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Sample records for human igg transport

  1. Maternofetal transplacental transport of recombinant IgG antibodies lacking effector functions

    DEFF Research Database (Denmark)

    Mathiesen, Line; Nielsen, Leif K; Andersen, Jan Terje

    2013-01-01

    type IgG1 and IgG3, and found transport across trophoblast-derived BeWo cells and ex vivo placenta perfusions with hierarchies as follows: IgG3ΔHinge:R435H>wild type IgG1≥IgG3ΔHinge, and IgG3ΔHinge:R435H=wild type IgG1=wild type IgG3>IgG3ΔHinge, respectively. Collectively, IgG3ΔHinge:R435H...

  2. Human IgG2 Antibody Disulfide Rearrangement in Vivo*

    OpenAIRE

    Liu, Y. Diana; CHEN, XIAOYU; Enk, Jian Zhang-van; Plant, Matt; Dillon, Thomas M.; Flynn, Gregory C.

    2008-01-01

    Proteins destined to circulate in the blood are first folded and assembled in the endoplasmic reticulum of secretory cells. For antibodies, like many other serum proteins, the folding and assembly steps involve the formation of disulfide bonds. Such bonds have been thought to be static features of proteins, stabilizing domains, and linking polypeptide chains, although some cases of extracellular disulfide bond cleavage have been noted. Recently, the human IgG2 antibody...

  3. Structural characterization of the Man5 glycoform of human IgG3 Fc

    Energy Technology Data Exchange (ETDEWEB)

    Shah, Ishan S.; Lovell, Scott; Mehzabeen, Nurjahan; Battaile, Kevin P.; Tolbert, Thomas J. (Kansas); (HWMRI)

    2017-12-01

    Immunoglobulin G (IgG) consists of four subclasses in humans: IgG1, IgG2, IgG3 and IgG4, which are highly conserved but have unique differences that result in subclass-specific effector functions. Though IgG1 is the most extensively studied IgG subclass, study of other subclasses is important to understand overall immune function and for development of new therapeutics. When compared to IgG1, IgG3 exhibits a similar binding profile to Fcγ receptors and stronger activation of complement. All IgG subclasses are glycosylated at N297, which is required for Fcγ receptor and C1q complement binding as well as maintaining optimal Fc conformation. We have determined the crystal structure of homogenously glycosylated human IgG3 Fc with a GlcNAc2Man5 (Man5) high mannose glycoform at 1.8 Å resolution and compared its structural features with published structures from the other IgG subclasses. Although the overall structure of IgG3 Fc is similar to that of other subclasses, some structural perturbations based on sequence differences were revealed. For instance, the presence of R435 in IgG3 (and H435 in the other IgG subclasses) has been implicated to result in IgG3-specific properties related to binding to protein A, protein G and the neonatal Fc receptor (FcRn). The IgG3 Fc structure helps to explain some of these differences. Additionally, protein-glycan contacts observed in the crystal structure appear to correlate with IgG3 affinity for Fcγ receptors as shown by binding studies with IgG3 Fc glycoforms. Finally, this IgG3 Fc structure provides a template for further studies aimed at engineering the Fc for specific gain of function.

  4. Increased levels of IgG antibodies against human HSP60 in patients with spondyloarthritis

    DEFF Research Database (Denmark)

    Hjelholt, Astrid; Carlsen, Thomas; Deleuran, Bent

    2013-01-01

    and Campylobacter jejuni. Disease severity was assessed by the clinical scorings Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI) and Bath Ankylosing Spondylitis Metrology Index (BASMI). Levels of IgG1 and IgG3 antibodies against human HSP60...... bacterial and human HSP60, also named HSPD1, are highly homologous, cross-reactivity has been suggested in disease initiation. In this study, levels of antibodies against bacterial and human HSP60 were analysed in SpA patients and healthy controls, and the association between such antibodies and disease...... severity in relation to HLA-B27 was evaluated.Serum samples from 82 patients and 50 controls were analysed by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin (Ig)G1, IgG2, IgG3 and IgG4 antibodies against human HSP60 and HSP60 from Chlamydia trachomatis, Salmonella enteritidis...

  5. Characterization of methylglyoxal-modified human IgG by physicochemical methods.

    Science.gov (United States)

    Khan, Mohd Adnan; Arif, Zarina; Moinuddin; Alam, Khursheed

    2017-10-16

    Human IgG is a defence protein and quite reactive to dicarbonyls. In this study, methylglyoxal-induced modification of IgG was examined by various biochemical and biophysical methods. The methylglyoxal-induced changes in IgG were monitored by UV-visible and fluorescence spectroscopy, Fourier transform infrared spectroscopy, 1-anilinonaphthalene-8-sulfonic acid (ANS), and thermal denaturation studies. Aggregate formation was studied by Thioflavin T (ThT), Congo red (CR) and scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Spectroscopic studies suggested gross changes in MGO-modified IgG. Fluorogenic AGEs appeared during modification and the MGO-modified IgG gained thermostability. The reaction produced oxidative stress in the medium because carbonyl content increased manifold and sulfhydryl groups decreased. Enhanced binding of the MGO-modified IgG by Congo red and Thioflavin T suggests crosslinking and aggregation. This was supported by SEM and TEM results.

  6. Hinge-Region O-Glycosylation of Human Immunoglobulin G3 (IgG3)*

    Science.gov (United States)

    Plomp, Rosina; Dekkers, Gillian; Rombouts, Yoann; Visser, Remco; Koeleman, Carolien A.M.; Kammeijer, Guinevere S.M.; Jansen, Bas C.; Rispens, Theo; Hensbergen, Paul J.; Vidarsson, Gestur; Wuhrer, Manfred

    2015-01-01

    Immunoglobulin G (IgG) is one of the most abundant proteins present in human serum and a fundamental component of the immune system. IgG3 represents ∼8% of the total amount of IgG in human serum and stands out from the other IgG subclasses because of its elongated hinge region and enhanced effector functions. This study reports partial O-glycosylation of the IgG3 hinge region, observed with nanoLC-ESI-IT-MS(/MS) analysis after proteolytic digestion. The repeat regions within the IgG3 hinge were found to be in part O-glycosylated at the threonine in the triple repeat motif. Non-, mono- and disialylated core 1-type O-glycans were detected in various IgG3 samples, both poly- and monoclonal. NanoLC-ESI-IT-MS/MS with electron transfer dissociation fragmentation and CE-MS/MS with CID fragmentation were used to determine the site of IgG3 O-glycosylation. The O-glycosylation site was further confirmed by the recombinant production of mutant IgG3 in which potential O-glycosylation sites had been knocked out. For IgG3 samples from six donors we found similar O-glycan structures and site occupancies, whereas for the same samples the conserved N-glycosylation of the Fc CH2 domain showed considerable interindividual variation. The occupancy of each of the three O-glycosylation sites was found to be ∼10% in six serum-derived IgG3 samples and ∼13% in two monoclonal IgG3 allotypes. PMID:25759508

  7. Micro-ELISA for the quantitation of human urinary IgG

    DEFF Research Database (Denmark)

    Fomsgaard, A; Feldt-Rasmussen, B; Deckert, M

    1987-01-01

    range was 5-200 micrograms/l. The relative standard deviations within and between assays were 5 and 9%, respectively. Recovery of IgG added to urine was 100-102% (n = 12), and dilution of urine was linear. The assay range allowed for the quantitation of IgG in human urine samples, covering the clinical...

  8. Regulated glycosylation patterns of IgG during alloimmune responses against human platelet antigens

    NARCIS (Netherlands)

    Wuhrer, Manfred; Porcelijn, Leendert; Kapur, Rick; Koeleman, Carolien A. M.; Deelder, André; de Haas, Masja; Vidarsson, Gestur

    2009-01-01

    Various biological activities of immunoglobulin G (IgG) including antibody-dependent cellular cytotoxicity (ADCC) are modulated by the structural features of the N-glycans in the Fc part. In this study, we describe a population of IgG1 alloantibodies which are formed during pregnancy against human

  9. Different adaptations of IgG effector function in human and nonhuman primates and implications for therapeutic antibody treatment.

    Science.gov (United States)

    Warncke, Max; Calzascia, Thomas; Coulot, Michele; Balke, Nicole; Touil, Ratiba; Kolbinger, Frank; Heusser, Christoph

    2012-05-01

    Safety of human therapeutic Abs is generally assessed in nonhuman primates. Whereas IgG1 shows identical FcγR interaction and effector function profile in both species, fundamental differences in the IgG2 and IgG4 Ab subclasses were found between the two species. Granulocytes, the main effector cells against IgG2- and IgG4-opsonized bacteria and parasites, do not express FcγRIIIb, but show higher levels of FcγRII in cynomolgus monkey. In humans, IgG2 and IgG4 adapted a silent Fc region with weak binding to FcγR and effector functions, whereas, in contrast, cynomolgus monkey IgG2 and IgG4 display strong effector function as well as differences in IgG4 Fab arm exchange. To balance this shift toward activation, the cynomolgus inhibitory FcγRIIb shows strongly increased affinity for IgG2. In view of these findings, in vitro and in vivo results for human IgG2 and IgG4 obtained in the cynomolgus monkey have to be cautiously interpreted, whereas effector function-related effects of human IgG1 Abs are expected to be predictable for humans.

  10. Human IgG subclass antibodies to the 19 kilodalton carboxy ...

    African Journals Online (AJOL)

    Human IgG subclass antibodies to the 19 kilodalton carboxy terminal fragment of Plasmodium Falciparum merozoite surface protein 1 (MSP1 19 ) and predominance of the MAD20 allelic type of MSP1 in Uganda.

  11. The MHC Class I-Like IgG Receptor Controls Perinatal IgG Transport, IgG Homeostasis, and Fate of IgG-Fc-Coupled Drugs

    National Research Council Canada - National Science Library

    Roopenian, Derry C; Christianson, Gregory J; Sproule, Thomas J; Brown, Aaron C; Akilesh, Shreeram; Jung, Nadja; Petkova, Stefka; Avanessian, Lia; Choi, Eun Young; Shaffer, Daniel J; Eden, Peter A; Anderson, Clark L

    2003-01-01

    * The Jackson Laboratory, Bar Harbor, ME 04609; and Ohio State University, Columbus, OH 43210 Abs of the IgG isotype are efficiently transported from mother to neonate and have an extended serum t 1/2 compared with Abs of other isotypes...

  12. Changes in Healthy Human IgG Fc-Glycosylation after Birth and during Early Childhood.

    Science.gov (United States)

    de Haan, Noortje; Reiding, Karli R; Driessen, Gertjan; van der Burg, Mirjam; Wuhrer, Manfred

    2016-06-03

    Glycosylation on the fragment crystallizable (Fc) region of immunoglobulin G (IgG) has a large influence on the interaction of the antibody with Fc gamma receptors (FcγRs). IgG consists of four subclasses that all have distinct affinities for the different FcγRs. Knowledge about the Fc-glycosylation in healthy human is valuable as reference for new biomarkers and in the design of biopharmaceuticals that rely on IgG Fc-glycosylation. Previously, subclass-specific characterization of IgG Fc-glycosylation was performed for healthy adults, pregnant women, and newborns. For young healthy children, however, the subclass-specific description of IgG Fc-glycosylation is still lacking. Therefore, we performed the IgG subclass-specific analysis of the Fc-glycosylation of 130 healthy humans between birth and 40 years of age, including 22 samples derived from the umbilical cords of newborns. The analysis was performed by a previously published matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) workflow, including a derivatization step for the linkage-specific stabilization of sialic acids. The characterization revealed that when children start to produce their own IgG they have a decreased galactosylation, sialylation, and bisection and an increased fucosylation compared with newborns. During childhood, the fucosylation and sialylation decrease, whereas bisection increases and galactosylation stays constant.

  13. [Multicenter study of commercial kits for the determination of human IgG subclasses, using the ELISA technique].

    Science.gov (United States)

    Pontet, F; Pressac, M; Musset, L; Tapon-Bretaudière, J; Jehl, C; Cailliez, M; Bienvenu, F

    1994-01-01

    We evaluated two commercially available sandwich type Elisa procedures for the measurement of IgG subclasses in human serum. Assay kits from The Binding Site and the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service were tested in six laboratories. The performance of spectrophotometers, pipettes and dilutors were assessed at each center. Within-run precision was estimated according to the Valtec method (Société Française de Biologie Clinique). The overall coefficient of variation ranged from 4 to 50% depending on subclass and kit. We also evaluated the IgG2 and IgG4 specificity using four sera containing a monoclonal IgG2 or IgG4 (kappa or lambda type). Using total IgG and immunoelectrophoresis as a comparative technique, IgG2 kappa and IgG4 kappa were both underestimated, IgG2 lambda was overestimated while IgG4 lambda compared favorably. Polyclonal IgG subclasses were frequently overestimated in these sera suggesting cross-reactions with either monoclonal IgG or other polyclonal IgG. Antigen excess was investigated and not encountered with either kit. Our results demonstrate that these procedures are insufficiently accurate or precise for routine clinical use.

  14. Human IgG1 antibodies suppress angiogenesis in a target-independent manner

    NARCIS (Netherlands)

    Bogdanovich, Sasha; Kim, Younghee; Mizutani, Takeshi; Yasuma, Reo; Tudisco, Laura; Cicatiello, Valeria; Bastos-Carvalho, Ana; Kerur, Nagaraj; Hirano, Yoshio; Baffi, Judit Z; Tarallo, Valeria; Li, Shengjian; Yasuma, Tetsuhiro; Arpitha, Parthasarathy; Fowler, Benjamin J; Wright, Charles B; Apicella, Ivana; Greco, Adelaide; Brunetti, Arturo; Ruvo, Menotti; Sandomenico, Annamaria; Nozaki, Miho; Ijima, Ryo; Kaneko, Hiroki; Ogura, Yuichiro; Terasaki, Hiroko; Ambati, Balamurali K; Leusen, Jeanette HW; Langdon, Wallace Y; Clark, Michael R; Armour, Kathryn L; Bruhns, Pierre; Verbeek, J Sjef; Gelfand, Bradley D; De Falco, Sandro; Ambati, Jayakrishna

    2016-01-01

    Aberrant angiogenesis is implicated in diseases affecting nearly 10% of the world's population. The most widely used anti-angiogenic drug is bevacizumab, a humanized IgG1 monoclonal antibody that targets human VEGFA. Although bevacizumab does not recognize mouse Vegfa, it inhibits angiogenesis in

  15. A Monosaccharide Residue Is Sufficient to Maintain Mouse and Human IgG Subclass Activity and Directs IgG Effector Functions to Cellular Fc Receptors

    Directory of Open Access Journals (Sweden)

    Daniela Kao

    2015-12-01

    Full Text Available Immunoglobulin G (IgG glycosylation modulates antibody activity and represents a major source of heterogeneity within antibody preparations. Depending on their glycosylation pattern, individual IgG glycovariants present in recombinant antibody preparations may trigger effects ranging from enhanced pro-inflammatory activity to increased anti-inflammatory activity. In contrast, reduction of IgG glycosylation beyond the central mannose core is generally believed to result in impaired IgG activity. However, this study reveals that a mono- or disaccharide structure consisting of one N-acetylglucosamine with or without a branching fucose residue is sufficient to retain the activity of the most active human and mouse IgG subclasses in vivo and further directs antibody activity to cellular Fcγ receptors. Notably, the activity of minimally glycosylated antibodies is not predicted by in vitro assays based on a monomeric antibody-Fcγ-receptor interaction analysis, whereas in vitro assay systems using immune complexes are more suitable to predict IgG activity in vivo.

  16. Increased levels of IgG antibodies against human HSP60 in patients with spondyloarthritis.

    Directory of Open Access Journals (Sweden)

    Astrid Hjelholt

    Full Text Available Spondyloarthritis (SpA comprises a heterogeneous group of inflammatory diseases, with strong association to human leukocyte antigen (HLA-B27. A triggering bacterial infection has been considered as the cause of SpA, and bacterial heat shock protein (HSP seems to be a strong T cell antigen. Since bacterial and human HSP60, also named HSPD1, are highly homologous, cross-reactivity has been suggested in disease initiation. In this study, levels of antibodies against bacterial and human HSP60 were analysed in SpA patients and healthy controls, and the association between such antibodies and disease severity in relation to HLA-B27 was evaluated.Serum samples from 82 patients and 50 controls were analysed by enzyme-linked immunosorbent assay (ELISA for immunoglobulin (IgG1, IgG2, IgG3 and IgG4 antibodies against human HSP60 and HSP60 from Chlamydia trachomatis, Salmonella enteritidis and Campylobacter jejuni. Disease severity was assessed by the clinical scorings Bath Ankylosing Spondylitis Disease Activity Index (BASDAI, Bath Ankylosing Spondylitis Functional Index (BASFI and Bath Ankylosing Spondylitis Metrology Index (BASMI. Levels of IgG1 and IgG3 antibodies against human HSP60, but not antibodies against bacterial HSP60, were elevated in the SpA group compared with the control group. Association between IgG3 antibodies against human HSP60 and BASMI was shown in HLA-B27⁺ patients. Only weak correlation between antibodies against bacterial and human HSP60 was seen, and there was no indication of cross-reaction. These results suggest that antibodies against human HSP60 is associated with SpA, however, the theory that antibodies against human HSP60 is a specific part of the aetiology, through cross-reaction to bacterial HSP60, cannot be supported by results from this study. We suggest that the association between elevated levels of antibodies against human HSP60 and disease may reflect a general activation of the immune system and an increased

  17. Human anti-rhesus D IgG1 antibody produced in transgenic plants

    DEFF Research Database (Denmark)

    Bouquin, Thomas; Thomsen, Mads; Nielsen, Leif Kofoed

    2002-01-01

    Transgenic plants represent an alternative to cell culture systems for producing cheap and safe antibodies for diagnostic and therapeutic use. To evaluate the functional properties of a 'plantibody', we generated transgenic Arabidopsis plants expressing full-length human IgG1 against the Rhesus D...

  18. Quantitative Analysis of the Interaction Strength and Dynamics of Human IgG4 Half Molecules by Native Mass Spectrometry

    NARCIS (Netherlands)

    Rose, Rebecca J.; Labrijn, Aran F.; van den Bremer, Ewald T. J.; Loverix, Stefan; Lasters, Ignace; van Berkel, Patrick H. C.; van de Winkel, Jan G. J.; Schuurman, Janine; Parren, Paul W. H. I.; Heck, Albert J. R.

    2011-01-01

    Native mass spectrometry (MS) is a powerful technique for studying noncovalent protein-protein interactions. Here, native MS was employed to examine the noncovalent interactions involved in homodimerization of antibody half molecules (HL) in hinge-deleted human IgG4 (IgG4 Delta hinge). By analyzing

  19. Human IgG3 with extended half-life does not improve Fc-gamma receptor-mediated cancer antibody therapies in mice.

    Directory of Open Access Journals (Sweden)

    Rens Braster

    Full Text Available Current anti-cancer therapeutic antibodies that are used in the clinic are predominantly humanized or fully human immunoglobulin G1 (IgG1. These antibodies bind with high affinity to the target antigen and are efficient in activating the immune system via IgG Fc receptors and/or complement. In addition to IgG1, three more isotypes are present in humans, of which IgG3 has been found to be superior compared to human IgG1 in inducing antibody dependent cell cytotoxicity (ADCC, phagocytosis or activation of complement in some models. Nonetheless, no therapeutic human IgG3 mAbs have been developed due to the short in vivo half-life of most known IgG3 allotypes. In this manuscript, we compared the efficacy of V-gene matched IgG1 and IgG3 anti-tumour mAb (TA99 in mice, using natural variants of human IgG3 with short- or long half-life, differing only at position 435 with an arginine or histidine, respectively.In vitro human IgG1 and IgG3 did not show any differences in opsonisation ability of B16F10-gp75 mouse melanoma cells. IgG1, however, was superior in inducing phagocytosis of tumour cells by mouse macrophages. Similarly, in a mouse peritoneal metastasis model we did not detect an improved effect of IgG3 in preventing tumour outgrowth. Moreover, replacing the arginine at position 435 for a histidine in IgG3 to enhance half-life did not result in better suppression of tumour outgrowth compared to wild type IgG3 when injected prior to tumour cell injection.In conclusion, human IgG3 does not have improved therapeutic efficacy compared to human IgG1 in a mouse tumour model.

  20. Serological analysis of human IgG and IgE anti-insulin antibodies by solid-phase radioimmunoassays

    Energy Technology Data Exchange (ETDEWEB)

    Hamilton, R.G.; Rendell, M.; Adkinson, N.F. Jr.

    1980-12-01

    A single solid-phase assay system which is useful for quantitative measurement of both IgG and IgE anti-insulin antibodies in human serum has been developed. Insulin-specific immunoglobulins are absorbed from human serum by excess quantities of insulin-agarose. After washes to remove unbound immunoglobulins, radioiodinated Staph A or rabbit anti-human IgE is added to detect bound IgG or IgE anbitodies, respectively.

  1. Specificity and Effector Functions of Human RSV-Specific IgG from Bovine Milk.

    Directory of Open Access Journals (Sweden)

    Gerco den Hartog

    Full Text Available BACKGROUND: Respiratory syncytial virus (RSV infection is the second most important cause of death in the first year of life, and early RSV infections are associated with the development of asthma. Breastfeeding and serum IgG have been shown to protect against RSV infection. Yet, many infants depend on bovine milk-based nutrition, which at present lacks intact immunoglobulins. OBJECTIVE: To investigate whether IgG purified from bovine milk (bIgG can modulate immune responses against human RSV. METHODS: ELISAs were performed to analyse binding of bIgG to human respiratory pathogens. bIgG or hRSV was coated to plates to assess dose-dependent binding of bIgG to human Fcγ receptors (FcγR or bIgG-mediated binding of myeloid cells to hRSV respectively. S. Epidermidis and RSV were used to test bIgG-mediated binding and internalisation of pathogens by myeloid cells. Finally, the ability of bIgG to neutralise infection of HEp2 cells by hRSV was evaluated. RESULTS: bIgG recognised human RSV, influenza haemagglutinin and Haemophilus influenza. bIgG bound to FcγRII on neutrophils, monocytes and macrophages, but not to FcγRI and FcγRIII, and could bind simultaneously to hRSV and human FcγRII on neutrophils. In addition, human neutrophils and dendritic cells internalised pathogens that were opsonised with bIgG. Finally, bIgG could prevent infection of HEp2 cells by hRSV. CONCLUSIONS: The data presented here show that bIgG binds to hRSV and other human respiratory pathogens and induces effector functions through binding to human FcγRII on phagocytes. Thus bovine IgG may contribute to immune protection against RSV.

  2. Antigen recognition by IgG4 antibodies in human trichinellosis

    Directory of Open Access Journals (Sweden)

    Pinelli E.

    2001-06-01

    Full Text Available The antibody isotype response to Trichinella spiralis excretory/secretory (ES products of muscle larva was examined using sera from patients with confirmed trichinellosis. Using Western blots we identify components of the ES antigen that are recognized by IgM and IgG antibodies. A 45 kDa component was strongly recognized by different antibody classes and subclasses. We observed a 45 kDa-specific lgG4 response that was detected exclusively using sera of patients with trichinellosis and not of patients with echinococcosis, filariasis, cysticercosis, ascariasis, strongyloidiasis or toxocariasis. These results are relevant for the diagnosis of human trichinellosis.

  3. Human plasma contains cross-reactive Abeta conformer-specific IgG antibodies.

    Science.gov (United States)

    O'Nuallain, Brian; Acero, Luis; Williams, Angela D; Koeppen, Helen P McWilliams; Weber, Alfred; Schwarz, Hans P; Wall, Jonathan S; Weiss, Deborah T; Solomon, Alan

    2008-11-25

    Two conformers of aggregated Abeta, i.e., fibrils and oligomers, have been deemed important in the pathogenesis of Alzheimer's disease. We now report that intravenous immune globulin (IVIG) derived from pools of human plasma contains IgGs that recognize conformational epitopes present on fibrils and oligomers, but not their soluble monomeric precursor. We have used affinity chromatography to isolate these antibodies and have shown that they cross-reacted with comparable nanomolar avidity with both types of Abeta aggregates; notably, binding was not inhibited by soluble Abeta monomers. Our studies provide further support for investigating the therapeutic use of IVIG in Alzheimer's disease.

  4. Human IgG antibody response to Glossina saliva: an epidemiologic marker of exposure to Glossina bites.

    Science.gov (United States)

    Poinsignon, Anne; Remoue, Franck; Rossignol, Marie; Cornelie, Sylvie; Courtin, David; Grébaut, Pascal; Garcia, Andre; Simondon, Francois

    2008-05-01

    The evaluation of human antibody response specific to arthropod saliva may be a useful marker of exposure to vector-borne disease. Such an immunologic tool, applied to the evaluation of the exposure to Glossina bites, could be integrated in the control of human African trypanosomiasis (HAT). The antibody (IgG) response specific to uninfected Glossina fuscipes fuscipes saliva was evaluated according to the vector exposure and trypanic status in individuals residing in an HAT-endemic area. A high level of anti-saliva IgG antibodies was only detected in exposed individuals, whether infected or not by Trypanosoma brucei gambiense. In addition, the evaluation of specific IgG response represented spatial heterogeneity according to studied sites. These results suggest that the evaluation of anti-saliva IgG could be an indicator of Glossina exposure and thus could be integrated in other available tools to identify populations presenting risks of HAT transmission.

  5. A neutralized human LMP1-IgG inhibits ENKTL growth by suppressing the JAK3/STAT3 signaling pathway.

    Science.gov (United States)

    Mao, Yuan; Wang, Jun; Zhang, Mingzhi; Fan, Weifei; Tang, Qi; Xiong, Siping; Tang, Xiaojun; Xu, Juqing; Wang, Lin; Yang, Shu; Liu, Suyao; Xu, Li; Chen, Yan; Xu, Lin; Yin, Rong; Zhu, Jin

    2017-02-14

    Latent membrane protein 1 (LMP1), which is associated with the development of different types of Epstein-Barr virus (EBV) related lymphoma, has been suggested to be an important oncoprotein. In this study, a human anti-LMP1 IgG antibody (LMP1-IgG) was constructed and characterized by ELISA, western blotting (WB), affinity and immunohistochemistry (IHC) analyses. CCK-8, MTT, apoptosis assays, antibody-dependent cell-mediated cytotoxicity (ADCC) and CDC (complement-dependent cytotoxicity) assays were performed to evaluate the inhibitory effects of LMP1-IgG on extranodal nasal-type natural killer (NK)/T-cell lymphoma (ENKTL). Then, the influence of LMP1-IgG on the JAK/STAT signaling pathway was investigated. The results showed that the successfully constructed LMP1-IgG inhibited proliferation, induced apoptosis, and activated ADCC and CDC of ENKTL in a concentration- and time- dependent manner. Moreover, phosphorylation of JAK3 and STAT3 was inhibited by LMP1-IgG. Our data indicate that LMP1-IgG may provide a novel and promising therapeutic strategy for the treatment of LMP1-positive ENKTL.

  6. Binding of human IgG to single-walled carbon nanotubes accelerated myeloperoxidase-mediated degradation in activated neutrophils.

    Science.gov (United States)

    Ding, Yun; Tian, Rong; Yang, Zhen; Chen, Jianfa; Lu, Naihao

    2016-11-01

    The binding of protein to carboxylated single-walled carbon nanotubes (SWCNTs) was believed to play an important role in the biological effects of nanotubes. However, the effects of protein-SWCNTs interactions on the oxidative degradation of nanotubes were not stressed enough. Here, we investigated the binding of human immunoglobulin G (IgG) to SWCNTs, and found that the preferred binding site was located in the Fc region of IgG. The hydrophobic and electrostatic interactions might be the crucial factors in stabilizing the binding of SWCNTs with IgG. Through the competitive binding of IgG and myeloperoxidase (MPO) to nanotube surfaces, the binding of IgG could impair MPO-induced SWCNTs biodegradation in vitro. However, both SWCNTs and IgG-SWCNTs were significantly degraded in zymosan-stimulated neutrophils, and the degradation degree was more for IgG-SWCNTs. These results suggest that the binding of IgG may be an important determinant for MPO-mediated SWCNTs biodegradation in activated human inflammatory cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Decreased levels of bisecting GlcNAc glycoforms of IgG are associated with human longevity.

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    L Renee Ruhaak

    Full Text Available BACKGROUND: Markers for longevity that reflect the health condition and predict healthy aging are extremely scarce. Such markers are, however, valuable in aging research. It has been shown previously that the N-glycosylation pattern of human immunoglobulin G (IgG is age-dependent. Here we investigate whether N-linked glycans reflect early features of human longevity. METHODOLOGY/PRINCIPAL FINDINGS: The Leiden Longevity Study (LLS consists of nonagenarian sibling pairs, their offspring, and partners of the offspring serving as control. IgG subclass specific glycosylation patterns were obtained from 1967 participants in the LLS by MALDI-TOF-MS analysis of tryptic IgG Fc glycopeptides. Several regression strategies were applied to evaluate the association of IgG glycosylation with age, sex, and longevity. The degree of galactosylation of IgG decreased with increasing age. For the galactosylated glycoforms the incidence of bisecting GlcNAc increased as a function of age. Sex-related differences were observed at ages below 60 years. Compared to males, younger females had higher galactosylation, which decreased stronger with increasing age, resulting in similar galactosylation for both sexes from 60 onwards. In younger participants (60 years, decreased levels of non-galactosylated glycoforms containing a bisecting GlcNAc reflected early features of longevity. CONCLUSIONS/SIGNIFICANCE: We here describe IgG glycoforms associated with calendar age at all ages and the propensity for longevity before middle age. As modulation of IgG effector functions has been described for various IgG glycosylation features, a modulatory effect may be expected for the longevity marker described in this study.

  8. Role of human-tissue transglutaminase IgG and anti-gliadin IgG antibodies in the diagnosis of coeliac disease in patients with selective immunoglobulin A deficiency.

    Science.gov (United States)

    Lenhardt, A; Plebani, A; Marchetti, F; Gerarduzzi, T; Not, T; Meini, A; Villanacci, V; Martelossi, S; Ventura, A

    2004-11-01

    Selective IgA deficiency is associated with coeliac disease, and studies have shown an increased prevalence of coeliac disease in these patients ranging from 0.71 to 30.7%, depending on the test used for screening. To determine the sensitivity of IgG anti-gliadin-antibodies and of IgG human-tissue-transglutaminase for diagnosing coeliac disease and assessing its prevalence in subjects with IgA deficiency. We tested serum samples from 126 IgA-deficient children (66 female, median age: 10.8 years). All samples were analysed to measure IgG anti-gliadin-antibodies and IgG anti-human-tissue-transglutaminase. Patients testing positive to either test underwent intestinal biopsy. Subjects testing positive for IgG anti-human-tissue-transglutaminase underwent genetic testing for the human leucocyte antigen heterodimer. Twenty-seven of 126 subjects tested positive for IgG anti-gliadin-antibodies (five of whom tested positive also for IgG anti-human-tissue-transglutaminase) and 18 (including the aforementioned five) for IgG anti-human-tissue-transglutaminase. Intestinal biopsy was performed in 37 of the 40 patients who tested positive (three subjects refused). Eleven had positive intestinal biopsies all of whom tested positive for IgG anti-human-tissue-transglutaminase, but only five of these tested positive also for IgG anti-gliadin-antibodies. All 22 patients testing positive for anti-gliadin-antibody alone had normal intestinal mucosa. All the patients who tested positive for IgG anti-human-tissue-transglutaminase and underwent genetic screening (15/18) had the coeliac-related human leucocyte antigen. Overall, coeliac disease was diagnosed in 11 of the 126 subjects with IgA deficiency (8.7%). The prevalence of coeliac disease in subjects with total IgA deficiency was 8.7%. Assay of IgG anti-human-tissue-transglutaminase can be recommended for screening coeliac disease in IgA-deficient subjects.

  9. Characterization of a novel hemolytic activity of human IgG fractions arising from diversity in protein and oligosaccharide components.

    Directory of Open Access Journals (Sweden)

    Shaoying Min

    Full Text Available Human IgG is a well-established multifunctional antigen specific immunoglobulin molecule of the adaptive immune system. However, an antigen nonspecific immunological function of human IgG has never been reported. In this study, human IgG was isolated using ammonium sulfate fractional precipitation and diethylaminoethanol (DEAE cellulose 52 ion exchange chromatography, from which h-IgG and hs-IgG fractions were purified on the basis of their differential binding to rabbit anti-shrimp hemocyanin antibody (h and rabbit anti-shrimp hemocyanin's small subunit antibody (hs, respectively. We found that h-IgG had a higher hemolytic activity than hs-IgG against erythrocytes from humans, rabbits, mice and chickens, whereas the control IgG showed negligible activity. h-IgG could interact directly with erythrocyte membranes, and this interaction was suppressed by high molecular weight osmoprotectants, showing that it may follow a colloid-osmotic mechanism. In comparative proteomics and glycomics studies, h-IgG and hs-IgG yielded 20 and 5 significantly altered protein spots, respectively, on a 2-D gel. The mean carbohydrate content of h-IgG and hs-IgG was approximately 3.6- and 2-fold higher than that of IgG, respectively, and the α-d-mannose/α-d-glucose content was in the order of h-IgG>hs-IgG>IgG. In this study, a novel antigen nonspecific immune property of human IgG was investigated, and the diversity in the protein constituents and glycosylation levels may have functional signficance.

  10. Characterization of a Novel Hemolytic Activity of Human IgG Fractions Arising from Diversity in Protein and Oligosaccharide Components

    Science.gov (United States)

    Zhang, Yueling; Ye, Xiangqun; Zhong, Mingqi; Cao, Jinsong; Zou, Haiying; Chen, Jiehui

    2014-01-01

    Human IgG is a well-established multifunctional antigen specific immunoglobulin molecule of the adaptive immune system. However, an antigen nonspecific immunological function of human IgG has never been reported. In this study, human IgG was isolated using ammonium sulfate fractional precipitation and diethylaminoethanol (DEAE) cellulose 52 ion exchange chromatography, from which h-IgG and hs-IgG fractions were purified on the basis of their differential binding to rabbit anti-shrimp hemocyanin antibody (h) and rabbit anti-shrimp hemocyanin's small subunit antibody (hs), respectively. We found that h-IgG had a higher hemolytic activity than hs-IgG against erythrocytes from humans, rabbits, mice and chickens, whereas the control IgG showed negligible activity. h-IgG could interact directly with erythrocyte membranes, and this interaction was suppressed by high molecular weight osmoprotectants, showing that it may follow a colloid-osmotic mechanism. In comparative proteomics and glycomics studies, h-IgG and hs-IgG yielded 20 and 5 significantly altered protein spots, respectively, on a 2-D gel. The mean carbohydrate content of h-IgG and hs-IgG was approximately 3.6- and 2-fold higher than that of IgG, respectively, and the α-d-mannose/α-d-glucose content was in the order of h-IgG>hs-IgG>IgG. In this study, a novel antigen nonspecific immune property of human IgG was investigated, and the diversity in the protein constituents and glycosylation levels may have functional signficance. PMID:24465658

  11. Anti-Lipid IgG Antibodies Are Produced via Germinal Centers in a Murine Model Resembling Human Lupus

    Science.gov (United States)

    Wong-Baeza, Carlos; Reséndiz-Mora, Albany; Donis-Maturano, Luis; Wong-Baeza, Isabel; Zárate-Neira, Luz; Yam-Puc, Juan Carlos; Calderón-Amador, Juana; Medina, Yolanda; Wong, Carlos; Baeza, Isabel; Flores-Romo, Leopoldo

    2016-01-01

    Anti-lipid IgG antibodies are produced in some mycobacterial infections and in certain autoimmune diseases [such as anti-phospholipid syndrome, systemic lupus erythematosus (SLE)]. However, few studies have addressed the B cell responses underlying the production of these immunoglobulins. Anti-lipid IgG antibodies are consistently found in a murine model resembling human lupus induced by chlorpromazine-stabilized non-bilayer phospholipid arrangements (NPA). NPA are transitory lipid associations found in the membranes of most cells; when NPA are stabilized they can become immunogenic and induce specific IgG antibodies, which appear to be involved in the development of the mouse model of lupus. Of note, anti-NPA antibodies are also detected in patients with SLE and leprosy. We used this model of lupus to investigate in vivo the cellular mechanisms that lead to the production of anti-lipid, class-switched IgG antibodies. In this murine lupus model, we found plasma cells (Gr1−, CD19−, CD138+) producing NPA-specific IgGs in the draining lymph nodes, the spleen, and the bone marrow. We also found a significant number of germinal center B cells (IgD−, CD19+, PNA+) specific for NPA in the draining lymph nodes and the spleen, and we identified in situ the presence of NPA in these germinal centers. By contrast, very few NPA-specific, extrafollicular reaction B cells (B220+, Blimp1+) were found. Moreover, when assessing the anti-NPA IgG antibodies produced during the experimental protocol, we found that the affinity of these antibodies progressively increased over time. Altogether, our data indicate that, in this murine model resembling human lupus, B cells produce anti-NPA IgG antibodies mainly via germinal centers. PMID:27746783

  12. Maternal-fetal immunoglobulin transport: Studies on the binding, internalization, and release of IgG by chick yolk sac tissue and cultured cells

    Energy Technology Data Exchange (ETDEWEB)

    Donaldson, J.G.

    1988-01-01

    Immunoglobulin G (IgG) is transported from the yolk across the endodermal cells of the yolk sac and into the fetal circulation during chick embryonic development, thus providing the chick with passive immunity until it becomes immunocompetent. Saturable, Fc-specific receptors are present on the endodermal cells and are believed to mediate this transfer. In this study, IgG receptors were shown to be present on the yolk sac endodermal cells throughout the 21 days of development, although most of the transport occurs during the last 3 days prior to hatching. Fluorescently conjugated IgG was internalized by a receptor mechanism into small apical vesicles in yolk sac endoderm throughout, but cells from 19 day yolk sacs internalized more conjugate than those from 14 day yolk sacs. This was confirmed and quantitated by assaying the internalization of {sup 125}I-IgG into yolk sac tissue. IgG was internalized by a receptor mediated mechanism, reaching a steady state level after 1 to 2 hours. Although both ages of yolk sac tissue possessed the same number of surface IgG receptors, as measured by equilibrium binding assays at 4{degree}C, 19 day yolk sac had the capacity to internalize six times as much IgG by a receptor mechanism as 14 day yolk sac.

  13. Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn

    Science.gov (United States)

    Pan, Hai; Chen, Kenneth; Chu, Liping; Kinderman, Francis; Apostol, Izydor; Huang, Gang

    2009-01-01

    Susceptibility of methionine residues to oxidation is a significant issue of protein therapeutics. Methionine oxidation may limit the product's clinical efficacy or stability. We have studied kinetics of methionine oxidation in the Fc portion of the human IgG2 and its impact on the interaction with FcRn and Protein A. Our results confirm previously published observations for IgG1 that two analogous solvent-exposed methionine residues in IgG2, Met 252 and Met 428, oxidize more readily than the other methionine residue, Met 358, which is buried inside the Fc. Met 397, which is not present in IgG1 but in IgG2, oxidizes at similar rate as Met 358. Oxidation of two labile methionines, Met 252 and Met 428, weakens the binding of the intact antibody with Protein A and FcRn, two natural protein binding partners. Both of these binding partners share the same binding site on the Fc. Additionally, our results shows that Protein A may serve as a convenient and inexpensive surrogate for FcRn binding measurements. PMID:19165723

  14. IgG Fc variant cross-reactivity between human and rhesus macaque FcγRs.

    Science.gov (United States)

    Boesch, Austin W; Miles, Adam R; Chan, Ying N; Osei-Owusu, Nana Y; Ackerman, Margaret E

    2017-04-01

    Non-human primate (NHP) studies are often an essential component of antibody development efforts before human trials. Because the efficacy or toxicity of candidate antibodies may depend on their interactions with Fcγ receptors (FcγR) and their resulting ability to induce FcγR-mediated effector functions such as antibody-dependent cell-meditated cytotoxicity and phagocytosis (ADCP), the evaluation of human IgG variants with modulated affinity toward human FcγR is becoming more prevalent in both infectious disease and oncology studies in NHP. Reliable translation of these results necessitates analysis of the cross-reactivity of these human Fc variants with NHP FcγR. We report evaluation of the binding affinities of a panel of human IgG subclasses, Fc amino acid point mutants and Fc glycosylation variants against the common allotypes of human and rhesus macaque FcγR by applying a high-throughput array-based surface plasmon resonance platform. The resulting data indicate that amino acid variation present in rhesus FcγRs can result in disrupted, matched, or even increased affinity of IgG Fc variants compared with human FcγR orthologs. These observations emphasize the importance of evaluating species cross-reactivity and developing an understanding of the potential limitations or suitability of representative in vitro and in vivo models before human clinical studies when either efficacy or toxicity may be associated with FcγR engagement.

  15. Human peptide transporters

    DEFF Research Database (Denmark)

    Nielsen, Carsten Uhd; Brodin, Birger; Jørgensen, Flemming Steen

    2002-01-01

    Peptide transporters are epithelial solute carriers. Their functional role has been characterised in the small intestine and proximal tubules, where they are involved in absorption of dietary peptides and peptide reabsorption, respectively. Currently, two peptide transporters, PepT1 and PepT2...... to peptide transporters, as well as their role in drug delivery and in potential future drug design and targeted tissue delivery of peptides and peptidomimetics....

  16. Isolation of high-affinity human IgE and IgG antibodies recognising Bet v 1 and Humicola lanuginosa lipase from combinatorial phage libraries

    DEFF Research Database (Denmark)

    Jakobsen, Charlotte G; Bødtger, Uffe; Kristensen, Peter

    2004-01-01

    . Human IgE and IgG libraries have been created from patients allergic to birch pollen or lipase. These libraries have been used to select binders recognising the major birch pollen allergen Bet v 1 and Humicola lanuginosa lipase. A panel of allergen-specific IgE and IgG antibodies were identified...

  17. Human stiff-person syndrome IgG induces anxious behavior in rats.

    Directory of Open Access Journals (Sweden)

    Christian Geis

    Full Text Available BACKGROUND: Anxiety is a heterogeneous behavioral domain playing a role in a variety of neuropsychiatric diseases. While anxiety is the cardinal symptom in disorders such as panic disorder, co-morbid anxious behavior can occur in a variety of diseases. Stiff person syndrome (SPS is a CNS disorder characterized by increased muscle tone and prominent agoraphobia and anxiety. Most patients have high-titer antibodies against glutamate decarboxylase (GAD 65. The pathogenic role of these autoantibodies is unclear. METHODOLOGY/PRINCIPAL FINDINGS: We re-investigated a 53 year old woman with SPS and profound anxiety for GABA-A receptor binding in the amygdala with (11C-flumazenil PET scan and studied the potential pathogenic role of purified IgG from her plasma filtrates containing high-titer antibodies against GAD 65. We passively transferred the IgG fraction intrathecally into rats and analyzed the effects using behavioral and in vivo electrophysiological methods. In cell culture, we measured the effect of patient IgG on GABA release from hippocampal neurons. Repetitive intrathecal application of purified patient IgG in rats resulted in an anxious phenotype resembling the core symptoms of the patient. Patient IgG selectively bound to rat amygdala, hippocampus, and frontal cortical areas. In cultured rat hippocampal neurons, patient IgG inhibited GABA release. In line with these experimental results, the GABA-A receptor binding potential was reduced in the patient's amygdala/hippocampus complex. No motor abnormalities were found in recipient rats. CONCLUSION/SIGNIFICANCE: The observations in rats after passive transfer lead us to propose that anxiety-like behavior can be induced in rats by passive transfer of IgG from a SPS patient positive for anti-GAD 65 antibodies. Anxiety, in this case, thus may be an antibody-mediated phenomenon with consecutive disturbance of GABAergic signaling in the amygdala region.

  18. B-cell display-based one-step method to generate chimeric human IgG monoclonal antibodies.

    Science.gov (United States)

    Lin, Waka; Kurosawa, Kohei; Murayama, Akiho; Kagaya, Eri; Ohta, Kunihiro

    2011-02-01

    The recent development of screening strategies based on the generation and display of large libraries of antibody fragments has allowed considerable advances for the in vitro isolation of monoclonal antibodies (mAbs). We previously developed a technology referred to as the 'ADLib (Autonomously Diversifying Library) system', which allows the rapid screening and isolation in vitro of antigen-specific monoclonal antibodies (mAbs) from libraries of immunoglobulin M (IgM) displayed by the chicken B-cell line DT40. Here, we report a novel application of the ADLib system to the production of chimeric human mAbs. We have designed gene knock-in constructs to generate DT40 strains that coexpress chimeric human IgG and chicken IgM via B-cell-specific RNA alternative splicing. We demonstrate that the application of the ADLib system to these strains allows the one-step selection of antigen-specific human chimeric IgG. In addition, the production of chimeric IgG can be selectively increased when we modulate RNA processing by overexpressing the polyadenylation factor CstF-64. This method provides a new way to efficiently design mAbs suitable for a wide range of purposes including antibody therapy.

  19. IgG to Various Beta-Glucans in a Human Adult Population

    NARCIS (Netherlands)

    Noss, I.; Wouters, I.M.; Smit, L.A.; Meijer, E.; Pronk, A.; Heederik, D.J.J.; Doekes, G.

    2012-01-01

    Background: Fungal β-(1,3)-glucans are pro-inflammatory agents, and exposures to β-(1,3)-glucans are associated with respiratory tract symptoms. IgG anti-(1,3)-glucan titers are measured in diagnosis of fungal infections. Although other β-glucan structures exist, like β-(1,6)-glucans, little is

  20. IgG to various beta-glucans in a human adult population

    NARCIS (Netherlands)

    Noss, I.; Wouters, I.M.; Smit, L.A.; Meijer, E.; Pronk, A.; Heederik, D.J.; Doekes, G.

    2012-01-01

    Background: Fungal β-(1,3)-glucans are pro-inflammatory agents, and exposures to β-(1,3)-glucans are associated with respiratory tract symptoms. IgG anti-(1,3)-glucan titers are measured in diagnosis of fungal infections. Although other β-glucan structures exist, like β-(1,6)-glucans, little is

  1. Three-dimensional structure of the human myeloma IgG2.

    Directory of Open Access Journals (Sweden)

    Sergey Ryazantsev

    Full Text Available Human immunoglobulin G, subclass 2 (hIgG2, plays an important role in immunity to bacterial pathogens and in numerous pathological conditions. However, there is a lack of information regarding the three-dimensional (3D structure of the hIgG2 molecule. We used electron microscopy (EM, differential scanning microcalorimetry (DSC and fluorescence for structural analysis of the hIgG2. DSC and fluorescence indicated two types of interaction between CH1 domain of Fab (antigen-binding fragment/subunit and CH2 domain of Fc (complement fixation fragment/subunit simultaneously present in the sample: close interaction, which increases the thermostability of both, CH1 and CH2 domains, and weak (or no interaction, which is typical for most IgGs but not hIgG2. Thermodynamics could not determine if both types of interactions are present within a single molecule. To address this question, EM was used. We employed a single-particle reconstruction and negative staining approach to reveal the three-dimensional structure of the hIgG2. A three-dimensional model of hIgG2 was created at 1.78 nm resolution. The hIgG2 is asymmetrical: one Fab subunit is in close proximity to the upper portion of the Fc subunit (CH2 domain and the other Fab is distant from Fc. The plane of Fab subunits is nearly perpendicular to Fc. EM structure of the hIgG2 is in good agreement with thermodynamic data: a Fab distant from Fc should exhibit a lower melting temperature while a Fab interacting with Fc should exhibit a higher melting temperature. Both types of Fab subunits exist within one molecule resembling an A/B hIgG2 isoform introduced earlier on physicochemical level by Dillon et al. (2008. In such an arrangement, the access to the upper portion of Fc subunit is partially blocked by a Fab subunit. That might explain for instance why hIgG2 mildly activates complement and binds poorly to Fc receptors. Understanding of the three-dimensional structure of the hIgG2 should lead to better

  2. [PHEMA/PEI]-Cu(II) based immobilized metal affinity chromatography cryogels: Application on the separation of IgG from human plasma.

    Science.gov (United States)

    Bakhshpour, Monireh; Derazshamshir, Ali; Bereli, Nilay; Elkak, Assem; Denizli, Adil

    2016-04-01

    The immobilized metal-affinity chromatography (IMAC) has gained significant interest as a widespread separation and purification tool for therapeutic proteins, nucleic acids and other biological molecules. The enormous potential of IMAC for proteins with natural surface exposed-histidine residues and for recombinant proteins with histidine clusters. Cryogels as monolithic materials have recently been proposed as promising chromatographic adsorbents for the separation of biomolecules in downstream processing. In the present study, IMAC cryogels have been synthesized and utilized for the adsorption and separation of immunoglobulin G (IgG) from IgG solution and whole human plasma. For this purpose, Cu(II)-ions were coupled to poly(hydroxyethyl methacrylate) PHEMA using poly(ethylene imine) (PEI) as the chelating ligand. In this study the cryogels formation optimized by the varied proportion of PEI from 1% to 15% along with different amounts of Cu (II) as chelating metal. The prepared cryogels were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis. The [PHEMA/PEI]-Cu(II) cryogels were assayed for their capability to bind the human IgG from aqueous solutions. The IMAC cryogels were found to have high affinity toward human IgG. The adsorption of human IgG was investigated onto the PHEMA/PEI cryogels with (10% PEI) and the concentration of Cu (II) varied as 10, 50, 100 and 150 mg/L. The separation of human IgG was achieved in one purification step at pH7.4. The maximum adsorption capacity was observed at the [PHEMA/PEI]-Cu(II) (10% PEI) with 72.28 mg/g of human IgG. The purification efficiency and human IgG purity were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Antiviral Functions of Human Immunodeficiency Virus Type 1 (HIV-1)-Specific IgG Antibodies: Effects of Antiretroviral Therapy and Implications for Therapeutic HIV-1 Vaccine Design.

    Science.gov (United States)

    French, Martyn A; Tjiam, M Christian; Abudulai, Laila N; Fernandez, Sonia

    2017-01-01

    Contemporary antiretroviral therapy (ART) is effective and tolerable for long periods of time but cannot eradicate human immunodeficiency virus type 1 (HIV-1) infection by either elimination of viral reservoirs or enhancement of HIV-1-specific immune responses. Boosting "protective" HIV-1-specific immune responses by active or passive immunization will therefore be necessary to control or eradicate HIV-1 infection and is currently the topic of intense investigation. Recently reported studies conducted in HIV patients and non-human primate (NHP) models of HIV-1 infection suggest that HIV-1-specific IgG antibody responses may contribute to the control of HIV-1 infection. However, production of IgG antibodies with virus neutralizing activity by vaccination remains problematic and while vaccine-induced natural killer cell-activating IgG antibodies have been shown to prevent the acquisition of HIV-1 infection, they may not be sufficient to control or eradicate established HIV-1 infection. It is, therefore, important to consider other functional characteristics of IgG antibody responses. IgG antibodies to viruses also mediate opsonophagocytic antibody responses against virions and capsids that enhance the function of phagocytic cells playing critical roles in antiviral immune responses, particularly conventional dendritic cells and plasmacytoid dendritic cells. Emerging evidence suggests that these antibody functions might contribute to the control of HIV-1 infection. In addition, IgG antibodies contribute to the intracellular degradation of viruses via binding to the cytosolic fragment crystallizable (Fc) receptor tripartite motif containing-21 (TRIM21). The functional activity of an IgG antibody response is influenced by the IgG subclass content, which affects binding to antigens and to Fcγ receptors on phagocytic cells and to TRIM21. The IgG subclass content and avidity of IgG antibodies is determined by germinal center (GC) reactions in follicles of lymphoid tissue

  4. Differential antibody isotype reactivity to specific antigens in human lymphatic filariasis: gp15/400 preferentially induces immunoglobulin E (IgE), IgG4, and IgG2.

    Science.gov (United States)

    Yazdanbakhsh, M; Paxton, W A; Brandenburg, A; Van Ree, R; Lens, M; Partono, F; Maizels, R M; Selkirk, M E

    1995-01-01

    Lymphatic filarial infection in humans is associated with a strong skewing of the immune response towards the TH2 arm, with prominent interleukin 4-producing cells and elevated levels of immunoglobulin G4 (IgG4) and IgE antibodies in peripheral blood. To determine how such a generalized TH2 imbalance governs responses to individual parasite antigens, the profiles of isotypes of antibodies to two recombinant proteins of Brugia spp. were studied. One molecule was the C-terminal portion of the filarial heat shock protein 70 (Bpa-26), representative of a cytoplasmic protein, and the second antigen was a single unit of the tandem repeats of a Brugia polypeptide (BpL-4), a secreted product which is prominently exposed to the immune system. Serum samples from 146 individuals resident in areas in which brugian filariasis is endemic were used, and it was found that whereas the levels of IgG1 and IgG3 responses to both Bpa-26 and BpL-4 were high, IgG4 and IgE antibodies to only BpL-4, not to Bpa-26, were prominent. Thus, an antigen which is chronically exposed to the immune system elicited a TH2-dependent isotype switch, as manifested by increased IgG4 and IgE responses. Moreover, IgG4 and IgE responses to BpL-4 showed a strong negative association, suggesting that mediators other than interleukin 4 must be responsible for such differential regulation of these two isotypes. When the data were analyzed as a function of clinical status, a striking association between elevated levels of IgG3 antibodies to Bpa-26 and manifestation of chronic obstructive disease was found; elephantiasis patients showed significantly higher levels of IgG3 antibodies to Bpa-26 than microfilaremics and asymptomatic amicrofilaremics. This indicates that an imbalance of isotypes of antibodies to particular filarial antigens might play a role in the pathogenesis of chronic disease. PMID:7558279

  5. An engineered diatom acting like a plasma cell secreting human IgG antibodies with high efficiency

    Directory of Open Access Journals (Sweden)

    Hempel Franziska

    2012-09-01

    Full Text Available Abstract Background Although there are many different expression systems for recombinant production of pharmaceutical proteins, many of these suffer from drawbacks such as yield, cost, complexity of purification, and possible contamination with human pathogens. Microalgae have enormous potential for diverse biotechnological applications and currently attract much attention in the biofuel sector. Still underestimated, though, is the idea of using microalgae as solar-fueled expression system for the production of recombinant proteins. Results In this study, we show for the first time that completely assembled and functional human IgG antibodies can not only be expressed to high levels in algal systems, but also secreted very efficiently into the culture medium. We engineered the diatom Phaeodactylum tricornutum to synthesize and secrete a human IgG antibody against the Hepatitis B Virus surface protein. As the diatom P. tricornutum is not known to naturally secrete many endogenous proteins, the secreted antibodies are already very pure making extensive purification steps redundant and production extremely cost efficient. Conclusions Microalgae combine rapid growth rates with all the advantages of eukaryotic expression systems, and offer great potential for solar-powered, low cost production of pharmaceutical proteins.

  6. Non-immune binding of human IgG to M-related proteins confers resistance to phagocytosis of group A streptococci in blood.

    Directory of Open Access Journals (Sweden)

    Harry S Courtney

    Full Text Available The non-immune binding of immunoglobulins by bacteria is thought to contribute to the pathogenesis of infections. M-related proteins (Mrp are group A streptococcal (GAS receptors for immunoglobulins, but it is not known if this binding has any impact on virulence. To further investigate the binding of immunoglobulins to Mrp, we engineered mutants of an M type 4 strain of GAS by inactivating the genes for mrp, emm, enn, sof, and sfbX and tested these mutants in IgG-binding assays. Inactivation of mrp dramatically decreased the binding of human IgG, whereas inactivation of emm, enn, sof, and sfbx had only minor effects, indicating that Mrp is a major IgG-binding protein. Binding of human immunoglobulins to a purified, recombinant form of Mrp indicated that it selectively binds to the Fc domain of human IgG, but not IgA or IgM and that it preferentially bound subclasses IgG₁>IgG₄>IgG₂>IgG₃. Recombinant proteins encompassing different regions of Mrp were engineered and used to map its IgG-binding domain to its A-repeat region and a recombinant protein with 3 A-repeats was a better inhibitor of IgG binding than one with a single A-repeat. A GAS mutant expressing Mrp with an in-frame deletion of DNA encoding the A-repeats had a dramatically reduced ability to bind human IgG and to grow in human blood. Mrp exhibited host specificity in binding IgG; human IgG was the best inhibitor of the binding of IgG followed by pig, horse, monkey, and rabbit IgG. IgG from goat, mouse, rat, cow, donkey, chicken, and guinea pig were poor inhibitors of binding. These findings indicate that Mrp preferentially binds human IgG and that this binding contributes to the ability of GAS to resist phagocytosis and may be a factor in the restriction of GAS infections to the human host.

  7. Structural Basis for Fc[gamma]RIIa Recognition of Human IgG and Formation of Inflammatory Signaling Complexes

    Energy Technology Data Exchange (ETDEWEB)

    Ramsland, Paul A.; Farrugia, William; Bradford, Tessa M.; Sardjono, Caroline Tan; Esparon, Sandra; Trist, Halina M.; Powell, Maree S.; Tan, Peck Szee; Cendron, Angela C.; Wines, Bruce D.; Scott, Andrew M.; Hogarth, P. Mark (Burnet); (Monash); (LICR); (Melbourne)

    2011-09-20

    The interaction of Abs with their specific FcRs is of primary importance in host immune effector systems involved in infection and inflammation, and are the target for immune evasion by pathogens. Fc{gamma}RIIa is a unique and the most widespread activating FcR in humans that through avid binding of immune complexes potently triggers inflammation. Polymorphisms of Fc{gamma}RIIa (high responder/low responder [HR/LR]) are linked to susceptibility to infections, autoimmune diseases, and the efficacy of therapeutic Abs. In this article, we define the three-dimensional structure of the complex between the HR (arginine, R134) allele of Fc{gamma}RIIa (Fc{gamma}RIIa-HR) and the Fc region of a humanized IgG1 Ab, hu3S193. The structure suggests how the HR/LR polymorphism may influence Fc{gamma}RIIa interactions with different IgG subclasses and glycoforms. In addition, mutagenesis defined the basis of the epitopes detected by FcR blocking mAbs specific for Fc{gamma}RIIa (IV.3), Fc{gamma}RIIb (X63-21), and a pan Fc{gamma}RII Ab (8.7). The epitopes detected by these Abs are distinct, but all overlap with residues defined by crystallography to contact IgG. Finally, crystal structures of LR (histidine, H134) allele of Fc{gamma}RIIa and Fc{gamma}RIIa-HR reveal two distinct receptor dimers that may represent quaternary states on the cell surface. A model is presented whereby a dimer of Fc{gamma}RIIa-HR binds Ag-Ab complexes in an arrangement that possibly occurs on the cell membrane as part of a larger signaling assembly.

