of Chemical Industry.

  2. Structure of recombinant human carboxylesterase 1 isolated from whole cabbage looper larvae

    International Nuclear Information System (INIS)

    Greenblatt, Harry M.; Otto, Tamara C.; Kirkpatrick, Melanie G.; Kovaleva, Elena; Brown, Susan; Buchman, George; Cerasoli, Douglas M.; Sussman, Joel L.

    2012-01-01

    Large quantities of recombinant human carboxylesterase 1 have been produced in an economical whole insect larvae system. The crystal structure of this enzyme is essentially identical to that produced by cell culture techniques. The use of whole insect larvae as a source of recombinant proteins offers a more cost-effective method of producing large quantities of human proteins than conventional cell-culture approaches. Human carboxylesterase 1 has been produced in and isolated from whole Trichoplusia ni larvae. The recombinant protein was crystallized and its structure was solved to 2.2 Å resolution. The results indicate that the larvae-produced enzyme is essentially identical to that isolated from cultured Sf21 cells, supporting the use of this expression system to produce recombinant enzymes for crystallization studies

  3. Transcriptional profiling of five isolated size-matched stages of human preantral follicles

    DEFF Research Database (Denmark)

    Kristensen, Stine Gry; Ebbesen, Pernille; Andersen, Claus Yding

    2015-01-01

    Little is known of the early stages of human follicular development and the complex processes that regulate follicular growth. To identify genes of potential importance, we analysed follicle-related transcripts in five populations of isolated size-matched human preantral follicles by microarray...... factors of NOTCH signalling, IGF2, orphan nuclear receptor LRH-1, and homeobox gene HOXA7, indicating potentially important regulatory roles for these genes during early human folliculogenesis. We also found that FSHR mRNA and protein were present in the earliest stages of preantral follicles, whereas LHR...

  4. Differences in Virulence Between Legionella pneumophila Isolates From Human and Non-human Sources Determined in Galleria mellonella Infection Model

    Directory of Open Access Journals (Sweden)

    Patrícia S. Sousa

    2018-04-01

    Full Text Available Legionella pneumophila is a ubiquitous bacterium in freshwater environments and in many man-made water systems capable of inducing pneumonia in humans. Despite its ubiquitous character most studies on L. pneumophila virulence focused on clinical strains and isolates from man-made environments, so little is known about the nature and extent of virulence variation in strains isolated from natural environments. It has been established that clinical isolates are less diverse than man-made and natural environmental strains, suggesting that only a subset of environmental isolates is specially adapted to infect humans. In this work we intended to determine if unrelated L. pneumophila strains, isolated from different environments and with distinct virulence-related genetic backgrounds, displayed differences in virulence, using the Wax Moth Galleria mellonella infection model. We found that all tested strains were pathogenic in G. mellonella, regardless of their origin. Indeed, a panoply of virulence-related phenotypes was observed sustaining the existence of significant differences on the ability of L. pneumophila strains to induce disease. Taken together our results suggest that the occurrence of human infection is not related with the increased capability of some strains to induce disease since we also found a concentration threshold above which L. pneumophila strains are equally able to cause disease. In addition, no link could be established between the sequence-type (ST and L. pneumophila pathogenicity. We envision that in man-made water distribution systems environmental filtering selection and biotic competition acts structuring L. pneumophila populations by selecting more resilient and adapted strains that can rise to high concentration if no control measures are implemented. Therefore, public health strategies based on the sequence based typing (STB scheme analysis should take into account that the major disease-associated clones of L

  5. Bacteriocinogenic potential and virulence traits of Enterococcus faecium and E. faecalis isolated from human milk

    Science.gov (United States)

    Khalkhali, Soodabeh; Mojgani, Naheed

    2017-01-01

    Background and Objectives: Human milk is a continuous supply of Lactic Acid bacteria (LAB), including enterococci with probiotic potentials. The aim of this study was to analyze two Enterococcus species, isolated from human milk for their probiotic potential, bacteriocin producing ability and virulence traits. Materials and Methods: Enterococcus faecium TA0033 and E. faecalis TA102 were tested for acid and bile tolerance, survival in simulated gastric and intestinal conditions. The antibacterial spectrum of the isolates was tested by agar well diffusion assay. The antagonistic agent was characterized by physico-chemical methods. The enterocin structural genes, virulence determinants, vancomycin resistance and biogenic amine genes, such as hdc1, hdc2, tdc, ldc and odc were also determined. Results: The tested isolates survived acidic conditions, high bile salt (1%), simulated gastric and intestinal conditions. The culture supernatant fluids of the two isolates inhibited the growth of Escherichia coli, Listeria monocytogenes, Salmonella typhi, Staphylococcus aureus, Shigella dysenteriae and Streptococcus agalactiae. The antagonistic activity was lost in the presence of proteolytic enzymes but tolerated the action of catalase, lysozyme and lipase. In contrast to enterocin TA102, enterocin TA0033 possessed bactericidal mode of action. Bacteriocin structural genes, entA and entB were present in the genome of the two isolates, while E. faecalis TA102 additionally harboured entP and bac31 genes. The phenotypic and genotypic virulence assessment studies indicated hyaluronidase (hyl) production and vancomycin resistance in E. faecalis TA102 while, none of the isolates harboured the biogenic amine genes. Conclusion: The presence of virulence genes in E. faecalis TA102 calls for careful monitoring of Enterococcus isolates for their safety parameters. PMID:29238458

  6. Inhibition of Primary Clinical Isolates of Human Parainfluenza Virus by DAS181 in Cell Culture and in a Cotton Rat Model

    OpenAIRE

    Jones, B. G.; Hayden, R.T.; Hurwitz, J. L.

    2013-01-01

    DAS181 is a novel drug in development for the treatment of influenza as well as human parainfluenza viruses (hPIV). Previous studies demonstrated that DAS181 inhibited laboratory strains of hPIV, but no tests were conducted with primary clinical isolates of hPIV. To fill this gap, we studied six primary isolates including hPIV-2 and hPIV-3. First tests showed that the amplification of all viruses in vitro was reproducibly inhibited with DAS181 drug concentrations ranging between 0.1 and 1 nM....

  7. Phage types of Salmonella enterica ssp. enterica serovar Typhimurium isolated from production animals and humans in Denmark

    DEFF Research Database (Denmark)

    Baggesen, Dorte Lau; Wegener, Henrik Caspar

    1994-01-01

    S. Typhimurium is one of the 2 most common salmonella serotypes causing human salmonellosis in Denmark. In order to illustrate the significance of different production animals as a source of infection, 1461 isolates were characterized by phage typing. The isolates originated from human patients a...

  8. HUMAN LIVER SLICES EXPRESS THE SAME LIDOCAINE BIOTRANSFORMATION RATE AS ISOLATED HUMAN HEPATOCYTES

    NARCIS (Netherlands)

    OLINGA, P; MEIJER, DKF; SLOOFF, MJH; GROOTHUIS, GMM; Merema, M.T.

    1993-01-01

    In order to investigate whether liver slices are a valuable tool for the assessment of drug metabolism in human liver, we compared the phase I metabolism of lidocaine in human liver slices and hepatocytes prepared from three human livers. Lidocaine is mainly metabolised to monoethylglycinexylidide

  9. Genetic relatedness between Japanese and European isolates of Clostridium difficile originating from piglets and their risk associated with human health

    Directory of Open Access Journals (Sweden)

    Masaru eUsui

    2014-10-01

    Full Text Available Clostridium difficile colonization in pig intestine has been a public health concern. We analyzed C. difficile prevalence among piglets in Japan to clarify their origin and extent of the associated risk by using molecular and microbiological methods for both swine and human clinical isolates and foreign isolates. C. difficile was isolated from 120 neonatal piglet faecal samples. Toxin gene profile, antimicrobial susceptibilities, PCR ribotype, and multiple-locus variable-number tandem-repeat analysis (MLVA type of swine isolates were determined and compared with those of human clinical and foreign isolates. One-hundred C. difficile strains were isolated from 69 (57.5% samples, and 61 isolates (61% were toxin gene-positive. Some isolates were resistant to antimicrobials, contributing to antibiotic-associated diarrhoea by C. difficile. These results suggest that C. difficile, prevalent among Japanese pigs, is a potential risk for antibiotic-associated diarrhoea. Furthermore, PCR ribotype 078 (12 isolates, which has been linked to multiple outbreaks worldwide, was the third-most frequently isolated of the 14 PCR ribotypes identified. Moreover, MLVA revealed that all 12 PCR ribotype 078 isolates were genetically related to European PCR ribotype 078 strains found in both humans and pigs. To date, in Japan, many breeding pigs have been imported from European countries. The genetic relatedness of C. difficile isolates of Japanese swine origin to those of European origin suggests that they were introduced into Japan via imported pigs.

  10. Isolation and isoenzyme characterization of Leishmania (Viannia braziliensis from a case of human cutaneous leishmaniasis in northeast centre of the state of São Paulo

    Directory of Open Access Journals (Sweden)

    MC Pinto

    2005-11-01

    Full Text Available The diagnosis of human cutaneous leishmaniasis in small towns is sometimes made without the species identification of the Leishmania, even in areas without previous epidemiological surveys. Here we report the isolation of a Leishmania strain from a patient of Rincão, state of São Paulo, that was identified by isoenzyme characterization as L. (Viannia braziliensis. Sand fly collections were made in the area where the patient live in order to investigate the likely vector species.

  11. Genetically similar strains of Escherichia coli O157:H7 isolated from sheep, cattle and human patients

    Directory of Open Access Journals (Sweden)

    Söderlund Robert

    2012-10-01

    Full Text Available Abstract Background Comparatively little is known about the prevalence or the molecular characteristics of the zoonotic pathogen E. coli O157:H7 in the sheep reservoir. To investigate this and determine the host specificity of subclones of the bacterium, we have conducted a slaughterhouse prevalence study in sheep and compared the collected isolates to O157:H7 previously isolated from cattle and human patients. Results Verotoxin-producing O157:H7 was found in 11/597 (1.8% of samples from sheep in Swedish slaughterhouses, 9/492 faecal (1.8% and 2/105 ear samples (1.9%. All positive sheep were eaeA, hlyA, cdtV-B, vtx1, and partial sequencing of vtx2. The observed profiles were similar to those of cattle strains investigated previously. Conclusions The same pathogenic subtypes of VTEC O157:H7, including the highly virulent clade 8, appear to be present in both sheep and cattle in Sweden, suggesting strains can circulate freely between ruminant reservoirs.

  12. Virulence of Mycobacterium avium Subsp. hominissuis Human Isolates in an in vitro Macrophage Infection Model

    Directory of Open Access Journals (Sweden)

    Laura Rindi

    2018-01-01

    Full Text Available Background: Mycobacterium avium subsp. hominissuis (MAH is an environmental opportunistic pathogen for humans and swine worldwide; in humans, the vast majority of MAH infections is due to strains belonging to specific genotypes, such as the internal transcribed spacer (ITS-sequevars Mav-A and Mav-B that mostly cause pulmonary infections in elderly patients and severe disseminated infections in acquired immunodeficiency syndrome patients, respectively. To test whether the different types of infections in distinct patients' populations might reflect a different virulence of the infecting genotypes, MAH human isolates, genotyped by ITS sequencing and MIRU-VNTR minisatellite analysis, were studied for the capacity to infect and replicate in human macrophages in vitro. Methods: Cultures of human peripheral blood mononuclear cells and phagocytic human leukemic cell line THP-1 cells were infected with each MAH isolate and intracellular colony-forming units (CFU were determined. Results: At 2 h after infection, i.e., immediately after cell entry, the numbers of intracellular bacteria did not differ between Mav-A and Mav-B organisms in both phagocytic cell types. At 5 days, Mav-A organisms, sharing highly related VNTR-MIRU genotypes, yielded numbers of intracellular CFUs significantly higher than Mav-B organisms in both phagocytic cell types. MIRU-VNTR-based minimum spanning tree analysis of the MAH isolates showed a divergent phylogenetic pathway of Mav-A and Mav-B organisms. Conclusion: Mav-A and Mav-B sequevars might have evolved different pathogenetic properties that might account for their association with different human infections.

  13. Isolation and identification of gene mediating radiation-induced apoptosis in human leukemia U937 cells

    International Nuclear Information System (INIS)

    Tong Xin; Luo Ying; Dong Yan; Sun Zhixian

    1998-01-01

    Objective: Increasing evidences suggest that Caspase family proteases play an important role in the effector mechanism of apoptotic cell death. Radiation (IR) can induce apoptosis in tumor cells, so it is very important to isolate and identify the member of the Caspase family proteases involved in IR-induced apoptosis, and this would contribute to the understanding of the mechanism responsible for apoptosis execution. Methods: A PCR approach to isolate genes for IR-induced apoptosis was developed. The approach used degenerated oligonucleotide encoding the highly conserved peptides that were present in all known Caspases. Results: Protease inhibitors special for Caspases could block the apoptotic cell death caused by IR, and Caspase-3 was isolated from irradiated human leukemia U937 cells. Conclusion: Caspases involve in IR-induced apoptosis, and Caspase-3 is the pivotal element of IR-induced apoptosis

  14. Anti-proliferation activity of terpenoids isolated from Euphorbia kansui in human cancer cells and their structure-activity relationship.

    Science.gov (United States)

    Hou, Jin-Jun; Shen, Yao; Yang, Zhou; Fang, Lin; Cai, Lu-Ying; Yao, Shuai; Long, Hua-Li; Wu, Wan-Ying; Guo, De-An

    2017-10-01

    Euphorbia kansui is a commonly used traditional Chinese medicine for the treatment of edema, pleural effusion, and asthma, etc. According to the previous researches, terpenoids in E. kansui possess various biological activities, e.g., anti-virus, anti-allergy, antitumor effects. In this work, twenty five terpenoids were isolated from E. kansui, including thirteen ingenane- and eight jatrophane-type diterpenoids (with two new compounds, kansuinin P and Q) and four triterpenoids. Eighteen of them were analyzed by MTS assay for in vitro anticancer activity in five human cancer cell lines. Structure-activity relationship for 12 ingenane-type diterpenoids in colorectal cancer Colo205 cells were preliminary studied. Significant anti-proliferation activities were observed in human melanoma cells breast cancer MDA-MB-435 cells and Colo205 cells. More than half of the isolated ingenane-type diterpenoids showed inhibitory activities in MDA-MB-435 cells. Eight ingenane- and one jatrophane-type diterpenoids possessed much lower IC 50 values in MDA-MB-435 cells than positive control staurosporine. Preliminary structure-activity relationship analysis showed that substituent on position 20 was important for the activity of ingenane-type diterpenoids in Colo205 cells and substituent on position 3 contributed more significant biological activity of the compounds than that on position 5 in both MDA-MB-435 and Colo205 cells. Copyright © 2017 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

  15. Whole genome sequencing of multidrug-resistant Salmonella enterica serovar Typhimurium isolated from humans and poultry in Burkina Faso.

    Science.gov (United States)

    Kagambèga, Assèta; Lienemann, Taru; Frye, Jonathan G; Barro, Nicolas; Haukka, Kaisa

    2018-01-01

    Multidrug-resistant Salmonella is an important cause of morbidity and mortality in developing countries. The aim of this study was to characterize and compare multidrug-resistant Salmonella enterica serovar Typhimurium isolates from patients and poultry feces. Salmonella strains were isolated from poultry and patients using standard bacteriological methods described in previous studies. The strains were serotype according to Kaufmann-White scheme and tested for antibiotic susceptibility to 12 different antimicrobial agents using the disk diffusion method. The whole genome of the S. Typhimurium isolates was analyzed using Illumina technology and compared with 20 isolates of S. Typhimurium for which the ST has been deposited in a global MLST database.The ResFinder Web server was used to find the antibiotic resistance genes from whole genome sequencing (WGS) data. For comparative genomics, publicly available complete and draft genomes of different S. Typhimurium laboratory-adapted strains were downloaded from GenBank. All the tested Salmonella serotype Typhimurium were multiresistant to five commonly used antibiotics (ampicillin, chloramphenicol, streptomycin, sulfonamide, and trimethoprim). The multilocus sequence type ST313 was detected from all the strains. Our sequences were very similar to S. Typhimurium ST313 strain D23580 isolated from a patient with invasive non-typhoid Salmonella (NTS) infection in Malawi, also located in sub-Saharan Africa. The use of ResFinder web server on the whole genome of the strains showed a resistance to aminoglycoside associated with carriage of the following resistances genes: strA , strB , and aadA1 ; resistance to β-lactams associated with carriage of a bla TEM-1B genes; resistance to phenicol associated with carriage of catA1 gene; resistance to sulfonamide associated with carriage of sul1 and sul2 genes; resistance to tetracycline associated with carriage of tet B gene; and resistance to trimethoprim associated to dfrA1 gene

  16. Phenotypic and genotypic evaluation of 18 Nocardia isolates from human clinical samples in Mexico.

    Science.gov (United States)

    Sánchez-Herrera, K; Sandoval, H; Couble, A; Mouniee, D; Ramírez-Durán, N; Uzcategui de Morillo, M; Serrano, J A; Bergeron, E; Boiron, P; Rodríguez-Nava, V

    2012-03-01

    Mexico has the largest number of clinical cases of actinomycetoma in North and South America. Species originally identified by less specific methods have been recently reclassified as other known species or as new species. To assess, by 16S rRNA gene sequencing and phenotypic methods, the species distribution of 18 human clinical isolates originally identified as N. brasiliensis, some of them isolated between 1947 and 1959 in Mexico City. Clinical isolates came from the Hospital General, "Dr. Manuel Gea Gonzalez", and Instituto Nacional de Diagnóstico y Referencia Epidemiológica (INDRE) in Mexico, D.F. The strains used in this study included 15 clinical strains isolated between 1947 and 1959 that were originally identified as N. brasiliensis and three more strains obtained in 2007 identified as Nocardia spp. The isolates were identified genotypically by sequencing the 16S rRNA gene, and their phenotypic profiles were obtained with the API Coryne(®) system. Antibiotic susceptibility patterns were tested according to the protocol of the Comité de l'antibiogramme de la Société française de microbiologie[4]. According to 16S rRNA gene, sequencing were identified among 18 human clinical isolates as Nocardia farcinica (n=11) and Nocardia brasiliensis (n=7). A high number of the strains were susceptible to the majority of the antibiotics tested. The phenotypic profiles of the strains were quite uniform for N. farcinica and some variability was observed for N. brasiliensis strains. N. farcinica was the most prevalent species identified. Modern methodologies should be applied in clinical laboratories to accurately identify etiological agents. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  17. Probiotic assessment of Enterococcus faecalis CP58 isolated from human gut.

    Science.gov (United States)

    Nueno-Palop, Carmen; Narbad, Arjan

    2011-02-28

    A total of seventy lactic acid bacteria (LAB) were isolated from the faeces of healthy humans and their identities were confirmed by sequencing of their 16S rDNA genes. Of these only 5 isolates were found to resist bile salts and indicated survival in the simulated in vitro digestion assay which reproduces the stomach and intestinal digestion indicating their tolerance to gastric enzymes and the low pH conditions. Species that showed the best resistance to these conditions were: Lactobacillus casei, Lactobacillus sp., uncultured bifidobacteria, Enterococcus faecalis and Streptococcus anginosus. These strains were investigated further to study their capacity to adhere to human intestinal Caco-2 cells. E. faecalis was the most adherent strain. Examination of the virulence determinants for this strain indicated that it was positive for efaAfs, gelE, agg, cpd, cob, ccf and cad, a profile that is similar to that of many E. faecalis isolates from food sources. The cytolysin biosynthetic genes cylA, cylB and cylM that are more associated with the clinical isolates of E. faecium were not detected in this strain. The antibiotic susceptibility tests indicated that the strain was sensitive to vancomycin, tetracycline, rifampicin and erythromycin but resistant only to kanamycin and chloramphenicol. These data suggest that the strain E. faecalis CP58 may be tested further for beneficial properties and developed as a new probiotic. Copyright © 2010 Elsevier B.V. All rights reserved.

  18. Isolation and characterization of adult human liver progenitors from ischemic liver tissue derived from therapeutic hepatectomies.

    Science.gov (United States)

    Stachelscheid, Harald; Urbaniak, Thomas; Ring, Alexander; Spengler, Berlind; Gerlach, Jörg C; Zeilinger, Katrin

    2009-07-01

    Recent evidence suggests that progenitor cells in adult tissues and embryonic stem cells share a high resistance to hypoxia and ischemic stress. To study the ischemic resistance of adult liver progenitors, we characterized remaining viable cells in human liver tissue after cold ischemic treatment for 24-168 h, applied to the tissue before cell isolation. In vitro cultures of isolated cells showed a rapid decline of the number of different cell types with increasing ischemia length. After all ischemic periods, liver progenitor-like cells could be observed. The comparably small cells exhibited a low cytoplasm-to-nucleus ratio, formed densely packed colonies, and showed a hepatobiliary marker profile. The cells expressed epithelial cell adhesion molecule, epithelial-specific (CK8/18) and biliary-specific (CK7/19) cytokeratins, albumin, alpha-1-antitrypsin, cytochrome-P450 enzymes, as well as weak levels of hepatocyte nuclear factor-4 and gamma-glutamyl transferase, but not alpha-fetoprotein or Thy-1. In vitro survival and expansion was facilitated by coculture with mouse embryonic fibroblasts. Hepatic progenitor-like cells exhibit a high resistance to ischemic stress and can be isolated from human liver tissue after up to 7 days of ischemia. Ischemic liver tissue from various sources, thought to be unsuitable for cell isolation, may be considered as a prospective source of hepatic progenitor cells.

  19. Isolation and clinical sample typing of human leptospirosis cases in Argentina.

    Science.gov (United States)

    Chiani, Yosena; Jacob, Paulina; Varni, Vanina; Landolt, Noelia; Schmeling, María Fernanda; Pujato, Nazarena; Caimi, Karina; Vanasco, Bibiana

    2016-01-01

    Leptospira typing is carried out using isolated strains. Because of difficulties in obtaining them, direct identification of infective Leptospira in clinical samples is a high priority. Multilocus sequence typing (MLST) proved highly discriminatory for seven pathogenic species of Leptospira, allowing isolate characterization and robust assignment to species, in addition to phylogenetic evidence for the relatedness between species. In this study we characterized Leptospira strains circulating in Argentina, using typing methods applied to human clinical samples and isolates. Phylogenetic studies based on 16S ribosomal RNA gene sequences enabled typing of 8 isolates (6 Leptospira interrogans, one Leptospira wolffii and one Leptospira broomii) and 58 out of 85 (68.2%) clinical samples (55 L. interrogans, 2 Leptospira meyeri, and one Leptospira kirschneri). MLST results for the L. interrogans isolates indicated that five were probably Canicola serogroup (ST37) and one was probably Icterohaemorrhagiae serogroup (ST17). Eleven clinical samples (21.6%), provided MLST interpretable data: five were probably Pyrogenes serogroup (ST13), four Sejroe (ST20), one Autumnalis (ST22) and one Canicola (ST37). To the best of our knowledge this study is the first report of the use of an MLST typing scheme with seven loci to identify Leptospira directly from clinical samples in Argentina. The use of clinical samples presents the advantage of the possibility of knowing the infecting strain without resorting to isolates. This study also allowed, for the first time, the characterization of isolates of intermediate pathogenicity species (L. wolffii and L. broomii) from symptomatic patients. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Antimicrobial susceptibility of Clostridium difficile isolated from animals and humans in Brazil

    Directory of Open Access Journals (Sweden)

    Rodrigo Otávio Silveira Silva

    2014-05-01

    Full Text Available The objective of this study was to evaluate antimicrobial susceptibility in Clostridium difficile strains isolated from animals and humans in Brazil. The 54 C. difficile strains used were isolated from stool samples from piglets (n=16, dogs (n=13, humans (n=13, foals (n=8 calves (n=2, an ocelot (n=1 and a maned wolf (n=1. Antimicrobial susceptibility was determined using the serial plate agar dilution method for penicillin, florfenicol, oxytetracycline, erythromycin, vancomycin, metronidazole and tylosin. The C. difficile strains assessed were susceptible to metronidazole and vancomycin. Florfenicol resistance was rarely observed; 52 (96.4% strains were sensitive to this antimicrobial. Five (9.3%, five (9.3%, 14 (25.9% and 20 (37.0% strains were resistant to oxytetracycline, penicillin, tylosin and erythromycin respectively.

  1. Relationship between plasma cholesterol levels and cholesterol esterification in isolated human mononuclear cells

    International Nuclear Information System (INIS)

    Dallongeville, J.; Davignon, J.; Lussier-Cacan, S.

    1990-01-01

    The authors studied the relationship between plasma lipoprotein concentrations and cholesterol esterification in freshly isolated human mononuclear cells from 27 normolipidemic and 32 hyperlipidemic individuals. Cells were either incubated for 5 hours with radiolabeled oleate immediately after isolation or were preincubated for 18 hours in the presence of exogenous cholesterol, and then incubated with [ 14 C]sodium-oleate-albumin complex. In the absence of exogenous cholesterol, control and hypercholesterolemic subjects had similarly low values of intracellular cholesterol esterification. In the presence of exogenous cholesterol, both hypertriglyceridemic and hypercholesterolemic subjects had higher cholesterol esterification than controls. There was a significant correlation between the rate of cholesterol esterification and plasma total cholesterol. These results suggest that plasma cholesterol levels may regulate mononuclear cell intra-cellular cholesterol esterification in humans

  2. Isolation of a novel human papillomavirus (type 51) from a cervical condyloma

    International Nuclear Information System (INIS)

    Nuovo, G.J.; Crum, C.P.; Levine, R.U.; Silverstein, S.J.; De Villiers, E.M.

    1988-01-01

    The authors cloned the DNA from a novel human papillomavirus (HPV) present in a cervical condyloma. When DNA from this isolate was hybridized at high stringency with HPV types 1 through 50 (HPV-1 through HPV-50), it showed weak homology with HPV-6 and -16 and stronger homology with HPV-26. A detailed restriction endonuclease map was prepared which showed marked differences from the maps for other HPVs that have been isolated from the female genital tract. Reassociation kinetic analysis revealed that HPV-26 and this new isolate were less than 10% homologous; hence, the new isolate is a noel strain of HPV. The approximate positions of the open reading frames of the new strain were surmised by hybridization with probes derived from individual open reading frames of HPV-16. In an analysis of 175 genital biopsies from patients with abnormal Papanicolaou smears, sequences hybridizing under highly stringent conditions to probes from this novel HPV type were found in 4.2, 6.1, and 2.4% of biopsies containing normal squamous epithelium, condylomata, and intraepithelial neoplasia, respectively. In addition, sequences homologous to probes from this novel isolate were detected in one of five cervical carcinomas examined

  3. Isolation and complete amino acid sequence of human thymopoietin and splenin

    International Nuclear Information System (INIS)

    Audhya, T.; Schlesinger, D.H.; Goldstein, G.

    1987-01-01

    Human thymopoietin and splenin were isolated from human thymus and spleen, respectively, by monitoring tissue fractionation with a bovine thymopoietin RIA cross-reactive with human thymopoietin and splenin. Bovine thymopoietin and splenin are 49-amino acid polypeptides that differ by only 2 amino acids at positions 34 and 43; the change at position 34 in the active-site region changes the receptor specificities and biological activities. The complete amino acid sequences of purified human thymopoietin and splenin were determined and shown to be 48-amino acid polypeptides differing at four positions. Ten amino acids, constant within each species for thymopoietin and splenin, differ between the human and bovine polypeptides. The pentapeptide active side of thymopoietin (residues 32-36) is constant between the human and bovine thymopoietins, but position 34 in the active site of splenin has changed from glutamic acid in bovine splenin to alanine in human splenin, accounting for the biological activity of the human but not the bovine splenin on the human T-cell line MOLT-4

  4. Genomic characterisation of Leptospira inadai serogroup Lyme isolated from captured rat in Brazil and comparative analysis with human reference strain

    Science.gov (United States)

    Moreno, Luisa Z; Miraglia, Fabiana; Loureiro, Ana P; Kremer, Frederico S; Eslabao, Marcus R; Dellagostin, Odir A; Lilenbaum, Walter; Vasconcellos, Silvio A; Heinemann, Marcos B; Moreno, Andrea M

    2018-01-01

    Leptospira inadai is classified as a species of the Leptospira intermediate group that has been poorly studied due to its apparent insignificance to human and animal health. Nevertheless, over the last two decades the species has been described in human cases in India and in carrier animals in Ecuador. Here, we present the first identification and genomic characterisation of L. inadai serogroup Lyme isolated from captured rodent in Brazil. Even though the M34/99 strain was not pathogenic for hamsters, it was able to establish renal colonisation. The M34/99 strain presented high similarity with L. inadai serogroup Lyme human reference indicating that animal strain could also infect humans, although it does not represent high risk of severe disease. An extrachromosomal sequence was also identified in M34/99 strain and presented high identity with previously described L. inadai phage LinZ_10, suggesting that phage-like extrachromosomal sequence may be another feature of this understudied species. PMID:29538491

  5. Genomic characterisation of Leptospira inadai serogroup Lyme isolated from captured rat in Brazil and comparative analysis with human reference strain.

    Science.gov (United States)

    Moreno, Luisa Z; Miraglia, Fabiana; Loureiro, Ana P; Kremer, Frederico S; Eslabao, Marcus R; Dellagostin, Odir A; Lilenbaum, Walter; Vasconcellos, Silvio A; Heinemann, Marcos B; Moreno, Andrea M

    2018-03-12

    Leptospira inadai is classified as a species of the Leptospira intermediate group that has been poorly studied due to its apparent insignificance to human and animal health. Nevertheless, over the last two decades the species has been described in human cases in India and in carrier animals in Ecuador. Here, we present the first identification and genomic characterisation of L. inadai serogroup Lyme isolated from captured rodent in Brazil. Even though the M34/99 strain was not pathogenic for hamsters, it was able to establish renal colonisation. The M34/99 strain presented high similarity with L. inadai serogroup Lyme human reference indicating that animal strain could also infect humans, although it does not represent high risk of severe disease. An extrachromosomal sequence was also identified in M34/99 strain and presented high identity with previously described L. inadai phage LinZ_10, suggesting that phage-like extrachromosomal sequence may be another feature of this understudied species.

  6. Insights into the Evolution of Host Association through the Isolation and Characterization of a Novel Human Periodontal Pathobiont, Desulfobulbus oralis

    Directory of Open Access Journals (Sweden)

    Karissa L. Cross

    2018-03-01

    Full Text Available The human oral microbiota encompasses representatives of many bacterial lineages that have not yet been cultured. Here we describe the isolation and characterization of previously uncultured Desulfobulbus oralis, the first human-associated representative of its genus. As mammalian-associated microbes rarely have free-living close relatives, D. oralis provides opportunities to study how bacteria adapt and evolve within a host. This sulfate-reducing deltaproteobacterium has adapted to the human oral subgingival niche by curtailing its physiological repertoire, losing some biosynthetic abilities and metabolic independence, and by dramatically reducing environmental sensing and signaling capabilities. The genes that enable free-living Desulfobulbus to synthesize the potent neurotoxin methylmercury were also lost by D. oralis, a notably positive outcome of host association. However, horizontal gene acquisitions from other members of the microbiota provided novel mechanisms of interaction with the human host, including toxins like leukotoxin and hemolysins. Proteomic and transcriptomic analysis revealed that most of those factors are actively expressed, including in the subgingival environment, and some are secreted. Similar to other known oral pathobionts, D. oralis can trigger a proinflammatory response in oral epithelial cells, suggesting a direct role in the development of periodontal disease.

  7. Genomic characterisation of Leptospira inadai serogroup Lyme isolated from captured rat in Brazil and comparative analysis with human reference strain

    Directory of Open Access Journals (Sweden)

    Luisa Z Moreno

    2018-03-01

    Full Text Available Leptospira inadai is classified as a species of the Leptospira intermediate group that has been poorly studied due to its apparent insignificance to human and animal health. Nevertheless, over the last two decades the species has been described in human cases in India and in carrier animals in Ecuador. Here, we present the first identification and genomic characterisation of L. inadai serogroup Lyme isolated from captured rodent in Brazil. Even though the M34/99 strain was not pathogenic for hamsters, it was able to establish renal colonisation. The M34/99 strain presented high similarity with L. inadai serogroup Lyme human reference indicating that animal strain could also infect humans, although it does not represent high risk of severe disease. An extrachromosomal sequence was also identified in M34/99 strain and presented high identity with previously described L. inadai phage LinZ_10, suggesting that phage-like extrachromosomal sequence may be another feature of this understudied species.

  8. Insights into the Evolution of Host Association through the Isolation and Characterization of a Novel Human Periodontal Pathobiont, Desulfobulbus oralis

    Science.gov (United States)

    Cross, Karissa L.; Chirania, Payal; Xiong, Weili; Elkins, James G.; Giannone, Richard J.; Griffen, Ann L.; Hettich, Robert L.; Joshi, Snehal S.; Mokrzan, Elaine M.; Martin, Roman K.; Leys, Eugene J.

    2018-01-01

    ABSTRACT The human oral microbiota encompasses representatives of many bacterial lineages that have not yet been cultured. Here we describe the isolation and characterization of previously uncultured Desulfobulbus oralis, the first human-associated representative of its genus. As mammalian-associated microbes rarely have free-living close relatives, D. oralis provides opportunities to study how bacteria adapt and evolve within a host. This sulfate-reducing deltaproteobacterium has adapted to the human oral subgingival niche by curtailing its physiological repertoire, losing some biosynthetic abilities and metabolic independence, and by dramatically reducing environmental sensing and signaling capabilities. The genes that enable free-living Desulfobulbus to synthesize the potent neurotoxin methylmercury were also lost by D. oralis, a notably positive outcome of host association. However, horizontal gene acquisitions from other members of the microbiota provided novel mechanisms of interaction with the human host, including toxins like leukotoxin and hemolysins. Proteomic and transcriptomic analysis revealed that most of those factors are actively expressed, including in the subgingival environment, and some are secreted. Similar to other known oral pathobionts, D. oralis can trigger a proinflammatory response in oral epithelial cells, suggesting a direct role in the development of periodontal disease. PMID:29535201

  9. Isolation, Characterization, Cryopreservation of Human Amniotic Stem Cells and Differentiation to Osteogenic and Adipogenic Cells.

    Directory of Open Access Journals (Sweden)

    Shiva Gholizadeh-Ghaleh Aziz

    Full Text Available Human stem cells and progenitor cells can be used to treat cancer and replace dysfunctional cells within a tissue or organ. The objective of this study was to identify the appropriate cells type in regenerative medicine and targeted therapy. As an alternative to embryonic and bone marrow stem cells, we examined human amniotic fluid stem cells (hAFSCs, one of the potential source of multipotent stem cells isolated from both cell pellet (using single-stage method, and supernatant of human amniotic fluid. Source of isolation and unique property of the cells emphasize that these cells are one of the promising new tools in therapeutic field. Double sources for isolation and availability of the left over samples in diagnostic laboratory at the same time have less legal and ethical concerns compared with embryonic stem cell studies. Cells were isolated, cultured for 18th passage for 6 months and characterized using qPCR and flow cytometry. Cells showed good proliferative ability in culture condition. The cells successfully differentiated into the adipogenic and osteogenic lineages. Based on these findings, amniotic fluid can be considered as an appropriate and convenient source of human amniotic fluid stem cells. These cells provide potential tools for therapeutic applications in the field of regenerative medicine. To get a better understanding of crosstalk between Oct4/NANOG with osteogenesis and adipogenesis, we used network analysis based on Common Targets algorithm and Common Regulators algorithm as well as subnetwork discovery based on gene set enrichment. Network analysis highlighted the possible role of MIR 302A and MIR let-7g. We demonstrated the high expression of MIR 302A and low expression of MIR let7g in hAFSCs by qPCR.

  10. Isolation of a human intestinal anaerobe, Bifidobacterium sp. strain SEN, capable of hydrolyzing sennosides to sennidins.

    OpenAIRE

    Akao, T; Che, Q M; Kobashi, K; Yang, L; Hattori, M; Namba, T

    1994-01-01

    A strictly anaerobic bacterium capable of metabolizing sennosides was isolated from human feces and identified as Bifidobacterium sp., named strain SEN. The bacterium hydrolyzed sennosides A and B to sennidins A and B via sennidin A and B 8-monoglucosides, respectively. Among nine species of Bifidobacterium having beta-glucosidase activity, only Bifidobacterium dentium and B. adolescentis metabolized sennoside B to sennidin B, suggesting that the sennoside-metabolizing bacteria produce a nove...

  11. Cryptolepine, isolated from Sida acuta, sensitizes human gastric adenocarcinoma cells to TRAIL-induced apoptosis.

    Science.gov (United States)

    Ahmed, Firoj; Toume, Kazufumi; Ohtsuki, Takashi; Rahman, Mahmudur; Sadhu, Samir Kumar; Ishibashi, Masami

    2011-01-01

    Bioassay guided separation of Sida acuta whole plants led to the isolation of an alkaloid, cryptolepine (1), along with two kaempferol glycosides (2-3). Compound 1 showed strong activity in overcoming TRAIL-resistance in human gastric adenocarcinoma (AGS) cells at 1.25, 2.5 and 5 μm. Combined treatment of 1 and TRAIL sensitized AGS cells to TRAIL-induced apoptosis at the aforementioned concentrations. Copyright © 2010 John Wiley & Sons, Ltd.

  12. Beta 3 and PDI proteins isolated from human platelets bind with ECwt rotavirus in vitro

    International Nuclear Information System (INIS)

    Mayorga, Diana; Rubio, Linda; Guerrero-Fonseca, Carlos A; Acosta-Losada, Orlando

    2010-01-01

    Commercial integrin Beta 3 is currently not available and commercial PDI is too expensive, which is making access difficult to these proteins needed for conducting experiments aimed at the establishment of possible interactions between integrin Beta 3 and PDI and wild type rotavirus strains. Objective. To explore a methodology allowing isolation of proteins Beta 3 and PDI from human platelets to be used as antigens in the generation of rabbit polyclonal antibodies useful in the assessment of interactions between these proteins and rotavirus ECwt. Materials and methods. Proteins Beta 3 and PDI from human platelet lysates were separated using preparative electrophoresis under reducing conditions and then eluted. Interactions of these proteins with rotavirus ECwt were analyzed using co-immunoprecipitation, Western blotting and capture ELISA. Results. Proteins from human platelet lysates were separated by preparative electrophoresis under reducing conditions. The identification of proteins Beta 3 and PDI present in a gel slice was performed through their reaction with commercial antibodies in a Western blotting analysis. Protein purity was established after electro elution from a gel slice. Polyclonal antibodies against protein Beta 3 were generated in rabbit. Incubation of eluted proteins Beta 3 and PDI with rotavirus ECwt showed in co-immunoprecipitation and ELISA assays that these proteins bound virus in vitro. The same binding was showed to occur when rotavirus was incubated with isolated small intestinal villi from suckling mice. Conclusions. Relatively high amounts of proteins Beta 3 and PDI were partially purified from human platelets by preparative electrophoresis. The isolation of these proteins allowed the generation of polyclonal antibodies against Beta 3 in addition to the establishment of the in vitro interaction of proteins Beta 3 and PDI with rotavirus ECwt. This interaction was also demonstrated in vivo after incubating the virus with isolated small

  13. Impact and Cost-effectiveness of 3 Doses of 9-Valent Human Papillomavirus (HPV) Vaccine Among US Females Previously Vaccinated With 4-Valent HPV Vaccine.

    Science.gov (United States)

    Chesson, Harrell W; Laprise, Jean-François; Brisson, Marc; Markowitz, Lauri E

    2016-06-01

    We estimated the potential impact and cost-effectiveness of providing 3-doses of nonavalent human papillomavirus (HPV) vaccine (9vHPV) to females aged 13-18 years who had previously completed a series of quadrivalent HPV vaccine (4vHPV), a strategy we refer to as "additional 9vHPV vaccination." We used 2 distinct models: (1) the simplified model, which is among the most basic of the published dynamic HPV models, and (2) the US HPV-ADVISE model, a complex, stochastic, individual-based transmission-dynamic model. When assuming no 4vHPV cross-protection, the incremental cost per quality-adjusted life-year (QALY) gained by additional 9vHPV vaccination was $146 200 in the simplified model and $108 200 in the US HPV-ADVISE model ($191 800 when assuming 4vHPV cross-protection). In 1-way sensitivity analyses in the scenario of no 4vHPV cross-protection, the simplified model results ranged from $70 300 to $182 000, and the US HPV-ADVISE model results ranged from $97 600 to $118 900. The average cost per QALY gained by additional 9vHPV vaccination exceeded $100 000 in both models. However, the results varied considerably in sensitivity and uncertainty analyses. Additional 9vHPV vaccination is likely not as efficient as many other potential HPV vaccination strategies, such as increasing primary 9vHPV vaccine coverage. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  14. Isolation and characterization of neural stem cells from human fetal striatum

    International Nuclear Information System (INIS)

    Li Xiaoxia; Xu Jinchong; Bai Yun; Wang Xuan; Dai Xin; Liu Yinan; Zhang Jun; Zou Junhua; Shen Li; Li Lingsong

    2005-01-01

    This paper described that neural stem cells (hsNSCs) were isolated and expanded rapidly from human fetal striatum in adherent culture. The population was serum- and growth factor-dependent and expressed neural stem cell markers. They were capable of multi-differentiation into neurons, astrocytes, and oligodendrocytes. When plated in the dopaminergic neuron inducing medium, human striatum neural stem cells could differentiate into tyrosine hydroxylase positive neurons. hsNSCs were morphologically homogeneous and possessed high proliferation ability. The population doubled every 44.28 h and until now it has divided for more than 82 generations in vitro. Normal human diploid karyotype was unchanged throughout the in vitro culture period. Together, this study has exploited a method for continuous and rapid expansion of human neural stem cells as pure population, which maintained the capacity to generate almost fifty percent neurons. The availability of such cells may hold great interest for basic and applied neuroscience

  15. Human Intestinal Cells Modulate Conjugational Transfer of Multidrug Resistance Plasmids between Clinical Escherichia coli Isolates

    DEFF Research Database (Denmark)

    Machado, Ana Manuel; Sommer, Morten

    2014-01-01

    Bacterial conjugation in the human gut microbiota is believed to play a major role in the dissemination of antibiotic resistance genes and virulence plasmids. However, the modulation of bacterial conjugation by the human host remains poorly understood and there is a need for controlled systems...... to study this process. We established an in vitro co-culture system to study the interaction between human intestinal cells and bacteria. We show that the conjugation efficiency of a plasmid encoding an extended spectrum beta-lactamase is reduced when clinical isolates of Escherichia coli are co...... of the intestinal cells exposed to bacteria leading to a two-fold reduction in conjugation efficiency. These results show that human gut epithelial cells can modulate bacterial conjugation and may have relevance to gene exchange in the gut....

  16. Isolation and Characterization of Current Human Coronavirus Strains in Primary Human Epithelial Cell Cultures Reveal Differences in Target Cell Tropism

    Science.gov (United States)

    Dijkman, Ronald; Jebbink, Maarten F.; Koekkoek, Sylvie M.; Deijs, Martin; Jónsdóttir, Hulda R.; Molenkamp, Richard; Ieven, Margareta; Goossens, Herman; Thiel, Volker

    2013-01-01

    The human airway epithelium (HAE) represents the entry port of many human respiratory viruses, including human coronaviruses (HCoVs). Nowadays, four HCoVs, HCoV-229E, HCoV-OC43, HCoV-HKU1, and HCoV-NL63, are known to be circulating worldwide, causing upper and lower respiratory tract infections in nonhospitalized and hospitalized children. Studies of the fundamental aspects of these HCoV infections at the primary entry port, such as cell tropism, are seriously hampered by the lack of a universal culture system or suitable animal models. To expand the knowledge on fundamental virus-host interactions for all four HCoVs at the site of primary infection, we used pseudostratified HAE cell cultures to isolate and characterize representative clinical HCoV strains directly from nasopharyngeal material. Ten contemporary isolates were obtained, representing HCoV-229E (n = 1), HCoV-NL63 (n = 1), HCoV-HKU1 (n = 4), and HCoV-OC43 (n = 4). For each strain, we analyzed the replication kinetics and progeny virus release on HAE cell cultures derived from different donors. Surprisingly, by visualizing HCoV infection by confocal microscopy, we observed that HCoV-229E employs a target cell tropism for nonciliated cells, whereas HCoV-OC43, HCoV-HKU1, and HCoV-NL63 all infect ciliated cells. Collectively, the data demonstrate that HAE cell cultures, which morphologically and functionally resemble human airways in vivo, represent a robust universal culture system for isolating and comparing all contemporary HCoV strains. PMID:23427150

  17. Alginate beads as a tool to handle, cryopreserve and culture isolated human primordial/primary follicles.

    Science.gov (United States)

    Camboni, A; Van Langendonckt, A; Donnez, J; Vanacker, J; Dolmans, M M; Amorim, C A

    2013-08-01

    One major concern of grafting cryopreserved ovarian tissue to restore fertility in cancer patients is the possibility of reintroducing tumor cells. Cryopreservation of isolated primordial/primary follicles (PFs) may circumvent this problem. The aim of our work was to compare dimethyl sulfoxide (ME2SO) and ethylene glycol (EG) as cryoprotectants (CPAs) for slow-freezing of isolated human PFs in alginate. Ovarian biopsies from four women were processed for follicle isolation. PFs were embedded in alginate (5-15 per group). Follicles were frozen-thawed using 1.4M ME2SO or 1.5M EG as CPAs. Fresh and cryopreserved isolated follicles were in vitro cultured (IVC) for 7 days. At different time periods (after isolation, cryopreservation and IVC), follicles were evaluated with live/dead assay (using fluorescent probes) and diameter measurement. Follicle viability was calculated according to the percentage of dead follicular cells and the presence of a live/dead oocyte. A total of 841 PFs were isolated, embedded in alginate and cryopreserved with ME2SO (n=424) or EG (n=259), or used as controls (n=158). After 7 days of IVC, a significant increase in follicle size was observed in the fresh and ME2SO groups, but not in the EG group. The percentage of totally viable PFs was not significantly different before or after seven days of culture in fresh (100% and 82%) or ME2SO (93.2% and 85.1%) tissue. The EG group showed significantly lower viability before (63.9%) and after IVC (66.2%) than the fresh and ME2SO groups. Our results show that 1.4M ME2SO yields better preservation of isolated PF viability after thawing and 7 days of IVC than 1.5M EG. Alginate constitutes an easy, safe hydrogel matrix to handle and cryopreserve isolated human follicles using ME2SO as a CPA. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Isolation, identification, and pathological effects of beach sand bacterial extract on human skin keratinocytes in vitro

    Directory of Open Access Journals (Sweden)

    Fazli Subhan

    2018-01-01

    Full Text Available Background Beaches are recreational spots for people. However, beach sand contains harmful microbes that affect human health, and there are no established methods for either sampling and identifying beach-borne pathogens or managing the quality of beach sand. Method This study was conducted with the aim of improving human safety at beaches and augmenting the quality of the beach experience. Beach sand was used as a resource to isolate bacteria due to its distinctive features and the biodiversity of the beach sand biota. A selected bacterial isolate termed FSRS was identified as Pseudomonas stutzeri using 16S rRNA sequencing and phylogenetic analysis, and the sequence was deposited in the NCBI GenBank database under the accession number MF599548. The isolated P. stutzeri bacterium was cultured in Luria–Bertani growth medium, and a crude extract was prepared using ethyl acetate to examine the potential pathogenic effect of P. stutzeri on human skin. A human skin keratinocyte cell line (HaCaT was used to assess cell adhesion, cell viability, and cell proliferation using a morphological analysis and a WST-1 assay. Result The crude P. stutzeri extract inhibited cell adhesion and decreased cell viability in HaCaT cells. We concluded that the crude extract of P. stutzeri FSRS had a strong pathological effect on human skin cells. Discussion Beach visitors frequently get skin infections, but the exact cause of the infections is yet to be determined. The beach sand bacterium P. stutzeri may, therefore, be responsible for some of the dermatological problems experienced by people visiting the beach.

  19. VTEC O157 subtypes associated with the most severe clinical symptoms in humans constitute a minor part of VTEC 0157 isolates from Danish Cattle

    DEFF Research Database (Denmark)

    Roldgaard, Bemt Bjørn; Scheutz, Flemming; Boel, Jeppe

    2004-01-01

    -positive VTEC 0 157 isolates (63 of bovine origin and 86 from human clinical cases) isolated between 1987 and 2001. All were analysed by vtx-PCR-RFLP and phage typing. The vtx-PCR-RFLP showed that isolates carrying the vtx2 gene was more than four times as prevalent among the human clinical isolates (55...

  20. Role of Heparan Sulfate in Cellular Infection of Integrin-Binding Coxsackievirus A9 and Human Parechovirus 1 Isolates.

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    Pirjo Merilahti

    Full Text Available Heparan sulfate/heparin class of proteoglycans (HSPG have been shown to function in cellular attachment and infection of numerous viruses including picornaviruses. Coxsackievirus A9 (CV-A9 and human parechovirus 1 (HPeV-1 are integrin-binding members in the family Picornaviridae. CV-A9 Griggs and HPeV-1 Harris (prototype strains have been reported not to bind to heparin, but it was recently shown that some CV-A9 isolates interact with heparin in vitro via VP1 protein with a specific T132R/K mutation. We found that the infectivity of both CV-A9 Griggs and HPeV-1 Harris was reduced by sodium chlorate and heparinase suggestive of HSPG interactions. We analyzed the T132 site in fifty-four (54 CV-A9 clinical isolates and found that only one of them possessed T132/R mutation while the other nine (9 had T132K. We then treated CV-A9 Griggs and HPeV-1 Harris and eight CV-A9 and six HPeV-1 clinical isolates with heparin and protamine. Although infectivity of Griggs strain was slightly reduced (by 25%, heparin treatment did not affect the infectivity of the CV-A9 isolates that do not possess the T132R/K mutation, which is in line with the previous findings. Some of the HPeV-1 isolates were also affected by heparin treatment, which suggested that there may be a specific heparin binding site in HPeV-1. In contrast, protamine (a specific inhibitor of heparin completely inhibited the infection of both prototypes and clinical CV-A9 and HPeV-1 isolates. We conclude that T132R/K mutation has a role in heparin binding of CV-A9, but we also show data, which suggest that there are other HSPG binding sites in CV-A9. In all, we suggest that HSPGs play a general role in both CV-A9 and HPeV-1 infections.

  1. Species identification and molecular typing of human Brucella isolates from Kuwait.

    Science.gov (United States)

    Mustafa, Abu S; Habibi, Nazima; Osman, Amr; Shaheed, Faraz; Khan, Mohd W

    2017-01-01

    Brucellosis is a zoonotic disease of major concern in Kuwait and the Middle East. Human brucellosis can be caused by several Brucella species with varying degree of pathogenesis, and relapses are common after apparently successful therapy. The classical biochemical methods for identification of Brucella are time-consuming, cumbersome, and provide information limited to the species level only. In contrast, molecular methods are rapid and provide differentiation at intra-species level. In this study, four molecular methods [16S rRNA gene sequencing, real-time PCR, enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus variable-number tandem-repeat analysis (MLVA)-8, MLVA-11 and MLVA-16 were evaluated for the identification and typing of 75 strains of Brucella isolated in Kuwait. 16S rRNA gene sequencing of all isolates showed 90-99% sequence identity with B. melitensis and real-time PCR with genus- and species- specific primers identified all isolates as B. melitensis. The results of ERIC-PCR suggested the existence of 75 ERIC genotypes of B. melitensis with a discriminatory index of 0.997. Cluster classification of these genotypes divided them into two clusters, A and B, diverging at ~25%. The maximum number of genotypes (n = 51) were found in cluster B5. MLVA-8 analysis identified all isolates as B. melitensis, and MLVA-8, MLVA-11 and MLVA-16 typing divided the isolates into 10, 32 and 71 MLVA types, respectively. Furthermore, the combined minimum spanning tree analysis demonstrated that, compared to MLVA types discovered all over the world, the Kuwaiti isolates were a distinct group of MLVA-11 and MLVA-16 types in the East Mediterranean Region.