  8. [PHEMA/PEI]–Cu(II) based immobilized metal affinity chromatography cryogels: Application on the separation of IgG from human plasma

    Energy Technology Data Exchange (ETDEWEB)

    Bakhshpour, Monireh; Derazshamshir, Ali; Bereli, Nilay [Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara (Turkey); Elkak, Assem [Laboratory of “Valorisation des Ressources Naturelles et Produits de Santé (VRNPS)”, Doctoral School of Sciences and Technology, Lebanese University, Rafic Hariri University Campus, Hadath (Lebanon); Denizli, Adil, E-mail: denizli@hacettepe.edu [Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara (Turkey)

    2016-04-01

    The immobilized metal-affinity chromatography (IMAC) has gained significant interest as a widespread separation and purification tool for therapeutic proteins, nucleic acids and other biological molecules. The enormous potential of IMAC for proteins with natural surface exposed-histidine residues and for recombinant proteins with histidine clusters. Cryogels as monolithic materials have recently been proposed as promising chromatographic adsorbents for the separation of biomolecules in downstream processing. In the present study, IMAC cryogels have been synthesized and utilized for the adsorption and separation of immunoglobulin G (IgG) from IgG solution and whole human plasma. For this purpose, Cu(II)-ions were coupled to poly(hydroxyethyl methacrylate) PHEMA using poly(ethylene imine) (PEI) as the chelating ligand. In this study the cryogels formation optimized by the varied proportion of PEI from 1% to 15% along with different amounts of Cu (II) as chelating metal. The prepared cryogels were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis. The [PHEMA/PEI]–Cu(II) cryogels were assayed for their capability to bind the human IgG from aqueous solutions. The IMAC cryogels were found to have high affinity toward human IgG. The adsorption of human IgG was investigated onto the PHEMA/PEI cryogels with (10% PEI) and the concentration of Cu (II) varied as 10, 50, 100 and 150 mg/L. The separation of human IgG was achieved in one purification step at pH 7.4. The maximum adsorption capacity was observed at the [PHEMA/PEI]–Cu(II) (10% PEI) with 72.28 mg/g of human IgG. The purification efficiency and human IgG purity were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). - Highlights: • Cu(II)-ions were coupled to PHEMA using PEI as the chelating ligand. • Cu(II) chelated [PHEMA/PEI] cryogels for IgG separation were produced. • Maximum IgG adsorption capacity

  9. Engineering upper hinge improves stability and effector function of a human IgG1.

    Science.gov (United States)

    Yan, Boxu; Boyd, Daniel; Kaschak, Timothy; Tsukuda, Joni; Shen, Amy; Lin, Yuwen; Chung, Shan; Gupta, Priyanka; Kamath, Amrita; Wong, Anne; Vernes, Jean-Michel; Meng, Gloria Y; Totpal, Klara; Schaefer, Gabriele; Jiang, Guoying; Nogal, Bartek; Emery, Craig; Vanderlaan, Martin; Carter, Paul; Harris, Reed; Amanullah, Ashraf

    2012-02-17

    Upper hinge is vulnerable to radical attacks that result in breakage of the heavy-light chain linkage and cleavage of the hinge of an IgG1. To further explore mechanisms responsible for the radical induced hinge degradation, nine mutants were designed to determine the roles that the upper hinge Asp and His play in the radical reactions. The observation that none of these substitutions could inhibit the breakage of the heavy-light chain linkage suggests that the breakage may result from electron transfer from Cys(231) directly to the heavy-light chain linkage upon radical attacks, and implies a pathway separate from His(229)-mediated hinge cleavage. On the other hand, the substitution of His(229) with Tyr showed promising advantages over the native antibody and other substitutions in improving the stability and function of the IgG1. This substitution inhibited the hinge cleavage by 98% and suggests that the redox active nature of Tyr did not enable it to replicate the ability of His to facilitate radical induced degradation. We propose that the lower redox potential of Tyr, a residue that may be the ultimate sink for oxidizing equivalents in proteins, is responsible for the inhibition. More importantly, the substitution increased the antibody's binding to FcγRIII receptors by 2-3-fold, and improved ADCC activity by 2-fold, while maintaining a similar pharmacokinetic profile with respect to the wild type. Implications of these observations for antibody engineering and development are discussed.

  10. Human IgG1 Responses to Surface Localised Schistosoma mansoni Ly6 Family Members Drop following Praziquantel Treatment.

    Directory of Open Access Journals (Sweden)

    Iain W Chalmers

    Full Text Available The heptalaminate-covered, syncytial tegument is an important anatomical adaptation that enables schistosome parasites to maintain long-term, intravascular residence in definitive hosts. Investigation of the proteins present in this surface layer and the immune responses elicited by them during infection is crucial to our understanding of host/parasite interactions. Recent studies have revealed a number of novel tegumental surface proteins including three (SmCD59a, SmCD59b and Sm29 containing uPAR/Ly6 domains (renamed SmLy6A SmLy6B and SmLy6D in this study. While vaccination with SmLy6A (SmCD59a and SmLy6D (Sm29 induces protective immunity in experimental models, human immunoglobulin responses to representative SmLy6 family members have yet to be thoroughly explored.Using a PSI-BLAST-based search, we present a comprehensive reanalysis of the Schistosoma mansoni Ly6 family (SmLy6A-K. Our examination extends the number of members to eleven (including three novel proteins and provides strong evidence that the previously identified vaccine candidate Sm29 (renamed SmLy6D is a unique double uPAR/Ly6 domain-containing representative. Presence of canonical cysteine residues, signal peptides and GPI-anchor sites strongly suggest that all SmLy6 proteins are cell surface-bound. To provide evidence that SmLy6 members are immunogenic in human populations, we report IgG1 (as well as IgG4 and IgE responses against two surface-bound representatives (SmLy6A and SmLy6B within a cohort of S. mansoni-infected Ugandan males before and after praziquantel treatment. While pre-treatment IgG1 prevalence for SmLy6A and SmLy6B differs amongst the studied population (7.4% and 25.3% of the cohort, respectively, these values are both higher than IgG1 prevalence (2.7% for a sub-surface tegumental antigen, SmTAL1. Further, post-treatment IgG1 levels against surface-associated SmLy6A and SmLy6B significantly drop (p = 0.020 and p < 0.001, respectively when compared to rising IgG

  11. Human-derived IgG level as an indicator for EBV-associated lymphoma model in Hu-PBL/SCID chimeras.

    Science.gov (United States)

    Tang, Yunlian; He, Rongfang; Zhang, Yang; Liu, Fang; Cheng, Ailan; Wu, Yimou; Gan, Runliang

    2011-05-09

    Epstein-Barr virus (EBV) has a close association with various types of human lymphomas. Animal models are essential to elucidate the pathogenesis of human EBV-associated lymphomas. The aim of the present study is to evaluate the association between human IgG concentration and EBV-associated lymphoma development in huPBL/SCID mice. Human peripheral blood lymphocytes (hu-PBL) from EBV-seropositive donors were inoculated intraperitoneally into SCID mouse. Immunohistochemical staining was used to examine differentiated antigens of tumor cells. EBV infection of the induced tumors was detected by in situ hybridization. IgG concentrations in the serums of 12 SCID mice were measured by unidirectional immunodiffusion assay. 21 out of 29 mice developed tumors in their body. Immunohistochemical staining showed that all induced tumors were LCA (leukocyte common antigen) positive, B-cell markers (CD20, CD79a) positive, and T-cell markers (both CD3 and CD45RO) negative. The tumors can be diagnosed as human B-cell lymphomas by these morphological and immunohistochemical features. In situ hybridization exhibited resultant tumor cells had EBV encoded small RNA-1 (EBER-1). Human-derived IgG could be found in the serum from SCID mice on the 15th day following hu-PBL transplantation, and IgG levels increased with the tumor development in 6 hu-PBL/SCID chimeras. Intraperitoneal transfer of hu-PBLs from EBV+ donors to SCID mice leads to high human IgG levels in mouse serum and B cell lymphomas. Our findings suggest that increasing levels of human-derived IgG in peripheral blood from hu-PBL/SCID mice could be used to monitor EBV-related human B-cell lymphoma development in experimental animals.

  12. Human-derived IgG level as an indicator for EBV-associated lymphoma model in Hu-PBL/SCID chimeras

    Directory of Open Access Journals (Sweden)

    Cheng Ailan

    2011-05-01

    Full Text Available Abstract Background Epstein-Barr virus (EBV has a close association with various types of human lymphomas. Animal models are essential to elucidate the pathogenesis of human EBV-associated lymphomas. The aim of the present study is to evaluate the association between human IgG concentration and EBV-associated lymphoma development in huPBL/SCID mice. Methods Human peripheral blood lymphocytes (hu-PBL from EBV-seropositive donors were inoculated intraperitoneally into SCID mouse. Immunohistochemical staining was used to examine differentiated antigens of tumor cells. EBV infection of the induced tumors was detected by in situ hybridization. IgG concentrations in the serums of 12 SCID mice were measured by unidirectional immunodiffusion assay. Results 21 out of 29 mice developed tumors in their body. Immunohistochemical staining showed that all induced tumors were LCA (leukocyte common antigen positive, B-cell markers (CD20, CD79a positive, and T-cell markers (both CD3 and CD45RO negative. The tumors can be diagnosed as human B-cell lymphomas by these morphological and immunohistochemical features. In situ hybridization exhibited resultant tumor cells had EBV encoded small RNA-1 (EBER-1. Human-derived IgG could be found in the serum from SCID mice on the 15th day following hu-PBL transplantation, and IgG levels increased with the tumor development in 6 hu-PBL/SCID chimeras. Conclusions Intraperitoneal transfer of hu-PBLs from EBV+ donors to SCID mice leads to high human IgG levels in mouse serum and B cell lymphomas. Our findings suggest that increasing levels of human-derived IgG in peripheral blood from hu-PBL/SCID mice could be used to monitor EBV-related human B-cell lymphoma development in experimental animals.

  13. Characterization of the basic charge variants of a human IgG1

    Science.gov (United States)

    Lu, Franklin; Derfus, Gayle; Kluck, Brian; Nogal, Bartek; Emery, Craig; Summers, Christie; Zheng, Kai; Bayer, Robert; Amanullah, Ashraf

    2011-01-01

    We report a case study of an IgG1 with a unique basic charge variant profile caused by C-terminal proline amidation on either one or two heavy chains. The proline amidation was sensitive to copper ion concentration in the production media during cell culture: the higher the Cu2+ ion concentration, the higher the level of proline amidation detected. This conclusion was supported by the analysis of samples that revealed direct correlation between the proline amidation level observed from peptide maps and the level of basic peaks measured by imaged capillary isoelectric focusing and a pH gradient ion-exchange chromatography method. The importance of these observations to therapeutic antibody production is discussed. PMID:22123059

  14. Snake venomics of monocled cobra (Naja kaouthia) and investigation of human IgG response against venom toxins.

    Science.gov (United States)

    Laustsen, Andreas H; Gutiérrez, José María; Lohse, Brian; Rasmussen, Arne R; Fernández, Julián; Milbo, Christina; Lomonte, Bruno

    2015-06-01

    The venom proteome of the monocled cobra, Naja kaouthia, from Thailand, was characterized by RP-HPLC, SDS-PAGE, and MALDI-TOF-TOF analyses, yielding 38 different proteins that were either identified or assigned to families. Estimation of relative protein abundances revealed that venom is dominated by three-finger toxins (77.5%; including 24.3% cytotoxins and 53.2% neurotoxins) and phospholipases A2 (13.5%). It also contains lower proportions of components belonging to nerve growth factor, ohanin/vespryn, cysteine-rich secretory protein, C-type lectin/lectin-like, nucleotidase, phosphodiesterase, metalloproteinase, l-amino acid oxidase, cobra venom factor, and cytidyltransferase protein families. Small amounts of three nucleosides were also evidenced: adenosine, guanosine, and inosine. The most relevant lethal components, categorized by means of a 'toxicity score', were α-neurotoxins, followed by cytotoxins/cardiotoxins. IgGs isolated from a person who had repeatedly self-immunized with a variety of snake venoms were immunoprofiled by ELISA against all venom fractions. Stronger responses against larger toxins, but lower against the most critical α-neurotoxins were obtained. As expected, no neutralization potential against N. kaouthia venom was therefore detected. Combined, our results display a high level of venom complexity, unveil the most relevant toxins to be neutralized, and provide prospects of discovering human IgGs with toxin neutralizing abilities through use of phage display screening. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Western blotting using Strongyloides ratti antigen for the detection of IgG antibodies as confirmatory test in human strongyloidiasis

    Directory of Open Access Journals (Sweden)

    Luciana Pereira Silva

    2003-07-01

    Full Text Available The present study was conducted to evaluate the frequency of antigenic components recognized by serum IgG antibodies in Western blotting (WB using a Strongyloides ratti larval extract for the diagnosis of human strongyloidiasis. In addition, the WB results were compared to the enzyme-linked immunosorbent assay (ELISA and the indirect immunofluorescence antibody test (IFAT results. Serum samples of 180 individuals were analyzed (80 with strongyloidiasis, 60 with other intestinal parasitoses, and 40 healthy individuals. S. ratti was obtained from fecal culture of experimentally infected Rattus rattus. For IFAT, S. ratti larvae were used as antigen and S. ratti larval antigenic extracts were employed in WB and ELISA. Eleven S. ratti antigenic components were predominantly recognized by IgG antibodies in sera of patients with strongyloidiasis. There was a positive concordance for the three tests in 87.5% of the cases of strongyloidiasis. The negative concordance in the three tests was 94% and 97.5%, in patients with other intestinal parasitoses and healthy individuals, respectively. In cases of positive ELISA and negative IFAT results, diagnosis could be confirmed by WB. ELISA, IFAT, and WB using S. ratti antigens showed a high rate of sensitivity and specificity. In conclusion, WB using S. ratti larval extract was able to recognize 11 immunodominant antigenic components, showing to be a useful tool to define the diagnosis in cases of equivocal serology.

  16. IgG subclass antibodies to human and bacterial HSP60 are not associated with disease activity and progression over time in axial spondyloarthritis

    DEFF Research Database (Denmark)

    Carlsen, Thomas Gelsing; Hjelholt, Astrid Johannesson; Jurik, Anne Grethe

    2013-01-01

    of three reactive arthritis-related bacteria; human HSP60; and the microorganisms Chlamydia trachomatis and C. pneumoniae were determined by ELISA. Serum samples collected from 2004 to 2006 and in 2010 and 2011 from 39 axial SpA patients were analyzed and compared with samples from 39 healthy controls...... in the same serum sample, IgG1 and IgG3, respectively, indicating that there was no cross-reaction. CONCLUSIONS: A significant association was observed between axial SpA and the presence of IgG1/IgG3 antibodies to human HSP60 and of IgG1 to S. enterica Enteritidis and C. trachomatis. Generation of antibodies......INTRODUCTION: Spondyloarthritis (SpA), an interrelated group of rheumatic diseases, has been suggested to be triggered by bacterial infections prior to the development of an autoimmune response that causes inflammation of the spinal and peripheral joints. Because human heat shock protein 60 (HSP60...

  17. Solid phase radioimmunoassay for quantitation of antigen-specific IgG in human sera with /sup 125/I-protein A from Staphylococcus aureus

    Energy Technology Data Exchange (ETDEWEB)

    Hamilton, R.G.; Sobotka, A.K.; Adkinson, N.F. Jr.

    1979-03-01

    Radiolabeled protein A from Staphylococcus aureus (Staph A) has been used to develop a solid phase, noncompetitive radioimmunoassay for quantitation of specific IgG antibody. The assay involves two incubations: First, agarose-insolubilized antigen is mixed with serum samples for 1 to 4 h during which specific antibody is bound; second, after a washing procedure, the solid phase immune complexes are incubated for 4 to 18 h with /sup 125/I-Staph A, during which the radiolabeled detection protein binds to the insolubilized specific IgG antibody. In a comparative study of the IgG antiphospholipase A antibody content of 23 human sera drawn from honeybee venom-sensitive patients, results of the Staph A assay correlated highly (r = 0.981, p < 0.001, N = 23) with those obtained from a liquid phase, competitive radioimmunoprecipitation (double antibody) assay. The two assays demonstrated comparable precision, sensitivity, and reproducibility. In contrast, the use of /sup 125/I-Staph A in the solid phase radioimmunoassay was superior to /sup 125/I rabbit anti-human IgG because of lower negative serum (blank) values, shorter time required to reach equilibrium binding, and greater precision and reproducibility. In principle, the /sup 125/ Staph A assay may be applied to IgG quantitation for crude allergen extracts as well as purified antigens. Furthermore, the sera of a number of mammalian species may be studied without further modification.

  18. Study of chronic hemolytic anaemia patients in Rio de Janeiro: prevalence of anti-human parvovirus B19 IgG antibodies and the developement aplastic crises

    Directory of Open Access Journals (Sweden)

    SANT'ANNA Anadayr L.M.

    2002-01-01

    Full Text Available The prevalence of anti-human parvovirus B19 IgG antibodies was determined in sera from 165 chronic hemolytic anemia patients, receiving medical care at Instituto Estadual de Hematologia (IEHE, Rio de Janeiro, during the year of 1994. This sample represents around 10% of the chronic hemolytic anemia patients attending at IEHE. Most of these patients (140 have sickle cell disease. Anti-B19 IgG antibodies were detected in 32.1% of patients. No statistically significant difference (p > 0.05 was seen between IgG antibody prevalence in male (27.8% and female (35.5% patients. Anti-B19 IgG antibodies were more frequent in older (37.6% than younger (28.2% than 20 years old patients, although this difference had no statistical significance (p > 0.05. Anti-B19 IgG antibody prevalence showed that 67.9% of patients enrolled in the study were susceptible to B19 acute infection. With the aim to detect acute B19 infection, patients follow up continued until February 1996. During this period four patients presented transient aplastic crisis due to human parvovirus B19 as confirmed by the detection of specific IgM antibodies. All four patients were younger than 20 years old, and 3 were younger than 10 years old. Three of them were sickle cell disease patients. Three of the four acute B19 infection occurred during 1994 springtime.

  19. Study of chronic hemolytic anaemia patients in Rio de Janeiro: prevalence of anti-human parvovirus B19 IgG antibodies and the development aplastic crises.

    Science.gov (United States)

    Sant'Anna, Anadayr L M; Garcia, Rita de Cássia N Cubel; Marzoche, Mônica; da Rocha, Heloisa Helena A Gallo; Paula, Maria Tereza M; Lobo, Clarisse C; Nascimento, Jussara P

    2002-01-01

    The prevalence of anti-human parvovirus B19 IgG antibodies was determined in sera from 165 chronic hemolytic anemia patients, receiving medical care at Instituto Estadual de Hematologia (IEHE), Rio de Janeiro, during the year of 1994. This sample represents around 10% of the chronic hemolytic anemia patients attending at IEHE. Most of these patients (140) have sickle cell disease. Anti-B19 IgG antibodies were detected in 32.1% of patients. No statistically significant difference (p > 0.05) was seen between IgG antibody prevalence in male (27.8%) and female (35.5%) patients. Anti-B19 IgG antibodies were more frequent in older (37.6%) than younger (28.2%) than 20 years old patients, although this difference had no statistical significance (p > 0.05). Anti-B19 IgG antibody prevalence showed that 67.9% of patients enrolled in the study were susceptible to B19 acute infection. With the aim to detect acute B19 infection, patients follow up continued until February 1996. During this period four patients presented transient aplastic crisis due to human parvovirus B19 as confirmed by the detection of specific IgM antibodies. All four patients were younger than 20 years old, and 3 were younger than 10 years old. Three of them were sickle cell disease patients. Three of the four acute B19 infection occurred during 1994 springtime.

  20. IgG antibodies against human papillomavirus type 16 E7 proteins in cervicovaginal washing fluid from patients with cervical neoplasia

    NARCIS (Netherlands)

    Tjiong, M. Y.; ter Schegget, J.; Tjiong-A-Hung, S. P.; Out, T. A.; van der Vange, N.; Burger, M. P. M.; Struyk, L.

    2000-01-01

    Little information is available about the cervicovaginal mucosal antibodies against human papillomavirus (HPV) proteins. In this study specific IgG antibodies against HPV 16 E7 protein were determined in paired samples of cervicovaginal washing fluid and serum from patients with cervical cancer (n =

  1. QUALITATIVE MEASUREMENTS OF IGE AND IGG IN HUMAN ASTHMATIC SERUM FOR MOLD REACTIVITY

    Science.gov (United States)

    Rational: Molds have the ability to induce allergic asthma-like responses in mouse models; however, their role in human disease is unclear. This study was to develop a screening tool for reactivity of human sera to mold extracts by using a minimal amount of sera for a qual...

  2. Evolution of IgG antibody response against Toxoplasma gondii tissue cyst in acute and chronic human infections

    Directory of Open Access Journals (Sweden)

    Margarita VILLAVEDRA

    1998-03-01

    Full Text Available The recognition profile of the tissue cysts antigens by IgG antibodies was studied during acute and chronic human toxoplasmic infection. Thus the IgG response against Toxoplasma gondii was investigated by immunoblotting in two patients accidentally infected with the RH strain as well as in group of naturally infected patients at acute and chronic phase. There was an overall coincidence of molecular mass among antigens of tachyzoites and tissue cysts recognized by these sera, however, they appear not to be the same molecules. The response against tissue cysts starts early during acute infection, and the reactivity of antibodies is strong against a wide range of antigens. Six bands (between 82 and 151 kDa were exclusively recognized by chronic phase sera but only the 132 kDa band was positive in more than 50% of the sera analysed. A mixture of these antigens could be used to discriminate between the two infection phases. The most important antigens recognized by the acute and the chronic phase sera were 4 clusters in the ranges 20-24 kDa, 34-39 kDa, 58-80 kDa and 105-130 kDa as well as two additional antigens of 18 and 29 kDa. Both accidentally infected patients and some of the naturally infected patients showed a weak specific response against tissue cyst antigens.O reconhecimento do perfil dos antígenos de cistos tissulares pelos anticorpos IgG foi estudado durante a infecção toxoplasmótica aguda e crônica. Assim a resposta de IgG contra Toxoplasma gondii foi investigada pelo "immunoblotting" em dois pacientes acidentalmente infectados com a variedade RH bem como em grupos de pacientes naturalmente infectados nas fases aguda e crônica. Houve uma coincidência global da massa molecular entre antígenos de taquizoitas e cistos tissulares reconhecidos por estes soros, todavia, eles parecem não ser as mesmas moléculas. A resposta contra cistos tissulares começa precocemente durante a infecção aguda e a reatividade de anticorpos é forte

  3. Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation

    DEFF Research Database (Denmark)

    van den Bremer, E. T. J.; Beurskens, F. J.; Voorhorst, M.

    2015-01-01

    G hexamerization at the cell surface. Here we demonstrate that C-terminal lysines may interfere with this process, leading to suboptimal C1q binding and CDC of cells opsonized with C-terminal lysine-containing IgG. After we removed these lysines with a carboxypeptidase, maximal complement activation was observed...... are produced in a pro-form in which charged C-termini interfere with IgG hexamer formation, C1q binding and CDC. To allow maximal complement activation, C-terminal lysine processing is required to release the antibody's full cytotoxic potential.......Human IgG is produced with C-terminal lysines that are cleaved off in circulation. The function of this modification was unknown and generally thought not to affect antibody function. We recently reported that efficient C1q binding and complement-dependent cytotoxicity (CDC) requires Ig...

  4. Human IgG subclass antibodies to the 19 kilodalton carboxy ...

    African Journals Online (AJOL)

    Objective: To determine the natural human humoral immune responses to the 19 kilodalton carboxy terminal fragment of Plasmodium falciparum merozoite surface protein 1 (MSP119), a malaria candidate vaccine antigen and to determine the prevalence of MAD20 and K1 alleles of P. falciparum MSP1. Design: Community ...

  5. NOG-hIL-4-Tg, a new humanized mouse model for producing tumor antigen-specific IgG antibody by peptide vaccination.

    Directory of Open Access Journals (Sweden)

    Yoshie Kametani

    Full Text Available Immunodeficient mice transplanted with human peripheral blood mononuclear cells (PBMCs are promising tools to evaluate human immune responses to vaccines. However, these mice usually develop severe graft-versus-host disease (GVHD, which makes estimation of antigen-specific IgG production after antigen immunization difficult. To evaluate antigen-specific IgG responses in PBMC-transplanted immunodeficient mice, we developed a novel NOD/Shi-scid-IL2rγnull (NOG mouse strain that systemically expresses the human IL-4 gene (NOG-hIL-4-Tg. After human PBMC transplantation, GVHD symptoms were significantly suppressed in NOG-hIL-4-Tg compared to conventional NOG mice. In kinetic analyses of human leukocytes, long-term engraftment of human T cells has been observed in peripheral blood of NOG-hIL-4-Tg, followed by dominant CD4+ T rather than CD8+ T cell proliferation. Furthermore, these CD4+ T cells shifted to type 2 helper (Th2 cells, resulting in long-term suppression of GVHD. Most of the human B cells detected in the transplanted mice had a plasmablast phenotype. Vaccination with HER2 multiple antigen peptide (CH401MAP or keyhole limpet hemocyanin (KLH successfully induced antigen-specific IgG production in PBMC-transplanted NOG-hIL-4-Tg. The HLA haplotype of donor PBMCs might not be relevant to the antibody secretion ability after immunization. These results suggest that the human PBMC-transplanted NOG-hIL-4-Tg mouse is an effective tool to evaluate the production of antigen-specific IgG antibodies.

  6. Neutrophils extracellular traps damage Naegleria fowleri trophozoites opsonized with human IgG.

    Science.gov (United States)

    Contis-Montes de Oca, A; Carrasco-Yépez, M; Campos-Rodríguez, R; Pacheco-Yépez, J; Bonilla-Lemus, P; Pérez-López, J; Rojas-Hernández, S

    2016-08-01

    Naegleria fowleri infects humans through the nasal mucosa causing a disease in the central nervous system known as primary amoebic meningoencephalitis (PAM). Polymorphonuclear cells (PMNs) play a critical role in the early phase of N. fowleri infection. Recently, a new biological defence mechanism called neutrophil extracellular traps (NETs) has been attracting attention. NETs are composed of nuclear DNA combined with histones and antibacterial proteins, and these structures are released from the cell to direct its antimicrobial attack. In this work, we evaluate the capacity of N. fowleri to induce the liberation of NETs by human PMN cells. Neutrophils were cocultured with unopsonized or IgG-opsonized N. fowleri trophozoites. DNA, histone, myeloperoxidase (MPO) and neutrophil elastase (NE) were stained, and the formation of NETs was evaluated by confocal microscopy and by quantifying the levels of extracellular DNA. Our results showed N. fowleri induce the liberation of NETs including release of MPO and NE by human PMN cells as exposure interaction time is increased, but N. fowleri trophozoites evaded killing. However, when trophozoites were opsonized, they were susceptible to the neutrophils activity. Therefore, our study suggests that antibody-mediated PMNs activation through NET formation may be crucial for antimicrobial responses against N. fowleri. © 2016 John Wiley & Sons Ltd.

  7. Use of larval, parasitic female and egg antigens from Strongyloides venezuelensis to detect parasite-specific IgG and immune complexes in immunodiagnosis of human strongyloidiasis.

    Science.gov (United States)

    Gonçalves, A L R; Nunes, D S; Gonçalves-Pires, M R F; Ueta, M T; Costa-Cruz, J M

    2012-06-01

    The aim of this study was to use larval, parasitic female and egg antigens from Strongyloides venezuelensis to detect parasite-specific IgG and immune complexes in human serum samples by enzyme-linked immunosorbent assay (ELISA). In total, 95 serum samples were analysed, consisting of 30 patients harbouring S. stercoralis larvae, 30 healthy subjects and 35 patients with other parasites. Sensitivity, specificity and diagnostic efficiency were calculated. A significant statistical difference was found in the detection of immune complexes and antibodies in patients harbouring S. stercoralis larvae from larval and eggs antigens, with higher positivity using larval antigen. The larval antigen showed the highest values for sensitivity, specificity and diagnostic efficiency in ELISA from detection of immune complexes. For the first time we used IgG anti-larvae, IgG anti-parasitic females or IgG anti-eggs for immune complex detection. We concluded that the association of antibody and immune complex detection could be used in the diagnosis of human strongyloidiasis.

  8. Monitoring the Systemic Human Memory B Cell Compartment of Melanoma Patients for Anti-Tumor IgG Antibodies

    Science.gov (United States)

    Gilbert, Amy E.; Karagiannis, Panagiotis; Dodev, Tihomir; Koers, Alexander; Lacy, Katie; Josephs, Debra H.; Takhar, Pooja; Geh, Jenny L. C.; Healy, Ciaran; Harries, Mark; Acland, Katharine M.; Rudman, Sarah M.; Beavil, Rebecca L.; Blower, Philip J.; Beavil, Andrew J.; Gould, Hannah J.; Spicer, James; Nestle, Frank O.; Karagiannis, Sophia N.

    2011-01-01

    Melanoma, a potentially lethal skin cancer, is widely thought to be immunogenic in nature. While there has been much focus on T cell-mediated immune responses, limited knowledge exists on the role of mature B cells. We describe an approach, including a cell-based ELISA, to evaluate mature IgG antibody responses to melanoma from human peripheral blood B cells. We observed a significant increase in antibody responses from melanoma patients (n = 10) to primary and metastatic melanoma cells compared to healthy volunteers (n = 10) (Pcell cultures (n = 1,800) compared to 2% of cultures from healthy controls (n = 600) produced antibodies that recognized melanoma cells. Lastly, a patient-derived melanoma-specific monoclonal antibody was selected for further study. This antibody effectively killed melanoma cells in vitro via antibody-mediated cellular cytotoxicity. These data demonstrate the presence of a mature systemic B cell response in melanoma patients, which is reduced with disease progression, adding to previous reports of tumor-reactive antibodies in patient sera, and suggesting the merit of future work to elucidate the clinical relevance of activating humoral immune responses to cancer. PMID:21559411

  9. High prevalence of human anti-bovine IgG antibodies as the major cause of false positive reactions in two-site immunoassays based on monoclonal antibodies

    DEFF Research Database (Denmark)

    Andersen, Ditte C; Koch, Claus; Jensen, Charlotte H

    2004-01-01

    A sandwich ELISA for quantification of the endometrial protein PP14 revealed false positive reactions in 81% of male sera (n = 54). The PP14 ELISA was based on two monoclonal antibodies (Mabs) with different epitope specificities--a catcher and a biotinylated indicator. The monoclonal antibodies...... were purified by protein G affinity chromatography from culture supernatant containing 10% (v/v) fetal calf serum (FCS). Human anti-animal IgG (bovine, mouse, horse, and swine) antibodies and human anti-bovine serum albumin antibodies were measured using an ELISA design, with direct bridging...... of the solid phase and biotinylated antigens. The false positive reactions were abolished by addition of 1% (v/v) bovine serum to the dilution buffer (DB). Human anti-bovine IgG antibodies (HABIA) were detected in 99 out of 104 sera from blood donors (50 females; 54 males). HABIA levels in male sera (n = 54...

  10. Modulation of single-cell IgG secretion frequency and rates in human memory B cells by CpG DNA, CD40L, IL-21, and cell division.

    Science.gov (United States)

    Henn, Alicia D; Rebhahn, Jonathan; Brown, Miguel A; Murphy, Alison J; Coca, Mircea N; Hyrien, Ollivier; Pellegrin, Tina; Mosmann, Tim; Zand, Martin S

    2009-09-01

    During the recall response by CD27(+) IgG class-switched human memory B cells, total IgG secreted is a function of the following: 1) the number of IgG-secreting cells (IgG-SC), and 2) the secretion rate of each cell. In this study, we report the quantitative ELISPOT method for simultaneous estimation of single-cell IgG secretion rates and secreting cell frequencies in human B cell populations. We found that CD27(+) IgM(-) memory B cells activated with CpG and cytokines had considerable heterogeneity in the IgG secretion rates, with two major secretion rate subpopulations. BCR cross-linking reduced the frequency of cells with high per-cell IgG secretion rates, with a parallel decrease in CD27(high) B cell blasts. Increased cell death may account for the BCR-stimulated reduction in high-rate IgG-SC CD27(high) B cell blasts. In contrast, the addition of IL-21 to CD40L plus IL-4-activated human memory B cells induced a high-rate IgG-SC population in B cells with otherwise low per-cell IgG secretion rates. The profiles of human B cell IgG secretion rates followed the same biphasic distribution and range irrespective of division class. This, along with the presence of non-IgG-producing, dividing B cells in CpG plus cytokine-activated B memory B cell populations, is suggestive of an on/off switch regulating IgG secretion. Finally, these data support a mixture model of IgG secretion in which IgG secreted over time is modulated by the frequency of IgG-SC and the distribution of their IgG secretion rates.

  11. Lysine conjugation properties in human IgGs studied by integrating high-resolution native mass spectrometry and bottom-up proteomics.

    Science.gov (United States)

    Gautier, Violette; Boumeester, Anja J; Lössl, Philip; Heck, Albert J R

    2015-08-01

    Antibody-drug conjugates (ADCs) are a novel class of biopharmaceuticals several of which are now being investigated in clinical studies. In ADCs, potent cytotoxic drugs are coupled via a linker to reactive residues in IgG monoclonal antibodies. Linkage to lysine residues in the IgGs, using N-hydroxysuccinimide ester based chemistry, is one of the possible options. To control drug load and specificity, proper knowledge is required about which lysine residues are most accessible and reactive. Here, we combine native MS and bottom-up proteomics to monitor the overall drug load and site-specific lysine reactivity, using N-hydroxysuccinimide-based tandem mass tags. High-resolution Orbitrap native MS enables us to monitor and quantify, due to the achieved baseline resolution, the sequential incorporation of up to 69 tandem mass tag molecules into human IgGs. Complementary, bottom-up proteomics facilitates the identification of some very reactive "hot-spot" conjugation sites. However, we also identify lysine residues that are highly resistant to chemical labeling. Our integrated approach gives insight into the conjugation properties of IgGs at both the intact protein and residue levels, providing fundamental information for controlling drug load and specificity in lysine-linked ADCs. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Enhanced insight into the autoimmune component of glaucoma: IgG autoantibody accumulation and pro-inflammatory conditions in human glaucomatous retina.

    Directory of Open Access Journals (Sweden)

    Oliver W Gramlich

    Full Text Available BACKGROUND: There is accumulating evidence that autoimmune components, such as autoantibodies and autoantibody depositions, play a role in the pathogenesis of neurodegenerative diseases like Alzheimeŕs disease or Multiple Sclerosis. Due to alterations of autoantibody patterns in sera and aqueous humor, an autoimmune component is also assumed in the pathogenesis of glaucoma, a common reason for irreversible blindness worldwide. So far there has been no convincing evidence that autoantibodies are accumulated in the retina of glaucoma patients and that the local immune homeostasis might be affected. METHODS AND RESULTS: Six human glaucomatous donor eyes and nine samples from donors with no recorded ocular disease were included. Antibody microarrays were used to examine the patterns of pro-inflammatory proteins and complement proteins. Analysis of TNF-α and interleukin levels revealed a slight up-regulation exclusively in the glaucomatous group, while complement protein levels were not altered. IgG autoantibody accumulations and/or cellular components were determined by immunohistology (n = 4 per group. A significantly reduced number of retinal ganglion cells was found in the glaucomatous group (healthy: 104±7 nuclei/mm, glaucoma: 67±9 nuclei/mm; p = 0.0007. Cell loss was accompanied by strong retinal IgG autoantibody accumulations, which were at least twice as high as in healthy subjects (healthy: 5.0±0.5 IgG deposits/100 cells, glaucoma: 9.4±1.9 IgG deposits/100 cells; p = 0.004. CD27(+ cells and CD27(+/IgG(+ plasma cells were observed in all glaucomatous subjects, but not in controls. CONCLUSION: This work provides serious evidence for the occurrence of IgG antibody deposition and plasma cells in human glaucomatous retina. Moreover, the results suggest that these IgG deposits occurred in a pro-inflammatory environment which seems to be maintained locally by immune-competent cells like microglia. Thereby, glaucoma features an

  13. Low doses of IgG from atopic individuals can modulate in vitro IFN-γ production by human intra-thymic TCD4 and TCD8 cells: An IVIg comparative approach.

    Science.gov (United States)

    Sgnotto, Fábio da Ressureição; Oliveira, Marília Garcia de; Lira, Aline Aparecida de Lima; Bento-de-Souza, Luciana; Duarte, Alberto José da Silva; Victor, Jefferson Russo

    2017-07-03

    The regulatory effect of allergic responses induced by IgG antibodies on human intra-thymic cells has not been reported in the literature. The aim of this study was to evaluate the possible differential effect of purified IgG from atopic and non-atopic individuals on human intra-thymic αβT cell cytokine production. Thymic tissues were obtained from 14 patients who were less than 7 d old. Additionally, blood samples were collected from atopic and non-atopic volunteers. Thymocytes and peripheral blood mononuclear cells were cultured with purified atopic or non-atopic IgG, and intracellular cytokine production was assessed. Purified IgG did not influence the frequency or viability of human intra-thymic αβT cells. Purified non-atopic IgG induced greater IFN-γ production by intra-thymic CD4+CD8+ T cells than did the mock treatment and atopic IgG. A similar effect of purified non-atopic IgG on TCD8 cells was observed compared with the mock treatment. Atopic IgG inhibited IFN-γ and TGF-β production by intra-thymic TCD4 cells. Treatment with intravenous immunoglobulin resulted in intermediate levels of IFN-γ and TGF-β in intra-thymic TCD4 cells compared with treatment with atopic and non-atopic IgG. Peripheral TCD4 cells from non-atopic individuals produced IFN-γ only in response to atopic IgG. This report describes novel evidence revealing that IgG from atopic individuals may influence intracellular IFN-γ production by intra-thymic αβT cells in a manner that may favor allergy development.

  14. Cross-species analysis of Fc engineered anti-Lewis-Y human IgG1 variants in human neonatal receptor transgenic mice reveal importance of S254 and Y436 in binding human neonatal Fc receptor

    Science.gov (United States)

    Burvenich, Ingrid J. G.; Farrugia, William; Lee, Fook T.; Catimel, Bruno; Liu, Zhanqi; Makris, Dahna; Cao, Diana; O'Keefe, Graeme J.; Brechbiel, Martin W.; King, Dylan; Spirkoska, Violeta; Allan, Laura C.; Ramsland, Paul A.; Scott, Andrew M.

    2016-01-01

    ABSTRACT IgG has a long half-life through engagement of its Fc region with the neonatal Fc receptor (FcRn). The FcRn binding site on IgG1 has been shown to contain I253 and H310 in the CH2 domain and H435 in the CH3 domain. Altering the half-life of IgG has been pursued with the aim to prolong or reduce the half-life of therapeutic IgGs. More recent studies have shown that IgGs bind differently to mouse and human FcRn. In this study we characterize a set of hu3S193 IgG1 variants with mutations in the FcRn binding site. A double mutation in the binding site is necessary to abrogate binding to murine FcRn, whereas a single mutation in the FcRn binding site is sufficient to no longer detect binding to human FcRn and create hu3S193 IgG1 variants with a half-life similar to previously studied hu3S193 F(ab')2 (t1/2β, I253A, 12.23 h; H310A, 12.94; H435A, 12.57; F(ab')2, 12.6 h). Alanine substitutions in S254 in the CH2 domain and Y436 in the CH3 domain showed reduced binding in vitro to human FcRn and reduced elimination half-lives in huFcRn transgenic mice (t1/2β, S254A, 37.43 h; Y436A, 39.53 h; wild-type, 83.15 h). These variants had minimal effect on half-life in BALB/c nu/nu mice (t1/2β, S254A, 119.9 h; Y436A, 162.1 h; wild-type, 163.1 h). These results provide insight into the interaction of human Fc by human FcRn, and are important for antibody-based therapeutics with optimal pharmacokinetics for payload strategies used in the clinic. PMID:27030023

  15. Cross-species analysis of Fc engineered anti-Lewis-Y human IgG1 variants in human neonatal receptor transgenic mice reveal importance of S254 and Y436 in binding human neonatal Fc receptor.

    Science.gov (United States)

    Burvenich, Ingrid J G; Farrugia, William; Lee, Fook T; Catimel, Bruno; Liu, Zhanqi; Makris, Dahna; Cao, Diana; O'Keefe, Graeme J; Brechbiel, Martin W; King, Dylan; Spirkoska, Violeta; Allan, Laura C; Ramsland, Paul A; Scott, Andrew M

    2016-01-01

    IgG has a long half-life through engagement of its Fc region with the neonatal Fc receptor (FcRn). The FcRn binding site on IgG1 has been shown to contain I253 and H310 in the CH2 domain and H435 in the CH3 domain. Altering the half-life of IgG has been pursued with the aim to prolong or reduce the half-life of therapeutic IgGs. More recent studies have shown that IgGs bind differently to mouse and human FcRn. In this study we characterize a set of hu3S193 IgG1 variants with mutations in the FcRn binding site. A double mutation in the binding site is necessary to abrogate binding to murine FcRn, whereas a single mutation in the FcRn binding site is sufficient to no longer detect binding to human FcRn and create hu3S193 IgG1 variants with a half-life similar to previously studied hu3S193 F(ab')2 (t1/2β, I253A, 12.23 h; H310A, 12.94; H435A, 12.57; F(ab')2, 12.6 h). Alanine substitutions in S254 in the CH2 domain and Y436 in the CH3 domain showed reduced binding in vitro to human FcRn and reduced elimination half-lives in huFcRn transgenic mice (t1/2β, S254A, 37.43 h; Y436A, 39.53 h; wild-type, 83.15 h). These variants had minimal effect on half-life in BALB/c nu/nu mice (t1/2β, S254A, 119.9 h; Y436A, 162.1 h; wild-type, 163.1 h). These results provide insight into the interaction of human Fc by human FcRn, and are important for antibody-based therapeutics with optimal pharmacokinetics for payload strategies used in the clinic.

  16. DARPA Antibody Technology Program Standardized Test Bed for Antibody Characterization: Characterization of an MS2 Human IgG Antibody Produced by AnaptysBio, Inc.

    Science.gov (United States)

    2016-02-01

    human IgG antibody produced by AnaptysBio DARPA ATP Standardized Test Bed for Antibody Characterization: Characterization of an MS2 Patricia E...Buckley, Patricia E.; Calm, Alena; Welsh, Heather; Thompson, Roy; Carney, James (ECBC); Warner, Candice; and Zacharko, Melody (Excet) 5d. PROJECT...corresponding aliquot was removed and placed in an ice bath . These samples were then tested for activity using ELISA. 2.7 ELISA ELISAs were

  17. A nylon ball solid-phase radioimmunoassay for specific antibodies in human sera. Application to measurement of IgG antibodies to pollen allergens.

    Science.gov (United States)

    Djurup, R; Søndergaard, I; Minuva, U; Weeke, B

    1983-09-16

    The principle of the radioallergosorbent test (RAST) has been used to measure IgG antibodies to timothy grass pollen allergens in sera from desensitized allergic subjects. 125I-labeled goat anti-human IgG was used as detector protein. Non-specific binding was eliminated by use of a non-porous nylon ball an antigen carrier and by use of a special buffer with high ionic strength and pH, containing 1% bovine gamma globulin and 5% normal rabbit serum as 'balance proteins'. At dilution 1:80 non-specific binding was only 0.28% and the binding ratio for a high-liter serum was about 10. By inhibition experiments the assay was demonstrated to be specific for IgG antibodies to timothy grass pollen. The results obtained with this assay correlated statistically significantly with those found th a double -antibody method (rs equal 0.68, n equal 20, t equal 3.93, P less than 0.001). Serum dilution curves were parallel, indicating that the assay is in allergen excess. The within-assay coefficient of variation ranged from 3.9 to 7.6%; the between-assay coefficient of variation from 8.4 to 19.5%. The assay is very simple to perform, requiring no centrifugation. The allergen-coated balls are stable for at least 3 months. The assay should be applicable to measurement of IgG antibodies and IgG subclass antibodies to any protein antigen of interest.

  18. Engineering the fragment crystallizable (Fc) region of human IgG1 multimers and monomers to fine-tune interactions with sialic acid-dependent receptors.

    Science.gov (United States)

    Blundell, Patricia A; Le, Ngoc Phuong Lan; Allen, Joel; Watanabe, Yasunori; Pleass, Richard J

    2017-08-04

    Multimeric fragment crystallizable (Fc) regions and Fc-fusion proteins are actively being explored as biomimetic replacements for IVIG therapy, which is deployed to manage many diseases and conditions but is expensive and not always efficient. The Fc region of human IgG1 (IgG1-Fc) can be engineered into multimeric structures (hexa-Fcs) that bind their cognate receptors with high avidity. The critical influence of the unique N-linked glycan attached at Asn-297 on the structure and function of IgG1-Fc is well documented; however, whether the N-linked glycan has a similarly critical role in multimeric, avidly binding Fcs, is unknown. Hexa-Fc contains two N-linked sites at Asn-77 (equivalent to Asn-297 in the Fc of IgG1) and Asn-236 (equivalent to Asn-563 in the tail piece of IgM). We report here that glycosylation at Asn-297 is critical for interactions with Fc receptors and complement and that glycosylation at Asn-563 is essential for controlling multimerization. We also found that introduction of an additional fully occupied N-linked glycosylation site at the N terminus at position 1 (equivalent to Asp-221 in the Fc of IgG1) dramatically enhances overall sialic acid content of the Fc multimers. Furthermore, replacement of Cys-575 in the IgM tail piece of multimers resulted in monomers with enhanced sialic acid content and differential receptor-binding profiles. Thus insertion of additional N-linked glycans into either the hinge or tail piece of monomers or multimers leads to molecules with enhanced sialylation that may be suitable for managing inflammation or blocking pathogen invasion. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. High-yield production of a human monoclonal IgG by rhizosecretion in hydroponic tobacco cultures.

    Science.gov (United States)

    Madeira, Luisa M; Szeto, Tim H; Henquet, Maurice; Raven, Nicole; Runions, John; Huddleston, Jon; Garrard, Ian; Drake, Pascal M W; Ma, Julian K-C

    2016-02-01

    Rhizosecretion of recombinant pharmaceuticals from in vitro hydroponic transgenic plant cultures is a simple, low cost, reproducible and controllable production method. Here, we demonstrate the application and adaptation of this manufacturing platform to a human antivitronectin IgG1 monoclonal antibody (mAb) called M12. The rationale for specific growth medium additives was established by phenotypic analysis of root structure and by LC-ESI-MS/MS profiling of the total protein content profile of the hydroponic medium. Through a combination of optimization approaches, mAb yields in hydroponic medium reached 46 μg/mL in 1 week, the highest figure reported for a recombinant mAb in a plant secretion-based system to date. The rhizosecretome was determined to contain 104 proteins, with the mAb heavy and light chains the most abundant. This enabled evaluation of a simple, scalable extraction and purification protocol and demonstration that only minimal processing was necessary prior to protein A affinity chromatography. MALDI-TOF MS revealed that purified mAb contained predominantly complex-type plant N-glycans, in three major glycoforms. The binding of M12 purified from hydroponic medium to vitronectin was comparable to its Chinese hamster ovary (CHO)-derived counterpart. This study demonstrates that in vitro hydroponic cultivation coupled with recombinant protein rhizosecretion can be a practical, low-cost production platform for monoclonal antibodies. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  20. Cetuximab in combination with anti-human IgG antibodies efficiently down-regulates the EGF receptor by macropinocytosis

    Energy Technology Data Exchange (ETDEWEB)

    Berger, Christian [Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo (Norway); Madshus, Inger Helene [Institute of Pathology, University of Oslo, Rikshospitalet, 0027 Oslo (Norway); Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo (Norway); Stang, Espen, E-mail: espsta@rr-research.no [Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo (Norway)

    2012-12-10

    The monoclonal antibody C225 (Cetuximab) blocks binding of ligand to the epidermal growth factor receptor (EGFR). In addition, it is known that incubation with C225 induces endocytosis of the EGFR. This endocytosis has previously been shown to be increased when C225 is combined with an additional monoclonal anti-EGFR antibody. However, the effects of antibody combinations on EGFR activation, endocytosis, trafficking and degradation have been unclear. By binding a secondary antibody to the C225-EGFR complex, we here demonstrate that a combination of antibodies can efficiently internalize and degrade the EGFR. Although the combination of antibodies activated the EGFR kinase and induced ubiquitination of the EGFR, the kinase activity was not required for internalization of the EGFR. In contrast to EGF-induced EGFR down-regulation, the antibody combination efficiently degraded the EGFR without initiating downstream proliferative signaling. The antibody-induced internalization of EGFR was found not to depend on clathrin and/or dynamin, but depended on actin polymerization, suggesting induction of macropinocytosis. Macropinocytosis may cause internalization of large membrane areas, and this could explain the highly efficient internalization of the EGFR induced by combination of antibodies. -- Highlight: Black-Right-Pointing-Pointer Cetuximab induced endocytosis of EGFR increases upon combination with anti-human IgG. Black-Right-Pointing-Pointer Antibody combination causes internalization of EGFR by macropinocytosis. Black-Right-Pointing-Pointer Antibody-induced internalization of EGFR is independent of EGFR kinase activity. Black-Right-Pointing-Pointer Antibody combination may have a zipper effect and cross-link EGFRs on neighboring cells.

  1. Enhancement of retroviral infection in vitro by anti-Le(y) IgG: reversal by humanization of monoclonal mouse antibody

    DEFF Research Database (Denmark)

    Hansen, J E; Sørensen, A M; Arendrup, M

    1993-01-01

    also enhanced infection, a human/mouse chimeric antibody and a fully humanized antibody had no enhancing effect on free virus infection. We suggest that binding of anti-Le(y) ABL 364 or its F(ab)2 fragment induced a conformational change in the gp120 oligomers facilitating the process of infection......, and that this function was abrogated by the IgG1 Fc of the chimeric and the humanized antibodies. The observations indicate that the non-paratope domains of antiviral antibodies can influence their function as neutralizing or enhancing for infection....

  2. Immuno-epidemiology of human Schistosoma haematobium infection: preferential IgG3 antibody responsiveness to a recombinant antigen dependent on age and parasite burden

    Directory of Open Access Journals (Sweden)

    Fernandez Cecilia

    2006-06-01

    Full Text Available Abstract Background Schistosomiasis is a major parasitic disease affecting over 200 million people in the developing world with a further 400 million people at risk of infection. The aim of this study was to identify a single antigen from adult Schistosoma haematobium worms and subsequently use this antigen to study the development of schistosome-acquired immunity in a human population. Methods The full-length cDNA sequence of a S. haematobium protein, a putative orthologue of the S. mansoni tegumental antigen Sm13, was obtained from a cDNA library of adult S. haematobium worms and named Sh13 following a small-scale expressed sequence tags (EST project. The recombinant Sh13 protein expressed in E. coli, was used to investigate immuno-epidemiological patterns in 147 Zimbabweans (7–18 years old exposed to S. haematobium. Results Sequence analysis of the full-length cDNA sequence of the S. haematobium protein Sh13, indicated that the protein has an N-terminal signal peptide and encodes an 85-amino acid mature protein with a highly conserved predicted transmembrane domain (86 % identity with the S. mansoni tegumental antigen Sm13. The recombinant Sh13 protein was used in ELISA assays to determine the reactivity of sera from the study participants. Antibody responses against Sh13 were predominantly IgG3 isotype compared to responses against crude worm antigens which were predominantly IgG1 and IgG4. The relationship between anti-Sh13 IgG3 levels and infection intensity varied significantly with host age. The youngest children (7–10 years old had relatively low levels of both infection and anti-Sh13 IgG3. In older children (11–12 years old rising infection levels were accompanied by a significant increase in anti-Sh13 IgG3 levels. Subsequently, infection intensity declined significantly in 13–18 year olds but levels of the antibody continued to rise. The changing relationship between infection intensity and anti-Sh13 IgG3 levels with host age

  3. Human IgG antigranulocyte antibodies: comparison of detection by quantitative antiglobulin consumption and by binding of /sup 125/I staph protein A

    Energy Technology Data Exchange (ETDEWEB)

    Blumfelder, T.; Logue, G.

    1981-01-01

    The amount of IgG in the serum of patients with suspected immune neutropenia that binds to normal paraformaldehyde-fixed human granulocytes was measured simultaneously by a quantitative antiglobulin consumption assay and by binding of 125I-staphylococcal protein A (SPA). There was a significant linear relationship between the results of these two assays for the sera of 42 different patients. However, SPA binding appeared more sensitive than the quantitative antiglobulin assay for determining IgG antigranulocyte antibodies in serum. In a patient with Felty's syndrome who underwent splenectomy, the results of both assays on sequential serum samples correlated with clinical improvement. Thus, SPA binding appears to be a sensitive and reliable technique for measuring antigranulocyte antibodies, and there is a close correlation between antibody measured by antiglobulin consumption and those detected by SPA binding.

  4. Role of IgG4 in histamine release from human basophil leucocytes. I. Sensitization of cells from normal donors

    DEFF Research Database (Denmark)

    Poulsen, L K; Stahl Skov, P; Mosbech, H

    1988-01-01

    Several conflicting reports on the ability of IgG4 to mediate type I allergic reactions have appeared lately. We have developed a model system for testing this possibility, using passive sensitization of basophil leucocytes from normal individuals. At first, the system was optimized with regard t...

  5. Human transportation needs in rural Oklahoma.

    Science.gov (United States)

    2012-09-01

    Mobility is extremely important, especially in rural areas, which have dispersed populations and locations. : This study was conducted among rural minority populations to evaluate human transportation needs of the : underserved rural population in Ok...

  6. Modulation of Single-Cell IgG Secretion Frequency and Rates in Human Memory B Cells by CpG DNA, CD40L, IL-21 and Cell Division∥

    Science.gov (United States)

    Henn, Alicia D.; Rebhahn, Jonathan; Brown, Miguel A.; Murphy, Alison J.; Coca, Mircea N.; Hyrien, Ollivier; Pellegrin, Tina; Mosmann, Tim; Zand, Martin S.

    2009-01-01

    During the recall response by CD27+ IgG class switched human memory B cells, total IgG secreted is a function of (1) the number of IgG secreting cells (IgG-SC) and (2) the secretion rate of each cell. Here we report the quantitative ELISPOT method (qELISPOT) for simultaneous estimation of single cell IgG secretion rates and secreting cell frequencies in human B cell populations. We found that CD27+ IgMneg memory B cells activated with CpG and cytokines had considerable heterogeneity in the IgG secretion rates, with two major secretion rate subpopulations. B cell receptor cross-linking reduced the frequency of cells with high per-cell IgG secretion rates, with a parallel decrease in CD27hi B cell blasts. Increased cell death may account for the BCR-stimulated reduction in high-rate IgG-SC CD27hi B cell blasts. In contrast, the addition of IL-21 to CD40L +IL-4 activated human memory B cells induced a high-rate IgG-SC population in B cells with otherwise low per-cell IgG secretion rates. The profiles of human B cell IgG secretion rates followed the same biphasic distribution and range irrespective of division class. This, along with the presence of non-IgG-producing, dividing B cells in CpG+ck-activated B memory B cell populations, is suggestive of an “On/Off switch” regulating IgG secretion. Finally, these data support a mixture model of IgG secretion in which IgG secreted over time is modulated by the frequency of IgG secreting cells and the distribution of their IgG secretion rates. This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This version of the manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence, it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this

  7. Serological diagnostics in the detection of IgG autoantibodies against human collagen VII in epidermolysis bullosa acquisita: a multicentre analysis.

    Science.gov (United States)

    Schmidt, T; Hoch, M; Lotfi Jad, S S; Solimani, F; Di Zenzo, G; Marzano, A V; Goebeler, M; Cozzani, E; Kern, J S; Sitaru, C; Lakoš Jukić, I; Sárdy, M; Uzun, S; Jedlickova, H; Gläser, R; Kaneda, M; Eming, R; Göpel, G; Ishii, N; Greene, B; Hashimoto, T; Hertl, M

    2017-07-13

    Epidermolysis bullosa acquisita (EBA) is a rare, potentially devastating autoimmune disease of the skin. IgG autoantibodies directed against type VII collagen (Col7), the major component of anchoring fibrils, induce skin fragility leading to cutaneous and mucocutaneous blister formation, which is mostly of a scarring phenotype. Thus, powerful and reproducible diagnostic assays are critical to establish the diagnosis of EBA early to avoid irreversible sequelae. The present international, retrospective multicentre study included a large cohort of patients with EBA and evaluated the diagnostic power of four different diagnostic assays for the detection of anti-Col7 IgG autoantibodies. Overall, 95 EBA sera and 200 control sera consisting of 100 bullous pemphigoid sera, 50 pemphigus vulgaris sera and 50 sera of healthy controls were tested for anti-Col7 IgG autoantibodies using indirect immunofluorescence (IIF), two commercial enzyme-linked immunosorbent assay (ELISA) systems and Western blot (WB) analysis. EBA sera were taken from patients with positive direct immunofluorescence and IgG reactivity in at least one of the immunoserological assays (IIF, ELISA, WB). A Col7-NC1/NC2 ELISA (MBL, Nagoya, Japan) showed the highest sensitivity (97·9%), followed by a Col7-NC1 ELISA (Euroimmun, Lübeck, Germany) (89·5%), WB with Col7-NC1 (85·3%), and IIF on saline-split human skin (74·7%). The specificities of both ELISA systems were comparable (NC1 98·7%, NC1/NC2 99·3%). Furthermore, WB was more sensitive than IIF, which was more specific. The two commercially available ELISA systems allow for a highly sensitive and specific diagnosis of EBA. The sensitivity of the Col7-NC1/NC2 ELISA is significantly higher compared with the ELISA based on the Col7-NC1 domain only. © 2017 British Association of Dermatologists.

  8. Transfer of IgG in the female genital tract by MHC class I-related neonatal Fc receptor (FcRn) confers protective immunity to vaginal infection

    Science.gov (United States)

    Palaniyandi, Senthilkumar; Zeng, Rongyu; Tuo, Wenbin; Roopenian, Derry C.; Zhu, Xiaoping

    2011-01-01

    IgG is a major Ig subclass in mucosal secretions of the human female genital tract, where it predominates over the IgA isotype. Despite the abundance of IgG, surprisingly little is known about where and how IgG enters the lumen of the genital tract and the exact role local IgG plays in preventing sexually transmitted diseases. We demonstrate here that the neonatal Fc receptor, FcRn, is expressed in female genital tract epithelial cells of humans and mice and binds IgG in a pH-dependent manner. In vitro we show that FcRn mediates bidirectional IgG transport across polarized human endometrial HEC-1-A monolayers and primary human genital epithelial cells. Furthermore, endosomal acidification appears to be a prerequisite for FcRn-mediated IgG transcytosis; IgG transcytosis was demonstrated in vivo by translocation of systemically administered IgG into the genital lumen in WT but not FcRn-KO mice. The biological relevance of FcRn-transported IgG was demonstrated by passive immunization using herpes simplex virus-2 (HSV-2)–specific polyclonal serum, which conferred significantly higher protection against intravaginal challenge infection by the HSV-2 186 strain in WT mice than in FcRn-KO mice. These studies demonstrate that FcRn-mediated transport is a mechanism by which IgG can act locally in the female genital tract in immune surveillance and in host defense against sexually transmitted diseases. PMID:21368166

  9. IgG, IgA and IgM concentration in human sera from different age groups: statistical evaluation.

    Science.gov (United States)

    Quintiliani, L; Taggi, F; Giuliani, E; Buzzonetti, A; D'Amico, F

    1976-07-31

    A statistical analysis is presented of the results obtained from the measure of the serum concentration of IgG, A, M in 773 blood donors. In each of the two groups identified by sex, four subgroups were established according to age in ten years intervals from 20 to 59. On the basis of the results of the analysis, different fiducial intervals of normal values (calculated as mean +/- 2 X standard deviation) have been proposed. 1) A unique interval for IgG: 87-234 U.I. 2) Two intervals for IgA: 54-236 U.I. for subjects under 40 years and 64-270 U.I. for subjects over 40 years. 3) Four intervals for IgM: one for males under 40 years: 65-364 U.I., one for males over 40 years: 57-314 U.I., one for females under 50 years: 47-363 U.I., and one for females over 50 years: 53-434 U.I.