  2. Regulation of lipoprotein lipase in primary cultures of isolated human adipocytes

    International Nuclear Information System (INIS)

    Kern, P.A.; Marshall, S.; Eckel, R.H.

    1985-01-01

    To study the regulation of adipose tissue lipoprotein lipase (LPL) in human adipocytes, omental adipose tissue was obtained from healthy subjects and digested in collagenase. The isolated adipocytes thus obtained were suspended in Medium 199 and cultured at 37 degrees C. Cell viability was demonstrated in adipocytes cultured for up to 72 h by constancy of cell number, cell size, trypan-blue exclusion, and specific 125 I-insulin binding. In addition, chloroquine induced an increase in cell-associated 125 I-insulin at 24, 48, and 72 h after preparation. Thus, isolated adipocytes retained their ability to bind, internalize, and degrade insulin. LPL was measured as activity secreted into the culture medium (CM), released from cells by heparin (HR), and extracted from cell digests. A broad range of heparin concentrations produced a prompt release of LPL from a rapidly replenishable pool of cellular activity. When cells were cultured in medium containing 10% fetal bovine serum, there was a marked stimulation of CM and HR. The secretory response to serum (CM) correlated strongly with HR 24 h after preparation. In addition, HR was found to correlate logarithmically and inversely with body mass index. Insulin, at 400 ng/ml only, increased HR by 36 +/- 10%, an effect simulated by lower concentrations of insulin-like growth factor-1 (IGF1). Thus, LPL is produced and regulated in isolated human adipocytes. The degree of adiposity and serum are important regulators of HR activity, whereas insulin is stimulatory only at a pharmacologic concentration. This effect of insulin may be mediated through the IGF1 receptor. Isolated human adipocytes represent a novel and useful system for the study of LPL and lipid metabolism as well as for other aspects of adipocyte biology

  3. Detection and Characterization of Clade 1 Reassortant H5N1 Viruses Isolated from Human Cases in Vietnam during 2013.

    Directory of Open Access Journals (Sweden)

    Sharmi W Thor

    Full Text Available Highly pathogenic avian influenza (HPAI H5N1 is endemic in Vietnamese poultry and has caused sporadic human infection in Vietnam since 2003. Human infections with HPAI H5N1 are of concern due to a high mortality rate and the potential for the emergence of pandemic viruses with sustained human-to-human transmission. Viruses isolated from humans in southern Vietnam have been classified as clade 1 with a single genome constellation (VN3 since their earliest detection in 2003. This is consistent with detection of this clade/genotype in poultry viruses endemic to the Mekong River Delta and surrounding regions. Comparison of H5N1 viruses detected in humans from southern Vietnamese provinces during 2012 and 2013 revealed the emergence of a 2013 reassortant virus with clade 1.1.2 hemagglutinin (HA and neuraminidase (NA surface protein genes but internal genes derived from clade 2.3.2.1a viruses (A/Hubei/1/2010-like; VN12. Closer analysis revealed mutations in multiple genes of this novel genotype (referred to as VN49 previously associated with increased virulence in animal models and other markers of adaptation to mammalian hosts. Despite the changes identified between the 2012 and 2013 genotypes analyzed, their virulence in a ferret model was similar. Antigenically, the 2013 viruses were less cross-reactive with ferret antiserum produced to the clade 1 progenitor virus, A/Vietnam/1203/2004, but reacted with antiserum produced against a new clade 1.1.2 WHO candidate vaccine virus (A/Cambodia/W0526301/2012 with comparable hemagglutination inhibition titers as the homologous antigen. Together, these results indicate changes to both surface and internal protein genes of H5N1 viruses circulating in southern Vietnam compared to 2012 and earlier viruses.

  4. Isolation of Human Colon Stem Cells Using Surface Expression of PTK7.

    Science.gov (United States)

    Jung, Peter; Sommer, Christian; Barriga, Francisco M; Buczacki, Simon J; Hernando-Momblona, Xavier; Sevillano, Marta; Duran-Frigola, Miquel; Aloy, Patrick; Selbach, Matthias; Winton, Douglas J; Batlle, Eduard

    2015-12-08

    Insertion of reporter cassettes into the Lgr5 locus has enabled the characterization of mouse intestinal stem cells (ISCs). However, low cell surface abundance of LGR5 protein and lack of high-affinity anti-LGR5 antibodies represent a roadblock to efficiently isolate human colonic stem cells (hCoSCs). We set out to identify stem cell markers that would allow for purification of hCoSCs. In an unbiased approach, membrane-enriched protein fractions derived from in vitro human colonic organoids were analyzed by quantitative mass spectrometry. Protein tyrosine pseudokinase PTK7 specified a cell population within human colonic organoids characterized by highest self-renewal and re-seeding capacity. Antibodies recognizing the extracellular domain of PTK7 allowed us to isolate and expand hCoSCs directly from patient-derived mucosa samples. Human PTK7+ cells display features of canonical Lgr5+ ISCs and include a fraction of cells that undergo differentiation toward enteroendocrine lineage that resemble crypt label retaining cells (LRCs). Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  5. An association of genotypes and antimicrobial resistance patterns among Salmonella isolates from pigs and humans in Taiwan.

    Directory of Open Access Journals (Sweden)

    Hung-Chih Kuo

    Full Text Available We collected 110 Salmonella enterica isolates from sick pigs and determined their serotypes, genotypes using pulsed-field gel electrophoresis (PFGE, and antimicrobial susceptibility to 12 antimicrobials and compared the data with a collection of 18,280 isolates obtained from humans. The pig isolates fell into 12 common serovars for human salmonellosis in Taiwan; S. Typhimurium, S. Choleraesuis, S. Derby, S. Livingstone, and S. Schwarzengrund were the 5 most common serovars and accounted for a total of 84% of the collection. Of the 110 isolates, 106 (96% were multidrug resistant (MDR and 48 (44% had PFGE patterns found in human isolates. S. Typhimurium, S. Choleraesuis, and S. Schwarzengrund were among the most highly resistant serovars. The majority of the 3 serovars were resistant to 8-11 of the tested antimicrobials. The isolates from pigs and humans sharing a common PFGE pattern displayed identical or very similar resistance patterns and Salmonella strains that caused severe infection in pigs were also capable of causing infections in humans. The results indicate that pigs are one of the major reservoirs to human salmonellosis in Taiwan. Almost all of the pig isolates were MDR, which highlights the necessity of strictly regulating the use of antimicrobials in the agriculture sector in Taiwan.

  6. An association of genotypes and antimicrobial resistance patterns among Salmonella isolates from pigs and humans in Taiwan.

    Science.gov (United States)

    Kuo, Hung-Chih; Lauderdale, Tsai-Ling; Lo, Dan-Yuan; Chen, Chiou-Lin; Chen, Pei-Chen; Liang, Shiu-Yun; Kuo, Jung-Che; Liao, Ying-Shu; Liao, Chun-Hsing; Tsao, Chi-Sen; Chiou, Chien-Shun

    2014-01-01

    We collected 110 Salmonella enterica isolates from sick pigs and determined their serotypes, genotypes using pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility to 12 antimicrobials and compared the data with a collection of 18,280 isolates obtained from humans. The pig isolates fell into 12 common serovars for human salmonellosis in Taiwan; S. Typhimurium, S. Choleraesuis, S. Derby, S. Livingstone, and S. Schwarzengrund were the 5 most common serovars and accounted for a total of 84% of the collection. Of the 110 isolates, 106 (96%) were multidrug resistant (MDR) and 48 (44%) had PFGE patterns found in human isolates. S. Typhimurium, S. Choleraesuis, and S. Schwarzengrund were among the most highly resistant serovars. The majority of the 3 serovars were resistant to 8-11 of the tested antimicrobials. The isolates from pigs and humans sharing a common PFGE pattern displayed identical or very similar resistance patterns and Salmonella strains that caused severe infection in pigs were also capable of causing infections in humans. The results indicate that pigs are one of the major reservoirs to human salmonellosis in Taiwan. Almost all of the pig isolates were MDR, which highlights the necessity of strictly regulating the use of antimicrobials in the agriculture sector in Taiwan.

  7. A Novel Method for Isolation of Pluripotent Stem Cells from Human Umbilical Cord Blood.

    Science.gov (United States)

    Monti, Manuela; Imberti, Barbara; Bianchi, Niccolò; Pezzotta, Anna; Morigi, Marina; Del Fante, Claudia; Redi, Carlo Alberto; Perotti, Cesare

    2017-09-01

    Very small embryonic-like cells (VSELs) are a population of very rare pluripotent stem cells isolated in adult murine bone marrow and many other tissues and organs, including umbilical cord blood (UCB). VSEL existence is still not universally accepted by the scientific community, so for this purpose, we sought to investigate whether presumptive VSELs (pVSELs) could be isolated from human UCB with an improved protocol based on the isolation of enriched progenitor cells by depletion of nonprogenitor cells with magnetic separation. Progenitor cells, likely including VSELs, cultured with retinoic acid were able to form dense colonies and cystic embryoid bodies and to differentiate toward the ecto-meso-endoderm lineages as shown by the positivity to specific markers. VSEL differentiative potential toward mesodermal lineage was further demonstrated in vitro upon exposure to an established inductive protocol, which induced the acquisition of renal progenitor cell phenotype. VSEL-derived renal progenitors showed regenerative potential in a cisplatin model of acute kidney injury by restoring renal function and tubular structure through induction of proliferation of endogenous renal cells. The data presented here foster the great debate that surrounds VSELs and, more in general, the existence of cells endowed with pluripotent features in adult tissues. In fact, the possibility to find and isolate subpopulations of cells that fully fit all the criteria utilized to define pluripotency remains, nowadays, almost unproven. Thus, efforts to better characterize the phenotype of these intriguing cells are crucial to understand their possible applications for regenerative and precision medicine purposes.

  8. Isolation of mineralizing Nestin+ Nkx6.1+ vascular muscular cells from the adult human spinal cord

    Directory of Open Access Journals (Sweden)

    Guillon Hélène

    2011-10-01

    Full Text Available Abstract Background The adult central nervous system (CNS contains different populations of immature cells that could possibly be used to repair brain and spinal cord lesions. The diversity and the properties of these cells in the human adult CNS remain to be fully explored. We previously isolated Nestin+ Sox2+ neural multipotential cells from the adult human spinal cord using the neurosphere method (i.e. non adherent conditions and defined medium. Results Here we report the isolation and long term propagation of another population of Nestin+ cells from this tissue using adherent culture conditions and serum. QPCR and immunofluorescence indicated that these cells had mesenchymal features as evidenced by the expression of Snai2 and Twist1 and lack of expression of neural markers such as Sox2, Olig2 or GFAP. Indeed, these cells expressed markers typical of smooth muscle vascular cells such as Calponin, Caldesmone and Acta2 (Smooth muscle actin. These cells could not differentiate into chondrocytes, adipocytes, neuronal and glial cells, however they readily mineralized when placed in osteogenic conditions. Further characterization allowed us to identify the Nkx6.1 transcription factor as a marker for these cells. Nkx6.1 was expressed in vivo by CNS vascular muscular cells located in the parenchyma and the meninges. Conclusion Smooth muscle cells expressing Nestin and Nkx6.1 is the main cell population derived from culturing human spinal cord cells in adherent conditions with serum. Mineralization of these cells in vitro could represent a valuable model for studying calcifications of CNS vessels which are observed in pathological situations or as part of the normal aging. In addition, long term propagation of these cells will allow the study of their interaction with other CNS cells and their implication in scar formation during spinal cord injury.

  9. Isolation and functional interrogation of adult human prostate epithelial stem cells at single cell resolution.

    Science.gov (United States)

    Hu, Wen-Yang; Hu, Dan-Ping; Xie, Lishi; Li, Ye; Majumdar, Shyama; Nonn, Larisa; Hu, Hong; Shioda, Toshi; Prins, Gail S

    2017-08-01

    Using primary cultures of normal human prostate epithelial cells, we developed a novel prostasphere-based, label-retention assay that permits identification and isolation of stem cells at a single cell level. Their bona fide stem cell nature was corroborated using in vitro and in vivo regenerative assays and documentation of symmetric/asymmetric division. Robust WNT10B and KRT13 levels without E-cadherin or KRT14 staining distinguished individual stem cells from daughter progenitors in spheroids. Following FACS to isolate label-retaining stem cells from label-free progenitors, RNA-seq identified unique gene signatures for the separate populations which may serve as useful biomarkers. Knockdown of KRT13 or PRAC1 reduced sphere formation and symmetric self-renewal highlighting their role in stem cell maintenance. Pathways analysis identified ribosome biogenesis and membrane estrogen-receptor signaling enriched in stem cells with NF-ĸB signaling enriched in progenitors; activities that were biologically confirmed. Further, bioassays identified heightened autophagy flux and reduced metabolism in stem cells relative to progenitors. These approaches similarly identified stem-like cells from prostate cancer specimens and prostate, breast and colon cancer cell lines suggesting wide applicability. Together, the present studies isolate and identify unique characteristics of normal human prostate stem cells and uncover processes that maintain stem cell homeostasis in the prostate gland. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  10. Isolation and functional interrogation of adult human prostate epithelial stem cells at single cell resolution

    Directory of Open Access Journals (Sweden)

    Wen-Yang Hu

    2017-08-01

    Full Text Available Using primary cultures of normal human prostate epithelial cells, we developed a novel prostasphere-based, label-retention assay that permits identification and isolation of stem cells at a single cell level. Their bona fide stem cell nature was corroborated using in vitro and in vivo regenerative assays and documentation of symmetric/asymmetric division. Robust WNT10B and KRT13 levels without E-cadherin or KRT14 staining distinguished individual stem cells from daughter progenitors in spheroids. Following FACS to isolate label-retaining stem cells from label-free progenitors, RNA-seq identified unique gene signatures for the separate populations which may serve as useful biomarkers. Knockdown of KRT13 or PRAC1 reduced sphere formation and symmetric self-renewal highlighting their role in stem cell maintenance. Pathways analysis identified ribosome biogenesis and membrane estrogen-receptor signaling enriched in stem cells with NF-ĸB signaling enriched in progenitors; activities that were biologically confirmed. Further, bioassays identified heightened autophagy flux and reduced metabolism in stem cells relative to progenitors. These approaches similarly identified stem-like cells from prostate cancer specimens and prostate, breast and colon cancer cell lines suggesting wide applicability. Together, the present studies isolate and identify unique characteristics of normal human prostate stem cells and uncover processes that maintain stem cell homeostasis in the prostate gland.

  11. Rare and unusual isolates of viridans streptococci from the human oral cavity

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    Shree Dhotre

    2016-01-01

    Full Text Available Context: The genus Streptococcus consists of more than 65 species. The taxonomic classification of these members is not well-defined. Among the viridans group streptococci (VGS, there are major taxonomic changes by the addition of many new species; whereas, most of the new strains are of animal origin and only a few have been reported to be isolated from humans. Rare and unusual species of VGS such as Streptococcus thoraltensis, S. pluranimalium and S. hyointestinalis are normally associated with different animals. Their isolation from human being is not yet reported. Aim: To find out the rare and unusual species of viridans group streptococci from human oral cavity. Settings and Design: A case-controlled study carried out at hospital-based dental services in a tertiary care hospital. Materials and Methods: Subgingival plaque samples of the tooth were collected from 80 patients (34 with periodontitis and 46 without periodontitis undergoing tooth extraction. Cultures were subcultured onto special media such as Tryptone Soya blood Agar supplemented with strepto supplement and Mutans-Sanguis Agar. Identification of strains and antimicrobial susceptibilities were measured as minimum inhibitory concentration using Vitek 2 (BioMérieux, Paris, France automated system. Results: We have identified three strains of VGS - S. thoraltensis, S. pluranimalium and S. hyointestinalis from subgingival plaque samples from patients with periodontitis. S. thoraltensis and S. pluranimalium were found to be resistant to most of the antibiotics. Conclusions: To the best of our knowledge, this is the first report of isolation of these rare and unusual strains from the human oral cavity.

  12. Complete genome sequence of the first human parechovirus type 3 isolated in Taiwan

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    Jenn-Tzong Chang

    2017-11-01

    Full Text Available The first human parechovirus 3 (HPeV3 VGHKS-2007 in Taiwan was identified from a clinical specimen from a male infant. The entire genome of the HPeV3 isolate was sequenced and compared to known HPeV3 sequences. Genome alignment data showed that HPeV3 VGHKS-2007 shares the highest nucleotide identity, 99%, with the Japanese strain of HPeV3 1361K-162589-Yamagata-2008. All HPeV3 isolates possess at least 97% amino acid identity. The analysis of the genome sequence of HPeV3 VGHKS-2007 will facilitate future investigations of the epidemiology and pathogenicity of HPeV3 infection.

  13. Human Behaviour and Responses Challenge towards Emergence of Infectious Diseases: E.coli clinical isolate

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    Ummi Mohlisi Mohd Asmawi

    2016-01-01

    Full Text Available Consumption of undercooked ground beef is the most common route of transmission of verotoxin-producing E.coli. It is estimated that non-O157 verotoxigenic E.coli (VTEC can cause diarrhea.The sample was isolated from Universiti Malaya Medical Centre. All the isolates were identified using agarose gel electrophoresis method. This study aims to detect the verotoxin genes and detect the link or involvement of plasmids with these verotoxin genes. Therefore, this study will contribute to shed new light on resolving the significant and global problem of diarrheal disease caused by this particular pathogenic organism and help in improvising novel therapeutic approaches to improve human healthcare.

  14. Characteristics of primary infection of a European human immunodeficiency virus type 1 clade B isolate in chimpanzees

    NARCIS (Netherlands)

    Bogers, W. M.; Koornstra, W. H.; Dubbes, R. H.; ten Haaft, P. J.; Verstrepen, B. E.; Jhagjhoorsingh, S. S.; Haaksma, A. G.; Niphuis, H.; Laman, J. D.; Norley, S.; Schuitemaker, H.; Goudsmit, J.; Hunsmann, G.; Heeney, J. L.; Wigzell, H.

    1998-01-01

    The aim of the study was to select, from a panel of candidate European human immunodeficiency virus type 1 (HIV-1) clade B primary virus isolates, one isolate based on replication properties in chimpanzee peripheral blood mononuclear cells (PBMC). Secondly, to evaluate the in vivo kinetics of

  15. Full genome sequences and molecular characterization of tick-borne encephalitis virus strains isolated from human patients.

    Science.gov (United States)

    Formanová, Petra; Černý, Jiří; Bolfíková, Barbora Černá; Valdés, James J; Kozlova, Irina; Dzhioev, Yuri; Růžek, Daniel

    2015-02-01

    Tick-borne encephalitis virus (TBEV) causes tick-borne encephalitis (TBE), one of the most important human neuroinfections across Eurasia. Up to date, only three full genome sequences of human European TBEV isolates are available, mostly due to difficulties with isolation of the virus from human patients. Here we present full genome characterization of an additional five low-passage TBEV strains isolated from human patients with severe forms of TBE. These strains were isolated in 1953 within Central Bohemia in the former Czechoslovakia, and belong to the historically oldest human TBEV isolates in Europe. We demonstrate here that all analyzed isolates are distantly phylogenetically related, indicating that the emergence of TBE in Central Europe was not caused by one predominant strain, but rather a pool of distantly related TBEV strains. Nucleotide identity between individual sequenced TBEV strains ranged from 97.5% to 99.6% and all strains shared large deletions in the 3' non-coding region, which has been recently suggested to be an important determinant of virulence. The number of unique amino acid substitutions varied from 3 to 9 in individual isolates, but no characteristic amino acid substitution typical exclusively for all human TBEV isolates was identified when compared to the isolates from ticks. We did, however, correlate that the exploration of the TBEV envelope glycoprotein by specific antibodies were in close proximity to these unique amino acid substitutions. Taken together, we report here the largest number of patient-derived European TBEV full genome sequences to date and provide a platform for further studies on evolution of TBEV since the first emergence of human TBE in Europe. Copyright © 2014 Elsevier GmbH. All rights reserved.

  16. Application of fluorescent amplified fragment length polymorphism for comparison of human and animal isolates of Yersinia enterocolitica

    DEFF Research Database (Denmark)

    Fearnley, C.; On, S.L.W.; Kokotovic, Branko

    2005-01-01

    An amplified fragment length polymorphism (AFLP) method, developed to genotype Yersinia enterocolitica, has been used to investigate 70 representative strains isolated from humans, pigs, sheep, and cattle in the United Kingdom. AFLP primarily distinguished Y enterocolitica strains according...

  17. Isolation of the functional human excision repair gene ERCC5 by intercosmid recombination

    International Nuclear Information System (INIS)

    Mudgett, J.S.; MacInnes, M.A.

    1990-01-01

    The complete human nucleotide exicision repair gene ERCC5 was isolated as a functional gene on overlapping cosmids. ERCC5 corrects the excision repair deficiency of Chinese hamster ovary cell line UV135, of complementation group 5. Cosmids that contained human sequences were obtained from a UV-resistant cell line derived from UV135 cells transformed with human genomic DNA. Individually, none of the cosmids complemented the UV135 repair defect; cosmid groups were formed to represent putative human genomic regions, and specific pairs of cosmids that effectively transformed UV135 cells to UV resistance were identified. Analysis of transformants derived from the active cosmid pairs showed that the functional 32-kbp ERCC5 gene was reconstructed by homologous intercosmid recombination. The cloned human sequences exhibited 100% concordance with the locus designated genetically as ERCC5 located on human chromosome 13q. Cosmid-transformed UV135 host cells repaired cytotoxic damage to levels about 70% of normal and repaired UV-irradiated shuttle vector DNA to levels about 82% of normal

  18. Isolation and characterization of cDNA clones for human erythrocyte β-spectrin

    International Nuclear Information System (INIS)

    Prchal, J.T.; Morley, B.J.; Yoon, S.H.; Coetzer, T.L.; Palek, J.; Conboy, J.G.; Kan, Y.W.

    1987-01-01

    Spectrin is an important structural component of the membrane skeleton that underlies and supports the erythrocyte plasma membrane. It is composed of nonidentical α (M/sub r/ 240,000) and β (M/sub r/ 220,000) subunits, each of which contains multiple homologous 106-amino acid segments. The authors report here the isolation and characterization of a human erythroid-specific β-spectrin cDNA clone that encodes parts of the β-9 through β-12 repeat segments. This cDNA was used as a hybridization probe to assign the β-spectrin gene to human chromosome 14 and to begin molecular analysis of the gene and its mRNA transcripts. RNA transfer blot analysis showed that the reticulocyte β-spectrin mRNA is 7.8 kilobases in length. Southern blot analysis of genomic DNA revealed the presence of restriction fragment length polymorphisms (RFLPs) within the β-spectrin gene locus. The isolation of human spectrin cDNA probes and the identification of closely linked RFLPs will facilitate analysis of mutant spectrin genes causing congenital hemolytic anemias associated with quantitative and qualitative spectrin abnormalities

  19. The isolated perfused human skin flap model: A missing link in skin penetration studies?

    Science.gov (United States)

    Ternullo, Selenia; de Weerd, Louis; Flaten, Gøril Eide; Holsæter, Ann Mari; Škalko-Basnet, Nataša

    2017-01-01

    Development of effective (trans)dermal drug delivery systems requires reliable skin models to evaluate skin drug penetration. The isolated perfused human skin flap remains metabolically active tissue for up to 6h during in vitro perfusion. We introduce the isolated perfused human skin flap as a close-to-in vivo skin penetration model. To validate the model's ability to evaluate skin drug penetration the solutions of a hydrophilic (calcein) and a lipophilic (rhodamine) fluorescence marker were applied. The skin flaps were perfused with modified Krebs-Henseleit buffer (pH7.4). Infrared technology was used to monitor perfusion and to select a well-perfused skin area for administration of the markers. Flap perfusion and physiological parameters were maintained constant during the 6h experiments and the amount of markers in the perfusate was determined. Calcein was detected in the perfusate, whereas rhodamine was not detectable. Confocal images of skin cross-sections shoved that calcein was uniformly distributed through the skin, whereas rhodamine accumulated in the stratum corneum. For comparison, the penetration of both markers was evaluated on ex vivo human skin, pig skin and cellophane membrane. The proposed perfused flap model enabled us to distinguish between the penetrations of the two markers and could be a promising close-to-in vivo tool in skin penetration studies and optimization of formulations destined for skin administration. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Products of neutrophils and eosinophils increase the responsiveness of human isolated bronchial tissue.

    Science.gov (United States)

    Hallahan, A R; Armour, C L; Black, J L

    1990-05-01

    This study examines the possibility that products of neutrophils and eosinophils could increase the responsiveness of human isolated bronchial tissue. Neutrophils and eosinophils were isolated from the peripheral blood of healthy volunteers. The cells were incubated with 1 microM calcium ionophore A23187 for 10-15 min then centrifuged, the supernatant collected and stored at -70 degrees C. Human bronchial rings (2-3 mm diameter, 3-4 mm long) were prepared from specimens resected at thoracotomy. The tissues were suspended in organ baths under a 1 g load and changes in tension measured isometrically. Stable contractions to bolus doses of histamine (0.1-10 microM) or to electrical field stimulation (40-100 V, 4-16 Hz, 1 ms for 20 s) were established. Supernatant from 106 neutrophils or 105 eosinophils was then added and tissue responsiveness reassessed. Neutrophil supernatant increased tissue responsiveness to histamine and electrical field stimulation by 54 +/- 17% (n = 5, p less than 0.05) and 18 +/- 7% (n = 6, p less than 0.05), respectively. Eosinophil supernatant increased the histamine response by 60 +/- 23% (n = 8, p less than 0.05) while tissue responsiveness to electrical field stimulation was unchanged (n = 3). Thus, as neutrophils and eosinophils can change the responsiveness of human bronchus in vitro it is possible that they do this in vivo and may not simply be temporally related to the development of bronchial hyperresponsiveness.

  1. Computer graphics of SEM images facilitate recognition of chromosome position in isolated human metaphase plates.

    Science.gov (United States)

    Hodge, L D; Barrett, J M; Welter, D A

    1995-04-01

    There is general agreement that at the time of mitosis chromosomes occupy precise positions and that these positions likely affect subsequent nuclear function in interphase. However, before such ideas can be investigated in human cells, it is necessary to determine first the precise position of each chromosome with regard to its neighbors. It has occurred to us that stereo images, produced by scanning electron microscopy, of isolated metaphase plates could form the basis whereby these positions could be ascertained. In this paper we describe a computer graphic technique that permits us to keep track of individual chromosomes in a metaphase plate and to compare chromosome positions in different metaphase plates. Moreover, the computer graphics provide permanent, easily manipulated, rapid recall of stored chromosome profiles. These advantages are demonstrated by a comparison of the relative position of group A-specific and groups D- and G-specific chromosomes to the full complement of chromosomes in metaphase plates isolated from a nearly triploid human-derived cell (HeLa S3) to a hypo-diploid human fetal lung cell.

  2. Molecular Identification of Hookworm Isolates in Humans, Dogs and Soil in a Tribal Area in Tamil Nadu, India.

    Directory of Open Access Journals (Sweden)

    Santosh George

    2016-08-01

    Full Text Available Hookworms (Necator americanus and Ancylostoma duodenale remain a major public health problem worldwide. Infections with hookworms (e.g., A. caninum, A. ceylanicum and A. braziliense are also prevalent in dogs, but the role of dogs as a reservoir for zoonotic hookworm infections in humans needs to be further explored.As part of an open-label community based cluster-randomized trial in a tribal area in Tamil Nadu (India; 2013-2015, a total of 143 isolates of hookworm eggs from human stool were speciated based on a previously described PCR-RFLP methodology. The presence of hookworm DNA was confirmed in 119 of 143 human samples. N. americanus (100% was the most prevalent species, followed by A. caninum (16.8% and A. duodenale (8.4%. Because of the high prevalence of A. caninum in humans, dog samples were also collected to assess the prevalence of A. caninum in dogs. In 68 out of 77 canine stool samples the presence of hookworms was confirmed using PCR-RFLP. In dogs, both A. caninum (76.4% and A. ceylanicum (27.9% were identified. Additionally, to determine the contamination of soil with zoonotic hookworm larvae, topsoil was collected from defecating areas. Hookworm DNA was detected in 72 out of 78 soil samples that revealed presence of hookworm-like nematode larvae. In soil, different hookworm species were identified, with animal hookworms being more prevalent (A. ceylanicum: 60.2%, A. caninum: 29.4%, A. duodenale: 16.6%, N. americanus: 1.4%, A. braziliense: 1.4%.In our study we regularly detected the presence of A. caninum DNA in the stool of humans. Whether this is the result of infection is currently unknown but it does warrant a closer look at dogs as a potential reservoir.

  3. Molecular Identification of Hookworm Isolates in Humans, Dogs and Soil in a Tribal Area in Tamil Nadu, India.

    Science.gov (United States)

    George, Santosh; Levecke, Bruno; Kattula, Deepthi; Velusamy, Vasanthakumar; Roy, Sheela; Geldhof, Peter; Sarkar, Rajiv; Kang, Gagandeep

    2016-08-01

    Hookworms (Necator americanus and Ancylostoma duodenale) remain a major public health problem worldwide. Infections with hookworms (e.g., A. caninum, A. ceylanicum and A. braziliense) are also prevalent in dogs, but the role of dogs as a reservoir for zoonotic hookworm infections in humans needs to be further explored. As part of an open-label community based cluster-randomized trial in a tribal area in Tamil Nadu (India; 2013-2015), a total of 143 isolates of hookworm eggs from human stool were speciated based on a previously described PCR-RFLP methodology. The presence of hookworm DNA was confirmed in 119 of 143 human samples. N. americanus (100%) was the most prevalent species, followed by A. caninum (16.8%) and A. duodenale (8.4%). Because of the high prevalence of A. caninum in humans, dog samples were also collected to assess the prevalence of A. caninum in dogs. In 68 out of 77 canine stool samples the presence of hookworms was confirmed using PCR-RFLP. In dogs, both A. caninum (76.4%) and A. ceylanicum (27.9%) were identified. Additionally, to determine the contamination of soil with zoonotic hookworm larvae, topsoil was collected from defecating areas. Hookworm DNA was detected in 72 out of 78 soil samples that revealed presence of hookworm-like nematode larvae. In soil, different hookworm species were identified, with animal hookworms being more prevalent (A. ceylanicum: 60.2%, A. caninum: 29.4%, A. duodenale: 16.6%, N. americanus: 1.4%, A. braziliense: 1.4%). In our study we regularly detected the presence of A. caninum DNA in the stool of humans. Whether this is the result of infection is currently unknown but it does warrant a closer look at dogs as a potential reservoir.

  4. A newly isolated probiotic Enterococcus faecalis strain from vagina microbiota enhances apoptosis of human cancer cells.

    Science.gov (United States)

    Nami, Y; Abdullah, N; Haghshenas, B; Radiah, D; Rosli, R; Yari Khosroushahi, A

    2014-08-01

    This study aimed to describe probiotic properties and bio-therapeutic effects of newly isolated Enterococcus faecalis from the human vaginal tract. The Enterococcus faecalis strain was originally isolated from the vaginal microbiota of Iranian women and was molecularly identified using 16SrDNA gene sequencing. Some biochemical methodologies were preliminarily used to characterize the probiotic potential of Ent. faecalis, including antibiotic susceptibility, antimicrobial activity, as well as acid and bile resistance. The bio-therapeutic effects of this strain's secreted metabolites on four human cancer cell lines (AGS, HeLa, MCF-7 and HT-29) and one normal cell line (HUVEC) were evaluated by cytotoxicity assay and apoptosis scrutiny. The characterization results demonstrated into the isolated bacteria strain revealed probiotic properties, such as antibiotic susceptibility, antimicrobial activity and resistance under conditions similar to those in the gastrointestinal tract. Results of bio-therapeutic efficacy assessments illustrated acceptable apoptotic effects on four human cancer cell lines and negligible side effects on assayed normal cell line. Our findings revealed that the apoptotic effect of secreted metabolites mainly depended on proteins secreted by Ent. faecalis on different cancer cells. These proteins can induce the apoptosis of cancer cells. The metabolites produced by this vaginal Ent. faecalis strain can be used as alternative pharmaceutical compounds with promising therapeutic indices because they are not cytotoxic to normal mammalian cells. Accordingly, the physicochemical, structural and functional properties of the secreted anticancer substances should be further investigated before using them as anticancer therapeutics. This study aim to screen total bacterial secreted metabolites as a wealthy source to find the new active compounds to introduce as anticancer therapeutics in the future. © 2014 The Society for Applied Microbiology.

  5. Featured Article: Isolation, characterization, and cultivation of human hepatocytes and non-parenchymal liver cells

    Science.gov (United States)

    Pfeiffer, Elisa; Kegel, Victoria; Zeilinger, Katrin; Hengstler, Jan G; Nüssler, Andreas K; Seehofer, Daniel

    2015-01-01

    Primary human hepatocytes (PHH) are considered to be the gold standard for in vitro testing of xenobiotic metabolism and hepatotoxicity. However, PHH cultivation in 2D mono-cultures leads to dedifferentiation and a loss of function. It is well known that hepatic non-parenchymal cells (NPC), such as Kupffer cells (KC), liver endothelial cells (LEC), and hepatic stellate cells (HSC), play a central role in the maintenance of PHH functions. The aims of the present study were to establish a protocol for the simultaneous isolation of human PHH and NPC from the same tissue specimen and to test their suitability for in vitro co-culture. Human PHH and NPC were isolated from tissue obtained by partial liver resection by a two-step EDTA/collagenase perfusion technique. The obtained cell fractions were purified by Percoll density gradient centrifugation. KC, LEC, and HSC contained in the NPC fraction were separated using specific adherence properties and magnetic activated cell sorting (MACS®). Identified NPC revealed a yield of 1.9 × 106 KC, 2.7 × 105 LEC and 4.7 × 105 HSC per gram liver tissue, showing viabilities >90%. Characterization of these NPC showed that all populations went through an activation process, which influenced the cell fate. The activation of KC strongly depended on the tissue quality and donor anamnesis. KC became activated in culture in association with a loss of viability within 4–5 days. LEC lost specific features during culture, while HSC went through a transformation process into myofibroblasts. The testing of different culture conditions for HSC demonstrated that they can attenuate, but not prevent dedifferentiation in vitro. In conclusion, the method described allows the isolation and separation of PHH and NPC in high quality and quantity from the same donor. PMID:25394621

  6. Recovery of extracellular vesicles from human breast milk is influenced by sample collection and vesicle isolation procedures

    Directory of Open Access Journals (Sweden)

    Marijke I. Zonneveld

    2014-08-01

    Full Text Available Extracellular vesicles (EV in breast milk carry immune relevant proteins and could play an important role in the instruction of the neonatal immune system. To further analyze these EV and to elucidate their function it is important that native populations of EV can be recovered from (stored breast milk samples in a reproducible fashion. However, the impact of isolation and storage procedures on recovery of breast milk EV has remained underexposed. Here, we aimed to define parameters important for EV recovery from fresh and stored breast milk. To compare various protocols across different donors, breast milk was spiked with a well-defined murine EV population. We found that centrifugation of EV down into density gradients largely improved density-based separation and isolation of EV, compared to floatation up into gradients after high-force pelleting of EV. Using cryo-electron microscopy, we identified different subpopulations of human breast milk EV and a not previously described population of lipid tubules. Additionally, the impact of cold storage on breast milk EV was investigated. We determined that storing unprocessed breast milk at −80°C or 4°C caused death of cells present in breast milk, leading to contamination of the breast milk EV population with storage-induced EV. Here, an alternative method is proposed to store breast milk samples for EV analysis at later time points. The proposed adaptations to the breast milk storage and EV isolation procedures can be applied for EV-based biomarker profiling of breast milk and functional analysis of the role of breast milk EV in the development of the neonatal immune system.

  7. Isolation of a human intestinal anaerobe, Bifidobacterium sp. strain SEN, capable of hydrolyzing sennosides to sennidins.

    Science.gov (United States)

    Akao, T; Che, Q M; Kobashi, K; Yang, L; Hattori, M; Namba, T

    1994-01-01

    A strictly anaerobic bacterium capable of metabolizing sennosides was isolated from human feces and identified as Bifidobacterium sp., named strain SEN. The bacterium hydrolyzed sennosides A and B to sennidins A and B via sennidin A and B 8-monoglucosides, respectively. Among nine species of Bifidobacterium having beta-glucosidase activity, only Bifidobacterium dentium and B. adolescentis metabolized sennoside B to sennidin B, suggesting that the sennoside-metabolizing bacteria produce a novel type of beta-glucosidase capable of hydrolyzing sennosides to sennidins. PMID:8161172

  8. Complete genome sequence of Yersinia pestis strain 91001, an isolate avirulent to humans

    DEFF Research Database (Denmark)

    Song, Yajun; Tong, Zongzhong; Wang, Jin

    2004-01-01

    pseudo-genes. Due to the rearrangements mediated by insertion elements, the structure of the 91001 chromosome shows dramatic differences compared with CO92 and KIM. Based on the analysis of plasmids and chromosome architectures, pseudogene distribution, nitrate reduction negative mechanism and gene...... comparison, we conclude that strain 91001 and other strains isolated from M. brandti might have evolved from ancestral Y. pestis in a different lineage. The large genome fragment deletions in the 91001 chromosome and some pseudogenes may contribute to its unique nonpathogenicity to humans and host...

  9. Streptococcus agalactiae in elephants - A comparative study with isolates from human and zoo animal and livestock origin.

    Science.gov (United States)

    Eisenberg, Tobias; Rau, Jörg; Westerhüs, Uta; Knauf-Witzens, Tobias; Fawzy, Ahmad; Schlez, Karen; Zschöck, Michael; Prenger-Berninghoff, Ellen; Heydel, Carsten; Sting, Reinhard; Glaeser, Stefanie P; Pulami, Dipen; van der Linden, Mark; Ewers, Christa

    2017-05-01

    Streptococcus (S.) agalactiae represents a significant pathogen for humans and animals. However, there are only a few elderly reports on S. agalactiae infections in wild and zoo elephants even though this pathogen has been isolated comparatively frequently in these endangered animal species. Consequently, between 2004 and 2015, we collected S. agalactiae isolates from African and Asian elephants (n=23) living in four different zoos in Germany. These isolates were characterised and compared with isolates from other animal species (n=20 isolates) and humans (n=3). We found that the isolates from elephants can be readily identified by classical biochemistry and MALDI-TOF mass spectrometry. Further characterisations for epidemiological issues were achieved using Fourier transform-infrared spectroscopy, capsule typing and molecular fingerprinting (PFGE, RAPD PCR). We could demonstrate that our elephant isolate collection contained at least six different lineages that were representative for their source of origin. Despite generally broad antimicrobial susceptibility of S. agalactiae, many showed tetracycline resistance in vitro. S. agalactiae plays an important role in bacterial infections not only in cattle and humans, but also in elephants. Comparative studies were able to differentiate S. agalactiae isolates from elephants into different infectious clusters based on their epidemiological background. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Genomic Relatedness of Chlamydia Isolates Determined by Amplified Fragment Length Polymorphism Analysis

    OpenAIRE

    Meijer, Adam; Morré, Servaas A.; Van Den Brule, Adriaan J. C.; Savelkoul, Paul H. M.; Ossewaarde, Jacobus M.

    1999-01-01

    The genomic relatedness of 19 Chlamydia pneumoniae isolates (17 from respiratory origin and 2 from atherosclerotic origin), 21 Chlamydia trachomatis isolates (all serovars from the human biovar, an isolate from the mouse biovar, and a porcine isolate), 6 Chlamydia psittaci isolates (5 avian isolates and 1 feline isolate), and 1 Chlamydia pecorum isolate was studied by analyzing genomic amplified fragment length polymorphism (AFLP) fingerprints. The AFLP procedure was adapted from a previously...

  11. Identification of Clinical Staphylococcal Isolates from Humans by Internal Transcribed Spacer PCR

    Science.gov (United States)

    Couto, Isabel; Pereira, Sandro; Miragaia, Maria; Sanches, Ilda Santos; de Lencastre, Hermínia

    2001-01-01

    The emergence of coagulase-negative staphylococci not only as human pathogens but also as reservoirs of antibiotic resistance determinants requires the deployment and development of methods for their rapid and reliable identification. Internal transcribed spacer-PCR (ITS-PCR) was used to identify a collection of 617 clinical staphylococcal isolates. The amplicons were resolved in high-resolution agarose gels and visually compared with the patterns obtained for the control strains of 29 staphylococcal species. Of the 617 isolates studied, 592 (95.95%) were identified by ITS-PCR and included 11 species: 302 isolates of Staphylococcus epidermidis, 157 of S. haemolyticus, 79 of S. aureus, 21 of S. hominis, 14 of S. saprophyticus, 8 of S. warneri, 6 of S. simulans, 2 of S. lugdunensis, and 1 each of S. caprae, S. carnosus, and S. cohnii. All species analyzed had unique ITS-PCR patterns, although some were very similar, namely, the group S. saprophyticus, S. cohnii, S. gallinarum, S. xylosus, S. lentus, S. equorum, and S. chromogenes, the pair S. schleiferi and S. vitulus, and the pair S. piscifermentans and S. carnosus. Four species, S. aureus, S. caprae, S. haemolyticus, and S. lugdunensis, showed polymorphisms on their ITS-PCR patterns. ITS-PCR proved to be a valuable alternative for the identification of staphylococci, offering, within the same response time and at lower cost, higher reliability than the currently available commercial systems. PMID:11526135

  12. Prospective Isolation of Murine and Human Bone Marrow Mesenchymal Stem Cells Based on Surface Markers

    Directory of Open Access Journals (Sweden)

    Yo Mabuchi

    2013-01-01

    Full Text Available Mesenchymal stem cells (MSCs are currently defined as multipotent stromal cells that undergo sustained in vitro growth and can give rise to cells of multiple mesenchymal lineages, such as adipocytes, chondrocytes, and osteoblasts. The regenerative and immunosuppressive properties of MSCs have led to numerous clinical trials exploring their utility for the treatment of a variety of diseases (e.g., acute graft-versus-host disease, Crohn’s disease, multiple sclerosis, osteoarthritis, and cardiovascular diseases including heart failure and myocardial infarction. On the other hand, conventionally cultured MSCs reflect heterogeneous populations that often contain contaminating cells due to the significant variability in isolation methods and the lack of specific MSC markers. This review article focuses on recent developments in the MSC research field, with a special emphasis on the identification of novel surface markers for the in vivo localization and prospective isolation of murine and human MSCs. Furthermore, we discuss the physiological importance of MSC subtypes in vivo with specific reference to data supporting their contribution to HSC niche homeostasis. The isolation of MSCs using selective markers (combination of PDGFRα and Sca-1 is crucial to address the many unanswered questions pertaining to these cells and has the potential to enhance their therapeutic potential enormously.

  13. Identification of clinical staphylococcal isolates from humans by internal transcribed spacer PCR.

    Science.gov (United States)

    Couto, I; Pereira, S; Miragaia, M; Sanches, I S; de Lencastre, H

    2001-09-01

    The emergence of coagulase-negative staphylococci not only as human pathogens but also as reservoirs of antibiotic resistance determinants requires the deployment and development of methods for their rapid and reliable identification. Internal transcribed spacer-PCR (ITS-PCR) was used to identify a collection of 617 clinical staphylococcal isolates. The amplicons were resolved in high-resolution agarose gels and visually compared with the patterns obtained for the control strains of 29 staphylococcal species. Of the 617 isolates studied, 592 (95.95%) were identified by ITS-PCR and included 11 species: 302 isolates of Staphylococcus epidermidis, 157 of S. haemolyticus, 79 of S. aureus, 21 of S. hominis, 14 of S. saprophyticus, 8 of S. warneri, 6 of S. simulans, 2 of S. lugdunensis, and 1 each of S. caprae, S. carnosus, and S. cohnii. All species analyzed had unique ITS-PCR patterns, although some were very similar, namely, the group S. saprophyticus, S. cohnii, S. gallinarum, S. xylosus, S. lentus, S. equorum, and S. chromogenes, the pair S. schleiferi and S. vitulus, and the pair S. piscifermentans and S. carnosus. Four species, S. aureus, S. caprae, S. haemolyticus, and S. lugdunensis, showed polymorphisms on their ITS-PCR patterns. ITS-PCR proved to be a valuable alternative for the identification of staphylococci, offering, within the same response time and at lower cost, higher reliability than the currently available commercial systems.

  14. Aerobic metabolism of human quadriceps muscle: in vivo data parallel measurements on isolated mitochondria

    DEFF Research Database (Denmark)

    Rasmussen, U.F.; Rasmussen, H.N.; Krustrup, Peter

    2001-01-01

    The aim of the present study was to examine whether parameters of isolated mitochondria could account for the in vivo maximum oxygen uptake ( O2 max) of human skeletal muscle. O2 max and work performance of the quadriceps muscle of six volunteers were measured in the knee extensor model (range 10......-18 mmol O2 · min 1 · kg 1 at work rates of 22-32 W/kg). Mitochondria were isolated from the same muscle at rest. Strong correlations were obtained between O2 max and a number of mitochondrial parameters (mitochondrial protein, cytochrome aa3, citrate synthase, and respiratory activities). The activities...... of citrate synthase, succinate dehydrogenase, and pyruvate dehydrogenase, measured in isolated mitochondria, corresponded to, respectively, 15, 3, and 1.1 times the rates calculated from O2 max. The respiratory chain activity also appeared sufficient. Fully coupled in vitro respiration, which is limited...

  15. Efficient Generation of Glucose-Responsive Beta Cells from Isolated GP2+ Human Pancreatic Progenitors

    Directory of Open Access Journals (Sweden)

    Jacqueline Ameri

    2017-04-01

    Full Text Available Stem cell-based therapy for type 1 diabetes would benefit from implementation of a cell purification step at the pancreatic endoderm stage. This would increase the safety of the final cell product, allow the establishment of an intermediate-stage stem cell bank, and provide a means for upscaling β cell manufacturing. Comparative gene expression analysis revealed glycoprotein 2 (GP2 as a specific cell surface marker for isolating pancreatic endoderm cells (PECs from differentiated hESCs and human fetal pancreas. Isolated GP2+ PECs efficiently differentiated into glucose responsive insulin-producing cells in vitro. We found that in vitro PEC proliferation declines due to enhanced expression of the cyclin-dependent kinase (CDK inhibitors CDKN1A and CDKN2A. However, we identified a time window when reducing CDKN1A or CDKN2A expression increased proliferation and yield of GP2+ PECs. Altogether, our results contribute tools and concepts toward the isolation and use of PECs as a source for the safe production of hPSC-derived β cells.

  16. Leptospira mayottensis sp. nov., a pathogenic species of the genus Leptospira isolated from humans.

    Science.gov (United States)

    Bourhy, Pascale; Collet, Louis; Brisse, Sylvain; Picardeau, Mathieu

    2014-12-01

    A group of strains representing species of the genus Leptospira, isolated from patients with leptospirosis in Mayotte (Indian Ocean), were previously found to be considerably divergent from other known species of the genus Leptospira. This was inferred from sequence analysis of rrs (16S rRNA) and other genetic loci and suggests that they belong to a novel species. Two strains from each serogroup currently identified within this novel species were studied. Spirochaete, aerobic, motile, helix-shaped strains grew well at 30-37 °C, but not at 13 °C or in the presence of 8-azaguanine. Draft genomes of the strains were also analysed to study the DNA relatedness with other species of the genus Leptospira. The new isolates formed a distinct clade, which was most closely related to Leptospira borgpetersenii, in multilocus sequence analysis using concatenated sequences of the genes rpoB, recA, fusA, gyrB, leuS and sucA. Analysis of average nucleotide identity and genome-to-genome distances, which have recently been proposed as reliable substitutes for classical DNA-DNA hybridization, further confirmed that these isolates should be classified as representatives of a novel species. The G+C content of the genomic DNA was 39.5 mol%. These isolates are considered to represent a novel species, for which the name Leptospira mayottensis sp. nov. is proposed, with 200901116(T) ( = CIP 110703(T) = DSM 28999(T)) as the type strain. © 2014 IUMS.

  17. Genetic diversity of Dirofilaria spp. isolated from subcutaneous and ocular lesions of human patients in Ukraine.

    Science.gov (United States)

    Rossi, Alice; Peix, Álvaro; Pavlikovskaya, Tamara; Sagach, Olga; Nikolaenko, Svetlana; Chizh, Nina; Kartashev, Vladimir; Simón, Fernando; Siles-Lucas, Mar

    2015-02-01

    This short communication describes the phylogenetic analysis of 48 Dirofilaria worms isolated from human patients in Ukraine. 102 cases were both of subcutaneous (47; 46.1%) and ocular (54; 52.9%) locations. Worms from 44 patients (15 subcutaneous and 29 ocular) were subjected to DNA extraction and amplification of a specific fragment of the 12S rRNA subunit, and sequences were used for phylogenetic analysis. Results showed that 13.8% of the ocular cases analyzed at molecular level were caused by Dirofilaria immitis. Very few cases of ocular human dirofilariosis due to D. immitis have been described in the literature to date, majority of them attributed to Dirofilaria repens. Our results show that ocular dirofilariosis cannot be excluded in areas of low endemicity for D. repens were D. immitis is also present. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Characterization of a 2016 Clinical Isolate of Zika Virus in Non-human Primates

    Directory of Open Access Journals (Sweden)

    Xiao-Feng Li

    2016-10-01

    Full Text Available Animal models are critical to understand disease and to develop countermeasures for the ongoing epidemics of Zika virus (ZIKV. Here we report a non-human primate model using a 2016 contemporary clinical isolate of ZIKV. Upon subcutaneous inoculation, rhesus macaques developed fever and viremia, with robust excretion of ZIKV RNA in urine, saliva, and lacrimal fluid. Necropsy of two infected animals revealed that systematic infections involving central nervous system and visceral organs were established at the acute phrase. ZIKV initially targeted the intestinal tracts, spleen, and parotid glands, and retained in spleen and lymph nodes till 10 days post infection. ZIKV-specific immune responses were readily induced in all inoculated animals. The non-human primate model described here provides a valuable platform to study ZIKV pathogenesis and to evaluate vaccine and therapeutics.

  19. Sensitive microculture method for isolation of human immunodeficiency virus type 1 from blood leukocytes.

    Science.gov (United States)

    Erice, A; Sannerud, K J; Leske, V L; Aeppli, D; Balfour, H H

    1992-02-01

    A study was conducted to compare our standard culture with a new microculture procedure for isolation of human immunodeficiency virus type 1 (HIV-1) from blood leukocytes. A total of 137 blood specimens from 102 HIV-1 antibody-positive individuals (52 were asymptomatic, 31 were symptomatic, and 19 had AIDS) were cultured in a microculture system in which 10(6) of the patients' peripheral blood mononuclear cells (PBMC) were cocultured with 10(6) phytohemagglutinin (PHA)-stimulated PBMC from an HIV-1 antibody-negative blood donor in 1.2 ml of culture medium. Results were compared with those of a historical control group of 139 standard HIV-1 cultures from 108 HIV-1 antibody-positive subjects (58 were asymptomatic, 36 were symptomatic, and 14 had AIDS). For standard cultures, 10 x 10(6) of the patients' PBMC were cocultured with 5 x 10(6) PHA-stimulated PBMC from an HIV-1 antibody-negative blood donor in 15 ml of culture medium. HIV-1 was isolated in 128 (93%) microcultures and 133 (96%) standard cultures. Both methods identified more than 75% of the positive cultures within 7 days and 100% of the positive cultures within 14 days. The isolation rates for HIV-1 in microcultures compared with standard cultures were 91 versus 93% (specimens from asymptomatic individuals), 93 versus 96% (specimens from symptomatic individuals), and 97 versus 100% (specimens from patients with AIDS). The median time to positivity for both culture methods was 7 days, and this correlated significantly with symptoms and CD4+ cell counts. The microculture method is a sensitive and less expensive system for isolation of HIV-1 from PBMC of HIV-1 antibody-positive individuals, and we recommend it as the culture method of choice, especially for children and patients with AIDS and severe anemia or leukopenia whose blood volume is an important consideration.