  10. Structural insights into the interaction of human IgG1 with FcγRI: no direct role of glycans in binding

    Energy Technology Data Exchange (ETDEWEB)

    Oganesyan, Vaheh, E-mail: oganesyanv@medimmune.com; Mazor, Yariv; Yang, Chunning; Cook, Kimberly E.; Woods, Robert M. [MedImmune LLC, 1 MedImmune Way, Gaithersburg, MD 20878 (United States); Ferguson, Andrew [AstraZeneca Pharmaceuticals, 35 Gatehouse Drive, Mailstop E3, Waltham, MA 02451 (United States); Bowen, Michael A.; Martin, Tom; Zhu, Jie; Wu, Herren; Dall’Acqua, William F., E-mail: oganesyanv@medimmune.com [MedImmune LLC, 1 MedImmune Way, Gaithersburg, MD 20878 (United States)

    2015-10-31

    In an effort to identify the critical structural features responsible for the high-affinity interaction of IgG1 Fc with FcγRI, the structure of the corresponding complex was solved at a resolution of 2.4 Å. The three-dimensional structure of a human IgG1 Fc fragment bound to wild-type human FcγRI is reported. The structure of the corresponding complex was solved at a resolution of 2.4 Å using molecular replacement; this is the highest resolution achieved for an unmutated FcγRI molecule. This study highlights the critical structural and functional role played by the second extracellular subdomain of FcγRI. It also explains the long-known major energetic contribution of the Fc ‘LLGG’ motif at positions 234–237, and particularly of Leu235, via a ‘lock-and-key’ mechanism. Finally, a previously held belief is corrected and a differing view is offered on the recently proposed direct role of Fc carbohydrates in the corresponding interaction. Structural evidence is provided that such glycan-related effects are strictly indirect.

  11. Study on interactions of human IgG with immobilized anti-IgG or recombinant Staphylococcal protein A using surface plasmon resonance spectrometry

    Directory of Open Access Journals (Sweden)

    Bakhmachuk A. O.

    2016-02-01

    Full Text Available Aim. Comparison of the IgG-binding activity of recombinant Staphylococcal protein A with introduced C-terminal cysteine residue (SPA-Cys or goat anti-human IgG antibodies (anti-IgG after their immobilization on a gold sensor surface of surface plasmon resonance (SPR spectrometer. Methods. SPA-Cys or anti-IgG were immobilized on a gold sensor surface to form two variants of a bioselective element of the immunosensor. SPR spectrometry was used for the detection of IgG-binding activity of the immobilized proteins. Results.The SPR sensor response to the immobilization of anti-IgG was more than two times higher than that at the immobilization of SPA-Cys. However, there is almost the double advantage for SPA-Cys in the number of immobilized molecules. Moreover, the bioselective element of the immunosensor based on SPA-Cys showed a much better capability of binding Ig than bioselective element based on anti-IgG. Conclusions.The study on the immobilization of SPA-Cys or anti-IgG on the sensor surface of SPR spectrometer, and the interactions of immobilized proteins with human IgG demonstrated obvious advantages of SPA-Cys.

  12. Human transporter database: comprehensive knowledge and discovery tools in the human transporter genes.

    Directory of Open Access Journals (Sweden)

    Adam Y Ye

    Full Text Available Transporters are essential in homeostatic exchange of endogenous and exogenous substances at the systematic, organic, cellular, and subcellular levels. Gene mutations of transporters are often related to pharmacogenetics traits. Recent developments in high throughput technologies on genomics, transcriptomics and proteomics allow in depth studies of transporter genes in normal cellular processes and diverse disease conditions. The flood of high throughput data have resulted in urgent need for an updated knowledgebase with curated, organized, and annotated human transporters in an easily accessible way. Using a pipeline with the combination of automated keywords query, sequence similarity search and manual curation on transporters, we collected 1,555 human non-redundant transporter genes to develop the Human Transporter Database (HTD (http://htd.cbi.pku.edu.cn. Based on the extensive annotations, global properties of the transporter genes were illustrated, such as expression patterns and polymorphisms in relationships with their ligands. We noted that the human transporters were enriched in many fundamental biological processes such as oxidative phosphorylation and cardiac muscle contraction, and significantly associated with Mendelian and complex diseases such as epilepsy and sudden infant death syndrome. Overall, HTD provides a well-organized interface to facilitate research communities to search detailed molecular and genetic information of transporters for development of personalized medicine.

  13. IgG Subclasses and Allotypes: From Structure to Effector Functions

    Science.gov (United States)

    Vidarsson, Gestur; Dekkers, Gillian; Rispens, Theo

    2014-01-01

    Of the five immunoglobulin isotypes, immunoglobulin G (IgG) is most abundant in human serum. The four subclasses, IgG1, IgG2, IgG3, and IgG4, which are highly conserved, differ in their constant region, particularly in their hinges and upper CH2 domains. These regions are involved in binding to both IgG-Fc receptors (FcγR) and C1q. As a result, the different subclasses have different effector functions, both in terms of triggering FcγR-expressing cells, resulting in phagocytosis or antibody-dependent cell-mediated cytotoxicity, and activating complement. The Fc-regions also contain a binding epitope for the neonatal Fc receptor (FcRn), responsible for the extended half-life, placental transport, and bidirectional transport of IgG to mucosal surfaces. However, FcRn is also expressed in myeloid cells, where it participates in both phagocytosis and antigen presentation together with classical FcγR and complement. How these properties, IgG-polymorphisms and post-translational modification of the antibodies in the form of glycosylation, affect IgG-function will be the focus of the current review. PMID:25368619

  14. Aquaporins Mediate Silicon Transport in Humans.

    Science.gov (United States)

    Garneau, Alexandre P; Carpentier, Gabriel A; Marcoux, Andrée-Anne; Frenette-Cotton, Rachelle; Simard, Charles F; Rémus-Borel, Wilfried; Caron, Luc; Jacob-Wagner, Mariève; Noël, Micheline; Powell, Jonathan J; Bélanger, Richard; Côté, François; Isenring, Paul

    2015-01-01

    In animals, silicon is an abundant and differentially distributed trace element that is believed to play important biological functions. One would thus expect silicon concentrations in body fluids to be regulated by silicon transporters at the surface of many cell types. Curiously, however, and even though they exist in plants and algae, no such transporters have been identified to date in vertebrates. Here, we show for the first time that the human aquaglyceroporins, i.e., AQP3, AQP7, AQP9 and AQP10 can act as silicon transporters in both Xenopus laevis oocytes and HEK-293 cells. In particular, heterologously expressed AQP7, AQP9 and AQP10 are all able to induce robust, saturable, phloretin-sensitive silicon transport activity in the range that was observed for low silicon rice 1 (lsi1), a silicon transporter in plant. Furthermore, we show that the aquaglyceroporins appear as relevant silicon permeation pathways in both mice and humans based on 1) the kinetics of substrate transport, 2) their presence in tissues where silicon is presumed to play key roles and 3) their transcriptional responses to changes in dietary silicon. Taken together, our data provide new evidence that silicon is a potentially important biological element in animals and that its body distribution is regulated. They should open up original areas of investigations aimed at deciphering the true physiological role of silicon in vertebrates.

  15. Aquaporins Mediate Silicon Transport in Humans.

    Directory of Open Access Journals (Sweden)

    Alexandre P Garneau

    Full Text Available In animals, silicon is an abundant and differentially distributed trace element that is believed to play important biological functions. One would thus expect silicon concentrations in body fluids to be regulated by silicon transporters at the surface of many cell types. Curiously, however, and even though they exist in plants and algae, no such transporters have been identified to date in vertebrates. Here, we show for the first time that the human aquaglyceroporins, i.e., AQP3, AQP7, AQP9 and AQP10 can act as silicon transporters in both Xenopus laevis oocytes and HEK-293 cells. In particular, heterologously expressed AQP7, AQP9 and AQP10 are all able to induce robust, saturable, phloretin-sensitive silicon transport activity in the range that was observed for low silicon rice 1 (lsi1, a silicon transporter in plant. Furthermore, we show that the aquaglyceroporins appear as relevant silicon permeation pathways in both mice and humans based on 1 the kinetics of substrate transport, 2 their presence in tissues where silicon is presumed to play key roles and 3 their transcriptional responses to changes in dietary silicon. Taken together, our data provide new evidence that silicon is a potentially important biological element in animals and that its body distribution is regulated. They should open up original areas of investigations aimed at deciphering the true physiological role of silicon in vertebrates.

  16. Structural Insights into Complexes of Glucose-Regulated Protein94 (Grp94) with Human Immunoglobulin G. Relevance for Grp94-IgG Complexes that Form In Vivo in Pathological Conditions

    Science.gov (United States)

    Pagetta, Andrea; Tramentozzi, Elisa; Tibaldi, Elena; Cendron, Laura; Zanotti, Giuseppe; Brunati, Anna Maria; Vitadello, Maurizio; Gorza, Luisa; Finotti, Paola

    2014-01-01

    While the mechanism by which Grp94 displays its chaperone function with client peptides in the cell has been elucidated extensively, much less is known about the nature and properties of how Grp94 can engage binding to proteins once it is exposed on the cell surface or liberated in the extra-cellular milieu, as occurs in pathological conditions. In this work, we wanted to investigate the molecular aspects and structural characteristics of complexes that Grp94 forms with human IgG, posing the attention on the influence that glycosylation of Grp94 might have on the binding capacity to IgG, and on the identification of sites involved in the binding. To this aim, we employed both native, fully glycosylated and partially glycosylated Grp94, and recombinant, non-glycosylated Grp94, as well as IgG subunits, in different experimental conditions, including the physiological setting of human plasma. Regardless of the species and type, Grp94 engages a similar, highly specific and stable binding with IgG that involves sites located in the N-terminal domain of Grp94 and the hinge region of whole IgG. Grp94 does not form stable complex with Fab, F(ab)2 or Fc. Glycosylation turns out to be an obstacle to the Grp94 binding to IgG, although this negative effect can be counteracted by ATP and spontaneously also disappears in time in a physiological setting of incubation. ATP does not affect at all the binding capacity of non-glycosylated Grp94. However, complexes that native, partially glycosylated Grp94 forms with IgG in the presence of ATP show strikingly different characteristics with respect to those formed in absence of ATP. Results have relevance for the mechanism regulating the formation of stable Grp94-IgG complexes in vivo, in the pathological conditions associated with the extra-cellular location of Grp94. PMID:24489700

  17. Recombinant human IgG antibodies recognizing distinct extracellular domains of EGF receptor exhibit different degrees of growth inhibitory effects on human A431 cancer cells.

    Science.gov (United States)

    Chang, Chialun; Takayanagi, Atsushi; Yoshida, Tetsuhiko; Shimizu, Nobuyoshi

    2013-05-01

    Recently, we isolated 4 distinct kinds of single chain antibody against human EGF receptor (EGFR) after screening the Keio phage display scFv library by using two methods of target-guided proximity labeling. In the current study, these monovalent scFv antibodies were converted to bivalent IgGs of humanized forms (hIgGs) by recombinant technology using the specially designed expression vectors followed by protein production in CHO cells. The resulting recombinant hIgGs were examined for their binding specificity using several different transformed human BJ cell lines that express deletion mutants of EGFR, each lacking one of 4 distinct extracellular domains (L1, L2, C1 and C2). Immuno-fluorescent microscopy and immuno-precipitation assay on these cells indicated that 4 distinct kinds of hIgGs bind to one of 3 different domains (L1, C1 and C2). Then, these hIgGs were further examined for biological effects on human A431 cancer cells, which overexpress EGFR. The results indicated that hIgG38 binding to L1 and hIgG45 binding to C2 substantially suppressed the EGF-induced phosphorylation of EGFR, resulting in the growth inhibition of A431 cancer cells. On the contrary, hIgG40 binding to C1 and hIgG42 binding to another site (epitope) of C2 exhibited no such inhibitory effects. Thus, the newly produced four recombinant hIgG antibodies recognize 4 different sites (epitopes) in 3 different extracellular domains of EGFR and exhibit different biological effects on cancer cells. These characteristics are somewhat different from the currently utilized therapeutic anti-EGFR antibodies. Hence, these hIgG antibodies will be invaluable as a research tool for the detailed molecular analysis of the EGFR-mediated signal transduction mechanism and more importantly a possible application as new therapeutic agents to treat certain types of cancers. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Human proton/oligopeptide transporter (POT) genes

    DEFF Research Database (Denmark)

    Botka, C. W.; Wittig, T. W.; Graul, R. C.

    2000-01-01

    The proton-dependent oligopeptide transporters (POT) gene family currently consists of approximately 70 cloned cDNAs derived from diverse organisms. In mammals, two genes encoding peptide transporters, PepT1 and PepT2 have been cloned in several species including humans, in addition to a rat...... human orthologue of rPHT1 (expression largely confined to rat brain and retina) was represented by numerous ESTs originating from many tissues. Assembly of these ESTs resulted in a contiguous sequence covering approximately 95% of the suspected coding region. The contig sequences and analyses revealed...... the presence of several possible splice variants of hPHT1. A second closely related human EST-contig displayed high identity to a recently cloned mouse cDNA encoding cyclic adenosine monophosphate (cAMP)-inducible 1 protein (gi:4580995). This contig served to identify a PAC clone containing deduced exons...

  19. Overview: ABC transporters and human disease.

    Science.gov (United States)

    Gottesman, M M; Ambudkar, S V

    2001-12-01

    ABC transporters are found in all known organisms, and approximately 1,100 different transporters belonging to this family have been described in the literature. The family is defined by homology within the ATP-binding cassette (ABC) region, which extends outside of the more typical Walker motifs found in all ATP-binding proteins. Most family members also contain transmembrane domains involved in recognition of substrates, which are transported across, into, and out of cell membranes, but some members utilize ABCs as engines to regulate ion channels. There are approximately 50 known ABC transporters in the human, and there are currently 13 genetic diseases associated with defects in 14 of these transporters. The most common genetic disease conditions include cystic fibrosis, Stargardt disease, age-related macular degeneration, adrenoleukodystrophy, Tangier disease, Dubin-Johnson syndrome and progressive familial intrahepatic cholestasis. At least 8 members of this family are involved in the transport of a variety of amphipathic compounds, including anticancer drugs, and some appear to contribute to the resistance of cancer cells to chemotherapy.

  20. The effects of selected drugs, including chlorpromazine and non-steroidal anti-inflammatory agents, on polyclonal IgG synthesis and interleukin 1 production by human peripheral blood mononuclear cells in vitro.

    Science.gov (United States)

    Martinez, F; Coleman, J W

    1989-01-01

    We tested a range of drugs for their effects on in vitro polyclonal IgG synthesis by human peripheral blood mononuclear cells (PBMC) stimulated with the lectin pokeweed mitogen (PWM). The test drugs were selected on the basis of reported disruptive effects on immune function in vivo. IgG production between day 4 and days 7 or 8 of culture was measured by biotin-streptavidin sandwich ELISA. The anti-psychotic agent chlorpromazine (0.55-1.7 microM) enhanced IgG synthesis to approximately double control levels. In contrast, the non-steroidal anti-inflammatory drugs (NSAIDs) indomethacin, piroxicam, ibuprofen and aspirin inhibited IgG synthesis by up to 50%, with a rank order of potency that reflects their activity as inhibitors of cyclo-oxygenase. Phenytoin, procainamide, propylthiouracil, methimazole, D-penicillamine and D-penicillamine-L-cysteine all failed to modulate IgG synthesis at non-toxic concentrations. The potentiation and inhibition of IgG synthesis by chlorpromazine and indomethacin, respectively, was observed only when the drug was present during the first 24 h of culture. Neither chlorpromazine nor indomethacin, at non-toxic concentrations, affected PHA- and PWM-stimulated proliferation of PBMC. In addition, chlorpromazine, indomethacin and piroxicam, at concentrations which produced maximal modulation of IgG synthesis, and D-penicillamine and D-penicillamine-L-cysteine at 10 microM failed to influence production of interleukin-1-like activity. We conclude that chlorpromazine and NSAIDs, although they exert opposite effects on IgG synthesis, act at an early stage of B cell differentiation that appears to be independent of interleukin 1 synthesis and early proliferative events. PMID:2788047

  1. Synthetic study on cystinyl peptides using solution and solid phase methodology: human IgG1 hinge region.

    Science.gov (United States)

    Niederhafner, P; Gut, V; Jezek, J; Budesínský, M; Kasicka, V; Wünsch, E; Hlavácek, J

    2010-08-01

    Synthetic study on cystinyl peptides using solution and solid phase methodology was carried out with the central hinge region of immunoglobulin IgG1. In the solid phase synthesis of hexadecapeptide 1c, the time necessary for the formation of disulfide bonds between linear precursors was shortened four times by the action of pure oxygen in buffered solution, in comparison with air oxidation. The product was thus obtained devoid of impurities from side reactions. In the preparation of the shortened bis-cystinyl analogs 2k and 3d of the natural hexadecapeptide 1c, both the classical and polyethylene glycol (PEG6000) solution methods were utilized using a disulfide synthon (Boc-Cys-OPfp)2 to obtain peptide chains in a natural parallel alignment. In the PEG6000 strategy, lysine as a linker on both sides of the polymer was attached to enhance the loading capacity. The leucine residue, instead of proline one, was introduced to the carboxy terminus to facilitate a specific enzymatic cleavage of the peptides from PEG6000 by thermolysine.

  2. Evaluation of IgG4 subclass antibody detection by peptide-based ELISA for the diagnosis of human paragonimiasis heterotrema.

    Science.gov (United States)

    Intapan, Pewpan M; Sanpool, Oranuch; Janwan, Penchom; Laummaunwai, Porntip; Morakote, Nimit; Kong, Yoon; Maleewong, Wanchai

    2013-12-01

    A synthetic peptide was prepared based on the antigenic region of Paragonimus westermani pre-procathepsin L, and its applicability for immunodiagnosis for human paragonimiasis (due to Paragonimus heterotremus) was tested using an ELISA to detect IgG4 antibodies in the sera of patients. Sera from other helminthiases, tuberculosis, and healthy volunteers were used as the references. This peptide-based assay system gave sensitivity, specificity, accuracy, and positive and negative predictive values of 100%, 94.6%, 96.2%, 100%, and 88.9%, respectively. Cross reactivity was frequently seen against the sera of fascioliasis (75%) and hookworm infections (50%). Since differential diagnosis between paragonimiasis and fascioliasis can be easily done by clinical presentation and fascioliasis serology, this cross reaction is not a serious problem. Sera from patients with other parasitoses (0-25%) rarely responded to this synthetic antigen. This synthetic peptide antigen seems to be useful for development of a standardized diagnostic system for paragonimiasis.

  3. Survival and digestibility of orally-administered immunoglobulin preparations containing IgG through the gastrointestinal tract in humans

    National Research Council Canada - National Science Library

    Jasion, Victoria S; Burnett, Bruce P

    2015-01-01

    .... Oral administration of Ig preparations from human serum as well as bovine colostrum and serum have been tested and proven to be safe as well as effective in human clinical trials for a variety...

  4. Sociodemographic factors associated with IgG and IgM seroprevalence for human cytomegalovirus infection in adult populations of Pakistan: a seroprevalence survey

    Directory of Open Access Journals (Sweden)

    Saira Ibrahim

    2016-10-01

    Full Text Available Abstract Background The seroprevalence of human cytomegalovirus (HCMV infection ranges from 30 to 90 % in developed countries. Reliable estimates of HCMV seroprevalence are not available for Pakistan. This study determined the seroprevalence and sociodemographic factors associated with HCMV infection in adult populations of Karachi, Pakistan. Methods A seroprevalence survey was conducted on 1000 adults, including residents of two semi-urban communities, and visitors to a government and a private hospital. Questionnaire-based interviews were conducted. Sera were analysed for HCMV-specific IgG and IgM. Chi-square or Fisher’s exact test was used for comparing sociodemographic variables against seropositivity of HCMV-IgG or IgM. Multiple logistic regression modeling was performed for IgG seroprevalence and adjusted odds ratios were computed. Results The seroprevalence of HCMV-IgG and IgM was 93.2 and 4.3 % respectively. 95.3 % of individuals who were IgM seropositive were also seropositive for IgG. Around 6 % (15/250 of women of childbearing age remained uninfected and were therefore susceptible to primary infection. HCMV-IgG seroprevalence was associated with being female (p = 0.001, increasing age (p = 0.002 and crowding index (p = 0.003 and also with lower levels of both education (p < 0.001 and income (p = 0.008. Seroprevalence also differed significantly by marital status (p = 0.008 and sampling location (p < 0.001. A logistic regression model for HCMV-IgG seroprevalence showed associations with being female (OR = 1.89; 95 % CI: 1.10–3.25, increasing age (OR = 3.95; 95 % CI: 1.79–8.71 and decreasing income (OR = 0.72; 95 % CI: 0.54–0.96. A strong association was observed between increased seroprevalence of HCMV-IgM and decreasing household size (p = 0.008. Conclusions Seroprevalence of HCMV is very high in Pakistan, although 6 % of women of childbearing age remain at risk of primary

  5. Sociodemographic factors associated with IgG and IgM seroprevalence for human cytomegalovirus infection in adult populations of Pakistan: a seroprevalence survey.

    Science.gov (United States)

    Ibrahim, Saira; Siddiqui, Anwar A; Siddiqui, Amna R; Ahmed, Waquaruddin; Moss, Paul A H; Lalani, El-Nasir M A

    2016-10-22

    The seroprevalence of human cytomegalovirus (HCMV) infection ranges from 30 to 90 % in developed countries. Reliable estimates of HCMV seroprevalence are not available for Pakistan. This study determined the seroprevalence and sociodemographic factors associated with HCMV infection in adult populations of Karachi, Pakistan. A seroprevalence survey was conducted on 1000 adults, including residents of two semi-urban communities, and visitors to a government and a private hospital. Questionnaire-based interviews were conducted. Sera were analysed for HCMV-specific IgG and IgM. Chi-square or Fisher's exact test was used for comparing sociodemographic variables against seropositivity of HCMV-IgG or IgM. Multiple logistic regression modeling was performed for IgG seroprevalence and adjusted odds ratios were computed. The seroprevalence of HCMV-IgG and IgM was 93.2 and 4.3 % respectively. 95.3 % of individuals who were IgM seropositive were also seropositive for IgG. Around 6 % (15/250) of women of childbearing age remained uninfected and were therefore susceptible to primary infection. HCMV-IgG seroprevalence was associated with being female (p = 0.001), increasing age (p = 0.002) and crowding index (p = 0.003) and also with lower levels of both education (p < 0.001) and income (p = 0.008). Seroprevalence also differed significantly by marital status (p = 0.008) and sampling location (p < 0.001). A logistic regression model for HCMV-IgG seroprevalence showed associations with being female (OR = 1.89; 95 % CI: 1.10-3.25), increasing age (OR = 3.95; 95 % CI: 1.79-8.71) and decreasing income (OR = 0.72; 95 % CI: 0.54-0.96). A strong association was observed between increased seroprevalence of HCMV-IgM and decreasing household size (p = 0.008). Seroprevalence of HCMV is very high in Pakistan, although 6 % of women of childbearing age remain at risk of primary infection. The IgM seropositivity observed in some individuals

  6. IgG abnormality in narcolepsy and idiopathic hypersomnia.

    Directory of Open Access Journals (Sweden)

    Susumu Tanaka

    Full Text Available BACKGROUND: A close association between narcolepsy and the Human Leukocyte Antigen (HLA-DQB1*0602 allele suggests the involvement of the immune system, or possibly an autoimmune process. We investigated serum IgG levels in narcolepsy. METHODOLOGY/PRINCIPAL FINDINGS: We measured the serum total IgG levels in 159 Japanese narcolepsy-cataplexy patients positive for the HLA-DQB1*0602 allele, 28 idiopathic hypersomnia patients with long sleep time, and 123 healthy controls (the HLA-DQB1*0602 allele present in 45 subjects. The serum levels of each IgG subclass were subsequently measured. The distribution of serum IgG was significantly different among healthy controls negative for the HLA-DQB1*0602 allele (11.66+/-3.55 mg/ml, healthy controls positive for the HLA-DQB1*0602 allele (11.45+/-3.43, narcolepsy patients (9.67+/-3.38, and idiopathic hypersomnia patients (13.81+/-3.80. None of the following clinical variables, age, disease duration, Epworth Sleepiness Scale, smoking habit and BMI at the time of blood sampling, were associated with IgG levels in narcolepsy or idiopathic hypersomnia. Furthermore we found the decrease in IgG1 and IgG2 levels, stable expression of IgG3, and the increase in the proportion of IgG4 in narcolepsy patients with abnormally low IgG levels. The increase in the proportion of IgG4 levels was also found in narcolepsy patients with normal serum total IgG levels. Idiopathic hypersomnia patients showed a different pattern of IgG subclass distribution with high IgG3 and IgG4 level, low IgG2 level, and IgG1/IgG2 imbalance. CONCLUSIONS/SIGNIFICANCE: Our study is the first to determine IgG abnormalities in narcolepsy and idiopathic hypersomnia by measuring the serum IgG levels in a large number of hypersomnia patients. The observed IgG abnormalities indicate humoral immune alterations in narcolepsy and idiopathic hypersomnia. Different IgG profiles suggest immunological differences between narcolepsy and idiopathic hypersomnia.

  7. Transport of human adenoviruses in porous media

    Science.gov (United States)

    Kokkinos, Petros; Syngouna, Vasiliki I.; Tselepi, Maria A.; Bellou, Maria; Chrysikopoulos, Constantinos V.; Vantarakis, Apostolos

    2015-04-01

    Groundwater may be contaminated with infective human enteric viruses from various wastewater discharges, sanitary landfills, septic tanks, agricultural practices, and artificial groundwater recharge. Coliphages have been widely used as surrogates of enteric viruses, because they share many fundamental properties and features. Although a large number of studies focusing on various factors (i.e. pore water solution chemistry, fluid velocity, moisture content, temperature, and grain size) that affect biocolloid (bacteria, viruses) transport have been published over the past two decades, little attention has been given toward human adenoviruses (hAdVs). The main objective of this study was to evaluate the effect of pore water velocity on hAdV transport in water saturated laboratory-scale columns packed with glass beads. The effects of pore water velocity on virus transport and retention in porous media was examined at three pore water velocities (0.39, 0.75, and 1.22 cm/min). The results indicated that all estimated average mass recovery values for hAdV were lower than those of coliphages, which were previously reported in the literature by others for experiments conducted under similar experimental conditions. However, no obvious relationship between hAdV mass recovery and water velocity could be established from the experimental results. The collision efficiencies were quantified using the classical colloid filtration theory. Average collision efficiency, α, values decreased with decreasing flow rate, Q, and pore water velocity, U, but no significant effect of U on α was observed. Furthermore, the surface properties of viruses and glass beads were used to construct classical DLVO potential energy profiles. The results revealed that the experimental conditions of this study were unfavorable to deposition and that no aggregation between virus particles is expected to occur. A thorough understanding of the key processes governing virus transport is pivotal for public

  8. Rabbit IgG directed to a synthetic C-terminal peptide of the major grass pollen allergen Lol p I inhibits human basophil histamine release induced by natural Lol p I

    NARCIS (Netherlands)

    van Ree, R.; Aalberse, R. C.

    1995-01-01

    The potential role of allergen-specific IgG antibodies as 'blocking' antibodies in allergen-induced human basophil histamine release was investigated. This was studied in a model with the major grass pollen allergen Lol p I and polyclonal rabbit antisera directed against this allergen and against a

  9. A new human IgG avidity test, using mixtures of recombinant antigens (rROP1, rSAG2, rGRA6), for the diagnosis of difficult-to-identify phases of toxoplasmosis.

    Science.gov (United States)

    Drapała, Dorota; Holec-Gąsior, Lucyna; Kur, Józef; Ferra, Bartłomiej; Hiszczyńska-Sawicka, Elżbieta; Lautenbach, Dariusz

    2014-07-01

    The preliminary diagnostic utility of two mixtures of Toxoplasma gondii recombinant antigens (rROP1+rSAG2 and rROP1+rGRA6) in IgG ELISA and IgG avidity test has been evaluated. A total of 173 serum samples from patients with toxoplasmosis and seronegative people were examined. The sensitivity of IgG ELISA for rROP1+rSAG2 and rROP1+rGRA6 was 91.1% and 76.7%, respectively, while the reactivity for sera from patients where acute toxoplasmosis was suspected was higher, at 100% and 95.4%, respectively, than for people with chronic infection, at 88.2% and 70.6%. In this study a different trend in avidity maturation of IgG antibodies for two mixtures of proteins in comparison with native antigen was observed. The results suggest that a new IgG avidity test using the mixtures of recombinant antigens may be useful for the diagnosis of difficult-to-identify phases of toxoplasmosis. For this reason, selected mixtures after the additional tests on groups of sera with well-defined dates of infection could be used as a better alternative to the native antigens of the parasite in the serodiagnosis of human T. gondii infection. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Pathogenetics of the human SLC26 transporters.

    Science.gov (United States)

    Dawson, P A; Markovich, D

    2005-01-01

    Over the past decade, 11 human genes belonging to the solute linked carrier (SLC) 26 family of transporters, have been identified. The SLC26 proteins, which include SAT-1, DTDST, DRA/CLD, pendrin, prestin, PAT-1/CFEX and Tat-1, are structurally related and have been shown to transport one or more of the following substrates: sulfate, chloride, bicarbonate, iodide, oxalate, formate, hydroxyl or fructose. Special interest has focused on four members of the SLC26 family that are associated with distinct recessive diseases: (i) Mutations in SLC26A2 lead to four different chondrodysplasias (diastrophic dysplasia, atelosteogenesis type II, achondrogenesis type IB and multiple epiphyseal dysplasia); (ii) SLC26A3 is associated with congenital chloride diarrhea; (iii) SLC26A4 is associated with Pendred syndrome and non-syndromic deafness, DFNB4; and (iv) SLC26A5 is defective in non-syndromic hearing impairment. This review article summarizes current information on the pathophysiological consequences of mutations in the human SLC26A2 to A5 genes.

  11. In vitro and in vivo properties of a fully human IgG1 monoclonal antibody that combats multidrug resistant Pseudomonas aeruginosa

    Science.gov (United States)

    ADAWI, AZMI; BISIGNANO, CARLO; GENOVESE, TIZIANA; FILOCAMO, ANGELA; KHOURI-ASSI, CAMELLIA; NEVILLE, ANAT; FEUERSTEIN, GIORA Z.; CUZZOCREA, SALVATORE; NEVILLE, LEWIS F.

    2012-01-01

    The development of an anti-bacterial drug in the form of a monoclonal antibody (mAb) targeting an exposed virulence factor, represents an innovative therapeutic strategy. Consequently, a fully human IgG1 mAb (LST-007) targeting Pseudomonas aeruginosa (PA) flagellin type b was recombinantly expressed and characterized in vitro and in an infection model driven by a multidrug resistant (MDR) PA strain. LST-007 demonstrated a highly specific binding towards whole PA bacteria harboring flagellin type b and its recombinant counterpart, with a KD of 7.4×10−10 M. In bioactivity assays, LST-007 or titers of Cmax sera derived from pharmacokinetic studies, markedly attenuated PA motility in an equipotent manner. In vivo, parenteral LST-007 (20 mg/kg) given as a single or double-dosing paradigm post-infection, afforded survival (up to 75% at Day 7) in a lethal model of pneumonia driven by the intratracheal (i.t.) instillation of an LD80 of the MDR PA isolate. This protective effect was markedly superior to that of imipenem (30% survival at Day 7) and totally devoid with an irrelevant, human isotype mAb. These data lay credence that LST-007 may be a valuable adjunct to the limited list of anti-bacterials that can tackle MDR PA strains, thereby warranting its continued development for eventual clinical evaluation. PMID:22735858

  12. Human anti-Aβ IgGs target conformational epitopes on synthetic dimer assemblies and the AD brain-derived peptide.

    Science.gov (United States)

    Welzel, Alfred T; Williams, Angela D; McWilliams-Koeppen, Helen P; Acero, Luis; Weber, Alfred; Blinder, Veronika; Mably, Alex; Bunk, Sebastian; Hermann, Corinna; Farrell, Michael A; Ehrlich, Hartmut J; Schwarz, Hans P; Walsh, Dominic M; Solomon, Alan; O'Nuallain, Brian

    2012-01-01

    Soluble non-fibrillar assemblies of amyloid-beta (Aβ) and aggregated tau protein are the proximate synaptotoxic species associated with Alzheimer's disease (AD). Anti-Aβ immunotherapy is a promising and advanced therapeutic strategy, but the precise Aβ species to target is not yet known. Previously, we and others have shown that natural human IgGs (NAbs) target diverse Aβ conformers and have therapeutic potential. We now demonstrate that these antibodies bound with nM avidity to conformational epitopes on plate-immobilized synthetic Aβ dimer assemblies, including synaptotoxic protofibrils, and targeted these conformers in solution. Importantly, NAbs also recognized Aβ extracted from the water-soluble phase of human AD brain, including species that migrated on denaturing PAGE as SDS-stable dimers. The critical reliance on Aβ's conformational state for NAb binding, and not a linear sequence epitope, was confirmed by the antibody's nM reactivity with plate-immobilized protofibrills, and weak uM binding to synthetic Aβ monomers and peptide fragments. The antibody's lack of reactivity against a linear sequence epitope was confirmed by our ability to isolate anti-Aβ NAbs from intravenous immunoglobulin using affinity matrices, immunoglobulin light chain fibrils and Cibacron blue, which had no sequence similarity with the peptide. These findings suggest that further investigations on the molecular basis and the therapeutic/diagnostic potential of anti-Aβ NAbs are warranted.

  13. IMC-A12, a human IgG1 monoclonal antibody to the insulin-like growth factor I receptor.

    Science.gov (United States)

    Rowinsky, Eric K; Youssoufian, Hagop; Tonra, James R; Solomon, Phillip; Burtrum, Douglas; Ludwig, Dale L

    2007-09-15

    Targeted monoclonal antibody therapy is an important strategy in cancer therapeutics. Among the most promising characteristics of therapeutic targets are those that modulate the growth and survival of malignant neoplasms and their sensitivity to anticancer therapies. The insulin-like growth factor-I receptor (IGF-IR) is overexpressed in many types of solid and hematopoietic malignancies, and has been implicated as a principal cause of heightened proliferative and survival signaling. IGF-IR has also been shown to confer resistance to cytotoxic, hormonal, and targeted therapies, suggesting that therapeutics targeting IGF-IR may be effective against a broad range of malignancies. IMC-A12 (ImClone Systems Incorporated), a fully human monoclonal IgG1 antibody that binds with high affinity to the IGF-IR, inhibits ligand-dependent receptor activation and downstream signaling. IMC-A12 also mediates robust internalization and degradation of the IGF-IR. In human tumor xenograft models, IGF-IR blockade by IMC-A12 results in rapid and profound growth inhibition of cancers of the breast, lung, colon, and pancreas, and many other neoplasms. Although promising single-agent activity has been observed, the most impressive effects of targeting the IGF-IR with IMC-A12 have been noted when this agent was combined with cytotoxic agents or other targeted therapeutics. The results with IMC-A12 to date suggest that it may be an effective therapeutic in a diverse array of oncologic indications.

  14. High-yield production of a human monoclonal IgG by rhizosecretion in hydroponic tobacco cultures

    NARCIS (Netherlands)

    Madeira, L.M.; Szeto, T.H.; Henquet, Maurice; Raven, Nicole; Runions, John; Huddleston, Jon; Garrard, Ian; Drake, P.M.W.; Ma, Julian K.C.

    2016-01-01

    Rhizosecretion of recombinant pharmaceuticals from in vitro hydroponic transgenic plant cultures is a simple, low cost, reproducible and controllable production method. Here, we demonstrate the application and adaptation of this manufacturing platform to a human antivitronectin IgG1

  15. Human IgG subclass antibodies to the 19 kilodalton carboxy terminal fragment of Plasmodium falciparum merozoite surface protein 1 (MSP1(19)) and predominance of the MAD20 allelic type of MSP1 in Uganda.

    Science.gov (United States)

    Apio, B; Nalunkuma, A; Okello, D; Riley, E; Egwang, T G

    2000-04-01

    To determine the natural human humoral immune responses to the 19 kilodalton carboxy terminal fragment of Plasmodium falciparum merozoite surface protein 1 (MSP1(19)), a malaria candidate vaccine antigen and to determine the prevalence of MAD20 and K1 alleles of P. falciparum MSP1. Community based cross-sectional study. Atopi Parish, Apac District, Uganda, 1995. Three hundred and seventy four Ugandans between <1 and 70 years old provided serum samples. IgG subclass antibodies by ELISA; MAD20 and K1 allelic types of MSP1 by PCR. Both the prevalence and the mean concentration of serum IgG1, and to a lesser extent IgG3, antibodies increased with age. IgG2 or IgG4 antibodies were virtually nonexistent. The cross-reactivity between the 4 sequence variants (E-KNG, E-TSR, Q-KNG and Q-TSR) of MSP1(19) was confirmed; however, a minority of sera preferentially recognised the KNG but not the TSR variants. All 33 P. falciparum isolates from different parts of Uganda carried the E-TSR (Mad20) allelic type and 3 isolates were mixed infections with E-TSR (MAD20) and Q-KNG (K1) allelic types, confirming the rarity of the K1 allele in Uganda. There is a robust IgG1 antibody response to the malaria vaccine candidate antigen MSP1(19) which begins at an early age. Future cohort studies are necessary to estblish the impact of these antibodies on clinical immunity to malaria. The MAD20 allelic type of MSP1 id predominant in Ugandan P. falciparum isolates.

  16. Modeling Oxygen Transport in the Human Placenta

    Science.gov (United States)

    Serov, Alexander; Filoche, Marcel; Salafia, Carolyn; Grebenkov, Denis

    Efficient functioning of the human placenta is crucial for the favorable pregnancy outcome. We construct a 3D model of oxygen transport in the placenta based on its histological cross-sections. The model accounts for both diffusion and convention of oxygen in the intervillous space and allows one to estimate oxygen uptake of a placentone. We demonstrate the existence of an optimal villi density maximizing the uptake and explain it as a trade-off between the incoming oxygen flow and the absorbing villous surface. Calculations performed for arbitrary shapes of fetal villi show that only two geometrical characteristics - villi density and the effective villi radius - are required to predict fetal oxygen uptake. Two combinations of physiological parameters that determine oxygen uptake are also identified: maximal oxygen inflow of a placentone and the Damköhler number. An automatic image analysis method is developed and applied to 22 healthy placental cross-sections demonstrating that villi density of a healthy human placenta lies within 10% of the optimal value, while overall geometry efficiency is rather low (around 30-40%). In a perspective, the model can constitute the base of a reliable tool of post partum oxygen exchange efficiency assessment in the human placenta. Also affiliated with Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA 90095, USA.

  17. Developmental Toxicity and Fertility Assessment in Rabbits with Tabalumab: A Human IgG4 Monoclonal Antibody.

    Science.gov (United States)

    Breslin, William J; Hilbish, Kim G; Martin, Jennifer A; Halstead, Carolyn A; Edwards, Tammy L

    2015-06-01

    Tabalumab is a human immunoglobulin G subclass 4 monoclonal antibody that has been under development for autoimmune disorders. Tabalumab has full neutralizing activity against both soluble and membrane B-cell activating factor, a B-cell survival factor. The objectives of these studies were to assess the effects of tabalumab on embryo-fetal development and on male (M) and female (F) fertility in rabbits, a pharmacologically relevant species. Doses were administered at 0 (vehicle control), 0.3 (embryo-fetal study only), 1.0, and 30 mg/kg. In the embryo-fetal study, pregnant rabbits does were given a single dose by intravenous injection on gestation day (GD) 7. In the fertility studies, tabalumab was administered by intravenous injection every 7 days starting 2 (F) or 4 (M) weeks before mating, during cohabitation, and until necropsy (M) or through GD 18 (F). Treated animals were mated with untreated partners. Parental clinical signs, body weight, food consumption, blood lymphocyte phenotyping, organ weights, morphologic pathology, ovarian and uterine observations, sperm parameters, and fertility indices were evaluated along with conceptus viability, weight, and morphology. Exposure assessments were made in all main study animals and satellite animals. No adverse parental, reproductive, or developmental effects were observed in any study at any dose. A pharmacodynamic response consisting of dose-dependent decreases in the percent and number of total B lymphocytes and increases in the percent and/or number of total T lymphocytes was observed in parental rabbits at 1.0 and 30 mg/kg. In conclusion, no adverse reproductive or developmental effects were observed in rabbits following exposure to tabalumab at doses as high as 30 mg/kg and exposures at least 14-fold greater than human exposure levels. © 2015 Wiley Periodicals, Inc.

  18. Development of a complete human IgG monoclonal antibody to transferrin receptor 1 targeted for adult T-cell leukemia/lymphoma.

    Science.gov (United States)

    Shimosaki, Shunsuke; Nakahata, Shingo; Ichikawa, Tomonaga; Kitanaka, Akira; Kameda, Takuro; Hidaka, Tomonori; Kubuki, Yoko; Kurosawa, Gene; Zhang, Lilin; Sudo, Yukio; Shimoda, Kazuya; Morishita, Kazuhiro

    2017-03-25

    Iron is an essential nutrient for normal cell growth, and reprogramming of iron metabolism is essential to tumor cell survival and progression. HTLV-1-associated adult T-cell leukemia/lymphoma (ATLL) has no effective therapy and high levels of cell surface transferrin receptor 1 (TFR1) expression have been reported in ATLL by us and other groups. In this study, to develop a novel molecular-targeted therapy against TFR1 to modulate iron metabolism, we initially determined the expression pattern of several iron-related genes along with TFR1 and found that ATLL cells presented characteristic of an iron-deficiency state such as high expression of iron-regulatory protein 2 (IRP2) and low expression of its E3 ubiquitin-ligase, FBXL5. Therefore, we developed human IgG monoclonal antibodies to human TFR1 using a phage display method (ICOS method) to block the incorporation of the transferrin (TF)-iron complex into ATLL cells for inhibiting cell growth. One of the mAbs, JST-TFR09, presented its greater affinity to TFR1 on ATLL cells in flow cytometry (FCM) analysis than those of commercially available anti-TFR1 antibodies and identified high expression of TFR1 in most of the acute-type ATLL cells. Moreover, JST-TFR09 could interfere with binding between TFR1 and TF, which resulted in effective blockade of TFR1 internalization and induction of cell apoptosis by the treatment of ATLL cells with JST-TFR09. JST-TFR09 showed dual activities through direct cell cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC), and the treatment of JST-TFR09 significantly suppressed cell growth of ATLL cells with induction of apoptosis in in vitro and in vivo experiments. Thus, JST-TFR09 described here may become a promising therapeutic antibody for the treatment of ATLL. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Exaggerated neointima formation in human C-reactive protein transgenic mice is IgG Fc receptor type I (Fc gamma RI)-dependent.

    Science.gov (United States)

    Xing, Dongqi; Hage, Fadi G; Chen, Yiu-Fai; McCrory, Mark A; Feng, Wenguang; Skibinski, Gregory A; Majid-Hassan, Erum; Oparil, Suzanne; Szalai, Alexander J

    2008-01-01

    Neointima formation after vascular injury is exaggerated in ovariectomized (OVX) human C-reactive protein transgenic mice (CRPtg) compared to nontransgenic mice (NTG). We tested the hypothesis that this CRP-mediated exacerbation requires IgG Fc receptors (Fc gamma Rs). OVX NTG, CRPtg, and CRPtg lacking Fc gamma RI, Fc gamma RIIb, Fc gamma RIII, or the common gamma chain (FcR gamma) had their common carotid artery ligated. Twenty-eight days later neointimal thickening in CRPtg/Fc gamma RI(-/-) and CRPtg/FcR gamma (-/-) was significantly less than in CRPtg and no worse than in NTG, whereas in CRPtg/Fc gamma RIIb(-/-) and CRPtg/Fc gamma RIII(-/-) neointimal thickness was equal to or greater than in CRPtg. Immunohistochemistry revealed human CRP in the neointima of CRPtg, but little or none was observed in those lacking Fc gamma RI or FcR gamma. Real-time reverse transcriptase-polymerase chain reaction demonstrated that Fc gamma R types I to III were expressed in the CRPtg arteries, with Fc gamma RI expression increasing by threefold after ligation injury. Levels of serum complement (C3), neointimal deposition of complement (C3d), and cellular composition (monocytes, macrophages, lymphocytes) in the neointima did not differ among the different CRPtg genotypes. However, by immunofluorescence a neointimal population of F4/80+CRP+ cells was revealed only in OVX CRPtg. The exaggerated response to vascular injury provoked by CRP in OVX CRPtg depends on Fc gamma RI and probably requires its expression by F4/80+ cells.

  20. Effect of rIFN-gamma on antibody-mediated cytotoxicity via human monocyte IgG Fc receptor II (CD32).

    Science.gov (United States)

    van Schie, R C; Verstraten, H G; Tax, W J; van den Berkmortel, F W; van de Winkel, J G; de Mulder, P H

    1992-09-01

    Human monocytes and macrophages express an isoform of IgG Fc receptor II (Fc gamma RII), Fc gamma RIIa. Two allotypic variants of this receptor could be distinguished with respect to their ability to bind murine (m)IgG1 complexes either strongly or weakly, defined as high-responder (HR) and low-responder (LR), respectively. We investigated the effect of recombinant (r)IFN-gamma on the ability of freshly isolated monocytes, and those cultured for 40 h and 9 days, to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). Using human erythrocytes (E) sensitized with mIgG1 as target cells, Fc gamma RII was studied selectively. Cells which had been cultured for 40 h exhibit a significantly decreased Fc gamma RII expression, and Fc gamma RII-mediated ADCC activity as compared with freshly isolated monocytes. Co-culture with rIFN-gamma (40 h) reversed this decrease. Short-term rIFN-gamma-cultured cells, and fresh cells express similar numbers of Fc gamma RII, and exhibit comparable Fc gamma RII-mediated ADCC activity. Phagocytic activity was not affected. Prolonged culture of monocytes for 9 days, co-cultured with rIFN-gamma either from day 0 or from day 7, did not affect expression or functional activity of Fc gamma RII. Furthermore, the effects were observed in both HR and LR individuals. Our results show that rIFN-gamma has strong effects on Fc gamma RII-mediated responses specifically during the early stages of monocyte maturation, most likely by affecting receptor expression levels.

  1. An integrated practical implementation of continuous aqueous two-phase systems for the recovery of human IgG: From the microdevice to a multistage bench-scale mixer-settler device.

    Science.gov (United States)

    Espitia-Saloma, Edith; Vâzquez-Villegas, Patricia; Rito-Palomares, Marco; Aguilar, Oscar

    2016-05-01

    Aqueous two-phase systems (ATPS) are a liquid-liquid extraction technology with clear process benefits; however, its lack of industrial embracement is still a challenge to overcome. Antibodies are a potential product to be recovered by ATPS in a commercial context. The objective of this work is to present a more integral approach of the different isolated strategies that have arisen in order to enable a practical, generic implementation of ATPS, using human immunoglobulin G (IgG) as experimental model. A microfluidic device is used for ATPS parameters preselection for product recovery. ATPS were continuously operated in a mixer-settler device in one stage, multistage and multistage with recirculation configuration. Single-stage pure IgG extraction with a polyethylene glycol (PEG) 3350-phophates ATPS within continuous operation allowed a 65% recovery. Further implementation of a multistage platform promoted a higher particle partitioning reaching a 90% recovery. The processing of IgG from a cell supernatant culture harvest in a multistage system with top phase recirculation resulted in 78% IgG recovery in bottom phase. This work conjugates three not widely spread methodologies for ATPS: microfluidics, continuous and multistage operation. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Does the Maternal Serum IgG Level during Pregnancy in Primary Antibody Deficiency Influence the IgG Level in the Newborn?

    Science.gov (United States)

    Nagendran, Vasantha; Emmanuel, Noel; Bansal, Amolak S

    2015-01-01

    Purpose. To find out if the serum IgG level in the newborn baby was affected by low maternal serum IgG during pregnancy in two newly diagnosed primary antibody deficient patients. Method. Infant cord blood IgG level was compared with maternal IgG level in 2 mothers with newly diagnosed primary antibody deficiency, who declined replacement IgG treatment during pregnancy. Results. Both mothers delivered healthy babies with normal IgG levels at birth. Conclusions. The normal IgG levels and sound health in these 2 babies in spite of low maternal IgG throughout pregnancy raise interesting discussion points about maternofoetal immunoglobulin transport mechanisms in primary antibody deficiency.

  3. Does the Maternal Serum IgG Level during Pregnancy in Primary Antibody Deficiency Influence the IgG Level in the Newborn?

    Directory of Open Access Journals (Sweden)

    Vasantha Nagendran

    2015-01-01

    Full Text Available Purpose. To find out if the serum IgG level in the newborn baby was affected by low maternal serum IgG during pregnancy in two newly diagnosed primary antibody deficient patients. Method. Infant cord blood IgG level was compared with maternal IgG level in 2 mothers with newly diagnosed primary antibody deficiency, who declined replacement IgG treatment during pregnancy. Results. Both mothers delivered healthy babies with normal IgG levels at birth. Conclusions. The normal IgG levels and sound health in these 2 babies in spite of low maternal IgG throughout pregnancy raise interesting discussion points about maternofoetal immunoglobulin transport mechanisms in primary antibody deficiency.

  4. The action of NIR (808nm) laser radiation and gold nanorods labeled with IgA and IgG human antibodies on methicillin-resistant and methicillin sensitive strains of Staphylococcus aureus

    Science.gov (United States)

    Tuchina, Elena S.; Petrov, Pavel O.; Ratto, Fulvio; Centi, Sonia; Pini, Roberto; Tuchin, Valery V.

    2015-03-01

    The effect of NIR laser radiation (808 nm) on methicillin-sensitive and methicillin resistant strains of Staphylococcus aureus incubated with gold nanorods is studied. Nanorods having length of 44 (± 4) nm and diameter of 10 (± 3) nm with the absorption maximum in the NIR (800 nm), functionalized with human immunoglobulins IgA and IgG, were synthesized and used in the studies. The killing ability up to 97% of the microorganism populations by using this nanotechnology was shown.

  5. The different effector function capabilities of the seven equine IgG subclasses have implications for vaccine strategies

    OpenAIRE

    Lewis, Melanie J.; Wagner, Bettina; Woof, Jenny M.

    2008-01-01

    Recombinant versions of the seven equine IgG subclasses were expressed in CHO cells. All assembled into intact immunoglobulins stabilised by disulphide bridges, although, reminiscent of human IgG4, a small proportion of equine IgG4 and IgG7 were held together by non-covalent bonds alone. All seven IgGs were N-glycosylated. In addition IgG3 appeared to be O-glycosylated and could bind the lectin jacalin. Staphylococcal protein A displayed weak binding for the equine IgGs in the order: IgG1 > I...

  6. Site-directed in vitro immunization leads to a complete human monoclonal IgG4λ that binds specifically to the CDR2 region of CTLA-4 (CD152 without interfering the engagement of natural ligands

    Directory of Open Access Journals (Sweden)

    Hsu Shu-Ching

    2007-08-01

    Full Text Available Abstract Background The ability to acquire fully human monoclonal antibodies (mAbs with pre-defined specificities is critical to the development of molecular tags for the analysis of receptor function in addition to promising immunotherapeutics. Yet most of the arriving affinity maturated and complete human immunoglobulin G (IgG molecules, which are actually derived from single human B cells, have not widely been used to study the conserved self antigens (Ags such as CD152 (cytotoxic T lymphocyte antigen-4, CTLA-4 because proper hosts are lacking. Results Here we developed an optimized protocol for site-directed in vitro immunizing peripheral blood mononuclear cells (PBMC by using a selected epitope of human CD152, an essential receptor involved in down-regulation of T cell activation. The resultant stable trioma cell lines constantly produce anti-CD152 mAb (γ4λhuCD152, which contains variable (V regions of the heavy chain and the light chain derived from the VH3 and Vλ human germline genes, respectively, and yet displays an unusual IgG4 isotype. Interestingly, γ4λhuCD152 has a basic pI not commonly found in myeloid monoclonal IgG4λs as revealed by the isoelectric focusing (IEF analysis. Furthermore, γ4λhuCD152 binds specifically, with nanomolar affinity, to an extracellular constituency encompassing the putative second complementarity determining region (CDR2 of CD152, whereby it can react to activated CD3+ cells. Conclusion In a context of specific cell depletion and conditioned medium,in vitro induction of human Abs against a conserved self Ag was successfully acquired and a relatively basic mAb, γ4λhuCD152, with high affinity to CDR2 of CD152 was thus obtained. Application of such a human IgG4λ mAb with designated CDR2 specificity may impact upon and prefer for CD152 labeling both in situ and ex situ, as it does not affect the binding of endogenous B7 ligands and can localize into the confined immunological synapse which may

  7. Shared IgG Infection Signatures vs. Hemorrhage-Restricted IgA Clusters in Human Dengue: A Phenotype of Differential Class-Switch via TGFβ1

    Directory of Open Access Journals (Sweden)

    Chung-Hao Huang

    2017-12-01

    Full Text Available Phenotypic manifestations of infectious diseases are closely related to individual immune responses. Methods to extract information from patients’ own immune reactions would be of great use for both diagnosis and treatment. Dengue fever is one of the diseases that clinical aggravations could occur paradoxically after humoral immunity appears. This property makes dengue fever an excellent disease model to explore. A principal component analyses (PCAs-based framework derived from a prior vaccination study was developed. The framework was verified by successful demonstrations of known IgG signatures from a Mexico Dengue data set. Afterward the pipeline was tested upon de novo IgG and IgA libraries of Dengue patients from southern Taiwan. We discovered four infection signatures within IgG repertoires, two of which were identical to previous reports. However, it was IgA but not IgG that could differentiate hemorrhagic from non-hemorrhagic patients. IgA repertoires were found more diversified among bleeders, from whom seven signature clusters were characterized. The expressions of transforming growth factor beta 1 (TGFβ1 and accordingly mediated class-switch activity of IgA were distinct only among the PCA-segregated bleeding group. In sum, intercontinental sharing of IgG signatures in dengue fever was demonstrated via a unified working flow. Differential regulation of IgA class-switch with associated diversity expansion plus existences of hemorrhage-restricted clusters were shown. The ability of the framework to find common IgG signatures would implicate applications to infections even from unknown pathogens. The clusters within IgA repertoires could offer perspectives to other IgA-related bleeding disorders such as Henoch-Schönlein purpura or IgA nephropathy. Substantiated grounds for IgA-specific effector function via TGFβ1-mediated class-switch would be a new factor to consider for infectious diseases.

  8. Prevaccination Rotavirus Serum IgG and IgA Are Associated With Lower Immunogenicity of Live, Oral Human Rotavirus Vaccine in South African Infants.

    Science.gov (United States)

    Moon, Sung-Sil; Groome, Michelle J; Velasquez, Daniel E; Parashar, Umesh D; Jones, Stephanie; Koen, Antoinette; van Niekerk, Nadia; Jiang, Baoming; Madhi, Shabir A

    2016-01-15

    Live oral rotavirus (RV) vaccines have shown modest efficacy among children in African countries for reasons that are not completely understood. We examined the possible inhibitory effect of preexisting antirotavirus antibodies on immunogenicity of monovalent RV vaccine (RV1). Mother-infant pairs were enrolled at presentation for their routine immunization visit in Soweto, South Africa, when infants were aged 5-8 weeks. Infant serum samples were obtained before the first and second doses of RV1 and 1 month after the second dose. Maternal serum and breast milk samples were obtained prior to administration of each dose of RV1 to infants. RV-specific immunoglobulin G (IgG), IgA, and neutralizing activity in sera of infants and serum or breast milk samples of mothers were measured using enzyme-linked immunosorbent assays or a microneutralization test. Of the 107 serum pairs from infants who were seronegative for RV IgA at enrollment, we observed a strong positive association between IgG titers in pre-dose 1 sera of infants and mothers and significant negative associations between IgG titers in pre-dose 1 sera of infants and seroconversion to RV1 post-dose 1. Similarly, mothers whose infants' IgA seroconverted after RV1 had significantly lower pre-dose 1 IgG titers in sera than those whose infants did not seroconvert. High levels of preexisting serum IgG, including transplacentally acquired maternal IgG, appeared to have an inhibitory effect on the immunogenicity of RV1 among infants and may, in part, contribute to lower efficacy of RV vaccines in this and other low-income settings. Published by Oxford University Press for the Infectious Diseases Society of America 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  9. Fully human IgG and IgM antibodies directed against the carcinoembryonic antigen (CEA Gold 4 epitope and designed for radioimmunotherapy (RIT of colorectal cancers

    Directory of Open Access Journals (Sweden)

    Pugnière Martine

    2004-10-01

    VG-IgM biodistribution was not possible in this mouse model in which IgM displays a very short half-life due to poly-Ig receptor expression in the liver. Conclusion Our human anti-CEA IgG2κ is a promising candidate for radioimmunotherapy in intact form, as F(ab'2 fragments, or as a bispecific antibody.

  10. Protein transport into the human endoplasmic reticulum

    NARCIS (Netherlands)

    Dudek, Johanna; Pfeffer, Stefan; Lee, Po-Hsien; Jung, Martin; Cavalié, Adolfo; Helms, Volkhard; Förster, Friedrich; Zimmermann, Richard

    2015-01-01

    Protein transport into the endoplasmic reticulum (ER) is essential for all eukaryotic cells and evolutionary related to protein transport into and across the cytoplasmic membrane of eubacteria and archaea. It is based on amino-terminal signal peptides in the precursor polypeptides plus various

  11. IgG recognizing 21-24 kDa and 30-33 kDa tachyzoite antigens show maximum avidity maturation during natural and accidental human toxoplasmosis

    Directory of Open Access Journals (Sweden)

    VILLAVEDRA Margarita

    1999-01-01

    Full Text Available We describe the avidity maturation of IgGs in human toxoplasmosis using sequential serum samples from accidental and natural infections. In accidental cases, avidity increased continuously throughout infection while naturally infected patients showed a different profile. Twenty-five percent of sera from chronic patients having specific IgM positive results could be appropriately classified using exclusively the avidity test data. To take advantage of the potentiality of this technique, antigens recognized by IgG showing steeper avidity maturation were identified using immunoblot with KSCN elution. Two clusters of antigens, in the ranges of 21-24 kDa and 30-33 kDa, were identified as the ones that fulfill the aforementioned avidity characteristics.

  12. Use of the Brucella IgM and IgG flow assays in the serodiagnosis of human brucellosis in an area endemic for brucellosis

    NARCIS (Netherlands)

    Irmak, Hasan; Buzgan, Turan; Evirgen, Omer; Akdeniz, Hayrettin; Demiroz, A. Pekcan; Abdoel, Theresia H.; Smits, Henk L.

    2004-01-01

    The clinical utility of two complementary tests for brucellosis, the Brucella IgM and IgG flow assays, was evaluated in a hospital in eastern Turkey. The results show that the flow assays are convenient diagnostic tests for use in endemic areas. A positive result in the flow assays was obtained in

  13. Enhancement of retroviral infection in vitro by anti-Le(y) IgG: reversal by humanization of monoclonal mouse antibody

    DEFF Research Database (Denmark)

    Hansen, J E; Sørensen, A M; Arendrup, M

    1993-01-01

    Monoclonal mouse IgG3 antibody (ABL 364) against the carbohydrate Le(y) antigen enhanced infection in vitro with HTLV-1 and with HIV-1 when propagated in both transformed and normal lymphocytes. Enhancement was independent of complement, occurred with both lymphocytes and monocytes as target cells...