  20. Isolation and characterisation of new putative probiotic bacteria from human colonic flora.

    Science.gov (United States)

    Raz, Irit; Gollop, Natan; Polak-Charcon, Sylvie; Schwartz, Betty

    2007-04-01

    The present study describes a novel bacterial isolate exhibiting high ability to synthesise and secrete butyrate. The novel isolated bacterium was obtained from human faeces and grown in selective liquid intestinal microflora medium containing rumen fluid under microaerobic conditions. Its probiotic properties were demonstrated by the ability of the isolate to survive high acidity and medium containing bile acids and the ability to adhere to colon cancer cells (Caco-2) in vitro. Phylogenetic identity to Enterococcus durans was established using specific primers for 16S rRNA (99% probability). PCR analyses with primers to the bacterial gene encoding butyrate kinase, present in the butyrogenic bacteria Clostridium, showed that this gene is present in E. durans. The in vivo immunoprotective and anti-inflammatory effects of E. durans were assessed in dextran sodium sulfate (DSS)-induced colitis in Balb/c mice. Administration of E. durans ameliorated histological, clinical and biochemical scores directly related to intestinal inflammation whereas the lactic acid bacterium Lactobacillus delbrueckii was ineffective in this regard. Colonic cDNA concentrations of IL-1beta and TNF-alpha were significantly down regulated in DSS-treated E. durans-fed mice but not in control or DSS-treated L. delbrueckii- fed mice. Fluorescent in situ hybridisation analyses of colonic tissue from mice fed E. durans, using a butyrate kinase probe, demonstrated that E. durans significantly adheres to the colonic tissue. The novel isolated bacterium described in the present paper, upon further characterisation, can be developed into a useful probiotic aimed at the treatment of patients suffering from ulcerative colitis.

  1. Isolated corneal papilloma-like lesion associated with human papilloma virus type 6.

    Science.gov (United States)

    Park, Choul Yong; Kim, Eo-Jin; Choi, Jong Sun; Chuck, Roy S

    2011-05-01

    To report a case of a corneal papilloma-like lesion associated with human papilloma virus type 6. A 48-year-old woman presented with a 2-year history of ocular discomfort and gradual visual deterioration in her right eye. Ophthalmic examination revealed an elevated, semitranslucent, well-defined vascularized mass approximately 4 × 2.5 mm in size localized to the right cornea. The surface of the mass appeared smooth and many small, shallow, and irregular elevations were noted. An excisional biopsy was performed. The underlying cornea was markedly thinned, and fine ramifying vasculature was also noted on the exposed corneal stroma. Typical koilocytic change was observed on the histopathologic examination. Polymerase chain reaction revealed the existence of human papilloma virus type 6 DNA. Here we describe a case of an isolated corneal papilloma-like lesion. Although the corneal extension of the limbal or the conjunctival papillomas has been commonly observed, an isolated corneal papilloma-like lesion with underlying stromal destruction has only rarely been reported.

  2. Inhibition of isolated human myometrium contractility by minoxidil and reversal by glibenclamide.

    Science.gov (United States)

    Prabhakaran, S S; Dhanasekar, K R; Thomas, E; Jose, R; Peedicayil, J; Samuel, P

    2010-03-01

    This study investigated the ability of the antihypertensive drug minoxidil to inhibit potassium chloride (KCl)-induced contractility of the isolated human myometrium. Twelve strips of myometrium obtained from 12 patients who underwent hysterectomy were triggered to contract with 55 mM KCl before and after incubation with 3 concentrations (1, 3 and 10 microM) of minoxidil. The percent inhibition by minoxidil on the extent of contraction, and the area under the contractile curve of KCl-induced contraction of the myometrial strips was determined. Furthermore, the effect of 10 microM glibenclamide on the inhibition generated by 3 microM minoxidil on KCl-induced contractility was studied. It was found that minoxidil produced a concentration-dependent inhibition of KCl-induced contractility of the myometrium and that glibenclamide reversed this inhibitory effect. These results suggest that the inhibitory effect of minoxidil on isolated human myometrium contractility may prove useful in clinical conditions requiring relaxation of the myometrium. 2010 Prous Science, S.A.U. or its licensors. All rights reserved.

  3. Complete genome sequence of a commensal bacterium, Hafnia alvei CBA7124, isolated from human feces.

    Science.gov (United States)

    Song, Hye Seon; Kim, Joon Yong; Kim, Yeon Bee; Jeong, Myeong Seon; Kang, Jisu; Rhee, Jin-Kyu; Kwon, Joseph; Kim, Ju Suk; Choi, Jong-Soon; Choi, Hak-Jong; Nam, Young-Do; Roh, Seong Woon

    2017-01-01

    Members of the genus Hafnia have been isolated from the feces of mammals, birds, reptiles, and fish, as well as from soil, water, sewage, and foods. Hafnia alvei is an opportunistic pathogen that has been implicated in intestinal and extraintestinal infections in humans. However, its pathogenicity is still unclear. In this study, we isolated H. alvei from human feces and performed sequencing as well as comparative genomic analysis to better understand its pathogenicity. The genome of H. alvei CBA7124 comprised a single circular chromosome with 4,585,298 bp and a GC content of 48.8%. The genome contained 25 rRNA genes (9 5S rRNA genes, 8 16S rRNA genes, and 8 23S rRNA genes), 88 tRNA genes, and 4043 protein-coding genes. Using comparative genomic analysis, the genome of this strain was found to have 72 strain-specific singletons. The genome also contained genes for antibiotic and antimicrobial resistance, as well as toxin-antitoxin systems. We revealed the complete genome sequence of the opportunistic gut pathogen, H. alvei CBA7124. We also performed comparative genomic analysis of the sequences in the genome of H. alvei CBA7124, and found that it contained strain-specific singletons, antibiotic resistance genes, and toxin-antitoxin systems. These results could improve our understanding of the pathogenicity and the mechanism behind the antibiotic resistance of H. alvei strains.

  4. Isolation and Characterization of Human Mesenchymal Stem Cells From Facet Joints and Interspinous Ligaments.

    Science.gov (United States)

    Kristjánsson, Baldur; Limthongkul, Worawat; Yingsakmongkol, Wicharn; Thantiworasit, Pattarawat; Jirathanathornnukul, Napaphat; Honsawek, Sittisak

    2016-01-01

    A descriptive in vitro study on isolation and differentiation of human mesenchymal stem cells (MSCs) derived from the facet joints and interspinous ligaments. To isolate cells from the facet joints and interspinous ligaments and investigate their surface marker profile and differentiation potentials. Lumbar spinal canal stenosis and ossification of the posterior longitudinal ligament are progressive conditions characterized by the hypertrophy and ossification of ligaments and joints within the spinal canal. MSCs are believed to play a role in the advancement of these diseases and the existence of MSCs has been demonstrated within the ligamentum flavum and posterior longitudinal ligament. The aim of this study was to investigate whether these cells could also be found within facet joints and interspinous ligaments. Samples were harvested from 10 patients undergoing spinal surgery. The MSCs from facet joints and interspinous ligaments were isolated using direct tissue explant technique. Cell surface antigen profilings were performed via flow cytometry. Their lineage differentiation potentials were analyzed. The facet joints and interspinous ligaments-derived MSCs have the tri-lineage potential to be differentiated into osteogenic, adipogenic, and chondrogenic cells under appropriate inductions. Flow cytometry analysis revealed both cell lines expressed MSCs markers. Both facet joints and interspinous ligaments-derived MSCs expressed marker genes for osteoblasts, adipocytes, and chondrocytes. The facet joints and interspinous ligaments may provide alternative sources of MSCs for tissue engineering applications. The facet joints and interspinous ligaments-derived MSCs are part of the microenvironment of the human ligaments of the spinal column and might play a crucial role in the development and progression of degenerative spine conditions.

  5. Specific binding of lactoferrin to Escherichia coli isolated from human intestinal infections

    International Nuclear Information System (INIS)

    Naidu, S.S.; Erdei, J.; Forsgren, A.; Naidu, A.S.; Czirok, E.; Gado, I.; Kalfas, S.; Thoren, A.

    1991-01-01

    The degrees of human lactoferrin (HLf) and bovine lactoferrin (BLf) binding in 169 Escherichia coli strains isolated from human intestinal infections, and in an additional 68 strains isolated from healthy individuals, were examined in a 125 I-labelled protein binding assay. The binding was expressed as a percentage calculated from the total labelled ligand added to bacteria. The HLf and BLf binding to E. coli was in the range 3.7 to 73.4% and 4.8 to 61.6%, respectively. Enterotoxigenic strains demonstrated a significantly higher HLf binding (median = 19%) than enteropathogenic, enteroinvasive, enterohaemorrhagic strains or normal intestinal E. coli isolates (medians 6 to 9). Enteropathogenic strains belonging to serotypes O44 and O127 demonstrated significantly higher HLf binding compared to O26, O55, O111, O119 and O126. No significant differences in the degree of HLf or BLf binding were found between aerobactin-producing and non-producing strains. The interaction was further characterized in a high Lf-binging EPEC strain, E34663 (serotype O127). The binding was stable in the pH range 4.0 to 7.5, did not dissociate in the presence of 2M NaCl or 2M urea, and reached saturation within two h. Unlabelled HLf and BLf displaced the 125 I-HLf binding to E34663 in a dose-dependent manner. Apo- and iron-saturated forms of Lf demonstrated similar binding to E34663. Among various unlabelled subephithelial matrix proteins and carbohydrates tested (in 10 4 -fold excess) only fibronectin and fibrinogen caused a moderate inhibition of 125 I-HLf binding. According to Scatchard plot analysis, 5,400 HLf-binding sites/cell, with an affinity constant (K a ) of 1.4 x 10 -7 M, were estimated in strain E34663. These data establish the presence of a specific Lf-binding mechanism in E. coli. (au)

  6. Antimicrobial susceptibilities of Listeria monocytogenes human strains isolated from 1970 to 2008 in Brazil

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    Cristhiane Moura Falavina dos Reis

    2011-04-01

    Full Text Available INTRODUCTION: Listeria monocytogenes is the causative agent of listeriosis, a foodborne illness that affects mainly pregnant women, the elderly and immunocompromised patients. The primary treatment is a combination of ampicillin with an aminoglycoside, in addition to a second-choice drug represented by chloramphenicol, erythromycin, tetracycline and rifampicin. The aim of this study was to analyze the antimicrobial susceptibility profile of strains isolated from human sources in the last four decades. METHODS: Sixty-eight strains were selected from the culture collection of the Laboratory of Bacterial Zoonoses/LABZOO/FIOCRUZ isolated in different regions of Brazil from 1970 to 2008 and primarily isolated from cerebrospinal fluid and blood culture. Susceptibility tests to antimicrobials drugs were evaluated using the criteria established by Soussy using the Kirby-Bauer method and E-Test strips were used to determine the minimum inhibitory concentration (MIC. RESULTS: Among the strains tested, serovar L4b (60.3% was the most prevalent, followed by serovar 1/2a (20.6%, 1/2b (13.2% and the more uncommon serovars 1/2c, 3b and 4ab (5.9%. All strains were susceptible to ampicillin, cephalothin, erythromycin, gentamicin, teicoplanin and vancomycin. Only one strain (1.5% showed resistance to rifampin, and two (3% were resistant to trimethoprim-sulfamethoxazole. MICs with values up to 2μg/ml reinforce the need for microbiological surveillance. CONCLUSIONS: The study demonstrated low prevalence of strains resistant to the antimicrobial drugs indicated in the treatment of human listeriosis. Monitoring antimicrobial resistance profile is still very important to determine adequate treatment, especially in immunocompromised patients.

  7. Prospectively Isolated Human Bone Marrow Cell-Derived MSCs Support Primitive Human CD34-Negative Hematopoietic Stem Cells.

    Science.gov (United States)

    Matsuoka, Yoshikazu; Nakatsuka, Ryusuke; Sumide, Keisuke; Kawamura, Hiroshi; Takahashi, Masaya; Fujioka, Tatsuya; Uemura, Yasushi; Asano, Hiroaki; Sasaki, Yutaka; Inoue, Masami; Ogawa, Hiroyasu; Takahashi, Takayuki; Hino, Masayuki; Sonoda, Yoshiaki

    2015-05-01

    Hematopoietic stem cells (HSCs) are maintained in a specialized bone marrow (BM) niche, which consists of osteoblasts, endothelial cells, and a variety of mesenchymal stem/stromal cells (MSCs). However, precisely what types of MSCs support human HSCs in the BM remain to be elucidated because of their heterogeneity. In this study, we succeeded in prospectively isolating/establishing three types of MSCs from human BM-derived lineage- and CD45-negative cells, according to their cell surface expression of CD271 and stage-specific embryonic antigen (SSEA)-4. Among them, the MSCs established from the Lineage(-) CD45(-) CD271(+) SSEA-4(+) fraction (DP MSC) could differentiate into osteoblasts and chondrocytes, but they lacked adipogenic differentiation potential. The DP MSCs expressed significantly higher levels of well-characterized HSC-supportive genes, including IGF-2, Wnt3a, Jagged1, TGFβ3, nestin, CXCL12, and Foxc1, compared with other MSCs. Interestingly, these osteo-chondrogenic DP MSCs possessed the ability to support cord blood-derived primitive human CD34-negative severe combined immunodeficiency-repopulating cells. The HSC-supportive actions of DP MSCs were partially carried out by soluble factors, including IGF-2, Wnt3a, and Jagged1. Moreover, contact between DP MSCs and CD34-positive (CD34(+) ) as well as CD34-negative (CD34(-) ) HSCs was important for the support/maintenance of the CD34(+/-) HSCs in vitro. These data suggest that DP MSCs might play an important role in the maintenance of human primitive HSCs in the BM niche. Therefore, the establishment of DP MSCs provides a new tool for the elucidation of the human HSC/niche interaction in vitro as well as in vivo. © 2014 AlphaMed Press.

  8. Avian influenza A (H9N2: computational molecular analysis and phylogenetic characterization of viral surface proteins isolated between 1997 and 2009 from the human population

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    Idrees Muhammad

    2010-11-01

    Full Text Available Abstract Background H9N2 avian influenza A viruses have become panzootic in Eurasia over the last decade and have caused several human infections in Asia since 1998. To study their evolution and zoonotic potential, we conducted an in silico analysis of H9N2 viruses that have infected humans between 1997 and 2009 and identified potential novel reassortments. Results A total of 22 hemagglutinin (HA and neuraminidase (NA nucleotide and deduced amino acid sequences were retrieved from the NCBI flu database. It was identified that mature peptide sequences of HA genes isolated from humans in 2009 had glutamine at position 226 (H3 of the receptor binding site, indicating a preference to bind to the human α (2-6 sialic acid receptors, which is different from previously isolated viruses and studies where the presence of leucine at the same position contributes to preference for human receptors and presence of glutamine towards avian receptors. Similarly, strains isolated in 2009 possessed new motif R-S-N-R in spite of typical R-S-S-R at the cleavage site of HA, which isn't reported before for H9N2 cases in humans. Other changes involved loss, addition, and variations in potential glycosylation sites as well as in predicted epitopes. The results of phylogenetic analysis indicated that HA and NA gene segments of H9N2 including those from current and proposed vaccine strains belong to two different Eurasian phylogenetic lineages confirming possible genetic reassortments. Conclusions These findings support the continuous evolution of avian H9N2 viruses towards human as host and are in favor of effective surveillance and better characterization studies to address this issue.

  9. Isolation of human anti-serum albumin Fab antibodies with an extended serum-half life.

    Science.gov (United States)

    Kang, Hyeon-Ju; Kim, Hye-Jin; Cha, Sang-Hoon

    2016-01-01

    The serum albumin (SA) has been exploited to generate long-acting biotherapeutics by taking advantage of the FcRn-mediated recycling mechanism in a direct or an indirect way. Since Fab fragments have been proven to be clinically safe for human usage, we assumed that human anti-SA Fab antibodies could have a great potential as a carrier molecule to extend the serum half-life of therapeutic proteins. We, herein, had attempted to isolate anti-SA Fab antibodies from HuDVFab-8L antibody library via a phage display technology, and identified eight discrete human Fab antibodies. One of the Fab antibodies, SL335, showed the strongest binding reactivity to human SA with nM range of affinity at both pH 6 and pH 7.4, and cross-reacted to SAs from various species including rat, mouse, canine and monkey. The in vivo pharmacokinetic assay using a rat model indicated that SL335 has approximately 10 fold longer serum half-life and 26 to 44-fold increase in AUC0 → ∞ compared to the negative control Fab molecule in both intravenous and subcutaneous administrations. Knowing that Fabs have proven to be safe in clinics for a long time, SL335 seems to have a great potential in generating long-acting protein drugs by tagging effector molecules with either chemical conjugation or genetic fusion. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Expert judgement on inadvertent human intrusion into the Waste Isolation Pilot Plant

    International Nuclear Information System (INIS)

    Hora, S.C.; von Winterfeldt, D.; Trauth, K.M.

    1991-12-01

    Four expert-judgment teams have developed analyses delineating possible future societies in the next 10,000 years in the vicinity of the Waste Isolation Pilot Plant (WIPP). Expert-judgment analysis was used to address the question of future societies because neither experimentation, observation, nor modeling can resolve such uncertainties. Each of the four, four-member teams, comprised of individuals with expertise in the physical, social, or political sciences, developed detailed qualitative assessments of possible future societies. These assessments include detailed discussions of the underlying physical and societal factors that would influence society and the likely modes of human-intrusion at the WIPP, as well as the probabilities of intrusion. Technological development, population growth, economic development, conservation of information, persistence of government control, and mitigation of danger from nuclear waste were the factors the teams believed to be most important. Likely modes of human-intrusion were categorized as excavation, disposal/storage, tunneling, drilling, and offsite activities. Each team also developed quantitative assessments by providing probabilities of various alternative futures, of inadvertent human intrusion, and in some cases, of particular modes of intrusion. The information created throughout this study will be used in conjunction with other types of information, including experimental data, calculations from physical principles and computer models, and perhaps other judgments, as input to ''performance assessment.'' The more qualitative results of this study will be used as input to another expert panel considering markers to deter inadvertent human intrusion at the WIPP

  11. Human osteoblast cells: isolation, characterization, and growth on polymers for musculoskeletal tissue engineering.

    Science.gov (United States)

    El-Amin, Saadiq F; Botchwey, Edward; Tuli, Richard; Kofron, Michelle D; Mesfin, Addisu; Sethuraman, Swaminathan; Tuan, Rocky S; Laurencin, Cato T

    2006-03-01

    We performed a detailed examination of the isolation, characterization, and growth of human osteoblast cells derived from trabecular bone. We further examined the morphology, phenotypic gene expression, mineralization,and growth of these human osteoblasts on polyester polymers used for musculoskeletal tissue engineering. Polylactic-co-glycolic acid [PLAGA (85:15, 50:50, 75:25)], and poly-lactic acid (L-PLA, D,L-PLA) were examined. The osteoblastic expression of key phenotypic markers osteocalcin, alkaline phosphatase, collagen, and bone sialoprotein at 4 and 8 weeks was examined. Reverse transcription-polymerase chain reaction studies revealed that trabecular-derived osteoblasts were positive for all markers evaluated with higher levels expressed over long-term culture. These cells also revealed mineralization and maturation as evidenced by energy dispersive X-ray analysis and scanning electron microscopy. Growth studies on PLAGA at 50:50,75:25, and 85:15 ratios and PLA in the L and DL isoforms revealed that human osteoblasts actively grew, with significantly higher cell numbers attached to scaffolds composed of PLAGA 50:50 in the short term and PLAGA 85:15 in the long term compared with PLA (p < 0.05). We believe human cell adhesion among these polymeric materials may be dependent on differences in cellular integrin expression and extracellular matrix protein elaboration. (c) 2005 Wiley Periodicals, Inc.

  12. Dihydrochalcone Compounds Isolated from Crabapple Leaves Showed Anticancer Effects on Human Cancer Cell Lines

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    Xiaoxiao Qin

    2015-11-01

    Full Text Available Seven dihydrochalcone compounds were isolated from the leaves of Malus crabapples, cv. “Radiant”, and their chemical structures were elucidated by UV, IR, ESI-MS, 1H-NMR and 13C-NMR analyses. These compounds, which include trilobatin (A1, phloretin (A2, 3-hydroxyphloretin (A3, phloretin rutinoside (A4, phlorizin (A5, 6′′-O-coumaroyl-4′-O-glucopyranosylphloretin (A6, and 3′′′-methoxy-6′′-O-feruloy-4′-O-glucopyranosyl-phloretin (A7, all belong to the phloretin class and its derivatives. Compounds A6 and A7 are two new rare dihydrochalcone compounds. The results of a MTT cancer cell growth inhibition assay demonstrated that phloretin and these derivatives showed significant positive anticancer activities against several human cancer cell lines, including the A549 human lung cancer cell line, Bel 7402 liver cancer cell line, HepG2 human ileocecal cancer cell line, and HT-29 human colon cancer cell line. A7 had significant effects on all cancer cell lines, suggesting potential applications for phloretin and its derivatives. Adding a methoxyl group to phloretin dramatically increases phloretin’s anticancer activity.

  13. Various methods for isolation of multipotent human periodontal ligament cells for regenerative medicine.

    Science.gov (United States)

    Tran, Ha Le Bao; Doan, Vu Nguyen; Le, Huong Thi Ngoc; Ngo, Lan Thi Quynh

    2014-08-01

    Periodontal ligament (PDL) is a specialized connective tissue that connects cementum and alveolar bone to maintain and support the teeth in situ and preserve tissue homeostasis. Recent studies have revealed the existence of stem cells in human dental tissues including periodontal ligament that play an important role, not only in the maintenance of the periodontium but also in promoting periodontal regeneration. In this study, human periodontal ligament cells (hPDLCs) were isolated by outgrowth and enzymatic dissociation methods. Expression of surface markers on PDLCs as human mesenchymal stem cells (MSCs) was identified by flow cytometry. In addition, proliferation and differentiation capacity of cultured cells to osteoblasts, adipocytes were evaluated. As a result, we successfully cultured cells from the human periodontal ligament tissues. PDLCs express mesenchymal stem cell (MSC) markers such as CD44, CD73, and CD90 and do not express CD34, CD45, and HLA-DR. PDLCs also possess the multipotential to differentiate into various types of cells, such as osteoblast and adipocytes, in vitro. Therefore, these cells have high potential to serve as materials for tissue engineering, especially dental tissue engineering.

  14. Effect of cAMP on short-circuit current in isolated human ciliary body.

    Science.gov (United States)

    Wu, Ren-yi; Ma, Ning; Hu, Qian-qian

    2013-07-01

    Cyclic adenosine monophosphate (cAMP) could activate chloride channels in bovine ciliary body and trigger an increase in the ionic current (short-circuit current, Isc) across the ciliary processes in pigs. The purpose of this study was to investigate how cAMP modulates Isc in isolated human ciliary processes and the possible involvement of chloride transport across the tissue in cAMP-induced Isc change. In an Ussing-type chamber system, the Isc changes induced by the cAMP analogue 8-bromo-cAMP and an adenylyl cyclase activator forskolin in isolated human ciliary processes were assessed. The involvement of Cl(-) component in the bath solution was investigated. The effect of Cl(-) channel (10 µmol/L niflumic acid and 1 mmol/L 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)), K(+) channel (10 mmol/L tetraethylammonium chloride (TEA)), or Na(+) channel blockers (1 mmol/L amiloride) on 8-bromo-cAMP-induced Isc change was also studied. Dose-dependently, 8-bromo-cAMP (10 nmol/L-30 µmol/L) or forskolin (10 nmol/L-3 µmol/L) increased Isc across the ciliary processes with an increase in negative potential difference on the non-pigmented epithelium (NPE) side of the tissue. Isc increase induced by 8-bromo-cAMP was more pronounced when the drug was applied on the NPE side than on the pigmented epithelium side. When the tissue was bathed in low Cl(-) solutions, the Isc increase was significantly inhibited. Finally, niflumic acid and DIDS, but not TEA or amiloride, significantly prevented the Isc increase induced by 8-bromo-cAMP. cAMP stimulates stroma-to-aqueous anionic transport in isolated human ciliary processes. Chloride is likely to be among the ions, the transportation of which across the tissue is triggered by cAMP, suggesting the potential role of cAMP in the process of aqueous humor formation in human eyes.

  15. [Isolation and identification of brain tumor stem cells from human brain neuroepithelial tumors].

    Science.gov (United States)

    Fang, Jia-sheng; Deng, Yong-wen; Li, Ming-chu; Chen, Feng-Hua; Wang, Yan-jin; Lu, Ming; Fang, Fang; Wu, Jun; Yang, Zhuan-yi; Zhou, Xang-yang; Wang, Fei; Chen, Cheng

    2007-01-30

    To establish a simplified culture system for the isolation of brain tumor stem cells (BTSCs) from the tumors of human neuroepithelial tissue, to observe the growth and differentiation pattern of BTSCs, and to investigate their expression of the specific markers. Twenty-six patients with brain neuroepithelial tumors underwent tumor resection. Two pieces of tumor tissues were taken from each tumor to be dissociated, triturated into single cells in sterile DMEM-F12 medium, and then filtered. The tumor cells were seeded at a concentration of 200,000 viable cells per mL into serum-free DMEM-F12 medium simply supplemented with B27, human basic fibroblast growth factor (20 microg/L), human epidermal growth factor (20 microg /L), insulin (4 U/L), L-glutamine, penicillin and streptomycin. After the primary brain tumor spheres (BTSs) were generated, they were triturated again and passed in fresh medium. Limiting dilution assay was performed to observe the monoclone formation. 5-bromodeoxyuridine (BrdU) incorporation test was performed to observe the proliferation of the BTS. The BTSCs were cultured in mitogen-free DMEM-F12 medium supplemented with 10% fetal bovine serum to observe their differentiation. Immunocytochemistry was used to examine the expression of CD133 and nestin, specific markers of BTSC, and the rate of CD133 positive cells. Only a minority of subsets of cells from the tumors of neuroepithelial tissue had the capacity to survive, proliferate, and generate free-floating neurosphere-like BTSs in the simplified serum-free medium. These cells attached to the poly-L-lysine coated coverslips in the serum-supplemented medium and differentiated. The BTSCs were CD133 and nestin positive. The rate of CD133 positive cells in the tumor specimens was (21 +/- 6.2)% - (38 +/- 7.0)%. A new simplified culture system for the isolation of BTSCs is established. The tumors of human neuroepithelial tissue contain CD133 and nestin positive tumor stem cells which can be isolated

  16. Age-dependent Fourier model of the shape of the isolated ex vivo human crystalline lens.

    Science.gov (United States)

    Urs, Raksha; Ho, Arthur; Manns, Fabrice; Parel, Jean-Marie

    2010-06-01

    To develop an age-dependent mathematical model of the zero-order shape of the isolated ex vivo human crystalline lens, using one mathematical function, that can be subsequently used to facilitate the development of other models for specific purposes such as optical modeling and analytical and numerical modeling of the lens. Profiles of whole isolated human lenses (n=30) aged 20-69, were measured from shadow-photogrammetric images. The profiles were fit to a 10th-order Fourier series consisting of cosine functions in polar-co-ordinate system that included terms for tilt and decentration. The profiles were corrected using these terms and processed in two ways. In the first, each lens was fit to a 10th-order Fourier series to obtain thickness and diameter, while in the second, all lenses were simultaneously fit to a Fourier series equation that explicitly include linear terms for age to develop an age-dependent mathematical model for the whole lens shape. Thickness and diameter obtained from Fourier series fits exhibited high correlation with manual measurements made from shadow-photogrammetric images. The root-mean-squared-error of the age-dependent fit was 205 microm. The age-dependent equations provide a reliable lens model for ages 20-60 years. The contour of the whole human crystalline lens can be modeled with a Fourier series. Shape obtained from the age-dependent model described in this paper can be used to facilitate the development of other models for specific purposes such as optical modeling and analytical and numerical modeling of the lens. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  17. Multispacer typing of Rickettsia isolates from humans and ticks in Tunisia revealing new genotypes.

    Science.gov (United States)

    Znazen, Abir; Khrouf, Fatma; Elleuch, Nihel; Lahiani, Dorra; Marrekchi, Chakib; M'Ghirbi, Youmna; Ben Jemaa, Mounir; Bouattour, Ali; Hammami, Adnene

    2013-12-31

    Rickettsioses are important remerging vector born infections. In Tunisia, many species have been described in humans and vectors. Genotyping is important for tracking pathogen movement between hosts and vectors. In this study, we characterized Rickettsia species detected in patients and vectors using multispacer typing (MST), proposed by Founier et al. and based on three intergenic spacers (dksA-xerC, rmpE- tRNA(fMet), mppA-pruC) sequencing. Our study included 25 patients hospitalized during 2009. Ticks and fleas were collected in the vicinity of confirmed cases. Serology was performed on serum samples by microimmunofluorescence using Rickettsia conorii and Rickettsia typhi antigens. To detect and identify Rickettsia species, PCR targeting ompA, ompB and gltA genes followed by sequencing was performed on 18 obtained skin biopsies and on all collected vectors. Rickettsia positive samples were further characterized using primers targeting three intergenic spacers (dksA-xerC, rmpE- tRNA(fMet) and mppA-purC). A rickettsial infection was confirmed in 15 cases (60%). Serology was positive in 13 cases (52%). PCR detected Rickettsia DNA in four biopsies (16%) allowing the identification of R. conorii subsp israelensis in three cases and R. conorii subsp conorii in one case. Among 380 collected ticks, nine presented positive PCR (2.4%) allowing the identification of six R. conorii subsp israelensis, two R. massiliae and one R. conorii subsp conorii. Among 322 collected fleas, only one was positive for R. felis. R. conorii subsp israelensis strains detected in humans and vectors clustered together and showed a new MST genotype. Similarly, R. conorii subsp conorii strains detected in a skin biopsy and a tick were genetically related and presented a new MST genotype. New Rickettsia spotted fever strain genotypes were found in Tunisia. Isolates detected in humans and vectors were genetically homogenous despite location differences in their original isolation suggesting

  18. Molecular characterisation of Sporothrix schenckii isolates from humans and cats involved in the sporotrichosis epidemic in Rio de Janeiro, Brazil

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    Rosani Santos Reis

    2009-08-01

    Full Text Available An epidemic of sporotrichosis, a subcutaneous mycosis caused by the fungus Sporothrix schenckii, is ongoing in Rio de Janeiro, Brazil, in which cases of human infection are related to exposure to cats. In an attempt to demonstrate the zoonotic character of this epidemic using molecular methodology, we characterised by DNA-based typing methods 19 human and 25 animal S. schenckii isolates from the epidemic, as well as two control strains. To analyse the isolates, the random amplified polymorphic DNA (RAPD technique was performed using three different primers, together with DNA fingerprinting using the minisatellite derived from the wild-type phage M13 core-sequence. The analyses generated amplicons with considerable polymorphism. Although isolates exhibited high levels of genetic relatedness, they could be clustered into 5-10 genotypes. The RAPD profiles of epidemic S. schenckii isolates could be distinguished from that of the United States isolate, displaying 20% similarity to each primer and 60% when amplified with the M13 primer. DNA fingerprinting of S. schenckii isolated from the nails (42.8% and the oral cavities (66% of cats were identical to related human samples, suggesting that there is a common infection source for animals and humans in this epidemic. It is clear that cats act as a vehicle for dissemination of S. schenckii.

  19. Molecular characterization and antimicrobial resistance of faecal and urinary Escherichia coli isolated from dogs and humans in Italy

    Directory of Open Access Journals (Sweden)

    Clara Tramuta

    2014-03-01

    Full Text Available During this study, 109 faecal Escherichia coli samples isolated from 61 dogs and 48 humans were characterised according to phylogenetic group, extraintestinal virulence factors and antibiotic resistance. The isolates from dogs were predominantly distributed within phylogroup B1 (36%, while the majority of human strains belonged to phylogroup B2 (54%. The prevalence of cnf1, hlyA, papC and sfa virulence genes was significantly associated with the group B2. Canine isolates showed multidrug resistance (MDR more frequently than human strains. Since group B2 contains most of the strains that cause extraintestinal infections, all 46 B2 faecal strains were confronted against an addition population of 57 urinary E. coli strains belonging to the same phylogroup. The comparison shows that there was no significant difference in the occurrence of virulence factors or in the distribution of antibiotic resistance between faecal and urinary E. coli isolates fromd dogs. At the same time, a highly significant association was detected between multiple resistence and the source of the strains and between MDR and E. coli isolated from urine in human. This study highlighted similar features of E. coli isolated across sources and hosts. The data suggest a high prevalence of antibiotic resistance in faecal strains, which may represent a serious health risk since these strains can function as a reservoir for uropathogenic E. coli.

  20. The use of protective barriers to deter inadvertent human intrusion into a mined geologic facility for the disposal of radioactive waste: A review of previous investigations and potential concepts

    International Nuclear Information System (INIS)

    Tolan, T.L.

    1993-06-01

    Sandia National Laboratories is evaluating the feasibility of developing protective barrier system for the Waste Isolation Pilot Plant (WIPP) to thwart inadvertent human intrusion into this radioactive-waste disposal system for a period of 9,900 years after assumed loss of active institutional controls. The protective barrier system would be part of a series of enduring passive institutional controls whose long-term function will be to reduce the likelihood of inadvertent human activities (e.g., exploratory drilling for resources) that could disrupt the WIPP disposal system

  1. Laparoscopy After Previous Laparotomy

    Directory of Open Access Journals (Sweden)

    Zulfo Godinjak

    2006-11-01

    Full Text Available Following the abdominal surgery, extensive adhesions often occur and they can cause difficulties during laparoscopic operations. However, previous laparotomy is not considered to be a contraindication for laparoscopy. The aim of this study is to present that an insertion of Veres needle in the region of umbilicus is a safe method for creating a pneumoperitoneum for laparoscopic operations after previous laparotomy. In the last three years, we have performed 144 laparoscopic operations in patients that previously underwent one or two laparotomies. Pathology of digestive system, genital organs, Cesarean Section or abdominal war injuries were the most common causes of previouslaparotomy. During those operations or during entering into abdominal cavity we have not experienced any complications, while in 7 patients we performed conversion to laparotomy following the diagnostic laparoscopy. In all patients an insertion of Veres needle and trocar insertion in the umbilical region was performed, namely a technique of closed laparoscopy. Not even in one patient adhesions in the region of umbilicus were found, and no abdominal organs were injured.

  2. Isolation and characterization of lactobacilli from human faeces and indigenous fermented foods for their potential application as probiotics.

    Science.gov (United States)

    Mandal, Hemanti; Jariwala, Ruchi; Bagchi, Tamishraha

    2016-04-01

    This study was conducted to select Lactobacillus strains from various sources on the basis of their probiotic attributes, such as acid and bile tolerance, binding to intestinal cells, and antimicrobial activity. Twelve isolates were obtained from human and food sources and were evaluated against standard probiotic Lactobacillus rhamnosus GG (LGG). Isolates were also studied for their antibiotic susceptibility. Isolate Lactobacillus fermentum GPI-6 showed the best survival profile at 0.3% and 1% bile salt, as compared with LGG. Isolates Lactobacillus plantarum GRI-2 and Lactobacillus salivarius GPI-4 showed no reduction in survival rate at pH 2.5. As expected, isolates showed strain-specific differences when comparing various attributes. Isolates GPI-4, GPI-7, and FA-5 showed better adhesion to HT-29, while isolate GPI-4 adhered better to Caco-2 cells than did LGG. However, when studying their ability to compete with Escherichia coli O26:H11, isolates GPI-6 and GPI-7 significantly inhibited E. coli adhesion to both HT-29 and Caco-2 cells compared with LGG. In conclusion, isolates GPI-4, GPI-7, and FA-5 showed excellent binding ability and antagonistic activity and better tolerance to acidic pH (pH 2.5) and to different bile salt concentrations in comparison with LGG, and hence, they could be considered as potential probiotic candidates.

  3. Gene editing and clonal isolation of human induced pluripotent stem cells using CRISPR/Cas9.

    Science.gov (United States)

    Yumlu, Saniye; Stumm, Jürgen; Bashir, Sanum; Dreyer, Anne-Kathrin; Lisowski, Pawel; Danner, Eric; Kühn, Ralf

    2017-05-15

    Human induced pluripotent stem cells (hiPSCs) represent an ideal in vitro platform to study human genetics and biology. The recent advent of programmable nucleases makes also the human genome amenable to experimental genetics through either the correction of mutations in patient-derived iPSC lines or the de novo introduction of mutations into otherwise healthy iPSCs. The production of specific and sometimes complex genotypes in multiple cell lines requires efficient and streamlined gene editing technologies. In this article we provide protocols for gene editing in hiPSCs. We presently achieve high rates of gene editing at up to three loci using a modified iCRISPR system. This system includes a doxycycline inducible Cas9 and sgRNA/reporter plasmids for the enrichment of transfected cells by fluorescence-activated cell sorting (FACS). Here we cover the selection of target sites, vector construction, transfection, and isolation and genotyping of modified hiPSC clones. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Effects of polysaccharide isolated from Streptococcus thermophilus CRL1190 on human gastric epithelial cells.

    Science.gov (United States)

    Marcial, Guillermo; Messing, Jutta; Menchicchi, Bianca; Goycoolea, Francisco M; Faller, Gerhard; Graciela, Font de Valdez; Hensel, Andreas

    2013-11-01

    EPS1190 was isolated from skim milk fermented with Stretococcus thermophilus CRL1190. The polysaccharide consisted of 33% glucose and 66% galactose with 1,4- and 1,4,6-galactose residues as main building blocks beside a high amount of 1,4-linked glucose. The polymer was characterized additionally concerning viscosity and zeta potential. EPS1190 stimulated cellular vitality and proliferation of human stomach AGS cells and human buccal KB cells significantly. EPS1190 stimulated phagocytosis rate of murine macrophages RAW264.7 significantly. NO-release or anti-inflammatory effects by inhibition of LPS-induced NO release were not observed. Confocal laser scanning microscopy revealed that EPS1190 is partially internalized into AGS cells via endosomes. The bioadhesive absorption of FITC-labeled EPS1190 into the mucus layer on the apical side of the epithelium using histological tissue sections from human stomach was observed. Specific interaction of EPS1190 with mucin can be excluded as shown by microviscosimetry studies. EPS1190 increased the adhesion of H. pylori to AGS cells, which resulted in increased secretion of proinflammatory cytokines TNFa, IL-6 and IL-8. Summarizing, EPS1190 seems to stimulate epithelial cell regeneration and immunological innate defense mechanisms, which again can rationalized the use of this polysaccharide as cytoprotective compound in probiotioc preparations. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Isolation and characterization of the human parathyroid hormone-like peptide gene

    International Nuclear Information System (INIS)

    Mangin, M.; Ikeda, K.; Dreyer, B.E.; Broadus, A.E.

    1989-01-01

    A parathyroid hormone-like peptide (PTH-LP) has recently been identified in human tumors associated with the syndrome of humoral hypercalcemia of malignancy. The peptide appears to be encoded by a single-copy gene that gives rise to multiple mRNAs that are heterogeneous at both their 5' and their 3' ends. Alternative RNA splicing is responsible for the 3' heterogeneity and results in mRNAs encoding three different peptides, each with a unique C terminus. The authors have isolated and characterized the human PTHLP gene. The gene is a complex transcriptional unit spanning more than 12 kilobases of DNA and containing six exons. Two 5' exons encode distinct 5' untranslated regions and are separated by a putative promoter element, indicating that the gene either has two promoters or is alternatively spliced from a single promoter upstream of the first exon. The middle portion of the PTHLP gene, comprising exons 2-4, has an organizational pattern of introns and exons identical to that of the parathyroid hormone gene, consistent with a common ancestral origin of these two genes. Exon 4 of the PTHLP gene encodes the region common to all three peptides and the C terminus of the shortest peptide, and exons 5 and 6 encode the unique C termini of the other two peptides. Northern analysis of mRNAs from four human tumors of different histological types reveals the preferential use of 3' splicing patterns of individual tumors

  6. Cells isolated from human periapical cysts express mesenchymal stem cell-like properties.

    Science.gov (United States)

    Marrelli, Massimo; Paduano, Francesco; Tatullo, Marco

    2013-01-01

    We provide a detailed description of mesenchymal stem cells (MSCs) isolated from human periapical cysts, which we have termed hPCy-MSCs. These cells have a fibroblast-like shape and adhere to tissue culture plastic surfaces. hPCy-MSCs possess high proliferative potential and self-renewal capacity properties. We characterised the immunophenotype of hPCy-MSCs (CD73(+), CD90(+), CD105(+), CD13(+), CD29(+), CD44(+), CD45(-), STRO-1(+), CD146(+)) by flow cytometry and immunofluorescence. hPCy-MSCs possess the potential to differentiate into osteoblast- and adipocyte-like cells in vitro. Multi-potentiality was evaluated with culture-specific staining and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis for osteo/odontogenic and adipogenic markers. This is the first report to indicate that human periapical cysts contain cells with MSC-like properties. Taken together, our findings indicate that human periapical cysts could be a rich source of MSCs.

  7. Revised description and classification of atypical isolates of Pasteurella multocida from bovine lungs based on genotypic characterization to include variants previously classified as biovar 2 of Pasteurella canis and Pasteurella avium

    DEFF Research Database (Denmark)

    Christensen, Henrik; Angen, Øystein; Olsen, John E.

    2004-01-01

    Strains deviating in key phenotypic characters, mainly isolated from cases of bovine pneumonia in five European countries, were genotyped in order to examine their genotypic relationship with Pasteurella multocida. Twenty-two strains of Pasteurella avium biovar 2, including variants in indole, xy...

  8. Orthogonal typing methods identify genetic diversity among Belgian Campylobacter jejuni strains isolated over a decade from poultry and cases of sporadic human illness.

    Science.gov (United States)

    Elhadidy, Mohamed; Arguello, Hector; Álvarez-Ordóñez, Avelino; Miller, William G; Duarte, Alexandra; Martiny, Delphine; Hallin, Marie; Vandenberg, Olivier; Dierick, Katelijne; Botteldoorn, Nadine

    2018-06-20

    Campylobacter jejuni is a zoonotic pathogen commonly associated with human gastroenteritis. Retail poultry meat is a major food-related transmission source of C. jejuni to humans. The present study investigated the genetic diversity, clonal relationship, and strain risk-analysis of 403 representative C. jejuni isolates from chicken broilers (n = 204) and sporadic cases of human diarrhea (n = 199) over a decade (2006-2015) in Belgium, using multilocus sequence typing (MLST), PCR binary typing (P-BIT), and identification of lipooligosaccharide (LOS) biosynthesis locus classes. A total of 123 distinct sequence types (STs), clustered in 28 clonal complexes (CCs) were assigned, including ten novel sequence types that were not previously documented in the international database. Sequence types ST-48, ST-21, ST-50, ST-45, ST-464, ST-2274, ST-572, ST-19, ST-257 and ST-42 were the most prevalent. Clonal complex 21 was the main clonal complex in isolates from humans and chickens. Among observed STs, a total of 35 STs that represent 72.2% (291/403) of the isolates were identified in both chicken and human isolates confirming considerable epidemiological relatedness; these 35 STs also clustered together in the most prevalent CCs. A majority of the isolates harbored sialylated LOS loci associated with potential neuropathic outcomes in humans. Although the concordance between MLST and P-BIT, determined by the adjusted Rand and Wallace coefficients, showed low congruence between both typing methods. The discriminatory power of P-BIT and MLST was similar, with Simpson's diversity indexes of 0.978 and 0.975, respectively. Furthermore, P-BIT could provide additional epidemiological information that would provide further insights regarding the potential association to human health from each strain. In addition, certain clones could be linked to specific clinical symptoms. Indeed, LOS class E was associated with less severe infections. Moreover, ST-572 was significantly

  9. Differences in virulence genes and genome patterns of mastitis-associated Staphylococcus aureus among goat, cow, and human isolates in Taiwan.

    Science.gov (United States)

    Chu, Chishih; Wei, Yajiun; Chuang, Shih-Te; Yu, Changyou; Changchien, Chih-Hsuan; Su, Yaochi

    2013-03-01

    A total of 117 mastitis-associated Staphylococcus aureus isolates from cow, goat, and human patients were analyzed for differences in antibiotic susceptibility, virulence genes, and genotypes using accessory gene regulator (agr) typing, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Multidrug-resistant (MDR) S. aureus were commonly found in all sources, though they were predominantly found in human and goat isolates. Almost 70% of the isolates were resistant to ampicillin and penicillin. Host-associated virulence genes were identified as follows: tst, a gene encoding toxic shock syndrome toxin, was found in goat isolates; lukED and lukM, genes encoding leukocidin, found in cow isolates; lukPV, a gene encoding leukocidin, found in human isolates; and eta, a gene encoding for exfoliative toxin, found in both human and cow isolates. All four types of hemolysin, α, β, γ, and δ, were identified in human isolates, three types (α, γ, and δ), were identified in cow isolates, and two types (α and δ) were identified in goat isolates. Agr-typing determined agr1 to be the main subtype in human and cow isolates. PFGE and MLST analysis revealed the presence of diverse genotypes among the three sources. In conclusion, mastitis-associated, genetically diverse strains of MDR S. aureus differed in virulence genes among human, cow, and goat isolates.

  10. CDC25A Protein Stability Represents a Previously Unrecognized Target of HER2 Signaling in Human Breast Cancer: Implication for a Potential Clinical Relevance in Trastuzumab Treatment

    Directory of Open Access Journals (Sweden)

    Emanuela Brunetto

    2013-06-01

    Full Text Available The CDC25A-CDK2 pathway has been proposed as critical for the oncogenic action of human epidermal growth factor receptor 2 (HER2 in mammary epithelial cells. In particular, transgenic expression of CDC25A cooperates with HER2 in promoting mammary tumors, whereas CDC25A hemizygous loss attenuates the HER2-induced tumorigenesis penetrance. On the basis of this evidence of a synergism between HER2 and the cell cycle regulator CDC25A in a mouse model of mammary tumorigenesis, we investigated the role of CDC25A in human HER2-positive breast cancer and its possible implications in therapeutic response. HER2 status and CDC25A expression were assessed in 313 breast cancer patients and we found statistically significant correlation between HER2 and CDC25A (P = .007. Moreover, an HER2-positive breast cancer subgroup with high levels of CDC25A and very aggressive phenotype was identified (P = .005. Importantly, our in vitro studies on breast cancer cell lines showed that the HER2 inhibitor efficacy on cell growth and viability relied also on CDC25A expression and that such inhibition induces CDC25A down-regulation through phosphatidylinositol 3-kinase/protein kinase B pathway and DNA damage response activation. In line with this observation, we found a statistical significant association between CDC25A overexpression and trastuzumab-combined therapy response rate in two different HER2-positive cohorts of trastuzumab-treated patients in either metastatic or neoadjuvant setting (P = .018 for the metastatic cohort and P = .021 for the neoadjuvant cohort. Our findings highlight a link between HER2 and CDC25A that positively modulates HER2- targeted therapy response, suggesting that, in HER2-positive breast cancer patients, CDC25A overexpression affects trastuzumab sensitivity.

  11. Characteristics of Quinolone Resistance in Escherichia coli Isolates from Humans, Animals, and the Environment in the Czech Republic

    Science.gov (United States)

    Röderova, Magdalena; Halova, Dana; Papousek, Ivo; Dolejska, Monika; Masarikova, Martina; Hanulik, Vojtech; Pudova, Vendula; Broz, Petr; Htoutou-Sedlakova, Miroslava; Sauer, Pavel; Bardon, Jan; Cizek, Alois; Kolar, Milan; Literak, Ivan

    2017-01-01

    Escherichia coli is a common commensal bacterial species of humans and animals that may become a troublesome pathogen causing serious diseases. The aim of this study was to characterize the quinolone resistance phenotypes and genotypes in E. coli isolates of different origin from one area of the Czech Republic. E. coli isolates were obtained from hospitalized patients and outpatients, chicken farms, retailed turkeys, rooks wintering in the area, and wastewaters. Susceptibility of the isolates grown on the MacConkey agar with ciprofloxacin (0.05 mg/L) to 23 antimicrobial agents was determined. The presence of plasmid-mediated quinolone resistance (PMQR) and ESBL genes was tested by PCR and sequencing. Specific mutations in gyrA, gyrB, parC, and parE were also examined. Multilocus sequence typing and pulsed-field gel electrophoresis were performed to assess the clonal relationship. In total, 1050 E. coli isolates were obtained, including 303 isolates from humans, 156 from chickens, 105 from turkeys, 114 from the rooks, and 372 from wastewater samples. PMQR genes were detected in 262 (25%) isolates. The highest occurrence was observed in isolates from retailed turkey (49% of the isolates were positive) and inpatients (32%). The qnrS1 gene was the most common PMQR determinant identified in 146 (56%) followed by aac(6′)-Ib-cr in 77 (29%), qnrB19 in 41 (16%), and qnrB1 in 9 (3%) isolates. All isolates with high level of ciprofloxacin resistance (>32 mg/L) carried double or triple mutations in gyrA combined with single or double mutations in parC. The most frequently identified substitutions were Ser(83)Leu; Asp(87)Asn in GyrA, together with Ser(80)Ile, or Glu(84)Val in ParC. Majority of these isolates showed resistance to beta-lactams and multiresistance phenotype was found in 95% isolates. Forty-eight different sequence types among 144 isolates analyzed were found, including five major clones ST131 (26), ST355 (19), ST48 (13), ST95 (10), and ST10 (5). No isolates

  12. Formation of harmful compounds in biotransformation of lilial by microorganisms isolated from human skin.

    Science.gov (United States)

    Esmaeili, Akbar; Afshari, Shima; Esmaeili, Davood

    2015-01-01

    The biotransformation of lilial results in an acid that is used in the dairy industry, in perfumery, as an intermediate in the manufacture of pharmaceuticals and cosmetics, and as a food additive for enhancing taste. This study investigates the biotransformation of lilial by Staphylococcus aureus and Staphylococcus epidermidis, two bacterial species isolated from human skin. Both species of Staphylococcus were isolated in samples taken from the skin of individuals living in a rural area of Iran. The pH of the culture medium was optimized, and after culturing the microorganisms, the bacteria were added to a flask containing a nutrient broth and incubated for several hours. The flasks of bacteria were combined with lilial, and various biochemical tests and diagnostics were performed, including Fourier transform infrared spectroscopy (FT-IR), ultraviolet-visible spectrophotometry (UV-Vis), and gas chromatography-mass spectroscopy (GC-MS). The S. aureus produced isobutyric acid (2-methylpropanoic acid) after 72 h (71% of the total products yielded during biotransformation), whereas the S. epidermidis produced terpenoid alcoholic media after 24 h (90% of total products obtained). The results obtained indicate that biotransformation of lilial by S. aureus is more desirable than by S. epidermidis due to the highly efficient production of a single product. Bourgeonal and liliol were two toxic compounds produced during biotransformation, which indicates that the use of lilial in cosmetics can be harmful to the skin.

  13. Isolation of uv-sensitive variants of human FL cells by a viral suicide method

    International Nuclear Information System (INIS)

    Shiomi, T.; Sato, K.

    1979-01-01

    A new method (viral suicide method) for the isolation of uv-sensitive mutants is described. Colonies of mutagenized human FL cells were infected with uv-irradiated Herpes simplex viruses and surviving ones which seemed to be deficient in host cell reactivation (HCR) were examined for their uv sensitivity. Nineteen of 238 clones examined were sensitive to uv irradiation at the time of the isolation. After recloning, four of these clones have been studied and two (UVS-1 and UVS-2) of them are stable in their uv sensitivity for 4 months in culture. uv sensitivity of UVS-1, UVS-2, and the parental FL cells are as follows: the extrapolation numbers (n) are 2.2, 2.1, and 1.8 and mean lethal doses (DO) are 2.9, 3.7, and 7.8 J/m 2 for UVS-1, UVS-2, and the parental FL cells, respectively. They are no more sensitive than FL cells to x-irradiation. The ability of HCR in UVS-2 cells is apparently lower than that in FL cells, whereas UVS-1 cells are the same as FL cells in the ability

  14. Chromatographic Monoliths for High-Throughput Immunoaffinity Isolation of Transferrin from Human Plasma

    Directory of Open Access Journals (Sweden)

    Irena Trbojević-Akmačić

    2016-06-01

    Full Text Available Changes in protein glycosylation are related to different diseases and have a potential as diagnostic and prognostic disease biomarkers. Transferrin (Tf glycosylation changes are common marker for congenital disorders of glycosylation. However, biological interindividual variability of Tf N-glycosylation and genes involved in glycosylation regulation are not known. Therefore, high-throughput Tf isolation method and large scale glycosylation studies are needed in order to address these questions. Due to their unique chromatographic properties, the use of chromatographic monoliths enables very fast analysis cycle, thus significantly increasing sample preparation throughput. Here, we are describing characterization of novel immunoaffinity-based monolithic columns in a 96-well plate format for specific high-throughput purification of human Tf from blood plasma. We optimized the isolation and glycan preparation procedure for subsequent ultra performance liquid chromatography (UPLC analysis of Tf N-glycosylation and managed to increase the sensitivity for approximately three times compared to initial experimental conditions, with very good reproducibility. This work is licensed under a Creative Commons Attribution 4.0 International License.

  15. Isolation of human salivary extracellular vesicles by iodixanol density gradient ultracentrifugation and their characterizations

    Directory of Open Access Journals (Sweden)

    Kazuya Iwai

    2016-05-01

    Full Text Available Diagnostic methods that focus on the extracellular vesicles (EVs present in saliva have been attracting great attention because of their non-invasiveness. EVs contain biomolecules such as proteins, messenger RNA (mRNA and microRNA (miRNA, which originate from cells that release EVs, making them an ideal source for liquid biopsy. Although there have been many reports on density-based fractionation of EVs from blood and urine, the number of reports on EVs from saliva has been limited, most probably because of the difficulties in separating EVs from viscous saliva using density gradient centrifugation. This article establishes a protocol for the isolation of EVs from human saliva using density gradient centrifugation. The fractionated salivary EVs were characterized by atomic force microscopy, western blot and reverse transcription polymerase chain reaction. The results indicate that salivary EVs have a smaller diameter (47.8±12.3 nm and higher density (1.11 g/ml than EVs isolated from conditioned cell media (74.0±23.5 nm and 1.06 g/ml, respectively. Additionally, to improve the throughput of density-based fractionation of EVs, the original protocol was further modified by using a fixed angle rotor instead of a swinging rotor. It was also confirmed that several miRNAs were expressed strongly in the EV-marker-expressing fractions.

  16. Characterization of antibiotic resistant enterococci isolated from untreated waters for human consumption in Portugal.

    Science.gov (United States)

    Macedo, Ana S; Freitas, Ana R; Abreu, Cristina; Machado, Elisabete; Peixe, Luísa; Sousa, João C; Novais, Carla

    2011-01-31

    Untreated drinking water is frequently overlooked as a source of antibiotic resistance in developed countries. To gain further insight on this topic, we isolated the indicator bacteria Enterococcus spp. from water samples collected in wells, fountains and natural springs supplying different communities across Portugal, and characterized their antibiotic resistance profile with both phenotypic and genetic approaches. We found various rates of resistance to seven antibiotic families. Over 50% of the isolates were resistant to at least ciprofloxacin, tetracyclines or quinupristin-dalfopristin and 57% were multidrug resistant to ≥3 antibiotics from different families. Multiple enterococcal species (E. faecalis, E. faecium, E. hirae, E. casseliflavus and other Enterococcus spp) from different water samples harbored genes encoding resistance to tetracyclines, erythromycin or gentamicin [tet(M)-46%, tet(L)-14%, tet(S)-5%, erm(B)-22%, aac(6´)-Ie-aph(2″)-12%] and putative virulence factors [gel-28%, asa1-16%]. The present study positions untreated drinking water within the spectrum of ecological niches that may be reservoirs of or vehicles for antibiotic resistant enterococci/genes. These findings are worthy of attention as spread of antibiotic resistant enterococci to humans and animals through water ingestion cannot be dismissed. Copyright © 2010 Elsevier B.V. All rights reserved.

  17. (-)-Epicatechin-induced relaxation of isolated human saphenous vein: Roles of K+ and Ca2+ channels.

    Science.gov (United States)

    Marinko, Marija; Jankovic, Goran; Nenezic, Dragoslav; Milojevic, Predrag; Stojanovic, Ivan; Kanjuh, Vladimir; Novakovic, Aleksandra

    2018-02-01

    In this study, we aimed to investigate relaxant effect of flavanol (-)-epicatechin on the isolated human saphenous vein (HSV), as a part of its cardioprotective action, and to define the mechanisms underlying this vasorelaxation. (-)-Epicatechin induced a concentration-dependent relaxation of HSV pre-contracted by phenylephrine. Among K + channel blockers, 4-aminopyridine, margatoxin, and iberiotoxin significantly inhibited relaxation of HSV, while glibenclamide considerably reduced effects of the high concentrations of (-)-epicatechin. Additionally, (-)-epicatechin relaxed contraction induced by 80 mM K + , whereas in the presence of nifedipine produced partial relaxation of HSV rings pre-contracted by phenylephrine. In Ca 2+ -free solution, (-)-epicatechin relaxed contraction induced by phenylephrine, but had no effect on contraction induced by caffeine. A sarcoplasmic reticulum Ca 2+ -ATPase inhibitor, thapsigargin, significantly reduced relaxation of HSV produced by (-)-epicatechin. These results demonstrate that (-)-epicatechin produces endothelium-independent relaxation of isolated HSV rings. Vasorelaxation to (-)-epicatechin probably involves activation of 4-aminopyridine- and margatoxin-sensitive K V channels, BK Ca channels, and at least partly, K ATP channels. In addition, not only the inhibition of extracellular Ca 2+ influx, but regulation of the intracellular Ca 2+ release, via inositol-trisphosphate receptors and reuptake into sarcoplasmic reticulum, via stimulation of Ca 2+ -ATPase, as well, most likely participate in (-)-epicatechin-induced relaxation of HSV. Copyright © 2017 John Wiley & Sons, Ltd.

  18. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

    Directory of Open Access Journals (Sweden)

    Tal Noy-Porat

    2016-03-01

    Full Text Available Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1 that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication.

  19. Complete Genome Sequence of Lactobacillus salivarius CECT 5713, a Probiotic Strain Isolated from Human Milk and Infant Feces▿

    Science.gov (United States)

    Jiménez, Esther; Martín, Rocío; Maldonado, Antonio; Martín, Virginia; Gómez de Segura, Aranzazu; Fernández, Leonides; Rodríguez, Juan M.

    2010-01-01

    Lactobacillus salivarius is a homofermentative lactic acid bacterium and is frequently isolated from mucosal surfaces of healthy humans. L. salivarius CECT 5713, a strain isolated simultaneously from breast milk and infant feces of a healthy mother-infant pair, has immunomodulatory, anti-inflammatory, and anti-infectious properties, as revealed by several in vitro and in vivo assays. Here, we report its complete and annotated genome sequence. PMID:20675488

  20. Prevalence, identification by a DNA microarray-based assay of human and food isolates Listeria spp. from Tunisia.

    Science.gov (United States)

    Hmaïed, F; Helel, S; Le Berre, V; François, J-M; Leclercq, A; Lecuit, M; Smaoui, H; Kechrid, A; Boudabous, A; Barkallah, I

    2014-02-01

    We aimed at evaluating the prevalence of Listeria species isolated from food samples and characterizing food and human cases isolates. Between 2005 and 2007, one hundred food samples collected in the markets of Tunis were analysed in our study. Five strains of Listeria monocytogenes responsible for human listeriosis isolated in hospital of Tunis were included. Multiplex PCR serogrouping and pulsed field gel electrophoresis (PFGE) applying the enzyme AscI and ApaI were used for the characterization of isolates of L. monocytogenes. We have developed a rapid microarray-based assay to a reliable discrimination of species within the Listeria genus. The prevalence of Listeria spp. in food samples was estimated at 14% by using classical biochemical identification. Two samples were assigned to L. monocytogenes and 12 to L. innocua. DNA microarray allowed unambiguous identification of Listeria species. Our results obtained by microarray-based assay were in accordance with the biochemical identification. The two food L. monocytogenes isolates were assigned to the PCR serogroup IIa (serovar 1/2a). Whereas human L. monocytogenes isolates were of PCR serogroup IVb, (serovars 4b). These isolates present a high similarity in PFGE. Food L. monocytogenes isolates were classified into two different pulsotypes. These pulsotypes were different from that of the five strains responsible for the human cases. We confirmed the presence of Listeria spp. in variety of food samples in Tunis. Increased food and clinical surveillance must be taken into consideration in Tunisia to identify putative infections sources. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  1. Isolation of Osteosarcoma-Associated Human Antibodies from a Combinatorial Fab Phage Display Library

    Directory of Open Access Journals (Sweden)

    Carmela Dantas-Barbosa

    2009-01-01

    Full Text Available Osteosarcoma, a highly malignant disease, is the most common primary bone tumor and is frequently found in children and adolescents. In order to isolate antibodies against osteosarcoma antigens, a combinatorial osteosarcoma Fab library displayed on the surface of phages was used. After three rounds of selection on the surface of tumor cells, several osteosarcoma-reactive Fabs were detected. From these Fabs, five were better characterized, and despite having differences in their VH (heavy chain variable domain and Vκ (kappa chain variable domain regions, they all bound to a protein with the same molecular mass. Further analysis by cell ELISA and immunocytochemistry suggested that the Fabs recognize a membrane-associated tumor antigen expressed in higher amounts in neoplasic cells than in normal tissue. These results suggest that the human Fabs selected in this work are a valuable tool for the study of this neoplasia.

  2. Human adipose-derived stromal/stem cell isolation, culture, and osteogenic differentiation.

    Science.gov (United States)

    Qureshi, Ammar T; Chen, Cong; Shah, Forum; Thomas-Porch, Caasy; Gimble, Jeffrey M; Hayes, Daniel J

    2014-01-01

    Annually, more than 200,000 elective liposuction procedures are performed in the United States and over a million worldwide. The ease of harvest and abundance make human adipose-derived stromal/stem cells (hASCs) isolated from lipoaspirates an attractive, readily available source of adult stem cells that have become increasingly popular for use in many studies. Here, we describe common methods for hASC culture, preservation, and osteogenic differentiation. We introduce methods of ceramic, polymer, and composite scaffold synthesis with a description of morphological, chemical, and mechanical characterization techniques. Techniques for scaffold loading are compared, and methods for determining cell loading efficiency and proliferation are described. Finally, we provide both qualitative and quantitative techniques for in vitro assessment of hASC osteogenic differentiation. © 2014 Elsevier Inc. All rights reserved.

  3. Monovalent cations transfer through isolated human amnion: a new pharmacological model

    Energy Technology Data Exchange (ETDEWEB)

    Bara, M.; Guiet-Bara, A.; Durlach, J.

    1985-04-01

    Transfer of monovalent cations through the isolated human amnion consists of different factors: paracellular, coupling, ATPase dependent cellular transfer, leak cellular transfer. Understanding this transfer permits testing of the action of various substances. Physiological substances (Mg, taurine) increase ionic transfer and there is a vicarious effect between Mg and taurine. The tocolytic agents MgSO/sub 4/ and ethanol do not exhibit a good effect on the transfer: decrease with ethanol; equality between entry and exit fluxes with MgSO/sub 4/. On the other hand, amphotericin B increases mother-to-fetus transfer. Polluting metals (Pb, Cd, Hg, As) dramatically reduce exchanges and almost completely inhibit amnion permeability. Ingestion of ethanol also exhibits a dramatic effect on the exchange between mother and fetus through the amnion. Study of ionic transfer in vitro can be considered a pharmacological model to investigate the modifications of mother-fetus exchanges by various substances.

  4. GAWK, a novel human pituitary polypeptide: isolation, immunocytochemical localization and complete amino acid sequence.

    Science.gov (United States)

    Benjannet, S; Leduc, R; Lazure, C; Seidah, N G; Marcinkiewicz, M; Chrétien, M

    1985-01-16

    During the course of reverse-phase high pressure liquid chromatography (RP-HPLC) purification of a postulated big ACTH (1) from human pituitary gland extracts, a highly purified peptide bearing no resemblance to any known polypeptide was isolated. The complete sequence of this 74 amino acid polypeptide, called GAWK, has been determined. Search on a computer data bank on the possible homology to any known protein or fragment, using a mutation data matrix, failed to reveal any homology greater than 30%. An antibody produced against a synthetic fragment allowed us to detect several immunoreactive forms. The antisera also enabled us to localize the polypeptide, by immunocytochemistry, in the anterior lobe of the pituitary gland.

  5. Isolation and characterization of the human CDX1 gene: A candidate gene for diastrophic dysplasia

    Energy Technology Data Exchange (ETDEWEB)

    Bonner, C.; Loftus, S.; Wasmuth, J.J. [Univ. of California, Irvine, CA (United States)

    1994-09-01

    Diastrophic dysplasia is an autosomal recessive disorder characterized by short stature, dislocation of the joints, spinal deformities and malformation of the hands and feet. Multipoint linkage analysis places the diastrophic dysplasia (DTD) locus in 5q31-5q34. Linkage disequilibrium mapping places the DTD locus near CSFIR in the direction of PDGFRB (which is tandem to CSFIR). This same study tentatively placed PDGFRB and DTD proximal to CSFIR. Our results, as well as recently reported work from other laboratories, suggest that PDGFRB (and possibly DTD) is distal rather than proximal to CSFIR. We have constructed a cosmid contig covering approximately 200 kb of the region containing CSFIR. Several exons have been {open_quotes}trapped{close_quotes} from these cosmids using exon amplification. One of these exons was trapped from a cosmid isolated from a walk from PDGFRB, approximately 80 kb from CSFIR. This exon was sequenced and was determined to be 89% identical to the nucleotide sequence of exon two of the murine CDX1 gene (100% amino acid identity). The exon was used to isolate the human CDX gene. Sequence analysis of the human CDX1 gene indicates a very high degree of homology to the murine gene. CDX1 is a caudal type homeobox gene expressed during gastrulation. In the mouse, expression during gastrulation begins in the primitive streak and subsequently localizes to the ectodermal and mesodermal cells of the primitive streak, neural tube, somites, and limb buds. Later in gastrulation, CDX1 expression becomes most prominent in the mesoderm of the forelimbs, and, to a lesser extent, the hindlimbs. CDX1 is an intriguing candidate gene for diastrophic dysplasia. We are currently screening DNA from affected individuals and hope to shortly determine whether CDX1 is involved in this disorder.

  6. Molecular characterization of Echinococcus granulosus isolates from Bulgarian human cystic echinococcosis patients.

    Science.gov (United States)

    Marinova, Irina; Spiliotis, Markus; Wang, Junhua; Muhtarov, Marin; Chaligiannis, Ilias; Sotiraki, Smaro; Rainova, Iskra; Gottstein, Bruno; Boubaker, Ghalia

    2017-03-01

    Although cystic echinococcosis (CE) is highly endemic in Bulgaria, there is still scarce information about species and/or genotypes of the Echinococcus granulosus complex that infect humans. Our study tackled the genetic diversity of E. granulosus complex in a cohort of 30 Bulgarian CE patients. Ten animal E. granulosus isolates from neighboring Greece were additionally included. Specimens were comparatively analyzed for partial sequences of five mitochondrial (mt) (cox I, nad I, rrnS, rrnL, and atp6) and three nuclear (nc) genes (act II, hbx 2, and ef-1α) using a PCR-sequencing approach. All 30 Bulgarian isolates were identified as E. granulosus sensu stricto (s.s.) and were showing identical sequences for each of the three examined partial nc gene markers. Based upon concatenated sequences from partial mtDNA markers, we detected 10 haplotypes: 6 haplotypes (H1-H6) clustering with E. granulosus s.s. (G1) and 4 haplotypes (H9-H13) grouping with E. granulosus s.s. (G3), with H1 and H10 being the most frequent in Bulgarian patients. The haplotypes H1, H4, and H11 were also present in Greek hydatid cyst samples of animal origin. In conclusion, E. granulosus s.s. (G1 and G3 genotypes) is the only causative agent found so far to cause human CE in Bulgaria. However, further studies including larger sample sizes and other additional geographic regions in Bulgaria will have to be performed to confirm our results.

  7. Using organotypic (raft) epithelial tissue cultures for the biosynthesis and isolation of infectious human papillomaviruses.

    Science.gov (United States)

    Ozbun, Michelle A; Patterson, Nicole A

    2014-08-01

    Papillomaviruses have a strict tropism for epithelial cells, and they are fully reliant on cellular differentiation for completion of their life cycles, resulting in the production of progeny virions. Thus, a permissive environment for full viral replication in vitro-wherein virion morphogenesis occurs under cooperative viral and cellular cues-requires the cultivation of epithelium. Presented in the first section of this unit is a protocol to grow differentiating epithelial tissues that mimic many important morphological and biochemical aspects of normal skin. The technique involves growing epidermal cells atop a dermal equivalent consisting of live fibroblasts and a collagen lattice. Epithelial stratification and differentiation ensues when the keratinocyte-dermal equivalent is placed at the air-liquid interface. The apparent floating nature of the cell-matrix in this method led to the nickname "raft" cultures. The general technique can be applied to normal low passage keratinocytes, to cells stably transfected with papillomavirus genes or genomes, or keratinocytes established from neoplastic lesions. However, infectious papillomavirus particles have only been isolated from organotypic epithelial cultures initiated with cells that maintain oncogenic human papillomavirus genomes in an extrachomosomal replicative form. The second section of this unit is dedicated to a virion isolation method that minimizes aerosol and skin exposure to these human carcinogens. Although the focus of the protocols is on the growth of tissues that yields infectious papillomavirus progeny, this culture system facilitates the investigation of these fastidious viruses during their complex replicative cycles, and raft tissues can be manipulated and harvested at any point during the process. Importantly, a single-step virus growth cycle is achieved in this process, as it is unlikely that progeny virions are released to initiate subsequent rounds of infection. Copyright © 2014 John Wiley

  8. Assessment and characterization of biofilm formation among human isolates of Streptococcus dysgalactiae subsp. equisimilis.

    Science.gov (United States)

    Genteluci, Gabrielle Limeira; Silva, Ligia Guedes; Souza, Maria Clara; Glatthardt, Thaís; de Mattos, Marcos Corrêa; Ejzemberg, Regina; Alviano, Celuta Sales; Figueiredo, Agnes Marie Sá; Ferreira-Carvalho, Bernadete Teixeira

    2015-12-01

    The capacity to form biofilm is considered a protective mechanism that allows the bacteria to survive and proliferate in hostile environments, facilitating the maintenance of the infectious process. Recently, biofilm has become a topic of interest in the study of the human pathogen group A Streptococcus (GAS). Although GAS has not been associated with infection on medical implants, the presence of microcolonies embedded in an extracellular matrix on infected tissues has been reported. Despite the similarity between GAS and Streptococcus dysgalactiae subspecies equisimilis (SDSE), there are no studies in the literature describing the production of biofilm by SDSE. In this work, we assessed and characterized biofilm development among SDSE human isolates of group C. The in vitro data showed that 59.3% of the 118 isolates tested were able to form acid-induced biofilm on glass, and 28% formed it on polystyrene surfaces. More importantly, biofilm was also formed in a foreign body model in mice. The biofilm structure was analyzed by confocal laser scanning microscopy, transmission electron microscopy, and scanning electron microscopy. Long fibrillar-like structures were observed by scanning electron microscopy. Additionally, the expression of a pilus associated gene of SDSE was increased for in vitro sessile cells compared with planktonics, and when sessile cells were collected from biofilms formed in the animal model compared with that of in vitro model. Results obtained from the immunofluorescence microscopy indicated the biofilm was immunogenic. Our data also suggested a role for proteins, exopolysaccharide and extracellular DNA in the formation and accumulation of biofilm by SDSE. Copyright © 2015 Elsevier GmbH. All rights reserved.

  9. Assessing the relative importance of isolated Ficus trees to insectivorous birds in an Indian human-modified tropical landscape

    DEFF Research Database (Denmark)

    Matthews, Thomas J.; Cottee-Jones, H. Eden W.; Bregman, Tom P.

    2017-01-01

    The destruction of forest for agricultural expansion has created a vast estate of human-modified land in tropical regions. One group of organisms that are particularly vulnerable to the loss of forest habitat are insectivorous birds. Despite this, few conservation strategies have been identified...... for this group in human-modified landscapes. We survey the use of 104 isolated trees by insectivorous birds in rural Assam, India. We used an information theoretic model comparison approach to determine the important variables driving insectivorous bird diversity within these isolated trees. Our work...... demonstrates that the conservation of large trees in human-modified landscapes may play an important role in maintaining bird diversity and ecological function beyond the forest edge. More specifically, we found that isolated Ficus trees hold assemblages with particularly high insectivore abundance, richness...

  10. Molecular and Morphological Characterizations of Echinococcus granulosus from Human and Animal Isolates in Kashan, Markazi Province, Iran

    Science.gov (United States)

    ARBABI, Mohsen; PIRESTANI, Majid; DELAVARI, Mahdi; HOOSHYAR, Hossein; ABDOLI, Amir; SARVI, Shahab

    2017-01-01

    Background: One of the most important zoonotic helminths in the world is known as Echinococcus granulosus. Different strains of the E. granulosus have been described based on morphological and molecular characterizations, however, there is limited information regarding the characteristics of the phenotypes and genotypes of E. granulosus in Iran. Methods: The present study was prepared to evaluate the phenotypic and genotypic diversity of E. granulosus isolates collected from human, goat, sheep, and cattle based on 19 standard morphometric parameters and mitochondrial and nuclear genes (CO1, ND1, and ITS1) in Kashan, Markazi Province, Iran during 2013–2014. Results: The biometric analysis for the 19 characters revealed that the 19 morphometric values of cattle isolates were exceptionally higher than human, goat, and sheep isolates (Pgranulosus travels between humans and other intermediate hosts of this parasite in the area study. PMID:28761477

  11. Detection of vitamin D binding protein on the surface of cytotrophoblasts isolated from human placentae

    International Nuclear Information System (INIS)

    Nestler, J.E.; McLeod, J.F.; Kowalski, M.A.; Strauss, J.F. III; Haddad, J.G. Jr.

    1987-01-01

    Vitamin D binding protein (DBP), a Mr 56,000-58,000 alpha 2-glycoprotein, is the major serum protein involved in the transport of vitamin D sterols. Recently it has been suggested that DBP may also be involved in immunoglobulin G binding to cells. Because the trophoblast is involved in the transport of molecules such as vitamin D and immunoglobulin G to the fetus, we asked whether DBP could be detected on the surface of human placental trophoblast cells. Cytotrophoblasts purified from human term placentae were fixed and made permeant with Triton X-100 and examined by indirect immunofluorescence after incubation with a monoclonal antibody to DBP. Greater than 90% of these cells stained positively, whereas no staining was observed with nonimmune antiserum. The presence of DBP on/in the surface of cytotrophoblasts could also be demonstrated by fluorescent cytometry. When cell surface-associated proteins of cytotrophoblasts were radioiodinated, a Mr 57,000 radiolabeled protein could be immunoisolated from the cell lysate with a purified monospecific polyclonal antibody to DBP. Immunoisolation of this radiolabeled protein was prevented by the addition of excess unlabeled human DBP to the cell lysate before incubation with antibody. This Mr 57,000 radiolabeled protein could also be isolated by affinity chromatography selecting for proteins that bind to globular actin. When cytotrophoblasts were incubated with [ 35 S]methionine for 3 or 18 h, active synthesis of DBP could not be demonstrated by immunoisolation techniques. These studies demonstrate the presence of DBP on the surface of well washed, human cytotrophoblasts. This DBP may be maternally derived, since active synthesis of DBP could not be demonstrated

  12. Conceptual aspects: analyses law, ethical, human, technical, social factors of development ICT, e-learning and intercultural development in different countries setting out the previous new theoretical model and preliminary findings

    NARCIS (Netherlands)

    Kommers, Petrus A.M.; Smyrnova-Trybulska, Eugenia; Morze, Natalia; Issa, Tomayess; Issa, Theodora

    2015-01-01

    This paper, prepared by an international team of authors focuses on the conceptual aspects: analyses law, ethical, human, technical, social factors of ICT development, e-learning and intercultural development in different countries, setting out the previous and new theoretical model and preliminary

  13. Application of Pulsed-Field Gel Electrophoresis and Binary Typing as Tools in Veterinary Clinical Microbiology and Molecular Epidemiologic Analysis of Bovine and Human Staphylococcus aureus Isolates

    Science.gov (United States)

    Zadoks, Ruth; van Leeuwen, Willem; Barkema, Herman; Sampimon, Otlis; Verbrugh, Henri; Schukken, Ynte Hein; van Belkum, Alex

    2000-01-01

    Thirty-eight bovine mammary Staphylococcus aureus isolates from diverse clinical, temporal, and geographical origins were genotyped by pulsed-field gel electrophoresis (PFGE) after SmaI digestion of prokaryotic DNA and by means of binary typing using 15 strain-specific DNA probes. Seven pulsed-field types and four subtypes were identified, as were 16 binary types. Concordant delineation of genetic relatedness was documented by both techniques, yet based on practical and epidemiological considerations, binary typing was the preferable method. Genotypes of bovine isolates were compared to 55 previously characterized human S. aureus isolates through cluster analysis of binary types. Genetic clusters containing strains of both human and bovine origin were found, but bacterial genotypes were predominantly associated with a single host species. Binary typing proved an excellent tool for comparison of S. aureus strains, including methicillin-resistant S. aureus, derived from different host species and from different databases. For 28 bovine S. aureus isolates, detailed clinical observations in vivo were compared to strain typing results in vitro. Associations were found between distinct genotypes and severity of disease, suggesting strain-specific bacterial virulence. Circumstantial evidence furthermore supports strain-specific routes of bacterial dissemination. We conclude that PFGE and binary typing can be successfully applied for genetic analysis of S. aureus isolates from bovine mammary secretions. Binary typing in particular is a robust and simple method and promises to become a powerful tool for strain characterization, for resolution of clonal relationships of bacteria within and between host species, and for identification of sources and transmission routes of bovine S. aureus. PMID:10790124

  14. Successful isolation, in vitro expansion and characterization of stem cells from Human Dental Pulp

    Directory of Open Access Journals (Sweden)

    Preethy SP

    2010-01-01

    Full Text Available BACKGROUND: Recent studies have shown that mesenchymal stem cells isolated from post natal human dental pulp, (Dental pulp stem cells-DPSCs which is from permanent teeth and SHED (stem cells from human exfoliated deciduous teeth,the Periodontal ligament stem cells (PDLSC and Stem cells from root Apical papilla(SCAPhave the potential to differentiate into cells of a variety of tissues including heart, muscle, cartilage, bone, nerve, salivary glands, teeth etc(1,2,3,4.This multipotential ability of DPSCs is being researched for clinical application for treating a variety of diseases like myocardial infarction, muscular dystrophy, neuro-degenerative disorders, cartilage replacement, tooth regeneration and for repair of bone defects to mention a few. Moreover, the isolation of stem cells from teeth is minimally invasive, readily accessible and the non immunogenic characteristic of dental stem cells has paved the way for efforts to store the exfoliated deciduous teeth or milk teeth which is usually discarded, for use in the future. In this study we have isolated and expanded in vitro, the cells obtained from human dental pulp. MATERIALS AND METHODS: After obtaining written informed consent, 24 teeth that were extracted for therapeutic or cosmetic reasons from 16 patients were used in this study. The specimens were transported from the clinic to NCRM lab taking 6 to 48 Hrs. For removal of the pulp tissue, the teeth were split obliquely at the Cementoenamel junction and the pulp tissue was isolated using brooches. The extracted pulp tissues were subjected to digestion using Collagenase type-I and type II at 37˚C for 15- 30 minutes. The digested cells were filtered with 70µm filter and centrifuged at 1800 rpm for 10 minutes. The pellet was then suspended in Dulbecco’s modified Eagle’s medium (DMEM/Ham’s F12 supplemented with 15% fetal bovine serum , 100 U/ml penicillin, 100 µg/ml streptomycin,2 m M L -glutamine, and 2 m M nonessential amino

  15. Isolation of bacterial extrachromosomal DNA from human dental plaque associated with periodontal disease, using transposon-aided capture (TRACA).

    Science.gov (United States)

    Warburton, Philip J; Allan, Elaine; Hunter, Stephanie; Ward, John; Booth, Veronica; Wade, William G; Mullany, Peter

    2011-11-01

    The human oral cavity is host to a complex microbial community estimated to comprise >700 bacterial species, of which at least half are thought to be not yet cultivable in vitro. To investigate the plasmids present in this community, we used a transposon-aided capture system, which allowed the isolation of plasmids from human oral supra- and subgingival plaque samples. Thirty-two novel plasmids and a circular molecule that could be an integrase-generated circular intermediate were isolated. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  16. Dynamics of co-existing Escherichia colilineages in situ of the infant gut and multiplex phenotypic targeted recovery of previously uncultivated bacteria from the human gut

    DEFF Research Database (Denmark)

    Gumpert, Heidi

    The work in this thesis explores the dynamic nature of Escherichia coli lineages co-existing in the human intestinal tract. The work is supported via full genomesequencing of co-existing E. coli strains isolated from infants enrolled in the ALLERGYFLORA study. Both sets of isolates examined here...... were selected due to an observed change in their antibiotic susceptibility profile. Via full genome sequencing, we identified that in both cases a conjugative plasmid harboring antibiotic resistance genes was transferred between co-existing E. coli lineages and is responsible for the change...... in antibiotic susceptibility. In one case, the transfer occurred in the absenceof antibiotic treatment and the transconjugant remained amongst the gut microbiota for months, providing evidence to the hypothesis that resistance genes are stably maintained once acquired. To our knowledge, this is the first...

  17. siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts.

    Science.gov (United States)

    Lokossou, Adjimon Gatien; Toufaily, Chirine; Vargas, Amandine; Barbeau, Benoit

    2016-09-20

    Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

  18. Characterization of Lactobacillus salivarius CECT 5713, a strain isolated from human milk: from genotype to phenotype.

    Science.gov (United States)

    Langa, Susana; Maldonado-Barragán, Antonio; Delgado, Susana; Martín, Rebeca; Martín, Virginia; Jiménez, Esther; Ruíz-Barba, José L; Mayo, Baltasar; Connor, Ruth I; Suárez, Juan Evaristo; Rodríguez, Juan M

    2012-06-01

    Lactobacillus salivarius CECT 5713, isolated from human milk, has immunomodulatory, anti-inflammatory and antiinfectious properties, as revealed by several in vitro and in vivo assays, which suggests a strong potential as a probiotic strain. In this work, the relationships between several genetic features of L. salivarius CECT 5713 and the corresponding phenotypes were evaluated. Although it contains a plasmid-encoded bacteriocin cluster, no bacteriocin biosynthesis was observed, possibly due to a 4-bp deletion at the beginning of the histidine kinase determinant abpK. The genome of L. salivarius CECT 5713 harbours two apparently complete prophages of 39.6 and 48 kbp. Upon induction, the 48-kbp prophage became liberated from the bacterial genome, but no DNA replication took place, which resulted in lysis of the cultures but not in phage progeny generation. The strain was sensitive to most antibiotics tested and no transmissible genes potentially involved in antibiotic resistance were detected. Finally, the genome of L. salivarius CECT 5713 contained four ORFs potentially involved in human molecular mimetism. Among them, protein 1230 was considered of particular relevance because of its similarity with dendritic cell-related proteins. Subsequently, in vitro assays revealed the ability of L. salivarius CECT 5713 to stimulate the maturation of immature dendritic cells and to inhibit the in vitro infectivity of HIV-1.

  19. Antitumour Effects of Isocurcumenol Isolated from Curcuma zedoaria Rhizomes on Human and Murine Cancer Cells

    Science.gov (United States)

    Lakshmi, S.; Padmaja, G.; Remani, P.

    2011-01-01

    Curcuma zedoaria belonging to the family Zingiberaceae has been used in the traditional system of medicine in India and Southwest Asia in treating many human ailments and is found to possess many biological activities. The rationale of the present study was to isolate, identify, and characterize antitumour principles from the rhizomes of Curcuma zedoaria, to assess its cytotoxic effects on human and murine cancer cells, to determine its apoptosis inducing capacity in cancer cells, and to evaluate its tumour reducing properties in in vivo mice models. Isocurcumenol was characterized as the active compound by spectroscopy and was found to inhibit the proliferation of cancer cells without inducing significant toxicity to the normal cells. Fluorescent staining exhibited the morphological features of apoptosis in the compound-treated cancer cells. In vivo tumour reduction studies revealed that a dose of 35.7 mg/kg body weight significantly reduced the ascitic tumour in DLA-challenged mice and increased the lifespan with respect to untreated control mice. PMID:27429805

  20. Nicotine promotes proliferation and collagen synthesis of chondrocytes isolated from normal human and osteoarthritis patients.

    Science.gov (United States)

    Ying, Xiaozhou; Cheng, Shaowen; Shen, Yue; Cheng, Xiaojie; An Rompis, Ferdinand; Wang, Wei; Lin, Zhongqin; Chen, Qingyu; Zhang, Wei; Kou, Dongquan; Peng, Lei; Tian, Xin Qiao; Lu, Chuan Zhu

    2012-01-01

    The aims of the study were to show the direct effect of nicotine with different concentrations (0, 25, 50, and 100 ng/ml) on chondrocytes isolated from normal human and osteoarthritis patients, respectively. Microscopic observation was performed during the culture with an inverted microscope. Methyl thiazolyl tetrazolium (MTT) assay method was adopted to observe the influence of nicotine on the proliferation of chondrocytes, and real-time PCR and ELISA were used to assay the mRNA and protein expression of type II collagen and aggrecan, respectively. We discovered that the OA chondrocytes were similar to fibroblasts in shape and grow slower than normal chondrocytes. The proliferation of the two kinds of chondrocytes was increased in a concentration-dependent manner and in a time-dependent manner (P<0.05). Also, we found that the mRNA level of type II collagen were upregulated under 25-100 ng/ml nicotine doses both in the two kinds of chondrocytes compared with control. The expression of protein levels of type II collagen were synthesized in line with the increase in mRNA. No effect was observed on aggrecan synthesis with any nicotine dose. We concluded that nicotine has the same effect on both chondrocytes, obtained either from osteoarthritis patients or from normal human, and the positive effect of smoking in OA may relate to the alteration in metabolism of chondrocytes.

  1. Novel tandem column method for the rapid isolation of radiostrontium from human urine

    International Nuclear Information System (INIS)

    Hawkins, Cory A.; Shkrob, Ilya A.; Mertz, Carol J.; Dietz, Mark L.; Kaminski, Michael D.

    2012-01-01

    Highlights: ► Method for separation and preconcentration of radiostrontium from human urine. ► Recoveries >98%, concentration factor of ca. 50, processing time of nearly 1 h. ► Retention model developed to assist optimization of separations on Diphonix ® column. ► Semi-automated sample preparation device developed. - Abstract: A method has been developed for the isolation of strontium from human urine for subsequent determination in sample volumes as low as 5–20 mL. This method involves the acidification of the sample using methanesulfonic acid and its decolorization using charcoal, treatment of the filtrate with Diphonix ® resin, and subsequent concentration of strontium on Sr resin. Data from retention model simulations provided the initial conditions which were then optimized by actual column separations. Diphonix ® resin was shown to be effective at removing alkali metal ions from the urine matrix under conditions that retain higher valence ions. The suggested processing method provides 99% recovery of Sr 2+ , a concentration factor of 50, and an expected per sample processing time of less than 1 h.

  2. Molecular characterization of pandemic H1N1 influenza viruses isolated from turkeys and pathogenicity of a human pH1N1 isolate in turkeys.

    Science.gov (United States)

    Berhane, Yohannes; Ojkic, Davor; Neufeld, James; Leith, Marsha; Hisanaga, Tamiko; Kehler, Helen; Ferencz, Arpad; Wojcinski, Helen; Cottam-Birt, Colleen; Suderman, Matthew; Handel, Katherine; Alexandersen, Soren; Pasick, John

    2010-12-01

    Suspected human-to-animal transmission of the 2009 pandemic H1N1 (pH1N1) virus has been reported in several animal species, including pigs, dogs, cats, ferrets, and turkeys. In this study we describe the genetic characterization of pH1N1 viruses isolated from breeder turkeys that was associated with a progressive drop in egg production. Sequence analysis of all eight gene segments from three viruses isolated from this outbreak demonstrated homology with other human and swine pH1N1 isolates. The susceptibility of turkeys to a human pH1N1 isolate was further evaluated experimentally. The 50% turkey infectious dose (TID50) for the human isolate A/Mexico/LnDRE/4487/2009 was determined by inoculating groups of 8-10-week-old turkeys with serial 10-fold dilutions of virus by oronasal and cloacal routes. We estimated the TID50 to be between 1 x 10(5) and 1 x 10(6) TCID50. The pathogenesis of pH1N1 in oronasally or cloacally inoculated juvenile turkeys was also examined. None of the turkeys exhibited clinical signs, and no significant difference in virus shedding or seroconversion was observed between the two inoculation groups. More than 50% of the turkeys in both oronasal and cloacal groups shed virus beginning at 2 days postinoculation (dpi). All birds that actively shed virus seroconverted by 14 dpi. Virus antigen was demonstrated by immunohistochemistry in the cecal tonsils and bursa of Fabricius in two of the birds that were infected by the cloacal route. Virus transmission to naive contact turkeys was at best doubtful. This report provides additional evidence that pH1N1 can cross the species barrier and cause disease outbreaks in domestic turkeys. However, it appears that the reproductive status of the host as well as environmental factors such as concurrent infections, stress, the presence or absence of litter, and stocking density may also contribute to efficient infection and transmission of this agent.

  3. Comparative characterisation of the biofilm-production abilities of Staphylococcus epidermidis isolated from human skin and platelet concentrates.

    Science.gov (United States)

    Taha, Mariam; Kohnen, Carissa; Mallya, Shruti; Kou, Yuntong; Zapata, Adriana; Ramirez-Arcos, Sandra

    2018-02-01

    Staphylococcus epidermidis is the predominant contaminant of platelet concentrates (PCs), a blood product used to treat patients with platelet deficiencies. This microorganism is able to form surface-attached aggregates (biofilms) in human skin. Herein, the abundance of S. epidermidis biofilm-producers in contaminated PCs compared to skin isolates was explored. Furthermore, the potential positive selection of S. epidermidis biofilm-producers during the blood donation process and PC manufacturing was investigated. Twenty-four S. epidermidis isolates obtained from contaminated PCs and 48 S. epidermidis isolates obtained from the venipuncture area of human volunteers were compared for their ability to form biofilms in laboratory media and in PCs using a semi quantitative crystal violet assay. Also, the presence of the biofilm-associated icaA and icaD genes was assessed by PCR-amplification.Results/Key findings.Biofilm production in laboratory media showed a higher number of S. epidermidis biofilm-producers in the skin-derived group (43.7 %) compared to the PC-derived isolates (25 %). However, all skin and PC isolates formed biofilms in PCs. The prevalence of ica-positive biofilm-producer isolates was similar in PC and skin isolates (16.6 and 18.8 %, respectively). In contrast, the abundance of ica-negative biofilm-producers was lower in PC isolates compared to skin isolates (8.3 vs 25 %, respectively). Positive selection of S. epidermidis biofilm-producers during blood donation and PC manufacturing was not observed. Interestingly, ica-negative biofilm-producers seem to be negatively affected by skin disinfection, blood processing and PC storage. Furthermore, this study shows that S. epidermidis adopts a biofilm-forming phenotype in PCs regardless of its genetic background or origin.

  4. Core Genome Multilocus Sequence Typing Scheme for Stable, Comparative Analyses of Campylobacter jejuni and C. coli Human Disease Isolates.

    Science.gov (United States)

    Cody, Alison J; Bray, James E; Jolley, Keith A; McCarthy, Noel D; Maiden, Martin C J

    2017-07-01

    Human campylobacteriosis, caused by Campylobacter jejuni and C. coli , remains a leading cause of bacterial gastroenteritis in many countries, but the epidemiology of campylobacteriosis outbreaks remains poorly defined, largely due to limitations in the resolution and comparability of isolate characterization methods. Whole-genome sequencing (WGS) data enable the improvement of sequence-based typing approaches, such as multilocus sequence typing (MLST), by substantially increasing the number of loci examined. A core genome MLST (cgMLST) scheme defines a comprehensive set of those loci present in most members of a bacterial group, balancing very high resolution with comparability across the diversity of the group. Here we propose a set of 1,343 loci as a human campylobacteriosis cgMLST scheme (v1.0), the allelic profiles of which can be assigned to core genome sequence types. The 1,343 loci chosen were a subset of the 1,643 loci identified in the reannotation of the genome sequence of C. jejuni isolate NCTC 11168, chosen as being present in >95% of draft genomes of 2,472 representative United Kingdom campylobacteriosis isolates, comprising 2,207 (89.3%) C. jejuni isolates and 265 (10.7%) C. coli isolates. Validation of the cgMLST scheme was undertaken with 1,478 further high-quality draft genomes, containing 150 or fewer contiguous sequences, from disease isolate collections: 99.5% of these isolates contained ≥95% of the 1,343 cgMLST loci. In addition to the rapid and effective high-resolution analysis of large numbers of diverse isolates, the cgMLST scheme enabled the efficient identification of very closely related isolates from a well-defined single-source campylobacteriosis outbreak. Copyright © 2017 Cody et al.

  5. Isolation of a hemidesmosome-rich fraction from a human squamous cell carcinoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Hirako, Yoshiaki, E-mail: s47526a@cc.nagoya-u.ac.jp [Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602 (Japan); Yonemoto, Yuki; Yamauchi, Tomoe [Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602 (Japan); Nishizawa, Yuji; Kawamoto, Yoshiyuki [Department of Biomedical Sciences, Chubu University, 1200 Matsumoto-cho, Kasugai 487-8501 (Japan); Owaribe, Katsushi [Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602 (Japan)

    2014-06-10

    Hemidesmosomes are cell-to-matrix adhesion complexes anchoring keratinocytes to basement membranes. For the first time, we present a method to prepare a fraction from human cultured cells that are highly enriched in hemidesmosomal proteins. Using DJM-1 cells derived from human squamous cell carcinoma, accumulation of hemidesmosomes was observed when these cells were cultured for more than 10 days in a commercial serum-free medium without supplemental calcium. Electron microscopy demonstrated that numerous electron-dense adhesion structures were present along the basal cell membranes of DJM-1 cells cultured under the aforementioned conditions. After removing cellular materials using an ammonia solution, hemidesmosomal proteins and deposited extracellular matrix were collected and separated by electrophoresis. There were eight major polypeptides, which were determined to be plectin, BP230, BP180, integrin α6 and β4 subunits, and laminin-332 by immunoblotting and mass spectrometry. Therefore, we designated this preparation as a hemidesmosome-rich fraction. This fraction contained laminin-332 exclusively in its unprocessed form, which may account for the promotion of laminin deposition, and minimal amounts of Lutheran blood group protein, a nonhemidesmosomal transmembrane protein. This hemidesmosome-rich fraction would be useful not only for biological research on hemidesmosomes but also for developing a serum test for patients with blistering skin diseases. - Highlights: • A defined condition promoted accumulation of hemidesmosomes in human cultured cells. • A fraction isolated from the cells contained eight major polypeptides. • The polypeptides were the five major hemidesmosome proteins and laminin-332. • The cultured cells deposited laminin-332 in its unprocessed form under the condition. • We report a method to prepare a fraction highly enriched in hemidesmosome proteins.

  6. Genotypic characteristics of hydatid cysts isolated from humans in East Azerbaijan Province (2011-2013

    Directory of Open Access Journals (Sweden)

    Amir Vahedi

    2014-08-01

    Full Text Available Introduction: Cystic echinococcosis (CE is one of the important helminthic diseases of human and animals, which causes by Echinococcus granulosus. Canids are its definite and grazers especially sheep, and cattle, and also wild herbivores are its intermediate hosts. Human can also be accidentally infected by a parasite. This study aimed to investigate genotypes of the hydatid cysts isolated from hydatidosis patients in order to confine the source of the infection, 2013. Methods: In this cross-sectional study 55 paraffin blocks of identified hydatid cysts have been undergone genotyping using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP technique. The ITS1 region of rDNA has been amplified using BD1 forward and 4s reverse primers. PCR products have been digested using HpaII and RsaI restriction endonucleases. RFLP products studied using gel electrophoresis. Data were analyzed using SPSS for Windows using the chi-square test. Results: About 29 (52.72%, 16 (29.1%, 3 (5.45%, 3 (5.45%, 1 (1.81%, 1 (1.81%, 1 (1.81% and 1 (1.81% out of 55 hydatid cysts were located in lung, liver, spleen, kidney, heart, pancreas, brain, and femore, respectively. The frequency of hydatidosis observed higher in patients from rural areas (P = 0.013; odds ratio = 0.599; 95% confidence interval: 0.28, 1.27. Based on RFLP results, the entire studied hydatid cysts identified as sheep strain (G1. Conclusion: According to the results of the present observation, it can be concluded that the majority of cases of human hydatidosis in East Azerbaijan Province are caused by sheep strain (G1 of E. granulosus, which indicates the sheep-doge cycle in the studied area.

  7. Probiotic properties of Lactobacillus rhamnosus and Lactobacillus paracasei isolated from human faeces.

    Science.gov (United States)

    Verdenelli, Maria Cristina; Ghelfi, Francesca; Silvi, Stefania; Orpianesi, Carla; Cecchini, Cinzia; Cresci, Alberto

    2009-09-01

    The possibility of using microbes to maintain health, and to prevent or treat disease is a topic as old as microbiology. The research of novel probiotic strains is important in order to satisfy the increasing request of the market and to obtain functional products in which the probiotic cultures are more active and with better probiotic characteristics than those already present on the market. In this study, the probiotic potential of Lactobacillus strains isolated from Italian elderly human faeces was investigated. The Lactobacillus strains were identified and examined for resistance to gastric acidity and bile toxicity, adhesion to HT-29 cells, antimicrobial activities, antibiotic susceptibility and plasmid profile. Survival of the strains through human intestine was examined in a 3 months human feeding trial. Two strains, Lactobacillus rhamnosus IMC 501 and Lactobacillus paracasei IMC 502, tolerated well low pH and bile acids. In antimicrobial activity assays, both strains showed inhibitory properties towards selected potential harmful microorganisms, particularly against Candida albicans. The two selected strains expressed high in vitro adherence to HT-29 cells increasing this characteristic when they are used in combination and they were resistant to vamcomycin, colistin sulphate, gentamicin, oxolinic acid and kanamycin. Moreover, the two strains could be recovered from stools of volunteers after the feeding trials. Lactobacillus rhamnosus IMC 501 and L. paracasei IMC 502 present favourable strain-specific properties for their utilisation as probiotics in functional foods and the high adhesion ability of the L. rhamnosus IMC 501 and L. paracasei IMC 502 used in combination, confirmed by both in vitro and in vivo study, indicate that the two bacterial strains could be used as health-promoting bacteria.

  8. Lipid-protein interactions in plasma membranes of fiber cells isolated from the human eye lens.

    Science.gov (United States)

    Raguz, Marija; Mainali, Laxman; O'Brien, William J; Subczynski, Witold K

    2014-03-01

    The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali, L., Raguz, M., O'Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Genotypic analyses of lactobacilli with a range of tannase activities isolated from human feces and fermented foods.

    Science.gov (United States)

    Nishitani, Yosuke; Sasaki, Eiki; Fujisawa, Tomohiko; Osawa, Ro

    2004-02-01

    A total of 77 tannase producing lactobacilli strains isolated from human feces or fermented foods were examined for their genotypic profiles and intensities of tannase production. With a PCR-based assay targeting recA gene, all strains except one isolate were assigned to either Lactobacillus plantarum, L. paraplantarum, or L. pentosus whereas a 16/23S rDNA targeted PCR-based assay identified all except 6 isolates (inclusive of the above one isolate) as one of the closely related species. Subsequent DNA/DNA hybridization assays revealed that these 6 exceptional isolates showed low homology (between 1.2% and 55.8% relative DNA binding) against type strains of the three species. Supplemental carbohydrate fermentation profiles on the 6 isolates indicated that two of them were identified as L. acidophilus, one as Pediococcus acidilactici, one as P. pentosaceus, and two remained unidentifiable. The evidence suggests that the 16/23S rDNA targeted PCR assay can be used as a reliable identification tool for the closely related lactobacilli, and that the tannase gene is widely distributed within members of the Lactobacillaceae family. Meanwhile, a randomly amplified polymorphism DNA (RAPD) analysis revealed that all except 8 isolates were well allocated in 4 major RAPD clusters, though not species specific, consisting of two L. plantarum predominant clusters, one L. paraplantarum predominant, and one L. pentosus predominant. The RAPD patterns of the 8 non-clustered isolates, which consisted of the 6 unidentifiable isolates and 2 isolates identified as L. pentosus, were tannase activities showed that there was a marked variation in the activities among the strains, which did not correlate with either species identification or clustering by RAPD.

  10. Multilocus sequence analysis of Echinococcus granulosus strains isolated from humans and animals in Iran.

    Science.gov (United States)

    Nikmanesh, Bahram; Mirhendi, Hossein; Mahmoudi, Shahram; Rokni, Mohammad Bagher

    2017-12-01

    Echinococcus granulosus is now considered a complex consisting of at least four species and ten genotypes. Different molecular targets have been described for molecular characterization of E. granulosus; however, in almost all studies only one or two of the targets have been used, and only limited data is available on the utilization of multiple loci. Therefore, we investigated the genetic diversity among 64 strains isolated from 138 cyst specimens of human and animal isolates, using a set of nuclear and mitochondrial genes; i.e., cytochrome c oxidase subunit 1 (cox1), NADH dehydrogenase subunit 1 (nad1), ATPase subunit 6 (atp6), 12S rRNA (12S), and Actin II (act II). In comparison to the use of molecular reference targets (nad1 + cox1), using singular target (act II or 12S or atp6) yielded lower discriminatory power. Act II and 12S genes could accurately discriminate the G6 genotype, but they were not able to differentiate between G1 and G3 genotypes. As the G1 and G3 genotypes belong to the E. granulosus sensu stricto, low intra-species variation was observed for act II and 12S. The atp6 gene could identify the G3 genotype but could not differentiate G6 and G1 genotypes. Using concatenated sequence of five genes (cox1 + nad1 + atp6 + 12S + act II), genotypes were identified accurately, and markedly higher resolution was obtained in comparison with the use of reference markers (nad1 + cox1) only. Application of multilocus sequence analysis (MLSA) to large-scale studies could provide valuable epidemiological data to make efficient control and management measures for cystic echinococcosis. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Distinct Trypanosoma cruzi isolates induce activation and apoptosis of human neutrophils.

    Directory of Open Access Journals (Sweden)

    Luísa M D Magalhães

    Full Text Available Neutrophils are critical players in the first line of defense against pathogens and in the activation of subsequent cellular responses. We aimed to determine the effects of the interaction of Trypanosoma cruzi with human neutrophils, using isolates of the two major discrete type units (DTUs associated with Chagas' disease in Latin America (clone Col1.7G2 and Y strain, DTU I and II, respectively. Thus, we used CFSE-stained trypomastigotes to measure neutrophil-T. cruzi interaction, neutrophil activation, cytokine expression and death, after infection with Col1.7G2 and Y strain. Our results show that the frequency of CFSE+ neutrophils, indicative of interaction, and CFSE intensity on a cell-per-cell basis were similar when comparing Col1.7G2 and Y strains. Interaction with T. cruzi increased neutrophil activation, as measured by CD282, CD284, TNF and IL-12 expression, although at different levels between the two strains. No change in IL-10 expression was observed after interaction of neutrophils with either strain. We observed that exposure to Y and Col1.7G2 caused marked neutrophil death. This was specific to neutrophils, since interaction of either strain with monocytes did not cause death. Our further analysis showed that neutrophil death was a result of apoptosis, which was associated with an upregulation of TNF-receptor, TNF and FasLigand, but not of Fas. Induction of TNF-associated neutrophil apoptosis by the different T. cruzi isolates may act as an effective common mechanism to decrease the host's immune response and favor parasite survival.