  14. DETECTION OF HUMAN ANTI-ZIKA VIRUS IgG BY ELISA USING AN ANTIGEN FROM in vitro INFECTED VERO CELLS: PRELIMINARY RESULTS

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    Laura Masami SUMITA

    Full Text Available SUMMARY Zika virus (ZKV infection is a huge public health problem in Brazil because of the increased incidence of microcephaly in neonates from infected mothers. Detection of specific IgG antibodies in maternal serum samples constitutes an important approach for diagnosing ZKV infection and evaluating its relationship with neonatal microcephaly. However, as there is no serological test produced in Brazil to detect IgM and IgG antibodies against ZKV, we sought to examine specific IgG in serum samples from patients or suspected mothers to detect previous infection and to test for specificity with regard to flaviviral infections occurring in the same area. Brazilian Zika virus native antigens were obtained from infected Vero cell layers or free virions in the culture medium and then used in ELISA. We tested sera from eight ZKV RNA-diagnosed infected patients (ZKVR, seven neonates with microcephaly and their mothers after delivery (MM, 140 dengue virus IgM-positive (DM and IgG (DG-positive patients, and 100 yellow fever (YF-vaccinated patients. According to the ELISA, ZKVR samples were mostly positive (7/8, and all the MM serum samples were positive for ZKV IgG (7/7. In contrast, cross-reactions for dengue or yellow fever-vaccinated patients were observed, including DM (48/95, DG (10/45 or YF (3/100 serum samples; however, these cross-reactions exhibited low antigen avidity so that 6 M urea largely removed this cross-reactivity, with only a few cross-reacting samples remaining (8/140. ELISA based on extracted virions was much more specific, with all ZKVR (8/8 and MM sera being positive for ZKV IgG (7/7 and only borderline cross-reactivity found for DM (6/95, DG (3/45 or YF (4/100-vaccinated serum samples. This technique (ELISA can identify specific IgG in ZKV-infected patients and may be helpful in diagnosing congenital infetions after maternal RNA virus clearance or in epidemiological studies.

  15. Antigen discovery in chronic human inflammatory central nervous system disease: panning phage-displayed antigen libraries identifies the targets of central nervous system-derived IgG in subacute sclerosing panencephalitis.

    Science.gov (United States)

    Burgoon, M P; Owens, G P; Carlson, S; Maybach, A L; Gilden, D H

    2001-11-15

    The presence of increased IgG in the brains of humans with infectious and inflammatory CNS diseases of unknown etiology such as multiple sclerosis may be a clue to the cause of disease. For example, the intrathecally synthesized oligoclonal bands in diseases such as subacute sclerosing panencephalitis (SSPE) or cryptococcal meningitis have been shown to represent Ab directed against the causative agents, measles virus (MV), or Cryptococcus neoformans, respectively. Using SSPE as a model system, we developed a strategy to identify the antigenic targets of the intrathecal disease-relevant IgG in chronic human inflammatory and demyelinating diseases of the CNS. Libraries of cDNA Ags were displayed on the surface of T7Select bacteriophage and biopanned on IgG extracted from the brain of an SSPE patient, or on a monospecific recombinant Fab identified from SSPE brain. After three or six rounds of biopanning on either Ab, positive phage-displayed Ags reacting with IgG were enriched to 35-77% of all panned clones. Sequence analysis of the positive clones identified fragments of the nucleocapsid protein of MV, the cause of SSPE. The sensitivity of the system was determined by diluting the positive clones from this SSPE phage-displayed library at a ratio of 10(-6) into another phage-displayed library that did not contain any detectable MV Ags; after six rounds of panning, the positive clones comprised 34% of all phage and were also shown to be MV nucleocapsid specific. This strategy will be useful to identify potentially rare Ags in diseases of unknown cause.

  16. Cloning and characterization of a functional human ¿-aminobutyric acid (GABA) transporter, human GAT-2

    DEFF Research Database (Denmark)

    Christiansen, Bolette; Meinild, Anne-Kristine; Jensen, Anders A.

    2007-01-01

    Plasma membrane gamma-aminobutyric acid (GABA) transporters act to terminate GABA neurotransmission in the mammalian brain. Intriguingly four distinct GABA transporters have been cloned from rat and mouse, whereas only three functional homologs of these transporters have been cloned from human....... The aim of this study therefore was to search for this fourth missing human transporter. Using a bioinformatics approach, we successfully identified and cloned the full-length cDNA of a so far uncharacterized human GABA transporter (GAT). The predicted protein displays high sequence similarity to rat GAT...... in human. Furthermore the availability of human GAT-2 enables the use of all human clones of the GABA transporters in drug development programs and functional characterization of novel inhibitors of GABA transport....

  17. Human IgG response to a salivary peptide, gSG6-P1, as a new immuno-epidemiological tool for evaluating low-level exposure to Anopheles bites

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    Simondon François

    2009-08-01

    Full Text Available Abstract Background Human populations exposed to low malaria transmission present particular severe risks of malaria morbidity and mortality. In addition, in a context of low-level exposure to Anopheles vector, conventional entomological methods used for sampling Anopheles populations are insufficiently sensitive and probably under-estimate the real risk of malaria transmission. The evaluation of antibody (Ab responses to arthropod salivary proteins constitutes a novel tool for estimating exposure level to insect bites. In the case of malaria, a recent study has shown that human IgG responses to the gSG6-P1 peptide represented a specific biomarker of exposure to Anopheles gambiae bites. The objective of this study was to investigate if this biomarker can be used to estimate low-level exposure of individuals to Anopheles vector. Methods The IgG Ab level to gSG6-P1 was evaluated at the peak and at the end of the An. gambiae exposure season in children living in Senegalese villages, where the Anopheles density was estimated to be very low by classical entomological trapping but where malaria transmission occurred during the studied season. Results Specific IgG responses to gSG6-P1 were observed in children exposed to very low-level of Anopheles bites. In addition, a significant increase in the specific IgG Ab level was observed during the Anopheles exposure season whereas classical entomological data have reported very few or no Anopheles during the studied period. Furthermore, this biomarker may also be applicable to evaluate the heterogeneity of individual exposure. Conclusion The results strengthen the hypothesis that the evaluation of IgG responses to gSG6-P1 during the season of exposure could reflect the real human contact with anthropophilic Anopheles and suggest that this biomarker of low exposure could be used at the individual level. This promising immuno-epidemiological marker could represent a useful tool to assess the risk to very low

  18. Direct evidence of generation and accumulation of β-sheet-rich prion protein in scrapie-infected neuroblastoma cells with human IgG1 antibody specific for β-form prion protein.

    Science.gov (United States)

    Kubota, Toshiya; Hamazoe, Yuta; Hashiguchi, Shuhei; Ishibashi, Daisuke; Akasaka, Kazuyuki; Nishida, Noriyuki; Katamine, Shigeru; Sakaguchi, Suehiro; Kuroki, Ryota; Nakashima, Toshihiro; Sugimura, Kazuhisa

    2012-04-20

    We prepared β-sheet-rich recombinant full-length prion protein (β-form PrP) (Jackson, G. S., Hosszu, L. L., Power, A., Hill, A. F., Kenney, J., Saibil, H., Craven, C. J., Waltho, J. P., Clarke, A. R., and Collinge, J. (1999) Science 283, 1935-1937). Using this β-form PrP and a human single chain Fv-displaying phage library, we have established a human IgG1 antibody specific to β-form but not α-form PrP, PRB7 IgG. When prion-infected ScN2a cells were cultured with PRB7 IgG, they generated and accumulated PRB7-binding granules in the cytoplasm with time, consequently becoming apoptotic cells bearing very large PRB7-bound aggregates. The SAF32 antibody recognizing the N-terminal octarepeat region of full-length PrP stained distinct granules in these cells as determined by confocal laser microscopy observation. When the accumulation of proteinase K-resistant PrP was examined in prion-infected ScN2a cells cultured in the presence of PRB7 IgG or SAF32, it was strongly inhibited by SAF32 but not at all by PRB7 IgG. Thus, we demonstrated direct evidence of the generation and accumulation of β-sheet-rich PrP in ScN2a cells de novo. These results suggest first that PRB7-bound PrP is not responsible for the accumulation of β-form PrP aggregates, which are rather an end product resulting in the triggering of apoptotic cell death, and second that SAF32-bound PrP lacking the PRB7-recognizing β-form may represent so-called PrP(Sc) with prion propagation activity. PRB7 is the first human antibody specific to β-form PrP and has become a powerful tool for the characterization of the biochemical nature of prion and its pathology.

  19. Human IgG response to a salivary peptide, gSG6-P1, as a new immuno-epidemiological tool for evaluating low-level exposure to Anopheles bites.

    Science.gov (United States)

    Poinsignon, Anne; Cornelie, Sylvie; Ba, Fatou; Boulanger, Denis; Sow, Cheikh; Rossignol, Marie; Sokhna, Cheikh; Cisse, Badara; Simondon, François; Remoue, Franck

    2009-08-13

    Human populations exposed to low malaria transmission present particular severe risks of malaria morbidity and mortality. In addition, in a context of low-level exposure to Anopheles vector, conventional entomological methods used for sampling Anopheles populations are insufficiently sensitive and probably under-estimate the real risk of malaria transmission. The evaluation of antibody (Ab) responses to arthropod salivary proteins constitutes a novel tool for estimating exposure level to insect bites. In the case of malaria, a recent study has shown that human IgG responses to the gSG6-P1 peptide represented a specific biomarker of exposure to Anopheles gambiae bites. The objective of this study was to investigate if this biomarker can be used to estimate low-level exposure of individuals to Anopheles vector. The IgG Ab level to gSG6-P1 was evaluated at the peak and at the end of the An. gambiae exposure season in children living in Senegalese villages, where the Anopheles density was estimated to be very low by classical entomological trapping but where malaria transmission occurred during the studied season. Specific IgG responses to gSG6-P1 were observed in children exposed to very low-level of Anopheles bites. In addition, a significant increase in the specific IgG Ab level was observed during the Anopheles exposure season whereas classical entomological data have reported very few or no Anopheles during the studied period. Furthermore, this biomarker may also be applicable to evaluate the heterogeneity of individual exposure. The results strengthen the hypothesis that the evaluation of IgG responses to gSG6-P1 during the season of exposure could reflect the real human contact with anthropophilic Anopheles and suggest that this biomarker of low exposure could be used at the individual level. This promising immuno-epidemiological marker could represent a useful tool to assess the risk to very low exposure to malaria vectors as observed in seasonal, urban

  20. Rabbit IgG directed to a synthetic C-terminal peptide of the major grass pollen allergen Lol p I inhibits human basophil histamine release induced by natural Lol p I.

    Science.gov (United States)

    van Ree, R; Aalberse, R C

    1995-03-01

    The potential role of allergen-specific IgG antibodies as 'blocking' antibodies in allergen-induced human basophil histamine release was investigated. This was studied in a model with the major grass pollen allergen Lol p I and polyclonal rabbit antisera directed against this allergen and against a synthetic peptide of its C terminus. When allergen and antibodies were allowed to preincubate, Lol p I induced histamine release was inhibited up to 85% by the antiserum against Lol p I. By omitting preincubation, and thereby more closely mimicking an in vivo situation, up to 55% inhibition was realized. This indicates that allergen-specific IgG can act as 'blocking' antibody without preincubation. Immunization of rabbits with a synthetic C-terminal peptide of Lol p I resulted in antibodies reactive with natural Lol p I. Despite their 100-fold lower avidity for Lol p I (as compared with antinatural Lol p I), these antibodies had the capacity to inhibit Lol p I induced histamine release for > 90% (up to 50% without preincubation). This indicates that it is possible to block histamine release induced by a major allergen with low-avidity IgG antibodies directed against a minor proportion of the allergen (25 amino acids). IgE antibodies from the donors studied were unreactive with this synthetic peptide, indicating that for blocking activity identical epitope specificity of IgE and IgG is not essential. This opens interesting perspectives for application of synthetic peptides in immunotherapy, distinct from their effects on T cell reactivity.

  1. Usefulness of concanavalin-A non-binding fraction of Strongyloides venezuelensis larvae to detect IgG and IgA in human strongyloidiasis.

    Science.gov (United States)

    Gonzaga, Henrique Tomaz; Ribeiro, Vanessa da Silva; Cunha-Júnior, Jair Pereira; Ueta, Marlene Tiduko; Costa-Cruz, Julia Maria

    2011-05-01

    Glycosylated components from Strongyloides have an important role in parasite establishment and host recognition of these substances. Considering the sugar-binding capacity of lectins, such as concanavalin-A (Con-A), IgG and IgA detection in serum samples from strongyloidiasis patients was tested using different antigenic preparations. The total saline extract (SE) of Strongyloides venezuelensis filariform larvae was fractionated in Con-A column to obtain Con-A unbound (Con-A UF) and Con-A bound (Con-A BF) fractions. Sensitivity (Se), specificity (Sp), area under the ROC curve (AUC), likelihood ratio (LR), and correlation coefficients were calculated. Con-A UF showed the highest diagnostic parameters for IgG detection (Se 95.0%, Sp 92.5%, AUC 0.99, LR 12.7) and high correlation (r = 0.700) with SE. Con-A fractions did not clearly demonstrate any usefulness for IgA detection. In conclusion, the results obtained demonstrate that Con-A UF is an important source of specific peptides efficient to detect IgG in strongyloidiasis immunodiagnosis. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Human behavior research and the design of sustainable transport systems

    Science.gov (United States)

    Schauer, James J.

    2011-09-01

    Transport currently represents approximately 19% of the global energy demand and accounts for about 23% of the global carbon dioxide emissions (IEA 2009). As the demand for mobility is expected to continue to increase in the coming decades, the stabilization of atmospheric carbon dioxide levels will require the evolution of transport, along with power generation, building design and manufacturing. The continued development of these sectors will need to include changes in energy sources, energy delivery, materials, infrastructure and human behavior. Pathways to reducing carbon from the transport sector have unique challenges and opportunities that are inherent to the human choices and behavioral patterns that mold the transportation systems and the associated energy needs. Technology, government investment, and regulatory policies have a significant impact on the formulation of transportation infrastructure; however, the role of human behavior and public acceptance on the efficiency and effectiveness of transport systems should not be underestimated. Although developed, rapidly developing, and underdeveloped nations face different challenges in the establishment of transport infrastructure that can meet transport needs while achieving sustainable carbon dioxide emissions, the constraints that establish the domain of possibilities are closely related for all nations. These constraints include capital investment, fuel supplies, power systems, and human behavior. Throughout the world, there are considerable efforts directed at advancing and optimizing the financing of sustainable infrastructures, the production of low carbon fuels, and the production of advanced power systems, but the foundational work on methods to understand human preferences and behavior within the context of transport and the valuation of reductions in carbon dioxide emissions is greatly lagging behind. These methods and the associated understanding of human behavior and the willingness to pay for

  3. Deoxynivalenol transport across the human placental barrier

    DEFF Research Database (Denmark)

    Nielsen, Jeanette K S; Vikström, Anna C; Turner, Paul

    2011-01-01

    with the ex vivo dual perfusion model. The concentration of DON on the foetal side after 4h was about 21% of that on the maternal side at t=0. These results support the data from the BeWo monolayer model in respect to the transport rate of DON, and are consistent with our hypothesis of foetal exposure to DON...

  4. IgG4 Cholangiopathy

    Directory of Open Access Journals (Sweden)

    Yoh Zen

    2012-01-01

    Full Text Available IgG4 cholangiopathy can involve any level of the biliary tree which exhibits sclerosing cholangitis or pseudotumorous hilar lesions. Most cases are associated with autoimmune pancreatitis, an important diagnostic clue. Without autoimmune pancreatitis, however, the diagnosis of IgG4-cholangiopathy is challenging. Indeed such cases have been treated surgically. IgG4-cholangiopathy should be diagnosed based on serological examinations including serum IgG4 concentrations, radiological features, and histological evidence of IgG4+ plasma cell infiltration. Steroid therapy is very effective even at disease relapse. A Th2-dominant immune response or the activation of regulatory T cells seems to be involved in the underlying immune reaction. It is still unknown why IgG4 levels are specifically elevated in patients with this disease. IgG4 might be secondarily overexpressed by Th2 or regulatory cytokines given the lack of evidence that IgG4 is an autoantibody.

  5. Biophysical and Functional Characterization of Rhesus Macaque IgG Subclasses

    Science.gov (United States)

    Boesch, Austin W.; Osei-Owusu, Nana Yaw; Crowley, Andrew R.; Chu, Thach H.; Chan, Ying N.; Weiner, Joshua A.; Bharadwaj, Pranay; Hards, Rufus; Adamo, Mark E.; Gerber, Scott A.; Cocklin, Sarah L.; Schmitz, Joern E.; Miles, Adam R.; Eckman, Joshua W.; Belli, Aaron J.; Reimann, Keith A.; Ackerman, Margaret E.

    2016-01-01

    Antibodies raised in Indian rhesus macaques [Macaca mulatta (MM)] in many preclinical vaccine studies are often evaluated in vitro for titer, antigen-recognition breadth, neutralization potency, and/or effector function, and in vivo for potential associations with protection. However, despite reliance on this key animal model in translation of promising candidate vaccines for evaluation in first in man studies, little is known about the properties of MM immunoglobulin G (IgG) subclasses and how they may compare to human IgG subclasses. Here, we evaluate the binding of MM IgG1, IgG2, IgG3, and IgG4 to human Fc gamma receptors (FcγR) and their ability to elicit the effector functions of human FcγR-bearing cells, and unlike in humans, find a notable absence of subclasses with dramatically silent Fc regions. Biophysical, in vitro, and in vivo characterization revealed MM IgG1 exhibited the greatest effector function activity followed by IgG2 and then IgG3/4. These findings in rhesus are in contrast with the canonical understanding that IgG1 and IgG3 dominate effector function in humans, indicating that subclass-switching profiles observed in rhesus studies may not strictly recapitulate those observed in human vaccine studies. PMID:28018355

  6. High-affinity IgG antibodies develop naturally in Ig-knockout rats carrying germline human IgH/Igκ/Igλ loci bearing the rat CH region.

    Science.gov (United States)

    Osborn, Michael J; Ma, Biao; Avis, Suzanne; Binnie, Ashleigh; Dilley, Jeanette; Yang, Xi; Lindquist, Kevin; Ménoret, Séverine; Iscache, Anne-Laure; Ouisse, Laure-Hélène; Rajpal, Arvind; Anegon, Ignacio; Neuberger, Michael S; Buelow, Roland; Brüggemann, Marianne

    2013-02-15

    Mice transgenic for human Ig loci are an invaluable resource for the production of human Abs. However, such mice often do not yield human mAbs as effectively as conventional mice yield mouse mAbs. Suboptimal efficacy in delivery of human Abs might reflect imperfect interaction between the human membrane IgH chains and the mouse cellular signaling machinery. To obviate this problem, in this study we generated a humanized rat strain (OmniRat) carrying a chimeric human/rat IgH locus (comprising 22 human V(H)s, all human D and J(H) segments in natural configuration linked to the rat C(H) locus) together with fully human IgL loci (12 Vκs linked to Jκ-Cκ and 16 Vλs linked to Jλ-Cλ). The endogenous Ig loci were silenced using designer zinc finger nucleases. Breeding to homozygosity resulted in a novel transgenic rat line exclusively producing chimeric Abs with human idiotypes. B cell recovery was indistinguishable from wild-type animals, and human V(D)J transcripts were highly diverse. Following immunization, the OmniRat strain performed as efficiently as did normal rats in yielding high-affinity serum IgG. mAbs, comprising fully human variable regions with subnanomolar Ag affinity and carrying extensive somatic mutations, are readily obtainable, similarly to conventional mAbs from normal rats.

  7. Purification and fluorescent labeling of the human serotonin transporter

    DEFF Research Database (Denmark)

    Rasmussen, Søren G F; Gether, Ulrik

    2005-01-01

    To establish a purification procedure for the human serotonin transporter (hSERT) we expressed in Sf9 insect cells an epitope-tagged version of the transporter containing a FLAG epitope at the N-terminus and a polyhistidine tail at the C-terminus (FLAG-hSERT-12H). For purification, the transporter...... was solubilized in digitonin followed by nickel affinity and subsequent concanavalin A chromatography. Using this procedure we were able to obtain an overall purification of 700-fold and a yield of approximately 0.1 mg/L of cell culture. The purified transporter displayed pharmacological properties similar...... quenching experiments revealed that the aqueous quencher iodide was able to cause marked quenching of the fluorescence of the IANBD labeled transporter with a K(SV) of 3.4 +/- 0.10 M(-)(1). In a mutant transporter with five cysteines mutated (5CysKO) we observed a significant reduction in this quenching (K...

  8. In Vitro Proliferation and Production of Cytokine and IgG by Human PBMCs Stimulated with Polysaccharide Extract from Plants Endemic to Gabon

    Directory of Open Access Journals (Sweden)

    Line Edwige Mengome

    2014-11-01

    Full Text Available Polysaccharides were extracted from seven plants endemic to Gabon to study their potential immunological activities. Peripheral blood mononuclear cell (PBMC (5 × 105 cells/mL proliferation, cytokine and immunoglobulin G (IgG assays were performed after stimulation with different concentrations of polysaccharide fractions compared with lipopolysaccharides (LPS and concanavalin A (ConA from healthy volunteers. The culture supernatants were used for cytokine and IgG detection by enzyme-linked immunosorbent assay (ELISA. The results show that pectin and hemicellulose extracts from Uvaria klainei, Petersianthus macrocarpus, Trichoscypha addonii, Aphanocalyx microphyllus, Librevillea klaineana, Neochevalierodendron stephanii and Scorodophloeus zenkeri induced production levels that were variable from one individual to another for IL-12 (3–40 pg/mL, IL-10 (6–443 pg/mL, IL-6 (7–370 pg/mL, GM-CSF (3–170 pg/mL and IFN-γ (5–80 pg/mL. Only hemicelluloses from Aphanocalyx microphyllus produce a small amount of IgG (OD = 0.034, while the proliferation of cells stimulated with these polysaccharides increased up to 318% above the proliferation of unstimulated cells. However, this proliferation of PBMCs was abolished when the pectin of some of these plants was treated with endopolygalacturonase (p < 0.05, but the trend of cytokine synthesis remained the same, both before and after enzymatic treatment or saponification. This study suggests that these polysaccharides stimulate cells in a structure-dependent manner. The rhamnogalacturonan-I (RGI fragment alone was not able to induce the proliferation of PBMC.

  9. Differential reactivity of salivary igA and igG against streptococcus mutans proteins in humans with different caries experience.

    Science.gov (United States)

    Gómez, Soledad I; Jaramillo, Lorenza M; Moreno, Gloria C; Roa, Nelly S; Rodríguez, Adriana

    2015-04-01

    Dental caries is an infectious disease which still constitutes a public health concern. It begins at an early age and is caused mainly Streptococcus mutans (S. mutans). The aim of this study was to characterize the salivary humor immune response to S. mutans proteins in patients with caries, with history of caries and without caries, in order to determine which S. mutans proteins participate in the immunological response in subjects with different caries experience. Saliva was collected by spontaneous salivation for 5 minutes from 60 subjects aged 18 to 30 years, classified according to their caries experience as: without caries (Group I), with active caries (Group II) and with history of caries (Group III). The antigens derived from S. mutans by sonication were recognized by salivary IgA and IgG by Western Blot. The results showed that all the individuals studied recognized S. mutans proteins with molecular weights in the range of 8 to 191 kDa, with similar recognition profiles for salivary IgA and IgG. Subjects without caries recognized the 29 kDa protein, also known as S. mutans Antigen A, via salivary IgA, differing from patients with caries and history of caries, who recognized it via IgG. The protective response against S. mutans is mediated by IgA. To conclude, a differential response to the 29 kDa protein between study individuals may be indicative of resistance to dental caries and may have a protective role in the induction of IgA antibodies against dental caries, as found in the group without caries, in contrast to subjects with active caries and history of caries.

  10. Overview Of Suborbital Human Transportation Concept Alpha

    Science.gov (United States)

    Adirim, H.; Pilz, N.; Marini, M.; Hendrick, P.; Schmid, M.; Behr, R.; Barth, T.; Tarfeld, F.; Wiegand, A.; Charbonnier, D.; Haya Ramos, R.; Steeland, J.; Mack, A.

    2011-05-01

    Within the EC co-funded project FAST20XX (Future high-Altitude high-Speed Transport 20XX), the European suborbital passenger transportation system concept ALPHA (Airplane Launched PHoenix Aircraft), which shall be based to a maximum extent on existing technologies and capabilities, is currently being investigated as collaborative project by a European consortium under coordination of ESA. The ALPHA concept incorporates an air-launch from a carrier aircraft, which shall be used as first stage. The ALPHA vehicle shall be capable of transporting up to four passengers plus one pilot to an altitude of at least 100 km. The ALPHA vehicle is a down-scaled version of the suborbital space transportation concept Hopper, which was already deeply investigated within the European FESTIP System Study and the German ASTRA program including the successfully flown experimental landing demonstrator Phoenix. This approach has allowed the use of existing aerodynamic vehicle data and has led to the adaptation of the external Hopper/Phoenix configuration for ALPHA. In FESTIP and ASTRA, the Hopper configuration showed sufficient stability margins. Due to the geometric similarity of the ALPHA and Hopper vehicles, a trimable and flyable configuration could be derived by means of ALPHA flight trajectory calculations. In its current configuration, the ALPHA vehicle has a length of ca. 9 m and a gross take-off mass of ca. 3.5 Mg. The launch, staging and separation of ALPHA shall be performed either as internal air-launch from the cargo bay of the carrier aircraft, as under-wing air-launch or as towed air-launch. After separation from the carrier aircraft, the ALPHA vehicle ignites its onboard rocket propulsion system. Since conventional liquid and solid propulsion did not seem suitable for ALPHA due to Their high cost, limited safety and toxicity, a low-cost, “green” and non-hazardous hybrid propulsion system based on liquid nitrous oxide in combination with a solid polymer fuel was

  11. Modelling of human transplacental transport as performed in Copenhagen, Denmark

    DEFF Research Database (Denmark)

    Mathiesen, Line; Mørck, Thit Aarøe; Zuri, Giuseppina

    2014-01-01

    Placenta perfusion models are very effective when studying the placental mechanisms in order to extrapolate to real-life situations. The models are most often used to investigate the transport of substances between mother and foetus, including the potential metabolism of these. We have studied...... the relationships between maternal and foetal exposures to various compounds including pollutants such as polychlorinated biphenyls, polybrominated flame retardants, nanoparticles as well as recombinant human antibodies. The compounds have been studied in the human placenta perfusion model and to some extent...... in vitro with an established human monolayer trophoblast cell culture model. Results from our studies distinguish placental transport of substances by physicochemical properties, adsorption to placental tissue, binding to transport and receptor proteins and metabolism. We have collected data from different...

  12. Quercetin inhibits hexose transport in a human diploid fibroblast

    Energy Technology Data Exchange (ETDEWEB)

    Salter, D.W.; Custead-Jones, S.; Cook, J.S.

    1978-01-01

    The flavonol quercetin, a phloretin analog, inhibits transport of 2-deoxyglucose and 3-O-methylglucose in a cultured human diploid fibroblast. This inhibition is related to transport itself and not to the reported effects of flavonoids on membrane-bound ATPases. From concentration-inhibition curves at several pH's we conclude that uncharged (acid) quercetin (pH = 7.65) is the inhibitory form of the molecule (K/sub I/ = 10 ..mu..m). Quercetin, unlike phloretin, is rapidly degraded in 0.1 N NaOH; the degradation products are weakly inhibitory to hexose transport.

  13. Species-specific and cross-reactive IgG1 antibody binding to viral capsid protein 1 (VP1 antigens of human rhinovirus species A, B and C.

    Directory of Open Access Journals (Sweden)

    Jua Iwasaki

    Full Text Available BACKGROUND: Human rhinoviruses (HRV are associated with upper and lower respiratory illnesses, including severe infections causing hospitalization in both children and adults. Although the clinical significance of HRV infections is now well established, no detailed investigation of the immune response against HRV has been performed. The purpose of this study was to assess the IgG1 antibody response to the three known HRV species, HRV-A, -B and -C in healthy subjects. METHODS: Recombinant polypeptides of viral capsid protein 1 (VP1 from two genotypes of HRV-A, -B and -C were expressed as glutathione S-transferase (GST fusion proteins and purified by affinity and then size exclusion chromatography. The presence of secondary structures similar to the natural antigens was verified by circular dichroism analysis. Total and species-specific IgG1 measurements were quantitated by immunoassays and immunoabsorption using sera from 63 healthy adults. RESULTS: Most adult sera reacted with the HRV VP1 antigens, at high titres. As expected, strong cross-reactivity between HRV genotypes of the same species was found. A high degree of cross-reactivity between different HRV species was also evident, particularly between HRV-A and HRV-C. Immunoabsorption studies revealed HRV-C specific titres were markedly and significantly lower than the HRV-A and HRV-B specific titres (P<0.0001. A truncated construct of HRV-C VP1 showed greater specificity in detecting anti-HRV-C antibodies. CONCLUSIONS: High titres of IgG1 antibody were bound by the VP1 capsid proteins of HRV-A, -B and -C, but for the majority of people, a large proportion of the antibody to HRV-C was cross-reactive, especially to HRV-A. The improved specificity found for the truncated HRV-C VP1 indicates species-specific and cross-reactive regions could be defined.

  14. Characterization of the basic charge variants of a human IgG1: effect of copper concentration in cell culture media.

    Science.gov (United States)

    Kaschak, Timothy; Boyd, Daniel; Lu, Franklin; Derfus, Gayle; Kluck, Brian; Nogal, Bartek; Emery, Craig; Summers, Christie; Zheng, Kai; Bayer, Robert; Amanullah, Ashraf; Yan, Boxu

    2011-01-01

    We report a case study of an IgG1 with a unique basic charge variant profile caused by C-terminal proline amidation on either one or two heavy chains. The proline amidation was sensitive to copper ion concentration in the production media during cell culture: the higher the Cu ( 2+) ion concentration, the higher the level of proline amidation detected. This conclusion was supported by the analysis of samples that revealed direct correlation between the proline amidation level observed from peptide maps and the level of basic peaks measured by imaged capillary isoelectric focusing and a pH gradient ion-exchange chromatography method. The importance of these observations to therapeutic antibody production is discussed.

  15. Response to 'Serological diagnostics in the detection of IgG autoantibodies against human collagen VII in epidermolysis bullosa acquisita - a multicenter analysis'.

    Science.gov (United States)

    Jonkman, M F; Meijer, J M; Diercks, G F H; Pas, H H

    2017-10-04

    In a recent retrospective serological study in 95 sera from epidermolysis bullosa acquisita (EBA) patients Schmidt et al. conclude that Col7A-NC1/NC2 ELISA (MBL, Japan) is superior to NC1 ELISA, Western blot and indirect immunofluorescence (IIF) on salt-split skin (SSS) with the highest sensitivity of 97.9%.1 This sensitivity is an overestimation from a biased sample, since the sera had been preselected from patients with IgG reactivity fitting EBA by at least one of the immune serological assays indirect immunofluorescence, ELISA, and Western blot. The deficiency in study design with an incorrect reference standard creates a bias in estimated test accuracy. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  16. Nucleobase Transport by Human Equilibrative Nucleoside Transporter 1 (hENT1)*

    Science.gov (United States)

    Yao, Sylvia Y. M.; Ng, Amy M. L.; Cass, Carol E.; Baldwin, Stephen A.; Young, James D.

    2011-01-01

    The human equilibrative nucleoside transporters hENT1 and hENT2 (each with 456 residues) are 40% identical in amino acid sequence and contain 11 putative transmembrane helices. Both transport purine and pyrimidine nucleosides and are distinguished functionally by a difference in sensitivity to inhibition by nanomolar concentrations of nitrobenzylmercaptopurine ribonucleoside (NBMPR), hENT1 being NBMPR-sensitive. Previously, we used heterologous expression in Xenopus oocytes to demonstrate that recombinant hENT2 and its rat ortholog rENT2 also transport purine and pyrimidine bases, h/rENT2 representing the first identified mammalian nucleobase transporter proteins (Yao, S. Y., Ng, A. M., Vickers, M. F., Sundaram, M., Cass, C. E., Baldwin, S. A., and Young, J. D. (2002) J. Biol. Chem. 277, 24938–24948). The same study also revealed lower, but significant, transport of hypoxanthine by h/rENT1. In the present investigation, we have used the enhanced Xenopus oocyte expression vector pGEMHE to demonstrate that hENT1 additionally transports thymine and adenine and, to a lesser extent, uracil and guanine. Fluxes of hypoxanthine, thymine, and adenine by hENT1 were saturable and inhibited by NBMPR. Ratios of Vmax (pmol/oocyte·min−1):Km (mm), a measure of transport efficiency, were 86, 177, and 120 for hypoxantine, thymine, and adenine, respectively, compared with 265 for uridine. Hypoxanthine influx was competitively inhibited by uridine, indicating common or overlapping nucleobase and nucleoside permeant binding pockets, and the anticancer nucleobase drugs 5-fluorouracil and 6-mercaptopurine were also transported. Nucleobase transport activity was absent from an engineered cysteine-less version hENT1 (hENT1C−) in which all 10 endogenous cysteine residues were mutated to serine. Site-directed mutagenesis identified Cys-414 in transmembrane helix 10 of hENT1 as the residue conferring nucleobase transport activity to the wild-type transporter. PMID:21795683

  17. Membrane Drug Transporters and Chemoresistance in Human Pancreatic Carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Hagmann, Wolfgang, E-mail: w.hagmann@dkfz.de; Faissner, Ralf [Clinical Cooperation Unit of Molecular Gastroenterology, DKFZ, Im Neuenheimer Feld 280, D-69120 Heidelberg (Germany); Schnolzer, Martina [Functional Proteome Analysis, DKFZ, Im Neuenheimer Feld 280, D-69120 Heidelberg (Germany); Lohr, Matthias [Clinical Cooperation Unit of Molecular Gastroenterology, DKFZ, Im Neuenheimer Feld 280, D-69120 Heidelberg (Germany); Department of Surgical Gastroenterology, CLINTEC, K53, Karolinska Institute, SE-14186 Stockholm (Sweden); Jesnowski, Ralf [Clinical Cooperation Unit of Molecular Gastroenterology, DKFZ, Im Neuenheimer Feld 280, D-69120 Heidelberg (Germany); Department of Medicine II, Medical Faculty of Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer 1-3, D-68167 Mannheim (Germany)

    2010-12-30

    Pancreatic cancer ranks among the tumors most resistant to chemotherapy. Such chemoresistance of tumors can be mediated by various cellular mechanisms including dysregulated apoptosis or ineffective drug concentration at the intracellular target sites. In this review, we highlight recent advances in experimental chemotherapy underlining the role of cellular transporters in drug resistance. Such contribution to the chemoresistant phenotype of tumor cells or tissues can be conferred both by uptake and export transporters, as demonstrated by in vivo and in vitro data. Our studies used human pancreatic carcinoma cells, cells stably transfected with human transporter cDNAs, or cells in which a specific transporter was knocked down by RNA interference. We have previously shown that 5-fluorouracil treatment affects the expression profile of relevant cellular transporters including multidrug resistance proteins (MRPs), and that MRP5 (ABCC5) influences chemoresistance of these tumor cells. Similarly, cell treatment with the nucleoside drug gemcitabine or a combination of chemotherapeutic drugs can variably influence the expression pattern and relative amount of uptake and export transporters in pancreatic carcinoma cells or select for pre-existing subpopulations. In addition, cytotoxicity studies with MRP5-overexpressing or MRP5-silenced cells demonstrate a contribution of MRP5 also to gemcitabine resistance. These data may lead to improved strategies of future chemotherapy regimens using gemcitabine and/or 5-fluorouracil.

  18. The Design of Transportation Equipment in Terms of Human Capabilities. The Role of Engineering Psychology in Transport Safety.

    Science.gov (United States)

    McFarland, Ross A.

    Human factors engineering is considered with regard to the design of safety factors for aviation and highway transportation equipment. Current trends and problem areas are identified for jet air transportation and for highway transportation. Suggested solutions to transportation safety problems are developed by applying the techniques of human…

  19. Human ATP-binding cassette (ABC transporter family

    Directory of Open Access Journals (Sweden)

    Vasiliou Vasilis

    2009-04-01

    Full Text Available Abstract There exist four fundamentally different classes of membrane-bound transport proteins: ion channels; transporters; aquaporins; and ATP-powered pumps. ATP-binding cassette (ABC transporters are an example of ATP-dependent pumps. ABC transporters are ubiquitous membrane-bound proteins, present in all prokaryotes, as well as plants, fungi, yeast and animals. These pumps can move substrates in (influx or out (efflux of cells. In mammals, ABC transporters are expressed predominantly in the liver, intestine, blood-brain barrier, blood-testis barrier, placenta and kidney. ABC proteins transport a number of endogenous substrates, including inorganic anions, metal ions, peptides, amino acids, sugars and a large number of hydrophobic compounds and metabolites across the plasma membrane, and also across intracellular membranes. The human genome contains 49 ABC genes, arranged in eight subfamilies and named via divergent evolution. That ABC genes are important is underscored by the fact that mutations in at least I I of these genes are already known to cause severe inherited diseases (eg cystic fibrosis and X-linked adrenoleukodystrophy [X-ALD]. ABC transporters also participate in the movement of most drugs and their metabolites across cell surface and cellular organelle membranes; thus, defects in these genes can be important in terms of cancer therapy, pharmacokinetics and innumerable pharmacogenetic disorders.

  20. Glucose transporter expression in human skeletal muscle fibers

    DEFF Research Database (Denmark)

    Gaster, M; Handberg, A; Beck-Nielsen, H

    2000-01-01

    amplification (TSA) technique to detect the localization of glucose transporter expression in human skeletal muscle. We found expression of GLUT-1, GLUT-3, and GLUT-4 in developing human muscle fibers showing a distinct expression pattern. 1) GLUT-1 is expressed in human skeletal muscle cells during gestation......, but its expression is markedly reduced around birth and is further reduced to undetectable levels within the first year of life; 2) GLUT-3 protein expression appears at 18 wk of gestation and disappears after birth; and 3) GLUT-4 protein is diffusely expressed in muscle cells throughout gestation, whereas...

  1. Human Equilibrative Nucleoside Transporter 1, as a Predictor of 5-Fluorouracil Resistance in Human Pancreatic Cancer

    National Research Council Canada - National Science Library

    MASANORI TSUJIE; SHOJI NAKAMORI; SHIN NAKAHIRA; YUJI TAKAHASHI; NOBUYASU HAYASHI; JIRO OKAMI; HIROAKI NAGANO; KEIZO DONO; KOJI UMESHITA; MASATO SAKON; MORITO MONDEN

    2007-01-01

    ...€²-difluoro-deoxycytidine) sensitivity in pancreatic cancer. Materials and Methods: The relationship between 5-FU and gemcitabine sensitivity and the mRNA levels of human equilibrative nucleoside transporter 1 (hENT1...

  2. Existence of different but overlapping IgG- and IgM-binding sties on the globular domain of human C1q

    DEFF Research Database (Denmark)

    Zlatarova, A.S.; Rouseva, M.; Roumenina, L.T.

    2006-01-01

    C1q is the first subcomponent of the classical complement pathway that binds antigen-bound IgG or IgM and initiates complement activation via association of serine proteases C1r and C1s. The globular domain of C1q (gC1q), which is the ligand-recognition domain, is a heterotrimeric structure...... composed of the C-terminal regions of A (ghA), B (ghB), and C (ghC) chains. The expression and functional characterization of ghA, ghB, and ghC modules have revealed that each chain has some structural and functional autonomy. Although a number of studies have tried to identify IgG-binding sites on the gC1......q domain, no such attempt has been made to localize IgM-binding site. On the basis of the information available via the gC1q crystal structure, molecular modeling, mutational studies, and bioinformatics, we have generated a series of substitution mutants of ghA, ghB, and ghC and examined...

  3. The subclass distribution of IgG autoantibodies in cicatricial pemphigoid and epidermolysis bullosa acquisita.

    Science.gov (United States)

    Bernard, P; Prost, C; Aucouturier, P; Durepaire, N; Denis, F; Bonnetblanc, J M

    1991-08-01

    To study the subclass distribution of autoantibodies and their complement-fixing capacity in cicatricial pemphigoid (CP) and epidermolysis bullosa acquisita (EBA) we studied the sera from 23 patients by both indirect immunofluorescence (IIF) on 4-microns cryostat sections of normal human skin and immunoblotting of epidermal or dermal extracts. Monoclonal antibodies of strict specificity for human IgG subclasses were used. Sera from 20 patients with BP served as controls. In addition, total IgG subclass levels were determined by indirect competitive ELISA in all sera. Complement binding capacity was studied by IIF using antibodies to C3 after incubation of skin section with autoantibodies and source of fresh complement. CP autoantibodies reacting with the 230-240 kD and/or the 180-kD epidermal bands showed an IgG4/IgG1 subclass restriction, with a predominance of IgG4 in 10 cases, of IgG1 in four. In BP sera, IgG4 and IgG1 autoantibodies were detected with a similar frequency (100% and 83%, respectively). In EBA sera, autoantibodies reacting with the 290 kD and 145 kD dermal bands also showed an IgG1/IgG4 restriction. Concordant results were obtained by IIF. However, the IIF method had a lower sensitivity for the detection of IgG4 CP antibodies and IgG1 EBA antibodies than immunoblotting. Finally, when CP antibodies were analyzed for their complement-binding activity, it was found that sera containing IgG4 autoantibodies alone never fixed complement whereas all complement-fixing CP sera had IgG1 autoantibodies, suggesting that only this subclass of antibodies is capable of fixing complement.

  4. Neuron-derived IgG Protects Neurons from Complement-dependent Cytotoxicity

    Science.gov (United States)

    Zhang, Jie; Li, Bingjie; McNutt, Michael A.

    2013-01-01

    Passive immunity of the nervous system has traditionally been thought to be predominantly due to the blood-brain barrier. This concept must now be revisited based on the existence of neuron-derived IgG. The conventional concept is that IgG is produced solely by mature B lymphocytes, but it has now been found to be synthesized by murine and human neurons. However, the function of this endogenous IgG is poorly understood. In this study, we confirm IgG production by rat cortical neurons at the protein and mRNA levels, with 69.0 ± 5.8% of cortical neurons IgG-positive. Injury to primary-culture neurons was induced by complement leading to increases in IgG production. Blockage of neuron-derived IgG resulted in more neuronal death and early apoptosis in the presence of complement. In addition, FcγRI was found in microglia and astrocytes. Expression of FcγR I in microglia was increased by exposure to neuron-derived IgG. Release of NO from microglia triggered by complement was attenuated by neuron-derived IgG, and this attenuation could be reversed by IgG neutralization. These data demonstrate that neuron-derived IgG is protective of neurons against injury induced by complement and microglial activation. IgG appears to play an important role in maintaining the stability of the nervous system. PMID:23979841

  5. Transport of paraquat by human organic cation transporters and multidrug and toxic compound extrusion family.

    Science.gov (United States)

    Chen, Ying; Zhang, Shuzhong; Sorani, Marco; Giacomini, Kathleen M

    2007-08-01

    Paraquat (N,N-dimethyl-4-4'-bipiridinium; PQ), a widely used herbicide, when ingested accidentally or intentionally can cause major organ toxicities in lung, liver, and kidney. Because PQ is primarily eliminated in the kidney, renal elimination, including tubular transport, plays a critical role in controlling systemic exposure to the herbicide. The goal of this study was to determine the molecular identities of the transporters involved in the renal elimination of PQ. Using stably transfected human embryonic kidney (HEK)-293 cells, we examined the role of human organic cation transporters (hOCTs, SLC22A1-3) and human multidrug and toxic compound extrusion (hMATE)1 in the cellular accumulation and cytotoxicity of PQ. We found that overexpression of hOCT2 but not hOCT1 and hOCT3 in HEK-293 cells significantly enhanced the accumulation and cytotoxicity of PQ (-fold increase for uptake was 12 +/- 0.5, p < 0.01; -fold increase of cytotoxicity was 18 +/- 1.5, p < 0.001). The kinetics of PQ transport was altered in cells expressing a genetic polymorphism of hOCT2 (A270S) in comparison with those expressing the reference hOCT2. In addition, the cellular accumulation and cytotoxicity of PQ were also enhanced in cells expressing hMATE1 (-fold increase for uptake was 18 +/- 3.7, p < 0.0001; -fold increase of cytotoxicity was 5.7 +/- 0.5, p < 0.0001). These results suggest that hOCT2 and hMATE1 mediate PQ transport. These transporters may play an important role in the accumulation and renal excretion of PQ, and they may serve as molecular targets for the prevention and treatment of PQ-induced nephrotoxicity.

  6. Organ Correlation in IgG4-Related Diseases

    OpenAIRE

    Koizumi, Satomi; Kamisawa, Terumi; Kuruma, Sawako; Tabata, Taku; Chiba, Kazuro; Iwasaki, Susumu; Kuwata, Go; Fujiwara, Takashi; FUJIWARA, Junko; Arakawa, Takeo; KOIZUMI, Koichi; Momma, Kumiko

    2015-01-01

    IgG4-related disease (IgG4-RD) is a potentially multiorgan disorder. In this study, clinical and serological features from 132 IgG4-RD patients were compared about organ correlations. Underlying pathologies comprised autoimmune pancreatitis (AIP) in 85 cases, IgG4-related sclerosing cholangitis (IgG4-SC) in 12, IgG4-related sialadenitis (IgG4-SIA) in 56, IgG4-related dacryoadenitis (IgG4-DAC) in 38, IgG4-related lymphadenopathy (IgG4-LYM) in 20, IgG4-related retroperitoneal fibrosis (IgG4-RF)...

  7. Neutrophil Fc gamma and complement receptors involved in binding soluble IgG immune complexes and in specific granule release induced by soluble IgG immune complexes.

    Science.gov (United States)

    Voice, J K; Lachmann, P J

    1997-10-01

    We examined the effect of soluble IgG immune complex (IC) characteristics on the binding of IC to human neutrophils and IC-induced specific granule release of neutrophils via Fc gamma receptors (CD16 and CD32) and complement receptors (CR1 and CR3). A set of soluble IgG IC varying in size, IgG subclass, antigen epitope density and complement (C) incorporation were formed between 5-iodo-4-hydroxy-3-nitrophenacetyl (NIP) coupled to bovine serum albumin (BSA) and chimeric mouse-human anti-NIP monoclonal antibodies (mAb) of all four IgG subclasses. High and low epitope density IC of all four IgG subclasses induced specific granule release with C, but in the absence of C only IgG1 and IgG3 IC were functionally active. The Fc gamma and C receptors responsible for IgG IC-induced specific granule release and IC binding were determined using mAb specific for the ligand binding sites of CD16, CD32 and CR3, and recombinant soluble CR1. Each defined IC displayed a unique pattern of receptor preference, dependent upon subclass and antigenic epitope density. IC binding and IC-induced specific granule release was not mediated by the same receptor, or combination of receptors. High and low epitope density IgG3 IC binding and induction of specific granule release was mediated predominantly via CD16. Other IC subclasses bound differently, i.e. IgG1 IC used CD16 and CR3; IgG2 and IgG4 predominantly used complement receptors; but all three induced specific granule release via CD32. In vivo these results may translate into differential activation of neutrophils by soluble IC dependent upon their characteristics, leading to subtle nuances in the etiology, pathology and control of the immune response in IC-related diseases.

  8. Increased transcapillary escape rate of albumin and IgG in essential hypertension

    DEFF Research Database (Denmark)

    Parving, H H; Jensen, H A; Westrup, M

    1977-01-01

    Transcapillary escape rates of albumin and IgG (fractions of intravascular mass of albumin and IgG that pass to the extravascular space per unit time) were determined simultaneously from the initial disappearance of intravenously injected 131I human albumin and 125I human IgG in seven untreated...... subjects suffering from essential hypertension. The average mean arterial blood pressure of these subjects 193/119 mmHg; four subjects had grade I-III funduscopic changes. Transcapillary escape rates of albumin (TERalb) and IgG (TERIgG) were found significantly increased in the hypertensive subjects...

  9. Hydrophobic photolabeling in membranes: The human erythrocyte glucose transporter

    Energy Technology Data Exchange (ETDEWEB)

    Lala, A.K.; Bhat, S. (Indian Institute of Technology Bombay, Powai (India))

    1990-10-01

    Human erythrocyte membranes were labeled with a hydrophobic photoactivable reagent, 2-({sup 3}H)Diazofluorene. Electrophoretic analysis of the protein fraction showed that several membrane spanning proteins like Band 3 (the anion transporter), Band 4.5 (the glucose transporter), and the sialoglycoproteins PAS 1, 2, and 3 have been labeled. To isolate the diazofluorene-labeled glucose transporter, the membrane preparation was solubilized with Triton X-100 and passed through a DEAE-cellulose column. The flow-through fraction was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radioactive analysis of the gel indicated that besides the Band 4.5, two more proteins corresponding to the Band 3 and Band 6 regions also coelute with the glucose transporter in the flow-through fraction. On the other hand, use of n-octyl glucoside gave a relatively better preparation. The 2-({sup 3}H)DAF-labeled glucose transporter isolated by the latter method on tryptic digestion indicated that the Mr 18,000 fragment corresponding to the C-terminal transmembrane fragment is labeled.

  10. Chloride transport in human fibroblasts is activated by hypotonic shock

    Energy Technology Data Exchange (ETDEWEB)

    Rugolo, M.; Mastocola, T.; Flamigni, A.; Lenaz, G. (Universita' di Bologna (Italy))

    1989-05-15

    Incubation of human skin fibroblasts in hypotonic media induced the activation of {sup 36}Cl- efflux which was roughly proportional to the decrease in the osmolality of the media. The efflux of {sup 36}Cl- was insensitive to DIDS plus furosemide and inhibited by addition of a Cl- channel blocker such as 5-nitro-2-(3-phenyl propylamino) benzoic acid (NPPB). We propose that a conductive pathway for Cl- transport, almost silent in isotonic conditions, is activated by exposing human fibroblasts to hypotonic shock, this conclusion being supported by evidence that also {sup 36}Cl- influx was enhanced by hypotonic medium.

  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. Transport and deposition of cohesive pharmaceutical powders in human airway

    Directory of Open Access Journals (Sweden)

    Wang Yuan

    2017-01-01

    Full Text Available Pharmaceutical powders used in inhalation therapy are in the size range of 1-5 microns and are usually cohesive. Understanding the cohesive behaviour of pharmaceutical powders during their transportation in human airway is significant in optimising aerosol drug delivery and targeting. In this study, the transport and deposition of cohesive pharmaceutical powders in a human airway model is simulated by a well-established numerical model which combines computational fluid dynamics (CFD and discrete element method (DEM. The van der Waals force, as the dominant cohesive force, is simulated and its influence on particle transport and deposition behaviour is discussed. It is observed that even for dilute particle flow, the local particle concentration in the oral to trachea region can be high and particle aggregation happens due to the van der Waals force of attraction. It is concluded that the deposition mechanism for cohesive pharmaceutical powders, on one hand, is dominated by particle inertial impaction, as proven by previous studies; on the other hand, is significantly affected by particle aggregation induced by van der Waals force. To maximum respiratory drug delivery efficiency, efforts should be made to avoid pharmaceutical powder aggregation in human oral-to-trachea airway.

  13. Uptake of purines in Plasmodium falciparum-infected human erythrocytes is mostly mediated by the human Equilibrative Nucleoside Transporter and the human Facilitative Nucleobase Transporter

    Directory of Open Access Journals (Sweden)

    Ranford-Cartwright Lisa C

    2010-01-01

    Full Text Available Abstract Background Plasmodium parasites are unable to synthesize purines de novo and have to salvage them from the host. Due to this limitation in the parasite, purine transporters have been an area of focus in the search for anti-malarial drugs. Although the uptake of purines through the human equilibrative nucleoside transporter (hENT1, the human facilitative nucleobase transporter (hFNT1 and the parasite-induced new permeation pathway (NPP has been studied, no information appears to exist on the relative contribution of these three transporters to the uptake of adenosine and hypoxanthine. Using the appropriate transporter inhibitors, the role of each of these salvage pathways to the overall purine transport in intraerythrocytic Plasmodium falciparum was systematically investigated. Methods The transport of adenosine, hypoxanthine and adenine into uninfected and P. falciparum-infected human erythrocytes was investigated in the presence or absence of classical inhibitors of the hFNT1, hENT1 and NPP. The effective inhibition of the various transporters by the classical inhibitors was verified using appropriate known substrates. The ability of high concentration of unlabelled substrates to saturate these transporters was also studied. Results Transport of exogenous purine into infected or uninfected erythrocytes occurred primarily through saturable transporters rather than through the NPP. Hypoxanthine and adenine appeared to enter erythrocytes mainly through the hFNT1 nucleobase transporter whereas adenosine entered predominantly through the hENT1 nucleoside transporter. The rate of purine uptake was approximately doubled in infected cells compared to uninfected erythrocytes. In addition, it was found that the rate of adenosine uptake was considerably higher than the rate of hypoxanthine uptake in infected human red blood cells (RBC. It was also demonstrated that furosemide inhibited the transport of purine bases through hFNT1. Conclusion

  14. Translating chimpanzee personality to humans: Investigating the transportability of chimpanzee-derived personality scales to humans.

    Science.gov (United States)

    Latzman, Robert D; Sauvigné, Katheryn C; Hopkins, William D

    2016-06-01

    There is a growing interest in the study of personality in chimpanzees with repeated findings of a similar structure of personality in apes to that found in humans. To date, however, the direct translational value of instruments used to assess chimpanzee personality to humans has yet to be explicitly tested. As such, in the current study we sought to determine the transportability of factor analytically-derived chimpanzee personality scales to humans in a large human sample (N = 301). Human informants reporting on target individuals they knew well completed chimpanzee-derived and human-derived measures of personality from the two most widely studied models of human personality: Big Five and Big Three. The correspondence between informant-reported chimpanzee- and human-derived personality scales was then investigated. Results indicated high convergence for corresponding scales across most chimpanzee- and human-derived personality scales. Findings from the current study provide evidence that chimpanzee-derived scales translate well to humans and operate quite similarly to the established human-derived personality scales in a human sample. This evidence of transportability lends support to the translational nature of chimpanzee personality research suggesting clear relevance of this growing literature to humans. Am. J. Primatol. 78:601-609, 2016. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  15. IgG4-related disease

    DEFF Research Database (Denmark)

    Detlefsen, Sönke; Klöppel, Günter

    2017-01-01

    alterations have been described under the terms autoimmune pancreatitis (AIP) and sclerosing cholangitis, respectively. These diseases are currently more precisely called IgG4-related pancreatitis (or type 1 AIP to distinguish it from type 2 AIP that is unrelated to IgG4-RD) and IgG4-related sclerosing...... disease (IgG4-RD). The histologic key findings are lymphoplasmacytic infiltration rich in IgG4-positive plasma cells combined with storiform fibrosis and obliterative phlebitis. Among the organs mainly affected by IgG4-RD are the pancreas and the extrahepatic bile ducts. The pancreatic and biliary...... cholangitis (IgG4-related SC). Clinically and grossly, both diseases commonly imitate pancreatic and biliary adenocarcinoma, tumors that are well known for their dismal prognosis. As IgG4-RD responds to steroid treatment, making a resection of a suspected tumor unnecessary, a biopsy is often required...

  16. Podocytes use FcRn to clear IgG from the glomerular basement membrane

    OpenAIRE

    Akilesh, Shreeram; Huber, Tobias B.; Wu, Hui; Wang, Gary; Hartleben, Björn; Kopp, Jeffrey B.; Miner, Jeffrey H.; Roopenian, Derry C.; Unanue, Emil. R.; Shaw, Andrey S.

    2008-01-01

    The glomerular filtration barrier prevents large serum proteins from being lost into the urine. It is not known, however, why the filter does not routinely clog with large proteins that enter the glomerular basement membrane (GBM). Here, we provide evidence that an active transport mechanism exists to remove immunoglobulins that accumulate at the filtration barrier. We found that FcRn, an IgG and albumin transport receptor, is expressed in podocytes and functions to internalize IgG from the G...

  17. An Integrated Hybrid Transportation Architecture for Human Mars Expeditions

    Science.gov (United States)

    Merrill, Raymond G.; Chai, Patrick R.; Qu, Min

    2015-01-01

    NASA's Human Spaceflight Architecture Team is developing a reusable hybrid transportation architecture that uses both chemical and electric propulsion systems on the same vehicle to send crew and cargo to Mars destinations such as Phobos, Deimos, the surface of Mars, and other orbits around Mars. By applying chemical and electrical propulsion where each is most effective, the hybrid architecture enables a series of Mars trajectories that are more fuel-efficient than an all chemical architecture without significant increases in flight times. This paper presents an integrated Hybrid in-space transportation architecture for piloted missions and delivery of cargo. A concept for a Mars campaign including orbital and Mars surface missions is described in detail including a system concept of operations and conceptual design. Specific constraints, margin, and pinch points are identified for the architecture and opportunities for critical path commercial and international collaboration are discussed.

  18. Distribution of vesicular glutamate transporters in the human brain

    Directory of Open Access Journals (Sweden)

    Erika eVigneault

    2015-03-01

    Full Text Available Glutamate is the major excitatory transmitter in the brain. Vesicular glutamate transporters (VGLUT1-3 are responsible for uploading glutamate into synaptic vesicles. VGLUT1 and VGLUT2 are considered as specific markers of canonical glutamatergic neurons, while VGLUT3 is found in neurons previously shown to use other neurotransmitters than glutamate. Although there exists a rich literature on the localization of these glutamatergic markers in the rodent brain, little is currently known about the distribution of VGLUT1-3 in the human brain. In the present study, using subtype specific probes and antisera, we examined the localization of the three vesicular glutamate transporters in the human brain by in situ hybridization, immunoautoradiography and immunohistochemistry. We found that the VGLUT1 transcript was highly expressed in the cerebral cortex, hippocampus and cerebellum, whereas VGLUT2 mRNA was mainly found in the thalamus and brainstem. VGLUT3 mRNA was localized in scarce neurons within the cerebral cortex, hippocampus, striatum and raphe nuclei. Following immunoautoradiographic labeling, intense VGLUT1- and VGLUT2-immunoreactivities were observed in all regions investigated (cerebral cortex, hippocampus, caudate-putamen, cerebellum, thalamus, amygdala, substantia nigra, raphe while VGLUT3 was absent from the thalamus and cerebellum. This extensive mapping of VGLUT1-3 in human brain reveals distributions that correspond for the most part to those previously described in rodent brains.