  12. [Prevalence of human papilloma virus isolated from cervix lesions in a female population from Transilvania].

    Science.gov (United States)

    Feticu, Lucia; Bocşan, I S; Bondor, Cosmina loana; Boboş, Cecilia

    2012-01-01

    Between the years 2008-2011 reverse hibridisation (INNO-LiPA HPV Genotyping Extra test) and genotyping 1a Roche (the kit: Linear array HPV genotyping test) were used for detection of Human Papilloma Virus (HPV) in the cervix secretions of 182 female patients aged 16-63 years, predominantly of urban origin. 99 patients (54.4%) were identified as being infected with various types of HPV, prevalent in urban (53 single infections and 46 multiple infections). HPV infection was not detected in 83 (45.6%) patients. Only 7 females from rural areas were tested (5 females had single or multiple HPV infections). 32 types of HPV were identificated: 15 HPV types with high risk (51, 82, 56, 18, 39, 45, 59, 68, 16, 31, 33, 35, 52, 58, 73), 14 types with low risk (42, 61, 62, 72, 81, 83, 84, CP6108, 70, 6, 11, 55, 74, 54), and 3 types with possible high risk (26, 53, 66). The type of HPV could not be identified in other two cases. The most frecvent types of HPV with high risk isolated were: the type 16. The types 51 and 58 of HPV with high risk and the type 84 with low risk are detected in single infections in urban and in rural. HPV clades involved in single infections are: 1 (1 case), 3 (5 cases), 5 (4 cases), 6 (5 cases), 7 (5 cases), 9 (21 cases), 10 (7 cases). The clades 11 (7 cases) and 13 (6 cases) were involved only in multiple infections detected in urban. The types 35, 39, 59, 68 of HPV with high risk were isolated from multple infections. In rural, multiple infections with two HPV were detected. The citological screening by Babe-Papanicolaou examination was made only in 9 cases: HPV was not detected in 4 cases (one female had ASC-US: atypical squamous cells of "undetermined significance"); in 5 positive cases were detected HPV 16, 31, 58, 6.

  13. Megasphaera indica sp. nov., an obligate anaerobic bacteria isolated from human faeces.

    Science.gov (United States)

    Lanjekar, V B; Marathe, N P; Ramana, V Venkata; Shouche, Y S; Ranade, D R

    2014-07-01

    Two coccoid, non-motile, obligately anaerobic, Gram-stain-negative bacteria, occurring singly or in pairs, or as short chains, with a mean size of 1.4-2.5 µm were isolated from the faeces of two healthy human volunteers, aged 26 and 56 years, and were designated NMBHI-10(T) and BLPYG-7, respectively. Both the strains were affiliated to the sub-branch Sporomusa of the class Clostridia as revealed by 16S rRNA gene sequence analysis. The isolates NMBHI-10(T) and BLPYG-7 showed 99.1 and 99.2% 16S rRNA gene sequence similarity, respectively, with Megasphaera elsdenii JCM 1772(T). DNA-DNA hybridization and phenotypic analysis showed that both the strains were distinct from their closest relative, M. elsdenii JCM 1772(T) (42 and 53% DNA-DNA relatedness with NMBHI-10(T) and BLPYG-7, respectively), but belong to the same species (DNA-DNA relatedness of 80.9 % between the isolates). According to DNA-DNA hybridization results, the coccoid strains belong to the same genospecies, and neither is related to any of the recognized species of the genus Megasphaera. Strains NMBHI-10(T) and BLPYG-7 grew in PYG broth at temperatures of between 15 and 40 °C (optimum 37 °C), but not at 45 °C. The strains utilized a range of carbohydrates as sources of carbon and energy including glucose, lactose, cellobiose, rhamnose, galactose and sucrose. Glucose fermentation resulted in the formation of volatile fatty acids, mainly caproic acid and organic acids such as succinic acid. Phylogenetic analysis, specific phenotypic characteristics and/or DNA G+C content also differentiated the strains from each other and from their closest relatives. The DNA G+C contents of strains NMBHI-10(T) and BLPYG-7 are 57.7 and 54.9 mol%, respectively. The major fatty acids were 12 : 0 FAME and 17 : 0 CYC FAME. On the basis of these data, we conclude that strains NMBHI-10(T) and BLPYG-7 should be classified as representing a novel species of the genus Megasphaera, for which the name Megsphaera indica sp. nov

  14. Alu polymerase chain reaction: A method for rapid isolation of human-specific sequences from complex DNA sources

    International Nuclear Information System (INIS)

    Nelson, D.L.; Ledbetter, S.A.; Corbo, L.; Victoria, M.F.; Ramirez-Solis, R.; Webster, T.D.; Ledbetter, D.H.; Caskey, C.T.

    1989-01-01

    Current efforts to map the human genome are focused on individual chromosomes or smaller regions and frequently rely on the use of somatic cell hybrids. The authors report the application of the polymerase chain reaction to direct amplification of human DNA from hybrid cells containing regions of the human genome in rodent cell backgrounds using primers directed to the human Alu repeat element. They demonstrate Alu-directed amplification of a fragment of the human HPRT gene from both hybrid cell and cloned DNA and identify through sequence analysis the Alu repeats involved in this amplification. They also demonstrate the application of this technique to identify the chromosomal locations of large fragments of the human X chromosome cloned in a yeast artificial chromosome and the general applicability of the method to the preparation of DNA probes from cloned human sequences. The technique allows rapid gene mapping and provides a simple method for the isolation and analysis of specific chromosomal regions

  15. Characterization of the virulence, growth temperature and antibiotic resistance of the Campylobacter jejuni IAL 2383 strain isolated from humans

    Science.gov (United States)

    Fonseca, B.B.; Rossi, D.A.; Maia, C.A.; Nalevaiko, P.C.; Melo, R.T.; Cuccato, L.P.; Beletti, M.E.

    2014-01-01

    The objective of this study was to characterize the C. jejuni IAL2383 strain isolated from humans in Brazil. Transcripts for the racR, dnaJ and ciaB genes were found and flaA, plda and cadF genes were present in the genome and bacteria was sensitive to most of the important antimicrobials used to treat humans. C. jejuni IAL2383 is a good experimental model to analyze the interactions with cells. PMID:24948944

  16. Isolation, propagation, and titration of human immunodeficiency virus type 1 from peripheral blood of infected individuals

    NARCIS (Netherlands)

    Schuitemaker, Hanneke; Kootstra, Neeltje A.

    2005-01-01

    HIV-1 can be isolated from peripheral blood mononuclear cells and is easily propagated on primary cells in vitro. Here we describe the method for bulk isolation of the HIV-1 quasispecies and a limiting dilution virus isolation protocol by which single coexisting clones can be obtained. In addition,

  17. Semisolid liver infusion tryptose supplemented with human urine allows growth and isolation of Trypanosoma cruzi and Trypanosoma rangeli clonal lineages

    Directory of Open Access Journals (Sweden)

    Emanuella Francisco Fajardo

    2016-06-01

    Full Text Available Abstract: INTRODUCTION This work shows that 3% (v/v human urine (HU in semisolid Liver Infusion Tryptose (SSL medium favors the growth of Trypanosoma cruzi and T. rangeli. METHODS Parasites were plated as individual or mixed strains on SSL medium and on SSL medium with 3% human urine (SSL-HU. Isolate DNA was analyzed using polymerase chain reaction (PCR and pulsed-field gel electrophoresis (PFGE. RESULTS SSL-HU medium improved clone isolation. PCR revealed that T. cruzi strains predominate on mixed-strain plates. PFGE confirmed that isolated parasites share the same molecular karyotype as parental cell lines. CONCLUSIONS SSL-HU medium constitutes a novel tool for obtaining T. cruzi and T. rangeli clonal lineages.

  18. Restoration of human B-cell differentiation into NOD-SCID mice engrafted with gene-corrected CD34+ cells isolated from Artemis or RAG1-deficient patients.

    Science.gov (United States)

    Lagresle-Peyrou, Chantal; Benjelloun, Fatine; Hue, Christophe; Andre-Schmutz, Isabelle; Bonhomme, Delphine; Forveille, Monique; Beldjord, Kheira; Hacein-Bey-Abina, Salima; De Villartay, Jean-Pierre; Charneau, Pierre; Durandy, Anne; Fischer, Alain; Cavazzana-Calvo, Marina

    2008-02-01

    Severe combined immunodeficiency (SCID) caused by mutation of the recombination-activating gene 1 (RAG1) or Artemis gene lead to the absence of B- and T-cell differentiation. The only curative treatment is allogeneic bone marrow (BM) transplantation, which displays a high survival rate when an HLA compatible donor is available but has a poorer prognosis when the donor is partially compatible. Consequently, gene therapy may be a promising alternative strategy for these diseases. Here, we report that lentiviral gene-corrected BM CD34(+) cells (isolated from Artemis- or RAG1-deficient patients) sustain human B-cell differentiation following injection into non-obese diabetic/SCID (NOD-SCID) mice previously infused with anti-interleukin-2 receptor beta chain monoclonal antibody. In most of the mice BM, engrafted with Artemis-transduced cells, human B-cell differentiation occurred until the mature stage. The B cells were functional as human immunoglobulin M (IgM) was present in the serum. Following injection with RAG1-transduced cells, human engraftment occurred in vivo but B-cell differentiation until the mature stage was less frequent. However, when it occurred, it was always associated with human IgM production. This overall approach represents a useful tool for evaluating gene transfer efficiency in human SCID forms affecting B-cell development (such as Artemis deficiency) and for testing new vectors for improving in vivo RAG1 complementation.

  19. Draft Genome Sequences of Two Propionibacterium acnes Strains Isolated from Progressive Macular Hypomelanosis Lesions of Human Skin

    DEFF Research Database (Denmark)

    Petersen, Rolf; Lomholt, Hans B.; Scholz, Christian F. P.

    2015-01-01

    Propionibacterium acnes is a Gram-positive bacterium that is prevalent on human skin. It has been associated with skin disorders such as acne vulgaris and progressive macular hypomelanosis (PMH). Here, we report draft genome sequences of two type III P. acnes strains, PMH5 and PMH7, isolated from...

  20. 76 FR 51374 - Direct Discovery of HLA Associated Influenza Epitopes Isolated From Human Cells for Vaccine and...

    Science.gov (United States)

    2011-08-18

    ... direct-discovery technology for use in FDA laboratories. C. Eligibility Information The technology...] Direct Discovery of HLA Associated Influenza Epitopes Isolated From Human Cells for Vaccine and... technology to molecularly characterize peptide epitopes that are processed and presented on soluble HLA...

  1. Comparative Activity of Pradofloxacin, Enrofloxacin, and Azithromycin against Bartonella henselae Isolates Collected from Cats and a Human

    Science.gov (United States)

    Biswas, Silpak; Maggi, Ricardo G.; Papich, Mark G.; Keil, Daniel; Breitschwerdt, Edward B.

    2010-01-01

    Using Bartonella henselae isolates from cats and a human, the activity of pradofloxacin was compared with those of enrofloxacin and azithromycin. By Etest and disc diffusion assay, pradofloxacin showed greater antimicrobial activity than did other antibiotics. We conclude that pradofloxacin may prove useful for the treatment of B. henselae infections. PMID:20007401

  2. Polyphasic taxonomic approach in the description of Alishewanella fetalis gen. nov., sp nov., isolated from a human foetus

    DEFF Research Database (Denmark)

    Vogel, Birte Fonnesbech; Venkateswaran, K.; Christensen, H.

    2000-01-01

    A taxonomically unique bacterium is described on the basis of a physiological and biochemical characterization, fatty acid profiling and sequence analyses of 16S rRNA and gyrase B (gyrB) genes. This non-motile, non-fermentative bacterium was isolated from a human foetus in Uppsala, Sweden...

  3. Distribution of Blastocystis subtypes isolated from humans from an urban community in Rio de Janeiro, Brazil

    Directory of Open Access Journals (Sweden)

    Carolina Valença Barbosa

    2017-10-01

    Full Text Available Abstract Background Blastocystis is a cosmopolitan protist parasite found in the human gastrointestinal tract and is highly prevalent in developing countries. Recent molecular studies have revealed extensive genetic diversity, which has been classified into different subtypes (STs based on sequence analysis of small subunit ribosomal RNA gene. Blastocystis is one of the most common fecal parasites in Brazil, but the diversity of subtypes remains unknown in the country. This study aimed to determine the distribution of Blastocystis STs in an urban community in Duque de Caxias, Rio de Janeiro, Brazil. Methods A total of 64 stool samples positive for Blastocystis in Pavlova’s medium were subtyped by PCR and sequenced using primers targeting the small subunit rRNA gene, in addition to phylogenetic analysis and subtype-specific PCR using sequence-tagged-site (STS primers. Results Endolimax nana (14%, Entamoeba complex (10.5%, Taenia sp. (0.6%, Trichuris trichiura (1.3% and Enterobius vermicularis (1.3% were detected in Blastocystis-positive samples. Of the 64 samples tested by PCR/DNA sequencing, 55 were identified as ST1 (42%, ST3 (49%, ST2 (7% and ST4 (2%, and the presence of mixed ST (ST1 + ST3 infection was detected in nine samples (14%. Conclusions DNA sequencing and phylogenetic analysis of Brazilian Blastocystis isolates identified four different subtypes. To our knowledge, this study provided the first genetic characterization of Blastocystis subtypes in an urban area of Rio de Janeiro, Brazil. We also identified ST4 for the first time in Brazil. Further studies are necessary to determine the distribution of STs across human populations in Rio de Janeiro.

  4. Differentiation of isolated human umbilical cord mesenchymal stem cells into neural stem cells

    Science.gov (United States)

    Chen, Song; Zhang, Wei; Wang, Ji-Ming; Duan, Hong-Tao; Kong, Jia-Hui; Wang, Yue-Xin; Dong, Meng; Bi, Xue; Song, Jian

    2016-01-01

    AIM To investigate whether umbilical cord human mesenchymal stem cell (UC-MSC) was able to differentiate into neural stem cell and neuron in vitro. METHODS The umbilical cords were obtained from pregnant women with their written consent and the approval of the Clinic Ethnics Committee. UC-MSC were isolated by adherent culture in the medium contains 20% fetal bovine serum (FBS), then they were maintained in the medium contain 10% FBS and induced to neural cells in neural differentiation medium. We investigated whether UC-MSC was able to differentiate into neural stem cell and neuron in vitro by using flow cytometry, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence (IF) analyzes. RESULTS A substantial number of UC-MSC was harvested using the tissue explants adherent method at about 2wk. Flow cytometric study revealed that these cells expressed common markers of MSCs, such as CD105 (SH2), CD73 (SH3) and CD90. After induction of differentiation of neural stem cells, the cells began to form clusters; RT-PCR and IF showed that the neuron specific enolase (NSE) and neurogenic differentiation 1-positive cells reached 87.3%±14.7% and 72.6%±11.8%, respectively. Cells showed neuronal cell differentiation after induced, including neuron-like protrusions, plump cell body, obviously and stronger refraction. RT-PCR and IF analysis showed that microtubule-associated protein 2 (MAP2) and nuclear factor-M-positive cells reached 43.1%±10.3% and 69.4%±19.5%, respectively. CONCLUSION Human umbilical cord derived MSCs can be cultured and proliferated in vitro and differentiate into neural stem cells, which may be a valuable source for cell therapy of neurodegenerative eye diseases. PMID:26949608

  5. Differentiation of isolated human umbilical cord mesenchymal stem cells into neural stem cells

    Directory of Open Access Journals (Sweden)

    Song Chen

    2016-01-01

    Full Text Available AIM: To investigate whether umbilical cord human mesenchymal stem cell (UC-MSC was able to differentiate into neural stem cell and neuron in vitro. METHODS: The umbilical cords were obtained from pregnant women with their written consent and the approval of the Clinic Ethnics Committee. UC-MSC were isolated by adherent culture in the medium contains 20% fetal bovine serum (FBS, then they were maintained in the medium contain 10% FBS and induced to neural cells in neural differentiation medium. We investigated whether UC-MSC was able to differentiate into neural stem cell and neuron in vitro by using flow cytometry, reverse transcriptase-polymerase chain reaction (RT-PCR and immunofluorescence (IF analyzes. RESULTS: A substantial number of UC-MSC was harvested using the tissue explants adherent method at about 2wk. Flow cytometric study revealed that these cells expressed common markers of MSCs, such as CD105 (SH2, CD73 (SH3 and CD90. After induction of differentiation of neural stem cells, the cells began to form clusters; RT-PCR and IF showed that the neuron specific enolase (NSE and neurogenic differentiation 1-positive cells reached 87.3%±14.7% and 72.6%±11.8%, respectively. Cells showed neuronal cell differentiation after induced, including neuron-like protrusions, plump cell body, obviously and stronger refraction. RT-PCR and IF analysis showed that microtubule-associated protein 2 (MAP2 and nuclear factor-M-positive cells reached 43.1%±10.3% and 69.4%±19.5%, respectively. CONCLUSION: Human umbilical cord derived MSCs can be cultured and proliferated in vitro and differentiate into neural stem cells, which may be a valuable source for cell therapy of neurodegenerative eye diseases.

  6. Development of a potential probiotic fresh cheese using two Lactobacillus salivarius strains isolated from human milk.

    Science.gov (United States)

    Cárdenas, Nivia; Calzada, Javier; Peirotén, Angela; Jiménez, Esther; Escudero, Rosa; Rodríguez, Juan M; Medina, Margarita; Fernández, Leónides

    2014-01-01

    Cheeses have been proposed as a good alternative to other fermented milk products for the delivery of probiotic bacteria to the consumer. The objective of this study was to assess the survival of two Lactobacillus salivarius strains (CECT5713 and PS2) isolated from human milk during production and storage of fresh cheese for 28 days at 4°C. The effect of such strains on the volatile compounds profile, texture, and other sensorial properties, including an overall consumer acceptance, was also investigated. Both L. salivarius strains remained viable in the cheeses throughout the storage period and a significant reduction in their viable counts was only observed after 21 days. Globally, the addition of the L. salivarius strains did not change significantly neither the chemical composition of the cheese nor texture parameters after the storage period, although cheeses manufactured with L. salivarius CECT5713 presented significantly higher values of hardness. A total of 59 volatile compounds were identified in the headspace of experimental cheeses, and some L. salivarius-associated differences could be identified. All cheeses presented good results of acceptance after the sensory evaluation. Consequently, our results indicated that fresh cheese can be a good vehicle for the two L. salivarius strains analyzed in this study.

  7. Development of a Potential Probiotic Fresh Cheese Using Two Lactobacillus salivarius Strains Isolated from Human Milk

    Directory of Open Access Journals (Sweden)

    Nivia Cárdenas

    2014-01-01

    Full Text Available Cheeses have been proposed as a good alternative to other fermented milk products for the delivery of probiotic bacteria to the consumer. The objective of this study was to assess the survival of two Lactobacillus salivarius strains (CECT5713 and PS2 isolated from human milk during production and storage of fresh cheese for 28 days at 4°C. The effect of such strains on the volatile compounds profile, texture, and other sensorial properties, including an overall consumer acceptance, was also investigated. Both L. salivarius strains remained viable in the cheeses throughout the storage period and a significant reduction in their viable counts was only observed after 21 days. Globally, the addition of the L. salivarius strains did not change significantly neither the chemical composition of the cheese nor texture parameters after the storage period, although cheeses manufactured with L. salivarius CECT5713 presented significantly higher values of hardness. A total of 59 volatile compounds were identified in the headspace of experimental cheeses, and some L. salivarius-associated differences could be identified. All cheeses presented good results of acceptance after the sensory evaluation. Consequently, our results indicated that fresh cheese can be a good vehicle for the two L. salivarius strains analyzed in this study.

  8. Isolation and Multiple Differentiation Potential Assessment of Human Gingival Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Yuan Gao

    2014-11-01

    Full Text Available The aim of this study was to isolate human mesenchymal stem cells (MSCs from the gingiva (GMSCs and confirm their multiple differentiation potentials, including the odontogenic lineage. GMSCs, periodontal ligament stem cells (PDLSCs and dermal stem cells (DSCs cultures were analyzed for cell shape, cell cycle, colony-forming unit-fibroblast (CFU-F and stem cell markers. Cells were then induced for osteogenic and adipogenic differentiation and analyzed for differentiation markers (alkaline phosphatase (ALP activity, mineralization nodule formation and Runx2, ALP, osteocalcin (OCN and collagen I expressions for the osteogenic differentiation, and lipid vacuole formation and PPARγ-2 expression for the adipogenic differentiation. Besides, the odontogenic differentiation potential of GMSCs induced with embryonic tooth germ cell-conditioned medium (ETGC-CM was observed. GMSCs, PDLSCs and DSCs were all stromal origin. PDLSCs showed much higher osteogenic differentiation ability but lower adipogenic differentiation potential than DSCs. GMSCs showed the medial osteogenic and adipogenic differentiation potentials between those of PDLSCs and DSCs. GMSCs were capable of expressing the odontogenic genes after ETGC-CM induction. This study provides evidence that GMSCs can be used in tissue engineering/regeneration protocols as an approachable stem cell source.

  9. Dermal-epidermal membrane systems by using human keratinocytes and mesenchymal stem cells isolated from dermis

    Energy Technology Data Exchange (ETDEWEB)

    Salerno, Simona, E-mail: s.salerno@itm.cnr.it [Institute on Membrane Technology, National Research Council of Italy, ITM-CNR, c/o University of Calabria, via P. Bucci cubo 17/C, I-87036, Rende (CS) (Italy); Messina, Antonietta [Institute on Membrane Technology, National Research Council of Italy, ITM-CNR, c/o University of Calabria, via P. Bucci cubo 17/C, I-87036, Rende (CS) (Italy); Giordano, Francesca [Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, I-87036 Rende, (CS) (Italy); Bader, Augustinus [Biomedical-Biotechnological Center, BBZ, University of Leipzig, D-04103 Leipzig (Germany); Drioli, Enrico [Institute on Membrane Technology, National Research Council of Italy, ITM-CNR, c/o University of Calabria, via P. Bucci cubo 17/C, I-87036, Rende (CS) (Italy); WCU Energy Engineering Department, Hanyang University, Seoul (Korea, Republic of); De Bartolo, Loredana, E-mail: l.debartolo@itm.cnr.it [Institute on Membrane Technology, National Research Council of Italy, ITM-CNR, c/o University of Calabria, via P. Bucci cubo 17/C, I-87036, Rende (CS) (Italy)

    2017-02-01

    Dermal-epidermal membrane systems were developed by co-culturing human keratinocytes with Skin derived Stem Cells (SSCs), which are Mesenchymal Stem Cells (MSCs) isolated from dermis, on biodegradable membranes of chitosan (CHT), polycaprolactone (PCL) and a polymeric blend of CHT and PCL. The membranes display physico-chemical, morphological, mechanical and biodegradation properties that could satisfy and fulfil specific requirements in skin tissue engineering. CHT membrane exhibits an optimal biodegradation rate for acute wounds; CHT-PCL for the chronic ones. On the other hand, PCL membrane in spite of its very slow biodegradation rate exhibits mechanical properties similar to in vivo dermis, a lower hydrophilic character, and a surface roughness, all properties that make it able to sustain cell adhesion and proliferation for in vitro skin models. Both CHT–PCL and PCL membranes guided epidermal and dermal differentiation of SSCs as pointed out by the expression of cytokeratins and the deposition of the ECM protein fibronectin, respectively. In the dermal-epidermal membrane systems, a more suitable microenvironment for the SSCs differentiation was promoted by the interactions and the mutual interplay with keratinocytes. Being skin tissue-biased stem cells committed to their specific final dermal and/or epidermal cell differentiation, SSCs are more suitable for skin tissue engineering than other adult MSCs with different origin. For this reason, they represent a useful autologous cell source for engineering skin substitutes for both in vivo and in vitro applications.

  10. Evaluation of resistance to low pH and bile salts of human Lactobacillus spp. isolates.

    Science.gov (United States)

    Fuochi, Virginia; Petronio, Giulio Petronio; Lissandrello, Edmondo; Furneri, Pio Maria

    2015-09-01

    There are nearly 100 trillion bacteria in the intestine that together form the intestinal microbiota. They are 'good' bacteria because they help to maintain a physiological balance and are called probiotics. Probiotics must have some important characteristics: be safe for humans, be resistant to the low pH in the stomach, as well as bile salts and pancreatic juice. Indeed, their survival is the most important factor, so that they can arrive alive in the intestine and are able to form colonies, at least temporarily. The aim of our study was the evaluation of resistance of Lactobacillus isolates from fecal and oral swabs compared to that found in a commercial product. Seven strains were randomly chosen: L. jensenii, L. gasseri, L. salivarius, L. fermentum, L. rhamnosus, L. crispatus, and L. delbrueckii. We observed a large variability in the results: L. gasseri and L. fermentum were the most resistance to low pH, while only L. gasseri showed the best survival rate to bile salts. Interestingly, the commercial product did not show tolerance to both low pH and bile salts. © The Author(s) 2015.

  11. Identification, characterization and description of Arcobacter faecis sp. nov., isolated from a human waste septic tank.

    Science.gov (United States)

    Whiteduck-Léveillée, Kerri; Whiteduck-Léveillée, Jenni; Cloutier, Michel; Tambong, James T; Xu, Renlin; Topp, Edward; Arts, Michael T; Chao, Jerry; Adam, Zaky; Lévesque, C André; Lapen, David R; Villemur, Richard; Khan, Izhar U H

    2016-03-01

    A study on the taxonomic classification of Arcobacter species was performed on the cultures isolated from various fecal sources where an Arcobacter strain AF1078(T) from human waste septic tank near Ottawa, Ontario, Canada was characterized using a polyphasic approach. Genetic investigations including 16S rRNA, atpA, cpn60, gyrA, gyrB and rpoB gene sequences of strain AF1078(T) are unique in comparison with other arcobacters. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the strain is most closely related to Arcobacter lanthieri and Arcobacter cibarius. Analyses of atpA, cpn60, gyrA, gyrB and rpoB gene sequences suggested that strain AF1078(T) formed a phylogenetic lineage independent of other species in the genus. Whole-genome sequence, DNA-DNA hybridization, fatty acid profile and phenotypic analysis further supported the conclusion that strain AF1078(T) represents a novel Arcobacter species, for which the name Arcobacter faecis sp. nov. is proposed, with type strain AF1078(T) (=LMG 28519(T); CCUG 66484(T)). Crown Copyright © 2015. Published by Elsevier GmbH. All rights reserved.

  12. Isolated human islets require hyperoxia to maintain islet mass, metabolism, and function.

    Science.gov (United States)

    Komatsu, Hirotake; Kang, Dongyang; Medrano, Leonard; Barriga, Alyssa; Mendez, Daniel; Rawson, Jeffrey; Omori, Keiko; Ferreri, Kevin; Tai, Yu-Chong; Kandeel, Fouad; Mullen, Yoko

    2016-02-12

    Pancreatic islet transplantation has been recognized as an effective treatment for Type 1 diabetes; however, there is still plenty of room to improve transplantation efficiency. Because islets are metabolically active they require high oxygen to survive; thus hypoxia after transplant is one of the major causes of graft failure. Knowing the optimal oxygen tension for isolated islets would allow a transplant team to provide the best oxygen environment during pre- and post-transplant periods. To address this issue and begin to establish empirically determined guidelines for islet maintenance, we exposed in vitro cultured islets to different partial oxygen pressures (pO2) and assessed changes in islet volume, viability, metabolism, and function. Human islets were cultured for 7 days in different pO2 media corresponding to hypoxia (90 mmHg), normoxia (160 mmHg), and hyerpoxia (270 or 350 mmHg). Compared to normoxia and hypoxia, hyperoxia alleviated the loss of islet volume, maintaining higher islet viability and metabolism as measured by oxygen consumption and glucose-stimulated insulin secretion responses. We predict that maintaining pre- and post-transplanted islets in a hyperoxic environment will alleviate islet volume loss and maintain islet quality thereby improving transplant outcomes. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. UL146 variability among clinical isolates of Human Cytomegalovirus from Japan

    Directory of Open Access Journals (Sweden)

    Francisco Aguayo

    2010-01-01

    Full Text Available Human Cytomegalovirus (HCMV is a herpesvirus associated with serious diseases in immunocompromised subjects. The region between ORF UL133 and UL151 from HCMV, named ULb' is frequently deleted in attenuated AD169 and in highly passaged laboratory strains. However, this region is conserved in low-passaged and more virulent HCMV, like the Toledo strain. The UL146 gene, which is located in the ULb' region, encodes a CXC-chemokine analogue. The diversity of UL146 gene was evaluated among fifty-six clinical isolates of HCMV from Japan. Results show that UL146 gene was successfully amplified by the polymerase chain reaction (PCR in only 17/56 strains (30%, while the success rate for UL145/UL147 gene was 18/56 strains (32%. After DNA sequencing, the 35 amplified strains were classified into 8 groups. When compared, variability of UL146 ranged from 25.1% to 52.9% at the DNA level and from 34.5% to 67% at the amino acid level. Seven groups had the interleukin-8 (IL-8 motif ERL (Glu-Leu-Arg CXC and one group had only the CXC motif, suggesting the absence of the IL-8 function of UL146. In conclusion, we found that UL146 gene of HCMV is hyper-variable in clinical strains from Japan suggesting the possibility of a different function in each sequence group.

  14. Isolation of low-molecular-weight lead-binding protein from human erythrocytes

    International Nuclear Information System (INIS)

    Raghavan, S.R.V.; Gonick, H.C.

    1977-01-01

    In blood, lead is mainly associated with erythrocytes and only a very small amount is found in plasma. Previously it was thought that the lead was bound to the erythrocyte cell membrane but more recently it has been observed that lead is bound primarily to the cell contents, ostensibly hemoglobin. In examining the lead-binding properties of normal human erythrocytes and those of lead-exposed industrial workers, we have found that, whereas lead binds only to hemoglobin in normal erythrocytes, there is also appreciable binding of lead to a low-molecular weight-protein in erythrocytes from lead-exposed workers. The synthesis of this protein may be induced by lead exposure. The 10,000 molecular weight protein may act as a storage site and mechanism for segregating lead in a non-toxic form

  15. Detection and viability of Campylobacter species isolates from different species of poultry and humans in Sokoto State, Nigeria

    Directory of Open Access Journals (Sweden)

    I. O. Nwankwo

    2016-04-01

    Full Text Available Aim: The study was conducted to determine the prevalence and viability of Campylobacter species isolates from different species of poultry and humans in Sokoto State, Nigeria. Materials and Methods: A cross-sectional study was performed in the live birds markets, humans on admission and at outpatient clinics in the randomly selected hospitals in Sokoto State. Isolation and characterization of Campylobacter species were performed using standard culture isolation techniques and biochemical characterization. A total of 798 (506 cloacal and 292 fecal swabs from poultry and humans, respectively, were collected and analyzed. The viability of 307 isolates stored in 15% glycerol and 85% tryptone broth at −20°C was determined after 7-13 months. Results: A total of 312 (39% were positive for Campylobacter species which comprises 119 (30%, 20 (30%, 3 (14%, 9 (56%, 1 (50%, and 160 (55% in chicken, guinea fowls, pigeons, ducks, turkey, and humans, respectively. The total of 38 (24%, 63 (39%, and 59 (37% humans and 29 (19%, 79 (52%, and 44 (29% poultry isolates were positive for Campylobacter jejuni, Campylobacter Coli, and Campylobacter Lari, respectively. A total of 261 (85% of the stored isolates were still viable on re-isolation with the viability rates of 41 (95%, 67 (85%, and 17 (59% at 7, 9, and 13 months of storage, respectively. There was a negative correlation between months of storage and viability rates. However, there was no significant statistical association (p>0.05 between prevalence rate and species of poultry. Conclusion: Campylobacter species have been detected with varying degree of prevalence in both poultry and humans and their ability to survive freezing at −20°C (95% for up to 7 months has been revealed in the study. This is not only a concern to food and livestock industries but also a concern to the public health at large, especially, in view of the study area being considered one of the largest livestock producers in Nigeria

  16. Isolation of human monocytes by double gradient centrifugation and their differentiation to macrophages in teflon-coated cell culture bags.

    Science.gov (United States)

    Menck, Kerstin; Behme, Daniel; Pantke, Mathias; Reiling, Norbert; Binder, Claudia; Pukrop, Tobias; Klemm, Florian

    2014-09-09

    Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

  17. Escherichia coli Isolates from Broiler Chicken Meat, Broiler Chickens, Pork, and Pigs Share Phylogroups and Antimicrobial Resistance with Community-Dwelling Humans and Patients with Urinary Tract Infection

    DEFF Research Database (Denmark)

    Jakobsen, L.; Kurbasic, A.; Skjot-Rasmussen, L.

    2010-01-01

    Escherichia coli is the most common cause of urinary tract infection (UTI). Phylogroup B2 and D isolates are associated with UTI. It has been proposed that E. coli causing UTI could have an animal origin. The objective of this study was to investigate the phylogroups and antimicrobial resistance......, and their possible associations in E. coli isolates from patients with UTI, community-dwelling humans, broiler chicken meat, broiler chickens, pork, and pigs in Denmark. A total of 964 geographically and temporally matched E. coli isolates from UTI patients (n = 102), community-dwelling humans (n = 109), Danish (n...... resistance data, we found that UTI isolates always grouped with isolates from meat and/or animals. We detected B2 and D isolates, that are associated to UTI, among isolates from broiler chicken meat, broiler chickens, pork, and pigs. Although B2 isolates were found in low prevalences in animals and meat...

  18. Evaluation of a Single Procedure Allowing the Isolation of Enteropathogenic Yersinia along with Other Bacterial Enteropathogens from Human Stools

    Science.gov (United States)

    Savin, Cyril; Leclercq, Alexandre; Carniel, Elisabeth

    2012-01-01

    Enteropathogenic Yersinia are among the most frequent agents of human diarrhea in temperate and cold countries. However, the incidence of yersiniosis is largely underestimated because of the peculiar growth characteristics of pathogenic Yersinia, which make their isolation from poly-contaminated samples difficult. The use of specific procedures for Yersinia isolation is required, but is expensive and time consuming, and therefore is not systematically performed in clinical pathology laboratories. A means to circumvent this problem would be to use a single procedure for the isolation of all bacterial enteropathogens. Since the Statens Serum Institut enteric medium (SSI) has been reported to allow the growth at 37°C of most Gram-negative bacteria, including Yersinia, our study aimed at evaluating its performances for Yersinia isolation, as compared to the commonly used Yersinia-specific semi-selective Cefsulodin-Irgasan-Novobiocin medium (CIN) incubated at 28°C. Our results show that Yersinia pseudotuberculosis growth was strongly inhibited on SSI at 37°C, and therefore that this medium is not suitable for the isolation of this species. All Yersinia enterocolitica strains tested grew on SSI, while some non-pathogenic Yersinia species were inhibited. The morphology of Y. enterocolitica colonies on SSI allowed their differentiation from various other Gram-negative bacteria commonly isolated from stool samples. However, in artificially contaminated human stools, the recovery of Y. enterocolitica colonies on SSI at 37°C was difficult and was 3 logs less sensitive than on CIN at 28°C. Therefore, despite its limitations, the use of a specific procedure (CIN incubated at 28°C) is still required for an efficient isolation of enteropathogenic Yersinia from stools. PMID:22911756

  19. Prevalence, antimicrobial susceptibility and multiplex PCR-serotyping of Listeria monocytogenes isolated from humans, foods and livestock in Iran.

    Science.gov (United States)

    Lotfollahi, Lida; Chaharbalesh, Ardalan; Ahangarzadeh Rezaee, Mohammad; Hasani, Alka

    2017-06-01

    Listeria monocytogenes is a foodborne pathogen causing listeriosis, which potentially affects all individuals, especially pregnant women and immunocompromised persons. The present study investigated the prevalence, antimicrobial susceptibility and serotypes distribution of the isolated L. monocytogenes from Iran. Twenty two (4.97%) of 442 human, food and livestock samples were found to be positive for L. monocytogenes. L. monocytogenes was identified in 8.8% of 125 human samples, 2.99% of 267 food and 6% of 50 livestock samples. The standard disk diffusion method and minimum inhibitory concentration (MIC) assay were used for antimicrobial susceptibility testing and multiplex PCR for serotyping. Among the 22 isolates tested, 6 (27.2%) displayed resistance to penicillin G, with all of the isolates and 2 (9%) of them showing intermediate susceptibility to clindamycin and rifampicin, respectively. According to the MIC assay, the rate of resistance to penicillin G was the same as that of disk diffusion method, but 16 (72.7%) of isolates showed intermediate susceptibility to clindamycin using E-test. In the multiplex PCR, 19 (86.4%) of isolates belonged to serotype 1/2c or 3c and the remaining 3 isolates were identified as (4b, 4d or 4e) and (1/2a or 3a), respectively. The occurrence of resistance to penicillin G, which can be used in the treatment of listeriosis, is very alarming and more prevalence of 1/2c serotype, in comparison to 3 other important ones (1/2a, 1/2b and 4b), in Iran has been reported for the first time. To the best of our knowledge, this is the first study showing the distribution of various serogroups of L. monocytogenes from human and livestock in Iran. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Mycobacterium grossiae sp. nov., a rapidly growing, scotochromogenic species isolated from human clinical respiratory and blood culture specimens.

    Science.gov (United States)

    Paniz-Mondolfi, Alberto Enrique; Greninger, Alexander L; Ladutko, Lynn; Brown-Elliott, Barbara A; Vasireddy, Ravikiran; Jakubiec, Wesley; Vasireddy, Sruthi; Wallace, Richard J; Simmon, Keith E; Dunn, Bruce E; Jackoway, Gary; Vora, Surabhi B; Quinn, Kevin K; Qin, Xuan; Campbell, Sheldon

    2017-11-01

    A previously undescribed, rapidly growing, scotochromogenic species of the genus Mycobacterium (represented by strains PB739 T and GK) was isolated from two clinical sources - the sputum of a 76-year-old patient with severe chronic obstructive pulmonary disease, history of tuberculosis exposure and Mycobacterium avium complex isolated years prior; and the blood of a 15-year-old male with B-cell acute lymphoblastic leukaemia status post bone marrow transplant. The isolates grew as dark orange colonies at 25-37 °C after 5 days, sharing features in common with other closely related species. Analysis of the complete 16S rRNA gene sequence (1492 bp) of strain PB739 T demonstrated that the isolate shared 98.8 % relatedness with Mycobacterium wolinskyi. Partial 429 bp hsp65 and 744 bp rpoB region V sequence analyses revealed that the sequences of the novel isolate shared 94.8 and 92.1 % similarity with those of Mycobacterium neoaurum and Mycobacterium aurum, respectively. Biochemical profiling, antimicrobial susceptibility testing, HPLC/gas-liquid chromatography analyses and multilocus sequence typing support the taxonomic status of these isolates (PB739 T and GK) as representatives of a novel species. Both isolates were susceptible to the Clinical and Laboratory Standards Institute recommended antimicrobials for susceptibility testing of rapidly growing mycobacteria including amikacin, ciprofloxacin, moxifloxacin, doxycycline/minocycline, imipenem, linezolid, clarithromycin and trimethropin/sulfamethoxazole. Both isolates PB739 T and GK showed intermediate susceptibility to cefoxitin. We propose the name Mycobacterium grossiae sp. nov. for this novel species and have deposited the type strain in the DSMZ and CIP culture collections. The type strain is PB739 T (=DSM 104744 T =CIP 111318 T ).

  1. Investigation of an isolated case of human Crimean–Congo hemorrhagic fever in Central Uganda, 2015

    Directory of Open Access Journals (Sweden)

    Stephen Balinandi

    2018-03-01

    Full Text Available Background: Crimean–Congo hemorrhagic fever (CCHF is the most geographically widespread tick-borne viral infection. Outbreaks of CCHF in sub-Saharan Africa are largely undetected and thus under-reported. On November 9, 2015, the National Viral Hemorrhagic Fever Laboratory at the Uganda Virus Research Institute received an alert for a suspect VHF case in a 33-year-old male who presented with VHF compatible signs and symptoms at Mengo Hospital in Kampala. Methods: A blood sample from the suspect patient was tested by RT-PCR for CCHF and found positive. Serological testing on sequential blood specimens collected from this patient showed increasing anti-CCHFV IgM antibody titers, confirming recent infection. Repeat sampling of the confirmed case post recovery showed high titers for anti-CCHFV-specific IgG. An epidemiological outbreak investigation was initiated following the initial RT-PCR positive detection to identify any additional suspect cases. Results: Only a single acute case of CCHF was detected from this outbreak. No additional acute CCHF cases were identified following field investigations. Environmental investigations collected 53 tick samples, with only 1, a Boophilus decoloratus, having detectable CCHFV RNA by RT-PCR. Full-length genomic sequencing on a viral isolate from the index human case showed the virus to be related to the DRC (Africa 2 lineage. Conclusions: This is the fourth confirmed CCHF outbreak in Uganda within 2 years after more than 50 years of no reported human CCHF cases in this country. Our investigations reaffirm the endemicity of CCHFV in Uganda, and show that exposure to ticks poses a significant risk for human infection. These findings also reflect the importance of having an established national VHF surveillance system and diagnostic capacity in a developing country like Uganda, in order to identify the first cases of VHF outbreaks and rapidly respond to reduce secondary cases. Additional efforts should focus on

  2. Leptospira Serovars for Diagnosis of Leptospirosis in Humans and Animals in Africa: Common Leptospira Isolates and Reservoir Hosts.

    Science.gov (United States)

    Mgode, Georgies F; Machang'u, Robert S; Mhamphi, Ginethon G; Katakweba, Abdul; Mulungu, Loth S; Durnez, Lies; Leirs, Herwig; Hartskeerl, Rudy A; Belmain, Steven R

    2015-12-01

    The burden of leptospirosis in humans and animals in Africa is higher than that reported from other parts of the world. However, the disease is not routinely diagnosed in the continent. One of major factors limiting diagnosis is the poor availability of live isolates of locally circulating Leptospira serovars for inclusion in the antigen panel of the gold standard microscopic agglutination test (MAT) for detecting antibodies against leptospirosis. To gain insight in Leptospira serovars and their natural hosts occurring in Tanzania, concomitantly enabling the improvement of the MAT by inclusion of fresh local isolates, a total of 52 Leptospira isolates were obtained from fresh urine and kidney homogenates, collected between 1996 and 2006 from small mammals, cattle and pigs. Isolates were identified by serogrouping, cross agglutination absorption test (CAAT), and molecular typing. Common Leptospira serovars with their respective animal hosts were: Sokoine (cattle and rodents); Kenya (rodents and shrews); Mwogolo (rodents); Lora (rodents); Qunjian (rodent); serogroup Grippotyphosa (cattle); and an unknown serogroup from pigs. Inclusion of local serovars particularly serovar Sokoine in MAT revealed a 10-fold increase in leptospirosis prevalence in Tanzania from 1.9% to 16.9% in rodents and 0.26% to 10.75% in humans. This indicates that local serovars are useful for diagnosis of human and animal leptospirosis in Tanzania and other African countries.

  3. Cleistopholine isolated from Enicosanthellum pulchrum exhibits apoptogenic properties in human ovarian cancer cells.

    Science.gov (United States)

    Nordin, Noraziah; Majid, Nazia Abdul; Mohan, Syam; Dehghan, Firouzeh; Karimian, Hamed; Rahman, Mashitoh Abdul; Ali, Hapipah Mohd; Hashim, Najihah Mohd

    2016-04-15

    Cleistopholine is a natural alkaloid present in plants with numerous biological activities. However, cleistopholine has yet to be isolated using modern techniques and the mechanism by which this alkaloid induces apoptosis in cancer cells remains to be elucidated. This study aims to isolate cleistopholine from the roots of Enicosanthellum pulchrum by using preparative-HPLC technique and explore the mechanism by which this alkaloid induces apoptosis in human ovarian cancer (CAOV-3) cells in vitro from 24 to 72 h. This compound may be developed as an anticancer agent that induces apoptosis in ovarian cancer cells. Cytotoxicity was assessed via the cell viability assay and changes in cell morphology were observed via the acridine orange/propidium iodide (AO/PI) assay. The involvement of apoptotic pathways was evaluated through caspase analysis and multiple cytotoxicity assays. Meanwhile, early and late apoptotic events via the Annexin V-FITC and DNA laddering assays, respectively. The mechanism of apoptosis was explored at the molecular level by evaluating the expression of specific genes and proteins. In addition, the proliferation of CAOV-3-cells treated with cleistopholine was analysed using the cell cycle arrest assay. The IC50 of cleistopholine (61.4 µM) was comparable with that of the positive control cisplatin (62.8 µM) at 24 h of treatment. Apoptos is was evidenced by cell membrane blebbing, chromatin compression and formation of apoptotic bodies. The initial phase of apoptosis was detected at 24 h by the increase in Annexin V-FITC binding to cell membranes. A DNA ladder was formed at 48 h, indicating DNA fragmentation in the final phase of apoptosis. The mitochondria participated in the process by stimulating the intrinsic pathway via caspase 9 with a reduction in mitochondrial membrane potential (MMP) and an increase in cytochrome c release. Cell death was further validated through the mRNA and protein overexpression of Bax, caspase 3 and caspase 9 in the

  4. Identification of Brucella spp. isolated from human brucellosis in Malaysia using high-resolution melt (HRM) analysis.

    Science.gov (United States)

    Mohamed Zahidi, Jama'ayah; Bee Yong, Tay; Hashim, Rohaidah; Mohd Noor, Azura; Hamzah, Siti Hawa; Ahmad, Norazah

    2015-04-01

    Molecular approaches have been investigated to overcome difficulties in identification and differentiation of Brucella spp. using conventional phenotypic methods. In this study, high-resolution melt (HRM) analysis was used for rapid identification and differentiation of members of Brucella genus. A total of 41 Brucella spp. isolates from human brucellosis were subjected to HRM analysis using 4 sets of primers, which identified 40 isolates as Brucella melitensis and 1 as Brucella canis. The technique utilized low DNA concentration and was highly reproducible. The assay is shown to be a useful diagnostic tool, which can rapidly differentiate Brucella up to species level. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Pathogenic strains of Yersinia enterocolitica isolated from domestic dogs (Canis familiaris) belonging to farmers are of the same subtype as pathogenic Y. enterocolitica strains isolated from humans and may be a source of human infection in Jiangsu Province, China.

    Science.gov (United States)

    Wang, Xin; Cui, Zhigang; Wang, Hua; Tang, Liuying; Yang, Jinchuan; Gu, Ling; Jin, Dong; Luo, Longze; Qiu, Haiyan; Xiao, Yuchun; Xiong, Haiping; Kan, Biao; Xu, Jianguo; Jing, Huaiqi

    2010-05-01

    We isolated 326 Yersinia enterocolitica strains from 5,919 specimens from patients with diarrhea at outpatient clinics, livestock, poultry, wild animals, insect vectors, food, and the environment in the cities of Nantong and Xuzhou in Jiangsu Province, China, from 2004 to 2008. The results showed that the 12 pathogenic strains were of the O:3 serotype. Six strains were isolated from domestic dogs (Canis familiaris) belonging to farmers and were found to be the primary carriers of pathogenic Y. enterocolitica strains, especially in Xuzhou. Pulsed-field gel electrophoresis analysis of the pathogenic strains from dogs belonging to farmers showed that they shared the same patterns as strains from diarrhea patients isolated in 1994. This indicates that the strains from domestic dogs have a close correlation with the strains causing human infections.

  6. Characterization of vancomycin-resistant and vancomycin-susceptible Enterococcus faecium isolates from humans, chickens and pigs by RiboPrinting and pulsed-field gel electrophoresis

    DEFF Research Database (Denmark)

    Hammerum, Anette Marie; Fussing, Vivian; Aarestrup, Frank Møller

    2000-01-01

    Forty-eight vancomycin-resistant and 35 vancomycin-sensitive Danish Enterococcus faecium isolates obtained from pigs, chickens and humans, as well as the human vanA reference isolate BM4147, were characterized by EcoRI RiboPrinting and Smal pulsed-field gel electrophoresis. RiboPrinting of the 84...

  7. Functional investigation of β-adrenoceptors in human isolated detrusor focusing on the novel selective β3-adrenoceptor agonist KUC-7322

    NARCIS (Netherlands)

    Igawa, Yasuhiko; Schneider, Tim; Yamazaki, Yoshinobu; Tatemichi, Satoshi; Homma, Yukio; Nishizawa, Osamu; Michel, Martin C.

    2012-01-01

    This study aimed to characterize the β-adrenoceptor (β-AR) subtype mediating relaxation of isolated human bladder strips and to explore relaxation by the novel β3-AR-selective agonist KUC-7322 for its relaxant effect on the human isolated detrusor and for its effect on the carbachol (CCh)-induced

  8. Streptococcus agalactiae isolates of serotypes Ia, III and V from human and cow are able to infect tilapia.

    Science.gov (United States)

    Chen, Ming; Wang, Rui; Luo, Fu-Guang; Huang, Yan; Liang, Wan-Wen; Huang, Ting; Lei, Ai-Ying; Gan, Xi; Li, Li-Ping

    2015-10-22

    Recent studies have shown that group B streptococcus (GBS) may be infectious across hosts. The purpose of this study is to investigate the pathogenicity of clinical GBS isolates with serotypes Ia, III and V from human and cow to tilapia and the evolutionary relationship among these GBS strains of different sources. A total of 27 clinical GBS isolates from human (n=10), cow (n=2) and tilapia (n=15) were analyzed using serotyping, multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Among them, 15 isolates were tested for their pathogenicity to tilapia. The results showed that five human GBS strains (2 serotype III, 2 serotype Ia and 1 serotype V) infected tilapia with mortality rate ranging from 56.67% to 100%, while the other five human GBS strains tested were unable to infect tilapia. In addition, two cow GBS strains C001 and C003 of serotype III infected tilapia. However, they had significantly lower pathogenicity than the five human strains. Furthermore, human GBS strains H005 and H008, which had very strong ability to infect tilapia, had the same PFGE pattern. MLST analysis showed that the five human and the two cow GBS strains that were able to infect tilapia belonged to clonal complexes CC19, CC23 and CC103. The study for the first time confirmed that human or cow GBS clonal complexes CC19, CC23 and CC103 containing strains with serotypes Ia, III and V could infect tilapia and induce clinical signs under experimental conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Human isolates of Salmonella enterica serovar Typhimurium from Taiwan displayed significantly higher levels of antimicrobial resistance than those from Denmark.