  19. Molecular determinants of acidic pH-dependent transport of human equilibrative nucleoside transporter 3.

    Science.gov (United States)

    Rahman, Md Fazlur; Askwith, Candice; Govindarajan, Rajgopal

    2017-09-08

    Equilibrative nucleoside transporters (ENTs) translocate hydrophilic nucleosides across cellular membranes and are essential for salvage nucleotide synthesis and purinergic signaling. Unlike the prototypic human ENT members hENT1 and hENT2, which mediate plasma membrane nucleoside transport at pH 7.4, hENT3 is an acidic pH-activated lysosomal transporter partially localized to mitochondria. Recent studies demonstrate that hENT3 is indispensable for lysosomal homeostasis, and that mutations in hENT3 can result in a spectrum of lysosomal storage-like disorders. However, despite hENT3's prominent role in lysosome pathophysiology, the molecular basis of hENT3-mediated transport is unknown. Therefore, we sought to examine the mechanistic basis of acidic pH-driven hENT3 nucleoside transport with site-directed mutagenesis, homology modeling, and [(3)H]adenosine flux measurements in mutant RNA-injected Xenopus oocytes. Scanning mutagenesis of putative residues responsible for pH-dependent transport via hENT3 revealed that the ionization states of Asp-219 and Glu-447, and not His, strongly determined the pH-dependent transport permissible-impermissible states of the transporter. Except for substitution with certain isosteric and polar residues, substitution of either Asp-219 or Glu-447 with any other residues resulted in robust activity that was pH-independent. Dual substitution of Asp-219 and Glu-447 to Ala sustained pH-independent activity over a broad range of physiological pH (pH 5.5-7.4), which also maintained stringent substrate selectivity toward endogenous nucleosides and clinically used nucleoside drugs. Our results suggest a putative pH-sensing role for Asp-219 and Glu-447 in hENT3 and that the size, ionization state, or electronegative polarity at these positions is crucial for obligate acidic pH-dependent activity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. A vaccine of L2 epitope repeats fused with a modified IgG1 Fc induced cross-neutralizing antibodies and protective immunity against divergent human papillomavirus types.

    Directory of Open Access Journals (Sweden)

    Xue Chen

    Full Text Available Current human papillomavirus (HPV major capsid protein L1 virus-like particles (VLPs-based vaccines in clinic induce strong HPV type-specific neutralizing antibody responses. To develop pan-HPV vaccines, here, we show that the fusion protein E3R4 consisting of three repeats of HPV16 L2 aa 17-36 epitope (E3 and a modified human IgG1 Fc scaffold (R4 induces cross-neutralizing antibodies and protective immunity against divergent HPV types. E3R4 was expressed as a secreted protein in baculovirus expression system and could be simply purified by one step Protein A affinity chromatography with the purity above 90%. Vaccination of E3R4 formulated with Freunds adjuvant not only induced cross-neutralizing antibodies against HPV pseudovirus types 16, 18, 45, 52, 58, 6, 11 and 5 in mice, but also protected mice against vaginal challenges with HPV pseudovirus types 16, 45, 52, 58, 11 and 5 for at least eleven months after the first immunization. Moreover, vaccination of E3R4 formulated with FDA approved adjuvant alum plus monophosphoryl lipid A also induced cross-neutralizing antibodies against HPV types 16, 18 and 6 in rabbits. Thus, our results demonstrate that delivery of L2 antigen as a modified Fc-fusion protein may facilitate pan-HPV vaccine development.

  1. Expression of Glucose Transporter Proteins in Human Diabetic Placenta.

    Science.gov (United States)

    Stanirowski, Paweł Jan; Szukiewicz, Dariusz; Pazura-Turowska, Monika; Sawicki, Włodzimierz; Cendrowski, Krzysztof

    2017-06-02

    Gestational diabetes mellitus and pregestational diabetes mellitus constitute carbohydrate metabolism disorders, which, if not diagnosed and adequately treated, lead to serious and often life-threatening pregnancy complications. According to a recently formulated hypothesis, some diabetes-related complications, such as fetal macrosomia, may be the result of disturbances in the transplacental transport of nutrients-in particular, excessive maternal-fetal glucose transfer. Throughout pregnancy, glucose flux across the placenta is mediated by the group of facilitative glucose transporters (GLUT), the expression of which in different placental compartments is the precondition for effective glucose uptake from maternal blood and its subsequent transfer to the fetal circulation. In diabetes-complicated pregnancies, the location, expression and activity of glucose transporters are modified to an extent that results in alterations in the maternal-fetal glucose exchange, potentially leading to an excessive supply of energy substrates to the fetus. This paper reviews the literature on the expression and activity of glucose transporter proteins-GLUT-1, GLUT-3, GLUT-4, GLUT-8, GLUT-9 and GLUT-12-in the human placenta, with a special focus on diabetes-complicated pregnancy. The characteristics of transporters in conditions of maternal normoglycemia and modifications occurring in the diabetic placenta are summarized, and the factors responsible for the regulation of the expression of selected isoforms are described. Finally, the impact of alterations in the placental expression of the aforementioned members of the GLUT family on intrauterine fetal development in pregnancies complicated by diabetes mellitus is discussed. Copyright © 2017 Diabetes Canada. Published by Elsevier Inc. All rights reserved.

  2. Human mobility in space from three modes of public transportation

    Science.gov (United States)

    Jiang, Shixiong; Guan, Wei; Zhang, Wenyi; Chen, Xu; Yang, Liu

    2017-10-01

    The human mobility patterns have drew much attention from researchers for decades, considering about its importance for urban planning and traffic management. In this study, the taxi GPS trajectories, smart card transaction data of subway and bus from Beijing are utilized to model human mobility in space. The original datasets are cleaned and processed to attain the displacement of each trip according to the origin and destination locations. Then, the Akaike information criterion is adopted to screen out the best fitting distribution for each mode from candidate ones. The results indicate that displacements of taxi trips follow the exponential distribution. Besides, the exponential distribution also fits displacements of bus trips well. However, their exponents are significantly different. Displacements of subway trips show great specialties and can be well fitted by the gamma distribution. It is obvious that human mobility of each mode is different. To explore the overall human mobility, the three datasets are mixed up to form a fusion dataset according to the annual ridership proportions. Finally, the fusion displacements follow the power-law distribution with an exponential cutoff. It is innovative to combine different transportation modes to model human mobility in the city.

  3. IgG4-related kidney disease.

    Science.gov (United States)

    Saeki, Takako; Kawano, Mitsuhiro

    2014-02-01

    IgG4-related kidney disease (IgG4-RKD) is a comprehensive term for renal lesions associated with IgG4-related disease, which is a recently recognized clinical entity characterized by a dense lymphoplasmacytic infiltrate rich in IgG4-positive plasma cells with fibrosis affecting several organs. Tubulointerstitial nephritis with increased IgG4-positive plasma cells and fibrosis is the most dominant feature of IgG4-RKD and may cause acute or chronic renal dysfunction, although some glomerular lesions such as membranous nephropathy are sometimes evident. Radiologically, several characteristic abnormalities are often demonstrated, sometimes mimicking malignancies. IgG4-RKD predominantly affects middle-aged to elderly men, and most patients have accompanying IgG4-related extrarenal lesions such as sialadenitis, lymphadenopathy, or type 1 autoimmune pancreatitis. Serology usually demonstrates high levels of serum total IgG and IgG4, and high levels of serum IgE and hypocomplementemia are also frequent features. Corticosteroid therapy is usually quite effective, leading to amelioration of the renal dysfunction and radiological and serological abnormalities. However, as any delay in treatment may result in irreversible renal failure, early diagnosis and appropriate therapy are very important. Despite these distinctive clinicopathological features of IgG4-RKD, its pathogenesis remains poorly understood. Awareness of this condition and accumulation of more cases worldwide are necessary.

  4. IgG Suppresses Antibody Responses in Mice Lacking C1q, C3, Complement Receptors 1 and 2, or IgG Fc-Receptors.

    Science.gov (United States)

    Bergström, Joakim J E; Heyman, Birgitta

    2015-01-01

    Antigen-specific IgG antibodies, passively administered to mice or humans together with large particulate antigens like erythrocytes, can completely suppress the antibody response against the antigen. This is used clinically in Rhesus prophylaxis, where administration of IgG anti-RhD prevents RhD-negative women from becoming immunized against RhD-positive fetal erythrocytes aquired transplacentally. The mechanisms by which IgG suppresses antibody responses are poorly understood. We have here addressed whether complement or Fc-receptors for IgG (FcγRs) are required for IgG-mediated suppression. IgG, specific for sheep red blood cells (SRBC), was administered to mice together with SRBC and the antibody responses analyzed. IgG was able to suppress early IgM- as well as longterm IgG-responses in wildtype mice equally well as in mice lacking FcγRIIB (FcγRIIB knockout mice) or FcγRI, III, and IV (FcRγ knockout mice). Moreover, IgG was able to suppress early IgM responses equally well in mice lacking C1q (C1qA knockout mice), C3 (C3 knockout mice), or complement receptors 1 and 2 (Cr2 knockout mice) as in wildtype mice. Owing to the previously described severely impaired IgG responses in the complement deficient mice, it was difficult to assess whether passively administered IgG further decreased their IgG response. In conclusion, Fc-receptor binding or complement-activation by IgG does not seem to be required for its ability to suppress antibody responses to xenogeneic erythrocytes.

  5. Monocyte Recruitment by HLA IgG‐Activated Endothelium: The Relationship Between IgG Subclass and FcγRIIa Polymorphisms

    National Research Council Canada - National Science Library

    Valenzuela, N. M; Trinh, K. R; Mulder, A; Morrison, S. L; Reed, E. F

    2015-01-01

    ...RIIa polymorphisms, the authors show the ability of different human IgG subclasses to cause endothelial activation and recruitment of monocytes, and evaluate therapeutic potential of IgG with two...

  6. Crystal structure of the human glucose transporter GLUT1

    Science.gov (United States)

    Deng, Dong; Xu, Chao; Sun, Pengcheng; Wu, Jianping; Yan, Chuangye; Hu, Mingxu; Yan, Nieng

    2014-06-01

    The glucose transporter GLUT1 catalyses facilitative diffusion of glucose into erythrocytes and is responsible for glucose supply to the brain and other organs. Dysfunctional mutations may lead to GLUT1 deficiency syndrome, whereas overexpression of GLUT1 is a prognostic indicator for cancer. Despite decades of investigation, the structure of GLUT1 remains unknown. Here we report the crystal structure of human GLUT1 at 3.2 Å resolution. The full-length protein, which has a canonical major facilitator superfamily fold, is captured in an inward-open conformation. This structure allows accurate mapping and potential mechanistic interpretation of disease-associated mutations in GLUT1. Structure-based analysis of these mutations provides an insight into the alternating access mechanism of GLUT1 and other members of the sugar porter subfamily. Structural comparison of the uniporter GLUT1 with its bacterial homologue XylE, a proton-coupled xylose symporter, allows examination of the transport mechanisms of both passive facilitators and active transporters.

  7. Evidence for a Homodimeric Structure of Human Monocarboxylate Transporter 8

    Science.gov (United States)

    Visser, W. Edward; Philp, Nancy J.; van Dijk, Thamar B.; Klootwijk, Wim; Friesema, Edith C. H.; Jansen, Jurgen; Beesley, Philip W.; Ianculescu, Alexandra G.; Visser, Theo J.

    2009-01-01

    The human monocarboxylate transporter 8 (hMCT8) protein mediates transport of thyroid hormone across the plasma membrane. Association of hMCT8 mutations with severe psychomotor retardation and disturbed thyroid hormone levels has established its physiological relevance, but little is still known about the basic properties of hMCT8. In this study we present evidence that hMCT8 does not form heterodimers with the ancillary proteins basigin, embigin, or neuroplastin, unlike other MCTs. In contrast, it is suggested that MCT8 exists as monomer and homodimer in transiently and stably transfected cells. Apparently hMCT8 forms stable dimers because the complex is resistant to denaturing conditions and dithiothreitol. Cotransfection of wild-type hMCT8 with a mutant lacking amino acids 267–360 resulted in formation of homo-and heterodimers of the variants, indicating that transmembrane domains 4–6 are not involved in the dimerization process. Furthermore, we explored the structural and functional role of the 10 Cys residues in hMCT8. All possible Cys>Ala mutants did not behave differently from wild-type hMCT8 in protein expression, cross-linking experiments with HgCl2 and transport function. Our findings indicate that individual Cys residues are not important for the function of hMCT8 or suggest that hMCT8 has other yet-undiscovered functions in which cysteines play an essential role. PMID:19797118

  8. Treatment with a human recombinant monoclonal IgG antibody against oxidized LDL in atherosclerosis-prone pigs reduces cathepsin S in coronary lesions

    DEFF Research Database (Denmark)

    Poulsen, Christian Bo; Al-Mashhadi, Ahmed Ludvigsen; Von Wachenfeldt, Karin

    2016-01-01

    Background Immunization with oxidized LDL (oxLDL) reduces atherosclerosis in rodents. We tested the hypothesis that treatment with a human recombinant monoclonal antibody against oxLDL will reduce the burden or composition of atherosclerotic lesions in hypercholesterolemic minipigs. Methods...... and results Thirty-eight hypercholesterolemic minipigs with defective LDL receptors were injected with an oxLDL antibody or placebo weekly for 12 weeks. An 18F-fluorodeoxyglucose positron emission tomography (FDG PET) scan (n = 9) was performed before inclusion and after 3 months of treatment. Blood samples.......03) with no difference in CD68 or CD163 positivity. Conclusions In hypercholesterolemic minipigs, treatment with a human recombinant monoclonal antibody against oxLDL reduced cathepsin S in coronary lesions without any effect on the burden of atherosclerosis or aortic FDG-PET signal....

  9. Malaria resistance genes are associated with the levels of IgG subclasses directed against Plasmodium falciparum blood-stage antigens in Burkina Faso

    Directory of Open Access Journals (Sweden)

    Afridi Sarwat

    2012-09-01

    Full Text Available Abstract Background HBB, IL4, IL12, TNF, LTA, NCR3 and FCGR2A polymorphisms have been associated with malaria resistance in humans, whereas cytophilic immunoglobulin G (IgG antibodies are thought to play a critical role in immune protection against asexual blood stages of the parasite. Furthermore, HBB, IL4, TNF, and FCGR2A have been associated with both malaria resistance and IgG levels. This suggests that some malaria resistance genes influence the levels of IgG subclass antibodies. Methods In this study, the effect of HBB, IL4, IL12, TNF, LTA, NCR3 and FCGR2A polymorphisms on the levels of IgG responses against Plasmodium falciparum blood-stage extract was investigated in 220 individuals living in Burkina Faso. The Pearson’s correlation coefficient among IgG subclasses was determined. A family-based approach was used to assess the association of polymorphisms with anti-P. falciparum IgG, IgG1, IgG2, IgG3 and IgG4 levels. Results After applying a multiple test correction, several polymorphisms were associated with IgG subclass or IgG levels. There was an association of i haemoglobin C with IgG levels; ii the FcγRIIa H/R131 with IgG2 and IgG3 levels; iii TNF-863 with IgG3 levels; iv TNF-857 with IgG levels; and, v TNF1304 with IgG3, IgG4, and IgG levels. Conclusion Taken together, the results support the hypothesis that some polymorphisms affect malaria resistance through their effect on the acquired immune response, and pave the way towards further comprehension of genetic control of an individual’s humoral response against malaria.

  10. Analytical theory of oxygen transport in the human placenta.

    Science.gov (United States)

    Serov, A S; Salafia, C M; Filoche, M; Grebenkov, D S

    2015-03-07

    We propose an analytical approach to solving the diffusion-convection equations governing oxygen transport in the human placenta. We show that only two geometrical characteristics of a placental cross-section, villi density and the effective villi radius, are needed to predict fetal oxygen uptake. We also identify two combinations of physiological parameters that determine oxygen uptake in a given placenta: (i) the maximal oxygen inflow of a placentone if there were no tissue blocking the flow and (ii) the ratio of transit time of maternal blood through the intervillous space to oxygen extraction time. We derive analytical formulas for fast and simple calculation of oxygen uptake and provide two diagrams of efficiency of oxygen transport in an arbitrary placental cross-section. We finally show that artificial perfusion experiments with no-hemoglobin blood tend to give a two-orders-of-magnitude underestimation of the in vivo oxygen uptake and that the optimal geometry for such setup alters significantly. The theory allows one to adjust the results of artificial placenta perfusion experiments to account for oxygen-hemoglobin dissociation. Combined with image analysis techniques, the presented model can give an easy-to-use tool for prediction of the human placenta efficiency. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Oral fluid, a substitute for serum to monitor measles IgG antibody?

    Directory of Open Access Journals (Sweden)

    Goyal A

    2009-01-01

    Full Text Available We have analyzed the suitability and potential of Oral Fluid (OF to substitute serum in estimating measles IgG antibodies, during community surveys, by comparing the Optical Density (OD of measles IgG antibodies in OF and serum of 100 apparently asymptomatic children. IgG antibody status was determined using commercially available - Measles IgG Capture ELISA. Sensitivity 89.5%, specificity 90.6% Concordance of 89%, coefficient of correlation r is equal to 0.97 (Karl Pearson′s and rho is equal to 0.86 (Spearman′s, was found between OD value of OF and serum. The study emphasizes the potential of OF to surrogate serum in estimating Measles IgG antibody among children. The OF collection is advantageous over blood as it is painless. It is suitable for non-technical staff, easy to transport and less bio-hazardous.

  12. A high sensitive label-free immunosensor for the determination of human serum IgG using overoxidized polypyrrole decorated with gold nanoparticle modified electrode.

    Science.gov (United States)

    Amouzadeh Tabrizi, Mahmoud; Shamsipur, Mojtaba; Mostafaie, Ali

    2016-02-01

    Herein, an electrochemical human immunoglobulin G (HIgG) immunosensor was fabricated using overoxidized polypyrrole decorated with gold nanoparticle (OPPy-Aunano) modified screen printed electrode (OPPy-Aunano/SPE). The results clearly demonstrate that OPPy-Aunano film is very permeable to [Fe(CN)6](3-/4-). Therefore, the fabrication of immunosensor based on permeability of OPPy-Aunano film to [Fe(CN)6](3-/4-) probe is a novel strategy for the fabrication of label free electrochemical immunosensor. The electrochemical impedance spectroscopy (EIS) techniques are used for the determination of HIgG in the concentration range 0.5 to 125.0ngmL(-1) and with a limit of detection of 0.02ngmL(-1), respectively. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Computational Model for Oxygen Transport and Consumption in Human Vitreous

    Science.gov (United States)

    Filas, Benjamen A.; Shui, Ying-Bo; Beebe, David C.

    2013-01-01

    Purpose. Previous studies that measured liquefaction and oxygen content in human vitreous suggested that exposure of the lens to excess oxygen causes nuclear cataracts. Here, we developed a computational model that reproduced available experimental oxygen distributions for intact and degraded human vitreous in physiologic and environmentally perturbed conditions. After validation, the model was used to estimate how age-related changes in vitreous physiology and structure alter oxygen levels at the lens. Methods. A finite-element model for oxygen transport and consumption in the human vitreous was created. Major inputs included ascorbate-mediated oxygen consumption in the vitreous, consumption at the posterior lens surface, and inflow from the retinal vasculature. Concentration-dependent relations were determined from experimental human data or estimated from animal studies, with the impact of all assumptions explored via parameter studies. Results. The model reproduced experimental data in humans, including oxygen partial pressure (Po2) gradients (≈15 mm Hg) across the anterior-posterior extent of the vitreous body, higher oxygen levels at the pars plana relative to the vitreous core, increases in Po2 near the lens after cataract surgery, and equilibration in the vitreous chamber following vitrectomy. Loss of the antioxidative capacity of ascorbate increases oxygen levels 3-fold at the lens surface. Homogeneous vitreous degeneration (liquefaction), but not partial posterior vitreous detachment, greatly increases oxygen exposure to the lens. Conclusions. Ascorbate content and the structure of the vitreous gel are critical determinants of lens oxygen exposure. Minimally invasive surgery and restoration of vitreous structure warrant further attention as strategies for preventing nuclear cataracts. PMID:24008409

  14. Serum IgG2 and tissue IgG2 plasma cell elevation in orbital IgG4-related disease (IgG4-RD): Potential use in IgG4-RD assessment.

    Science.gov (United States)

    Chan, Anita S Y; Mudhar, Hardeep; Shen, Sunny Yu; Lang, Stephanie S; Fernando, Malee; Hilmy, Maryam Hazly; Guppy, Naomi Jayne; Rennie, Ian; Dunkley, Lisa; Al Jajeh, Issam

    2017-03-28

    To determine the role of serum and tissue IgG2 in orbital biopsies with the histological features of IgG4-related disease (IgG4-RD) in comparison with non-IgG4-related orbital inflammatory disorders (OID), including autoimmune disorders. This is an international (Sheffield, UK, and Singapore) collaborative, retrospective case review of 69 patients (38 from Singapore National Eye Centre and 31 from Royal Hallamshire Hospital, Sheffield) with orbital inflammatory biopsies between 2002 and 2016. Clinical information and histology were reviewed and cases were classified into three groups: Group 1: IgG4-RD orbital inflammation (n=43); Group 2: idiopathic OID (n=12) and Group 3: autoimmune OID (n=14). Serum IgG1, IgG2, IgG3 and IgG4 levels were collated where available and immunohistochemistry (IHC) for tissue IgG2 plasma cells was performed. Dual IHC showed IgG2 plasma cells as a distinct population from IgG4 plasma cells. Significant (twofold) serum IgG2 elevation was noted among IgG4-RD (group 1), idiopathic (group 2) and autoimmune OID (group 3). Similarly, significant elevation of tissue IgG2 plasma cells was also seen among IgG4-RD (group 1), idiopathic and autoimmune OID (groups 2 and 3). Significant elevations of serum IgG2 and tissue IgG2 plasma cells are present in orbital IgG4-RD in comparison with non-IgG4 orbital inflammation (idiopathic and autoimmune OID), suggesting that IgG2 may play a role in IgG4-RD. A serum IgG2 cut-off >5.3 g/L was found to be 80% sensitive and 91.7% specific for orbital IgG4-RD, with an accuracy of 0.90. Tissue IgG2 and IgG4 subclass reporting may provide additional insight regarding the 'IgG4-RD' pathogenesis. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  15. Quality management for the international transportation of non-human primates

    Directory of Open Access Journals (Sweden)

    David B. Elmore

    2008-03-01

    Full Text Available Safe and humane transportation of live animals requires dedicated, informed personnel who carefully plan and attend to the details of appropriate animal care and handling throughout the shipping process. Specifically, although transportation of non-human primates shares goals common to all live animal transport, it also poses unique challenges stemming from the nature of these animals. Some of these unique challenges of transporting non-human primates, include the impact of public perception of non-human primates as cargo, maintaining biosecurity of non-human primate cargo, safety of both the non-human primate and public contacts, meeting the vital husbandry needs of varying species of non-human primates and compliance with numerous regulatory agencies, which may have overlapping responsibilities. This discussion will focus on these important considerations, as they relate to the legal international transportation of non-human primates for scientific use.

  16. Efficient production of bispecific IgG of different isotypes and species of origin in single mammalian cells.

    Science.gov (United States)

    Dillon, Michael; Yin, Yiyuan; Zhou, Jianhui; McCarty, Luke; Ellerman, Diego; Slaga, Dionysos; Junttila, Teemu T; Han, Guanghui; Sandoval, Wendy; Ovacik, Meric A; Lin, Kedan; Hu, Zhilan; Shen, Amy; Corn, Jacob E; Spiess, Christoph; Carter, Paul J

    Bispecific IgG production in single host cells has been a much sought-after goal to support the clinical development of these complex molecules. Current routes to single cell production of bispecific IgG include engineering heavy chains for heterodimerization and redesign of Fab arms for selective pairing of cognate heavy and light chains. Here, we describe novel designs to facilitate selective Fab arm assembly in conjunction with previously described knobs-into-holes mutations for preferential heavy chain heterodimerization. The top Fab designs for selective pairing, namely variants v10 and v11, support near quantitative assembly of bispecific IgG in single cells for multiple different antibody pairs as judged by high-resolution mass spectrometry. Single-cell and in vitro-assembled bispecific IgG have comparable physical, in vitro biological and in vivo pharmacokinetics properties. Efficient single-cell production of bispecific IgG was demonstrated for human IgG1, IgG2 and IgG4 thereby allowing the heavy chain isotype to be tailored for specific therapeutic applications. Additionally, a reverse chimeric bispecific IgG2a with humanized variable domains and mouse constant domains was generated for preclinical proof-of-concept studies in mice. Efficient production of a bispecific IgG in stably transfected mammalian (CHO) cells was shown. Individual clones with stable titer and bispecific IgG composition for >120 days were readily identified. Such long-term cell line stability is needed for commercial manufacture of bispecific IgG. The single-cell bispecific IgG designs developed here may be broadly applicable to biotechnology research, including screening bispecific IgG panels, and to support clinical development.

  17. Predicted congestions never occur. On the gap between transport modeling and human behavior

    Directory of Open Access Journals (Sweden)

    Harald FREY

    2011-01-01

    Full Text Available This paper presents an introduction to meso-scale transport modeling and issues of human behaviour in transport systems. Along with other examples of the human ability to learn in transport systems we look at the comparison of real life data and the prediction of modeling tools for the closure of Vienna’s inner ring road during the 2008 European Football Championship (EURO 2008. Some light is shed on the scientific question, whether currently used modeling tools are able to adequately reproduce the real-life behaviour of human beings in the transport system and should be used for transport policy decision making.

  18. Cutoff Values of Serum IgG4 and Histopathological IgG4+ Plasma Cells for Diagnosis of Patients with IgG4-Related Disease

    Directory of Open Access Journals (Sweden)

    Yasufumi Masaki

    2012-01-01

    Full Text Available IgG4-related disease is a new disease classification established in Japan in the 21st century. Patients with IgG4-related disease display hyper-IgG4-gammaglobulinemia, massive infiltration of IgG4+ plasma cells into tissue, and good response to glucocorticoids. Since IgG4 overexpression is also observed in other disorders, it is necessary to diagnose IgG4-related disease carefully and correctly. We therefore sought to determine cutoff values for serum IgG4 and IgG4/IgG and for IgG4+/IgG+ plasma cells in tissue diagnostic of IgG4-related disease. Patients and Methods. We retrospectively analyzed serum IgG4 concentrations and IgG4/IgG ratio and IgG4+/IgG+ plasma cell ratio in tissues of 132 patients with IgG4-related disease and 48 patients with other disorders. Result. Serum IgG4 >135  mg/dl demonstrated a sensitivity of 97.0% and a specificity of 79.6% in diagnosing IgG4-related disease, and serum IgG4/IgG ratios >8% had a sensitivity and specificity of 95.5% and 87.5%, respectively. IgG4+cell/IgG+ cell ratio in tissues >40% had a sensitivity and specificity of 94.4% and 85.7%, respectively. However, the number of IgG4+ cells was reduced in severely fibrotic parts of tissues. Conclusion. Although a recent unanimous consensus of all relevant researchers in Japan recently established the diagnostic criteria for IgG4-related disease, findings such as ours indicate that further discussion is needed.

  19. A phase I pharmacologic study of necitumumab (IMC-11F8), a fully human IgG1 monoclonal antibody directed against EGFR in patients with advanced solid malignancies.

    Science.gov (United States)

    Kuenen, Bart; Witteveen, Petronella O; Ruijter, Rita; Giaccone, Giuseppe; Dontabhaktuni, Aruna; Fox, Floyd; Katz, Terry; Youssoufian, Hagop; Zhu, Junming; Rowinsky, Eric K; Voest, Emile E

    2010-03-15

    This study aimed to determine a maximum tolerated dose (MTD) and recommended dose for disease-directed studies of necitumumab (IMC-11F8), a fully human IgG(1) monoclonal antibody directed at the epidermal growth factor receptor, and to characterize the safety profile, pharmacokinetics, preliminary antitumor activity, and immunogenicity of necitumumab. Patients with advanced solid malignancies were treated with 100 to 1,000 mg (flat dosing) necitumumab followed by a 2-week pharmacokinetics sampling period, before beginning 6-week cycles of therapy. Sixty patients received necitumumab weekly (29 patients) or every other week (31 patients). Two patients receiving 1,000 mg every 2 weeks experienced dose-limiting toxicities (DLT; grade 3 headache), accompanied by grade 3 nausea and vomiting in one patient. Occurring hours after the initial dose, these DLTs established 800 mg as the MTD. Mild dose-related skin toxicity was the most common drug-related toxicity (80%). One patient in each arm experienced grade 3 acneform rash, which responded to oral antibiotics and topical therapy. Toxicity was similar on both schedules. Necitumumab exhibited saturable elimination and nonlinear pharmacokinetics. At 800 mg (both arms), its half-life was approximately 7 days. All patients treated with >or=600 mg necitumumab achieved target trough concentrations (>or=40 microg/mL). Antibodies against necitumumab were not detected. Partial response and stable disease were experienced by 2 and 16 patients, respectively. Well tolerated, necitumumab is associated with preliminary evidence of antitumor activity, and achieves biologically relevant concentrations throughout the dosing period. The recommended dose of necitumumab for further clinical development is 800 mg (flat dose) weekly or every 2 weeks based on the clinical setting.

  20. IgG from amyotrophic lateral sclerosis affects tubular calcium channels of skeletal muscle.

    Science.gov (United States)

    Delbono, O; García, J; Appel, S H; Stefani, E

    1991-06-01

    Amyotrophic lateral sclerosis (ALS) is a devastating human disease of upper and lower motoneurons. We studied the action of the immunoglobulin G (IgG) from ALS and disease control patients on dihydropyridine (DHP)-sensitive Ca2+ channels in single mammalian skeletal muscle fibers with the double Vaseline gap technique. The peak of the Ca2+ current (ICa) and the charge movement were reduced when the fibers were incubated in ALS IgG. These effects were lost when the IgG was boiled or adsorbed with skeletal tubular membranes. ALS IgG reduced skeletal muscle ICa in a similar fashion as nifedipine; the ICa blockade was voltage dependent, and the associated charge movement was reduced. These observations suggest that IgG from ALS patients reacts with the skeletal muscle DHP-sensitive Ca2+ channels or some associated regulatory moiety.

  1. Histopathologie der IgG4-RD

    DEFF Research Database (Denmark)

    Detlefsen, S; Klöppel, G

    2016-01-01

    ). As IgG4-RD responds well to steroid treatment but imitates a tumor in many organs, particularly in the pancreas, a biopsy for confirmation of the diagnosis is often warranted. The histological criteria for IgG4-RD as defined in 2011 are based on the following main features: 1) dense lymphoplasmacytic...

  2. The characteristics of specific IgG to phthalic anhydride (PA)-albumin conjugate.

    Science.gov (United States)

    Hatanaka, S; Imamura, I; Terazawa, Y; Kuroda, K; Endo, G

    1997-10-01

    Phthalic anhydride (PA), used in the chemical industry, binds to proteins and causes allergic reactions. It is important to study the characteristics of antibody to PA-protein. We produced specific IgG against PA-rabbit serum albumin (RSA) by administering subcutaneous injections of PA-RSA conjugate to two rabbits. Both rabbits' sera had high titers of IgG not only to PA-RSA but also to PA-human serum albumin (HSA) and HSA. In enzyme-linked immunosorbent assay (ELISA) and ELISA HSA inhibition, specific IgG to PA-HSA revealed cross-reactivity to three other phthalyl anhydride conjugates, hexahydrophthalic anhydride (HHPA)-HSA, methylhexahydrophthalic anhydride (MHHPA)-HSA, and methyltetrahydrophthalic anhydride (MTHPA)-HSA, in both sera. Titers of IgG to HHPA-HSA, MHHPA-HSA, and MTHPA-HSA were not significantly different. On affinity chromatography, highly specific IgG to PA hapten alone was purified. In the serum not binding to PA column, specific IgG to PA-HSA was significantly less than in original serum, but levels of specific IgG to other phthalyl anhydride-HSA were unchanged. Rabbits immunized with PA-RSA produced at least two types of IgG: one is to PA hapten alone and the other may be against new antigenic determinants (NADs) on HSA.

  3. Transport sector decarbonisation - a social sciences and humanities annotated bibliography

    OpenAIRE

    Buchmann, Katrin; Rosalyn A. V. Robison; Foulds, Chris

    2017-01-01

    The challenge: \\ud * By 2014, transport had overtaken power companies as the sector with the highest carbon emissions across the European Union (EU). \\ud * From 1990 to 2014, EU road transport emissions rose by 17% and aviation emissions by 82%. Road transport accounted for 70% of EU transport emissions in 2014. \\ud Aim: \\ud * European energy policy has so far mainly relied on research from Science, Technology, Engineering and Mathematics (STEM) disciplines. Energy-related Social Sciences and...

  4. Diagnostic Value of Serum IgG4 for IgG4-Related Disease

    OpenAIRE

    Hao, Mingju; Liu, Min; Fan, Gaowei; YANG, XIN; Li, Jinming

    2016-01-01

    Abstract Many studies about serum IgG4 for the diagnosis of IgG4-related disease (IgG4-RD) have been reported. However, these studies had relatively small sample sizes and the diagnostic accuracy values varied much between them. The aim of this study was to perform a meta-analysis to evaluate the diagnostic value of serum IgG4 for IgG4-RD. We conducted a search of relevant articles using MEDLINE, EMBASE, Web of Science, SCOPUS, and Cochrane Library databases published before December 2015. St...

  5. Comprehensive diagnostic criteria for IgG4-related disease (IgG4-RD), 2011.

    Science.gov (United States)

    Umehara, Hisanori; Okazaki, Kazuichi; Masaki, Yasufumi; Kawano, Mitsuhiro; Yamamoto, Motohisa; Saeki, Takako; Matsui, Shoko; Yoshino, Tadashi; Nakamura, Shigeo; Kawa, Shigeyuki; Hamano, Hideaki; Kamisawa, Terumi; Shimosegawa, Toru; Shimatsu, Akira; Nakamura, Seiji; Ito, Tetsuhide; Notohara, Kenji; Sumida, Takayuki; Tanaka, Yoshiya; Mimori, Tsuneyo; Chiba, Tsutomu; Mishima, Michiaki; Hibi, Toshifumi; Tsubouchi, Hirohito; Inui, Kazuo; Ohara, Hirotaka

    2012-02-01

    IgG4-related disease (IgG4-RD) is a novel clinical disease entity characterized by elevated serum IgG4 concentration and tumefaction or tissue infiltration by IgG4+ plasma cells. Although IgG4-RD is not rare and is clinically important, its clinical diagnostic criteria have not been established. Comprehensive diagnostic criteria for IgG4-RD, including the involvement of various organs, are intended for the practical use of general physicians and nonspecialists. Two IgG4-RD study groups, the Umehara and Okazaki teams, were organized by the Ministry of Health, Labor and Welfare Japan. As IgG4-RD comprises a wide variety of diseases, these groups consist of physicians and researchers in various disciplines, including rheumatology, hematology, gastroenterology, nephrology, pulmonology, ophthalmology, odontology, pathology, statistics, and basic and molecular immunology throughout Japan, with 66 and 56 members of the Umehara and Okazaki teams, respectively. Collaborations of the two study groups involved detailed analyses of clinical symptoms, laboratory results, and biopsy specimens of patients with IgG4-RD, resulting in the establishment of comprehensive diagnostic criteria for IgG4-RD. Although many patients with IgG4-RD have lesions in several organs, either synchronously or metachronously, and the pathological features of each organ differ, consensus has been reached on two diagnostic criteria for IgG4RD: (1) serum IgG4 concentration >135 mg/dl, and (2) >40% of IgG+ plasma cells being IgG4+ and >10 cells/high powered field of biopsy sample. Although the comprehensive diagnostic criteria are not sufficiently sensitive for the diagnosis of type 1 IgG4-related autoimmune pancreatitis (IgG4-related AIP), they are adequately sensitive for IgG4-related Mikulicz's disease (MD) and kidney disease (KD). In addition, the comprehensive diagnostic criteria, combined with organ-specific diagnostic criteria, have increased the sensitivity of diagnosis to 100% for IgG4-related MD

  6. Multispecific amphipathic substrate transport by an organic anion transporter of human liver

    NARCIS (Netherlands)

    Bossuyt, [No Value; Muller, M; Meier, PJ

    1996-01-01

    Background: Hepatic uptake of differently charged amphipathic endo- and xenobiotics is thought to occur via distinct carrier-mediated transport systems, Alternatively, a single rat organic anion transporting polypeptide (oatp) has recently been demonstrated to mediate hepatocellular uptake of

  7. Functional characterization of the high affinity IgG Receptor : making heads and tails of FcγRI

    NARCIS (Netherlands)

    van der Poel, C.E.

    2011-01-01

    This thesis focuses on human FcγRI, a high affinity receptor for antibodies of the IgG isotype. IgG is the most abundant antibody type in blood and all currently FDA approved therapeutic antibodies are of the IgG isotype. FcγRI, a member of the activating Fcγ receptors, exists as a complex of a

  8. Cellular Localization and Trafficking of the Human ABCG1 Transporter

    Science.gov (United States)

    Neufeld, Edward B.; O’Brien, Katherine; Walts, Avram D.; Stonik, John A.; Demosky, Steven J.; Malide, Daniela; Combs, Christian A.; Remaley, Alan T.

    2014-01-01

    We have developed a suitable heterologous cell expression system to study the localization, trafficking, and site(s) of function of the human ABCG1 transporter. Increased plasma membrane (PM) and late endosomal (LE) cholesterol generated by ABCG1 was removed by lipoproteins and liposomes, but not apoA-I. Delivery of ABCG1 to the PM and LE was required for ABCG1-mediated cellular cholesterol efflux. ABCG1 LEs frequently contacted the PM, providing a collisional mechanism for transfer of ABCG1-mobilized cholesterol, similar to ABCG1-mediated PM cholesterol efflux to lipoproteins. ABCG1-mobilized LE cholesterol also trafficked to the PM by a non-vesicular pathway. Transfer of ABCG1-mobilized cholesterol from the cytoplasmic face of LEs to the PM and concomitant removal of cholesterol from the outer leaflet of the PM bilayer by extracellular acceptors suggests that ABCG1 mobilizes cholesterol on both sides of the lipid bilayer for removal by acceptors. ABCG1 increased uptake of HDL into LEs, consistent with a potential ABCG1-mediated cholesterol efflux pathway involving HDL resecretion. Thus, ABCG1 at the PM mobilizes PM cholesterol and ABCG1 in LE/LYS generates mobile pools of cholesterol that can traffic by both vesicular and non-vesicular pathways to the PM where it can also be transferred to extracellular acceptors with a lipid surface. PMID:25405320

  9. Ligand induced conformational changes of the human serotonin transporter revealed by molecular dynamics simulations.

    Directory of Open Access Journals (Sweden)

    Heidi Koldsø

    Full Text Available The competitive inhibitor cocaine and the non-competitive inhibitor ibogaine induce different conformational states of the human serotonin transporter. It has been shown from accessibility experiments that cocaine mainly induces an outward-facing conformation, while the non-competitive inhibitor ibogaine, and its active metabolite noribogaine, have been proposed to induce an inward-facing conformation of the human serotonin transporter similar to what has been observed for the endogenous substrate, serotonin. The ligand induced conformational changes within the human serotonin transporter caused by these three different types of ligands, substrate, non-competitive and competitive inhibitors, are studied from multiple atomistic molecular dynamics simulations initiated from a homology model of the human serotonin transporter. The results reveal that diverse conformations of the human serotonin transporter are captured from the molecular dynamics simulations depending on the type of the ligand bound. The inward-facing conformation of the human serotonin transporter is reached with noribogaine bound, and this state resembles a previously identified inward-facing conformation of the human serotonin transporter obtained from molecular dynamics simulation with bound substrate, but also a recently published inward-facing conformation of a bacterial homolog, the leucine transporter from Aquifex Aoelicus. The differences observed in ligand induced behavior are found to originate from different interaction patterns between the ligands and the protein. Such atomic-level understanding of how an inhibitor can dictate the conformational response of a transporter by ligand binding may be of great importance for future drug design.

  10. IgG4-Related Disease Manifesting as Interstitial Nephritis Accompanied by Hypophysitis

    Science.gov (United States)

    Matsuda, Ken; Saito, Ayako; Takeuchi, Yoichi; Fukami, Hirotaka; Sato, Hiroyuki; Nagasawa, Tasuku

    2017-01-01

    Patient: Male, 85 Final Diagnosis: IgG4-related disease Symptoms: Renal failure Medication: — Clinical Procedure: — Specialty: Nephrology Objective: Rare disease Background: IgG4-related disease is a systemic disease with marked infiltration of IgG4-positive plasma cells into affected organs and elevated serum IgG4. On clinical examination, swelling, nodules, and hypertrophic lesions might appear simultaneously or metachronously in different organs. Case Report: An 85-year-old man with sudden-onset polydipsia and polyuria insipidus was transported to our hospital because of hypothermia and general malaise. Laboratory tests revealed renal failure and central diabetes insipidus. According to his serum IgG4 level, the patient was diagnosed with possible IgG4-related kidney disease accompanied by IgG4-related hypophysitis. Abdominal contrast-enhanced computed tomography, hypophysis magnetic resonance imaging, and histological examination of the kidney were performed. Glucocorticoid therapy was administered and his renal function improved gradually. However, his central diabetes insipidus did not improve. Conclusions: Glucocorticoid therapy showed different therapeutic effects on the kidney and posterior lobe of the hypophysis. It is possible that glucocorticoid therapy needs to be supported by other immunomodulatory therapies to have an effect on all affected organs. PMID:28552902

  11. Targeting human prostate cancer with In-111-labeled D2B IgG, F(ab ')(2) and Fab fragments in nude mice with PSMA-expressing xenografts

    NARCIS (Netherlands)

    Lutje, Susanne; van Rij, Catharina M.; Franssen, Gerben M.; Fracasso, Giulio; Helfrich, Wijnand; Eek, Annemarie; Oyen, Wim J.; Colombatti, Marco; Boerman, Otto C.

    2015-01-01

    D2B is a new monoclonal antibody directed against an extracellular domain of prostate-specific membrane antigen (PSMA), which is overexpressed in prostate cancer. The potential of D2B IgG, and F(ab)(2) and Fab fragments of this antibody for targeting prostate cancer was determined in mice bearing

  12. IgG subclass deficiencies in children: Facts and fiction.

    Science.gov (United States)

    Wahn, Volker; von Bernuth, Horst

    2017-09-01

    The chance to analyse the four IgG subclasses arose with the publication of Terry and Fahey(1) . Since then, a lot of new information on the role of subclasses and their deficiency states in humans has been obtained. This review tries to analyse critically our current knowledge of subclass deficiencies in children. © 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.

  13. Inhibition of human GLUT1 and GLUT5 by plant carbohydrate products; insights into transport specificity

    OpenAIRE

    George Thompson, Alayna M.; Iancu, Cristina V.; Thi Thanh Hanh Nguyen; Doman Kim; Jun-yong Choe

    2015-01-01

    Glucose transporters GLUT1 (transports glucose) and GLUT5 (transports fructose), in addition to their functions in normal metabolism, have been implicated in several diseases including cancer and diabetes. While GLUT1 has several inhibitors, none have been described for GLUT5. By transport activity assays we found two plant products, rubusoside (from Rubus suavissimus) and astragalin-6-glucoside (a glycosylated derivative of astragalin, from Phytolacca americana) that inhibited human GLUT5. T...

  14. Anti-ApoA-1 IgG serum levels predict worse poststroke outcomes.

    Science.gov (United States)

    Carbone, Federico; Satta, Nathalie; Montecucco, Fabrizio; Virzi, Julien; Burger, Fabienne; Roth, Aline; Roversi, Gloria; Tamborino, Carmine; Casetta, Ilaria; Seraceni, Silva; Trentini, Alessandro; Padroni, Marina; Dallegri, Franco; Lalive, Patrice H; Mach, François; Fainardi, Enrico; Vuilleumier, Nicolas

    2016-09-01

    Autoantibodies to apolipoprotein A-1 (anti-ApoA-1 IgG) were shown to predict major adverse cardiovascular events and promote atherogenesis. However, their potential relationship with clinical disability and ischaemic lesion volume after acute ischaemic stroke (AIS) remains unexplored. We included n = 76 patients admitted for AIS and we investigated whether baseline serum anti-ApoA-1 IgG levels could predict (i) AIS-induced clinical disability [assessed by the modified Rankin Scale (mRS)], and (ii) AIS-related ischaemic lesion volume [assessed by Computed Tomography (CT)]. We also evaluated the possible pro-apoptotic and pro-necrotic effects of anti-ApoA-1 IgG on human astrocytoma cell line (U251) using flow cytometry. High levels of anti-ApoA-1 IgG were retrieved in 15·8% (12/76) of patients. Increased baseline levels of anti-ApoA-1 IgG were independently correlated with worse mRS [β = 0·364; P = 0·002; adjusted odds ratio (OR): 1·05 (95% CI 1·01-1·09); P = 0·017] and CT-assessed ischaemic lesion volume [β = 0·333; P IgG. Incubating human astrocytoma cells with anti-ApoA-1 IgG dose dependently induced necrosis and apoptosis of U251 cells in vitro. Anti-ApoA-1 IgG serum levels at AIS onset are associated with poorer clinical recovery and worse brain lesion volume 3 months after AIS. These observations could be partly explained by the deleterious effect of anti-ApoA-1 IgG on human brain cell survival in vitro and may have clinical implication in the prediction of poor outcome in AIS. © 2016 Stichting European Society for Clinical Investigation Journal Foundation.

  15. Adverse effects of IgG therapy.

    Science.gov (United States)

    Berger, Melvin

    2013-01-01

    IgG is widely used for patients with immune deficiencies and in a broad range of autoimmune and inflammatory disorders. Up to 40% of intravenous infusions of IgG may be associated with adverse effects (AEs), which are mostly uncomfortable or unpleasant but often are not serious. The most common infusion-related AE is headache. More serious reactions, including true anaphylaxis and anaphylactoid reactions, occur less frequently. Most reactions are related to the rate of infusion and can be prevented or treated just by slowing the infusion rate. Medications such as nonsteroidal anti-inflammatory drugs, antihistamines, or corticosteroids also may be helpful in preventing or treating these common AEs. IgA deficiency with the potential of IgG or IgE antibodies against IgA increases the risk of some AEs but should not be viewed as a contraindication if IgG therapy is needed. Potentially serious AEs include renal dysfunction and/or failure, thromboembolic events, and acute hemolysis. These events usually are multifactorial, related to combinations of constituents in the IgG product as well as risk factors for the recipient. Awareness of these factors should allow minimization of the risks and consequences of these AEs. Subcutaneous IgG is absorbed more slowly into the circulation and has a lower incidence of AEs, but awareness and diligence are necessary whenever IgG is administered. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  16. Adenosine triphosphate-dependent copper transport in human liver

    NARCIS (Netherlands)

    vandenBerg, GJ; Wolters, H; Veld, GI; Slooff, MJH; Heymans, GSA; Kuipers, F; Vonk, RJ

    Background/Aim: The recent cloning and sequencing of the Wilson disease gene indicates that hepatic copper (Cu) transport is mediated by a P-type ATPase. The location of this Cu-transporting protein within the hepatocyte is not known; in view of its proposed function and current concepts of hepatic

  17. Thyroid hormone transporters and deiodinases in the developing human hypothalamus

    NARCIS (Netherlands)

    Friesema, E.C.; Visser, T.J.; Borgers, A.J.F.; Kalsbeek, A.; Swaab, D.F.; Fliers, E.; Alkemade, A.

    2012-01-01

    OBJECTIVE: Thyroid hormone (TH) signaling in brain cells is dependent on transport of TH across the plasma membrane followed by intracellular deiodination and binding to the nuclear TH receptors. The aim of this study is to investigate the expression of the specific TH transporters monocarboxylate

  18. Legal Policy Of Indonesia's Air Transportations Human Resources Development In Facing ASEAN Economic Society 2015

    OpenAIRE

    Lestari, Endang Puji

    2016-01-01

    Legal policy of development and the provision of law flight human resources belong to national transportation policy subs-system. Legal policy contained in Act No. 7 of 2007 about RPJP puts the development of air transportation as one of the parts of the national facilities and infrastructure development. In the regime of flight law, human resources management is arranged in separated sections that belong to the national system development. The scope of human resources development and setting...

  19. Mutational scanning of the human serotonin transporter reveals fast translocating serotonin transporter mutants

    DEFF Research Database (Denmark)

    Kristensen, Anders S; Larsen, Mads B; Johnsen, Laust B

    2004-01-01

    The serotonin transporter (SERT) belongs to a family of sodium-chloride-dependent transporters responsible for uptake of amino acids and biogenic amines from the extracellular space. SERT represents a major pharmacological target in the treatment of several clinical conditions, including depression...

  20. Transplacental transport of IgG antibodies specific for pertussis, diphtheria, tetanus, haemophilus influenzae type b, and Neisseria meningitidis serogroup C is lower in preterm compared with term infants

    NARCIS (Netherlands)

    van den Berg, Jolice P.; Westerbeek, Elisabeth A. M.; Berbers, Guy A. M.; van Gageldonk, Pieter G. M.; van der Klis, Fiona R. M.; van Elburg, Ruurd M.

    2010-01-01

    Maternal antibodies, transported through the placenta during pregnancy, contribute to the protection of infants from infectious diseases during the first months of life. The aim of this study was to measure the concentration of antibodies against several vaccine-preventable diseases in paired

  1. ATP-dependent transport of statins by human and rat MRP2/Mrp2

    Energy Technology Data Exchange (ETDEWEB)

    Ellis, Lucy C.J., E-mail: Luc_ellis@yahoo.co.uk [Section of Translational Medicine, Division of Applied Biology, Polwarth Building, Foresterhill, Aberdeen AB25 2ZD (United Kingdom); Hawksworth, Gabrielle M. [Section of Translational Medicine, Division of Applied Biology, Polwarth Building, Foresterhill, Aberdeen AB25 2ZD (United Kingdom); Weaver, Richard J. [Biologie Servier, Drug Safety Research Centre, 905 Route de Saran, 45520 Gidy (France)

    2013-06-01

    Multidrug resistance associated protein-2, MRP2 (human), Mrp2 (rat) are an efflux transporter, responsible for the transport of numerous endogenous and xenobiotic compounds including taurocholate, methotrexate and carboxydichlorofluorescein (CDF). The present study aims to characterise transport of statins by human and rat MRP2/Mrp2 using membrane and vesicle preparations. All statins tested (simvastatin, pravastatin, pitavastatin, fluvastatin, atorvastatin, lovastatin and rosuvastatin) stimulated vanadate-sensitive ATPase activity in membranes expressing human or rat MRP2/Mrp2, suggesting that all statins are substrates of human and rat MRP2/Mrp2. The substrate affinity (Km) of all statins for MRP2/Mrp2 was comparable and no correlation between lipophilicity (logD{sub 7.0}) and Km was seen. All statins also inhibited uptake of the fluorescent Mrp2 substrate, CDF (1 μM) into vesicles expressing human or rat MRP2/Mrp2 with similar IC{sub 50} values. Fitting of the inhibitory data to the hill slope equation, gave hill coefficients (h) of greater than one, suggesting that transport involved more than one binding site for inhibitors of MPR2 and Mrp2. We conclude that statins were transported by both human and rat MRP2/Mrp2 with similar affinity. Statins were also shown to compete with other substrates for transport by MRP2/Mrp2 and that this transport involved more than one binding site on the Mrp2/MRP2 protein. - Highlights: • We characterised MRP2 (human)/Mrp2 (rat)-mediated transport of statins. • We show statins were transported by human and rat MRP2/Mrp2. • Statins competed with a known substrate for transport by MRP2/Mrp2. • Competition involved more than one binding site on the MRP2/Mrp2 protein.

  2. Chronic glomerulonephritis associated with IgG subclass deficiency.

    Science.gov (United States)

    Kamei, Koichi; Nakagawa, Atsuko; Otsuka, Yasufumi; Nakayama, Makiko; Kobayashi, Shinichi; Matsuoka, Kentaro; Iijima, Kazumoto

    2007-08-01

    We experienced two patients with IgG subclass deficiency who suffered from chronic glomerulonephritis (GN). Patient 1 was a 17-year-old girl with IgG subclass deficiency (combined deficiency of IgG2 and IgG4). Renal biopsy was performed when she was aged 16 years, and she was diagnosed with membranoproliferative GN. Patient 2 was a 16-year-old girl with IgG subclass deficiency (combined deficiency of IgG2, IgG3, and IgG4). Renal biopsy was performed when she was aged 15 years, and she was diagnosed with membranous nephropathy. We examined the glomerular deposition patterns of their IgG subclasses. Furthermore, we compared their clinical and laboratory findings with those of three patients with IgG subclass deficiency without GN. Patients with GN suffered infections more frequently than those without GN. The serum levels of IgG (especially IgG1) and IgM were higher in patients with GN than in those without GN. In patient 1 IgG1 and IgG3 were deposited in a mesangiocapillary pattern, but, in patient 2, only IgG1 was deposited in a capillary pattern. Thus, the different patterns of IgG subclass deficiency between the two patients may be responsible for their different glomerular pathologies. To the best of our knowledge, this is the first report of chronic GN in patients with IgG subclass deficiency.

  3. Human immunodeficiency virus impairs reverse cholesterol transport from macrophages.

    Directory of Open Access Journals (Sweden)

    Zahedi Mujawar

    2006-10-01

    Full Text Available Several steps of HIV-1 replication critically depend on cholesterol. HIV infection is associated with profound changes in lipid and lipoprotein metabolism and an increased risk of coronary artery disease. Whereas numerous studies have investigated the role of anti-HIV drugs in lipodystrophy and dyslipidemia, the effects of HIV infection on cellular cholesterol metabolism remain uncharacterized. Here, we demonstrate that HIV-1 impairs ATP-binding cassette transporter A1 (ABCA1-dependent cholesterol efflux from human macrophages, a condition previously shown to be highly atherogenic. In HIV-1-infected cells, this effect was mediated by Nef. Transfection of murine macrophages with Nef impaired cholesterol efflux from these cells. At least two mechanisms were found to be responsible for this phenomenon: first, HIV infection and transfection with Nef induced post-transcriptional down-regulation of ABCA1; and second, Nef caused redistribution of ABCA1 to the plasma membrane and inhibited internalization of apolipoprotein A-I. Binding of Nef to ABCA1 was required for down-regulation and redistribution of ABCA1. HIV-infected and Nef-transfected macrophages accumulated substantial amounts of lipids, thus resembling foam cells. The contribution of HIV-infected macrophages to the pathogenesis of atherosclerosis was supported by the presence of HIV-positive foam cells in atherosclerotic plaques of HIV-infected patients. Stimulation of cholesterol efflux from macrophages significantly reduced infectivity of the virions produced by these cells, and this effect correlated with a decreased amount of virion-associated cholesterol, suggesting that impairment of cholesterol efflux is essential to ensure proper cholesterol content in nascent HIV particles. These results reveal a previously unrecognized dysregulation of intracellular lipid metabolism in HIV-infected macrophages and identify Nef and ABCA1 as the key players responsible for this effect. Our findings

  4. Transport of gaseous pollutants around a human body in quiescent indoor environment

    DEFF Research Database (Denmark)

    Licina, Dusan; Melikov, Arsen Krikor; Mioduszewski, Pawel

    2014-01-01

    (CBL) to transport the pollution in quiescent indoor environment. A human body is resembled by a thermal manikin with a body shape and surface temperature distribution of a real person. The objective of the study is to examine the impact of the pollutant location around the human body on the pollution...... concentration levels in the breathing zone. The results show that the location of the pollution source has a considerable influence of the breathing zone concentrations. This is contributed to the human CBL, as it pulls the pollution emitted close to the human body and transports it to the breathing zone...... the human body should be recognized in ventilation design practice....

  5. Drug Transporter Expression and Activity in Human Hepatoma HuH-7 Cells

    Directory of Open Access Journals (Sweden)

    Elodie Jouan

    2016-12-01

    Full Text Available Human hepatoma cells may represent a valuable alternative to the use of human hepatocytes for studying hepatic drug transporters, which is now a regulatory issue during drug development. In the present work, we have characterized hepatic drug transporter expression, activity and regulation in human hepatoma HuH-7 cells, in order to determine the potential relevance of these cells for drug transport assays. HuH-7 cells displayed notable multidrug resistance-associated protein (MRP activity, presumed to reflect expression of various hepatic MRPs, including MRP2. By contrast, they failed to display functional activities of the uptake transporters sodium taurocholate co-transporting polypeptide (NTCP, organic anion-transporting polypeptides (OATPs and organic cation transporter 1 (OCT1, and of the canalicular transporters P-glycoprotein and breast cancer resistance protein (BCRP. Concomitantly, mRNA expressions of various sinusoidal and canalicular hepatic drug transporters were not detected (NTCP, OATP1B1, organic anion transporter 2 (OAT2, OCT1 and bile salt export pump or were found to be lower (OATP1B3, OATP2B1, multidrug and toxin extrusion protein 1, BCRP and MRP3 in hepatoma HuH-7 cells than those found in human hepatocytes, whereas other transporters such as OAT7, MRP4 and MRP5 were up-regulated. HuH-7 cells additionally exhibited farnesoid X receptor (FXR- and nuclear factor erythroid 2-related factor 2 (Nrf2-related up-regulation of some transporters. Such data indicate that HuH-7 cells, although expressing rather poorly some main hepatic drug transporters, may be useful for investigating interactions of drugs with MRPs, notably MRP2, and for studying FXR- or Nrf2-mediated gene regulation.

  6. Ecological Design of Cooperative Human-Machine Interfaces for Safety of Intelligent Transport Systems

    Directory of Open Access Journals (Sweden)

    Orekhov Aleksandr

    2016-01-01

    Full Text Available The paper describes research results in the domain of cooperative intelligent transport systems. The requirements for human-machine interface considering safety issue of for intelligent transport systems (ITSare analyzed. Profiling of the requirements to cooperative human-machine interface (CHMI for such systems including requirements to usability and safety is based on a set of standards for ITSs. An approach and design technique of cooperative human-machine interface for ITSs are suggested. The architecture of cloud-based CHMI for intelligent transport systems has been developed. The prototype of software system CHMI4ITSis described.

  7. The inhibitory effects of five alkaloids on the substrate transport mediated through human organic anion and cation transporters.

    Science.gov (United States)

    Shams, Tahiatul; Lu, Xiaoxi; Zhu, Ling; Zhou, Fanfan

    2018-02-01

    1. Human solute carrier transporters (SLCs) are important membrane proteins mediate the cellular transport of many endogenous and exogenous substances. Organic anion/cation transporters (OATs/OCTs) and organic anion transporting polypeptides (OATPs) are essential SLCs involved in drug influx. Drug-drug/herb interactions through competing for specific SLCs often lead to unsatisfied therapeutic outcomes and/or unwanted side effects. In this study, we comprehensively investigated the inhibitory effects of five clinically relevant alkaloids (dendrobine, matrine, oxymatrine, tryptanthrin and chelerythrine) on the substrate transport through several OATs/OCTs and OATPs. 2. We performed transport functional assay and kinetic analysis on the HEK-293 cells over-expressing each SLC gene. 3. Our data showed tryptanthrin significantly inhibited the transport activity of OAT3 (IC 50  = 0.93 ± 0.22 μM, K i  = 0.43 μM); chelerythrine acted as a potent inhibitor to the substrate transport mediated through OATP1A2 (IC 50  = 0.63 ± 0.43 μM, K i  = 0.60 μM), OCT1 (IC 50  = 13.60 ± 2.81 μM) and OCT2 (IC 50  =10.80 ± 1.16 μM). 4. Our study suggested tryptanthrin and chelerythrine could potently impact on the drug transport via specific OATs/OCTs. Therefore, the co-administration of these alkaloids with drugs could have clinical consequences due to drug-drug/herb interactions. Precautions should be warranted in the multi-drug therapies involving these alkaloids.

  8. Gene Expression Profiles Suggest Iron Transport Pathway in the Lactating Human Epithelial Cell.

    Science.gov (United States)

    Cai, Chenxi; Eck, Peter; Friel, James K

    2017-03-01

    The molecular background of iron excretion into breast milk has not been determined in humans. We determined the expression of known iron transporters in mRNA extracted from human milk fat globules to deduce which known transporters are responsible for iron excretion into human milk. The expression of iron transporters in mRNA from human milk fat globules and mouse mammary epithelial cell lines was determined by quantitative real-time polymerase chain reaction. The expression of the transferrin receptor 1 (TFRC), divalent metal transporter 1 (SLC11A2), transferrin (TF), and lactoferrin (LTF) was confirmed in RNA isolated from the human milk fat globule. Similar expression was observed in the mouse mammary epithelial cell line HC11 in resting and lactating phenotypes. No iron export protein could be determined in the RNA isolated from fat globules in human breast milk and a human mammary epithelial cell line. The lack of iron exporters in the human mammary epithelia, in conjunction with the presence of lactoferrin suggests that transmembrane transport is not a major route of iron excretion into human milk.

  9. Endogenous IgG affects the cell biology of RPE cells and involves the TLR4 pathway.

    Science.gov (United States)

    Niu, Na; Zhang, Jie; McNutt, Michael A

    2013-10-25

    RPE is a key component of the blood-ocular barrier (BOB) and is equipped with immunological molecules such as toll-like receptors (TLRs) and complement receptors, which together orchestrate the innate and adaptive immunity of the eye. Immunoglobulin G (IgG) in the aqueous humor and vitreous body has traditionally been thought to be derived from serum via transcytosis across the BOB. Our previous work validated production of endogenous IgG by RPE cells locally. However, the function and role of this IgG in the intraocular immunity is poorly understood. After confirming IgG production in a human RPE cell line (ARPE-19) with immunofluorescence, in situ hybridization, and RT-PCR, we further investigated the function of endogenous IgG in RPE biology with MTS, flow cytometry, and cell invasion analysis after downregulation of IgG by siRNA. Involvement of the TLR4 pathway was also studied using Western blot, ELISA and confocal microscopy. Endogenous IgG is crucial for support of proliferation, mitosis, migration, and inhibition of apoptosis of RPE. Moreover, production of endogenous IgG by RPE is regulated by the TLR4 pathway in a concentration- and duration-dependent manner, and IgG affects the activation of the TLR4 pathway in a synergistic manner. Activation of the FcγR I pathway and production of IL-10 could be induced by IgG derived from RPE. These data suggest that endogenous IgG may be a molecule that is essential for the physiological function of RPE, and suggest IgG is important for regulating intraocular immune responses under physiologic and pathologic conditions.

  10. Agglutinating mouse IgG3 compares favourably with IgMs in typing of the blood group B antigen: Functionality and stability studies.

    Science.gov (United States)

    Klaus, Tomasz; Bzowska, Monika; Kulesza, Małgorzata; Kabat, Agnieszka Martyna; Jemioła-Rzemińska, Małgorzata; Czaplicki, Dominik; Makuch, Krzysztof; Jucha, Jarosław; Karabasz, Alicja; Bereta, Joanna

    2016-08-03

    Mouse immunoglobulins M (IgMs) that recognize human blood group antigens induce haemagglutination and are used worldwide for diagnostic blood typing. Contrary to the current belief that IgGs are too small to simultaneously bind antigens on two different erythrocytes, we obtained agglutinating mouse IgG3 that recognized antigen B of the human ABO blood group system. Mouse IgG3 is an intriguing isotype that has the ability to form Fc-dependent oligomers. However, F(ab')2 fragments of the IgG3 were sufficient to agglutinate type B red blood cells; therefore, IgG3-triggered agglutination did not require oligomerization. Molecular modelling indicated that mouse IgG3 has a larger range of Fab arms than other mouse IgG subclasses and that the unique properties of mouse IgG3 are likely due to the structure of its hinge region. With a focus on applications in diagnostics, we compared the stability of IgG3 and two IgMs in formulated blood typing reagents using an accelerated storage approach and differential scanning calorimetry. IgG3 was much more stable than IgMs. Interestingly, the rapid decrease in IgM activity was caused by aggregation of the molecules and a previously unknown posttranslational proteolytic processing of the μ heavy chain. Our data point to mouse IgG3 as a potent diagnostic tool.

  11. The two Na+ sites in the human serotonin transporter play distinct roles in the ion coupling and electrogenicity of transport.

    Science.gov (United States)

    Felts, Bruce; Pramod, Akula Bala; Sandtner, Walter; Burbach, Nathan; Bulling, Simon; Sitte, Harald H; Henry, L Keith

    2014-01-17

    Neurotransmitter transporters of the SLC6 family of proteins, including the human serotonin transporter (hSERT), utilize Na(+), Cl(-), and K(+) gradients to induce conformational changes necessary for substrate translocation. Dysregulation of ion movement through monoamine transporters has been shown to impact neuronal firing potentials and could play a role in pathophysiologies, such as depression and anxiety. Despite multiple crystal structures of prokaryotic and eukaryotic SLC transporters indicating the location of both (or one) conserved Na(+)-binding sites (termed Na1 and Na2), much remains uncertain in regard to the movements and contributions of these cation-binding sites in the transport process. In this study, we utilize the unique properties of a mutation of hSERT at a single, highly conserved asparagine on TM1 (Asn-101) to provide several lines of evidence demonstrating mechanistically distinct roles for Na1 and Na2. Mutations at Asn-101 alter the cation dependence of the transporter, allowing Ca(2+) (but not other cations) to functionally replace Na(+) for driving transport and promoting 5-hydroxytryptamine (5-HT)-dependent conformational changes. Furthermore, in two-electrode voltage clamp studies in Xenopus oocytes, both Ca(2+) and Na(+) illicit 5-HT-induced currents in the Asn-101 mutants and reveal that, although Ca(2+) promotes substrate-induced current, it does not appear to be the charge carrier during 5-HT transport. These findings, in addition to functional evaluation of Na1 and Na2 site mutants, reveal separate roles for Na1 and Na2 and provide insight into initiation of the translocation process as well as a mechanism whereby the reported SERT stoichiometry can be obtained despite the presence of two putative Na(+)-binding sites.

  12. The Two Na+ Sites in the Human Serotonin Transporter Play Distinct Roles in the Ion Coupling and Electrogenicity of Transport*

    Science.gov (United States)

    Felts, Bruce; Pramod, Akula Bala; Sandtner, Walter; Burbach, Nathan; Bulling, Simon; Sitte, Harald H.; Henry, L. Keith

    2014-01-01

    Neurotransmitter transporters of the SLC6 family of proteins, including the human serotonin transporter (hSERT), utilize Na+, Cl−, and K+ gradients to induce conformational changes necessary for substrate translocation. Dysregulation of ion movement through monoamine transporters has been shown to impact neuronal firing potentials and could play a role in pathophysiologies, such as depression and anxiety. Despite multiple crystal structures of prokaryotic and eukaryotic SLC transporters indicating the location of both (or one) conserved Na+-binding sites (termed Na1 and Na2), much remains uncertain in regard to the movements and contributions of these cation-binding sites in the transport process. In this study, we utilize the unique properties of a mutation of hSERT at a single, highly conserved asparagine on TM1 (Asn-101) to provide several lines of evidence demonstrating mechanistically distinct roles for Na1 and Na2. Mutations at Asn-101 alter the cation dependence of the transporter, allowing Ca2+ (but not other cations) to functionally replace Na+ for driving transport and promoting 5-hydroxytryptamine (5-HT)-dependent conformational changes. Furthermore, in two-electrode voltage clamp studies in Xenopus oocytes, both Ca2+ and Na+ illicit 5-HT-induced currents in the Asn-101 mutants and reveal that, although Ca2+ promotes substrate-induced current, it does not appear to be the charge carrier during 5-HT transport. These findings, in addition to functional evaluation of Na1 and Na2 site mutants, reveal separate roles for Na1 and Na2 and provide insight into initiation of the translocation process as well as a mechanism whereby the reported SERT stoichiometry can be obtained despite the presence of two putative Na+-binding sites. PMID:24293367

  13. Predominant role of plasma membrane monoamine transporters in monoamine transport in 1321N1, a human astrocytoma-derived cell line.

    Science.gov (United States)

    Naganuma, Fumito; Yoshikawa, Takeo; Nakamura, Tadaho; Iida, Tomomitsu; Harada, Ryuichi; Mohsen, Attayeb S; Miura, Yamato; Yanai, Kazuhiko

    2014-05-01

    Monoamine neurotransmitters should be immediately removed from the synaptic cleft to avoid excessive neuronal activity. Recent studies have shown that astrocytes and neurons are involved in monoamine removal. However, the mechanism of monoamine transport by astrocytes is not entirely clear. We aimed to elucidate the transporters responsible for monoamine transport in 1321N1, a human astrocytoma-derived cell line. First, we confirmed that 1321N1 cells transported dopamine, serotonin, norepinephrine, and histamine in a time- and dose-dependent manner. Kinetics analysis suggested the involvement of low-affinity monoamine transporters, such as organic cation transporter (OCT) 2 and 3 and plasma membrane monoamine transporter (PMAT). Monoamine transport in 1321N1 cells was not Na(+) /Cl(-) dependent but was inhibited by decynium-22, an inhibitor of low-affinity monoamine transporters, which supported the importance of low-affinity transporters. RT-PCR assays revealed that 1321N1 cells expressed OCT3 and PMAT but no other neurotransmitter transporters. Another human astrocytoma-derived cell line, U251MG, and primary human astrocytes also exhibited the same gene expression pattern. Gene-knockdown assays revealed that 1321N1 and primary human astrocytes could transport monoamines predominantly through PMAT and partly through OCT3. These results might indicate that PMAT and OCT3 in human astrocytes are involved in monoamine clearance. © 2014 International Society for Neurochemistry.

  14. IgG4 related sclerosing mastitis: expanding the morphological spectrum of IgG4 related diseases.

    Science.gov (United States)

    Chougule, Abhijit; Bal, Amanjit; Das, Ashim; Singh, Gurpreet

    2015-01-01

    IgG4 related disease (IgG4RD) is a recently recognised condition characterised by mass forming lesions associated with storiform fibrosis, obliterative phlebitis, lymphoplasmacytic infiltrate rich in IgG4 positive plasma cells and elevated serum IgG4 levels. Although rare, mammary involvement has been reported as IgG4 related sclerosing mastitis, the morphological counterpart of a growing family of IgG4 related diseases. A total of 17 cases belonging to mass forming benign inflammatory breast lesions such as plasma cell mastitis, granulomatous lobular mastitis, non-specific mastitis and inflammatory pseudotumour were investigated as a possible member of IgG4 related sclerosing mastitis. Clinical, radiological, histopathological and immunohistochemistry findings were noted in all cases. Cases diagnosed as inflammatory pseudotumour showed all the histopathological features of IgG4RD along with increased number of IgG4 positive plasma cells and IgG4/IgG ratio >40%. However, only a few IgG4 positive cells were seen in plasma cell mastitis, granulomatous lobular mastitis and non-specific mastitis cases. These cases also did not fulfill the morphological criteria for the diagnosis of IgG4 related diseases. IgG4RD should be excluded in plasma cell rich lesions diagnosed on core biopsies by IgG4 immunostaining. This can avoid unnecessary surgery as IgG4 related diseases respond to simple and effective steroid treatment.

  15. IgG4-related kidney disease--A review.

    Science.gov (United States)

    Pradhan, Dinesh; Pattnaik, Niharika; Silowash, Russell; Mohanty, Sambit Kumar

    2015-10-01

    IgG4-related disease (IgG4-RD) is a recently recognized systemic autoimmune disorder characterized by high levels of serum IgG4 and dense infiltration of IgG4-positive plasma cells in multiple organs. The condition was first described as a disease of the pancreas, and has since been recognized in various organ systems including the kidneys. IgG4 related kidney disease (IgG4-RKD) signifies any form of renal involvement by IgG4-RD. The most common renal involvement by IgG4-RD is tubulointerstitial nephritis. Glomerular disease, in particular membranous glomerulonephritis, may also be seen. Other co-existent glomerular diseases such as IgA nephropathy, membranoproliferative glomerulonephritis, and mesangioproliferative immune complex glomerulonephritis may be identified. IgG4-related plasma cell arteritis has also been noted in the kidney. As with IgG4-RD in general, IgG4 related kidney disease (IgG4-RKD) usually occurs in middle-aged to elderly men. Common findings in IgG4-RKD are plasma cell-rich interstitial inflammatory infiltrate either in a focal or diffuse pattern with increased IgG4+ plasma cells, expansile swirling interstitial fibrosis, high levels of serum IgG and IgG4, hypocomplementemia, high serum IgE levels and/or peripheral blood eosinophilia. By immunofluorescence, most of the cases show IgG4 dominant tubular basement membrane immune complex deposits. Similar to IgG4-RD, IgG4-RKD often shows a rapid response to steroid therapy. In this review, we discuss the current knowledge on IgG4-RKD and its clinical relevance. Copyright © 2015 Elsevier GmbH. All rights reserved.

  16. Transcellular sodium transport in cultured cystic fibrosis human nasal epithelium

    DEFF Research Database (Denmark)

    Willumsen, Niels J.; Boucher, Richard C.

    1991-01-01

    Cystic fibrosis (CF) airway epithelia exhibit raised transepithelial Na+ transport rates, as determined by open-circuit isotope fluxes and estimates of the amiloride-sensitive equivalent short-circuit current (Ieq). To study the contribution of apical and basolateral membrane paths to raised Na...

  17. Multiple Drug Transport Pathways through human P-Glycoprotein(†)

    Science.gov (United States)

    McCormick, James W.; Vogel, Pia D.; Wise, John G.

    2015-01-01

    P-glycoprotein (P-gp) is a plasma membrane efflux pump that is commonly associated with therapy resistances in cancers and infectious diseases. P-gp can lower the intracellular concentrations of many drugs to subtherapeutic levels by translocating them out of the cell. Because of the broad range of substrates transported by P-gp, overexpression of P-gp causes multidrug resistance. We reported previously on dynamic transitions of P-gp as it moved through conformations based on crystal structures of homologous ABCB1 proteins using in silico targeted molecular dynamics techniques. We expanded these studies here by docking transport substrates to drug binding sites of P-gp in conformations open to the cytoplasm, followed by cycling the pump through conformations that opened to the extracellular space. We observed reproducible transport of two substrates, daunorubicin and verapamil, by an average of 11 to 12 Å through the plane of the membrane as P-gp progressed through a catalytic cycle. Methyl-pyrophosphate, a ligand that should not be transported by P-gp, did not show this movement through P-gp. Drug binding to either of two subsites on P-gp appeared to determine the initial pathway used for drug movement through the membrane. The specific side-chain interactions with drugs within each pathway seemed to be, at least in part, stochastic. The docking and transport properties of a P-gp inhibitor, tariquidar, were also studied. A mechanism of inhibition by tariquidar is presented that involves stabilization of an outward open conformation with tariquidar bound in intracellular loops or at the drug binding domain of P-gp. PMID:26125482

  18. Ion transport in the zebrafish kidney from a human disease angle: possibilities, considerations, and future perspectives.

    Science.gov (United States)

    Kersten, Simone; Arjona, Francisco J

    2017-01-01

    Unique experimental advantages, such as its embryonic/larval transparency, high-throughput nature, and ease of genetic modification, underpin the rapid emergence of the zebrafish (Danio rerio) as a preeminent model in biomedical research. Particularly in the field of nephrology, the zebrafish provides a promising model for studying the physiological implications of human solute transport processes along consecutive nephron segments. However, although the zebrafish might be considered a valuable model for numerous renal ion transport diseases and functional studies of many channels and transporters, not all human renal electrolyte transport mechanisms and human diseases can be modeled in the zebrafish. With this review, we explore the ontogeny of zebrafish renal ion transport, its nephron structure and function, and thereby demonstrate the clinical translational value of this model. By critical assessment of genomic and amino acid conservation of human proteins involved in renal ion handling (channels, transporters, and claudins), kidney and nephron segment conservation, and renal electrolyte transport physiology in the zebrafish, we provide researchers and nephrologists with an indication of the possibilities and considerations of the zebrafish as a model for human renal ion transport. Combined with advanced techniques envisioned for the future, implementation of the zebrafish might expand beyond unraveling pathophysiological mechanisms that underlie distinct genetic or environmentally, i.e., pharmacological and lifestyle, induced renal transport deficits. Specifically, the ease of drug administration and the exploitation of improved genetic approaches might argue for the adoption of the zebrafish as a model for preclinical personalized medicine for distinct renal diseases and renal electrolyte transport proteins. Copyright © 2017 the American Physiological Society.

  19. Inhibition of complement activation by IgG4 antibodies.

    Science.gov (United States)

    van der Zee, J S; van Swieten, P; Aalberse, R C

    1986-05-01

    Prolonged exposure to antigens may result in high IgG4 antibody titres as was shown in a previous paper (Aalberse et al., 1983b). In novice bee keepers, a shift in the IgG1/IgG4 ratio of the response against phospholipase-A (PLA; a major component of bee venom) occurred. This resulted in an IgG4-dominated response after approximately 2 years of bee-keeping experience. Subject of the present study was the influence of relatively high concentrations of IgG4 antibodies on the biological activity of immune complexes. In the PLA antigen model, it was demonstrated that IgG4-containing immune complexes do not activate complement and that IgG4 antibodies effectively inhibit immune precipitation and complement activation by IgG1 antibodies. Evidence is provided that IgG4 antibodies inhibit binding of C1q to IgG1 in mixed, IgG1- and IgG4-containing complexes. It is proposed that IgG4 antibodies protect against the biological effects of the complement-fixing IgG subclasses. For this reason, determination of the total IgG response or just determination of antibody activity in the complement-fixing isotypes is insufficient in immune-complex diseases. The modulating effect of the non-complement-fixing isotypes should be taken into account to predict the biological activity of the immune complexes.

  20. Cholangiocarcinoma with respect to IgG4 Reaction

    Directory of Open Access Journals (Sweden)

    Kenichi Harada

    2014-01-01

    Full Text Available IgG4 reactions marked by infiltration of IgG4-positive plasma cells in affected organs occur in cancer patients and in patients with IgG4-related diseases. Extrahepatic cholangiocarcinomas including gall bladder cancer are often accompanied by significant IgG4 reactions; these reactions show a negative correlation with CD8-positive cytotoxic T cells, suggesting that the evasion of immune surveillance is associated with cytotoxic T cells. The regulatory cytokine IL-10 may induce IgG4-positive plasma cell differentiation or promote B cell switching to IgG4 in the presence of IL-4. Cholangiocarcinoma cells may function as nonprofessional antigen presenting cells that indirectly induce IgG4 reactions via the IL-10-producing cells and/or these may act as Foxp3-positive and IL-10-producing cells that directly induce IgG4 reactions. Moreover, IgG4-related disease is a high-risk factor for cancer development; IgG4-related sclerosing cholangitis (IgG4-SC cases associated with cholangiocarcinoma or its precursor lesion biliary intraepithelial neoplasia (BilIN have been reported. IgG4-positive cell infiltration is an important finding of IgG4-SC but is not a histological hallmark of IgG4-SC. For the diagnosis of IgG4-SC, its differentiation from cholangiocarcinoma remains important.

  1. Interactions of bilastine, a new oral H₁ antihistamine, with human transporter systems.

    Science.gov (United States)

    Lucero, Maria Luisa; Gonzalo, Ana; Ganza, Alvaro; Leal, Nerea; Soengas, Itziar; Ioja, Eniko; Gedey, Szilvia; Jahic, Mirza; Bednarczyk, Dallas

    2012-06-01

    Membrane transporters play a significant role in facilitating transmembrane drug movement. For new pharmacological agents, it is important to evaluate potential interactions (e.g., substrate specificity and/or inhibition) with human transporters that may affect their pharmacokinetics, efficacy, or toxicity. Bilastine is a new nonsedating H₁ antihistamine indicated for the treatment of allergic rhinoconjunctivitis and urticaria. The in vitro inhibitory effects of bilastine were assessed on 12 human transporters: four efflux [multidrug resistance protein 1 (MDR1) or P-glycoprotein, breast cancer resistance protein (BCRP), multidrug resistance associated protein 2 (MRP2), and bile salt export pump) and eight uptake transporters (sodium taurocholate cotransporting polypeptide, organic cation transporter (OCT)1, organic anion transporter (OAT)1, OAT3, OCT2, OATP2B1, OATP1B1, and OATP1B3). Only mild inhibition was found for MDR1-, OCT1-, and OATP2B1-mediated transport of probe substrates at the highest bilastine concentration assayed (300 μM; half-maximal inhibitory concentration: ≥300 μM). Bilastine transport by MDR1, BCRP, OAT1, OAT3, and OCT2 was also investigated in vitro. Only MDR1 active transport of bilastine was relevant, whereas it did not appear to be a substrate of OCT2, OAT1, or OAT3, nor was it transported substantially by BCRP. Drug-drug interactions resulting from bilastine inhibition of drug transporters that would be generally regarded as clinically relevant are unlikely. Additionally, bilastine did not appear to be a substrate of human BCRP, OAT1, OAT3, or OCT2 and thus is not a potential victim of inhibitors of these transporters. On the other hand, based on in vitro evaluation, clinically relevant interactions with MDR1 inhibitors are anticipated.

  2. Human equilibrative nucleoside transporter 1, as a predictor of 5-fluorouracil resistance in human pancreatic cancer.

    Science.gov (United States)

    Tsujie, Masanori; Nakamori, Shoji; Nakahira, Shin; Takahashi, Yuji; Hayashi, Nobuyasu; Okami, Jiro; Nagano, Hiroaki; Dono, Keizo; Umeshita, Koji; Sakon, Masato; Monden, Morito

    2007-01-01

    The purpose of this study was to find a novel biomarker to predict 5-fluorouracil (5-FU) or gemcitabine (2',2'-difluoro-deoxycytidine) sensitivity in pancreatic cancer. The relationship between 5-FU and gemcitabine sensitivity and the mRNA levels of human equilibrative nucleoside transporter 1 (hENT1), thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) was investigated using seven types of human pancreatic carcinoma cell line (AsPC1, BxPC3, MiaPaCa-2, PSN1, Panc1, PCI6, and KMP-4). Quantitative mRNA expression was measured by LightCycler. A [3H] gemcitabine cellular uptake assay was performed to examine the inhibition of hENT1 by nitrobenzylmercaptoprine ribonucleoside (NBMPR). The expression levels of hENT1 mRNA significantly correlated with the IC50 value of 5-FU in all seven lines and also correlated with gemcitabine resistance in six lines (except AsPC1). No significant association was observed between TS or DPD mRNA levels and 5-FU sensitivity. In the PSN1 cells, [3H] gemcitabine uptake via hENT1 was significantly inhibited by NBMPR, and 5-FU sensitivity was significantly increased when the cells were pretreated with NBMPR. Our results suggest that hENT1 plays an important role in 5-FU resistance and that hENT1 mRNA levels might be a useful marker to predict 5-FU sensitivity in pancreatic cancer.

  3. The Contribution of Allergen-Specific IgG to the Development of Th2-Mediated Airway Inflammation

    Directory of Open Access Journals (Sweden)

    Jesse W. Williams

    2012-01-01

    Full Text Available In both human asthmatics and animal models of allergy, allergen-specific IgG can contribute to Th2-mediated allergic inflammation. Mouse models have elucidated an important role for IgG and Fc-gamma receptor (FcγR signaling on antigen presenting cells (APC for the induction of airway inflammation. These studies suggest a positive feedback loop between IgG produced by the adaptive B cell response and FcγR signaling on innate immune cells. Studies of IgG and FcγRs in humans with asthma or allergic lung disease have been more controversial. Some reports have identified associations between allergen-specific IgG and severity of allergic responses, while other studies have found associations of IgG subclass IgG4 with allergic tolerance. In this paper, we review the literature to help define the nature of IgG and FcγR signaling on innate immune cells and how it contributes to the development of allergic immune responses.

  4. Estradiol augments while progesterone inhibits arginine transport in human endothelial cells through modulation of cationic amino acid transporter-1.

    Science.gov (United States)

    Bentur, Ohad S; Schwartz, Doron; Chernichovski, Tamara; Ingbir, Merav; Weinstein, Talia; Chernin, Gil; Schwartz, Idit F

    2015-08-15

    Decreased generation of nitric oxide (NO) by endothelial NO synthase (eNOS) characterizes endothelial dysfunction (ECD). Delivery of arginine to eNOS by cationic amino acid transporter-1 (CAT-1) was shown to modulate eNOS activity. We found in female rats, but not in males, that CAT-1 activity is preserved with age and in chronic renal failure, two experimental models of ECD. In contrast, during pregnancy CAT-1 is inhibited. We hypothesize that female sex hormones regulate arginine transport. Arginine uptake in human umbilical vein endothelial cells (HUVEC) was determined following incubation with either 17β-estradiol (E2) or progesterone. Exposure to E2 (50 and 100 nM) for 30 min resulted in a significant increase in arginine transport and reduction in phosphorylated CAT-1 (the inactive form) protein content. This was coupled with a decrease in phosphorylated MAPK/extracellular signal-regulated kinase (ERK) 1/2. Progesterone (1 and 100 pM for 30 min) attenuated arginine uptake and increased phosphorylated CAT-1, phosphorylated protein kinase Cα (PKCα), and phosphorylated ERK1/2 protein content. GO-6976 (PKCα inhibitor) prevented the progesterone-induced decrease in arginine transport. Coincubation with both progesterone and estrogen for 30 min resulted in attenuated arginine transport. While estradiol increases arginine transport and CAT-1 activity through modulation of constitutive signaling transduction pathways involving ERK, progesterone inhibits arginine transport and CAT-1 via both PKCα and ERK1/2 phosphorylation, an effect that predominates over estradiol. Copyright © 2015 the American Physiological Society.

  5. The importance of drug transporters in human pluripotent stem cells and in early tissue differentiation.

    Science.gov (United States)

    Apáti, Ágota; Szebényi, Kornélia; Erdei, Zsuzsa; Várady, György; Orbán, Tamás I; Sarkadi, Balázs

    2016-01-01

    Drug transporters are large transmembrane proteins which catalyse the movement of a wide variety of chemicals, including drugs as well as xeno- and endobiotics through cellular membranes. The major groups of these proteins include the ATP-binding cassette transporters which in eukaryotes work as ATP-fuelled drug 'exporters' and the Solute Carrier transporters, with various transport directions and mechanisms. In this review, we discuss the key ATP-binding cassette and Solute Carrier drug transporters which have been reported to contribute to the function and/or protection of undifferentiated human stem cells and during tissue differentiation. We review the various techniques for studying transporter expression and function in stem cells, and the role of drug transporters in foetal and placental tissues is also discussed. We especially focus on the regulation of transporter expression by factors modulating cell differentiation properties and on the function of the transporters in adjustment to environmental challenges. The relatively new and as yet unexplored territory of transporters in stem cell biology may rapidly expand and bring important new information regarding the metabolic and epigenetic regulation of 'stemness' and the early differentiation properties. Drug transporters are clearly important protective and regulatory components in stem cells and differentiation.

  6. Modeling oxygen transport in human placental terminal villi.

    Science.gov (United States)

    Gill, J S; Salafia, C M; Grebenkov, D; Vvedensky, D D

    2011-12-21

    Oxygen transport from maternal blood to fetal blood is a primary function of the placenta. Quantifying the effectiveness of this exchange remains key in identifying healthy placentas because of the great variability in capillary number, caliber and position within the villus-even in placentas deemed clinically "normal". By considering villous membrane to capillary membrane transport, stationary oxygen diffusion can be numerically solved in terminal villi represented by digital photomicrographs. We aim to provide a method to determine whether and if so to what extent diffusional screening may operate in placental villi. Segmented digital photomicrographs of terminal villi from the Pregnancy, Infection and Nutrition study in North Carolina 2002 are used as a geometric basis for solving the stationary diffusion equation. Constant maternal villous oxygen concentration and perfect fetal capillary membrane absorption are assumed. System efficiency is defined as the ratio of oxygen flux into a villus and the sum of the capillary areas contained within. Diffusion screening is quantified by comparing numerical and theoretical maximum oxygen fluxes. A strong link between various measures of villous oxygen transport efficiency and the number of capillaries within a villus is established. The strength of diffusional screening is also related to the number of capillaries within a villus. Our measures of diffusional efficiency are shown to decrease as a function of the number of capillaries per villus. This low efficiency, high capillary number relationship supports our hypothesis that diffusional screening is present in this system. Oxygen transport per capillary is reduced when multiple capillaries compete for diffusing oxygen. A complete picture of oxygen fluxes, capillary and villus areas is obtainable and presents an opportunity for future work. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. Training-induced changes in membrane transport proteins of human skeletal muscle

    DEFF Research Database (Denmark)

    Juel, C.

    2006-01-01

    that the same type of training affects many transport proteins, suggesting that all transport proteins increase with training, and that both sprint and endurance training in humans increase the density of most membrane transport proteins. There seems to be an upper limit for these changes: intense training......Training improves human physical performance by inducing structural and cardiovascular changes, metabolic changes, and changes in the density of membrane transport proteins. This review focuses on the training-induced changes in proteins involved in sarcolemmal membrane transport. It is concluded...... for 6-8 weeks substantially increases the density of membrane proteins, whereas years of training (as performed by athletes) have no further effect. Studies suggest that training-induced changes at the protein level are important functionally. The underlying factors responsible for these changes...

  8. EFFICIENCY OF HUMAN RESOURCES MANAGEMENT IN RAILWAY TRANSPORT

    OpenAIRE

    DANIEL DANECI-PATRAU; LAURA PATACHE

    2011-01-01

    In analyzing the efficiency of human resources management, it is used a system of indicators that aim at, on one hand, provisioning with human resources concerning the number of employees, qualification, structure or personnel mobility, and on the another hand, their use, under the aspect of working time use, and of efficiency time use. This paper presents a type of dynamic analysis for the human potential in the Cãile Ferate Române (CFR) Co. Regional Infrastructure of Constanta, under the as...

  9. IgG1 and IgG4 antibody responses to the Anopheles gambiae salivary protein gSG6 in the sympatric ethnic groups Mossi and Fulani in a malaria hyperhendemic area of Burkina Faso.

    Directory of Open Access Journals (Sweden)

    Cinzia Rizzo

    Full Text Available Human antibody response to the Anopheles gambiae salivary protein gSG6 has recently emerged as a potentially useful tool for malaria epidemiological studies and for the evaluation of vector control interventions. However, the current understanding of the host immune response to mosquito salivary proteins and of the possible crosstalk with early response to Plasmodium parasites is still very limited. We report here the analysis of IgG1 and IgG4 subclasses among anti-gSG6 IgG responders belonging to Mossi and Fulani from Burkina Faso, two ethnic groups which are known for their differential humoral response to parasite antigens and for their different susceptibility to malaria. The IgG1 antibody response against the gSG6 protein was comparable in the two groups. On the contrary, IgG4 titers were significantly higher in the Fulani where, in addition, anti-gSG6 IgG4 antibodies appeared in younger children and the ratio IgG4/IgG1 stayed relatively stable throughout adulthood. Both gSG6-specific IgG1 and IgG4 antibodies showed a tendency to decrease with age whereas, as expected, the IgG response to the Plasmodium circumsporozoite protein (CSP exhibited an opposite trend in the same individuals. These observations are in line with the idea that the An. gambiae gSG6 salivary protein induces immune tolerance, especially after intense and prolonged exposure as is the case for the area under study, suggesting that gSG6 may trigger in exposed individuals a Th2-oriented immune response.

  10. Brain barriers and functional interfaces with sequential appearance of ABC efflux transporters during human development

    DEFF Research Database (Denmark)

    Møllgård, Kjeld; Dziegielewska, Katarzyna M.; Holst, Camilla B.

    2017-01-01

    Adult brain is protected from entry of drugs and toxins by specific mechanisms such as ABC (ATP-binding Cassette) efflux transporters. Little is known when these appear in human brain during development. Cellular distribution of three main ABC transporters (ABCC1, ABCG2, ABCB1) was determined...... at blood-brain barriers and interfaces in human embryos and fetuses in first half of gestation. Antibodies against claudin-5 and-11 and antibodies to α-fetoprotein were used to describe morphological and functional aspects of brain barriers. First exchange interfaces to be established, probably at 4...... three transporters. Results provide evidence for sequential establishment of brain exchange interfaces and spatial and temporal timetable for three main ABC transporters in early human brain....

  11. Catalase activity of IgG antibodies from the sera of healthy donors and patients with schizophrenia.

    Science.gov (United States)

    Ermakov, Evgeny A; Smirnova, Ludmila P; Bokhan, Nikolay A; Semke, Arkadiy V; Ivanova, Svetlana A; Buneva, Valentina N; Nevinsky, Georgy A

    2017-01-01

    We present first evidence showing that some electrophoretically homogeneous IgGs from the sera of patients with schizophrenia (36.4%) and their Fab and F(ab)2 fragments as well as from healthy donors (33.3%) possess catalase activity. The relative catalase activity of IgGs from the sera of individual schizophrenia patients (and healthy donors) significantly varied from patient to patient, but the activity of IgGs from healthy donors is on average 15.8-fold lower than that for schizophrenia patients. After extensive dialysis of purified IgGs against EDTA chelating metal ions, the relative catalase activity of IgGs decreases on average approximately 2.5-3.7-fold; all IgGs possess metal-dependent and independent catalase activity. The addition of external Me2+ ions to dialyzed and non-dialyzed IgGs leads to a significant increase in their activity. The best activator of dialyzed and non-dialyzed IgGs is Co2+, the activation by Cu2+, Mn2+, and Ni2+ ions were rare and always lower than by Co2+. Every IgG preparation demonstrates several individual sets of very well expressed pH optima in the pH range from 4.0 to 9.5. These data speak for the individual repertoire of catalase IgGs in every person and an extreme diversity of abzymes in their pH optima and activation by different metal ions. It is known that antioxidant enzymes such as superoxide dismutases, catalases, and glutathione peroxidases represent critical defense mechanisms preventing oxidative modifications of DNA, proteins, and lipids. Catalase activity of human IgGs could probably also play a major role in the protection of organisms from oxidative stress and toxic compounds.

  12. IgG4-Related Disease: A New Etiology Underlying Diffuse Intracranial Dilating Vasculopathy.

    Science.gov (United States)

    Marlin, Evan S; Dornbos, David; Ikeda, Daniel S; Lehman, Norman L; Powers, Ciarán J

    2017-11-01

    Diffuse intracranial aneurysmal vasculopathy is a rare condition, previously described in patients with human immunodeficiency virus infection. IgG4-related disease (IgG4-RD) is a recognized inflammatory disease of systemic organs, leading to fibrosis of connective tissues. It also has been linked to inflammatory dilating aortic aneurysms, coronary vascular disease, hypophysitis, orbital pseudotumor, and pachymeningitis. It has not yet been described as a cause of diffuse intracranial dilating vasculopathy. Histologically, this disease is characterized by IgG4-plasma cell infiltration, fibrosis, and phlebitis. A 40-year-old woman presented with acute heart failure, valvular insufficiency, and mycotic coronary aneurysms, concerning for endocarditis. Infectious workup was negative. Concurrent neurovascular workup revealed intracranial aneurysms, appearing mycotic in origin. Despite aggressive treatment for more than 5 years, she suffered multiple episodes of subarachnoid hemorrhage from a progressive dilating intracranial vasculopathy. Serum IgG levels and aneurysm wall pathology were consistent with IgG4-RD. This is the first reported case of a diffuse intracranial dilating vasculopathy secondary to IgG4-RD. Recognition of similar pathologic findings in clinical presentation and radiologic workup should prompt further rheumatologic workup and possible immunosuppressive therapies. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Impact of altered endogenous IgG on unspecific mAb clearance.

    Science.gov (United States)

    Fuhrmann, Saskia; Kloft, Charlotte; Huisinga, Wilhelm

    2017-08-01

    Immunodeficient mice are crucial models to evaluate the efficacy of monoclonal antibodies (mAbs). When studying mAb pharmacokinetics (PK), protection from elimination by binding to the neonatal Fc receptor (FcRn) is known to be a major process influencing the unspecific clearance of endogenous and therapeutic IgG. The concentration of endogenous IgG in immunodeficient mice, however is reduced, and this effect on the FcRn protection mechanism and subsequently on unspecific mAb clearance is unknown, yet of great importance for the interpretation of mAb PK data. We used a PBPK modelling approach to elucidate the influence of altered endogenous IgG concentrations on unspecific mAb clearance. To this end, we used PK data in immunodeficient mice, i.e. nude and severe combined immunodeficiency mice. To avoid impact of target-mediated clearance processes, we focussed on mAbs without affinity to a target antigen in these mice. In addition, intravenous immunoglobulin (IVIG) data of immunocompetent mice was used to study the impact of increased total IgG concentrations on unspecific therapeutic antibody clearance. The unspecific clearance is linear, whenever therapeutic IgG concentrations, i.e. mAb and IVIG concentrations are lower than FcRn; it can be non-linear if therapeutic IgG concentrations are larger than FcRn and endogenous IgG concentrations (e.g., under IVIG therapy). Unspecific mAb clearance of immunodeficient mice is effectively linear (under mAb doses as typically used in human). Studying the impact of reduced endogenous IgG concentrations on unspecific mAb clearance is of great relevance for the extrapolation to clinical species, e.g., when predicting mAb PK in immunosuppressed cancer patients.

  14. Atomic force microscopy analysis of IgG films at hydrophobic surfaces: a promising method to probe IgG orientations and optimize ELISA tests performance.

    Science.gov (United States)

    de Thier, Pierre; Bacharouche, Jalal; Duval, Jérôme F L; Skali-Lami, Salaheddine; Francius, Grégory

    2015-02-01

    IgG films are widely used in the field of immunoassays, especially in (double) antibody-sandwich ELISA tests where capture antibodies are coated on surfaces like polystyrene or hydrophobic self-assembled monolayers (h-SAMs). It is critical to analyze-at a molecular scale and under liquid conditions-the structure of the deposited IgG film in order to quantitatively address the efficiency of the ELISA test in terms of antigen detection. In this communication, we report an atomic force microscopy (AFM) analysis evidencing a strong relationship between immunological activities of mouse monoclonal anti-human interleukin-2 (IL-2) and 6 (IL-6) antibodies, thickness and roughness of the IgG monolayer adsorbed onto h-SAMs, and surface concentration of IgG molecules. Indirect information may be further obtained on antibody orientation. Collating the results obtained by AFM and those from ELISA tests leads us to conclude that antibodies like anti-IL-6 forming flat monolayers should be more efficient under ELISA detection conditions. In addition, the concentration of IgG in the coating suspension should be optimized to obtain a monolayer heavily populated by "end-on" adsorbed molecules, an orientation that is desirable for enhancing ELISA tests performance. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Heterogeneity and diversity of IgM and IgG lupus anticoagulants in an individual with systemic lupus erythematosus.

    Science.gov (United States)

    Nakamura, N; Azuma, C; Akamizu, T; Sugawa, H; Matsuda, F; Mitsuda, N; Honjo, T; Mori, T; Yamaji, K

    1994-09-30

    From one patient with systemic lupus erythematosus retaining lupus anticoagulant (LAC), we established 6 Epstein-Barr virus-transformed human B cell clones secreting antibodies that affect the coagulation assay. Two and 4 of the clones secreted IgM and IgG antibodies, respectively. Although all 6 antibodies displayed anticardiolipin activity in ELISA, the increased binding activity in the presence of beta 2-glycoprotein I was limited only to the IgG antibodies. Five antibodies (two IgM and three IgG) had LAC activity which prolonged the activated partial thromboplastin time (APTT), whereas one IgG antibody shortened the APTT. Two of the IgG producing clones had an identical Ig heavy chain gene rearrangement despite their opposite effects on the coagulation assay. These results demonstrated the heterogeneity of LACs and diversity among their physiological functions.

  16. IgG4-associated cholangitis.

    Science.gov (United States)

    Nath, Vikas; Lewin, Jack; Subramony, Charu; Shenoy, Veena

    2014-12-01

    We report a young female patient with IgG4-associated cholangitis (IAC) who presented with common bile duct (CBD) stricture and review the features that distinguish IAC from both primary sclerosing cholangitis (PSC) and other types of secondary sclerosing cholangitis (SSC). IAC is a biliary manifestation of IgG4-related sclerosing disease, an autoimmune condition characterized by elevated serum IgG4 and infiltrates containing lymphocytes and IgG4-positive plasma cells, accompanied by sclerosis. On endoscopic retrograde cholangiopancreatography, IAC consists of segmental biliary strictures with a predilection for the distal CBD, whereas in PSC the strictures are more band-like; other types of SSC often demonstrate unifocal ductal obstructions, sometimes associated with choleliths. On histologic examination, the bile duct wall in IAC contains a denser lymphocytic infiltrate and sparser sclerosis than in PSC; other types of SSC can be distinguished histologically by the types of inflammatory cells present. Unlike those of PSC, IAC-related strictures are reversible with corticosteroids.

  17. Peanut agglutinin and β-cyclodextrin functionalized polymer monolith: Microextraction of IgG galactosylation coupled with online MS detection.

    Science.gov (United States)

    Zheng, Haijiao; Zhu, Tenggao; Li, Xiqian; Ma, Jiutong; Jia, Qiong

    2017-08-29

    A facile online method coupling polymer monolithic microextraction (PMME) with mass spectrometry (MS) was developed for the detection of Immunoglobulin G (IgG) galactosylation glycopeptides. A peanut agglutinin-β-cyclodextrin (PNA-β-CD) functionalized poly(hydroxyethyl methylacrylate-ethyleneglycol dimethacrylate) monolith was designed via a click reaction. Thanking to the specificity of PNA-β-CD for the targets, the material exhibited enhanced enrichment selectivity and extraction efficiency for IgG galactosylation glycopeptides. Under optimal conditions, the developed method gave a linear range of 0.005-5 pmol for IgG glycopeptides with the regression coefficient greater than 0.9990, and the detection limit of IgG galactosylation glycopeptides as low as 0.5 fmol was achieved. The PMME-MS method was applied to IgG galactosylation glycopeptides in real samples including human serum and acute myelogenous leukemia (AML) cell lysate. A series of unique IgG galactosylation glycopeptides were captured by the monolith in the complex samples, indicating satisfactory enrichment ability for IgG galactosylation glycopeptides. The quick and integrated online PMME-MS method exhibited high selectivity for IgG galactosylation, demonstrating its perspectives on the development and broad applications of MS in studying galactosylation proteins regulated biological processes. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Changes in complementarity-determining regions significantly alter IgG binding to the neonatal Fc receptor (FcRn) and pharmacokinetics.

    Science.gov (United States)

    Piche-Nicholas, Nicole M; Avery, Lindsay B; King, Amy C; Kavosi, Mania; Wang, Mengmeng; O'Hara, Denise M; Tchistiakova, Lioudmila; Katragadda, Madan

    2017-10-09

    A large body of data exists demonstrating that neonatal Fc receptor (FcRn) binding of an IgG via its Fc CH2-CH3 interface trends with the pharmacokinetics (PK) of IgG. We have observed that PK of IgG molecules vary widely, even when they share identical Fc domains. This led us to hypothesize that domains distal from the Fc could contribute to FcRn binding and affect PK. In this study, we explored the role of these IgG domains in altering the affinity between IgG and FcRn. Using a surface plasmon resonance-based assay developed to examine the steady-state binding affinity (KD) of IgG molecules to FcRn, we dissected the contributions of IgG domains in modulating the affinity between FcRn and IgG. Through analysis of a broad collection of therapeutic antibodies containing more than 50 unique IgG molecules, we demonstrated that variable domains, and in particular complementarity-determining regions (CDRs), significantly alter binding affinity to FcRn in vitro. Furthermore, a panel of IgG molecules differing only by 1-5 mutations in CDRs altered binding affinity to FcRn in vitro, by up to 79-fold, and the affinity values correlated with calculated isoelectric point values of both variable domains and CDR-L3. In addition, tighter affinity values trend with faster in vivo clearance of a set of IgG molecules differing only by 1-3 mutations in human FcRn transgenic mice. Understanding the role of CDRs in modulation of IgG affinity to FcRn in vitro and their effect on PK of IgG may have far-reaching implications in the optimization of IgG therapeutics.

  19. Transports of delight how technology materializes human imagination

    CERN Document Server

    Hancock, Peter

    2017-01-01

    This inspiring book shows how the spiritual side of life, with its thoughts, feelings, and aspirations, is intimately bound up with our material technologies. From the wonder of Gothic Cathedrals, to the quiet majesty of lighter than air flight, to the ultimate in luxury of the north Atlantic steamers, Peter Hancock explores how these sequential heights of technology have enabled our dreams of being transported to new and uncharted realms to become reality. Sometimes literally, sometimes figuratively, technology has always been there to make material the visions of our imagination. This book shows how this has essentially been true for all technologies from Stonehenge to space station. But technology is far from perfect. Indeed, the author argues here that some of the most public and tragic of its failures still remain instructive, emblematic, and even inspiring. He reports on examples such as a Cathedral of the Earth (Beauvais), a Cathedral of the Seas (Titanic), and a Cathedral of the Air (Hindenburg) and t...

  20. Ion transport in epithelial spheroids derived from human airway cells

    DEFF Research Database (Denmark)

    Pedersen, P S; Frederiksen, O; Holstein-Rathlou, N H

    1999-01-01

    -CF nasal polyps developed free-floating, monolayered epithelial spheres, with the apical, ciliated cell membrane facing the bath and the basolateral cell membrane pointing toward a fluid-filled lumen. Microelectrode impalement of both non-CF and CF spheroids revealed lumen-positive transepithelial...... electrical potential differences (PDs) that were inhibited by amiloride, indicating that the spheroids were inflated due to amiloride-sensitive Na+ absorption followed by water. Transformation to a Cl- secretory state was achieved by addition of ATP to the bath, leading to the development of a diphenylamine......-2-carboxylate-sensitive PD. A cAMP-induced increase in PD was seen in non-CF spheroids only. In response to hydrocortisone treatment, Na+ transport reflected by amiloride-sensitive PD increased and more so in CF than in non-CF spheres. We concluded that this preparation is a useful model...

  1. Genetic variants of the human dipeptide transporter PEPT1

    DEFF Research Database (Denmark)

    Anderle, Pascale; Nielsen, Carsten Uhd; Pinsonneault, Julia

    2006-01-01

    We tested whether genetic polymorphisms affect activity of the dipeptide transporter PEPT1, which mediates bioavailability of peptidomimetic drugs. All 23 exons and adjoining intronic sections of PEPT1 (SLC15A1) were sequenced in 247 individuals of various ethnic origins (Coriell collection). Of 38...... single nucleotide polymorphisms (SNPs), 21 occurred in intronic and non-coding regions and 17 in exonic coding region, of which nine were nonsynonymous. Eight nonsynonymous variants were cloned into expression vectors and functionally characterized after transient transfection into Cos7 and Chinese...... formation of a splice variant (PEPT1-RF). PEPT1-RF mRNA levels ranged from 2 to 44% of total PEPT1-related mRNA, with potential consequences for drug absorption. Together with previous results, this study reveals a relatively low level of genetic variability in polymorphisms affecting both protein function...

  2. MuSK myasthenia gravis IgG4 disrupts the interaction of LRP4 with MuSK but both IgG4 and IgG1-3 can disperse preformed agrin-independent AChR clusters.

    Directory of Open Access Journals (Sweden)

    Inga Koneczny

    Full Text Available A variable proportion of patients with generalized myasthenia gravis (MG have autoantibodies to muscle specific tyrosine kinase (MuSK. During development agrin, released from the motor nerve, interacts with low density lipoprotein receptor-related protein-4 (LRP4, which then binds to MuSK; MuSK interaction with the intracellular protein Dok7 results in clustering of the acetylcholine receptors (AChRs on the postsynaptic membrane. In mature muscle, MuSK helps maintain the high density of AChRs at the neuromuscular junction. MuSK antibodies are mainly IgG4 subclass, which does not activate complement and can be monovalent, thus it is not clear how the antibodies cause disruption of AChR numbers or function to cause MG. We hypothesised that MuSK antibodies either reduce surface MuSK expression and/or inhibit the interaction with LRP4. We prepared MuSK IgG, monovalent Fab fragments, IgG1-3 and IgG4 fractions from MuSK-MG plasmas. We asked whether the antibodies caused endocytosis of MuSK in MuSK-transfected cells or if they inhibited binding of LRP4 to MuSK in co-immunoprecipitation experiments. In parallel, we investigated their ability to reduce AChR clusters in C2C12 myotubes induced by a agrin, reflecting neuromuscular development, and b by Dok7- overexpression, producing AChR clusters that more closely resemble the adult neuromuscular synapse. Total IgG, IgG4 or IgG1-3 MuSK antibodies were not endocytosed unless cross-linked by divalent anti-human IgG. MuSK IgG, Fab fragments and IgG4 inhibited the binding of LRP4 to MuSK and reduced agrin-induced AChR clustering in C2C12 cells. By contrast, IgG1-3 antibodies did not inhibit LRP4-MuSK binding but, surprisingly, did inhibit agrin-induced clustering. Moreover, both IgG4 and IgG1-3 preparations dispersed agrin-independent AChR clusters in Dok7-overexpressing C2C12 cells. Thus interference by IgG4 antibodies of the LRP4-MuSK interaction will be one pathogenic mechanism of MuSK antibodies, but IgG1

  3. MuSK myasthenia gravis IgG4 disrupts the interaction of LRP4 with MuSK but both IgG4 and IgG1-3 can disperse preformed agrin-independent AChR clusters.

    Science.gov (United States)

    Koneczny, Inga; Cossins, Judith; Waters, Patrick; Beeson, David; Vincent, Angela

    2013-01-01

    A variable proportion of patients with generalized myasthenia gravis (MG) have autoantibodies to muscle specific tyrosine kinase (MuSK). During development agrin, released from the motor nerve, interacts with low density lipoprotein receptor-related protein-4 (LRP4), which then binds to MuSK; MuSK interaction with the intracellular protein Dok7 results in clustering of the acetylcholine receptors (AChRs) on the postsynaptic membrane. In mature muscle, MuSK helps maintain the high density of AChRs at the neuromuscular junction. MuSK antibodies are mainly IgG4 subclass, which does not activate complement and can be monovalent, thus it is not clear how the antibodies cause disruption of AChR numbers or function to cause MG. We hypothesised that MuSK antibodies either reduce surface MuSK expression and/or inhibit the interaction with LRP4. We prepared MuSK IgG, monovalent Fab fragments, IgG1-3 and IgG4 fractions from MuSK-MG plasmas. We asked whether the antibodies caused endocytosis of MuSK in MuSK-transfected cells or if they inhibited binding of LRP4 to MuSK in co-immunoprecipitation experiments. In parallel, we investigated their ability to reduce AChR clusters in C2C12 myotubes induced by a) agrin, reflecting neuromuscular development, and b) by Dok7- overexpression, producing AChR clusters that more closely resemble the adult neuromuscular synapse. Total IgG, IgG4 or IgG1-3 MuSK antibodies were not endocytosed unless cross-linked by divalent anti-human IgG. MuSK IgG, Fab fragments and IgG4 inhibited the binding of LRP4 to MuSK and reduced agrin-induced AChR clustering in C2C12 cells. By contrast, IgG1-3 antibodies did not inhibit LRP4-MuSK binding but, surprisingly, did inhibit agrin-induced clustering. Moreover, both IgG4 and IgG1-3 preparations dispersed agrin-independent AChR clusters in Dok7-overexpressing C2C12 cells. Thus interference by IgG4 antibodies of the LRP4-MuSK interaction will be one pathogenic mechanism of MuSK antibodies, but IgG1-3 Mu

  4. Decreased HHV-6 IgG in Alzheimer’s Disease

    OpenAIRE

    Westman, Gabriel; Blomberg, Jonas; Yun, Zhibing; Lannfelt, Lars; Ingelsson, Martin; Eriksson, Britt-Marie

    2017-01-01

    Human herpesviruses have previously been implicated in the pathogenesis of Alzheimer’s disease (AD) but whether they are causal, facilitating, or confounding factors is yet to be established. A total of 50 AD subjects and 52 non-demented (ND) controls were analyzed in a multiplex assay for IgG reactivity toward herpes simplex virus (HSV), varicella zoster virus (VZV), cytomegalovirus (CMV), and human herpesvirus 6 (HHV-6). The HHV-6 IgG reactivity was significantly lower in AD subjects compar...

  5. Expression and Regulation of Drug Transporters and Metabolizing Enzymes in the Human Gastrointestinal Tract.

    Science.gov (United States)

    Drozdzik, M; Oswald, S

    2016-01-01

    Orally administered drugs must pass through the intestinal wall and then through the liver before reaching systemic circulation. During this process drugs are subjected to different processes that may determine the therapeutic value. The intestinal barrier with active drug metabolizing enzymes and drug transporters in enterocytes plays an important role in the determination of drug bioavailability. Accumulating information demonstrates variable distribution of drug metabolizing enzymes and transporters along the human gastrointestinal tract (GI), that creates specific barrier characteristics in different segments of the GI. In this review, expression of drug metabolizing enzymes and transporters in the healthy and diseased human GI as well as their regulatory aspects: genetic, miRNA, DNA methylation are outlined. The knowledge of unique interplay between drug metabolizing enzymes and transporters in specific segments of the GI tract allows more precise definition of drug release sites within the GI in order to assure more complete bioavailability and prediction of drug interactions.

  6. Transportation

    Science.gov (United States)

    2006-01-01

    container. It now permits free transit of shipping containers from their western ports, if transported by rail directly to the U.S. ( Mireles , 2005, p...Transportation Industry Study Seminar. Mireles , Richard, Castillo. (2005, January). A Cure for West Coast Congestion. Logistics Today, Vol. 46, Issue 1. 1

  7. Human transporters, PEPT1/2, facilitate melatonin transportation into mitochondria of cancer cells: An implication of the therapeutic potential.

    Science.gov (United States)

    Huo, Xiaokui; Wang, Chao; Yu, Zhenlong; Peng, Yulin; Wang, Shumei; Feng, Shengnan; Zhang, Shouji; Tian, Xiangge; Sun, Chengpeng; Liu, Kexin; Deng, Sa; Ma, Xiaochi

    2017-05-01

    Melatonin is present in virtually all organisms from bacteria to mammals, and it exhibits a broad spectrum of biological functions, including synchronization of circadian rhythms and oncostatic activity. Several functions of melatonin are mediated by its membrane receptors, but others are receptor-independent. For the latter, melatonin is required to penetrate membrane and enters intracellular compartments. However, the mechanism by which melatonin enters cells remains debatable. In this study, it was identified that melatonin and its sulfation metabolites were the substrates of oligopeptide transporter (PEPT) 1/2 and organic anion transporter (OAT) 3, respectively. The docking analysis showed that the binding of melatonin to PEPT1/2 was attributed to their low binding energy and suitable binding conformation in which melatonin was embedded in the active site of PEPT1/2 and fitted well with the cavity in three-dimensional space. PEPT1/2 transporters play a pivotal role in melatonin uptake in cells. Melatonin's membrane transportation via PEPT1/2 renders its oncostatic effect in malignant cells. For the first time, PEPT1/2 were identified to localize in the mitochondrial membrane of human cancer cell lines of PC3 and U118. PEPT1/2 facilitated the transportation of melatonin into mitochondria. Melatonin accumulation in mitochondria induced apoptosis of PC3 and U118 cells. Thus, PEPT1/2 can potentially be used as a cancer cell-targeted melatonin delivery system to improve the therapeutic effects of melatonin in cancer treatment. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. Electrogenic Binding of Intracellular Cations Defines a Kinetic Decision Point in the Transport Cycle of the Human Serotonin Transporter.

    Science.gov (United States)

    Hasenhuetl, Peter S; Freissmuth, Michael; Sandtner, Walter

    2016-12-09

    The plasmalemmal monoamine transporters clear the extracellular space from their cognate substrates and sustain cellular monoamine stores even during neuronal activity. In some instances, however, the transporters enter a substrate-exchange mode, which results in release of intracellular substrate. Understanding what determines the switch between these two transport modes demands time-resolved measurements of intracellular (co-)substrate binding and release. Here, we report an electrophysiological investigation of intracellular solute-binding to the human serotonin transporter (SERT) expressed in HEK-293 cells. We measured currents induced by rapid application of serotonin employing varying intracellular (co-)substrate concentrations and interpreted the data using kinetic modeling. Our measurements revealed that the induction of the substrate-exchange mode depends on both voltage and intracellular Na(+) concentrations because intracellular Na(+) release occurs before serotonin release and is highly electrogenic. This voltage dependence was blunted by electrogenic binding of intracellular K(+) and, notably, also H(+) In addition, our data suggest that Cl(-) is bound to SERT during the entire catalytic cycle. Our experiments, therefore, document an essential role of electrogenic binding of K(+) or of H(+) to the inward-facing conformation of SERT in (i) cancelling out the electrogenic nature of intracellular Na(+) release and (ii) in selecting the forward-transport over the substrate-exchange mode. Finally, the kinetics of intracellular Na(+) release and K(+) (or H(+)) binding result in a voltage-independent rate-limiting step where SERT may return to the outward-facing state in a KCl- or HCl-bound form. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Simultaneous measurement of glucose transport and utilization in the human brain

    OpenAIRE

    Shestov, Alexander A.; Uzay E. Emir; Kumar, Anjali; Henry, Pierre-Gilles; Seaquist, Elizabeth R.; Öz, Gülin

    2011-01-01

    Glucose is the primary fuel for brain function, and determining the kinetics of cerebral glucose transport and utilization is critical for quantifying cerebral energy metabolism. The kinetic parameters of cerebral glucose transport, KMt and Vmaxt, in humans have so far been obtained by measuring steady-state brain glucose levels by proton (1H) NMR as a function of plasma glucose levels and fitting steady-state models to these data. Extraction of the kinetic parameters for cerebral glucose tra...

  10. Transport of Antihypertensive Peptide RVPSL, Ovotransferrin 328-332, in Human Intestinal Caco-2 Cell Monolayers.

    Science.gov (United States)

    Ding, Long; Wang, Liying; Zhang, Yan; Liu, Jingbo

    2015-09-23

    The objective of this study was to investigate the transepithelial transport of RVPSL (Arg-Val-Pro-Ser-Leu), an egg-white-derived peptide with angiotensin I-converting enzyme (ACE) inhibitory and antihypertensive activity, in human intestinal Caco-2 cell monolayers. Results revealed that RVPSL could be passively transported across Caco-2 cell monolayers. However, during the process of transport, 36.31% ± 1.22% of the initial RVPSL added to the apical side was degraded, but this degradation decreased to 23.49% ± 0.68% when the Caco-2 cell monolayers were preincubated with diprotin A (P Caco-2 cell monolayers was studied by mutation of RVPSL. It was found that N-terminal Pro residues were more beneficial for transport of pentapeptides across Caco-2 cell monolayers than Arg and Val. Furthermore, RVPSL could be more easily transported as smaller peptides, especially in the form of dipeptides and tripeptides.

  11. A case of IgG4-related tubulointerstitial nephritis concurrent with Henoch-Schönlein purpura nephritis

    Directory of Open Access Journals (Sweden)

    Tamai Rukako

    2011-03-01

    Full Text Available Abstract We describe a 72-year-old man, who had been suffered from Henoch-Schönlein purpura (HSP several times, presented with hematoproteinuria with granular cast, and general lymphadenopathy. The immunological examination of the serum showed polyclonal hypergammagloburinemia with high value of IgG4. The renal biopsy revealed interstitial inflammatory cell infiltration, including infiltration of lymphocytes and plasma cells, and segmental glomerulonephritis. Direct immunofluorescence microscopy revealed apparent positive staining with anti-human IgA, and anti-human IgG in glomeruli, anti-human IgG4 antibody staining showed many positive plasma cells in the interstitium. The patient was diagnosed with HSP nephritis that was complicated by IgG4-related nephropathy. As a result of the treatment with 30mg prednisolone, the swelling of the LNs decreased, but the patient continued to have persistent hematoproteinuria.