    Science.gov (United States)

    Torpdahl, Mia; Lauderdale, Tsai-Ling; Liang, Shiu-Yun; Li, Ishien; Wei, Sung-Hsi; Chiou, Chien-Shun

    2013-02-01

    Salmonella enterica serovar Typhimurium is a major zoonotic pathogen with a high prevalence of antimicrobial resistance. This pathogen can disseminate across borders and spread far distances via the food trade and international travel. In this study, we compared the genotypes and antimicrobial resistance of 378 S. Typhimurium isolates collected in Taiwan and Denmark between 2009 and 2010. Genotyping revealed that many S. Typhimurium strains were concurrently circulating in Taiwan, Denmark and other countries in 2009 and 2010. When compared to the isolates collected from Denmark, the isolates from Taiwan displayed a significantly higher level of resistance to 11 of the 12 tested antimicrobials. Seven genetic clusters (A-G) were designated for the isolates. A high percentage of the isolates in genetic clusters C, F and G were multidrug-resistant. Of the isolates in cluster C, 79.2% were ASSuT-resistant, characterized by resistance to ampicillin, streptomycin, sulfamethoxazole, and tetracycline. In cluster F, 84.1% of the isolates were ACSSuT-resistant (resistant to ASSuT and chloramphenicol). Cluster G was unique to Taiwan and characterized in most isolates by the absence of three VNTRs (ST20, ST30 and STTR6) as well as a variety of multidrug resistance profiles. This cluster exhibited very high to extremely high levels of resistance to several first-line drugs, and among the seven clusters, it displayed the highest levels of resistance to cefotaxime and ceftazidime, ciprofloxacin and gentamicin. The high prevalence of antimicrobial resistance in S. Typhimurium from Taiwan highlights the necessity to strictly regulate the use of antimicrobials in the agriculture and human health care sectors. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Neutral endopeptidase up-regulation in isolated human umbilical artery: involvement in desensitization of bradykinin-induced vasoconstrictor effects.

    Science.gov (United States)

    Pelorosso, Facundo Germán; Halperin, Ana Verónica; Palma, Alejandro Martín; Nowak, Wanda; Errasti, Andrea Emilse; Rothlin, Rodolfo Pedro

    2007-02-01

    Previous reports show that bradykinin B(2) receptors mediate contractile responses induced by bradykinin (BK) in human umbilical artery (HUA). However, although it has been reported that BK-induced responses can desensitize in several inflammatory models, the effects of prolonged in vitro incubation on BK-induced vasoconstriction in HUA have not been studied. In isolated HUA rings, BK-induced responses after a 5-h in vitro incubation showed a marked desensitization compared with responses at 2 h. Inhibition of either angiotensin-converting enzyme (ACE) or neutral endopeptidase (NEP), both BK-inactivating enzymes, failed to modify responses to BK at 2 h. After 5 h, ACE inhibition produced only a slight potentiation of BK-induced responses. In contrast, BK-induced vasoconstriction at 5 h was markedly potentiated by NEP inhibition. Moreover, NEP activity, measured by hydrolysis of its synthetic substrate (Z-Ala-Ala-Leu-p-nitroanilide), showed a 2.4-fold increase in 5-h incubated versus 2-h incubated tissues, which was completely reversed by cycloheximide (CHX) treatment. Furthermore, CHX significantly potentiated BK-induced responses, suggesting that NEP-mediated kininase activity increase at 5 h depends on de novo protein synthesis. In addition, under NEP inhibition, CHX treatment failed to produce an additional potentiation of BK-induced vasoconstriction. Still, NEP up-regulation was confirmed by Western blot, showing a 2.1-fold increase in immunoreactive NEP in 5-h incubated versus 2-h incubated HUA. In summary, the present study provides strong pharmacological evidence that NEP is up-regulated and plays a key role in desensitization of BK-induced vasoconstriction after prolonged in vitro incubation in HUA. Our results provide new insights into the possible mechanisms involved in BK-induced response desensitization during sustained inflammatory conditions.

  11. Comparison of potentials between stem cells isolated from human anterior cruciate ligament and bone marrow for ligament tissue engineering.

    Science.gov (United States)

    Cheng, Ming-Te; Liu, Chien-Lin; Chen, Tain-Hsiung; Lee, Oscar K

    2010-07-01

    We have previously isolated and identified stem cells from human anterior cruciate ligament (ACL). The purpose of this study was to evaluate the differences in proliferation, differentiation, and extracellular matrix (ECM) formation abilities between bone marrow stem cells (BMSCs) and ACL-derived stem cells (LSCs) from the same donors when cultured with different growth factors, including basic fibroblast growth factor (bFGF), epidermal growth factor, and transforming growth factor-beta 1 (TGF-beta1). Ligament tissues and bone marrow aspirate were obtained from patients undergoing total knee arthroplasty and ACL reconstruction surgeries. Proliferation, colony formation, and population doubling capacity as well as multilineage differentiation potentials of LSCs and BMSCs were compared. Gene expression and ECM production for ligament engineering were also evaluated. It was found that BMSCs possessed better osteogenic differentiation potential than LSCs, while similar adipogenic and chondrogenic differentiation abilities were observed. Proliferation rates of both LSCs and BMSCs were enhanced by bFGF and TGF-beta1. TGF-beta1 treatment significantly increased the expression of type I collagen, type III collagen, fibronectin, and alpha-smooth muscle actin in LSCs, but TGF-beta1 only upregulated type I collagen and tenascin-c in BMSCs. Protein quantification further confirmed the results of differential gene expression and suggested that LSCs and BMSCs increase ECM production upon TGF-beta1 treatment. In summary, in comparison with BMSCs, LSCs proliferate faster and maintain an undifferentiated state with bFGF treatment, whereas under TGF-beta1 treatment, LSCs upregulate major tendinous gene expression and produce a robust amount of ligament ECM protein, making LSCs a potential cell source in future applications of ACL tissue engineering.

  12. Effect of gamma rays on antibiotic resistance of Staphylococcus aureus and Pseudomonas aeruginosa isolated from human skin

    International Nuclear Information System (INIS)

    Shokier, H. A.; EI-Adly, A.A.; Hussein, H.; Shabon, M. H.; EI-Shanshoury, I.H.

    2010-01-01

    Seventy one samples were randomly collected from patients suffering from different bacterial skin infections. Forty isolates could not grow on the artificial media after second subculture while 31 isolates were able to survive. Twenty six of them were identified as Staphylococcus aureus and 5 were identified as Pseudomonas aeruginosa. The isolated strains were tested for their susceptibilities to gentamycin, ampicillin, ciprofloxacin and amoxicillin antibiotics .Up to 88.4% of S. aureus and of 80% of P.aeruginosa isolates were found to be resistant to ampicillin. On the other. hand, about 30.7% of S. aureus and 20% of P. aeruginosa were resistant to ciprofloxacin reveals the lowest antibiotic resistance . The antibiotic sensitivity was retested for the most resistant bacterial isolates after irradiated by different doses of gamma radiation (0.5,1, 2 Gy). The previous doses increased S .aureus inhibition zone to gentamycin, from 7.5 mm for unirradiated cells to 25 mm for irradiated one. While ciprofloxacin inhibition zone increased from 1.5 cm to 3 cm in doses of 0.5 to 2.0 Gy. S. aureus sensitivity to amoxicillin increased from 0.0 to 1.0 cm inhibition zone with increase in dose from 0.5 to 2.0 Gy.While the previous doses had no effect on ampicillin resistance. The same doses increased P. aeruginosa isolate resistance. Very low doses of gamma irradiation increased S.aureus and P. aeruginosa capsule production, also increased the release rate of capsule content in both types of bacteria.

  13. Optimized exosome isolation protocol for cell culture supernatant and human plasma

    Directory of Open Access Journals (Sweden)

    Richard J. Lobb

    2015-07-01

    Full Text Available Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a

  14. Campylobacter troglodytis sp. nov., Isolated from Feces of Human-Habituated Wild Chimpanzees (Pan troglodytes schweinfurthii) in Tanzania ▿

    Science.gov (United States)

    Kaur, Taranjit; Singh, Jatinder; Huffman, Michael A.; Petrželková, Klára J.; Taylor, Nancy S.; Xu, Shilu; Dewhirst, Floyd E.; Paster, Bruce J.; Debruyne, Lies; Vandamme, Peter; Fox, James G.

    2011-01-01

    The transmission of simian immunodeficiency and Ebola viruses to humans in recent years has heightened awareness of the public health significance of zoonotic diseases of primate origin, particularly from chimpanzees. In this study, we analyzed 71 fecal samples collected from 2 different wild chimpanzee (Pan troglodytes) populations with different histories in relation to their proximity to humans. Campylobacter spp. were detected by culture in 19/56 (34%) group 1 (human habituated for research and tourism purposes at Mahale Mountains National Park) and 0/15 (0%) group 2 (not human habituated but propagated from an introduced population released from captivity over 30 years ago at Rubondo Island National Park) chimpanzees, respectively. Using 16S rRNA gene sequencing, all isolates were virtually identical (at most a single base difference), and the chimpanzee isolates were most closely related to Campylobacter helveticus and Campylobacter upsaliensis (94.7% and 95.9% similarity, respectively). Whole-cell protein profiling, amplified fragment length polymorphism analysis of genomic DNA, hsp60 sequence analysis, and determination of the mol% G+C content revealed two subgroups among the chimpanzee isolates. DNA-DNA hybridization experiments confirmed that both subgroups represented distinct genomic species. In the absence of differential biochemical characteristics and morphology and identical 16S rRNA gene sequences, we propose to classify all isolates into a single novel nomenspecies, Campylobacter troglodytis, with strain MIT 05-9149 as the type strain; strain MIT 05-9157 is suggested as the reference strain for the second C. troglodytis genomovar. Further studies are required to determine whether the organism is pathogenic to chimpanzees and whether this novel Campylobacter colonizes humans and causes enteric disease. PMID:21278267

  15. Campylobacter troglodytis sp. nov., isolated from feces of human-habituated wild chimpanzees (Pan troglodytes schweinfurthii) in Tanzania.

    Science.gov (United States)

    Kaur, Taranjit; Singh, Jatinder; Huffman, Michael A; Petrzelková, Klára J; Taylor, Nancy S; Xu, Shilu; Dewhirst, Floyd E; Paster, Bruce J; Debruyne, Lies; Vandamme, Peter; Fox, James G

    2011-04-01

    The transmission of simian immunodeficiency and Ebola viruses to humans in recent years has heightened awareness of the public health significance of zoonotic diseases of primate origin, particularly from chimpanzees. In this study, we analyzed 71 fecal samples collected from 2 different wild chimpanzee (Pan troglodytes) populations with different histories in relation to their proximity to humans. Campylobacter spp. were detected by culture in 19/56 (34%) group 1 (human habituated for research and tourism purposes at Mahale Mountains National Park) and 0/15 (0%) group 2 (not human habituated but propagated from an introduced population released from captivity over 30 years ago at Rubondo Island National Park) chimpanzees, respectively. Using 16S rRNA gene sequencing, all isolates were virtually identical (at most a single base difference), and the chimpanzee isolates were most closely related to Campylobacter helveticus and Campylobacter upsaliensis (94.7% and 95.9% similarity, respectively). Whole-cell protein profiling, amplified fragment length polymorphism analysis of genomic DNA, hsp60 sequence analysis, and determination of the mol% G+C content revealed two subgroups among the chimpanzee isolates. DNA-DNA hybridization experiments confirmed that both subgroups represented distinct genomic species. In the absence of differential biochemical characteristics and morphology and identical 16S rRNA gene sequences, we propose to classify all isolates into a single novel nomenspecies, Campylobacter troglodytis, with strain MIT 05-9149 as the type strain; strain MIT 05-9157 is suggested as the reference strain for the second C. troglodytis genomovar. Further studies are required to determine whether the organism is pathogenic to chimpanzees and whether this novel Campylobacter colonizes humans and causes enteric disease.

  16. Receptor-binding properties of modern human influenza viruses primarily isolated in Vero and MDCK cells and chicken embryonated eggs

    International Nuclear Information System (INIS)

    Mochalova, Larisa; Gambaryan, Alexandra; Romanova, Julia; Tuzikov, Alexander; Chinarev, Alexander; Katinger, Dietmar; Katinger, Herman; Egorov, Andrej; Bovin, Nicolai

    2003-01-01

    To study the receptor specificity of modern human influenza H1N1 and H3N2 viruses, the analogs of natural receptors, namely sialyloligosaccharides conjugated with high molecular weight (about 1500 kDa) polyacrylamide as biotinylated and label-free probes, have been used. Viruses isolated from clinical specimens were grown in African green monkey kidney (Vero) or Madin-Darby canine kidney (MDCK) cells and chicken embryonated eggs. All Vero-derived viruses had hemagglutinin (HA) sequences indistinguishable from original viruses present in clinical samples, but HAs of three of seven tested MDCK-derived isolates had one or two amino acid substitutions. Despite these host-dependent mutations and differences in the structure of HA molecules of individual strains, all studied Vero- and MDCK-isolated viruses bound to Neu5Ac α2-6Galβ1-4GlcNAc (6'SLN) essentially stronger than to Neu5Acα2-6Galβ1-4Glc (6'SL). Such receptor-binding specificity has been typical for earlier isolated H1N1 human influenza viruses, but there is a new property of H3N2 viruses that has been circulating in the human population during recent years. Propagation of human viruses in chicken embryonated eggs resulted in a selection of variants with amino acid substitutions near the HA receptor-binding site, namely Gln226Arg or Asp225Gly for H1N1 viruses and Leu194Ile and Arg220Ser for H3N2 viruses. These HA mutations disturb the observed strict 6'SLN specificity of recent human influenza viruses

  17. Christensenella timonensis, a new bacterial species isolated from the human gut

    Directory of Open Access Journals (Sweden)

    S. Ndongo

    2016-09-01

    Full Text Available We propose a new species, Christensenella timonensis, strain Marseille-P2437T (CSUR P2437T, which was isolated from gut microbiota of a 66-year-old patient as a part of culturomics study. C. timonensis represents the second species isolated within the Christensenella genus.

  18. Isolation and sequence of complementary DNA encoding human extracellular superoxide dismutase

    International Nuclear Information System (INIS)

    Hjalmarsson, K.; Marklund, S.L.; Engstroem, A.; Edlund, T.

    1987-01-01

    A complementary DNA (cDNA) clone from a human placenta cDNA library encoding extracellular superoxide dismutase has been isolated and the nucleotide sequence determined. The cDNA has a very high G + C content. EC-SOD is synthesized with a putative 18-amino acid signal peptide, preceding the 222 amino acids in the mature enzyme, indicating that the enzyme is a secretory protein. The first 95 amino acids of the mature enzyme show no sequence homology with other sequenced proteins and there is one possible N-glycosylation site (Asn-89). The amino acid sequence from residues 96-193 shows strong homology (∼ 50%) with the final two-thirds of the sequences of all know eukaryotic CuZn SODs, whereas the homology with the P. leiognathi CuZn SOD is clearly lower. The ligands to Cu and Zn, the cysteines forming the intrasubunit disulfide bridge in the CuZn SODs, and the arginine found in all CuZn SODs in the entrance to the active site can all be identified in EC-SOD. A comparison with bovine CuZn SOD, the three-dimensional structure of which is known, reveals that the homologies occur in the active site and the divergencies are in the part constituting the subunit contact area in CuZn SOD. Amino acid sequence 194-222 in the carboxyl-terminal end of EC-SOD is strongly hydrophilic and contains nine amino acids with a positive charge. This sequence probably confers the affinity of EC-SOD for heparin and heparan sulfate. An analysis of the amino acid sequence homologies with CuZn SODs from various species indicates that the EC-SODs may have evolved form the CuZn SODs before the evolution of fungi and plants

  19. Isolation and identification of compounds from Kalanchoe pinnata having human alphaherpesvirus and vaccinia virus antiviral activity.

    Science.gov (United States)

    Cryer, Matthew; Lane, Kyle; Greer, Mary; Cates, Rex; Burt, Scott; Andrus, Merritt; Zou, Jiping; Rogers, Paul; Hansen, Marc D H; Burgado, Jillybeth; Panayampalli, Subbian Satheshkumar; Day, Craig W; Smee, Donald F; Johnson, Brent F

    2017-12-01

    Kalanchoe pinnata (Lam.) Pers. (Crassulaceae) is a succulent plant that is known for its traditional antivirus and antibacterial usage. This work examines two compounds identified from the K. pinnata plant for their antivirus activity against human alphaherpesvirus (HHV) 1 and 2 and vaccinia virus (VACV). Compounds KPB-100 and KPB-200 were isolated using HPLC and were identified using NMR and MS. Both compounds were tested in plaque reduction assay of HHV-2 wild type (WT) and VACV. Both compounds were then tested in virus spread inhibition and virus yield reduction (VYR) assays of VACV. KPB-100 was further tested in viral cytopathic effect (CPE) inhibition assay of HHV-2 TK-mutant and VYR assay of HHV-1 WT. KPB-100 and KPB-200 inhibited HHV-2 at IC 50 values of 2.5 and 2.9 μg/mL, respectively, and VACV at IC 50 values of 3.1 and 7.4 μg/mL, respectively, in plaque reduction assays. In virus spread inhibition assay of VACV KPB-100 and KPB-200 yielded IC 50 values of 1.63 and 13.2 μg/mL, respectively, and KPB-100 showed a nearly 2-log reduction in virus in VYR assay of VACV at 20 μg/mL. Finally, KPB-100 inhibited HHV-2 TK- at an IC 50 value of 4.5 μg/mL in CPE inhibition assay and HHV-1 at an IC 90 of 3.0 μg/mL in VYR assay. Both compounds are promising targets for synthetic optimization and in vivo study. KPB-100 in particular showed strong inhibition of all viruses tested.

  20. Free hemoglobin enhances tumor necrosis factor-alpha production in isolated human monocytes.

    Science.gov (United States)

    Carrillo, Eddy H; Gordon, Laura E; Richardson, J David; Polk, Hiram C

    2002-03-01

    A systemic inflammatory response (SIR) is seen in approximately 75% of patients with complex blunt liver injuries treated nonoperatively. Many feel this response is caused by blood, bile, and necrotic tissue accumulation in the peritoneal cavity. Our current treatment for these patients is a delayed laparoscopic washout of the peritoneal cavity, resulting in a dramatic resolution of the SIR. Spectrophotometric analysis of the intraperitoneal fluid has confirmed the presence of high concentrations of free hemoglobin (Hb). We hypothesize that free Hb enhances the local peritoneal response by increasing tumor necrosis factor-alpha (TNF-alpha) production by monocytes, contributing to the local inflammatory response and SIR. Monocytes from five healthy volunteers were isolated and cultured in RPMI-1640 for 24 hours. Treatment groups included saline controls, lipopolysaccharide ([LPS], 10 ng/mL, from Escherichia coli), human Hb (25 microg/mL), and Hb + LPS. Supernatants were analyzed by enzyme-linked immunosorbent assay. Student's t test with Mann-Whitney posttest was used for statistical analysis with p < or = 0.05 considered significant. Free Hb significantly increased TNF-alpha production 915 +/- 223 pg/mL versus saline (p = 0.02). LPS and Hb + LPS further increased TNF-alpha production (2294 pg/mL and 2501 pg/mL, respectively, p < 0.001) compared with saline controls. These data confirm that free Hb is a proinflammatory mediator resulting in the production of significant amounts of TNF-alpha. These in vitro findings support our clinical data in which timely removal of intraperitoneal free hemoglobin helps prevent its deleterious local and systemic inflammatory effects in patients with complex liver injuries managed nonoperatively.

  1. MSCs can be differentially isolated from maternal, middle and fetal segments of the human umbilical cord.

    Science.gov (United States)

    Lim, Jezamine; Razi, Zainul Rashid Mohamad; Law, Jiaxian; Nawi, Azmawati Mohammed; Idrus, Ruszymah Binti Haji; Ng, Min Hwei

    2016-12-01

    Human Wharton's jelly-derived mesenchymal stromal cells (hWJMSCs) are possibly the most suitable allogeneic cell source for stromal cell therapy and tissue engineering applications because of their hypo-immunogenic and non-tumorigenic properties, easy availability and minimal ethical concerns. Furthermore, hWJMSCs possess unique properties of both adult mesenchymal stromal cells and embryonic stromal cells. The human umbilical cord (UC) is approximately 50-60 cm long and the existing studies in the literature have not provided information on which segment of the UC was studied. In this study, hWJMSCs derived from three anatomical segments of the UC are compared. Three segments of the whole UC, each 3 cm in length, were identified anatomically as the maternal, middle and fetal segments. The hWJMSCs from the different segments were analyzed via trypan blue exclusion assay to determine the growth kinetics and cell viability, flow cytometry for immunophenotyping and immunofluorescence and reverse transcriptase polymerase chain reaction (RT-PCR) for expression of stromal cell transcriptional factors. Furthermore, the trilineage differentiation potential (osteogenic, adipogenic and chondrogenic) of these cells was also assessed. hWJMSCs isolated from the maternal and fetal segments displayed greater viability and possessed a significantly higher proliferation rate compared with cells from the middle segment. Immunophenotyping revealed that hWJMSCs derived from all three segments expressed the MSC markers CD105, CD73, CD90, CD44, CD13 and CD29, as well as HLA-ABC and HLA-DR, but were negative for hematopoietic markers CD14, CD34 and CD45. Analysis of the embryonic markers showed that all three segments expressed Nanog and Oct 3/4, but only the maternal and fetal segments expressed SSEA 4 and TRA-160. Cells from all three segments were able to differentiate into chondrogenic, osteogenic and adipogenic lineages with the middle segments showing much lower differentiation

  2. Evaluation of probiotic properties of Lactobacillus plantarum WLPL04 isolated from human breast milk.

    Science.gov (United States)

    Jiang, Meiling; Zhang, Fen; Wan, Cuixiang; Xiong, Yonghua; Shah, Nagendra P; Wei, Hua; Tao, Xueying

    2016-03-01

    Lactobacillus plantarum WLPL04, a specific strain isolated from human breast milk, was investigated for its survival capacity (acid and bile salt tolerance, survival in simulated gastrointestinal tract, inhibition of pathogens, antibiotic susceptibility, yield of exopolysaccharides) and probiotic properties (antiadhesion of pathogens, protection from harmful effect of sodium dodecyl sulfate, and antiinflammatory stress on Caco-2 cells). The results showed that Lb. plantarum WLPL04 had broad-spectrum activity against gram-positive strains (Listeria monocytogenes CMCC54007, Bacillus cereus ATCC14579, and Staphylococcus aureus CMCC26003) and gram-negative strains (Pseudomonas aeruginosa MCC10104, Shigella sonnei ATCC25931, Enterobacter sakazakii ATCC29544, Salmonella typhimurium ATCC13311, and Escherichia coli O157:H7). Antibiotic susceptibility tests showed that Lb. plantarum WLPL04 was susceptible to 8 of 14 antibiotics (e.g., erythromycin and nitrofurantoin) and resistant to 6 of 14 antibiotics (e.g., kanamycin and bacitracin). Lactobacillus plantarum WLPL04 was able to survive at pH 2.5 for 3h and at 0.45% bile salt for 12h, suggesting that it can survive well in the gastrointestinal tract. In addition, the exopolysaccharide yield of Lb. plantarum WLPL04 reached 426.73 ± 65.56 mg/L at 24h. With strategies of competition, inhibition, and displacement, Lb. plantarum WLPL04 reduced the adhesion of E. coli O157:H7 (35.51%), Sal. typhimurium ATCC 13311 (8.10%), and Staph. aureus CMCC 26003 (40.30%) on Caco-2 cells by competition, and subsequently by 59.80, 62.50, and 42.60%, respectively, for the 3 pathogens through inhibition, and by 75.23, 39.97, and 52.88%, respectively, through displacement. Lactobacillus plantarum WLPL04 attenuated the acute stress induced by sodium dodecyl sulfate on Caco-2 cells and significantly inhibited the expression of inflammatory cytokines (IL-6, IL-8 and tumor necrosis factor-α) on Caco-2 cells but increased IL-10 expression in vitro

  3. Isolation and Characterization of a Human Intestinal Bacterium Eggerthella sp. AUH-JLD49s for the Conversion of (-)-3'-Desmethylarctigenin.

    Science.gov (United States)

    Wang, Ye; Yu, Fei; Liu, Ming-Yue; Zhao, Yi-Kai; Wang, Dong-Ming; Hao, Qing-Hong; Wang, Xiu-Ling

    2017-05-24

    Arctiin is the most abundant bioactive compound contained in the Arctium lappa plant. In our previous study, we isolated one single bacterium capable of bioconverting arctigenin, an aglycone of arctiin, to 3'-desmethylarctigenin (3'-DMAG) solely. However, to date, a specific bacterium capable of producing other arctiin metabolites has not been reported. In this study, we isolated one single bacterium, which we named Eggerthella sp. AUH-JLD49s, capable of bioconverting 3'-DMAG under anaerobic conditions. The metabolite of 3'-DMAG by strain AUH-JLD49s was identified as 3'-desmethyl-4'-dehydroxyarctigenin (DMDH-AG) based on electrospray ionization mass spectrometry (ESI-MS) and 1 H and 13 C nuclear magnetic resonance spectroscopy. The bioconversion kinetics and bioconversion capacity of strain AUH-JLD49s were investigated. In addition, the metabolite DMDH-AG showed an inhibitory effect on cell growth of human colon cancer cell line HCT116 and human breast cancer cell line MDA-MB-231.

  4. Broiler chickens, broiler chicken meat, pigs and pork as sources of ExPEC related virulence genes and resistance in Escherichia coli isolates from community-dwelling humans and UTI patients.

    Science.gov (United States)

    Jakobsen, Lotte; Spangholm, Daniel J; Pedersen, Karl; Jensen, Lars B; Emborg, Hanne-Dorthe; Agersø, Yvonne; Aarestrup, Frank M; Hammerum, Anette M; Frimodt-Møller, Niels

    2010-08-15

    Urinary tract infection (UTI) is one of the most common bacterial infections. UTI is primarily caused by extraintestinal pathogenic Escherichia coli (ExPEC) from the patients' own fecal flora. The ExPEC often belong to phylogroups B2 and D, the groups which include potent human ExPEC isolates causing UTI, bacteremia, and meningitis. The external sources of these ExPEC in the human intestine are unknown. The food supply may transmit ExPEC to humans. However, evidence of this hypothesis is limited. To assess this hypothesis, the objective of our study was to investigate the presence of ExPEC related virulence genes in E. coli isolates from UTI patients, community-dwelling humans, meat, and production animals. Accordingly, we included 964 geographically and temporally matched E. coli isolates from UTI patients (n=102), community-dwelling humans (n=109), fresh Danish (n=197) and imported broiler chicken meat (n=86), broiler chickens (n=138), fresh Danish (n=177) and imported pork (n=10), and pigs (n=145) in the study. All isolates were investigated for the presence of eight ExPEC related genes (kpsM II, papA, papC, iutA, sfaS, focG, afa, hlyD) using PCR. To investigate any similarities between isolates from the different origins, we performed a cluster analysis including antimicrobial resistance data previously published. We detected seven of the eight ExPEC related genes in isolates from broiler chicken meat, broiler chickens, pork and pigs. Our findings suggest that broiler chicken meat, broiler chickens, pork and pigs could be the source of strains with these ExPEC related virulence genes in community-dwelling humans and UTI patients. Especially detection of ExPEC related virulence genes in isolates belonging to phylogroups B2 and D is very concerning and may have a significant medical impact. The cluster analysis of virulence gene and antimicrobial resistance profiles showed strong similarities between UTI patient, community-dwelling human isolates, meat, and

  5. Infection of human and non-human cells by a highly fusogenic primary CD4-independent HIV-1 isolate with a truncated envelope cytoplasmic tail

    International Nuclear Information System (INIS)

    Saha, Kunal; Yan Hui; Nelson, Julie A.E.; Zerhouni-Layachi, Bouchra

    2005-01-01

    Truncation of the envelope cytoplasmic tail has enabled FIV, SIV, and some laboratory HIV-1 strains to acquire broader cellular tropism and enhanced fusogenicity. Here we have characterized a primary CD4-independent HIV-1 isolate (92UG046-T8) with a truncated cytoplasmic tail that was able to infect and induce syncytia in primary lymphocytes from human, chimpanzee, and monkey, as well as CD4-negative cell lines from human and monkey. Increased syncytia were also noticeable with 293 cells expressing the cloned envelope from the 92UG046-T8 isolate suggesting envelope-mediated cellular fusion. Except pooled serum from HIV-1-infected individuals, monoclonal anti-envelope antibodies or antibodies/antagonists against CD4, CXCR4, and CCR5 were not able to prevent infection by the 92UG046-T8 isolate. This is the first report showing a primary HIV-1 variant with truncated cytoplasmic tail which is highly fusogenic and can infect a broad range of cells from human and non-human origins. In vivo evolution of similar HIV-1 mutants may have important implications in AIDS pathogenesis

  6. Draft genome sequencing of giardia intestinalis assemblage B isolate GS: is human giardiasis caused by two different species?

    Directory of Open Access Journals (Sweden)

    Oscar Franzén

    2009-08-01

    Full Text Available Giardia intestinalis is a major cause of diarrheal disease worldwide and two major Giardia genotypes, assemblages A and B, infect humans. The genome of assemblage A parasite WB was recently sequenced, and the structurally compact 11.7 Mbp genome contains simplified basic cellular machineries and metabolism. We here performed 454 sequencing to 16x coverage of the assemblage B isolate GS, the only Giardia isolate successfully used to experimentally infect animals and humans. The two genomes show 77% nucleotide and 78% amino-acid identity in protein coding regions. Comparative analysis identified 28 unique GS and 3 unique WB protein coding genes, and the variable surface protein (VSP repertoires of the two isolates are completely different. The promoters of several enzymes involved in the synthesis of the cyst-wall lack binding sites for encystation-specific transcription factors in GS. Several synteny-breaks were detected and verified. The tetraploid GS genome shows higher levels of overall allelic sequence polymorphism (0.5 versus <0.01% in WB. The genomic differences between WB and GS may explain some of the observed biological and clinical differences between the two isolates, and it suggests that assemblage A and B Giardia can be two different species.

  7. Purification of human growth hormone. Isolation of the 22k variant. Assays of biological and radioimmunological activities

    International Nuclear Information System (INIS)

    Camillo, M.A.P.

    1985-01-01

    An extraction method for hGH from frozen human pituitaries with high yield: 8,7 mg per processed gland, is described. Isoelectric focusing studies made it possible to isolate and integral variant of the total preparation of hGH. After liophylization the protein was analysed in polyacrylamide gel electrophoresis showing only one protein band that corresponds to the 22K variant. (M.A.C.) [pt

  8. Antimicrobial susceptibility of lactic acid bacteria isolated from human and food-producing animal feces in Khon Kaen Province, Thailand.

    Science.gov (United States)

    Sornplang, Pairat; Sakulsawasdiphan, Kattinet; Piyadeatsoontorn, Sudthidol; Surasorn, Benyapha

    2016-12-01

    The aim of this study was to investigate the susceptibility of 93 Lactobacillus strains to seven antimicrobial agents, i.e., penicillin G, amoxicillin-clavulanic acid, vancomycin, tetracycline, streptomycin, ciprofloxacin, and sulfamethoxazole-trimethoprim, by disk diffusion test. The Lactobacillus strains were isolated from fecal samples taken from 90 healthy, food-producing animals (fattening pigs, free-grazing ducks, and beef cattle) and 30 healthy human subjects (1- to 6-year-olds) in Khon Kaen. The minimum inhibitory concentration (MIC) values of tetracycline and ciprofloxacin against all strains were determined using the E-test. All 93 Lactobacillus isolates were identified at the species level using 16S rRNA gene sequencing. The most common species of Lactobacillus isolated from fattening pigs, free-grazing ducks, beef cattle, and humans were L. reuteri (30 %), L. salivarius (46.7 %), L. acetotolerans (20 %), and L. gasseri (33.3 %), respectively. A total of 83 Lactobacillus strains were resistant to the examined antibiotics. Some strains were resistant to two to six types of antibiotics. More than 50 % of Lactobacillus species were intrinsically resistant to vancomycin, streptomycin, ciprofloxacin, and sulfamethoxazole-trimethoprim. The prevalence of acquired resistance to tetracycline was observed for Lactobacillus isolates from fattening pigs, humans, free-grazing ducks, and beef cattle at 92.3, 85.7, 77.8, and 68.4 %, respectively. These results demonstrate the impact of antibiotic use in human and veterinary medicine on antibiotic treatment efficacy and may support the spread of transferable antibiotic resistant genes to other bacteria via the food chain.

  9. Draft genome sequence of a human-associated isolate of Haloferax alexandrinus strain Arc-hr, an extremely halophilic archaea.

    Science.gov (United States)

    Khelaifia, S; Caputo, A; Djossou, F; Raoult, D

    2017-01-01

    We report the draft genome sequence of Haloferax alexandrinus strain Arc-hr (CSUR P798), isolated from the human gut of a 10-year-old Amazonian individual. Its 3 893 626 bp genome exhibits a 66.00% GC content. The genome of the strain Arc-hr contains 37 genes identified as ORFans, seven genes associated to halocin and 11 genes associated with polyketide synthases or nonribosomal peptide synthetases.

  10. A variety of human monoclonal antibodies against epidermal growth factor receptor isolated from a phage antibody library.

    Science.gov (United States)

    Kurosawa, Gene; Kondo, Mariko; Kurosawa, Yoshikazu

    2016-11-04

    When the technology for constructing human antibody (Ab) libraries using a phage-display system was developed, many researchers in Ab-related fields anticipated that it would be widely applied to the development of pharmaceutical drugs against various diseases, including cancers. However, successful examples of such applications are very limited. Moreover, researchers who utilize phage-display technology now show divergent ways of thinking about phage Ab libraries. For example, there is debate about what should be the source of V H and V L genes for the construction of libraries to cover the whole repertoire of Abs present in the human body. In the immune system, the introduction of mutations into V genes followed by selection based on binding activity, termed Ab maturation, is required for the production of Abs exhibiting high affinity to the antigen (Ag). Therefore, introduction of mutations and selection are required for isolation of Abs with high affinity after isolation of clones from phage Ab libraries. We constructed a large human Ab library termed AIMS, developed a screening method termed ICOS, and succeeded in isolating many human monoclonal Abs (mAbs) that specifically and strongly bind to various tumor-associated Ags. Eight anti-EGFR mAbs were included, which we characterized. These mAbs showed various different activities against EGFR-expressing cancer cells. In this paper, we describe these data and discuss the possibility and necessity that the mAbs isolated from the AIMS library might be developed as therapeutic drugs against cancers without introduction of mutations. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  11. A Method to Identify and Isolate Pluripotent Human Stem Cells and Mouse Epiblast Stem Cells Using Lipid Body-Associated Retinyl Ester Fluorescence

    OpenAIRE

    Thangaselvam Muthusamy; Odity Mukherjee; Radhika Menon; Megha Prakash Bangalore; Mitradas M. Panicker

    2014-01-01

    Summary We describe the use of a characteristic blue fluorescence to identify and isolate pluripotent human embryonic stem cells and human-induced pluripotent stem cells. The blue fluorescence emission (450–500 nm) is readily observed by fluorescence microscopy and correlates with the expression of pluripotency markers (OCT4, SOX2, and NANOG). It allows easy identification and isolation of undifferentiated human pluripotent stem cells, high-throughput fluorescence sorting and subsequent propa...

  12. Phosphoproteome analysis of functional mitochondria isolated from resting human muscle reveals extensive phosphorylation of inner membrane protein complexes and enzymes

    DEFF Research Database (Denmark)

    Zhao, Xiaolu; Leon, Ileana R; Bak, Steffen

    2011-01-01

    . In skeletal muscle, mitochondrial dysfunction is linked to insulin resistance in humans with obesity and type 2 diabetes. We performed a phosphoproteomic study of functional mitochondria isolated from human muscle biopsies with the aim to obtain a comprehensive overview of mitochondrial phosphoproteins...... in insulin resistance. We also assigned phosphorylation sites in mitochondrial proteins involved in amino acid degradation, importers and transporters, calcium homeostasis, and apoptosis. Bioinformatics analysis of kinase motifs revealed that many of these mitochondrial phosphoproteins are substrates....... Future comparative phosphoproteome analysis of mitochondria from healthy and diseased individuals will provide insights into the role of abnormal phosphorylation in pathologies, such as type 2 diabetes....

  13. Intramacrophage survival of uropathogenic Escherichia coli: Differences between diverse clinical isolates and between mouse and human macrophages

    DEFF Research Database (Denmark)

    Bokil, Nilesh J.; Totsika, Makrina; Carey, Alison J.

    2011-01-01

    assays, CFT073 was able to survive within primary mouse bone marrow-derived macrophages (BMM) up to 24h post-infection. Three additional well-characterized clinical UPEC isolates associated with distinct UTI symptomatologies displayed variable long-term survival within BMM. UPEC strains UTI89 and VR50...... or initial uptake of bacteria. E. coli UTI89 localized to a Lamp1+ vesicular compartment within BMM. In contrast to survival within mouse BMM, intracellular bacterial loads of VR50 were low in both human monocyte-derived macrophages (HMDM) and in human T24 bladder epithelial cells. Collectively, these data...

  14. Unsaponifiable fraction isolated from grape (Vitis vinifera L.) seed oil attenuates oxidative and inflammatory responses in human primary monocytes.

    Science.gov (United States)

    Millan-Linares, Maria C; Bermudez, Beatriz; Martin, Maria E; Muñoz, Ernesto; Abia, Rocio; Millan, Francisco; Muriana, Francisco J G; Montserrat-de la Paz, Sergio

    2018-04-25

    Grape (Vitis vinifera L.) seed has well-known potential for production of oil as a byproduct of winemaking and is a rich source of bioactive compounds. Herein, we report that the unsaponifiable fraction (UF) isolated from grape seed oil (GSO) possesses anti-oxidative and anti-inflammatory properties towards human primary monocytes. The UF isolated from GSO was phytochemically characterized by GC-MS and HPLC. Freshly obtained human monocytes were used to analyse the effects of GSOUF (10-100 μg mL-1) on oxidative and inflammatory responses using FACS analysis, RT-qPCR, and ELISA procedures. GSOUF skewed the monocyte plasticity towards the anti-inflammatory non-classical CD14+CD16++ monocytes and reduced the inflammatory competence of LPS-treated human primary monocytes diminishing TNF-α, IL-1β, and IL-6 gene expression and secretion. In addition, GSOUF showed a strong reactive oxygen species (ROS)-scavenging activity, reducing significantly nitrite levels with a significant decrease in Nos2 gene expression. Our results suggest that the UF isolated from GSO has significant potential for the management of inflammatory and oxidative conditions and offer novel benefits derived from the consumption of GSO in the prevention of inflammation-related diseases.

  15. Application of Fluorescent Amplified Fragment Length Polymorphism for Comparison of Human and Animal Isolates of Yersinia enterocolitica

    Science.gov (United States)

    Fearnley, Catherine; On, Stephen L. W.; Kokotovic, Branko; Manning, Georgina; Cheasty, Tom; Newell, Diane G.

    2005-01-01

    An amplified fragment length polymorphism (AFLP) method, developed to genotype Yersinia enterocolitica, has been used to investigate 70 representative strains isolated from humans, pigs, sheep, and cattle in the United Kingdom. AFLP primarily distinguished Y. enterocolitica strains according to their biotype, with strains dividing into two distinct clusters: cluster A, comprising largely the putatively pathogenic biotypes (BT2 to -4), and cluster B, comprising the putatively nonpathogenic biotype 1A strains and a single BT1B isolate. Within these two clusters, subclusters formed largely on the basis of serotype. However, AFLP profiles also allowed differentiation of strains within these serotype-related subclusters, indicating the high discriminatory power of the technique for Y. enterocolitica. Investigation of the relationship between strain AFLP profile and host confirmed that pigs are, and provides further proof that sheep may be, potential sources of human infection with putatively pathogenic strains. However, the results suggest that some strains causing human disease do not come from veterinary sources identifiable at this time. The distribution of some BT1A isolates within cluster A raises questions about the relationship between virulence potential and biotype. PMID:16151073

  16. ‘Lachnoclostridium massiliosenegalense’, a new bacterial species isolated from the human gut microbiota

    Directory of Open Access Journals (Sweden)

    M. Tidjani Alou

    2016-11-01

    Full Text Available We report the main characteristics of ‘Lachnoclostridium massiliosenegalense’ strain mt23T (=CSUR P299 =DSM 102084, a new bacterial species isolated from the gut microbiota of a healthy young girl from Senegal.

  17. Newly recognized Leptospira species ("Leptospira inadai" serovar lyme) isolated from human skin.

    OpenAIRE

    Schmid, G P; Steere, A C; Kornblatt, A N; Kaufmann, A F; Moss, C W; Johnson, R C; Hovind-Hougen, K; Brenner, D J

    1986-01-01

    Leptospira strain 10, which represents a new Leptospira species, was isolated from a skin biopsy of a patient with Lyme disease. Although pathogenic for laboratory animals, the organism was not considered to have a significant role in the patient's illness.

  18. ‘Lactobacillus timonensis’ sp. nov., a new bacterial species isolated from the human gut

    Directory of Open Access Journals (Sweden)

    P. Afouda

    2017-09-01

    Full Text Available Here, we describe the main characteristics of ‘Lactobacillus timonenis’ sp. nov., strain Marseille-P3825T (CSUR=P3825, isolated from a stool sample of a healthy Beninese woman.

  19. Evaluation of resorbable membrane in treatment of human gingival isolated buccal recession

    Directory of Open Access Journals (Sweden)

    Sumit Narang

    2011-01-01

    Conclusion: Resorbable membrane is a versatile treatment modality for coverage of isolated buccal gingival recession. Although membrane exposure occurred in four patients, it did not interfere with post operative healing.

  20. Comparison of genotypes and serotypes of Campylobacter jejuni isolated from Danish wild mammals and birds and from broiler flocks and humans

    DEFF Research Database (Denmark)

    Petersen, L.; Nielsen, E.M.; Engberg, J.

    2001-01-01

    (MRPs) of C. jejuni isolates from different sources. The serotype distribution in wildlife was significantly different from the known distributions in broilers and humans. Considerable sero- and genotype diversity was found within the wildlife collection, although two major groups of isolates within...... serotype O:12 and the O:4 complex were found. Common clonal lines among wildlife, chicken, and/or human isolates were identified within serotype O:12 and the O:4 complex. However, MRPs of O:12 and O:38 strains isolated from wildlife and other sources indicated that some clonal lines propagated in a wide...

  1. Integrated culture platform based on a human platelet lysate supplement for the isolation and scalable manufacturing of umbilical cord matrix-derived mesenchymal stem/stromal cells.

    Science.gov (United States)

    de Soure, António M; Fernandes-Platzgummer, Ana; Moreira, Francisco; Lilaia, Carla; Liu, Shi-Hwei; Ku, Chen-Peng; Huang, Yi-Feng; Milligan, William; Cabral, Joaquim M S; da Silva, Cláudia L

    2017-05-01

    Umbilical cord matrix (UCM)-derived mesenchymal stem/stromal cells (MSCs) are promising therapeutic candidates for regenerative medicine settings. UCM MSCs have advantages over adult cells as these can be obtained through a non-invasive harvesting procedure and display a higher proliferative capacity. However, the high cell doses required in the clinical setting make large-scale manufacturing of UCM MSCs mandatory. A commercially available human platelet lysate-based culture supplement (UltraGRO TM , AventaCell BioMedical) (5%(v/v)) was tested to effectively isolate UCM MSCs and to expand these cells under (1) static conditions, using planar culture systems and (2) stirred culture using plastic microcarriers in a spinner flask. The MSC-like cells were isolated from UCM explant cultures after 11 ± 2 days. After five passages in static culture, UCM MSCs retained their immunophenotype and multilineage differentiation potential. The UCM MSCs cultured under static conditions using UltraGRO TM -supplemented medium expanded more rapidly compared with UCM MSCs expanded using a previously established protocol. Importantly, UCM MSCs were successfully expanded under dynamic conditions on plastic microcarriers using UltraGRO TM -supplemented medium in spinner flasks. Upon an initial 54% cell adhesion to the beads, UCM MSCs expanded by >13-fold after 5-6 days, maintaining their immunophenotype and multilineage differentiation ability. The present paper reports the establishment of an easily scalable integrated culture platform based on a human platelet lysate supplement for the effective isolation and expansion of UCM MSCs in a xenogeneic-free microcarrier-based system. This platform represents an important advance in obtaining safer and clinically meaningful MSC numbers for clinical translation. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  2. Isolation and characterization of current human coronavirus strains in primary human epithelial cell cultures reveal differences in target cell tropism

    NARCIS (Netherlands)

    Dijkman, Ronald; Jebbink, Maarten F.; Koekkoek, Sylvie M.; Deijs, Martin; Jónsdóttir, Hulda R.; Molenkamp, Richard; Ieven, Margareta; Goossens, Herman; Thiel, Volker; van der Hoek, Lia

    2013-01-01

    The human airway epithelium (HAE) represents the entry port of many human respiratory viruses, including human coronaviruses (HCoVs). Nowadays, four HCoVs, HCoV-229E, HCoV-OC43, HCoV-HKU1, and HCoV-NL63, are known to be circulating worldwide, causing upper and lower respiratory tract infections in

  3. Isolation and cellular properties of mesenchymal cells derived from the decidua of human term placenta.

    Science.gov (United States)

    Kanematsu, Daisuke; Shofuda, Tomoko; Yamamoto, Atsuyo; Ban, Chiaki; Ueda, Takafumi; Yamasaki, Mami; Kanemura, Yonehiro

    2011-09-01

    The clinical promise of cell-based therapies is generally recognized, and has driven an intense search for good cell sources. In this study, we isolated plastic-adherent cells from human term decidua vera, called decidua-derived-mesenchymal cells (DMCs), and compared their properties with those of bone marrow-derived-mesenchymal stem cells (BM-MSCs). The DMCs strongly expressed the mesenchymal cell marker vimentin, but not cytokeratin 19 or HLA-G, and had a high proliferative potential. That is, they exhibited a typical fibroblast-like morphology for over 30 population doublings. Cells phenotypically identical to the DMCs were identified in the decidua vera, and genotyping confirmed that the DMCs were derived from the maternal components of the fetal adnexa. Flow cytometry analysis showed that the expression pattern of CD antigens on the DMCs was almost identical to that on BM-MSCs, but some DMCs expressed the CD45 antigen, and over 50% of them also expressed anti-fibroblast antigen. In vitro, the DMCs showed good differentiation into chondrocytes and moderate differentiation into adipocytes, but scant evidence of osteogenesis, compared with the BM-MSCs. Gene expression analysis showed that, compared with BM-MSCs, the DMCs expressed higher levels of TWIST2 and RUNX2 (which are associated with early mesenchymal development and/or proliferative capacity), several matrix metalloproteinases (MMP1, 3, 10, and 12), and cytokines (BMP2 and TGFB2), and lower levels of MSX2, interleukin 26, and HGF. Although DMCs did not show the full multipotency of BM-MSCs, their higher proliferative ability indicates that their cultivation would require less maintenance. Furthermore, the use of DMCs avoids the ethical concerns associated with the use of embryonic tissues, because they are derived from the maternal portion of the placenta, which is otherwise discarded. Thus, the unique properties of DMCs give them several advantages for clinical use, making them an interesting and

  4. Clostridium punense sp. nov., an obligate anaerobe isolated from healthy human faeces.

    Science.gov (United States)

    Lanjekar, Vikram Bholanath; Marathe, Nachiket Prakash; Shouche, Yogesh Shreepad; Ranade, Dilip Ramchandra

    2015-12-01

    An obligately anaerobic, rod-shaped (0.5-1.0 × 2.0-10.0 μm), Gram-stain-positive bacterium, occurring mainly singly or in pairs, and designated BLPYG-8T, was isolated from faeces of a healthy human volunteer aged 56 years. Cells were non-motile. Oval, terminal spores were formed that swell the cells. The strain was affiliated with the genus Clostridium sensu stricto (Clostridium rRNA cluster I) as revealed by 16S rRNA gene sequence analysis. Strain BLPYG-8T showed 97.3 to 97.4 % 16S rRNA gene sequence similarity with Clostridium sulfidigenes DSM 18982T, Clostridium subterminale DSM 6970T and Clostridium thiosulfatireducens DSM 13105T. DNA-DNA hybridization and phenotypic analysis showed that the strain was distinct from its closest relatives, C. sulfidigenes DSM 18982T, C. subterminale DSM 6970T, C. thiosulfatireducens DSM 13105T with 54.2, 53.9 and 53.3 % DNA-DNA relatedness, respectively. Strain BLPYG-8T grew in PYG broth at temperatures between 20 and 40 °C (optimum 37 °C). The strain utilized a range of amino acids as well as carbohydrates as a source of carbon and energy. Glucose fermentation resulted in the formation of volatile fatty acids mainly acetic acid, n-butyric acid and organic acids such as succinic and lactic acid. The DNA G+C content of strain BLPYG-8T was 44.1 mol%. The major fatty acids (>10 %) were C14 : 0, iso-C15 : 0, C16 : 1ω7c and C16 : 0. Phylogenetic analysis and specific phenotypic characteristics and/or DNA G+C content differentiated the strain from its closest relatives. On the basis of these data, strain BLPYG-8T represents a novel species of the genus Clostridium, for which the name Clostridium punense sp. nov. is proposed. The type strain is BLPYG-8T ( = DSM 28650T = CCUG 64195T = MCC 2737T).

  5. Pathogenicity of Human ST23 Streptococcus agalactiae to Fish and Genomic Comparison of Pathogenic and Non-pathogenic Isolates

    Directory of Open Access Journals (Sweden)

    Rui Wang

    2017-10-01

    Full Text Available Streptococcus agalactiae, or Group B Streptococcus (GBS, is a major pathogen causing neonatal sepsis and meningitis, bovine mastitis, and fish meningoencephalitis. CC23, including its namesake ST23, is not only the predominant GBS strain derived from human and cattle, but also can infect a variety of homeothermic and poikilothermic species. However, it has never been characterized in fish. This study aimed to determine the pathogenicity of ST23 GBS to fish and explore the mechanisms causing the difference in the pathogenicity of ST23 GBS based on the genome analysis. Infection of tilapia with 10 human-derived ST23 GBS isolates caused tissue damage and the distribution of pathogens within tissues. The mortality rate of infection was ranged from 76 to 100%, and it was shown that the mortality rate caused by only three human isolates had statistically significant difference compared with fish-derived ST7 strain (P < 0.05, whereas the mortality caused by other seven human isolates did not show significant difference compared with fish-derived ST7 strain. The genome comparison and prophage analysis showed that the major genome difference between virulent and non-virulent ST23 GBS was attributed to the different prophage sequences. The prophage in the P1 region contained about 43% GC and encoded 28–39 proteins, which can mediate the acquisition of YafQ/DinJ structure for GBS by phage recombination. YafQ/DinJ belongs to one of the bacterial toxin–antitoxin (TA systems and allows cells to cope with stress. The ST23 GBS strains carrying this prophage were not pathogenic to tilapia, but the strains without the prophage or carrying the pophage that had gene mutation or deletion, especially the deletion of YafQ/DinJ structure, were highly pathogenic to tilapia. In conclusion, human ST23 GBS is highly pathogenic to fish, which may be related to the phage recombination.