  12. H{sup +}/peptide transporter (PEPT2) is expressed in human epidermal keratinocytes and is involved in skin oligopeptide transport

    Energy Technology Data Exchange (ETDEWEB)

    Kudo, Michiko; Katayoshi, Takeshi; Kobayashi-Nakamura, Kumiko [DHC Corporation Laboratories, Division 2, 2-42 Hamada, Mihama-ku, Chiba 261-0025 (Japan); Akagawa, Mitsugu [Department of Biological Chemistry, Division of Applied Life Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 599-8531 (Japan); Tsuji-Naito, Kentaro, E-mail: knaito@dhc.co.jp [DHC Corporation Laboratories, Division 2, 2-42 Hamada, Mihama-ku, Chiba 261-0025 (Japan)

    2016-07-08

    Peptide transporter 2 (PEPT2) is a member of the proton-coupled oligopeptide transporter family, which mediates the cellular uptake of oligopeptides and peptide-like drugs. Although PEPT2 is expressed in many tissues, its expression in epidermal keratinocytes remains unclear. We investigated PEPT2 expression profile and functional activity in keratinocytes. We confirmed PEPT2 mRNA expression in three keratinocyte lines (normal human epidermal keratinocytes (NHEKs), immortalized keratinocytes, and malignant keratinocytes) by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. In contrast to PEPT1, PEPT2 expression in the three keratinocytes was similar or higher than that in HepG2 cells, used as PEPT2-positive cells. Immunolocalization analysis using human skin showed epidermal PEPT2 localization. We studied keratinocyte transport function by measuring the oligopeptide content using liquid chromatography/tandem mass spectrometry. Glycylsarcosine uptake in NHEKs was pH-dependent, suggesting that keratinocytes could absorb small peptides in the presence of an inward H{sup +} gradient. We also performed a skin-permeability test of several oligopeptides using skin substitute, suggesting that di- and tripeptides pass actively through the epidermis. In conclusion, PEPT2 is expressed in keratinocytes and involved in skin oligopeptide uptake. -- Highlights: •PEPT2 is expressed in keratinocytes, which are more common than other skin cells. •Immunolocalization analysis using human skin revealed epidermal PEPT2 localization. •Keratinocytes could absorb small peptides in the presence of an inward H{sup +} gradient. •Di- and tripeptide pass actively through the epidermis.

  13. Hubungan IgG (IgG1 Dan IgG2) Dengan Stadium Dalam Penanggulangan Karsinoma Payudara

    OpenAIRE

    Hutagalung, Humala Nagatua

    2006-01-01

    In breast cancer the stage (TNM) determines the treatment as well as the prognosis. The transformation of a normal cell to a malignancy can result that the tumors express the antigens (unique tumor-specific antigens and tumors associated antigens ) which permit the immunologic reaction. Serologic analysis of antibody responses (IgG and subclasses) may be much more common. The objective of the study is 1. to analyse whether there is a relationship between the stages and the shift of th...

  14. Inhibition of human GLUT1 and GLUT5 by plant carbohydrate products; insights into transport specificity.

    Science.gov (United States)

    George Thompson, Alayna M; Iancu, Cristina V; Nguyen, Thi Thanh Hanh; Kim, Doman; Choe, Jun-yong

    2015-08-26

    Glucose transporters GLUT1 (transports glucose) and GLUT5 (transports fructose), in addition to their functions in normal metabolism, have been implicated in several diseases including cancer and diabetes. While GLUT1 has several inhibitors, none have been described for GLUT5. By transport activity assays we found two plant products, rubusoside (from Rubus suavissimus) and astragalin-6-glucoside (a glycosylated derivative of astragalin, from Phytolacca americana) that inhibited human GLUT5. These plants are utilized in traditional medicine: R. suavissimus for weight loss and P. americana for cancer treatment, but the molecular interactions of these products are unknown. Rubusoside also inhibited human GLUT1, but astragalin-6-glucoside did not. In silico analysis of rubusoside:protein interactions pinpointed a major difference in substrate cavity between these transporters, a residue that is a tryptophan in GLUT1 but an alanine in GLUT5. Investigation of mutant proteins supported the importance of this position in ligand specificity. GLUT1W388A became susceptible to inhibition by astragalin-6-glucoside and resistant to rubusoside. GLUT5A396W transported fructose and also glucose, and maintained inhibition by rubusoside and astragalin-6-glucoside. Astragalin-6-glucoside can serve as a starting point in the design of specific inhibitors for GLUT5. The application of these studies to understanding glucose transporters and their interaction with substrates and ligands is discussed.

  15. Regulation of human hepatic drug transporter activity and expression by diesel exhaust particle extract.

    Directory of Open Access Journals (Sweden)

    Marc Le Vee

    Full Text Available Diesel exhaust particles (DEPs are common environmental air pollutants primarily affecting the lung. DEPs or chemicals adsorbed on DEPs also exert extra-pulmonary effects, including alteration of hepatic drug detoxifying enzyme expression. The present study was designed to determine whether organic DEP extract (DEPe may target hepatic drug transporters that contribute in a major way to drug detoxification. Using primary human hepatocytes and transporter-overexpressing cells, DEPe was first shown to strongly inhibit activities of the sinusoidal solute carrier (SLC uptake transporters organic anion-transporting polypeptides (OATP 1B1, 1B3 and 2B1 and of the canalicular ATP-binding cassette (ABC efflux pump multidrug resistance-associated protein 2, with IC50 values ranging from approximately 1 to 20 μg/mL and relevant to environmental exposure situations. By contrast, 25 μg/mL DEPe failed to alter activities of the SLC transporter organic cation transporter (OCT 1 and of the ABC efflux pumps P-glycoprotein and bile salt export pump (BSEP, whereas it only moderately inhibited those of sodium taurocholate co-transporting polypeptide and of breast cancer resistance protein (BCRP. Treatment by 25 μg/mL DEPe was next demonstrated to induce expression of BCRP at both mRNA and protein level in cultured human hepatic cells, whereas it concomitantly repressed mRNA expression of various transporters, including OATP1B3, OATP2B1, OCT1 and BSEP. Such changes in transporter expression were found to be highly correlated to those caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, a reference activator of the aryl hydrocarbon receptor (AhR pathway. This suggests that DEPe, which is enriched in known ligands of AhR like polycyclic aromatic hydrocarbons, alters drug transporter expression via activation of the AhR cascade. Taken together, these data established human hepatic transporters as targets of organic chemicals containing in DEPs, which may contribute

  16. Assignment of the creatine transporter gene (SLC6A8) to human chromosome Xq28 telomeric to G6PD

    Energy Technology Data Exchange (ETDEWEB)

    Gregor, P. [National Inst. on Drug Abuse, Baltimore, MD (United States); Nash, S.R.; Caron, M.G. [Duke Univ., Durham, NC (United States)] [and others

    1995-01-01

    The creatine-phosphocreatine shuttle has important functions in the temporal and spatial maintenance of the energy supply to skeletal and cardiac muscle. Muscle cells do not synthesize creatine, but take it up via a specific sodium-dependent transporter - the creatine transporter. Thus, the creatine transporter has an important role in muscular physiology. Furthermore, inhibition of creatine transport in experimental animals causes muscle weakness. Recently, creatine transporter cDNAs have been isolated and characterized from rabbit and human. In this communication we report mapping of the creatine transporter gene to human chromosome Xq28. 12 refs., 1 fig.

  17. Inhibition of complement activation by IgG4 antibodies

    NARCIS (Netherlands)

    van der Zee, J. S.; van Swieten, P.; Aalberse, R. C.

    1986-01-01

    Prolonged exposure to antigens may result in high IgG4 antibody titres as was shown in a previous paper (Aalberse et al., 1983b). In novice bee keepers, a shift in the IgG1/IgG4 ratio of the response against phospholipase-A (PLA; a major component of bee venom) occurred. This resulted in an

  18. Effects of ionic strength on passive and iontophoretic transport of cationic permeant across human nail.

    Science.gov (United States)

    Smith, Kelly A; Hao, Jinsong; Li, S Kevin

    2009-06-01

    Transport across the human nail under hydration can be modeled as hindered transport across aqueous pore pathways. As such, nail permselectivity to charged species can be manipulated by changing the ionic strength of the system in transungual delivery to treat nail diseases. The present study investigated the effects of ionic strength upon transungual passive and iontophoretic transport. Transungual passive and anodal iontophoretic transport experiments of tetraethylammonium ion (TEA) were conducted under symmetric conditions in which the donor and receiver had the same ionic strength in vitro. Experiments under asymmetric conditions were performed to mimic the in vivo conditions. Prior to the transport studies, TEA uptake studies were performed to assess the partitioning of TEA into the nail. Permselectivity towards TEA was inversely related to ionic strength in both passive and iontophoretic transport. The permeability and transference number of TEA were higher at lower ionic strengths under the symmetric conditions due to increased partitioning of TEA into the nail. Transference numbers were smaller under the asymmetric conditions compared with their symmetric counterparts. The results demonstrate significant ionic strength effects upon the partitioning and transport of a cationic permeant in transungual transport, which may be instrumental in the development of transungual delivery systems.

  19. Esterification of Quercetin Increases Its Transport Across Human Caco-2 Cells.

    Science.gov (United States)

    Hu, Jiang-Ning; Zou, Xian-Guo; He, Yi; Chen, Fang; Deng, Ze-Yuan

    2016-07-01

    Plant polyphenols showed useful biochemical characteristics in vitro; however, the assessments of their clinical applications in vivo are restricted by their limited bioavailability due to their strong resistance to 1st-pass effects during absorption. In order to improve the bioavailability of quercetin (QU), the ester derivative of QU (3,3',4',5,7-pentahydroxy flavones, TAQU) was synthesized, followed by examining the oil-water partition coefficient as well as the transport mechanisms of QU and its ester derivative (TAQU) using human Caco-2 cells. The transport characteristics of QU and TAQU transport under different conditions (different concentrations, time, pH, temperature, tight junctions, and potential transporters) were systematically investigated. Results showed that QU had a lower permeability coefficient (2.82 × 10(-6) cm/s) for apical-to-basolateral (AP-BL) transport over 5 to 50 μM, whereas the transport rate for AP to BL flux of TAQU (5.23 × 10(-6) cm/s) was significantly greater than that of QU. Paracellular pathways were not involved during the transport of both QU and TAQU. QU was poorly absorbed by active transport, whereas TAQU was mostly absorbed by passive diffusion. Efflux transporters, P-glycoproteins, multidrug resistance proteins were proven to participate in the transport process of QU, but not in that of TAQU. These results suggested that improving the lipophicity of QU by esterification could increase the transport of QU across Caco-2 cells. © 2016 Institute of Food Technologists®

  20. Flavonoids Are Inhibitors of Human Organic Anion Transporter 1 (OAT1)–Mediated Transport

    Science.gov (United States)

    An, Guohua; Wang, Xiaodong

    2014-01-01

    Organic anion transporter 1 (OAT1) has been reported to be involved in the nephrotoxicity of many anionic xenobiotics. As current clinically used OAT1 inhibitors are often associated with safety issues, identifying potent OAT1 inhibitors with little toxicity is of great value in reducing OAT1-mediated drug nephrotoxicity. Flavonoids are a class of polyphenolic compounds with exceptional safety records. Our objective was to evaluate the effects of 18 naturally occurring flavonoids, and some of their glycosides, on the uptake of para-aminohippuric acid (PAH) in both OAT1-expressing and OAT1-negative LLC-PK1 cells. Most flavonoid aglycones produced substantial decreases in PAH uptake in OAT1-expressing cells. Among the flavonoids screened, fisetin, luteolin, morin, and quercetin exhibited the strongest effect and produced complete inhibition of OAT1-mediated PAH uptake at a concentration of 50 μM. Further concentration-dependent studies revealed that both morin and luteolin are potent OAT1 inhibitors, with IC50 values of flavonoid aglycones, all flavonoid glycosides had negligible or small effects on OAT1. In addition, the role of OAT1 in the uptake of fisetin, luteolin, morin, and quercetin was investigated and fisetin was found to be a substrate of OAT1. Taken together, our results indicate that flavonoids are a novel class of OAT1 modulators. Considering the high consumption of flavonoids in the diet and in herbal products, OAT1-mediated flavonoid-drug interactions may be clinically relevant. Further investigation is warranted to evaluate the nephroprotective role of flavonoids in relation to drug-induced nephrotoxicity mediated by the OAT1 pathway. PMID:25002746

  1. Asma y deficiencia de subclases de IgG Asthma and IgG subclases deficiency

    Directory of Open Access Journals (Sweden)

    Lucía Santamaría Ortiz

    1995-04-01

    Full Text Available

    Se estudiaron 45 pacientes asmáticos adultos de difícil manejo, de más de 5 años de evolución, 37 de ellos esteroide dependientes y 8 no dependientes, con asma alérgica o intrínseca y algunos con Infecciones respiratorias recurrentes de predominio viral. Por nefelometría se midieron los niveles séricos de las IgsG, M y A, y por ELISA se determinó la IgE total. Se encontraron 4 pacientes con deficiencia de IgG total, en el grupo de los esteroide dependientes. Mediante ELISA tipo sandwich y con anticuerpos monoclonales específicos para las sub clases de IgG se investigaron los niveles sé ricos de IgG1, 2, 3 y 4. En el 55.6% de los enfermos se encontraron una O más deficiencias de sub clases. No hubo diferencias significativas entre los grupos esteroide y no esteroide dependientes, ni entre los asmáticos alérgicos e intrínsecos, ni entre los con infección recurrente o sin ella. predominó la deficiencia de IgG1; en total el 46.7% de los pacientes tenían deficiencia aislada o combinada de IgG1, el 31.1% de IgG2, el 24.4% de IgG3 y el 17.8% de Igd4. La alta incidencia de deficiencia de sub clases podría deberse a la acción de los esteroides o a una alteración en la regulación de la síntesis de Igs producida por un defecto Inmune primario. Esta deficiencia sería la responsable del comportamiento agresivo de la enfermedad.

    We studied 45 adult asthmatic patients with difficult to care disease and who had more than five years of evolution; they suffered from elther allergic or intrinsic asthma and some had experienced recurrent respiratory tract infections. predominantly of viral etiology. Serum levels of IgA, IgG and IgM were measured by nephelometry and total lgE was determined by an Enzyme-Linked immunosorbent Assay (ELISA. Total lg

  2. Plant-based production of two chimeric monoclonal IgG antibodies directed against immunodominant epitopes of Vibrio cholerae lipopolysaccharide.

    Science.gov (United States)

    Levinson, Kara J; Giffen, Samantha R; Pauly, Michael H; Kim, Do H; Bohorov, Ognian; Bohorova, Natasha; Whaley, Kevin J; Zeitlin, Larry; Mantis, Nicholas J

    2015-07-01

    We have produced and characterized two chimeric human IgG1 monoclonal antibodies that bind different immunodominant epitopes on Vibrio cholerae lipopolysaccharide (LPS). MAb 2D6 IgG1 recognizes Ogawa O-polysaccharide antigen, while mAb ZAC-3 IgG1 recognizes core/lipid A moiety of Ogawa and Inaba LPS. Both antibodies were expressed using a Nicotiana benthamiana-based rapid antibody-manufacturing platform (RAMP) and evaluated in vitro for activities associated with immunity to V. cholerae, including vibriocidal activity, bacterial agglutination and motility arrest. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Expression pattern of the human ABC transporters in pluripotent embryonic stem cells and in their derivatives.

    Science.gov (United States)

    Erdei, Zsuzsa; Lőrincz, Réka; Szebényi, Kornélia; Péntek, Adrienn; Varga, Nóra; Likó, István; Várady, György; Szakács, Gergely; Orbán, Tamás I; Sarkadi, Balázs; Apáti, Agota

    2014-09-01

    ATP-binding cassette (ABC) transporters have key roles in various physiological functions as well as providing chemical defense and stress tolerance in human tissues. In this study, we have examined the expression pattern of all ABC proteins in pluripotent human embryonic stem cells (hESCs) and in their differentiated progenies. We paid special attention to the cellular expression and localization of multidrug transporter ABC proteins. Stem cell differentiation was carried out without chemical induction or cell sorting, and specialized cell types were separated mechanically. Cellular features regarding pluripotency and tissue identity, as well as ABC transporter expression were studied by flow cytomtery, immuno-microscopy, and qPCR-based low-density arrays. Pluripotent hESCs and differentiated cell types (cardiomyocytes, neuronal cells, and mesenchymal stem cells) were distinguished by morphology, immunostaining markers, and selected mRNA expression patterns. We found that the mRNA expression levels of the 48 human ABC proteins also clearly distinguished the pluripotent and the respective differentiated cell types. When multidrug and lipid transporter ABC protein expression was examined by using well characterized specific antibodies by flow cytometry and confocal microscopy, the protein expression data corresponded well to the mRNA expression results. Moreover, the cellular localization of these important human ABC transporter proteins could be established in the pluripotent and differentiated hESC derived samples. These studies provide valuable information regarding ABC protein expression in human stem cells and their differentiated offspring. The results may also help to obtain further information concerning the specialized cellular functions of selected ABC transporters. © 2014 Clinical Cytometry Society.

  4. Maternal-fetal transport kinetics of manganese in perfused human placental lobule in vitro.

    Science.gov (United States)

    Nandakumaran, Moorkath; Al-Sannan, Baydaa; Al-Sarraf, Hameed; Al-Shammari, Majed

    2016-01-01

    There have been no detailed reports relating to maternal-fetal transport kinetics of manganese, an essential trace element in the human pregnancies, and hence we have attempted to study the transport kinetics of this trace element in the human placenta in vitro. Human placentae from normal uncomplicated pregnancies were collected postpartum. Manganese chloride solution (GFS Chem Inc., Columbus, OH), 10 times the physiological concentrations, along with antipyrine (Sigma Chem Co., St. Louis, MO) as reference marker were then injected as a single bolus (100 µl) into the maternal arterial circulation of perfused placental lobules and perfusate samples collected from maternal and fetal circulations over a period of five minutes. National Culture and Tissue Collection medium, diluted with Earle's buffered salt solution was used as the perfusate and serial perfusate samples from fetal venous perfusate collected for a period of 30 min. Concentration of manganese in perfusate samples was assessed by atomic absorption spectrophotometry, while that of antipyrine was assessed by spectrophotometry. Transport kinetics of substances studied were computed using established permeation parameters. Differential transport rates of manganese and antipyrine in 12 perfusions differed significantly for 25.75, 90% efflux fractions (ANOVA test, p manganese averaged 54.9% of bolus dose in 12 perfusions, whereas that of antipyrine averaged 89% of bolus dose, representing 61.80% of reference marker TF. The difference observed in TF values of manganese and antipyrine was statistically significant (Student's t-test, p manganese compared to reference marker were significantly different (ANOVA test, p manganese in human placenta in vitro. Considering the restricted transfer of this essential trace element despite its small molecular weight, we hypothesize possibility of active transport of manganese across the human placental membrane. Further studies relating to manganese placental

  5. Competition between Serum IgG, IgM, and IgA Anti-Glycan Antibodies

    Science.gov (United States)

    Muthana, Saddam M.; Xia, Li; Campbell, Christopher T.; Zhang, Yalong; Gildersleeve, Jeffrey C.

    2015-01-01

    Anti-glycan antibodies are an abundant subpopulation of serum antibodies with critical functions in many immune processes. Changes in the levels of these antibodies can occur with the onset of disease, exposure to pathogens, or vaccination. As a result, there has been significant interest in exploiting anti-glycan antibodies as biomarkers for many diseases. Serum contains a mixture of anti-glycan antibodies that can recognize the same antigen, and competition for binding can potentially influence the detection of antibody subpopulations that are more relevant to disease processes. The most abundant antibody isotypes in serum are IgG, IgM, and IgA, but little is known regarding how these different isotypes compete for the same glycan antigen. In this study, we developed a multiplexed glycan microarray assay and applied it to evaluate how different isotypes of anti-glycan antibodies (IgA, IgG, and IgM) compete for printed glycan antigens. While IgG and IgA antibodies typically outcompete IgM for peptide or protein antigens, we found that IgM outcompete IgG and IgA for many glycan antigens. To illustrate the importance of this effect, we provide evidence that IgM competition can account for the unexpected observation that IgG of certain antigen specificities appear to be preferentially transported from mothers to fetuses. We demonstrate that IgM in maternal sera compete with IgG resulting in lower than expected IgG signals. Since cord blood contains very low levels of IgM, competition only affects maternal IgG signals, making it appear as though certain IgG antibodies are higher in cord blood than matched maternal blood. Taken together, the results highlight the importance of competition for studies involving anti-glycan antibodies. PMID:25807519

  6. Seroprevalence of cytomegalovirus IgG antibodies among pregnant women in Japan from 2009-2014.

    Science.gov (United States)

    Shigemi, Daisuke; Yamaguchi, Satoru; Otsuka, Toshiaki; Kamoi, Seiryu; Takeshita, Toshiyuki

    2015-11-01

    Human cytomegalovirus (CMV) is a major cause of congenital infection. The seroprevalence of maternal CMV IgG antibodies among Japanese women is decreasing. In this study, we assessed the rate of and risk factors for CMV infection. This article includes a description of a method for the prevention of CMV infection. Medical records of 7,074 women who delivered a baby at our hospital were retrospectively reviewed. For seronegative patients, preventive educational materials were provided, and CMV IgG antibody levels were reassessed during late pregnancy. Congenital infection in neonates from seroconverted mothers was determined by urine analysis. The overall CMV IgG seropositivity rate was 69.1%. The prevalence of CMV IgG increased with age and parity. In a multivariate logistic regression analysis, parity remained an independent determinant of CMV IgG seropositivity. The seroconversion rate for CMV IgG antibody during pregnancy was 0.37%. Neonatal congenital CMV infection occurred in 37.5% of seroconverted women. The risk of primary CMV infection in mothers during their first pregnancy was 7.0%, with an average follow-up period of 2.1 years. We found that parity was an independent determinant of CMV IgG seropositivity, suggesting that child-rearing may be a high risk factor for maternal CMV infection. The provision of information on hygiene may be an effective and inexpensive method for preventing CMV infection. Copyright © 2015 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

  7. Allergen-specific IgG antibody signaling through FcγRIIb promotes food tolerance.

    Science.gov (United States)

    Burton, Oliver T; Tamayo, Jaciel M; Stranks, Amanda J; Koleoglou, Kyle J; Oettgen, Hans C

    2017-05-04

    Patients with food allergy produce high-titer IgE antibodies that bind to mast cells through FcεRI and trigger immediate hypersensitivity reactions on antigen encounter. Food-specific IgG antibodies arise in the setting of naturally resolving food allergy and accompany the acquisition of food allergen unresponsiveness in oral immunotherapy. In this study we sought to delineate the effects of IgG and its inhibitory Fc receptor, FcγRIIb, on both de novo allergen sensitization in naive animals and on established immune responses in the setting of pre-existing food allergy. Allergen-specific IgG was administered to mice undergoing sensitization and desensitization to the model food allergen ovalbumin. Cellular and molecular mechanisms were interrogated by using mast cell- and FcγRIIb-deficient mice. The requirement for FcγRII in IgG-mediated inhibition of human mast cells was investigated by using a neutralizing antibody. Administration of specific IgG to food allergy-prone IL4raF709 mice during initial food exposure prevented the development of IgE antibodies, TH2 responses, and anaphylactic responses on challenge. When given as an adjunct to oral desensitization in mice with established IgE-mediated hypersensitivity, IgG facilitated tolerance restoration, favoring expansion of forkhead box protein 3-positive regulatory T cells along with suppression of existing TH2 and IgE responses. IgG and FcγRIIb suppress adaptive allergic responses through effects on mast cell function. These findings suggest that allergen-specific IgG antibodies can act to induce and sustain immunologic tolerance to foods. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  8. Podocytes use FcRn to clear IgG from the glomerular basement membrane.

    Science.gov (United States)

    Akilesh, Shreeram; Huber, Tobias B; Wu, Hui; Wang, Gary; Hartleben, Björn; Kopp, Jeffrey B; Miner, Jeffrey H; Roopenian, Derry C; Unanue, Emil R; Shaw, Andrey S

    2008-01-22

    The glomerular filtration barrier prevents large serum proteins from being lost into the urine. It is not known, however, why the filter does not routinely clog with large proteins that enter the glomerular basement membrane (GBM). Here, we provide evidence that an active transport mechanism exists to remove immunoglobulins that accumulate at the filtration barrier. We found that FcRn, an IgG and albumin transport receptor, is expressed in podocytes and functions to internalize IgG from the GBM. Mice lacking FcRn accumulated IgG in the GBM as they aged, and tracer studies showed delayed clearance of IgG from the kidneys of FcRn-deficient mice. Supporting a role for this pathway in disease, saturating the clearance mechanism potentiated the pathogenicity of nephrotoxic sera. These studies support the idea that podocytes play an active role in removing proteins from the GBM and suggest that genetic or acquired impairment of the clearance machinery is likely to be a common mechanism promoting glomerular diseases.

  9. Hydrogen peroxide stimulates the active transport of serotonin into human platelets

    Energy Technology Data Exchange (ETDEWEB)

    Bosin, T.R. (Indiana Univ., Bloomington (United States))

    1991-03-11

    The effect of hydrogen peroxide on the active transport of serotonin (5-HT) by human platelets was investigated. Platelets were exposed to either a single dose of H{sub 2}O{sub 2} or to H{sub 2}O{sub 2} generated by the glucose/glucose oxidase or xanthine/xanthine oxidase enzyme systems. H{sub 2}{sub 2} produced a rapid, dose-dependent and time-dependent increase in 5-HT transport which was maximal after a 2 min incubation and decreased with continued incubation. Catalase completely prevented H{sub 2}O{sub 2}-induced stimulation and fluoxetine totally blocked 5-HT uptake into stimulated platelets. The glucose/glucose oxidase and the xanthine/xanthine oxidase generating systems produced a similar response to that of H{sub 2}O{sub 2}. In the xanthine/xanthine oxidase system, superoxide dismutase failed to alter the stimulation, while catalase effectively prevented the response. The kinetics of 5-HT transport indicated that H{sub 2}O{sub 2} treatment did not alter the K{sub m} of 5-HT transport but significantly increased the maximal rate of 5-HT transport. These data demonstrated that exposure of human platelets to H{sub 2}O{sub 2} resulted in a stimulation of the active transport of 5-HT and suggested that H{sub 2}O{sub 2} may function to regulate this process.

  10. Isotonic transport by the Na+-glucose cotransporter SGLT1 from humans and rabbit

    DEFF Research Database (Denmark)

    Zeuthen, T; Meinild, A K; Loo, D D

    2001-01-01

    1. In order to study its role in steady state water transport, the Na+-glucose cotransporter (SGLT1) was expressed in Xenopus laevis oocytes; both the human and the rabbit clones were tested. The transport activity was monitored as a clamp current and the flux of water followed optically as the c......1. In order to study its role in steady state water transport, the Na+-glucose cotransporter (SGLT1) was expressed in Xenopus laevis oocytes; both the human and the rabbit clones were tested. The transport activity was monitored as a clamp current and the flux of water followed optically...... as the change in oocyte volume. 2. SGLT1 has two modes of water transport. First, it acts as a molecular water pump: for each 2 Na+ and 1 sugar molecule 264 water molecules were cotransported in the human SGLT1 (hSGLT1), 424 for the rabbit SGLT1 (rSGLT1). Second, it acts as a water channel. 3. The cotransport...

  11. Transepithelial transport of ambroxol hydrochloride across human intestinal Caco-2 cell monolayers.

    Science.gov (United States)

    Stetinová, Vera; Smetanová, Libuse; Kholová, Dagmar; Svoboda, Zbynek; Kvetina, Jaroslav

    2009-09-01

    This study aimed i) to characterize the transepithelial transport of the mucolytic agent ambroxol hydrochloride across the intestinal barrier, ii) to classify the ambroxol according to Biopharmaceutics Classification System (BCS) and iii) to predict ambroxol absorption in humans. Transport of ambroxol (100, 300 and 1000 micromol/l) was studied in a human colon carcinoma cell line Caco-2 in apical to basolateral and basolateral to apical direction, under iso-pH 7.4 and pH-gradient (6 vs. 7.4) conditions. The relative contribution of the paracellular route was estimated using Ca2+-free transport medium. Ambroxol samples from receiver compartments were analysed by HPLC with UV detection (242 nm). Results showed that ambroxol transport is linear with time, pH-dependent and direction-independent, displays non-saturable (first-order) kinetics. Thus, the transport seems to be transcellular mediated by passive diffusion. Estimated high solubility and high permeability (P(app) = 45 x 10(-6) cm/s) of ambroxol rank it among well absorbed compounds and class I of BCS. It can be expected that the oral dose fraction of ambroxol absorbed in human intestine is high.

  12. Analysis of human factors effects on the safety of transporting radioactive waste materials: Technical report

    Energy Technology Data Exchange (ETDEWEB)

    Abkowitz, M.D.; Abkowitz, S.B.; Lepofsky, M.

    1989-04-01

    This report examines the extent of human factors effects on the safety of transporting radioactive waste materials. It is seen principally as a scoping effort, to establish whether there is a need for DOE to undertake a more formal approach to studying human factors in radioactive waste transport, and if so, logical directions for that program to follow. Human factors effects are evaluated on driving and loading/transfer operations only. Particular emphasis is placed on the driving function, examining the relationship between human error and safety as it relates to the impairment of driver performance. Although multi-modal in focus, the widespread availability of data and previous literature on truck operations resulted in a primary study focus on the trucking mode from the standpoint of policy development. In addition to the analysis of human factors accident statistics, the report provides relevant background material on several policies that have been instituted or are under consideration, directed at improving human reliability in the transport sector. On the basis of reported findings, preliminary policy areas are identified. 71 refs., 26 figs., 5 tabs.

  13. Frog oocytes to unveil the structure and supramolecular organization of human transport proteins.

    Directory of Open Access Journals (Sweden)

    Marc J Bergeron

    Full Text Available Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes. The method was validated for four human and one murine transporter. Negative stain transmission electron microscopy (TEM and single particle analysis (SPA of two of these transporters, i.e., the potassium-chloride cotransporter 4 (KCC4 and the aquaporin-1 (AQP1 water channel, revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly. This is the first time a cation-chloride cotransporter (SLC12 family member is isolated, and its shape, dimensions, low-resolution structure and oligomeric state determined by TEM, i.e., by a direct method. Finally, we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein. This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression.

  14. Overcoming Monocarboxylate Transporter 8 (MCT8-Deficiency to Promote Human Oligodendrocyte Differentiation and Myelination

    Directory of Open Access Journals (Sweden)

    Jae Young Lee

    2017-11-01

    Full Text Available Cell membrane thyroid hormone (TH transport can be facilitated by the monocarboxylate transporter 8 (MCT8, encoded by the solute carrier family 16 member 2 (SLC16A2 gene. Human mutations of the gene, SLC16A2, result in the X-linked-inherited psychomotor retardation and hypomyelination disorder, Allan-Herndon-Dudley syndrome (AHDS. We posited that abrogating MCT8-dependent TH transport limits oligodendrogenesis and myelination. We show that human oligodendrocytes (OL, derived from the NKX2.1-GFP human embryonic stem cell (hESC reporter line, express MCT8. Moreover, treatment of these cultures with DITPA (an MCT8-independent TH analog, up-regulates OL differentiation transcription factors and myelin gene expression. DITPA promotes hESC-derived OL myelination of retinal ganglion axons in co-culture. Pharmacological and genetic blockade of MCT8 induces significant OL apoptosis, impairing myelination. DITPA treatment limits OL apoptosis mediated by SLC16A2 down-regulation primarily signaling through AKT phosphorylation, driving myelination. Our results highlight the potential role of MCT8 in TH transport for human OL development and may implicate DITPA as a promising treatment for developmentally-regulated myelination in AHDS.

  15. Diffusion and transport in the human interphase cell nucleus - FCS experiments compared to simulations.

    NARCIS (Netherlands)

    M. Wachsmuth (Malte); T.A. Knoch (Tobias); C. Münkel (Christian); J. Langowski (Jörg)

    2001-01-01

    markdownabstractDespite the succesful linear sequencing of the human genome the three-dimensional arrangement of chromatin, functional, and structural components is still largely unknown. Molecular transport and diffusion are important for processes like gene regulation, replication, or repair and

  16. Identification of the first highly selective inhibitor of human GABA transporter GAT3

    DEFF Research Database (Denmark)

    Damgaard, Maria; Al-Khawaja, Anas; Vogensen, Stine B.

    2015-01-01

    Screening a library of small-molecule compounds using a cell line expressing human GABA transporter 3 (hGAT3) in a [(3)H]GABA uptake assay identified isatin derivatives as a new class of hGAT3 inhibitors. A subsequent structure-activity relationship (SAR) study led to the identification of hGAT3...

  17. Operation, Safety and Human: Critical Factors for the Success of Railway Transportation

    NARCIS (Netherlands)

    Rajabali Nejad, Mohammadreza; Martinetti, Alberto; van Dongen, Leonardus Adriana Maria

    2016-01-01

    This paper focuses on three categories of performance indicators for railway transportation: the excellence of operation, system safety and human factors. These are among the most critical indicators for delivering high quality services. This paper discusses the main issues, challenges and future

  18. Simultaneous measurement of glucose transport and utilization in the human brain

    Science.gov (United States)

    Shestov, Alexander A.; Emir, Uzay E.; Kumar, Anjali; Henry, Pierre-Gilles; Seaquist, Elizabeth R.

    2011-01-01

    Glucose is the primary fuel for brain function, and determining the kinetics of cerebral glucose transport and utilization is critical for quantifying cerebral energy metabolism. The kinetic parameters of cerebral glucose transport, KMt and Vmaxt, in humans have so far been obtained by measuring steady-state brain glucose levels by proton (1H) NMR as a function of plasma glucose levels and fitting steady-state models to these data. Extraction of the kinetic parameters for cerebral glucose transport necessitated assuming a constant cerebral metabolic rate of glucose (CMRglc) obtained from other tracer studies, such as 13C NMR. Here we present new methodology to simultaneously obtain kinetic parameters for glucose transport and utilization in the human brain by fitting both dynamic and steady-state 1H NMR data with a reversible, non-steady-state Michaelis-Menten model. Dynamic data were obtained by measuring brain and plasma glucose time courses during glucose infusions to raise and maintain plasma concentration at ∼17 mmol/l for ∼2 h in five healthy volunteers. Steady-state brain vs. plasma glucose concentrations were taken from literature and the steady-state portions of data from the five volunteers. In addition to providing simultaneous measurements of glucose transport and utilization and obviating assumptions for constant CMRglc, this methodology does not necessitate infusions of expensive or radioactive tracers. Using this new methodology, we found that the maximum transport capacity for glucose through the blood-brain barrier was nearly twofold higher than maximum cerebral glucose utilization. The glucose transport and utilization parameters were consistent with previously published values for human brain. PMID:21791622

  19. Simultaneous measurement of glucose transport and utilization in the human brain.

    Science.gov (United States)

    Shestov, Alexander A; Emir, Uzay E; Kumar, Anjali; Henry, Pierre-Gilles; Seaquist, Elizabeth R; Öz, Gülin

    2011-11-01

    Glucose is the primary fuel for brain function, and determining the kinetics of cerebral glucose transport and utilization is critical for quantifying cerebral energy metabolism. The kinetic parameters of cerebral glucose transport, K(M)(t) and V(max)(t), in humans have so far been obtained by measuring steady-state brain glucose levels by proton ((1)H) NMR as a function of plasma glucose levels and fitting steady-state models to these data. Extraction of the kinetic parameters for cerebral glucose transport necessitated assuming a constant cerebral metabolic rate of glucose (CMR(glc)) obtained from other tracer studies, such as (13)C NMR. Here we present new methodology to simultaneously obtain kinetic parameters for glucose transport and utilization in the human brain by fitting both dynamic and steady-state (1)H NMR data with a reversible, non-steady-state Michaelis-Menten model. Dynamic data were obtained by measuring brain and plasma glucose time courses during glucose infusions to raise and maintain plasma concentration at ∼17 mmol/l for ∼2 h in five healthy volunteers. Steady-state brain vs. plasma glucose concentrations were taken from literature and the steady-state portions of data from the five volunteers. In addition to providing simultaneous measurements of glucose transport and utilization and obviating assumptions for constant CMR(glc), this methodology does not necessitate infusions of expensive or radioactive tracers. Using this new methodology, we found that the maximum transport capacity for glucose through the blood-brain barrier was nearly twofold higher than maximum cerebral glucose utilization. The glucose transport and utilization parameters were consistent with previously published values for human brain.

  20. Identification of interspecies difference in efflux transporters of hepatocytes from dog, rat, monkey and human.

    Science.gov (United States)

    Li, Meng; Yuan, Haodan; Li, Na; Song, Guotao; Zheng, Yi; Baratta, Mike; Hua, Fengmei; Thurston, Archie; Wang, Joanne; Lai, Yurong

    2008-09-02

    The large interspecies differences of hepatobiliary transport present a challenge for the allometric prediction of human biliary excretion for drug candidates primarily cleared via hepatobiliary secretion. In the present study, we determined the metabolic stabilities of common fluorescent substrates of hepatobiliary efflux transporters and developed a rapid efflux assay to determine the functional activities of MRP/Mrp, BCRP/Bcrp and P-gp in hepatocytes of four species. The specificities of transporter-mediated dye efflux were confirmed by selective transporter inhibitors. Among tested species, transporter-specific dye efflux kinetics was consistent between freshly isolated and cryopreserved hepatocytes. Hepatocyte elimination half-lives of MRP/Mrp substrates GS-MF and calcein were observed in the rank order of human>monkey>dog>rat. The fourfold higher MRP/Mrp substrate efflux rate of rat hepatocytes compared to human is likely due to the species-specific functional differences of MRP2/Mrp2 expressed on the canalicular membrane. We also observed efficient BCRP-mediated pheophorbide A (PhA) efflux by human and dog hepatocytes, while PhA extrusion in monkey and rat hepatocytes appeared limited. P-gp function measured by DiOC2(3) efflux was minimal in hepatocytes of all origins and no significant species differences were detected. Our results demonstrated marked differences in hepatocyte MRP/Mrp and BCRP/Bcrp activities across species, indicating that they may contribute to the species differences of in vivo hepatobiliary excretion. These results also suggest the potential utility of primary hepatocytes, either fresh or cryopreserved, as an in vitro model to predict interspecies differences in the biliary transport of MRP/Mrp and BCRP/Bcrp substrates.

  1. A prominent lack of IgG1-Fc fucosylation of platelet alloantibodies in pregnancy

    NARCIS (Netherlands)

    Kapur, Rick; Kustiawan, Iwan; Vestrheim, Anne; Koeleman, Carolien A. M.; Visser, Remco; Einarsdottir, Helga K.; Porcelijn, Leendert; Jackson, Dave; Kumpel, Belinda; Deelder, André M.; Blank, Dennis; Skogen, Björn; Killie, Mette Kjaer; Michaelsen, Terje E.; de Haas, Masja; Rispens, Theo; van der Schoot, C. Ellen; Wuhrer, Manfred; Vidarsson, Gestur

    2014-01-01

    Immunoglobulin G (IgG) formed during pregnancy against human platelet antigens (HPAs) of the fetus mediates fetal or neonatal alloimmune thrombocytopenia (FNAIT). Because antibody titer or isotype does not strictly correlate with disease severity, we investigated by mass spectrometry variations in

  2. A prominent lack of IgG1-Fc fucosylation of platelet alloantibodies in pregnancy.

    NARCIS (Netherlands)

    Kapur, R.; Kustiawan, I.; Vestrheim, A.; Koeleman, C.A.; Visser, R.; Einarsdottir, H.K.; Porcelijn, L.; Jackson, D.; Kumpel, B.; Deelder, A.M.; Blank, D.; Skogen, B.; Killie, M.K.; Michaelsen, T.E.; de Haas, M.; Rispens, T.; van der Schoot, C.E.; Wuhrer, M.; Vidarsson, G.

    2014-01-01

    Immunoglobulin G (IgG) formed during pregnancy against human platelet antigens (HPAs) of the fetus mediates fetal or neonatal alloimmune thrombocytopenia (FNAIT). Because antibody titer or isotype does not strictly correlate with disease severity, we investigated by mass spectrometry variations in

  3. Proteolytic activity of IgGs from blood serum of wistar rats at experimental rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Yu. Ya. Kit

    2014-10-01

    Full Text Available The aim of this work was to study the proteolytic activity of IgGs purified from blood serum of Wistar rats at experimental rheumatoid arthritis (ERA induced by an injection of bovine collagen of type II. Twenty rats were immunized with a preparation of bovine collagen II (Sigma-Aldrich, USA in the presence of complete Freund’s adjuvant. ERA development was determined by inflammation in limbs of treated animals. IgG preparations were isolated from blood serum of immunized and non-immunized animals by precipitation of antibodies with 33% ammonium sulfate followed by chromatography on the Protein G-Sepharose column. Human histone H1, bovine collagen II, calf thymus histones, myelin basic protein (MBP, bovine serum albumin (BSA, and bovine casein were used as substrates of the proteolytic activity of IgGs. It was found that IgG preparations from blood serum of rats with ERA were capable of cleaving histone H1 and MBP, however, they were catalytically inactive towards collagen II, casein, BSA, and core histones. IgGs from blood serum of non-immunized rats were proteolytically inactive towards all used protein substrates. Thus, we demonstrated that immunization of rats with bovine collagen II induced IgG-antibodies possessing the proteolytic activity towards histone H1 and MBP. This activity might be associated with the development of inflammatory processes in the immunized rats.

  4. Neuromyelitis optica IgG in the cerebrospinal fluid induces astrocytopathy in optic nerve

    DEFF Research Database (Denmark)

    Soelberg, Kerstin; Lillevang, Søren Thue; Mørch, Marlene

    was coincident with deposition of complement. Histopathological lesions were markedly enhanced with extensive/long-segment astrocytopathy of optic nerve and optic chiasm involvement in AQP4- IgG+ C + anti-CD59a treated mice. Such pathology was not seen in mice receiving normal human IgG, C and anti-CD59a......-ON. The predilection of the optic nerve in NMOSD may partly be explained by the dense expression of AQP4 in the optic nerve. We previously reported that AQP4-IgG in cerebrospinal fluid (CSF) becomes widely distributed in the brain and causes complementdependent astrocyte injury in periventricular areas and brain...... of brain-specific antibodies in cerebrospinal fluid. Journal of neuroimmunology 2013;254:76-82....

  5. Common variable immunodeficiency and IgG subclass deficiency in central Alabama hemochromatosis probands homozygous for HFE C282Y.

    Science.gov (United States)

    Barton, James C; Bertoli, Luigi F; Acton, Ronald T

    2003-01-01

    Eight hemochromatosis probands with HFE C282Y homozygosity had frequent, severe, or unusual infections and common variable immunodeficiency (CVID) or immunoglobulin (Ig) G subclass deficiency (IgGSD). Thus, we performed serum Ig isotyping and other characterization of 43 additional unselected probands, 5 human leukocyte antigen (HLA)-identical siblings, and 240 consecutive CVID or IgGSD index patients. C282Y allele frequencies were estimated in 58 CVID or IgGSD index patients without hemochromatosis phenotypes and in 341 controls. HLA-A and -B haplotypes and frequencies were determined in all 51 probands, 186 CVID or IgGSD index patients without hemochromatosis phenotypes, and 751 controls. Thirteen unselected probands (30%) had CVID or IgGSD. Among all 21 hemochromatosis probands with CVID (n = 4) or IgGSD (n = 17), Ig subclass deficiency patterns were IgG(1) (n = 5), IgG(1) and IgG(3) (n = 6), IgG(3) (n = 9), and IgG(1), IgG(3), and IgG(4) (n = 1). IgG(2) or IgA deficiency was not detected; one proband had IgM deficiency. Mean values of total IgG, IgG(1), and IgG(3) were significantly lower in probands with CVID or IgGSD. Mean values of age, transferrin saturation, and ferritin at diagnosis and phlebotomy units required to induce iron depletion were similar in probands with or without CVID or IgGSD; phlebotomy had no apparent effect on IgG levels. C282Y frequencies were similar in CVID or IgGSD index cases without hemochromatosis phenotypes and in controls. There was concordance of Ig and hemochromatosis phenotypes in probands and respective HLA-identical siblings. Eight of 240 CVID or IgGSD index patients had hemochromatosis phenotypes and C282Y homozygosity (3 vs 0.7% and 0.2% controls; P IgG abnormalities characteristic of CVID or IgGSD are common in hemochromatosis probands, and that the prevalence of hemochromatosis is increased in CVID and IgGSD index cases. These observations could be explained by the increased frequencies of HLA-A*03-B*07 in C282Y

  6. Transportes

    Directory of Open Access Journals (Sweden)

    Hidalgo Fernández-Cano, Amalio

    1960-01-01

    Full Text Available El movimiento de materiales dentro de la Factoría está atendido por tres principales medios de transporte, en consonancia con las características del material y de los desplazamientos. Así se han establecido: sistemas de cintas transportadoras, una red ferroviaria de ancho normal y una completa malla de caminos enlazando funcionalmente las instalaciones.

  7. Persistent human Borna disease virus infection modifies the acetylome of human oligodendroglia cells towards higher energy and transporter levels.

    Science.gov (United States)

    Liu, Xia; Liu, Siwen; Bode, Liv; Liu, Chengyu; Zhang, Liang; Wang, Xiao; Li, Dan; Lei, Yang; Peng, Xiaojun; Cheng, Zhongyi; Xie, Peng

    2015-11-01

    Borna disease virus (BDV) is a neurotropic RNA virus persistently infecting mammalian hosts including humans. Lysine acetylation (Kac) is a key protein post-translational modification (PTM). The unexpectedly broad regulatory scope of Kac let us to profile the entire acetylome upon BDV infection. The acetylome was profiled through stable isotope labeling for cell culture (SILAC)-based quantitative proteomics. The quantifiable proteome was annotated using bioinformatics. We identified and quantified 791 Kac sites in 473 Kac proteins in human BDV Hu-H1-infected and non-infected oligodendroglial (OL) cells. Bioinformatic analysis revealed that BDV infection alters the acetylation of metabolic proteins, membrane-associated proteins and transmembrane transporter activity, and affects the acetylation of several lysine acetyltransferases (KAT). Upon BDV persistence the OL acetylome is manipulated towards higher energy and transporter levels necessary for shuttling BDV proteins to and from nuclear replication sites. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. A proposed study on the transplacental transport of parabens in the human placental perfusion model.

    Science.gov (United States)

    Mathiesen, Line; Zuri, Giuseppina; Andersen, Maria H; Knudsen, Lisbeth E

    2013-12-01

    Human exposure to parabens as a preservative used in personal care products is of increasing concern, as there is evidence from in vivo and in vitro studies of hormone disruption in association with exposure to parabens. Transport across the placenta could be critical for risk assessment, but the available data are sparse. The aim is to develop a method for estimating fetal exposure, via the placenta, to the most commonly-used parabens, by using a human placental perfusion model. The use of human tissue is vital for determining human fetal exposure, because animal studies are of little relevance, since the placenta exhibits significant interspecies variation. An HPLC model is currently being established to simultaneously quantify four different parabens, namely, methylparaben, ethylparaben, propylparaben and butylparaben, and their main metabolite, p-hydroxybenzoic acid. With this model, we aim to determine the transport kinetics of these parabens across the human placenta, and to investigate placental metabolism, including differences in transport due to molecular characteristics. This will facilitate assessment of the risks associated with the use of paraben-containing products during pregnancy. 2013 FRAME.

  9. Flow cytometric-based protocols for assessing anti-MT-2 IgG1 reactivity: High-dimensional data handling to define predictors for clinical follow-up of Human T-cell Leukemia virus type-1 infection.

    Science.gov (United States)

    Coelho-Dos-Reis, Jordana Grazziela; Peruhype-Magalhães, Vanessa; Pascoal-Xavier, Marcelo Antônio; de Souza Gomes, Matheus; do Amaral, Laurence Rodrigues; Cardoso, Ludmila Melo; Jonathan-Gonçalves, Juan; Ribeiro, Ágata Lopes; Starling, Ana Lúcia Borges; Ribas, João Gabriel; Gonçalves, Denise Utsch; de Freitas Carneiro-Proietti, Anna Bárbara; Teixeira-Carvalho, Andréa; Martins-Filho, Olindo Assis

    2017-05-01

    The present work provides an innovative methodological approach to assess the anti-HTLV-1 IgG1 reactivity with practical application in clinical laboratory. Serum from non-infected healthy controls (NI) and HTLV-1-infected patients, categorized as asymptomatic (AS), putatively progressing to HTLV-1 associated myelopathy/tropical spastic paraparesis - HAM/TSP (pHAM) or with clinical diagnosis of HAM/TSP (HT) were assayed in two-parallel flow cytometry platforms, referred as: Fix and Fix&Perm protocols. Operating-characteristics analysis indicated that a single pair of attributes ("serum dilution/cut-off") for Fix and Fix&Perm protocols presented excellent performance for the diagnosis of HTLV-1 infection. Conversely, Fix and Fix&Perm protocols displayed weak/moderate overall performances when applied with prognosis purposes of HTLV-1 infection. A panoramic snapshot provided by the reactivity boards revealed clearly the higher sensitivity of Fix&Perm protocol for detecting seropositivity for HT, suggesting that stepwise combinatory criteria would improve the global performance of using a single pair of attributes. Three data mining strategies were tested, including endpoint titer analysis, heatmap assemblage and decision tree analysis. Bi-dimensional heatmap analysis demonstrated that, while the clustering profile of NI vs HTLV-1+ revealed segregation in opposite poles, AS vs HT presented discrete segregation but still displaying an intertwined distribution pattern. The combination of methods for segregating AS from HT displayed a moderate but superior global accuracy (85.7%; LOOCV=71.4%). The comprehensive data analysis support that the combination of methods have improved the performance to the differential diagnosis of AS and HT, with direct association with laboratorial records, including serum cytokine levels and proviral load. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Human genetic variation influences vitamin C homeostasis by altering vitamin C transport and antioxidant enzyme function.

    Science.gov (United States)

    Michels, Alexander J; Hagen, Tory M; Frei, Balz

    2013-01-01

    New evidence for the regulation of vitamin C homeostasis has emerged from several studies of human genetic variation. Polymorphisms in the genes encoding sodium-dependent vitamin C transport proteins are strongly associated with plasma ascorbate levels and likely impact tissue cellular vitamin C status. Furthermore, genetic variants of proteins that suppress oxidative stress or detoxify oxidatively damaged biomolecules, i.e., haptoglobin, glutathione-S-transferases, and possibly manganese superoxide dismutase, affect ascorbate levels in the human body. There also is limited evidence for a role of glucose transport proteins. In this review, we examine the extent of the variation in these genes, their impact on vitamin C status, and their potential role in altering chronic disease risk. We conclude that future epidemiological studies should take into account genetic variation in order to successfully determine the role of vitamin C nutriture or supplementation in human vitamin C status and chronic disease risk.

  11. IgG responses to the gSG6-P1 salivary peptide for evaluating human exposure to Anopheles bites in urban areas of Dakar region, Sénégal

    National Research Council Canada - National Science Library

    Drame, Papa M; Machault, Vanessa; Diallo, Abdoulaye; Cornélie, Sylvie; Poinsignon, Anne; Lalou, Richard; Sembène, Mbacké; Dos Santos, Stéphanie; Rogier, Christophe; Pagès, Frédéric; Le Hesran, Jean-Yves; Remoué, Franck

    2012-01-01

    Urban malaria can be a serious public health problem in Africa. Human-landing catches of mosquitoes, a standard entomological method to assess human exposure to malaria vector bites, can lack sensitivity in areas where exposure is low...

  12. Identification of functional amino acid residues involved in polyamine and agmatine transport by human organic cation transporter 2.

    Directory of Open Access Journals (Sweden)

    Kyohei Higashi

    Full Text Available Polyamine (putrescine, spermidine and spermine and agmatine uptake by the human organic cation transporter 2 (hOCT2 was studied using HEK293 cells transfected with pCMV6-XL4/hOCT2. The Km values for putrescine and spermidine were 7.50 and 6.76 mM, and the Vmax values were 4.71 and 2.34 nmol/min/mg protein, respectively. Spermine uptake by hOCT2 was not observed at pH 7.4, although it inhibited both putrescine and spermidine uptake. Agmatine was also taken up by hOCT2, with Km value: 3.27 mM and a Vmax value of 3.14 nmol/min/mg protein. Amino acid residues involved in putrescine, agmatine and spermidine uptake by hOCT2 were Asp427, Glu448, Glu456, Asp475, and Glu516. In addition, Glu524 and Glu530 were involved in putrescine and spermidine uptake activity, and Glu528 and Glu540 were weakly involved in putrescine uptake activity. Furthermore, Asp551 was also involved in the recognition of spermidine. These results indicate that the recognition sites for putrescine, agmatine and spermidine on hOCT2 strongly overlap, consistent with the observation that the three amines are transported with similar affinity and velocity. A model of spermidine binding to hOCT2 was constructed based on the functional amino acid residues.

  13. Identification of Functional Amino Acid Residues Involved in Polyamine and Agmatine Transport by Human Organic Cation Transporter 2

    Science.gov (United States)

    Higashi, Kyohei; Imamura, Masataka; Fudo, Satoshi; Uemura, Takeshi; Saiki, Ryotaro; Hoshino, Tyuji; Toida, Toshihiko; Kashiwagi, Keiko; Igarashi, Kazuei

    2014-01-01

    Polyamine (putrescine, spermidine and spermine) and agmatine uptake by the human organic cation transporter 2 (hOCT2) was studied using HEK293 cells transfected with pCMV6-XL4/hOCT2. The Km values for putrescine and spermidine were 7.50 and 6.76 mM, and the Vmax values were 4.71 and 2.34 nmol/min/mg protein, respectively. Spermine uptake by hOCT2 was not observed at pH 7.4, although it inhibited both putrescine and spermidine uptake. Agmatine was also taken up by hOCT2, with Km value: 3.27 mM and a Vmax value of 3.14 nmol/min/mg protein. Amino acid residues involved in putrescine, agmatine and spermidine uptake by hOCT2 were Asp427, Glu448, Glu456, Asp475, and Glu516. In addition, Glu524 and Glu530 were involved in putrescine and spermidine uptake activity, and Glu528 and Glu540 were weakly involved in putrescine uptake activity. Furthermore, Asp551 was also involved in the recognition of spermidine. These results indicate that the recognition sites for putrescine, agmatine and spermidine on hOCT2 strongly overlap, consistent with the observation that the three amines are transported with similar affinity and velocity. A model of spermidine binding to hOCT2 was constructed based on the functional amino acid residues. PMID:25019617

  14. Metformin increases insulin-stimulated glucose transport in insulin-resistant human skeletal muscle.

    Science.gov (United States)

    Galuska, D; Zierath, J; Thörne, A; Sonnenfeld, T; Wallberg-Henriksson, H

    1991-05-01

    The effect of metformin (0.1 mM) on glucose transport was investigated in healthy control and in insulin-resistant human skeletal muscle. Muscle samples (200-400 mg) were obtained from the rectus abdominis muscle (abdominal surgery) or from the vastus lateralis portion of the quadriceps femoris muscle (open biopsy technique) from 8 healthy controls (age 38 +/- 4 yrs, BMI 23 +/- 1) and from 6 insulin-resistant subjects (age 53 +/- 5 yrs, BMI 30 +/- 2). Metformin had no effect on basal or insulin-stimulated (100 microU/ml) 3-0-methylglucose transport in incubated muscle strips from healthy subjects. Muscle tissue from the insulin resistant group did not respond to 100 microU/ml of insulin (0.73 +/- 0.17 for basal and 0.81 +/- 0.22 mumol x ml-1 x h-1 for insulin-stimulation, NS). Basal glucose transport was unaffected by metformin, whereas insulin-stimulated (100 microU/ml) glucose transport was increased by 63% in the insulin-resistant muscles (0.73 +/- 0.17 in the absence vs 1.19 +/- 0.18 mumol x ml-1 x h-1 in the presence of metformin, p less than 0.05). In conclusion, metformin abolishes insulin-resistance in human skeletal muscle by normalizing insulin-stimulated glucose transport accross the muscle cell membrane. The mechanism for this effect remains to be elucidated.

  15. Interacting Physical and Biological Processes Affecting Nutrient Transport Through Human Dominated Landscapes

    Science.gov (United States)

    Finlay, J. C.

    2015-12-01

    Human activities increasingly dominate biogeochemical cycles of limiting nutrients on Earth. Urban and agricultural landscapes represent the largest sources of excess nutrients that drive water quality degradation. The physical structure of both urban and agricultural watersheds has been extensively modified, and these changes have large impacts on water and nutrient transport. Despite strong physical controls over nutrient transport in human dominated landscapes, biological processes play important roles in determining the fates of both nitrogen and phosphorus. This talk uses examples from research in urban and agricultural watersheds in the Midwestern USA to illustrate interactions of physical and biological controls over nutrient cycles that have shifted nitrogen (N) and phosphorus (P) sources and cycling in unexpected ways in response to management changes. In urban watersheds, efforts to improve water quality have been hindered by legacy sources of phosphorus added to storm water through transport to drainage systems by vegetation. Similarly, reductions in field erosion in agricultural watersheds have not led to major reductions in phosphorus transport, because of continued release of biological sources of P. Where management of phosphorus has been most effective in reducing eutrophication of lakes, decreases in N removal processes have led to long term increases in N concentration and transport. Together, these examples show important roles for biological processes affecting nutrient movement in highly modified landscapes. Consideration of the downstream physical and biological responses of management changes are thus critical toward identification of actions that will most effectively reduce excess nutrients watersheds and coastal zones.

  16. Monocarboxylate Transporter 8 Modulates the Viability and Invasive Capacity of Human Placental Cells and Fetoplacental Growth in Mice

    NARCIS (Netherlands)

    E. Vasilopoulou (Elisavet); L.S. Loubière (Laurence); H. Heuer (Heike); M. Trajkovic-Arsic (Marija); V.M. Darras (Veerle); T.J. Visser (Theo); G.E. Lash (Gendie); G.S. Whitley (Guy); C.J. McCabe (Christopher); J.A. Franklyn (Jayne); M.D. Kilby (Mark); S.Y. Chan (Shiao)

    2013-01-01

    textabstractMonocarboxylate transporter 8 (MCT8) is a well-established thyroid hormone (TH) transporter. In humans, MCT8 mutations result in changes in circulating TH concentrations and X-linked severe global neurodevelopmental delay. MCT8 is expressed in the human placenta throughout gestation,

  17. Chemical form of selenium affects its uptake, transport and glutathione peroxidase activity in the human intestinal Caco-2 cell model

    Science.gov (United States)

    Determining the effect of selenium (Se) chemical form on uptake and transport in human intestinal cells is critical to assess Se bioavailability. In the present study, we measured the uptake and transport of various Se compounds in the human intestinal Caco-2 cell model. We found that two sources...