  6. Human and Animal Isolates of Yersinia enterocolitica Show Significant Serotype-Specific Colonization and Host-Specific Immune Defense Properties

    Science.gov (United States)

    Schaake, Julia; Kronshage, Malte; Uliczka, Frank; Rohde, Manfred; Knuuti, Tobias; Strauch, Eckhard; Fruth, Angelika; Wos-Oxley, Melissa

    2013-01-01

    Yersinia enterocolitica is a human pathogen that is ubiquitous in livestock, especially pigs. The bacteria are able to colonize the intestinal tract of a variety of mammalian hosts, but the severity of induced gut-associated diseases (yersiniosis) differs significantly between hosts. To gain more information about the individual virulence determinants that contribute to colonization and induction of immune responses in different hosts, we analyzed and compared the interactions of different human- and animal-derived isolates of serotypes O:3, O:5,27, O:8, and O:9 with murine, porcine, and human intestinal cells and macrophages. The examined strains exhibited significant serotype-specific cell binding and entry characteristics, but adhesion and uptake into different host cells were not host specific and were independent of the source of the isolate. In contrast, survival and replication within macrophages and the induced proinflammatory response differed between murine, porcine, and human macrophages, suggesting a host-specific immune response. In fact, similar levels of the proinflammatory cytokine macrophage inflammatory protein 2 (MIP-2) were secreted by murine bone marrow-derived macrophages with all tested isolates, but the equivalent interleukin-8 (IL-8) response of porcine bone marrow-derived macrophages was strongly serotype specific and considerably lower in O:3 than in O:8 strains. In addition, all tested Y. enterocolitica strains caused a considerably higher level of secretion of the anti-inflammatory cytokine IL-10 by porcine than by murine macrophages. This could contribute to limiting the severity of the infection (in particular of serotype O:3 strains) in pigs, which are the primary reservoir of Y. enterocolitica strains pathogenic to humans. PMID:23959720

  7. New genotypes of Orientia tsutsugamushi isolated from humans in Eastern Taiwan.

    Directory of Open Access Journals (Sweden)

    Hui-Hua Yang

    Full Text Available Scrub typhus, an acute febrile illness, is caused by the obligate intracellular bacterium Orientia tsutsugamushi. In our study, O. tsutsugamushi was rapidly detected and typed by polymerase chain reaction (PCR and restriction fragment length polymorphism (RFLP analysis of the 56-kDa type-specific antigen (TSA gene. To investigate the genotypes of clinical variants of O. tsutsugamushi, we collected 3223 blood samples from eastern Taiwanese patients with suspected scrub typhus from 2002 to 2008. In total, 505 samples were found to be positive for scrub typhus infection by PCR, and bacteria were isolated from 282 of them. Four prototype genotype strains (Karp, Kato, Kawasaki and Gilliam and eleven different Taiwanese genotype isolates (Taiwan-A, -B, -C, -D, -E, -G, -H, -J, -N, -O and -P were identified by RPLF analysis. Taiwan-H, the major genotype in eastern Taiwan, exhibited prevalence and isolation rates of 47.3% (239/505 and 42.6% (120/282, respectively. We also assessed the genetic relatedness of the 56-kDa TSA gene among eight Taiwan-H isolates, thirteen other Taiwanese isolates and 104 DNA sequences deposited in the GenBank database using MEGA version 5.0 and PHYLIP version 3.66. We found that the Taiwan-H isolates formed into a new cluster, which was designated the Taiwan Gilliam-variant (TG-v cluster to distinguish it from the Japanese Gilliam-variant (JG-v cluster. According to Simplot analysis, TG-v is a new recombinant strain among Gilliam, Ikeda and Kato. Moreover, the Gilliam-Kawasaki cluster had the highest percentage of RFLP cases and was the most frequently isolated type in eastern Taiwan (50.1%, 253/505; 44.0%, 124/282. These findings shed light on the genetic evolution of O. tsutsugamushi into different strains and may be useful in vaccine development and epidemic disease control in the future.

  8. New Genotypes of Orientia tsutsugamushi Isolated from Humans in Eastern Taiwan

    Science.gov (United States)

    Lin, Chin-Hui; Chen, Tren-Yi; Chen, Li-Kuang

    2012-01-01

    Scrub typhus, an acute febrile illness, is caused by the obligate intracellular bacterium Orientia tsutsugamushi. In our study, O. tsutsugamushi was rapidly detected and typed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis of the 56-kDa type-specific antigen (TSA) gene. To investigate the genotypes of clinical variants of O. tsutsugamushi, we collected 3223 blood samples from eastern Taiwanese patients with suspected scrub typhus from 2002 to 2008. In total, 505 samples were found to be positive for scrub typhus infection by PCR, and bacteria were isolated from 282 of them. Four prototype genotype strains (Karp, Kato, Kawasaki and Gilliam) and eleven different Taiwanese genotype isolates (Taiwan-A, -B, -C, -D, -E, -G, -H, -J, -N, -O and -P) were identified by RPLF analysis. Taiwan-H, the major genotype in eastern Taiwan, exhibited prevalence and isolation rates of 47.3% (239/505) and 42.6% (120/282), respectively. We also assessed the genetic relatedness of the 56-kDa TSA gene among eight Taiwan-H isolates, thirteen other Taiwanese isolates and 104 DNA sequences deposited in the GenBank database using MEGA version 5.0 and PHYLIP version 3.66. We found that the Taiwan-H isolates formed into a new cluster, which was designated the Taiwan Gilliam-variant (TG-v) cluster to distinguish it from the Japanese Gilliam-variant (JG-v) cluster. According to Simplot analysis, TG-v is a new recombinant strain among Gilliam, Ikeda and Kato. Moreover, the Gilliam-Kawasaki cluster had the highest percentage of RFLP cases and was the most frequently isolated type in eastern Taiwan (50.1%, 253/505; 44.0%, 124/282). These findings shed light on the genetic evolution of O. tsutsugamushi into different strains and may be useful in vaccine development and epidemic disease control in the future. PMID:23071693

  9. Isolation and characterization of human cDNA clones encoding the α and the α' subunits of casein kinase II

    International Nuclear Information System (INIS)

    Lozeman, F.J.; Litchfield, D.W.; Piening, C.; Takio, Koji; Walsh, K.A.; Krebs, E.G.

    1990-01-01

    Casein kinase II is a widely distributed protein serine/threonine kinase. The holoenzyme appears to be a tetramer, containing two α or α' subunits (or one of each) and two β subunits. Complementary DNA clones encoding the subunits of casein kinase II were isolated from a human T-cell λgt 10 library using cDNA clones isolated from Drosophila melanogasten. One of the human cDNA clones (hT4.1) was 2.2 kb long, including a coding region of 1176 bp preceded by 156 bp (5' untranslated region) and followed by 871 bp (3' untranslated region). The hT4.1 close was nearly identical in size and sequence with a cDNA clone from HepG2 human hepatoma cultured cells. Another of the human T-cell cDNA clones (hT9.1) was 1.8 kb long, containing a coding region of 1053 bp preceded by 171 by (5' untranslated region) and followed by 550 bp (3' untranslated region). Amino acid sequences deduced from these two cDNA clones were about 85% identical. Most of the difference between the two encoded polypeptides was in the carboxy-terminal region, but heterogeneity was distributed throughout the molecules. Partial amino acid sequence was determined in a mixture of α and α' subunits from bovine lung casein kinase II. The bovine sequences aligned with the 2 human cDNA-encoded polypeptides with only 2 discrepancies out of 535 amino acid positions. This confirmed that the two human T-cell cDNA clones encoded the α and α' subunits of casein kinase II. These studies show that there are two distinct catalytic subunits for casein II (α and α') and that the sequence of these subunits is largely conserved between the bovine and the human

  10. Nocardia vulneris sp. nov., isolated from wounds of human patients in North America.

    Science.gov (United States)

    Lasker, Brent A; Bell, Melissa; Klenk, Hans-Peter; Spröer, Cathrin; Schumann, Cathrin; Schumann, Peter; Brown, June M

    2014-09-01

    Nocardia species are ubiquitous in the environment with an increasing number of species isolated from clinical sources. From 2005 to 2009, eight isolates (W9042, W9247, W9290, W9319, W9846, W9851T, W9865, and W9908) were obtained from eight patients from three states in the United States and Canada; all were from males ranging in age from 47 to 81 years old; and all were obtained from finger (n = 5) or leg (n = 3) wounds. Isolates were characterized by polyphasic analysis using molecular, phenotypic, morphologic and chemotaxonomic methods. Sequence analysis of 16S rRNA gene sequences showed the eight isolates are 100 % identical to each other and belong in the genus Nocardia. The nearest phylogenetically related neighbours were found to be the type strains for Nocardia altamirensis (99.33 % sequence similarity), Nocardia brasiliensis (99.37 %), Nocardia iowensis (98.95 %) and Nocardia tenerifensis (98.44 %). The G+C content of isolate W9851T was determined to be 68.4 mol %. The DNA-DNA relatedness between strain W9851T and the N. brasiliensis type strain was 72.8 % and 65.8 % when measured in the laboratory and in silico from genome sequences, respectively, and 95.6 % ANI. Whole-cell peptidoglycan was found to contain meso-diaminopimelic acid; MK-8-(H4)ω-cyc was identified as the major menaquinone; the major fatty acids were identified as C16:0, 10 Me C18:0, and C18:1 w9c, the predominant phospholipids were found to include diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides; whole-cell sugars detected were arabinose and galactose; and mycolic acids ranging from 38 to 60 carbon atoms were found to be present. These chemotaxonomic analyses are consistent with assignment of the isolates to the genus Nocardia. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectra of the clinical isolates showed genus and species level profiles that were different from other Nocardia species. All

  11. Genetics and human rights. Two histories: Restoring genetic identity after forced disappearance and identity suppression in Argentina and after compulsory isolation for leprosy in Brazil.

    Science.gov (United States)

    Penchaszadeh, Victor B; Schuler-Faccini, Lavinia

    2014-03-01

    Over the past three decades, there has been an accelerated development of genetic technology, leading to its use in human genetic identification for many purposes. Additionally, it has been made explicit that identity is a fundamental human right. A number of historical circumstances have connected these developments. Personal identity is increasingly associated with the preservation and defense of human rights and is a tool to repair the violation of these rights, particularly the right to identity. In this article, we report the use of genetics to support the right to identity in two historical circumstances. First, we report the search, localization, DNA testing and genetic identification of 110 individuals who were appropriated as babies by the Argentine military dictatorship of 1976-1983 in the context of savage repression and egregious violations of human rights, including forced disappearance and suppression of identity. Second, we report on the repair of right-to-identity violations of hundreds of individuals that occurred during the process of compulsory isolation of patients with leprosy in Brazil through the Program "Reencontro", which has led to the genetic identification of 158 pairs of individuals who previously did not have proof that they were siblings. The high value placed on genetic identification by victims of identity suppression did not counter the prevailing view that genetic factors were not more important than other factors (social, emotional, educational, cultural, spiritual) in determining the complex phenomenon of personal identity. The use of genetic identification as a tool to redress and repair human rights violations is a novel application of human genetics for the benefit of mankind.

  12. Genetics and human rights. Two histories: Restoring genetic identity after forced disappearance and identity suppression in Argentina and after compulsory isolation for leprosy in Brazil

    Science.gov (United States)

    Penchaszadeh, Victor B.; Schuler-Faccini, Lavinia

    2014-01-01

    Over the past three decades, there has been an accelerated development of genetic technology, leading to its use in human genetic identification for many purposes. Additionally, it has been made explicit that identity is a fundamental human right. A number of historical circumstances have connected these developments. Personal identity is increasingly associated with the preservation and defense of human rights and is a tool to repair the violation of these rights, particularly the right to identity. In this article, we report the use of genetics to support the right to identity in two historical circumstances. First, we report the search, localization, DNA testing and genetic identification of 110 individuals who were appropriated as babies by the Argentine military dictatorship of 1976–1983 in the context of savage repression and egregious violations of human rights, including forced disappearance and suppression of identity. Second, we report on the repair of right-to-identity violations of hundreds of individuals that occurred during the process of compulsory isolation of patients with leprosy in Brazil through the Program “Reencontro”, which has led to the genetic identification of 158 pairs of individuals who previously did not have proof that they were siblings. The high value placed on genetic identification by victims of identity suppression did not counter the prevailing view that genetic factors were not more important than other factors (social, emotional, educational, cultural, spiritual) in determining the complex phenomenon of personal identity. The use of genetic identification as a tool to redress and repair human rights violations is a novel application of human genetics for the benefit of mankind. PMID:24764764

  13. Genetics and human rights: Two histories: restoring genetic identity after forced disappearance and identity suppression in Argentina and after compulsory isolation for leprosy in Brazil

    Directory of Open Access Journals (Sweden)

    Victor B. Penchaszadeh

    2014-01-01

    Full Text Available Over the past three decades, there has been an accelerated development of genetic technology, leading to its use in human genetic identification for many purposes. Additionally, it has been made explicit that identity is a fundamental human right. A number of historical circumstances have connected these developments. Personal identity is increasingly associated with the preservation and defense of human rights and is a tool to repair the violation of these rights, particularly the right to identity. In this article, we report the use of genetics to support the right to identity in two historical circumstances. First, we report the search, localization, DNA testing and genetic identification of 110 individuals who were appropriated as babies by the Argentine military dictatorship of 1976-1983 in the context of savage repression and egregious violations of human rights, including forced disappearance and suppression of identity. Second, we report on the repair of right-to-identity violations of hundreds of individuals that occurred during the process of compulsory isolation of patients with leprosy in Brazil through the Program "Reencontro", which has led to the genetic identification of 158 pairs of individuals who previously did not have proof that they were siblings. The high value placed on genetic identification by victims of identity suppression did not counter the prevailing view that genetic factors were not more important than other factors (social, emotional, educational, cultural, spiritual in determining the complex phenomenon of personal identity. The use of genetic identification as a tool to redress and repair human rights violations is a novel application of human genetics for the benefit of mankind.

  14. Corynebacterium pilbarense sp. nov., a non-lipophilic corynebacterium isolated from a human ankle aspirate.

    Science.gov (United States)

    Aravena-Roman, M; Spröer, C; Sträubler, B; Inglis, T; Yassin, A F

    2010-07-01

    A non-lipophilic coryneform bacterium isolated from an anaerobic Bactec bottle inoculated with an ankle aspirate from a male patient was characterized by phenotypic and molecular taxonomic methods. Chemotaxonomic investigations revealed the presence of short-chain mycolic acids in the cell wall of the bacterium, a feature consistent with members of the genus Corynebacterium. Comparative 16S rRNA gene sequence analysis demonstrated that the isolate displayed 92.0-99.0 % gene sequence similarity with members of the genus Corynebacterium, with Corynebacterium ureicelerivorans as the most closely related phylogenetic species (99.0 % gene sequence similarity). However, the isolate could be genomically separated from C. ureicelerivorans on the basis of DNA-DNA hybridization studies (39.5 % relatedness). Furthermore, the isolate could also be differentiated from C. ureicelerivorans and other species of the genus Corynebacterium on the basis of biochemical properties. Based on both phenotypic and phylogenetic evidence, it is proposed that this isolate be classified as representing a novel species, Corynebacterium pilbarense sp. nov. (type strain IMMIB WACC 658(T)=DSM 45350(T)=CCUG 57942(T)).

  15. Leptospira venezuelensis sp. nov., a new member of the intermediate group isolated from rodents, cattle and humans.

    Science.gov (United States)

    Puche, Rafael; Ferrés, Ignacio; Caraballo, Lizeth; Rangel, Yaritza; Picardeau, Mathieu; Takiff, Howard; Iraola, Gregorio

    2018-02-01

    Three strains, CLM-U50 T , CLM-R50 and IVIC-Bov1, belonging to the genus Leptospira, were isolated in Venezuela from a patient with leptospirosis, a domestic rat (Rattus norvegicus) and a cow (Bos taurus), respectively. The initial characterisation of these strains based on the rrs gene (16S rRNA) suggested their designation as a novel species within the 'intermediates' group of the genus Leptospira. Further phylogenomic characterisation based on single copy core genes was consistent with their separation into a novel species. The average nucleotide identity between these three strains was >99 %, but below 89 % with respect to any previously described leptospiral species, also supporting their designation as a novel species. Given this evidence, these three isolates were considered to represent a novel species, for which the name Leptospiravenezuelensis sp. nov. is proposed, with CLM-U50 T (=CIP 111407 T =DSM 105752 T ) as the type strain.

  16. Effect of sampling and short isolation methodologies on the recovery of human pathogenic Yersinia enterocolitica from pig tonsils.

    Science.gov (United States)

    Van Damme, Inge; Berkvens, Dirk; De Zutter, Lieven

    2012-07-01

    The objective of this study was to determine the effect of sampling (swab samples compared to destructive samples) on isolation rates of human pathogenic Yersinia enterocolitica from pig tonsils. Moreover, the relative efficiency of different rapid, routinely applicable isolation methods was evaluated. Therefore, swab and destructive samples from tonsils of 120 pigs at slaughter were analyzed in parallel using direct plating and different enrichment methods. Salmonella-Shigella-desoxycholate-calcium chloride (SSDC) agar, cefsulodin-irgasan-novobiocin (CIN) agar, and Yersinia enterocolitica chromogenic medium (YeCM) were used as selective agar media. For enrichment, irgasan-ticarcillin-potassium chlorate (ITC) broth and peptone-sorbitol-bile (PSB) broth were incubated at 25°C for 48 h. Overall, 55 tonsils (45.8%) were positive for Y. enterocolitica bioserotype 4/O:3. Recovery was significantly higher using the destructive method compared to the swabbing method. Direct plating resulted in 47 and 28 Y. enterocolitica-positive destructive and swab samples, respectively. Alkali treatment of PSB and ITC enrichment broths significantly increased recovery of pathogenic Y. enterocolitica from destructive tonsil samples. The performance of YeCM for qualitative and quantitative isolation of pathogenic Y. enterocolitica from pig tonsils was equal to SSDC and CIN. In conclusion, direct plating and ISO 10273: 2003 with minor modifications are suitable and rapid methods for isolation of pathogenic Y. enterocolitica from destructive tonsil samples.

  17. Combined lipidomic and proteomic analysis of isolated human islets exposed to palmitate reveals time-dependent changes in insulin secretion and lipid metabolism.

    Directory of Open Access Journals (Sweden)

    Kirsten Roomp

    Full Text Available Studies on the pathophysiology of type 2 diabetes mellitus (T2DM have linked the accumulation of lipid metabolites to the development of beta-cell dysfunction and impaired insulin secretion. In most in vitro models of T2DM, rodent islets or beta-cell lines are used and typically focus is on specific cellular pathways or organs. Our aim was to, firstly, develop a combined lipidomics and proteomics approach for lipotoxicity in isolated human islets and, secondly, investigate if the approach could delineate novel and/ or confirm reported mechanisms of lipotoxicity. To this end isolated human pancreatic islets, exposed to chronically elevated palmitate concentrations for 0, 2 and 7 days, were functionally characterized and their levels of multiple targeted lipid and untargeted protein species determined. Glucose-stimulated insulin secretion from the islets increased on day 2 and decreased on day 7. At day 7 islet insulin content decreased and the proinsulin to insulin content ratio doubled. Amounts of cholesterol, stearic acid, C16 dihydroceramide and C24:1 sphingomyelin, obtained from the lipidomic screen, increased time-dependently in the palmitate-exposed islets. The proteomic screen identified matching changes in proteins involved in lipid biosynthesis indicating up-regulated cholesterol and lipid biosynthesis in the islets. Furthermore, proteins associated with immature secretory granules were decreased when palmitate exposure time was increased despite their high affinity for cholesterol. Proteins associated with mature secretory granules remained unchanged. Pathway analysis based on the protein and lipid expression profiles implicated autocrine effects of insulin in lipotoxicity. Taken together the study demonstrates that combining different omics approaches has potential in mapping of multiple simultaneous cellular events. However, it also shows that challenges exist for effectively combining lipidomics and proteomics in primary cells. Our

  18. Species distribution & antifungal susceptibility pattern of oropharyngeal Candida isolates from human immunodeficiency virus infected individuals

    Directory of Open Access Journals (Sweden)

    Partha Pratim Das

    2016-01-01

    Results: From the 59 culture positive HIV seropositive cases, 61 Candida isolates were recovered; Candidaalbicans (n=47, 77.0%, C. dubliniensis (n=9, 14.7%, C. parapsilosis (n=2, 3.2%, C. glabrata (n=2, 3.2%, and C. famata (n=1, 1.6%. Candida colonization in HIV-seropositive individuals was significantly higher than that of HIV-seronegative (control group. Antifungal susceptibility testing revealed (n=6, 9.3% C. albicans isolates resistant to voriconazole and fluconazole by disk-diffusion method whereas no resistance was seen by Fungitest method. Interpretation & conclusions: C. albicans was the commonest Candida species infecting or colonizing HIV seropositive individuals. Oropharyngeal Candida isolates had high level susceptibility to all the major antifungals commonly in use. Increased level of immunosuppression in HIV-seropositives and drug resistance of non-albicans Candida species makes identification and susceptibility testing of Candida species necessary in different geographical areas of the country.

  19. Identifying Effective Enzyme Activity Targets for Recombinant Class I and Class II Collagenase for Successful Human Islet Isolation.

    Science.gov (United States)

    Balamurugan, Appakalai N; Green, Michael L; Breite, Andrew G; Loganathan, Gopalakrishnan; Wilhelm, Joshua J; Tweed, Benjamin; Vargova, Lenka; Lockridge, Amber; Kuriti, Manikya; Hughes, Michael G; Williams, Stuart K; Hering, Bernhard J; Dwulet, Francis E; McCarthy, Robert C

    2016-01-01

    Isolation following a good manufacturing practice-compliant, human islet product requires development of a robust islet isolation procedure where effective limits of key reagents are known. The enzymes used for islet isolation are critical but little is known about the doses of class I and class II collagenase required for successful islet isolation. We used a factorial approach to evaluate the effect of high and low target activities of recombinant class I (rC1) and class II (rC2) collagenase on human islet yield. Consequently, 4 different enzyme formulations with divergent C1:C2 collagenase mass ratios were assessed, each supplemented with the same dose of neutral protease. Both split pancreas and whole pancreas models were used to test enzyme targets (n = 20). Islet yield/g pancreas was compared with historical enzymes (n = 42). Varying the Wunsch (rC2) and collagen degradation activity (CDA, rC1) target dose, and consequently the C1:C2 mass ratio, had no significant effect on tissue digestion. Digestions using higher doses of Wunsch and CDA resulted in comparable islet yields to those obtained with 60% and 50% of those activities, respectively. Factorial analysis revealed no significant main effect of Wunsch activity or CDA for any parameter measured. Aggregate results from 4 different collagenase formulations gave 44% higher islet yield (>5000 islet equivalents/g) in the body/tail of the pancreas (n = 12) when compared with those from the same segment using a standard natural collagenase/protease mixture (n = 6). Additionally, islet yields greater than 5000 islet equivalents/g pancreas were also obtained in whole human pancreas. A broader C1:C2 ratio can be used for human islet isolation than has been used in the past. Recombinant collagenase is an effective replacement for the natural enzyme and we have determined that high islet yield can be obtained even with low doses of rC1:rC2, which is beneficial for the survival of islets.

  20. Molecular identification and polymorphism determination of cutaneous and visceral leishmaniasis agents isolated from human and animal hosts in Iran.

    Science.gov (United States)

    Hajjaran, Homa; Mohebali, Mehdi; Mamishi, Setareh; Vasigheh, Farzaneh; Oshaghi, Mohammad Ali; Naddaf, Saied Reza; Teimouri, Aref; Edrissian, Gholam Hossein; Zarei, Zabiholah

    2013-01-01

    Amplification of internal transcript spacer 1 of ribosomal RNA (ITS1-RNA) gene followed by RFLP analysis and sequencing was used to identify the causing agents of cutaneous and visceral leishmaniasis (CL and VL) in humans and animal reservoir hosts from various geographical areas in Iran. We also used random amplified polymorphic DNA (RAPD-PCR) to obtain polymorphisms among isolates of Leishmania spp. Totally, 362 suspected human and animal cases including 173 CL, 49 VL, 60 rodents, and 80 domestic dogs were examined for Leishmania infection. From 112 culture-positive samples prepared from CL cases, 75 (67%) were infected with L. major and 37 (33%) with L. tropica. Of the 60 rodents examined, 25 (41.6%) harbored the Leishmania infection; 21 were infected with L. major and 4 with L. turanica. From 49 suspected VL, 29 were positive by direct agglutination test (DAT), whereas microscopy detected parasite in bone marrow of 25 and culture in 28 of the patients. Two VL patients were infected with L. tropica and 26 with L. infantum. Of the 80 domestic dogs, 56 showed anti-Leishmania antibodies with DAT. Of these, 55 were positive by both microscopy and culture. Molecular identity, obtained only for 47 samples, revealed L. infantum in 43 and L. tropica in 4 dogs. The polymorphisms among L. tropica and L. major isolates were 3.6% and 7.3%; the rate among human and canine VL isolates was 2.8% and 9.8%, respectively. Our results showed that at least four different Leishmania species with various polymorphisms circulate among humans and animal hosts in Iran.

  1. Molecular Identification and Polymorphism Determination of Cutaneous and Visceral Leishmaniasis Agents Isolated from Human and Animal Hosts in Iran

    Directory of Open Access Journals (Sweden)

    Homa Hajjaran

    2013-01-01

    Full Text Available Amplification of internal transcript spacer 1 of ribosomal RNA (ITS1-RNA gene followed by RFLP analysis and sequencing was used to identify the causing agents of cutaneous and visceral leishmaniasis (CL and VL in humans and animal reservoir hosts from various geographical areas in Iran. We also used random amplified polymorphic DNA (RAPD-PCR to obtain polymorphisms among isolates of Leishmania spp. Totally, 362 suspected human and animal cases including 173 CL, 49 VL, 60 rodents, and 80 domestic dogs were examined for Leishmania infection. From 112 culture-positive samples prepared from CL cases, 75 (67% were infected with L. major and 37 (33% with L. tropica. Of the 60 rodents examined, 25 (41.6% harbored the Leishmania infection; 21 were infected with L. major and 4 with L. turanica. From 49 suspected VL, 29 were positive by direct agglutination test (DAT, whereas microscopy detected parasite in bone marrow of 25 and culture in 28 of the patients. Two VL patients were infected with L. tropica and 26 with L. infantum. Of the 80 domestic dogs, 56 showed anti-Leishmania antibodies with DAT. Of these, 55 were positive by both microscopy and culture. Molecular identity, obtained only for 47 samples, revealed L. infantum in 43 and L. tropica in 4 dogs. The polymorphisms among L. tropica and L. major isolates were 3.6% and 7.3%; the rate among human and canine VL isolates was 2.8% and 9.8%, respectively. Our results showed that at least four different Leishmania species with various polymorphisms circulate among humans and animal hosts in Iran.

  2. Draft genome sequence of the first human isolate of the ruminant pathogen Mycoplasma capricolum subsp. capricolum

    DEFF Research Database (Denmark)

    Seersholm, Frederik Valeur; Fischer, Anne; Heller, Martin

    2015-01-01

    Mycoplasma capricolum subsp. capricolum is a well-known pathogen of small ruminants. A recent human case of septicemia involving this agent raised the question of its potential pathogenicity to humans. We present the first draft genome sequence of a human Mycoplasma capricolum subsp. capricolum...

  3. Comparative Genotypes, Staphylococcal Cassette Chromosome mec (SCCmec) Genes and Antimicrobial Resistance amongst Staphylococcus epidermidis and Staphylococcus haemolyticus Isolates from Infections in Humans and Companion Animals

    OpenAIRE

    McManus, Brenda A.; Coleman, David C.; Deasy, Emily C.; Brennan, Gráinne I.; O’ Connell, Brian; Monecke, Stefan; Ehricht, Ralf; Leggett, Bernadette; Leonard, Nola; Shore, Anna C.

    2015-01-01

    This study compares the characteristics of Staphylococcus epidermidis (SE) and Staphylococcus haemolyticus (SH) isolates from epidemiologically unrelated infections in humans (Hu) (28 SE-Hu; 8 SH-Hu) and companion animals (CpA) (12 SE-CpA; 13 SH-CpA). All isolates underwent antimicrobial susceptibility testing, multilocus sequence typing and DNA microarray profiling to detect antimicrobial resistance and SCCmec-associated genes. All methicillin-resistant (MR) isolates (33/40 SE, 20/21 SH) und...

  4. Comparative Prevalence of Immune Evasion Complex Genes Associated with β-Hemolysin Converting Bacteriophages in MRSA ST5 Isolates from Swine, Swine Facilities, Humans with Swine Contact, and Humans with No Swine Contact.

    Directory of Open Access Journals (Sweden)

    Samantha J Hau

    Full Text Available Livestock associated methicillin-resistant Staphylococcus aureus (LA-MRSA draws concern from the public health community because in some countries these organisms may represent the largest reservoir of MRSA outside hospital settings. Recent studies indicate LA-MRSA strains from swine are more genetically diverse than the first reported sequence type ST398. In the US, a diverse population of LA-MRSA is found including organisms of the ST398, ST9, and ST5 lineages. Occurrence of ST5 MRSA in swine is of particular concern since ST5 is among the most prevalent lineages causing clinical infections in humans. The prominence of ST5 in clinical disease is believed to result from acquisition of bacteriophages containing virulence or host-adapted genes including the immune-evasion cluster (IEC genes carried by β-hemolysin converting bacteriophages, whose absence in LA-MRSA ST398 is thought to contribute to reduced rates of human infection and transmission associated with this lineage. The goal of this study was to investigate the prevalence of IEC genes associated with β-hemolysin converting bacteriophages in MRSA ST5 isolates obtained from agricultural sources, including swine, swine facilities, and humans with short- or long-term swine exposure. To gain a broader perspective, the prevalence of these genes in LA-MRSA ST5 strains was compared to the prevalence in clinical MRSA ST5 strains from humans with no known exposure to swine. IEC genes were not present in any of the tested MRSA ST5 strains from agricultural sources and the β-hemolysin gene was intact in these strains, indicating the bacteriophage's absence. In contrast, the prevalence of the β-hemolysin converting bacteriophage in MRSA ST5 strains from humans with no exposure to swine was 90.4%. The absence of β-hemolysin converting bacteriophage in LA-MRSA ST5 isolates is consistent with previous reports evaluating ST398 strains and provides genetic evidence indicating LA-MRSA ST5 isolates

  5. Comparative Prevalence of Immune Evasion Complex Genes Associated with β-Hemolysin Converting Bacteriophages in MRSA ST5 Isolates from Swine, Swine Facilities, Humans with Swine Contact, and Humans with No Swine Contact

    Science.gov (United States)

    Hau, Samantha J.; Sun, Jisun; Davies, Peter R.; Frana, Timothy S.; Nicholson, Tracy L.

    2015-01-01

    Livestock associated methicillin-resistant Staphylococcus aureus (LA-MRSA) draws concern from the public health community because in some countries these organisms may represent the largest reservoir of MRSA outside hospital settings. Recent studies indicate LA-MRSA strains from swine are more genetically diverse than the first reported sequence type ST398. In the US, a diverse population of LA-MRSA is found including organisms of the ST398, ST9, and ST5 lineages. Occurrence of ST5 MRSA in swine is of particular concern since ST5 is among the most prevalent lineages causing clinical infections in humans. The prominence of ST5 in clinical disease is believed to result from acquisition of bacteriophages containing virulence or host-adapted genes including the immune-evasion cluster (IEC) genes carried by β-hemolysin converting bacteriophages, whose absence in LA-MRSA ST398 is thought to contribute to reduced rates of human infection and transmission associated with this lineage. The goal of this study was to investigate the prevalence of IEC genes associated with β-hemolysin converting bacteriophages in MRSA ST5 isolates obtained from agricultural sources, including swine, swine facilities, and humans with short- or long-term swine exposure. To gain a broader perspective, the prevalence of these genes in LA-MRSA ST5 strains was compared to the prevalence in clinical MRSA ST5 strains from humans with no known exposure to swine. IEC genes were not present in any of the tested MRSA ST5 strains from agricultural sources and the β-hemolysin gene was intact in these strains, indicating the bacteriophage’s absence. In contrast, the prevalence of the β-hemolysin converting bacteriophage in MRSA ST5 strains from humans with no exposure to swine was 90.4%. The absence of β-hemolysin converting bacteriophage in LA-MRSA ST5 isolates is consistent with previous reports evaluating ST398 strains and provides genetic evidence indicating LA-MRSA ST5 isolates may harbor a

  6. An in vitro study of antifungal drug susceptibility of Candida species isolated from human immunodeficiency virus seropositive and human immunodeficiency virus seronegative individuals in Lucknow population Uttar Pradesh.

    Science.gov (United States)

    Dar, Mohammad Shafi; Sreedar, Gadiputi; Shukla, Abhilasha; Gupta, Prashant; Rehan, Ahmad Danish; George, Jiji

    2015-01-01

    Candidiasis is the most common opportunistic infection in human immunodeficiency virus (HIV) seropositive patients, starting from asymptomatic colonization to pathogenic forms and gradual colonization of non-albicans in patients with advanced immunosuppression leads to resistance for azole group of antifungal drugs with high rate of morbidity and mortality. To isolate the Candida species and determine of antifungal drug susceptibility against fluconazole, itraconazole, nystatin, amphotericin B, and clotrimazolein HIV seropositive and control individuals, with or without clinical oropharyngeal candidiasis (OPC). Includes samples from faucial region of 70 subjects with and without clinical candidiasis in HIV seropositive and controls were aseptically inoculated onto Sabaraud's Dextrose Agar media and yeasts were identified for the specific species by Corn Meal Agar, sugar fermentation and heat tolerance tests. Antifungal drug susceptibility of the isolated species was done against above-mentioned drugs by E-test and disc diffusion method. The commonly isolated species in HIV seropositive and controls were Candida albicans, Candida glabrata and Candida tropicalis Candida guilliermondii and Candida dubliniensis isolated only in HIV seropositive patients. Susceptibility against selected antifungal drugs was observed more in HIV-negative individuals whereas susceptible dose-dependent and resistance were predominant in HIV-positive patients. Resistance is the major problem in the therapy of OPC, especially in HIV seropositive patients due to aggressive and prolonged use of antifungal agents, therefore, our study emphasizes the need for antifungal drug susceptibility testing whenever antifungal treatment is desired, especially in HIV-infected subjects.

  7. Molecular analysis of Leptospira spp. isolated from humans by restriction fragment length polymorphism, real-time PCR and pulsed-field gel electrophoresis.

    Science.gov (United States)

    Turk, Nenad; Milas, Zoran; Mojcec, Vesna; Ruzic-Sabljic, Eva; Staresina, Vilim; Stritof, Zrinka; Habus, Josipa; Postic, Daniele

    2009-11-01

    A total of 17 Leptospira clinical strains isolated from humans in Croatia were serologically and genetically analysed. For serovar identification, the microscopic agglutination test (MAT) and pulsed-field gel electrophoresis (PFGE) were used. To identify isolates on genomic species level, PCR-based restriction fragment length polymorphism (RFLP) and real-time PCR were performed. MAT revealed the following serogroup affinities: Grippotyphosa (seven isolates), Icterohaemorrhagiae (eight isolates) and Javanica (two isolates). RFLP of PCR products from a 331-bp-long fragment of rrs (16S rRNA gene) digested with endonucleases MnlI and DdeI and real-time PCR revealed three Leptospira genomic species. Grippotyphosa isolates belonged to Leptospira kirschneri, Icterohaemorrhagiae isolates to Leptospira interrogans and Javanica isolates to Leptospira borgpetersenii. Genomic DNA from 17 leptospiral isolates was digested with NotI and SgrAI restriction enzymes and analysed by PFGE. Results showed that seven isolates have the same binding pattern to serovar Grippotyphosa, eight isolates to serovar Icterohaemorrhagiae and two isolates to serovar Poi. Results demonstrate the diversity of leptospires circulating in Croatia. We point out the usefulness of a combination of PFGE, RFLP and real-time PCR as appropriate molecular methods in molecular analysis of leptospires.

  8. Antibiotics and heavy metals resistance patterns of Enterococcus faecalis and faecium bacteria isolated from the human and the livestock sources

    Directory of Open Access Journals (Sweden)

    Yaser Sharifi

    2015-12-01

    Full Text Available Background: Enterococci have emerged as a major cause of nosocomial infections and within this group, Enterococcus faecalis and Enterococcus faecium cause the majority of human and livestock enterococcal infections. In this article, we tried to determine antibiotics and metals resistance patterns of E. faecalis and E. faecium strains. Methods: One hundred sixty different strains of E. faecalis and E. faecium were collected from livestock sewage and the human fecal waste during 15 months. Then bacterial antibiotics sensitivity tests were carried out using the Agar disc diffusion method. Results: Generally, 100% of E. faecalis strains separated from human and livestock sources (i.e. sheep showed penicillin (P/ kanamycin (K/ nitrofurantoin (N/ loracarbef (L/ Ciprofloxacin (Cc/ ampicillin (AN/ nalidixic acid (NA/ sulfamethoxazole (S antibiotics resistance patterns. In addition, 55% of isolated E. faecium showed P/S/AN/NA antibiotics resistance patterns. Each strain showed a resistance to at least two aminoglycoside antibiotics. However, E. faecalis strains from human and the livestock sources showed 94% and 100% of resistance to nitrofurantoin, respectively. The effects of different metal concentrations was evaluated in both strains. The agar dilution method was applied in this stage. Hg at 0.05 mmol/L of minimum inhibitory concentration (MIC showed toxicity to both the human and livestock Enterococcus strains. Cadmium at 1 mmol/L and 0.5 mmol/L concentrations had the most toxicity to E. faecalis and E. faecium strains, respectively. Obviously, toxicity to bacteria is less than other metals. As a result, Zn/Ni/Cu/Co resistance pattern is suggested for both strains. Finally, antibiotics and heavy metals resistance patterns were monitored simultaneously. Conclusion: Almost all E. faecalis strains isolated from humans and livestock showed antibiotics and heavy metals resistance patterns of P/K/L/Cc/S/AN/NA/Zn/Cu/Co simultaneously. Moreover, 55% of E

  9. From outer space to Earth-The social significance of isolated and confined environment research in human space exploration

    Science.gov (United States)

    Tachibana, Koji; Tachibana, Shoichi; Inoue, Natsuhiko

    2017-11-01

    Human space exploration requires massive budgets every fiscal year. Especially under severe financial constraint conditions, governments are forced to justify to society why spending so much tax revenue for human space exploration is worth the cost. The value of human space exploration might be estimated in many ways, but its social significance and cost-effectiveness are two key ways to gauge that worth. Since these measures should be applied country by country because sociopolitical conditions differ in each country and must be taken into consideration, the study on the social significance of human space exploration must take the coloration of a case-study. This paper, focusing on the case of Japan with surveying Japanese literary and national documents as well as taking its sociopolitical conditions into account, examines the social significance of human space exploration. First, we give an overview of the circumstances surrounding Japan's human space exploration program. Derived from the statements of such relevant parties as scholars, journalists, policy makers, and astronauts, this overview indicates that the main concerns about human space exploration in Japan are its social significance and cost-effectiveness (Section 1). Next, an overview of behavioral science-an essential field for human space exploration (referred to in this paper as space behavioral science) that provides support for astronauts-is presented from the perspective of stress research in isolated and confined environments (Section 2). We then give two examples of where such knowledge from space behavioral science research has been applied to terrestrial isolated and confined environments. One is JAXA's support in 2009 for people who were vulnerable to infection by a new strain of flu and accordingly placed in an isolated and confined facility under the Infectious Disease Law and the Quarantine Law. The other is NASA's support in 2010 for Chilean mine workers who were trapped 700 m

  10. β-Glucan induces reactive oxygen species production in human neutrophils to improve the killing of Candida albicans and Candida glabrata isolates from vulvovaginal candidiasis.

    Directory of Open Access Journals (Sweden)

    Patricia de Souza Bonfim-Mendonça

    Full Text Available Vulvovaginal candidiasis (VVC is among the most prevalent vaginal diseases. Candida albicans is still the most prevalent species associated with this pathology, however, the prevalence of other Candida species, such as C. glabrata, is increasing. The pathogenesis of these infections has been intensely studied, nevertheless, no consensus has been reached on the pathogenicity of VVC. In addition, inappropriate treatment or the presence of resistant strains can lead to RVVC (vulvovaginal candidiasis recurrent. Immunomodulation therapy studies have become increasingly promising, including with the β-glucans. Thus, in the present study, we evaluated microbicidal activity, phagocytosis, intracellular oxidant species production, oxygen consumption, myeloperoxidase (MPO activity, and the release of tumor necrosis factor α (TNF-α, interleukin-8 (IL-8, IL-1β, and IL-1Ra in neutrophils previously treated or not with β-glucan. In all of the assays, human neutrophils were challenged with C. albicans and C. glabrata isolated from vulvovaginal candidiasis. β-glucan significantly increased oxidant species production, suggesting that β-glucan may be an efficient immunomodulator that triggers an increase in the microbicidal response of neutrophils for both of the species isolated from vulvovaginal candidiasis. The effects of β-glucan appeared to be mainly related to the activation of reactive oxygen species and modulation of cytokine release.

  11. Further genetic characterization of the two Trypanosoma cruzi Berenice strains (Be-62 and Be-78) isolated from the first human case of Chagas disease (Chagas, 1909).

    Science.gov (United States)

    Cruz, R E; Macedo, A M; Barnabé, C; Freitas, J M; Chiari, E; Veloso, V M; Carneiro, C M; Bahia, M T; Tafuri, Washington L; Lana, M

    2006-03-01

    We describe here an extension of a previous genetic characterization of Trypanosoma cruzi strains (Be-62 and Be-78) isolated from the patient Berenice, the first human case of Chagas disease [Chagas, C., 1909. Nova Tripanomíase humana. Estudos sobre morfologia e o ciclo evolutivo do Schizotrypanum cruzi, n. gen., n. sp., agente etiolójico da nova entidade morbida do homem. Mem. Inst. Oswaldo Cruz 1, 159-218]. We wanted to verify the composition of T. cruzi populations originated from these two isolates. In the present work, 22 enzymatic loci (MLEE), nine RAPD primers and 7 microsatellite loci were analyzed. Clones from both strains were also characterized to verify whether these strains are mono or polyclonal. Be-62 and Be-78 strains were different in 3 out of 22 enzymatic systems, in 3 out of 9 RAPD primers tested and in all microsatellite loci investigated. However, our data suggests that both strains are phylogenetically closely related, belonging to genetic group 32 from Tibayrenc and Ayala [Tibayrenc, M., Ayala, F.J., 1988. Isoenzime variability in Trypanosoma cruzi, the agent of Chagas' disease: genetical, taxonomical, and epidemiological significance. Evolution 42, 277-292], equivalent to zymodeme 2 and T. cruzi II major lineage which, in Brazil, comprises parasites from the domestic cycle of the disease. Microsatellite analyses showed differences between the parental strains but suggested that both populations are monoclonal since each strain and their respective clones showed the same amplification products.

  12. MLVA genotyping of Brucella melitensis and Brucella abortus isolates from different animal species and humans and identification of Brucella suis vaccine strain S2 from cattle in China.

    Directory of Open Access Journals (Sweden)

    Hai Jiang

    Full Text Available In China, brucellosis is an endemic disease and the main sources of brucellosis in animals and humans are infected sheep, cattle and swine. Brucella melitensis (biovars 1 and 3 is the predominant species, associated with sporadic cases and outbreak in humans. Isolates of B. abortus, primarily biovars 1 and 3, and B. suis biovars 1 and 3 are also associated with sporadic human brucellosis. In this study, the genetic profiles of B. melitensis and B. abortus isolates from humans and animals were analyzed and compared by multi-locus variable-number tandem-repeat analysis (MLVA. Among the B. melitensis isolates, the majority (74/82 belonged to MLVA8 genotype 42, clustering in the 'East Mediterranean' group. Two B. melitensis biovar 1 genotype 47 isolates, belonging to the 'Americas' group, were recovered; both were from the Himalayan blue sheep (Pseudois nayaur, a wild animal. The majority of B. abortus isolates (51/70 were biovar 3, genotype 36. Ten B. suis biovar 1 field isolates, including seven outbreak isolates recovered from a cattle farm in Inner Mongolia, were genetically indistinguishable from the vaccine strain S2, based on MLVA cluster analysis. MLVA analysis provided important information for epidemiological trace-back. To the best of our knowledge, this is the first report to associate Brucella cross-infection with the vaccine strain S2 based on molecular comparison of recovered isolates to the vaccine strain. MLVA typing could be an essential assay to improve brucellosis surveillance and control programs.

  13. Ultrastructural study of the mycelial phase of clinical isolates of Sporothrix schenckii obtained from feline, canine and human cases of sporotrichosis

    Directory of Open Access Journals (Sweden)

    Isabel Martins Madrid

    2011-09-01

    Full Text Available Using transmission electron microscopy, we studied the presence of melanin and cell wall thickness of clinical isolates of Sporothrix schenckii obtained from cats, dogs and humans as compared to reference strains. We detected differences regarding presence of the melanin among the clinical isolates of S. schenckii and a correlation between presence of melanin and cell wall thickness.

  14. “Lachnoclostridium touaregense,” a new bacterial species isolated from the human gut microbiota

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    M. Tidjani Alou

    2016-11-01

    Full Text Available We report the main characteristics of “Lachnoclostridium touaregense” strain Marseille-P2415T (= CSUR P2415 = DSM 102219, a new bacterial species isolated from the gut microbiota of a healthy young girl from Niger.

  15. ‘Bacteroides cutis,’ a new bacterial species isolated from human skin

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    S. Belkacemi

    2018-03-01

    Full Text Available We report the main characteristics of ‘Bacteroides cutis’ sp. nov., strain Marseille-P4118T (= CSUR P4118, a new species within the genus Bacteroides. This strain was isolated from a skin sample of a 75-year-old man from Marseille. Keywords: Bacteroides cutis, culturomics, intensive care unit patient, skin microbiota, taxonogenomics

  16. ‘Peptoniphilus pacaensis’ sp. nov., a new species isolated from human female genital tract

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    K. Diop

    2016-09-01

    Full Text Available We relate here the primary characteristics of ‘Peptoniphilus pacaensis’ strain KHD5 (= CSUR P2271, a new member of the Peptoniphilus genus. Strain KHD5 was isolated from a vaginal sample of a 33-year-old woman exhibiting a bacterial vaginosis.

  17. Pacaella massiliensis gen. nov., sp. nov., a new bacterial species isolated from the human gut

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    S. Ndongo

    2017-03-01

    Full Text Available Herein, we report the main characteristics of a new species named Pacaella massiliensis gen. nov., sp. nov., strain Marseille-P2670T (CSUR P2670 that was isolated from the gut microbiota of a 45-year-old French patient.

  18. ‘Tidjanibacter massiliensis’ gen. nov., sp. nov., a new bacterial species isolated from human colon

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    M. Mailhe

    2017-05-01

    Full Text Available We report the summary of main characteristics of Tidjanibacter massiliensis strain Marseille-P3084T, a new bacterial species isolated from the liquid sample of the colon of a patient with a history of irritable bowel syndrome.

  19. Isolation of pre-antral follicles from human ovarian medulla tissue

    DEFF Research Database (Denmark)

    Kristensen, Stine Gry; Rasmussen, Annette; Byskov, Anne Grete

    2011-01-01

    Cryopreservation of ovarian tissue for fertility preservation is based on the ovarian cortex that contains the vast majority of the follicular reserve, while the remaining tissue, the medulla is discarded. The present study describes the development of a gentle method for isolating pre...

  20. Characterization of clinical and environmental Mycobacterium avium spp. isolates and their interaction with human macrophages

    Science.gov (United States)

    Members of the Mycobacterium avium complex (MAC) are naturally occurring bacteria in the environment. A link has been suggested between M. avium strains in drinking water and clinical isolates from infected individuals. There is a need to develop new screening methodologies tha...

  1. Draft Genome Sequence of "Terrisporobacter othiniensis" Isolated from a Blood Culture from a Human Patient

    DEFF Research Database (Denmark)

    Lund, Lars Christian; Sydenham, Thomas Vognbjerg; Høgh, Silje Vermedal

    2015-01-01

    "Terrisporobacter othiniensis" (proposed species) was isolated from a blood culture. Genomic DNA was sequenced using a MiSeq benchtop sequencer (Illumina) and assembled using the SPAdes genome assembler. This resulted in a draft genome sequence comprising 3,980,019 bp in 167 contigs containing 3...

  2. Isolation of anonymous, polymorphic DNA fragments from human chromosome 22q12-qter

    NARCIS (Netherlands)

    J.P. Dumanski (Jan); A.H.M. Geurts van Kessel (Ad); M. Ruttledge (Martin); A. Wladis (Andreas); N. Sugawa (Noriaki); V.P. Collins (Peter); M. Nordenskjöld

    1990-01-01

    textabstractA series of 195 random chromosome 22-specific probes, equivalent to approximately 1% of the size of this chromosome, have been isolated from a chromosome 22-specific bacteriophage lambda genomic library. These probes were mapped to four different regions of chromosome 22 on a panel of

  3. Genetic analysis and antigenic characterization of swine origin influenza viruses isolated from humans in the United States, 1990-2010.

    Science.gov (United States)

    Shu, Bo; Garten, Rebecca; Emery, Shannon; Balish, Amanda; Cooper, Lynn; Sessions, Wendy; Deyde, Varough; Smith, Catherine; Berman, LaShondra; Klimov, Alexander; Lindstrom, Stephen; Xu, Xiyan

    2012-01-05

    Swine influenza viruses (SIV) have been recognized as important pathogens for pigs and occasional human infections with swine origin influenza viruses (SOIV) have been reported. Between 1990 and 2010, a total of twenty seven human cases of SOIV infections have been identified in the United States. Six viruses isolated from 1990 to 1995 were recognized as classical SOIV (cSOIV) A(H1N1). After 1998, twenty-one SOIV recovered from human cases were characterized as triple reassortant (tr_SOIV) inheriting genes from classical swine, avian and human influenza viruses. Of those twenty-one tr_SOIV, thirteen were of A(H1N1), one of A(H1N2), and seven of A(H3N2) subtype. SOIV characterized were antigenically and genetically closely related to the subtypes of influenza viruses circulating in pigs but distinct from contemporary influenza viruses circulating in humans. The diversity of subtypes and genetic lineages in SOIV cases highlights the importance of continued surveillance at the animal-human interface. Copyright © 2011. Published by Elsevier Inc.

  4. Use of multiple-locus variable-number of tandem repeats analysis (MLVA) to investigate genetic diversity of Salmonella enterica subsp. enterica serovar Typhimurium isolates from human, food, and veterinary sources

    DEFF Research Database (Denmark)

    Mateva, Gergana; Pedersen, Karl; Sørensen, Gitte

    2017-01-01

    -locus variable-number of tandem repeats analysis (MLVA) and compared results with antimicrobial resistance (AMR) determinations for 100 S. Typhimurium strains isolated in Bulgaria during 2008-2012 (50 veterinary/food and 50 human isolates). Results showed that isolates were divided into 80 and 34 groups using......). No clustering of isolates related to susceptibility/resistance to antimicrobials, source of isolation, or year of isolation was observed. Some MLVA types were found in both human and veterinary/food isolates, indicating a possible route of transmission. A majority (83%) of the isolates were found...

  5. Antimicrobial Resistance status and prevalence rates of Extended Spectrum Beta-Lactamase (ESBL producers isolated from a mixed human population.

    Directory of Open Access Journals (Sweden)

    Ruth A. Afunwa

    2011-05-01

    Full Text Available Owing to the increasing epidemiological and therapeutic challenges associated with infections due to ESBL producers, ESBL prevalence rate among some bacteria isolates from healthy and non-healthy human population in a metropolitan Nigerian setting was evaluated.A total of one hundred and forty-five (145 bacteria strains were isolated from a total of four hundred and sixty (460 samples collected from urine, wound, throat and anal swabs of 220 healthy volunteers in the community and from 240 patients in 2 secondary and 2 tertiary hospitals (altogether, 4 in Enugu metropolis. The presumptive confirmatory test used for ESBL detection was the Double Disc Synergy Test (DDST method. Conjugation and plasmid curing studies were also done for resistance factor determination.Of the 145 isolates, 20 were ESBL producers with 35% of these ESBL producers being of community origin and 65% from hospitals. This translates to 4.8% and 9% incidences (comparably higher than established prevalence of 4.4% and 7.5 respectively for community and hospital infections respectively. The ESBL isolates showed high resistance to tetracycline, gentamicin, pefloxacin, ceftriaxone, cefuroxime, ciprofloxacin and Augmentin® (Amoxicilin and clavulanic acid combination. Conjugation studies for Resistance plasmid transfer showed non-transference of resistance determinants between the ESBL transconjugants and recipient strains. Correspondingly, the plasmid curing studies revealed that the acridine orange could not effect a cure on the isolates as they still retained high resistance to the antibiotics after the treatment.This study confirms the growing incidences/pool of ESBL strains in Nigeria and call for widespread and continuous monitoring towards an effective management of the potential therapeutic hurdle posed by this trend.

  6. Effect of the conditions of isolation on the physicochemical properties of human serum albumin in the norm and with pathology

    Science.gov (United States)

    Ivanov, A. I.; Zhbankov, R. G.; Korolenko, E. A.; Korolik, E. V.; Meleshchenko, L. A.; Sarnatskaya, V. V.; Nikolaev, V. G.; Nikolaichik, V. V.; Yushko, L. A.

    1997-01-01

    Differential scanning calorimetry and IR spectrosocopy were used to investigate the effect of the procedure of isolation of human serum albumin on its physicochemical characteristics. It is shown that fractionation of blood plasma with ethylene glycol followed by ion exchange chromatography can be used to obtain albumin of normal donors that is similar to the albumin in the nonfractionated plasma according to melting thermograms. Endotherms of human serum albumin samples that were obtained by affinity chromatography and preparative electrophoresis are bimodal, unlike the monophasic for albumin obtained by polyethylene glycol precipitation. These changes result from a higher content of nonetherified fatty acids in the albumin samples obtained by affinity chromatography and from modification of the secondary protein structure in the samples obtained by electrophoresis. Analysis of melting thermograms of serum albumin from patients with uremia, chronic hepatitis, and peritonitis shows that fractionation of blood with polyethylene glycol preserves the thermodynamic characteristics of the various pathological serum albumins to the greatest extent. The present results demonstrate the advantage of polyethylene glycol fractionation for isolation of native preparations of normal and “pathological” human serum albumin.

  7. Closely related methicillin-resistant Staphylococcus aureus isolates from retail meat, cows with mastitis, and humans in Japan.

    Science.gov (United States)

    Sato, Tomomi; Usui, Masaru; Konishi, Noriko; Kai, Akemi; Matsui, Hidehito; Hanaki, Hideaki; Tamura, Yutaka

    2017-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a pervasive healthcare-acquired (HA) pathogen with recent emergence as a community-acquired (CA) pathogen. To elucidate whether meat mediates MRSA transmission between animals and humans in Japan, this study examined MRSA isolates from retail meat (n = 8), cows with mastitis (n = 7), and humans (HA-MRSA = 46 and CA-MRSA = 54) by molecular typing, virulence gene analyses, and antimicrobial susceptibility testing. MRSA isolates from retail meat were classified into sequence type (ST) 8/spa type t1767 (n = 4), ST8/t4133 (n = 1), ST59/t3385 (n = 1), ST88/t375 (n = 1), and ST509/t375 (n = 1). All seven MRSA isolates from cows with mastitis were ST8/t1767. 46 HA-MRSA were clonal complex (CC) 5, divided into t002 (n = 30), t045 (n = 12), and t7455 (n = 4). 54 CA-MRSA were classified into 6 different CCs: CC1 (n = 14), CC5 (n = 7), CC8 (n = 29), CC45 (n = 1), CC89 (n = 1), CC509 (n = 1), and into 16 different spa types including newly identified t17177, t17193, and t17194. The majority were CC8/t1767 (n = 16). CC of one CA-MRSA isolate (spa type t1767) was not classified. Among 41 CC8 MRSA (five from meat, seven from cows with mastitis, and 29 CA-MRSA), 14 ST8/SCCmec IVl isolates (three from meat, one from a cow with mastitis, and 10 CA-MRSA) had identical pulsed-field gel electrophoresis patterns and similar spa type (t1767, t4133, and t17177), and were typed as CA-MRSA/J (ST8/SCCmec IVl, positive for sec + sel + tst but negative for Panton-Valentine leukocidin and the arginine catabolic mobile element). These results suggest that there is a transmission cycle of CA-MRSA/J among meat, cows, and humans in Japan, although it is unclear whether the origin is cow.

  8. Isolation of human fungi from soil and identification of two endemic areas of Cryptococcus neoformans and Coccidioides immitis

    Directory of Open Access Journals (Sweden)

    Héctor Rubinstein

    1989-02-01

    Full Text Available The present study was carried out in two different areas of Province of Cordoba, Argentina, where there was a suspicious of endemic mycosis. The previous data were the presence of a clinical case of pulmonary cryptococcosis in one area (Alta Gracia and the previous findings of a high incidence of coccidioidin and cryptococcin reactors in the population of the second one (Villa Dolores. In both areas soil samples for fungi were studied and Cryptococcus neoformans was found in 2/25 samples from Alta Gracia. In Villa Dolores Coccidioides immitis was isolated in 2/40 samples, and C. neoformans in 1/40 samples. Delayed hypersensitivity test with cryptococcin was determined in the population from Alta Gracia and it was found to be 5.3%. Positive cutaneous tests with coccidioidin (33.8% and cryptococcin (31.9% in Villa Dolores were obtained. With these findings two endemic areas of systemic mycoses in Cordoba, Argentina were delimited.

  9. Three novel, multiple recombinant types of species of human mastadenovirus D (HAdV-D 73, 74 & 75) isolated from diarrhoeal faeces of immunocompromised patients.

    Science.gov (United States)

    Hage, Elias; Dhingra, Akshay; Liebert, Uwe G; Bergs, Sandra; Ganzenmueller, Tina; Heim, Albert

    2017-11-02

    Species D is the largest of the seven species of human mastadenoviruses (HAdV), but few of its multiple types are associated with asevere disease, e.g. epidemic keratoconjunctivitis. Many other types are hardly ever associated with significant diseases in immunocompetent patients, but have been isolated from the diarrhoeal faeces of terminal AIDS patients suggesting their role as opportunistic pathogens. Three novel HAdV-D strains were isolated from the faeces of three immunocompromised adult patients (clinical diagnoses: lymphoma, myelodysplastic syndrome and AIDS CDC3B, respectively). These strains were not typeable by imputed serology of the hexon and fibre gene and therefore complete genomic sequences were generated by next-generation sequencing (NGS). All three strains were multiple recombinants and fulfilled the criteria for designation as types 73, 74 and 75 with the penton/hexon/fibre genotype codes P67H45F27, P70H74F51 and P75H26F29, respectively. A novel genomic backbone and also a novel hexon neutralization epitope sequence were discovered in type 74, and a novel penton sequence in type 75. At the complete genome level, types 73, 74 and 75 were closely related neither to each other nor to type 70, which was previously isolated in the same region. However, these four HAdV-D types were closely related to each other in single genes and gene regions, e.g. penton, E1 and E4 due to recombination events in their phylogeny. In conclusion, regional co-circulation of opportunistic HAdV-D types facilitated co- and super-infections, which are essential for homologous recombination, and thus resulted in the evolution of novel genotypes by lateral gene transfer.

  10. A Phase 3, multicenter, open-label, switchover trial to assess the safety and efficacy of taliglucerase alfa, a plant cell-expressed recombinant human glucocerebrosidase, in adult and pediatric patients with Gaucher disease previously treated with imiglucerase.

    Science.gov (United States)

    Pastores, Gregory M; Petakov, Milan; Giraldo, Pilar; Rosenbaum, Hanna; Szer, Jeffrey; Deegan, Patrick B; Amato, Dominick J; Mengel, Eugen; Tan, Ee Shien; Chertkoff, Raul; Brill-Almon, Einat; Zimran, Ari

    2014-12-01

    Taliglucerase alfa is a β-glucosidase enzyme replacement therapy (ERT) approved in the US and other countries for the treatment of Gaucher disease (GD) in adults and is approved in pediatric and adult patients in Australia and Canada. It is the first approved plant cell-expressed recombinant human protein. A Phase 3, multicenter, open-label, 9-month study assessed safety and efficacy of switching to taliglucerase alfa in adult and pediatric patients with GD treated with imiglucerase for at least the previous 2years. Patients with stable disease were offered taliglucerase alfa treatment using the same dose (9-60U/kg body weight) and regimen of administration (every 2weeks) as imiglucerase. This report summarizes results from 26 adult and 5 pediatric patients who participated in the trial. Disease parameters (spleen and liver volumes, hemoglobin concentration, platelet count, and biomarker levels) remained stable through 9months of treatment in adults and children following the switch from imiglucerase. All treatment-related adverse events were mild or moderate in severity and transient in nature. Exploratory parameters of linear growth and development showed positive outcomes in pediatric patients. These findings provide evidence of the efficacy and safety profile of taliglucerase alfa as an ERT for GD in patients previously treated with imiglucerase. This trial was registered at www.clinicaltrials.gov as # NCT00712348. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Comparison of the vasodilator responses of isolated human and rat middle meningeal arteries to migraine related compounds

    DEFF Research Database (Denmark)

    Grände, Gustaf; Labruijere, Sieneke; Haanes, Kristian Agmund

    2014-01-01

    , telcagepant) were applied to the isolated arteries, and both induced a significant decrease of the effect of exogenously administrated CGRP. In experiments on rat middle meningeal arteries, pre-contracted with PGF2α, similar tendencies were seen. When the pre-contraction was switched to K+ in a separate...... series of experiments, CGRP and sildenafil significantly relaxed the arteries. CONCLUSIONS: Still no definite answer can be given as to why pain is experienced during an attack of migraine. No clear correlation was found between the efficacy of a substance as a meningeal artery vasodilator in human...

  12. Isolation of Human Islets for Autologous Islet Transplantation in Children and Adolescents with Chronic Pancreatitis

    Directory of Open Access Journals (Sweden)

    Rita Bottino

    2012-01-01

    Full Text Available Chronic pancreatitis is an inflammatory disease of the pancreas that causes permanent changes in the function and structure of the pancreas. It is most commonly a complication of cystic fibrosis or due to a genetic predisposition. Chronic pancreatitis generally presents symptomatically as recurrent abdominal pain, which becomes persistent over time. The pain eventually becomes disabling. Once specific medical treatments and endoscopic interventions are no longer efficacious, total pancreatectomy is the alternative of choice for helping the patient achieve pain control. While daily administrations of digestive enzymes cannot be avoided, insulin-dependent diabetes can be prevented by transplanting the isolated pancreatic islets back to the patient. The greater the number of islets infused, the greater the chance to prevent or at least control the effects of surgical diabetes. We present here a technical approach for the isolation and preservation of the islets proven to be efficient to obtain high numbers of islets, favoring the successful treatment of young patients.

  13. Presence of Glycopeptide-Encoding Plasmids in Enterococcal Isolates from Food and Humans in Denmark

    DEFF Research Database (Denmark)

    Migura, Lourdes Garcia; Valenzuela, Antonio Jesus Sanchez; Jensen, Lars Bogø

    2011-01-01

    developed techniques for classification of plasmids. Replicons associated with sex pheromone-inducible plasmids were detected in all GR E. faecalis, whereas GR Enterococcus faecium contained plasmids known to be widely distributed among enterococci. vanA resistance is common in E. faecium isolates from meat...... and animals in Europe and is rarely found in E. faecalis. This article describes the first characterization of MGE from vanA mediated E. faecalis, thus linking this resistance genotype to pheromone responding plasmids....

  14. Chitinolytic enzymes from bacterium inhabiting human gastrointestinal tract - critical parameters of protein isolation from anaerobic culture

    Czech Academy of Sciences Publication Activity Database

    Dušková, Jarmila; Tishchenko, Galina; Ponomareva, E.; Šimůnek, Jiří; Koppová, Ingrid; Skálová, Tereza; Štěpánková, Andrea; Hašek, Jindřich; Dohnálek, Jan

    2011-01-01

    Roč. 58, č. 2 (2011), s. 261-263 ISSN 0001-527X R&D Projects: GA ČR GA310/09/1407; GA ČR GA305/07/1073 Institutional research plan: CEZ:AV0Z40500505; CEZ:AV0Z50450515 Keywords : chitinolytic enzymes * anaerobic cultivation * protein isolation Subject RIV: EE - Microbiology, Virology Impact factor: 1.491, year: 2011 http://www.actabp.pl/pdf/2_2011/261.pdf

  15. Distribution of phylogroups and co-resistance to antimicrobial agents in ampicillin resistant Escherichia coli isolated from healthy humans and from patients with bacteraemia

    DEFF Research Database (Denmark)

    Haugaard, A.; Hammerum, A. M.; Porsbo, Lone Jannok

    inhibitory concentration to antimicrobial agents and examined by PCR to determine their phylogroups. The phylotyping grouped the faecal samples into A (13%), B1 (10%), B2 (42%), D (19%), NT (16%) while the blood isolates grouped into A (16%), B1 (0%), B2 (48%), D (32%) and NT (3%). The frequency...... of resistance in faecal and blood isolates (F/B) was: tetracycline (48%/48%), gentamicin (0%/10%), ciprofloxacin (3%,13%), sulfonamide (68%/77%) and trimethoprim (39%/39%). Conclusion: B2 was the most prevalent phylogroup found both in faecal isolates collected from healthy humans and in blood isolates from...

  16. Epicardial excitation pattern as observed in the isolated revived and perfused fetal human heart

    NARCIS (Netherlands)

    Durrer, D.; Büller, J.; Graaff, P.; Lo, G.I.; Meijler, F.L.

    1961-01-01

    The resuscitated fetal human heart can be used as an experimental tooI for the investigation of the excitatory process in the human heart. During perfusion the configuration of the epicardial electrocardiograms does not change appreciably. For accurate recording permitting a detailed analysis, the

  17. Improved methodology for the affinity isolation of human protein complexes expressed at near endogenous levels

    DEFF Research Database (Denmark)

    Domanski, Michal; Molloy, Kelly; Jiang, Hua

    2012-01-01

    An efficient and reliable procedure for the capture of affinity-tagged proteins and associated complexes from human cell lines is reported. Through multiple optimizations, high yield and low background affinity-purifications are achieved from modest quantities of human cells expressing endogenous...

  18. Treponema putidum sp. nov., a medium-sized proteolytic spirochaete isolated from lesions of human periodontitis and acute necrotizing ulcerative gingivitis.

    Science.gov (United States)

    Wyss, C; Moter, A; Choi, B-K; Dewhirst, F E; Xue, Yi; Schüpbach, P; Göbel, U B; Paster, B J; Guggenheim, B

    2004-07-01

    So far, little phenotypic heterogeneity has been detected in cultured oral treponemes with trypsin-like proteolytic activity, and all have been assigned to the species Treponema denticola. However, comparisons of protein patterns and antigen expression in our collection of proteolytic oral treponemes occasionally identified isolates with a unique phenotype; e.g. strain OMZ 830 (=ATCC 700768), which qualified as a 'pathogen-related oral spirochaete' due to the presence of a approximately 37 kDa protein reactive with the Treponema pallidum FlaA-specific mAb H9-2. In addition to such single isolates, a homogeneous group of seven independent strains is described that were highly motile, medium-sized, proteolytic but asaccharolytic spirochaetes and were cultured from human gingivitis, periodontitis and acute necrotizing ulcerative gingivitis in medium OMIZ-Pat supplemented with 1% human serum and antibiotics. Growth of these spirochaetes in OMIZ-Pat was not dependent on, but was stimulated by, human or bovine serum. Carbohydrates were neither required nor stimulatory for growth. The protein and antigen patterns of total cell extracts of these organisms separated by SDS-PAGE were distinct from those of all previously cultured spirochaetes, with highest similarity to T. denticola. The novel spirochaete has a 2 : 4 : 2 arrangement of the periplasmic flagella, similar to T. denticola. However, the flagellin pattern as detected by immunostaining or glycan staining of Western blots readily distinguished the novel group from T. denticola. Also, distinct from reference strains of T. denticola, none of the novel isolates displayed sialidase or dentilisin activities, both of which are expressed by most strains of T. denticola. Trypsin-like activity and other enzymes as detected by API ZYM test were similar to those of T. denticola. The status of a novel species is supported by the 16S rRNA gene sequence, with 98.5% similarity to its closest cultured relative, T. denticola. The

  19. Phenotypic, Genotypic, and Antimicrobial Characteristics of Streptococcus halichoeri Isolates from Humans, Proposal To Rename Streptococcus halichoeri as Streptococcus halichoeri subsp. halichoeri, and Description of Streptococcus halichoeri subsp. hominis subsp. nov., a Bacterium Associated with Human Clinical Infections.

    Science.gov (United States)

    Shewmaker, P L; Whitney, A M; Humrighouse, B W

    2016-03-01

    Phenotypic, genotypic, and antimicrobial characteristics of six phenotypically distinct human clinical isolates that most closely resembled the type strain of Streptococcus halichoeri isolated from a seal are presented. Sequencing of the 16S rRNA, rpoB, sodA, and recN genes; comparative whole-genome analysis; conventional biochemical and Rapid ID 32 Strep identification methods; and antimicrobial susceptibility testing were performed on the human isolates, the type strain of S. halichoeri, and type strains of closely related species. The six human clinical isolates were biochemically indistinguishable from each other and showed 100% 16S rRNA, rpoB, sodA, and recN gene sequence similarity. Comparative 16S rRNA gene sequencing analysis revealed 98.6% similarity to S. halichoeri CCUG 48324(T), 97.9% similarity to S. canis ATCC 43496(T), and 97.8% similarity to S. ictaluri ATCC BAA-1300(T). A 3,530-bp fragment of the rpoB gene was 98.8% similar to the S. halichoeri type strain, 84.6% to the S. canis type strain, and 83.8% to the S. ictaluri type strain. The S. halichoeri type strain and the human clinical isolates were susceptible to the antimicrobials tested based on CLSI guidelines for Streptococcus species viridans group with the exception of tetracycline and erythromycin. The human isolates were phenotypically distinct from the type strain isolated from a seal; comparative whole-genome sequence analysis confirmed that the human isolates were S. halichoeri. On the basis of these results, a novel subspecies, Streptococcus halichoeri subsp. hominis, is proposed for the human isolates and Streptococcus halichoeri subsp. halichoeri is proposed for the gray seal isolates. The type strain of the novel subspecies is SS1844(T) = CCUG 67100(T) = LMG 28801(T). Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  20. Rapid isolation of antibody from a synthetic human antibody library by repeated fluorescence-activated cell sorting (FACS.

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    Sung Sun Yim

    Full Text Available Antibodies and their derivatives are the most important agents in therapeutics and diagnostics. Even after the significant progress in the technology for antibody screening from huge libraries, it takes a long time to isolate an antibody, which prevents a prompt action against the spread of a disease. Here, we report a new strategy for isolating desired antibodies from a combinatorial library in one day by repeated fluorescence-activated cell sorting (FACS. First, we constructed a library of synthetic human antibody in which single-chain variable fragment (scFv was expressed in the periplasm of Escherichia coli. After labeling the cells with fluorescent antigen probes, the highly fluorescent cells were sorted by using a high-speed cell sorter, and these cells were reused without regeneration in the next round of sorting. After repeating this sorting, the positive clones were completely enriched in several hours. Thus, we screened the library against three viral antigens, including the H1N1 influenza virus, Hepatitis B virus, and Foot-and-mouth disease virus. Finally, the potential antibody candidates, which show K(D values between 10 and 100 nM against the target antigens, could be successfully isolated even though the library was relatively small (∼ 10(6. These results show that repeated FACS screening without regeneration of the sorted cells can be a powerful method when a rapid response to a spreading disease is required.

  1. The first report of the vanC1 gene in Enterococcus faecium isolated from a human clinical specimen

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    Mingyue Sun

    2014-09-01

    Full Text Available The vanC1 gene, which is chromosomally located, confers resistance to vancomycin and serves as a species marker for Enterococcus gallinarum. Enterococcus faecium TJ4031 was isolated from a blood culture and harbours the vanC1gene. Polymerase chain reaction (PCR assays were performed to detect vanXYc and vanTc genes. Only the vanXYc gene was found in the E. faecium TJ4031 isolate. The minimum inhibitory concentrations of vancomycin and teicoplanin were 2 µg/mL and 1 µg/mL, respectively. Real-time reverse transcription-PCR results revealed that the vanC1and vanXYc genes were not expressed. Pulsed-field gel electrophoresis and southern hybridisation results showed that the vanC1 gene was encoded in the chromosome. E. faecalis isolated from animals has been reported to harbour vanC1gene. However, this study is the first to report the presence of the vanC1gene in E. faecium of human origin. Additionally, our research showed the vanC1gene cannot serve as a species-specific gene of E. gallinarum and that it is able to be transferred between bacteria. Although the resistance marker is not expressed in the strain, our results showed that E. faecium could acquire the vanC1gene from different species.

  2. Isolation and Characterization of Human Dental Pulp Stem Cells from Cryopreserved Pulp Tissues Obtained from Teeth with Irreversible Pulpitis.

    Science.gov (United States)

    Malekfar, Azin; Valli, Kusum S; Kanafi, Mohammad Mahboob; Bhonde, Ramesh R

    2016-01-01

    Human dental pulp stem cells (DPSCs) are becoming an attractive target for therapeutic purposes because of their neural crest origin and propensity. Although DPSCs can be successfully cryopreserved, there are hardly any reports on cryopreservation of dental pulp tissues obtained from teeth diagnosed with symptomatic irreversible pulpitis during endodontic treatment and isolation and characterization of DPSCs from such cryopreserved pulp. The aim of this study was to cryopreserve the said pulp tissues to propagate and characterize isolated DPSCs. A medium consisting of 90% fetal bovine serum and 10% dimethyl sulfoxide was used for cryopreservation of pulp tissues. DPSCs were isolated from fresh and cryopreserved pulp tissues using an enzymatic method. Cell viability and proliferation were determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. DPSC migration and interaction were analyzed with the wound healing assay. Mesenchymal characteristics of DPSCs were verified by flow cytometric analysis of cell surface CD markers. The osteogenic and adipogenic potential of DPSCs was shown by von Kossa and oil red O staining methods, respectively, and the polymerase chain reaction method. We found no significant difference in CD marker expression and osteogenic and adipogenic differentiation potential of DPSCs obtained from fresh and cryopreserved dental pulp tissue. Our study shows that dental pulp can be successfully cryopreserved without losing normal characteristics and differentiation potential of their DPSCs, thus making them suitable for dental banking and future therapeutic purposes. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  3. Geographic distribution of methyltransferases of Helicobacter pylori: evidence of human host population isolation and migration

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    Vítor Jorge MB

    2009-09-01

    Full Text Available Abstract Background Helicobacter pylori colonizes the human stomach and is associated with gastritis, peptic ulcer, and gastric cancer. This ubiquitous association between H. pylori and humans is thought to be present since the origin of modern humans. The H. pylori genome encodes for an exceptional number of restriction and modifications (R-M systems. To evaluate if R-M systems are an adequate tool to determine the geographic distribution of H. pylori strains, we typed 221 strains from Africa, America, Asia, and Europe, and evaluated the expression of different 29 methyltransferases. Results Independence tests and logistic regression models revealed that ten R-M systems correlate with geographical localization. The distribution pattern of these methyltransferases may have been originated by co-divergence of regional H. pylori after its human host migrated out of Africa. The expression of specific methyltransferases in the H. pylori population may also reflect the genetic and cultural background of its human host. Methyltransferases common to all strains, M. HhaI and M. NaeI, are likely conserved in H. pylori, and may have been present in the bacteria genome since the human diaspora out of Africa. Conclusion This study indicates that some methyltransferases are useful geomarkers, which allow discrimination of bacterial populations, and that can be added to our tools to investigate human migrations.

  4. Characteristics of Shigatoxin-Producing Escherichia coli Strains Isolated during 2010–2014 from Human Infections in Switzerland

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    Lisa Fierz

    2017-08-01

    Full Text Available Objectives: The aim of this study was to characterize a collection of 95 Shigatoxin-producing E.coli (STEC isolated from human patients in Switzerland during 2010–2014.Methods: We performed O and H serotyping and molecular subtyping.Results: The five most common serogroups were O157, O145, O26, O103, and O146. Of the 95 strains, 35 (36.8% carried stx1 genes only, 43 strains (45.2% carried stx2 and 17 (17.9% harbored combinations of stx1 and stx2 genes. Stx1a (42 strains and stx2a (32 strains were the most frequently detected stx subtypes. Genes for intimin (eae, hemolysin (hly, iron-regulated adhesion (iha, and the subtilase cytotoxin subtypes subAB1, subAB2-1, subAB2-2, or subAB2-3 were detected in 70.5, 83.2, 74.7, and 20% of the strains, respectively. Multilocus sequence typing assigned the majority (58.9% of the isolates to five different clonal complexes (CC, 11, 32, 29, 20, and 165, respectively. CC11 included all O157:[H7] and O55:[H7] isolates. CC32 comprised O145:[H28] isolates, and O145:[H25] belonged to sequence type (ST 342. CC29 contained isolates of the O26:[H11], O111:[H8] and O118:[Hnt] serogroups, and CC20 encompassed isolates of O51:H49/[Hnt] and O103:[H2]. CC165 included isolates typed O80:[H2]-ST301, all harboring stx2d, eae-ξ, hly, and 66.7% additionally harboring iha. All O80:[H2]-ST301 strains harbored at least 7 genes carried by pS88, a plasmid associated with extraintestinal virulence. Compared to data from Switzerland from the years 2000–2009, an increase of the proportion of non-O157 STEC infections was observed as well as an increase of infections due to STEC O146. By contrast, the prevalence of the highly virulent German clone STEC O26:[H11]-ST29 decreased from 11.3% during 2000–2009 to 1.1% for the time span 2010–2014. The detection of O80:[H2]-ST301 harboring stx2d, eae-ξ, hly, iha, and pS88 related genes suggests an ongoing emergence in Switzerland of an unusual, highly pathogenic STEC serotype

  5. Isolation of reovirus T3D mutants capable of infecting human tumor cells independent of junction adhesion molecule-A.

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    Diana J M van den Wollenberg

    Full Text Available Mammalian Reovirus is a double-stranded RNA virus with a distinctive preference to replicate in and lyse transformed cells. On that account, Reovirus type 3 Dearing (T3D is clinically evaluated as oncolytic agent. The therapeutic efficacy of this approach depends in part on the accessibility of the reovirus receptor Junction Adhesion Molecule-A (JAM-A on the target cells. Here, we describe the isolation and characterization of reovirus T3D mutants that can infect human tumor cells independent of JAM-A. The JAM-A-independent (jin mutants were isolated on human U118MG glioblastoma cells, which do not express JAM-A. All jin mutants harbour mutations in the S1 segments close to the region that encodes the sialic acid-binding pocket in the shaft of the spike protein. In addition, two of the jin mutants encode spike proteins with a Q336R substitution in their head domain. The jin mutants can productively infect a wide range of cell lines that resist wt reovirus T3D infection, including chicken LMH cells, hamster CHO cells, murine endothelioma cells, human U2OS and STA-ET2.1 cells, but not primary human fibroblasts. The jin-mutants rely on the presence of sialic-acid residues on the cell surface for productive infection, as is evident from wheat germ agglutinin (WGA inhibition experiments, and from the jin-reovirus resistance of CHO-Lec2 cells, which have a deficiency of sialic-acids on their glycoproteins. The jin mutants may be useful as oncolytic agents for use in tumors in which JAM-A is absent or inaccessible.

  6. Phytol isolated from watermelon (Citrullus lanatus) sprouts induces cell death in human T-lymphoid cell line Jurkat cells via S-phase cell cycle arrest.

    Science.gov (United States)

    Itoh, Tomohiro; Ono, Akito; Kawaguchi, Kaori; Teraoka, Sayaka; Harada, Mayo; Sumi, Keitaro; Ando, Masashi; Tsukamasa, Yasuyuki; Ninomiya, Masayuki; Koketsu, Mamoru; Hashizume, Toshiharu

    2018-05-01

    The phytol isolated from watermelon (Citrullus lanatus) sprouts inhibited the growth of a human T-cell leukemia line Jurkat cell and suppressed tumor progression in a xenograft model of human lung adenocarcinoma epithelial cell line A549 in nude mice. To elucidate the mechanisms underlying the phytol-induced cell death in the present study, we examined the changes in cell morphology, DNA fragmentation, and intracellular reactive oxygen species (ROS) levels and performed flow cytometric analysis to evaluate cell cycle stage. There were no significant changes in apoptosis, autophagy, and necrosis marker in cells treated with the phytol. But, we found, for the first time, that phytol remarkably induced S-phase cell cycle arrest accompanied with intracellular ROS production. Western blot analyses showed that phytolinduced S-phase cell cycle arrest was mediated through the decreased expression of cyclins A and D and the downregulations of MAPK and PI3K/Akt. The tumor volume levels in mice treated with phytol were lower than those of non-treatment groups, and it showed very similar suppression compared with those of mice treated with cyclophosphamide. Based on the data of in vitro and in vivo studies and previous studies, we suggest phytol as a potential therapeutic compound for cancer. Copyright © 2018 Elsevier Ltd. All rights reserved.

  7. Immunomodulatory Properties of Streptococcus and Veillonella Isolates from the Human Small Intestine Microbiota

    NARCIS (Netherlands)

    Bogert, van den B.; Meijerink, M.; Zoetendal, E.G.; Wells, J.M.; Kleerebezem, M.

    2014-01-01

    The human small intestine is a key site for interactions between the intestinal microbiota and the mucosal immune system. Here we investigated the immunomodulatory properties of representative species of commonly dominant small-intestinal microbial communities, including six streptococcal strains

  8. Binding of human endothelium to Ulex europaeus I-coated Dynabeads: application to the isolation of microvascular endothelium.

    Science.gov (United States)

    Jackson, C J; Garbett, P K; Nissen, B; Schrieber, L

    1990-06-01

    A major problem encountered when isolating human microvascular endothelium is the presence of contaminating cells such as fibroblasts that rapidly over-grow the endothelial cells. We describe here a simple, rapid technique for purifying endothelial cells derived from the microvasculature of neonatal foreskin and osteoarthritic and rheumatoid arthritic synovium. This technique is based on the selective binding of the lectin Ulex europaeus I (UEA I) to the endothelial cell surface via fucose residues. Initially UEA I was covalently bound to tosyl-activated super-paramagnetic polystyrene beads (Dynabeads) by incubation for 24 h at room temperature. Cells were isolated by extracting microvascular segments from enzyme-treated (trypsin and Pronase) cubes of tissue. The mixed population of cells obtained were purified by incubating them at 4 degrees C for 10 min with the UEA I-coated Dynabeads. Endothelium bound to the beads whilst contaminating cells were removed by five washes with HBSS using a magnetic particle concentrator. The endothelial cells thus obtained grew to confluence as a cobblestone-like monolayer and expressed von Willebrand factor antigen. The cells were released from the Dynabeads by the competitive binding of fucose (10 min at 4 degrees C). This new method is simple and reproducible and allows pure human microvascular endothelial cells to be cultured within 2 h of obtaining a specimen.

  9. Human pancreatic islet transplantation: an update and description of the establishment of a pancreatic islet isolation laboratory.

    Science.gov (United States)

    Rheinheimer, Jakeline; Bauer, Andrea C; Silveiro, Sandra P; Estivalet, Aline A F; Bouças, Ana P; Rosa, Annelise R; Souza, Bianca M de; Oliveira, Fernanda S de; Cruz, Lavínia A; Brondani, Letícia A; Azevedo, Mirela J; Lemos, Natália E; Carlessi, Rodrigo; Assmann, Taís S; Gross, Jorge L; Leitão, Cristiane B; Crispim, Daisy

    2015-04-01

    Type 1 diabetes mellitus (T1DM) is associated with chronic complications that lead to high morbidity and mortality rates in young adults of productive age. Intensive insulin therapy has been able to reduce the likelihood of the development of chronic diabetes complications. However, this treatment is still associated with an increased incidence of hypoglycemia. In patients with "brittle T1DM", who have severe hypoglycemia without adrenergic symptoms (hypoglycemia unawareness), islet transplantation may be a therapeutic option to restore both insulin secretion and hypoglycemic perception. The Edmonton group demonstrated that most patients who received islet infusions from more than one donor and were treated with steroid-free immunosuppressive drugs displayed a considerable decline in the initial insulin independence rates at eight years following the transplantation, but showed permanent C-peptide secretion, which facilitated glycemic control and protected patients against hypoglycemic episodes. Recently, data published by the Collaborative Islet Transplant Registry (CITR) has revealed that approximately 50% of the patients who undergo islet transplantation are insulin independent after a 3-year follow-up. Therefore, islet transplantation is able to successfully decrease plasma glucose and HbA1c levels, the occurrence of severe hypoglycemia, and improve patient quality of life. The goal of this paper was to review the human islet isolation and transplantation processes, and to describe the establishment of a human islet isolation laboratory at the Endocrine Division of the Hospital de Clínicas de Porto Alegre - Rio Grande do Sul, Brazil.

  10. Bacillus niameyensis sp. nov., a new bacterial species isolated from human gut

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    M. Tidjani Alou

    2015-11-01

    Full Text Available Bacillus niameyensis sp. nov. strain SIT3T (= CSUR P1266 = DSM 29725 is the type strain of B. niameyensis sp. nov. This Gram-positive strain was isolated from the digestive flora of a child with kwashiorkor and is a facultative anaerobic rod and a member of the Bacillaceae family. This organism is hereby described alongside its complete genome sequence and annotation. The 4  286  116 bp long genome (one chromosome but no plasmid contains 4130 protein-coding and 66 RNA genes including five rRNA genes.

  11. Simple and efficient method for isolation and cultivation of endoscopically obtained human colonocytes

    DEFF Research Database (Denmark)

    Seidelin, Jakob B; Horn, Thomas; Nielsen, Ole H

    2003-01-01

    and where the diagnosis of irritable bowel syndrome was later reached, were included. Seven colon biopsies were taken and incubated at varying time periods of 10-120 min and temperatures of 4-37 degrees C in a chelating buffer. The epithelium was then harvested and cultivated under three different......Few comparative and validated reports exist on the isolation and growth of colonoscopically obtained colonic epithelium. The aim of this study was to develop and validate a simple method for the cultivation of colonoscopically obtained colonocytes. Forty patients, who underwent routine colonoscopy...

  12. Selective isolation and differentiation of a stromal population of human embryonic stem cells with osteogenic potential

    DEFF Research Database (Denmark)

    Harkness, Linda M; Mahmood, Amer; Ditzel, Nicholas

    2011-01-01

    cultured in osteogenic differentiation media, up regulation of osteoblastic lineage markers (DLX5, MSX2, RUNX2, SPARC, ALP, COL1a1, BGLAP, IBSP, DCN, LOX-L4) and production of in vitro mineralized matrix was detected. hESC-stromal cells loaded on a carrier and implanted either subcutaneously...... or in a critical size calvarial defect in immune deficient mice for 10weeks, resulted in new bone formation and partial repair of the calvarial defect. In conclusion, hESC-stromal can be isolated from hESC cultures and represent a good source for obtaining cells with osteogenic differentiation potential suitable...

  13. Isolation and characterization of a neoplastic epithelial cell line derived from irradiated human submaxillary gland

    International Nuclear Information System (INIS)

    Shirasuna, Kanemitsu; Sato, Mitsunobu; Yura, Yoshiaki; Yanagawa, Tetuo; Kubo, Kazuko

    1979-01-01

    Submaxillary tissues taken from a patient whose oral base was irradiated for squamous cell carcinoma were cultured in order to isolate transformed epithelial cells in vitro. The cells showed a fine structure similar to an intermediate duct cell. When they were transplanted in nude mice, salivary tumors developed. It is epidemiologically known that irradiation induces salivary tumors. In this study, the risk of inducement was revealed and a salivary epithelial cell line was used as a model for the analysis of salivary tumors. (Ichikawa, K.)

  14. Intramacrophage survival of uropathogenic Escherichia coli: Differences between diverse clinical isolates and between mouse and human macrophages

    KAUST Repository

    Bokil, Nilesh J.

    2011-11-01

    Uropathogenic E. coli (UPEC) are the primary cause of urinary tract infections. Recent studies have demonstrated that UPEC can invade and replicate within epithelial cells, suggesting that this bacterial pathogen may occupy an intracellular niche within the host. Given that many intracellular pathogens target macrophages, we assessed the interactions between UPEC and macrophages. Colonization of the mouse bladder by UPEC strain CFT073 resulted in increased expression of myeloid-restricted genes, consistent with the recruitment of inflammatory macrophages to the site of infection. In in vitro assays, CFT073 was able to survive within primary mouse bone marrow-derived macrophages (BMM) up to 24h post-infection. Three additional well-characterized clinical UPEC isolates associated with distinct UTI symptomatologies displayed variable long-term survival within BMM. UPEC strains UTI89 and VR50, originally isolated from patients with cystitis and asymptomatic bacteriuria respectively, showed elevated bacterial loads in BMM at 24h post-infection as compared to CFT073 and the asymptomatic bacteriuria strain 83972. These differences did not correlate with differential effects on macrophage survival or initial uptake of bacteria. E. coli UTI89 localized to a Lamp1 + vesicular compartment within BMM. In contrast to survival within mouse BMM, intracellular bacterial loads of VR50 were low in both human monocyte-derived macrophages (HMDM) and in human T24 bladder epithelial cells. Collectively, these data suggest that some UPEC isolates may subvert macrophage anti-microbial pathways, and that host species differences may impact on intracellular UPEC survival. © 2011 Elsevier GmbH.

  15. Isolation and characterization of mesenchymal stem cells derived from dental pulp and follicle tissue of human third molar tooth

    Directory of Open Access Journals (Sweden)

    Yadegary Z

    2011-04-01

    Full Text Available "nBackground and Aims: In the last decade, several studies have reported the isolation of stem cell population from different dental sources, while their mesenchymal nature is still controversial. The aim of this study was to isolate stem cells from mature human dental pulp and follicle and to determine their mesenchymal nature before differentiation based on the ISCT (International Society for Cellular Therapy criteria."nMaterials and Methods: In this experimental study, intact human third molars extracted due to prophylactic or orthodontic reasons were collected from patients aged 18-25. After tooth extraction, dental pulp and follicle were stored at 4°C in RPMI 1640 medium containing antibiotics. Dental pulp and follicle were prepared in a sterile condition and digested using an enzyme solution containing 4mg/ml collagenase I and dispase (ratio: 1:1. The cells were then cultivated in α-MEM medium. Passage-3 cells were analyzed by flow cytometry for the expression of CD34, CD45, CD 73, CD90 and CD105 surface markers."nResults: Dental pulp and follicle were observed to grow in colony forming units, mainly composed of a fibroblast-like cell population. Flow cytometry results showed that dental pulp and follicle are highly positive for CD73, CD90 and CD105 (mesenchymal stem cell markers and are negative for hematopoietic markers such as CD34 and CD 45."nConclusion: In this study we were able to successfully confirm that dental pulp and follicle stem cells isolated from permanent third molars have a mesenchymal nature before differentiation. Therefore, these two sources can be considered as an easy accessible source of mesenchymal stem cells for stem cell research and tissue engineering.

  16. Overexpression and characterization of a novel transgalactosylic and hydrolytic β-galactosidase from a human isolate Bifidobacterium breve B24.

    Science.gov (United States)

    Yi, Sung Hun; Alli, Inteaz; Park, Kwan Hwa; Lee, Byonghoon

    2011-10-01

    After the complete gene of a β-galactosidase from human isolate Bifidobacterium breve B24 was isolated by PCR and overexpressed in E. coli, the recombinant β-galactosidase was purified to homogeneity and characterized for the glycoside transferase (GT) and glycoside hydrolase (GH) activities on lactose. One complete ORF encoding 691 amino acids (2,076 bp) was the structural gene, LacA (galA) of the β-gal gene. The recombinant enzyme shown by activity staining and gel-filtration chromatography was composed of a homodimer of 75 kDa with a total molecular mass of 150 kDa. The K(m) value for lactose (95.58 mM) was 52.5-fold higher than the corresponding K(m) values for the synthetic substrate ONPG (1.82 mM). This enzyme with the optimum of pH 7.0 and 45°C could synthesize approximately 42.00% of GOS from 1M of lactose. About 97.00% of lactose in milk was also quickly hydrolyzed by this enzyme (50 units) at 45°C for 5h to produce 46.30% of glucose, 46.60% of galactose and 7.10% of GOS. The results suggest that this recombinant β-galactosidase derived from a human isolate B. breve B24 may be suitable for both the hydrolysis and synthesis of galacto-oligosaccharides (GOS) in milk and lactose processing. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. Milk fat globule membrane isolate induces apoptosis in HT-29 human colon cancer cells.

    Science.gov (United States)

    Zanabria, Romina; Tellez, Angela M; Griffiths, Mansel; Corredig, Milena

    2013-02-01

    A native milk fat globule membrane (MFGM) isolate obtained from raw milk was assessed for its anticarcinogenic capacity using a colon cancer cell line (HT-29). To prevent microbial contamination and eliminate the presence of lipopolysaccharide (LPS) in the milk used for MFGM isolation, the milk was obtained from the mammary glands of cows using a catheter. Cell proliferation assays demonstrated a reduction of exponentially growing cancer cells of up to 53%, expressed as DNA synthesis (BrdU test), after 72 h stimulation with 100 μg of MFGM protein per mL. Using a similar MFGM concentration, the sulforhodamine B assay resulted in 57% reduction of cell density after 48 h incubation. This bioactivity was comparable to that of known anticancer drugs, 0.1 mM melphalan and 20 μM C2-ceramide, which achieved a cell division reduction of 25 and 40%, respectively, under the same experimental conditions. The toxic effect of the MFGM extracts on HT-29 cells was confirmed by the significant reduction in lactate dehydrogenase enzyme (LDH) by the residual viable cells. An increase of caspase-3 activity (up to 26%) led to the conclusion that MFGM has an apoptotic effect on HT-29 cancer cells.

  18. Third-Generation Cephalosporin-Resistant Non-Typhoidal Salmonella Isolated from Human Feces in Japan.

    Science.gov (United States)

    Saito, Satomi; Koori, Yoshio; Ohsaki, Yusuke; Osaka, Shunsuke; Oana, Kozue; Nagano, Yukiko; Arakawa, Yoshichika; Nagano, Noriyuki

    2017-05-24

    β-lactamase genes were detected and characterized from 10 non-typhoidal Salmonella (NTS) clinical isolates resistant to third-generation cephalosporins collected between 2012 and 2014 in Japan. Five strains showed cefotaxime minimum inhibitory concentration (MIC) ≥ 64 μg/ml and positive clavulanic acid inhibition results. The bla CTX-M-2 was detected in 3 strains (serotypes Stanley and Muenchen), whereas bla TEM-52 (serotype Manhattan) and bla SHV-12 (serotype Infantis) were each found in 1 strain. bla CMY-2 was detected in the remaining 5 strains (serotypes Infantis, Rissen, Newport, and Saintpaul) with cefotaxime MICs of 4-32 μg/ml and positive cloxacillin- and 3-aminophenylboronic acid- based inhibition tests. ISEcp1 was located upstream of the bla CMY-2 in 4 strains and of the bla CTX-M-2 in 1 strain. Incompatibility (Inc)A/C, IncP, and IncI1 plasmids were present in the strains harboring bla CMY-2 , which were detected predominantly in this study. Acquisition of resistance to third-generation cephalosporins by invasive NTS may limit therapeutic options for severe systemic infections and causing serious public health problems. Though such resistant clinical isolates are still rare in Salmonella species in Japan, our findings reveal the presence of cephem-resistant NTS in food handlers, thus emphasizing the necessity of more systematic nationwide investigations.

  19. Peptoniphilus catoniae sp. nov., isolated from a human faecal sample from a traditional Peruvian coastal community.

    Science.gov (United States)

    Patel, Nisha B; Tito, Raul Y; Obregón-Tito, Alexandra J; O'Neal, Lindsey; Trujillo-Villaroel, Omar; Marin-Reyes, Luis; Troncoso-Corzo, Luzmila; Guija-Poma, Emilio; Lewis, Cecil M; Lawson, Paul A

    2016-05-01

    A novel Gram-stain-positive, coccus-shaped, obligately anaerobic bacterium was isolated from a faecal sample obtained from an individual in a traditional community located off the southern coast of Peru. Comparative 16S rRNA gene sequence analysis showed the novel bacterium belonged to the genus Peptoniphilus but showed no particular relationship with any species, demonstrating less than 91 % 16S rRNA gene sequence similarity with all members of the genus. The major cellular fatty acids of the novel isolate were determined to be C10 : 0, C14 : 0, C16 : 0, C18 : 1ω9c and C18 : 2ω6,9c/anteiso-C18 : 0. The DNA G+C content was 34.4 mol%. End-products of metabolism from peptone-yeast-glucose broth (PYG) were determined to be acetate and butyrate. Based on the phenotypic, chemotaxonomic and phylogenetic results, the organism represents a novel species of the genus Peptoniphilus, for which the name Peptoniphilus catoniae sp. nov. is proposed. The type strain is M6.X2DT ( = DSM 29874T = CCUG 66798T).

  20. Comparative virulotyping of extended-spectrum cephalosporin-resistant E. coli isolated from broilers, humans on broiler farms and in the general population and UTI patients.

    Science.gov (United States)

    van Hoek, Angela H A M; Stalenhoef, Janneke E; van Duijkeren, Engeline; Franz, Eelco

    2016-10-15

    During the last decade extended-spectrum cephalosporin (ESC)-resistant Escherichia coli from food-producing animals, especially from broilers, have become a major public health concern because of the potential transmission of these resistant bacteria or their plasmid-encoded resistance genes to humans. The objective of this study was to compare ESC-resistant E. coli isolates from broilers (n=149), humans in contact with these broilers (n=44), humans in the general population (n=63), and patients with a urinary tract infection (UTI) (n=10) with respect to virulence determinants, phylogenetic groups and extended-spectrum β-lactamase (ESBL)/plasmidic-AmpC (pAmpC) genes. The most prevalent ESBL/pAmpC genes among isolates from broilers and individuals on broiler farms were bla CTX-M-1 , bla CMY-2 and bla SHV-12 . In isolates from humans in the general population bla CTX-M-1 , bla CTX-M-14 and bla CTX-M-15 were found most frequently, whereas in UTI isolates bla CTX-M-15 predominated. The marker for enteroaggregative E. coli, aggR, was only identified in a broiler and human isolates from the general population. The extraintestinal virulence genes afa and hlyD were exclusively present in human isolates in the general population and UTI isolates. Multivariate analysis, based on ESBL/pAmpC resistance genes, virulence profiles and phylogenetic groups, revealed that most UTI isolates formed a clearly distinct group. Isolates from broilers and humans associated with broiler farms clustered together. In contrast, isolates from the general population showed some overlap with the former two groups but primarily formed a separate group. These results indicate than transmission occurs between broilers and humans on broiler farms, but also indicate that the role of broilers as a source of foodborne transmission of ESC-resistant E. coli to the general population and subsequently causative agents of human urinary tract infections is likely relatively small. Copyright © 2016 Elsevier

  1. Isolation and characterization of full-length cDNA clones coding for cholinesterase from fetal human tissues

    International Nuclear Information System (INIS)

    Prody, C.A.; Zevin-Sonkin, D.; Gnatt, A.; Goldberg, O.; Soreq, H.

    1987-01-01

    To study the primary structure and regulation of human cholinesterases, oligodeoxynucleotide probes were prepared according to a consensus peptide sequence present in the active site of both human serum pseudocholinesterase and Torpedo electric organ true acetylcholinesterase. Using these probes, the authors isolated several cDNA clones from λgt10 libraries of fetal brain and liver origins. These include 2.4-kilobase cDNA clones that code for a polypeptide containing a putative signal peptide and the N-terminal, active site, and C-terminal peptides of human BtChoEase, suggesting that they code either for BtChoEase itself or for a very similar but distinct fetal form of cholinesterase. In RNA blots of poly(A) + RNA from the cholinesterase-producing fetal brain and liver, these cDNAs hybridized with a single 2.5-kilobase band. Blot hybridization to human genomic DNA revealed that these fetal BtChoEase cDNA clones hybridize with DNA fragments of the total length of 17.5 kilobases, and signal intensities indicated that these sequences are not present in many copies. Both the cDNA-encoded protein and its nucleotide sequence display striking homology to parallel sequences published for Torpedo AcChoEase. These finding demonstrate extensive homologies between the fetal BtChoEase encoded by these clones and other cholinesterases of various forms and species

  2. Defined Conditions for the Isolation and Expansion of Basal Prostate Progenitor Cells of Mouse and Human Origin

    Directory of Open Access Journals (Sweden)

    Thomas Höfner

    2015-03-01

    Full Text Available Methods to isolate and culture primary prostate epithelial stem/progenitor cells (PESCs have proven difficult and ineffective. Here, we present a method to grow and expand both murine and human basal PESCs long term in serum- and feeder-free conditions. The method enriches for adherent mouse basal PESCs with a Lin−SCA-1+CD49f+TROP2high phenotype. Progesterone and sodium selenite are additionally required for the growth of human Lin−CD49f+TROP2high PESCs. The gene-expression profiles of expanded basal PESCs show similarities to ESCs, and NF-kB function is critical for epithelial differentiation of sphere-cultured PESCs. When transplanted in combination with urogenital sinus mesenchyme, expanded mouse and human PESCs generate ectopic prostatic tubules, demonstrating their stem cell activity in vivo. This novel method will facilitate the molecular, genomic, and functional characterization of normal and pathologic prostate glands of mouse and human origin.

  3. Genetic analysis of human and swine influenza A viruses isolated in Northern Italy during 2010-2015.

    Science.gov (United States)

    Chiapponi, C; Ebranati, E; Pariani, E; Faccini, S; Luppi, A; Baioni, L; Manfredi, R; Carta, V; Merenda, M; Affanni, P; Colucci, M E; Veronesi, L; Zehender, G; Foni, E

    2018-02-01

    Influenza A virus (IAV) infection in swine plays an important role in the ecology of influenza viruses. The emergence of new IAVs comes through different mechanisms, with the genetic reassortment of genes between influenza viruses, also originating from different species, being common. We performed a genetic analysis on 179 IAV isolates from humans (n. 75) and pigs (n. 104) collected in Northern Italy between 2010 and 2015, to monitor the genetic exchange between human and swine IAVs. No cases of human infection with swine strains were noticed, but direct infections of swine with H1N1pdm09 strains were detected. Moreover, we pointed out a continuous circulation of H1N1pdm09 strains in swine populations evidenced by the introduction of internal genes of this subtype. These events contribute to generating new viral variants-possibly endowed with pandemic potential-and emphasize the importance of continuous surveillance at both animal and human level. © 2017 The Authors. Zoonoses and Public Health published by Blackwell Verlag GmbH.

  4. Evidence that human milk isolated cyclophilin B corresponds to a truncated form.

    Science.gov (United States)

    Mariller, C; Allain, F; Kouach, M; Spik, G

    1996-03-07

    Cyclophilin B (CyPB) is a member of the cyclophilin family (cyclosporin A-binding proteins) with specific N- and C-terminal extensions. In contrast to cyclophilin A, CyPB owns a signal sequence leading to its translocation in the endoplasmic reticulum. CyPB was reported to be present in human blood and milk, suggesting it is secreted. For this purpose, CyPB was purified to homogeneity from human milk and compared to recombinant CyPB expressed in E. coli. Ion spray mass spectrometry revealed that CyPB secreted in human milk exhibits a lower molecular mass than the one expected. Identification of phenylalanine as the C-terminus amino-acid residue of human milk CyPB indicates that the difference in molecular mass may be explained by the absence of the five C-terminal amino-acid residues AIAKE. These results suggest that in the sequence VEKPFAIAKE known to be responsible for retention of CyPB in the endoplasmic reticulum, the sequence AIAKE is more particularly necessary. Our findings raise the possibility that the CyPB may be processed to promote its release. As recombinant CyPB was shown to bind specifically to Jurkat cells, a lymphoblastic T-cell line, we then wanted to investigate the binding of human milk CyPB to these cells. Despite lacking the five C-terminal amino-acid residues, human milk CyPB is able to inhibit the binding of recombinant CyPB to Jurkat T cells.

  5. Molecular characterization of Salmonella enterica serotype Enteritidis isolates from food and human samples by serotyping, antimicrobial resistance, plasmid profiling, (GTG5-PCR and ERIC-PCR

    Directory of Open Access Journals (Sweden)

    F. Fardsanei

    2016-11-01

    Full Text Available In recent years, Salmonella enterica serovar Enteritidis has been a primary cause of human salmonellosis in many countries. The major objective of this study was to investigate genetic diversity among Salmonella Enteritidis strains from different origins (food and human by Enterobacterial Repetitive Intergenic Consensus (ERIC -PCR, as well as to assess their plasmid profiling and anti