  18. Functional and cytometric examination of different human lung epithelial cell types as drug transport barriers.

    Science.gov (United States)

    Min, Kyoung Ah; Rosania, Gus R; Kim, Chong-Kook; Shin, Meong Cheol

    2016-03-01

    To develop inhaled medications, various cell culture models have been used to examine the transcellular transport or cellular uptake properties of small molecules. For the reproducible high throughput screening of the inhaled drug candidates, a further verification of cell architectures as drug transport barriers can contribute to establishing appropriate in vitro cell models. In the present study, side-by-side experiments were performed to compare the structure and transport function of three lung epithelial cells (Calu-3, normal human bronchial primary cells (NHBE), and NL-20). The cells were cultured on the nucleopore membranes in the air-liquid interface (ALI) culture conditions, with cell culture medium in the basolateral side only, starting from day 1. In transport assays, paracellular transport across all three types of cells appeared to be markedly different with the NHBE or Calu-3 cells, showing low paracellular permeability and high TEER values, while the NL-20 cells showed high paracellular permeability and low TEER. Quantitative image analysis of the confocal microscope sections further confirmed that the Calu-3 cells formed intact cell monolayers in contrast to the NHBE and NL-20 cells with multilayers. Among three lung epithelial cell types, the Calu-3 cell cultures under the ALI condition showed optimal cytometric features for mimicking the biophysical characteristics of in vivo airway epithelium. Therefore, the Calu-3 cell monolayers could be used as functional cell barriers for the lung-targeted drug transport studies.

  19. DHM in human-centered product design: a case-study on public transport vehicle.

    Science.gov (United States)

    Santos, V; Guimarães, C P; Franca, G A N; Cid, G L; Paranhos, A G

    2012-01-01

    The goal of this paper is to present the advantages on the use of 3D Digital Human Models (DHM) on the design of public transport vehicles. In this case, the subjects were scanned using the WBX Cyberware 3D Whole Body Scanner, with functional and daily postures according to the use of public transportation and some especial cases, such as a mother with her offspring or a business man with his valise, so the volume of the person would be taken in consideration. A data collection was created to simulate several situations of the daily use of the vehicle.

  20. Genetic variants of the human H+/dipeptide transporter PEPT2

    DEFF Research Database (Denmark)

    Pinsonneault, Julia; Nielsen, Carsten Uhd; Sadée, Wolfgang

    2004-01-01

    . We have conducted a haplotype analysis of 27 single nucleotide polymorphisms located in or near exons of the human gene encoding hPEPT2 (SLC15A2), using genotyping data from 247 genomic DNA samples from the Coriell collection. Our analysis reveals that hPEPT2 has a >6-kilobase sequence block......PEPT2 is a high-affinity H+/dipeptide transporter expressed in kidney, brain, lung, and mammary gland. The physiological role of PEPT2 in kidney is to reabsorb small peptides generated by luminal peptidases. PEPT2 is also a transporter for peptide-like drugs such as penicillins and cephalosporins...

  1. Transplacental Nutrient Transport Mechanisms of Intrauterine Growth Restriction in Rodent Models and Humans

    Directory of Open Access Journals (Sweden)

    Elke Winterhager

    2017-11-01

    Full Text Available Although the causes of intrauterine growth restriction (IUGR have been intensively investigated, important information is still lacking about the role of the placenta as a link from adverse maternal environment to adverse pregnancy outcomes of IUGR and preterm birth. IUGR is associated with an increased risk of cardiovascular, metabolic, and neurological diseases later in life. Determination of the most important pathways that regulate transplacental transport systems is necessary for identifying marker genes as diagnostic tools and for developing drugs that target the molecular pathways. Besides oxygen, the main nutrients required for appropriate fetal development and growth are glucose, amino acids, and fatty acids. Dysfunction in transplacental transport is caused by impairments in both placental morphology and blood flow, as well as by factors such as alterations in the expression of insulin-like growth factors and changes in the mTOR signaling pathway leading to a change in nutrient transport. Animal models are important tools for systematically studying such complex events. Debate centers on whether the rodent placenta is an appropriate tool for investigating the alterations in the human placenta that result in IUGR. This review provides an overview of the alterations in expression and activity of nutrient transporters and alterations in signaling associated with IUGR and compares these findings in rodents and humans. In general, the data obtained by studies of the various types of rodent and human nutrient transporters are similar. However, direct comparison is complicated by the fact that the results of such studies are controversial even within the same species, making the interpretation of the results challenging. This difficulty could be due to the absence of guidelines of the experimental design and, especially in humans, the use of trophoblast cell culture studies instead of clinical trials. Nonetheless, developing new therapy

  2. Human vascular endothelial cells transport foreign exosomes from cow's milk by endocytosis.

    Science.gov (United States)

    Kusuma, Rio Jati; Manca, Sonia; Friemel, Taylor; Sukreet, Sonal; Nguyen, Christopher; Zempleni, Janos

    2016-05-15

    Encapsulation of microRNAs in exosomes confers protection against degradation and a vehicle for shuttling of microRNAs between cells and tissues, and cellular uptake by endocytosis. Exosomes can be found in foods including milk. Humans absorb cow's milk exosomes and deliver the microRNA cargo to peripheral tissues, consistent with gene regulation by dietary nucleic acids across species boundaries. Here, we tested the hypothesis that human vascular endothelial cells transport milk exosomes by endocytosis, constituting a step crucial for the delivery of dietary exosomes and their cargo to peripheral tissues. We tested this hypothesis by using human umbilical vein endothelial cells and fluorophore-labeled exosomes isolated from cow's milk. Exosome uptake followed Michaelis-Menten kinetics (Vmax = 0.057 ± 0.004 ng exosome protein × 40,000 cells/h; Km = 17.97 ± 3.84 μg exosomal protein/200 μl media) and decreased by 80% when the incubation temperature was lowered from 37°C to 4°C. When exosome surface proteins were removed by treatment with proteinase K, or transport was measured in the presence of the carbohydrate competitor d-galactose or measured in the presence of excess unlabeled exosomes, transport rates decreased by 45% to 80% compared with controls. Treatment with an inhibitor of endocytosis, cytochalasin D, caused a 50% decrease in transport. When fluorophore-labeled exosomes were administered retro-orbitally, exosomes accumulated in liver, spleen, and lungs in mice. We conclude that human vascular endothelial cells transport bovine exosomes by endocytosis and propose that this is an important step in the delivery of dietary exosomes and their cargo to peripheral tissues. Copyright © 2016 the American Physiological Society.

  3. Comprehensive Analysis of the Therapeutic IgG4 Antibody Pembrolizumab: Hinge Modification Blocks Half Molecule Exchange In Vitro and In Vivo.

    Science.gov (United States)

    Yang, Xiaoyu; Wang, Fengqiang; Zhang, Ying; Wang, Larry; Antonenko, Svetlana; Zhang, Shuli; Zhang, Yi Wei; Tabrizifard, Mohammad; Ermakov, Grigori; Wiswell, Derek; Beaumont, Maribel; Liu, Liming; Richardson, Daisy; Shameem, Mohammed; Ambrogelly, Alexandre

    2015-12-01

    IgG4 antibodies are evolving as an important class of cancer immunotherapies. However, human IgG4 can undergo Fab arm (half molecule) exchange with other IgG4 molecules in vivo. The hinge modification by a point mutation (S228P) prevents half molecule exchange of IgG4. However, the experimental confirmation is still expected by regulatory agencies. Here, we report for the first time the extensive analysis of half molecule exchange for a hinge-modified therapeutic IgG4 molecule, pembrolizumab (Keytruda) targeting programmed death 1 (PD1) receptor that was approved for advanced melanoma. Studies were performed in buffer or human serum using multiple exchange partners including natalizumab (Tysabri) and human IgG4 pool. Formation of bispecific antibodies was monitored by fluorescence resonance energy transfer, exchange with Fc fragments, mixed mode chromatography, immunoassays, and liquid chromatography-mass spectrometry. The half molecule exchange was also examined in vivo in SCID (severe combined immunodeficiency) mice. Both in vitro and in vivo results indicate that the hinge modification in pembrolizumab prevented half molecule exchange, whereas the unmodified counterpart anti-PD1 wt showed active exchange activity with other IgG4 antibodies or self-exchange activity with its own molecules. Our work, as an example expected for meeting regulatory requirements, contributes to establish without ambiguity that hinge-modified IgG4 antibodies are suitable for biotherapeutic applications. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  4. Development of an IgG4-RD Responder Index.

    Science.gov (United States)

    Carruthers, Mollie N; Stone, John H; Deshpande, Vikram; Khosroshahi, Arezou

    2012-01-01

    IgG4-related disease (IgG4-RD) is a multiorgan inflammatory disease in which diverse organ manifestations are linked by common histopathological and immunohistochemical features. Prospective studies of IgG4-RD patients are required to clarify the natural history, long-term prognosis, and treatment approaches in this recently recognized condition. Patients with IgG4-RD have different organ manifestations and are followed by multiple specialties. Divergent approaches to the assessment of patients can complicate the interpretation of studies, emphasizing the critical need for validated outcome measures, particularly assessments of disease activity and response to treatment. We developed a prototype IgG4-RD Responder Index (IgG4-RD RI) based on the approach used in the development of the Birmingham Vasculitis Activity Score for Wegener's granulomatosis (BVAS/WG). The IgG4-RD RI was refined by members of the International IgG4-RD Symposium Organizing Committee in a paper case exercise. The revised instrument was applied retrospectively to fifteen IgG4-RD patients at our institution. Those scores were compared to physician's global assessment scale for the same visits. This paper describes the philosophy and goals of the IgG4-RD RI, the steps in the development of this instrument to date, and future plans for validation of this instrument as an outcome measure.

  5. Immunoglobulin G (IgG) subclass deficiency in Thai children.

    Science.gov (United States)

    Visitsunthorn, Nualanong; Hengcrawit, Wiriya; Jirapongsananuruk, Orathai; Luangwedchakam, Voravich

    2011-12-01

    Patients with Immunoglobulin G (IgG) subclass deficiency may suffer from recurrent infections, mainly sino-pulmonary infection. To determine the epidemiology of IgG subclass deficiency in Thai children at a tertiary care hospital and to compare the differences between children who were diagnosed with IgG subclass deficiency by using low level criteria [less than 2 standard deviation (SD) of normal levels for age] and by using low percentage criteria (proportion of each IgG subclasses/total IgG). The study was a descriptive study of 55 children up to 15 years old with recurrent infections diagnosed as having IgG subclass deficiency but no acquired or other primary immune deficiencies except for IgA and/or IgM deficiency. Isolated IgG3 subclass deficiency was the most common IgG subclass deficiency (56.4%). IgG3 subclass deficiency, either isolated or combined with other IgG subclass deficiency, was found in 85.5% of the cases. The common age of onset was between birth and five years of age. The most common presenting symptom was recurrent sinusitis (83.6%). Majority of the cases (89.3%) were diagnosed by low percentage criteria while 12.7% were diagnosed by low level criteria. All cases with low levels of IgG subclass antibodies also had low percentages. There were no statistically significant differences in the clinical manifestations and management methods between the children who were diagnosed by low level and low percentage. IgG3 subclass deficiency was the most common IgG subclass deficiency in Thai children. The most common presenting symptom was recurrent sinusitis. Although the diagnosis could be made in the patients with recurrent upper respiratory infection by using low level criteria, but the diagnosis should be considered when the low percentage criteria are met.

  6. Explaining the power-law distribution of human mobility through transportation modality decomposition.

    Science.gov (United States)

    Zhao, Kai; Musolesi, Mirco; Hui, Pan; Rao, Weixiong; Tarkoma, Sasu

    2015-03-16

    Human mobility has been empirically observed to exhibit Lévy flight characteristics and behaviour with power-law distributed jump size. The fundamental mechanisms behind this behaviour has not yet been fully explained. In this paper, we propose to explain the Lévy walk behaviour observed in human mobility patterns by decomposing them into different classes according to the different transportation modes, such as Walk/Run, Bike, Train/Subway or Car/Taxi/Bus. Our analysis is based on two real-life GPS datasets containing approximately 10 and 20 million GPS samples with transportation mode information. We show that human mobility can be modelled as a mixture of different transportation modes, and that these single movement patterns can be approximated by a lognormal distribution rather than a power-law distribution. Then, we demonstrate that the mixture of the decomposed lognormal flight distributions associated with each modality is a power-law distribution, providing an explanation to the emergence of Lévy Walk patterns that characterize human mobility patterns.

  7. A stochastic model for microtubule motors describes the in vivo cytoplasmic transport of human adenovirus.

    Directory of Open Access Journals (Sweden)

    Mattia Gazzola

    2009-12-01

    Full Text Available Cytoplasmic transport of organelles, nucleic acids and proteins on microtubules is usually bidirectional with dynein and kinesin motors mediating the delivery of cargoes in the cytoplasm. Here we combine live cell microscopy, single virus tracking and trajectory segmentation to systematically identify the parameters of a stochastic computational model of cargo transport by molecular motors on microtubules. The model parameters are identified using an evolutionary optimization algorithm to minimize the Kullback-Leibler divergence between the in silico and the in vivo run length and velocity distributions of the viruses on microtubules. The present stochastic model suggests that bidirectional transport of human adenoviruses can be explained without explicit motor coordination. The model enables the prediction of the number of motors active on the viral cargo during microtubule-dependent motions as well as the number of motor binding sites, with the protein hexon as the binding site for the motors.

  8. Components of foods inhibit a drug exporter, human multidrug and toxin extrusion transporter 1.

    Science.gov (United States)

    Kawasaki, Tatsuya; Ito, Hideyuki; Omote, Hiroshi

    2014-01-01

    Human multidrug and toxic compounds extrusion transporter 1 (hMATE1/SLC47A1) is a H(+)-coupled organic cation exporter responsible for the final step of excretion of various xenobiotics at the kidney and liver. In this study, effects of dietary constituents on hMATE1 mediated drug transport were examined to evaluate possible food-drug interactions. Bergamottin inhibited hMATE1 mediated tetraethyl ammonium transport activity, with a Ki of 98.7 µM. Coumarins, flavonols, and catechin inhibited hMATE1 activity. Among 23 compounds tested, isorhamnetin was the strongest inhibitor of hMATE1 with the Ki of 0.32 µM in a competitive manner. Since isorhamnetin is abundant in Ginkgo biloba that is widely used for herbal supplements, the findings suggest the potential hMATE1 related food-drug interactions.

  9. Epitope-Specific Suppression of IgG Responses by Passively Administered Specific IgG: Evidence of Epitope Masking.

    Science.gov (United States)

    Bergström, Joakim J E; Xu, Hui; Heyman, Birgitta

    2017-01-01

    Specific IgG, passively administered together with particulate antigen, can completely prevent induction of antibody responses to this antigen. The ability of IgG to suppress antibody responses to sheep red blood cells (SRBCs) is intact in mice lacking FcγRs, complement factor 1q, C3, or complement receptors 1 and 2, suggesting that Fc-dependent effector functions are not involved. Two of the most widely discussed explanations for the suppressive effect are increased clearance of IgG-antigen complexes and/or that IgG "hides" the antigen from recognition by specific B cells, so-called epitope masking. The majority of data on how IgG induces suppression was obtained through studies of the effects on IgM-secreting single spleen cells during the first week after immunization. Here, we show that IgG also suppresses antigen-specific extrafollicular antibody-secreting cells, germinal center B-cells, long-lived plasma cells, long-term IgG responses, and induction of memory antibody responses. IgG anti-SRBC reduced the amount of SRBC in the spleens of wild-type, but not of FcγR-deficient mice. However, no correlation between suppression and the amount of SRBC in the spleen was observed, suggesting that increased clearance does not explain IgG-mediated suppression. Instead, we found compelling evidence for epitope masking because IgG anti-NP administered with NP-SRBC suppressed the IgG anti-NP, but not the IgG anti-SRBC response. Vice versa, IgG anti-SRBC administered with NP-SRBC, suppressed only the IgG anti-SRBC response. In conclusion, passively transferred IgG suppressed all measured parameters of an antigen-specific antibody/B cell response and an important mechanism of action is likely to be epitope masking.

  10. Thermostabilization of the Human Serotonin Transporter in an Antidepressant-Bound Conformation.

    Directory of Open Access Journals (Sweden)

    Evan M Green

    Full Text Available Serotonin is a ubiquitous chemical transmitter with particularly important roles in the gastrointestinal, cardiovascular and central nervous systems. Modulation of serotonergic signaling occurs, in part, by uptake of the transmitter by the serotonin transporter (SERT. In the brain, SERT is the target for numerous antidepressants including tricyclic antidepressants and selective serotonin reuptake inhibitors (SSRIs. Despite the importance of SERT in human physiology, biochemical, biophysical and high-resolution structural studies have been hampered due to the instability of SERT in detergent micelles. To identify a human SERT (hSERT construct suitable for detailed biochemical and structural studies, we developed an efficient thermostability screening protocol and rapidly screened 219 mutations for thermostabilization of hSERT in complex with the SSRI paroxetine. We discovered three mutations-Y110A, I291A and T439S -that, when combined into a single construct, deemed TS3, yielded a hSERT variant with an apparent melting temperature (Tm 19°C greater than that of the wild-type transporter, albeit with a loss of transport activity. Further investigation yielded a double mutant-I291A and T439S-defined as TS2, with a 12°C increase in Tm and retention of robust transport activity. Both TS2 and TS3 were more stable in short-chain detergents in comparison to the wild-type transporter. This thermostability screening protocol, as well as the specific hSERT variants, will prove useful in studies of other integral membrane receptors and transporters and in the investigation of structure and function relationships in hSERT.

  11. Persistent human Borna disease virus infection modifies the acetylome of human oligodendroglia cells towards higher energy and transporter levels

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xia [Shanghai Key Laboratory of Forensic Medicine, Institute of Forensic Science, Ministry of Justice, Shanghai 200063 (China); Liu, Siwen [Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing 400016 (China); Bode, Liv [Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing 400016 (China); Liu, Chengyu [Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing 400016 (China); Zhang, Liang [Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing 400016 (China); Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Wang, Xiao [Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing 400016 (China); Li, Dan [Department of Pathology, Faculty of Basic Medicine, Chongqing Medical University, Chongqing 400016 (China); Lei, Yang [Department of Internal Medicine, University-Town Hospital of Chongqing Medical University, Chongqing 400016 (China); Peng, Xiaojun [Jingjie PTM BioLab (Hangzhou) Co. Ltd, Hangzhou 310018 (China); Cheng, Zhongyi [Advanced Institute of Translational Medicine, Tongji University, Shanghai 200092 (China); and others

    2015-11-15

    Background: Borna disease virus (BDV) is a neurotropic RNA virus persistently infecting mammalian hosts including humans. Lysine acetylation (Kac) is a key protein post-translational modification (PTM). The unexpectedly broad regulatory scope of Kac let us to profile the entire acetylome upon BDV infection. Methods: The acetylome was profiled through stable isotope labeling for cell culture (SILAC)-based quantitative proteomics. The quantifiable proteome was annotated using bioinformatics. Results: We identified and quantified 791 Kac sites in 473 Kac proteins in human BDV Hu-H1-infected and non-infected oligodendroglial (OL) cells. Bioinformatic analysis revealed that BDV infection alters the acetylation of metabolic proteins, membrane-associated proteins and transmembrane transporter activity, and affects the acetylation of several lysine acetyltransferases (KAT). Conclusions: Upon BDV persistence the OL acetylome is manipulated towards higher energy and transporter levels necessary for shuttling BDV proteins to and from nuclear replication sites. - Highlights: • We used SILAC-based proteomics to analyze the acetylome of BDV infected OL cells. • We quantified 791Kac sites in 473 proteins. • Bioinformatic analysis revealed altered acetylation of metabolic proteins et al. • BDV manipulates the OL acetylome towards higher energy and transporter levels. • BDV infection is associated with enriched phosphate-associated metabolic processes.

  12. Immunogenicity of different stressed IgG monoclonal antibody formulations in immune tolerant transgenic mice

    Science.gov (United States)

    Filipe, Vasco; Jiskoot, Wim; Basmeleh, Abdul Hafid; Halim, Andhyk; Schellekens, Huub; Brinks, Vera

    2012-01-01

    The presence of protein aggregates in biopharmaceutical formulations is of great concern for safety and efficacy reasons. The aim of this study was to correlate the type and amount of IgG monoclonal antibody aggregates with their immunogenic potential. IgG degradation was obtained by freeze-thawing cycles, pH-shift cycles, heating, shaking and metal-catalyzed oxidation. The size, amount, morphology and type of intermolecular bonds of aggregates, as well as structural changes and epitope integrity were characterized. These formulations were injected in mice transgenic (TG) for human genes for Ig heavy and light chains and their non-transgenic (NTG) counterparts. Anti-drug antibody (ADA) titers were determined by bridging ELISA. Both unstressed IgG and freeze-thawed formulation did not induce measurable ADA levels. A mild antibody response was obtained in a fairly small percentage of mice, when injected with shaken, pH-shifted and heated formulations. The metal-catalyzed oxidized IgG formulation was the most immunogenic one, in both ADA titers and number of responders. The overall titers of NTG responders were significantly higher than the ones produced by TG mice, whereas there was no significant difference between the overall number of TG and NTG responders. This study reinforces the important role of protein aggregates on immunogenicity of therapeutic proteins and provides new insight into the immunogenic potential of different types of IgG aggregates. The results indicate that the quality of the IgG aggregates has more impact on the development of an immune response than their quantity or size. PMID:22951518

  13. A novel bacterial transport mechanism of Acinetobacter baumannii via activated human neutrophils through interleukin-8.

    Science.gov (United States)

    Kamoshida, Go; Tansho-Nagakawa, Shigeru; Kikuchi-Ueda, Takane; Nakano, Ryuichi; Hikosaka, Kenji; Nishida, Satoshi; Ubagai, Tsuneyuki; Higashi, Shouichi; Ono, Yasuo

    2016-12-01

    Hospital-acquired infections as a result of Acinetobacter baumannii have become problematic because of high rates of drug resistance. Although neutrophils play a critical role in early protection against bacterial infection, their interactions with A. baumannii remain largely unknown. To elucidate the interactions between A. baumannii and human neutrophils, we cocultured these cells and analyzed them by microscopy and flow cytometry. We found that A. baumannii adhered to neutrophils. We next examined neutrophil and A. baumannii infiltration into Matrigel basement membranes by an in vitro transmigration assay. Neutrophils were activated by A. baumannii, and invasion was enhanced. More interestingly, A. baumannii was transported together by infiltrating neutrophils. Furthermore, we observed by live cell imaging that A. baumannii and neutrophils moved together. In addition, A. baumannii-activated neutrophils showed increased IL-8 production. The transport of A. baumannii was suppressed by inhibiting neutrophil infiltration by blocking the effect of IL-8. A. baumannii appears to use neutrophils for transport by activating these cells via IL-8. In this study, we revealed a novel bacterial transport mechanism that A. baumannii exploits human neutrophils by adhering to and inducing IL-8 release for bacterial portage. This mechanism might be a new treatment target. © Society for Leukocyte Biology.

  14. The Ussing Chamber Assay to Study Drug Metabolism and Transport in the Human Intestine.

    Science.gov (United States)

    Kisser, Beatrice; Mangelsen, Eva; Wingolf, Caroline; Partecke, Lars Ivo; Heidecke, Claus-Dieter; Tannergren, Christer; Oswald, Stefan; Keiser, Markus

    2017-06-22

    The Ussing chamber is an old but still powerful technique originally designed to study the vectorial transport of ions through frog skin. This technique is also used to investigate the transport of chemical agents through the intestinal barrier as well as drug metabolism in enterocytes, both of which are key determinants for the bioavailability of orally administered drugs. More contemporary model systems, such as Caco-2 cell monolayers or stably transfected cells, are more limited in their use compared to the Ussing chamber because of differences in expression rates of transporter proteins and/or metabolizing enzymes. While there are limitations to the Ussing chamber assay, the use of human intestinal tissue remains the best laboratory test for characterizing the transport and metabolism of compounds following oral administration. Detailed in this unit is a step-by-step protocol for preparing human intestinal tissue, for designing Ussing chamber experiments, and for analyzing and interpreting the findings. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  15. Using full-mission simulation for human factors research in air transport operations

    Science.gov (United States)

    Orlady, Harry W.; Hennessy, Robert W.; Obermayer, Richard; Vreuls, Donald; Murphy, Miles R.

    1988-01-01

    This study examined state-of-the-art mission oriented simulation and its use in human factors research. Guidelines were developed for doing full-mission human factors research on crew member behavior during simulated air transport operations. The existing literature was reviewed. However, interviews with experienced investigators provided the most useful information. The fundamental scientific and practical issues of behavioral research in a simulation environment are discussed. Guidelines are presented for planning, scenario development, and the execution of behavioral research using full-mission simulation in the context of air transport flight operations . Research is recommended to enhance the validity and productivity of full-mission research by: (1) validating the need for high-fidelity simulation of all major elements in the operational environment, (2) improving methods for conducting full-mission research, and (3) examining part-task research on specific problems through the use of vehicles which contain higher levels of abstraction (and lower fidelity) of the operational environment.

  16. Physiology of SLC12 transporters: lessons from inherited human genetic mutations and genetically engineered mouse knockouts.

    Science.gov (United States)

    Gagnon, Kenneth B; Delpire, Eric

    2013-04-15

    Among the over 300 members of the solute carrier (SLC) group of integral plasma membrane transport proteins are the nine electroneutral cation-chloride cotransporters belonging to the SLC12 gene family. Seven of these transporters have been functionally described as coupling the electrically silent movement of chloride with sodium and/or potassium. Although in silico analysis has identified two additional SLC12 family members, no physiological role has been ascribed to the proteins encoded by either the SLC12A8 or the SLC12A9 genes. Evolutionary conservation of this gene family from protists to humans confirms their importance. A wealth of physiological, immunohistochemical, and biochemical studies have revealed a great deal of information regarding the importance of this gene family to human health and disease. The sequencing of the human genome has provided investigators with the capability to link several human diseases with mutations in the genes encoding these plasma membrane proteins. The availability of bacterial artificial chromosomes, recombination engineering techniques, and the mouse genome sequence has simplified the creation of targeting constructs to manipulate the expression/function of these cation-chloride cotransporters in the mouse in an attempt to recapitulate some of these human pathologies. This review will summarize the three human disorders that have been linked to the mutation/dysfunction of the Na-Cl, Na-K-2Cl, and K-Cl cotransporters (i.e., Bartter's, Gitleman's, and Andermann's syndromes), examine some additional pathologies arising from genetically modified mouse models of these cotransporters including deafness, blood pressure, hyperexcitability, and epithelial transport deficit phenotypes.

  17. Antigen-Specific IgG ameliorates allergic airway inflammation via Fcγ receptor IIB on dendritic cells

    Directory of Open Access Journals (Sweden)

    Karasuyama Hajime

    2011-04-01

    Full Text Available Abstract Background There have been few reports on the role of Fc receptors (FcRs and immunoglobulin G (IgG in asthma. The purpose of this study is to clarify the role of inhibitory FcRs and antigen presenting cells (APCs in pathogenesis of asthma and to evaluate antigen-transporting and presenting capacity by APCs in the tracheobronchial mucosa. Methods In FcγRIIB deficient (KO and C57BL/6 (WT mice, the effects of intratracheal instillation of antigen-specific IgG were analysed using the model with sensitization and airborne challenge with ovalbumin (OVA. Thoracic lymph nodes instilled with fluorescein-conjugated OVA were analysed by fluorescence microscopy. Moreover, we analysed the CD11c+ MHC class II+ cells which intaken fluorescein-conjugated OVA in thoracic lymph nodes by flow cytometry. Also, lung-derived CD11c+ APCs were analysed by flow cytometry. Effects of anti-OVA IgG1 on bone marrow dendritic cells (BMDCs in vitro were also analysed. Moreover, in FcγRIIB KO mice intravenously transplanted dendritic cells (DCs differentiated from BMDCs of WT mice, the effects of intratracheal instillation of anti-OVA IgG were evaluated by bronchoalveolar lavage (BAL. Results In WT mice, total cells and eosinophils in BAL fluid reduced after instillation with anti-OVA IgG1. Anti-OVA IgG1 suppressed airway inflammation in hyperresponsiveness and histology. In addition, the number of the fluorescein-conjugated OVA in CD11c+ MHC class II+ cells of thoracic lymph nodes with anti-OVA IgG1 instillation decreased compared with PBS. Also, MHC class II expression on lung-derived CD11c+ APCs with anti-OVA IgG1 instillation reduced. Moreover, in vitro, we showed that BMDCs with anti-OVA IgG1 significantly decreased the T cell proliferation. Finally, we demonstrated that the lacking effects of anti-OVA IgG1 on airway inflammation on FcγRIIB KO mice were restored with WT-derived BMDCs transplanted intravenously. Conclusion Antigen-specific IgG ameliorates

  18. Standardization of Assays That Detect Anti-Rubella Virus IgG Antibodies

    Science.gov (United States)

    Grangeot-Keros, Liliane; Vauloup-Fellous, Christelle

    2015-01-01

    SUMMARY Rubella virus usually causes a mild infection in humans but can cause congenital rubella syndrome (CRS). Vaccination programs have significantly decreased primary rubella virus infection and CRS; however, vaccinated individuals usually have lower levels of rubella virus IgG than those with natural infections. Rubella virus IgG is quantified with enzyme immunoassays that have been calibrated against the World Health Organization (WHO) international standard and report results in international units per milliliter. It is recognized that the results reported by these assays are not standardized. This investigation into the reasons for the lack of standardization found that the current WHO international standard (RUB-1-94) fails by three key metrological principles. The standard is not a pure analyte but is composed of pooled human immunoglobulin. It was not calibrated by certified reference methods; rather, superseded tests were used. Finally, no measurement uncertainty estimations have been provided. There is an analytical and clinical consequence to the lack of standardization of rubella virus IgG assays, which leads to misinterpretation of results. The current approach to standardization of rubella virus IgG assays has not achieved the desired results. A new approach is required. PMID:26607813

  19. Implementation and Reconfiguration of Robot Operating System on Human Follower Transporter Robot

    Directory of Open Access Journals (Sweden)

    Addythia Saphala

    2015-10-01

    Full Text Available Robotic Operation System (ROS is an im- portant platform to develop robot applications. One area of applications is for development of a Human Follower Transporter Robot (HFTR, which  can  be  considered  as a custom mobile robot utilizing differential driver steering method and equipped with Kinect sensor. This study discusses the development of the robot navigation system by implementing Simultaneous Localization and Mapping (SLAM.

  20. Sensitivity Analysis of Hybrid Propulsion Transportation System for Human Mars Expeditions

    Science.gov (United States)

    Chai, Patrick R.; Joyce, Ryan T.; Kessler, Paul D.; Merrill, Raymond G.; Qu, Min

    2017-01-01

    The National Aeronautics and Space Administration continues to develop and refine various transportation options to successfully field a human Mars campaign. One of these transportation options is the Hybrid Transportation System which utilizes both solar electric propulsion and chemical propulsion. The Hybrid propulsion system utilizes chemical propulsion to perform high thrust maneuvers, where the delta-V is most optimal when ap- plied to save time and to leverage the Oberth effect. It then utilizes solar electric propulsion to augment the chemical burns throughout the interplanetary trajectory. This eliminates the need for the development of two separate vehicles for crew and cargo missions. Previous studies considered single point designs of the architecture, with fixed payload mass and propulsion system performance parameters. As the architecture matures, it is inevitable that the payload mass and the performance of the propulsion system will change. It is desirable to understand how these changes will impact the in-space transportation system's mass and power requirements. This study presents an in-depth sensitivity analysis of the Hybrid crew transportation system to payload mass growth and solar electric propulsion performance. This analysis is used to identify the breakpoints of the current architecture and to inform future architecture and campaign design decisions.

  1. Functional expression of a human GDP-L-fucose transporter in Escherichia coli.

    Science.gov (United States)

    Förster-Fromme, Karin; Schneider, Sarah; Sprenger, Georg A; Albermann, Christoph

    2017-02-01

    To investigate the translocation of nucleotide-activated sugars from the cytosol across a membrane into the endoplasmatic reticulum or the Golgi apparatus which is an important step in the synthesis of glycoproteins and glycolipids in eukaryotes. The heterologous expression of the recombinant and codon-adapted human GDP-L-fucose antiporter gene SLC35C1 (encoding an N-terminal OmpA-signal sequence) led to a functional transporter protein located in the cytoplasmic membrane of Escherichia coli. The in vitro transport was investigated using inverted membrane vesicles. SLC35C1 is an antiporter specific for GDP-L-fucose and depending on the concomitant reverse transport of GMP. The recombinant transporter FucT1 exhibited an activity for the transport of 3 H-GDP-L-fucose with a V max of 8 pmol/min mg with a K m of 4 µM. The functional expression of SLC35C1 in GDP-L-fucose overproducing E. coli led to the export of GDP-L-fucose to the culture supernatant. The export of GDP-L-fucose by E. coli provides the opportunity for the engineering of a periplasmatic fucosylation reaction in recombinant bacterial cells.

  2. Transepithelial transport of putrescine across monolayers of the human intestinal epithelial cell line, Caco-2

    Science.gov (United States)

    Milovic, Vladan; Turchanowa, Lyudmila; Stein, Jürgen; Caspary, Wolfgang F.

    2001-01-01

    AIM: To study the transepithelial transport characteristics of the polyamine putrescine in human intestinal Caco-2 cell monolayers to elucidate the mechanisms of the putrescine intestinal absorption. METHODS: The transepithelial transport and the cellular accumulation of putrescine was measured using Caco-2 cell monolayers grown on permeable filters. RESULTS: Transepithelial transport of putrescine in physiological concentrations ( > 0.5 mM) from the apical to basolateral side was linear. Intracellular accumulation of putrescine was higher in confluent than in fully differentiated Caco-2 cells, but still negligible (less than 0.5%) of the overall transport across the monolayers in apical to basolateral direction.EGF enhanced putrescine accumulation in Caco-2 cells by four fold, as well as putrescine conversion to spermidine and spermine by enhancing the activity of S adenosylmethionine decarboxylase. However, EGF did not have any significant influence on putrescine flux across the Caco- 2 cell monolayers. Excretion of putrescine from Caco-2 cells into the basolateral medium did not exceed 50 picomoles, while putrescine passive flux from the apical to the basolateral chamber, contributed hundreds of micromoles polyamines to the basolateral chamber. CONCLUSION: Transepithelial transport of putrescine across Caco-2 cell monolayers occurs in passive diffusion, and is not influenced when epithelial cells are stimulated to proliferate by a potent mitogen such as EGF. PMID:11819759

  3. Numerical modeling of photon migration in human neck based on the radiative transport equation

    CERN Document Server

    Fujii, Hiroyuki; Nadamoto, Ken; Okada, Eiji; Yamada, Yukio; Hoshi, Yoko; Watanabe, Masao

    2016-01-01

    Biomedical optical imaging has a possibility of a comprehensive diagnosis of thyroid cancer in conjunction with ultrasound imaging. For improvement of the optical imaging, this study develops a higher order scheme for solving the time-dependent radiative transport equation (RTE) by use of the finite-difference and discrete-ordinate methods. The accuracy and efficiency of the developed scheme are examined by comparison with the analytical solutions of the RTE in homogeneous media. Then, the developed scheme is applied to describing photon migration in the human neck model. The numerical simulations show complex behaviors of photon migration in the human neck model due to multiple diffusive reflection near the trachea.

  4. Adenosine receptors in rat and human pancreatic ducts stimulate chloride transport

    DEFF Research Database (Denmark)

    Novak, Ivana; Hede, Susanne; Hansen, Mette

    2007-01-01

    these could be involved in secretory processes, which involve cystic fibrosis transmembrane regulator (CFTR) Cl(-) channels or Ca(2+)-activated Cl(-) channels and [Formula: see text] transporters. Reverse transcriptase polymerase chain reaction analysis on rat pancreatic ducts and human duct cell......, it was found that 58% of PANC-1 cells responded to adenosine, whereas only 9% of CFPAC-1 cells responded. Adenosine elicited Ca(2+) signals only in a few rat and human duct cells, which did not seem to correlate with Cl(-) signals. A(2A) receptors were localized in the luminal membranes of rat pancreatic ducts...

  5. Technology and human purpose: the problem of solids transport on the Earth's surface

    Directory of Open Access Journals (Sweden)

    P. K. Haff

    2012-11-01

    Full Text Available Displacement of mass of limited deformability ("solids" on the Earth's surface is opposed by friction and (the analog of form resistance – impediments relaxed by rotational motion, self-powering of mass units, and transport infrastructure. These features of solids transport first evolved in the biosphere prior to the emergence of technology, allowing slope-independent, diffusion-like motion of discrete objects as massive as several tons, as illustrated by animal foraging and movement along game trails. However, high-energy-consumption technology powered by fossil fuels required a mechanism that could support fast advective transport of solids, i.e., long-distance, high-volume, high-speed, unidirectional, slope-independent transport across the land surface of materials like coal, containerized fluids, minerals, and economic goods. Pre-technology nature was able to sustain regional- and global-scale advection only in the limited form of piggybacking on geophysical flows of water (river sediment and air (dust. The appearance of a mechanism for sustained advection of solids independent of fluid flows and gravity appeared only upon the emergence of human purpose. Purpose enables solids advection by, in effect, simulating a continuous potential gradient, otherwise lacking, between discrete and widely separated fossil-fuel energy sources and sinks. Invoking purpose as a mechanism in solids advection is an example of the need to import anthropic principles and concepts into the language and methodology of modern Earth system dynamics. As part of the emergence of a generalized solids advection mechanism, several additional transport requirements necessary to the function of modern large-scale technological systems were also satisfied. These include spatially accurate delivery of advected payload, targetability to essentially arbitrarily located destinations (such as cities, and independence of structure of advected payload from transport mechanism. The

  6. Transport of gaseous pollutants by convective boundary layer around a human body

    DEFF Research Database (Denmark)

    Licina, Dusan; Melikov, Arsen Krikor; Sekhar, Chandra

    2015-01-01

    This study investigates the ability of the human convective boundary layer to transport pollution in a quiescent indoor environment. The impact of the source location in the vicinity of a human body is examined in relation to pollution distribution in the breathing zone and the thickness...... of the pollution boundary layer. The study, in addition, evaluates the effects of the room air temperature, table positioning, and seated body inclination. The human body is represented by a thermal manikin that has a body shape, size, and surface temperature that resemble those of a real person. The results show...... at the upper back or behind the chair. The results also indicate that a decrease in personal exposure to pollutants released from or around the human body increases the extent to which the pollution spreads to the surroundings. Reducing the room air temperature or backward body inclination intensifies...

  7. Simulation of light transport in arthritic- and non-arthritic human fingers

    Science.gov (United States)

    Milanic, Matija; Paluchowski, Lukasz A.; Randeberg, Lise L.

    2014-03-01

    Rheumatoid arthritis is a disease that frequently leads to joint destruction. It has high incidence rates worldwide, and the disease significantly reduces patient's quality of life due to pain, swelling and stiffness of the affected joints. Early diagnosis is necessary to improve course of the disease, therefore sensitive and accurate diagnostic tools are required. Optical imaging techniques have capability for early diagnosis and monitoring of arthritis. As compared to conventional diagnostic techniques optical technique is a noninvasive, noncontact and fast way of collecting diagnostic information. However, a realistic model of light transport in human joints is needed for understanding and developing of such optical diagnostic tools. The aim of this study is to develop a 3D numerical model of light transport in a human finger. The model will guide development of a hyperspectral imaging (HSI) diagnostic modality for arthritis in human fingers. The implemented human finger geometry is based on anatomical data. Optical data of finger tissues are adjusted to represent either an arthritic or an unaffected finger. The geometry and optical data serve as input into a 3D Monte Carlo method, which calculate diffuse reflectance, transmittance and absorbed energy distributions. The parameters of the model are optimized based on HIS-measurements of human fingers. The presented model serves as an important tool for understanding and development of HSI as an arthritis diagnostic modality. Yet, it can be applied to other optical techniques and finger diseases.

  8. A proposed study on the transplacental transport of parabens in the human placental perfusion model

    DEFF Research Database (Denmark)

    Mathiesen, Line; Zuri, Giuseppina; Andersen, Maria H

    2013-01-01

    Human exposure to parabens as a preservative used in personal care products is of increasing concern, as there is evidence from in vivo and in vitro studies of hormone disruption in association with exposure to parabens. Transport across the placenta could be critical for risk assessment, but the......Human exposure to parabens as a preservative used in personal care products is of increasing concern, as there is evidence from in vivo and in vitro studies of hormone disruption in association with exposure to parabens. Transport across the placenta could be critical for risk assessment......, but the available data are sparse. The aim is to develop a method for estimating fetal exposure, via the placenta, to the most commonly-used parabens, by using a human placental perfusion model. The use of human tissue is vital for determining human fetal exposure, because animal studies are of little relevance......, since the placenta exhibits significant interspecies variation. An HPLC model is currently being established to simultaneously quantify four different parabens, namely, methylparaben, ethylparaben, propylparaben and butylparaben, and their main metabolite, p-hydroxybenzoic acid. With this model, we aim...

  9. IgG subclasses determine pathways of anaphylaxis in mice.

    Science.gov (United States)

    Beutier, Héloïse; Gillis, Caitlin M; Iannascoli, Bruno; Godon, Ophélie; England, Patrick; Sibilano, Riccardo; Reber, Laurent L; Galli, Stephen J; Cragg, Mark S; Van Rooijen, Nico; Mancardi, David A; Bruhns, Pierre; Jönsson, Friederike

    2017-01-01

    Animal models have demonstrated that allergen-specific IgG confers sensitivity to systemic anaphylaxis that relies on IgG Fc receptors (FcγRs). Mouse IgG2a and IgG2b bind activating FcγRI, FcγRIII, and FcγRIV and inhibitory FcγRIIB; mouse IgG1 binds only FcγRIII and FcγRIIB. Although these interactions are of strikingly different affinities, these 3 IgG subclasses have been shown to enable induction of systemic anaphylaxis. We sought to determine which pathways control the induction of IgG1-, IgG2a-, and IgG2b-dependent passive systemic anaphylaxis. Mice were sensitized with IgG1, IgG2a, or IgG2b anti-trinitrophenyl mAbs and challenged with trinitrophenyl-BSA intravenously to induce systemic anaphylaxis that was monitored by using rectal temperature. Anaphylaxis was evaluated in mice deficient for FcγRs injected with mediator antagonists or in which basophils, monocytes/macrophages, or neutrophils had been depleted. FcγR expression was evaluated on these cells before and after anaphylaxis. Activating FcγRIII is the receptor primarily responsible for all 3 models of anaphylaxis, and subsequent downregulation of this receptor was observed. These models differentially relied on histamine release and the contribution of mast cells, basophils, macrophages, and neutrophils. Strikingly, basophil contribution and histamine predominance in mice with IgG1- and IgG2b-induced anaphylaxis correlated with the ability of inhibitory FcγRIIB to negatively regulate these models of anaphylaxis. We propose that the differential expression of inhibitory FcγRIIB on myeloid cells and its differential binding of IgG subclasses controls the contributions of mast cells, basophils, neutrophils, and macrophages to IgG subclass-dependent anaphylaxis. Collectively, our results unravel novel complexities in the involvement and regulation of cell populations in IgG-dependent reactions in vivo. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier

  10. Modelling Interactions between forest pest invasions and human decisions regarding firewood transport restrictions.

    Directory of Open Access Journals (Sweden)

    Lee-Ann Barlow

    Full Text Available The invasion of nonnative, wood-boring insects such as the Asian longhorned beetle (A. glabripennis and the emerald ash borer (A. planipennis is a serious ecological and economic threat to Canadian deciduous and mixed-wood forests. Humans act as a major vector for the spread of these pests via firewood transport, although existing models do not explicitly capture human decision-making regarding firewood transport. In this paper we present a two-patch coupled human-environment system model that includes social influence and long-distance firewood transport and examines potential strategies for mitigating pest spread. We found that increasing concern regarding infestations (f significantly reduced infestation. Additionally it resulted in multiple thresholds at which the intensity of infestation in a patch was decreased. It was also found that a decrease in the cost of firewood purchased in the area where it is supposed to be burned (Cl resulted in an increased proportion of local-firewood strategists, and a 67% decrease in Cl from $6.75 to $4.50 was sufficient to eliminate crosspatch infestation. These effects are synergistic: increasing concern through awareness and education campaigns acts together with reduced firewood costs, thereby reducing the required threshold of both awareness and economic incentives. Our results indicate that the best management strategy includes a combination of public education paired with firewood subsidization.

  11. Modelling Interactions between forest pest invasions and human decisions regarding firewood transport restrictions.

    Science.gov (United States)

    Barlow, Lee-Ann; Cecile, Jacob; Bauch, Chris T; Anand, Madhur

    2014-01-01

    The invasion of nonnative, wood-boring insects such as the Asian longhorned beetle (A. glabripennis) and the emerald ash borer (A. planipennis) is a serious ecological and economic threat to Canadian deciduous and mixed-wood forests. Humans act as a major vector for the spread of these pests via firewood transport, although existing models do not explicitly capture human decision-making regarding firewood transport. In this paper we present a two-patch coupled human-environment system model that includes social influence and long-distance firewood transport and examines potential strategies for mitigating pest spread. We found that increasing concern regarding infestations (f) significantly reduced infestation. Additionally it resulted in multiple thresholds at which the intensity of infestation in a patch was decreased. It was also found that a decrease in the cost of firewood purchased in the area where it is supposed to be burned (Cl) resulted in an increased proportion of local-firewood strategists, and a 67% decrease in Cl from $6.75 to $4.50 was sufficient to eliminate crosspatch infestation. These effects are synergistic: increasing concern through awareness and education campaigns acts together with reduced firewood costs, thereby reducing the required threshold of both awareness and economic incentives. Our results indicate that the best management strategy includes a combination of public education paired with firewood subsidization.

  12. The transepithelial transport mechanism of polybrominated diphenyl ethers in human intestine determined using a Caco-2 cell monolayer.

    Science.gov (United States)

    Yu, Yingxin; Wang, Mengmeng; Zhang, Kaiqiong; Yang, Dan; Zhong, Yufang; An, Jing; Lei, Bingli; Zhang, Xinyu

    2017-04-01

    Oral ingestion plays an important role in human exposure to polybrominated diphenyl ethers (PBDEs). The uptake of PBDEs primarily occurs in the small intestine. The aim of the present study is to investigate the transepithelial transport characteristics and mechanisms of PBDEs in the small intestine using a Caco-2 cell monolayer model. The apparent permeability coefficients of PBDEs indicated that tri- to hepta-BDEs were poorly absorbed compounds. A linear increase in transepithelial transport was observed with various concentrations of PBDEs, which suggested that passive diffusion dominated their transport at the concentration range tested. In addition, the pseudo-first-order kinetics equation can be applied to the transepithelial transport of PBDEs. The rate-determining step in transepithelial transport of PBDEs was trans-cell transport including the trans-pore process. The significantly lower transepithelial transport rates at low temperature for bidirectional transepithelial transport suggested that an energy-dependent transport mechanism was involved. The efflux transporters (P-glycoprotein, multidrug resistance-associated protein, and breast cancer resistance protein) and influx transporters (organic cation transporters) participated in the transepithelial transport of PBDEs. In addition, the transepithelial transport of PBDEs was pH sensitive; however, more information is required to understand the influence of pH. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Performance characteristics of the new ARCHITECT Toxo IgG and Toxo IgG Avidity assays.

    Science.gov (United States)

    Sickinger, Eva; Gay-Andrieu, Françoise; Jonas, Gesa; Schultess, Jan; Stieler, Myriam; Smith, Darwin; Hausmann, Michael; Stricker, René; Stricker, Reto; Dhein, Jens; Braun, Hans-Bertram

    2008-11-01

    The ARCHITECT Toxo IgG and IgG Avidity assays have been developed as a fully automated panel for immune status determination and acute infection exclusion. Resolved relative specificity and sensitivity of the ARCHITECT Toxo IgG assay were 99.6% (1359/1365) and 99.7% (1096/1099) as determined on pregnant females, blood donor, and diagnostic specimens. Seroconversion sensitivity of the ARCHITECT assay was comparable with the AxSYM Toxo IgG assay. The ARCHITECT Toxo IgG Avidity assay detected 100.0% (124/124) of acute phase specimens (ARCHITECT Toxo IgG assay, using recombinant antigens, showed excellent specificity and sensitivity for acute phase as well as past infection specimens. The ARCHITECT Toxoplasmosis panel can be reliably used to rule out acute Toxoplasma gondii infection in pregnant women.

  14. Transport of the Glucosamine-Derived Browning Product Fructosazine (Polyhydroxyalkylpyrazine) Across the Human Intestinal Caco-2 Cell Monolayer: Role of the Hexose Transporters.

    Science.gov (United States)

    Bhattacherjee, Abhishek; Hrynets, Yuliya; Betti, Mirko

    2017-06-14

    The transport mechanism of fructosazine, a glucosamine self-condensation product, was investigated using a Caco-2 cell model. Fructosazine transport was assessed by measuring the bidirectional permeability coefficient across Caco-2 cells. The mechanism of transport was evaluated using phlorizin, an inhibitor of sodium-dependent glucose cotransporters (SGLT) 1 and 2, phloretin and quercetin, inhibitors of glucose transporters (GLUT) 1 and 2, transcytosis inhibitor wortmannin, and gap junction disruptor cytochalasin D. The role of hexose transporters was further studied using downregulated or overexpressed cell lines. The apparent permeability (P a,b ) of fructosazine was 1.30 ± 0.02 × 10 -6 cm/s. No significant (p > 0.05) effect was observed in fructosazine transport by adding wortmannin and cytochalasin D. The presence of phlorizin, phloretin, and quercetin decreased fructosazine transport. The downregulated GLUT cells line was unable to transport fructosazine. In human intestinal epithelial Caco-2 cells, GLUT1 or GLUT2 and SGLT are mainly responsible for fructosazine transport.

  15. The serum igg subclass levels in healthy infants of 13–62 weeks of age

    NARCIS (Netherlands)

    Zegers, B.J.M.; Giessen, M. van der; Reerink-Brongers, E.E.; Stoop, J.W.

    1980-01-01

    The levels of IgG1, IgG2, IgG3, and IgG4 were determined in serum samples of 160 infants aged 13–62 weeks, and of their mothers. In addition the serum IgM, IgG, IgA, and IgD levels of the infants are presented. The results show that IgM, IgG1, and IgG3 slightly increase during the first year of

  16. Intracellular acidification increases adenosine transport in human umbilical vein endothelial cells.

    Science.gov (United States)

    Celis, Natalia; Araos, Joaquín; Sanhueza, Carlos; Toledo, Fernando; Beltrán, Ana R; Pardo, Fabián; Leiva, Andrea; Ramírez, Marco A; Sobrevia, Luis

    2017-03-01

    Adenosine is taken up via human equilibrative nucleoside transporters 1 (hENT1) and 2 (hENT2) at a physiological extracellular pH (pHo ∼7.4) in human umbilical vein endothelial cells (HUVECs). Acidic pHo increases the uptake of adenosine and 5-hydroxytryptamine (5HT) via hENT4 in this cell type. However, modulation of hENT1 and hENT2 transport activity by the pHi is unknown. We investigated whether hENT1 and hENT2-adenosine transport was regulated by acidic pHi. HUVECs loaded with a pH sensitive probe were subjected to 0.1-20 mmol/L NH 4 Cl pulse assay to generate 6.9-6.2 pHi. Before pHi started to recover, adenosine transport kinetics (0-500 μmol/L, 37 °C) in the absence or presence 1 or 10 μmol/L S-(4-nitrobenzyl)-6-thio-inosine (NBTI), 2 mmol/L hypoxanthine, 2 mmol/L adenine, 100 μmol/L 5HT, or 500 μmol/L adenosine, was measured. Overall adenosine transport (i.e., hENT1+hENT2) was semisaturable and partially inhibited by 1 μmol/L, but abolished by 10 μmol/L NBTI in cells non-treated or treated with NH 4 Cl. The initial velocity and non-saturable, lineal component for overall transport were increased after NH 4 Cl pulse. hENT1 and hENT2-mediated adenosine transport maximal capacity was increased by acidic pHi. hENT1 activity was more sensitive than hENT2 activity to acidic pHi. hENT1 and hENT2-adenosine transport is differentially regulated by acidic pHi in HUVECs. These findings are important in pathologies associated with pHi alterations such as gestational diabetes mellitus. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Review: Selenium contamination, fate, and reactive transport in groundwater in relation to human health

    Science.gov (United States)

    Bailey, Ryan T.

    2017-06-01

    Selenium (Se) is an essential micro-nutrient for humans, but can be toxic at high levels of intake. Se deficiency and Se toxicity are linked with serious diseases, with some regions worldwide experiencing Se deficiency due to Se-poor rocks and soils and other areas dealing with Se toxicity due to the presence of Se-enriched geologic materials. In addition, Se is consumed primarily through plants that take up Se from soil and through animal products that consume these plants. Hence, the soil and groundwater system play important roles in determining the effect of Se on human health. This paper reviews current understanding of Se fate and transport in soil and groundwater systems and its relation to human health, with a focus on alluvial systems, soil systems, and the interface between alluvial systems and Cretaceous shale that release Se via oxidation processes. The review focuses first on the relation between Se and human health, followed by a summary of Se distribution in soil-aquifer systems, with an emphasis on the quantitative relationship between Se content in soil and Se concentration in underlying groundwater. The physical, chemical, and microbial processes that govern Se fate and transport in subsurface systems then are presented, followed by numerical modeling techniques used to simulate these processes in study regions and available remediation strategies for either Se-deficient or Se-toxic regions. This paper can serve as a guide to any field, laboratory or modeling study aimed at assessing Se fate and transport in groundwater systems and its relation to human health.

  18. The N-Glycosylation of Mouse Immunoglobulin G (IgG)-Fragment Crystallizable Differs Between IgG Subclasses and Strains.

    Science.gov (United States)

    de Haan, Noortje; Reiding, Karli R; Krištić, Jasminka; Hipgrave Ederveen, Agnes L; Lauc, Gordan; Wuhrer, Manfred

    2017-01-01

    N-linked glycosylation of the fragment crystallizable (Fc)-region of immunoglobulin G (IgG) is known to have a large influence on the activity of the antibody, an effect reported to be IgG subclass specific. This situation applies both to humans and mice. The mouse is often used as experimental animal model to study the effects of Fc-glycosylation on IgG effector functions, and results are not uncommonly translated back to the human situation. However, while human IgG Fc-glycosylation has been extensively characterized in both health and disease, this is not the case for mice. To characterize the glycosylation profile of murine IgG-Fc and in addition evaluate the systematic glycosylation differences between mouse strains, sexes, and IgG subclasses, we used nanoliquid chromatography mass spectrometry (nanoLC-MS(/MS)) to look at the subclass-specific IgG Fc-glycopeptides of male and female mice from the strains BALB/c, C57BL/6, CD-1, and Swiss Webster. The structural analysis revealed the presence of predominantly fucosylated, diantennary glycans, with varying amounts of galactosylation and α2,6-sialylation. In addition, we report glycosylation features not previously reported in an Fc-specific way on murine IgG, including monoantennary, hybrid, and high mannose structures, as well as diantennary structures without a core fucose, with a bisecting N-acetylglucosamine, or with α1,3-galactosylation. Pronounced differences were detected between strains and the IgG subclasses within each strain. Especially the large spread in galactosylation and sialylation levels found between both strains and subclasses may vastly influence IgG effector functions. Mouse strain-based and subclass-specific glycosylation differences should be taken into account when designing and interpreting immunological and glycobiological mouse studies involving IgG effector functions.

  19. The N-Glycosylation of Mouse Immunoglobulin G (IgG-Fragment Crystallizable Differs Between IgG Subclasses and Strains

    Directory of Open Access Journals (Sweden)

    Noortje de Haan

    2017-05-01

    Full Text Available N-linked glycosylation of the fragment crystallizable (Fc-region of immunoglobulin G (IgG is known to have a large influence on the activity of the antibody, an effect reported to be IgG subclass specific. This situation applies both to humans and mice. The mouse is often used as experimental animal model to study the effects of Fc-glycosylation on IgG effector functions, and results are not uncommonly translated back to the human situation. However, while human IgG Fc-glycosylation has been extensively characterized in both health and disease, this is not the case for mice. To characterize the glycosylation profile of murine IgG-Fc and in addition evaluate the systematic glycosylation differences between mouse strains, sexes, and IgG subclasses, we used nanoliquid chromatography mass spectrometry (nanoLC-MS(/MS to look at the subclass-specific IgG Fc-glycopeptides of male and female mice from the strains BALB/c, C57BL/6, CD-1, and Swiss Webster. The structural analysis revealed the presence of predominantly fucosylated, diantennary glycans, with varying amounts of galactosylation and α2,6-sialylation. In addition, we report glycosylation features not previously reported in an Fc-specific way on murine IgG, including monoantennary, hybrid, and high mannose structures, as well as diantennary structures without a core fucose, with a bisecting N-acetylglucosamine, or with α1,3-galactosylation. Pronounced differences were detected between strains and the IgG subclasses within each strain. Especially the large spread in galactosylation and sialylation levels found between both strains and subclasses may vastly influence IgG effector functions. Mouse strain-based and subclass-specific glycosylation differences should be taken into account when designing and interpreting immunological and glycobiological mouse studies involving IgG effector functions.

  20. Hatchery workers' IgG antibody profiles to airborne bacteria.

    Science.gov (United States)

    Brauner, Paul; Gromöller, Silvana; Pfeifer, Yvonne; Wilharm, Gottfried; Jäckel, Udo

    2017-04-01

    Occupational exposure to high concentrations of airborne bacteria in poultry production is related to an increased risk of respiratory disorders. However, etiology and in particular microorganisms' potential role in pathogenesis still needs to be elucidated. Thus, detection of specific antibodies against occupational microbial antigens may lead to identification of potentially harmful species. For the purpose of IgG titer determination, indirect immunofluorescence on various bacterial isolates from duck hatchery air was combined with image-based quantification of fluorescence intensity. Moreover, in addition to established assays with pure bacterial cultures, a new approach utilized complex bioaerosol samples for detection of anti-microbial antibodies in human sera by determination of percentages of antibody-bound cells in different serum dilutions. Mean titers in sera from hatchery workers and a non-exposed control group did not display significant differences for most tested isolates and application of comprehensive cluster analysis to entire titer data revealed no structure reflecting workers and controls group. Furthermore, determination of immunoreactivity to the complete microbial community in workplace air displayed similar proportions of antibody-bound cells in both groups. Although no general differences in immunoreaction patterns were observed, mean titers to a Proteus mirabilis isolate and to 3 of 4 distinct Acinetobacter baumannii isolates were higher in the group of hatchery workers than in the reference group indicating a potential applicability as exposure markers. We conclude, despite long term bioaerosol exposure, hatchery workers' IgG antibody profiles to tested antigens did not differ substantially from those of the control group. However, increased workers' titers to A. baumannii and clinical relevance of this species should lead to further investigations regarding potential involvement in pathogenesis of occupational respiratory disorders

  1. Effective Cellular Uptake and Efflux of Thyroid Hormone by Human Monocarboxylate Transporter 10

    Science.gov (United States)

    Friesema, Edith C. H.; Jansen, Jurgen; Jachtenberg, Jan-willem; Visser, W. Edward; Kester, Monique H. A.; Visser, Theo J.

    2008-01-01

    Cellular entry of thyroid hormone is mediated by plasma membrane transporters, among others a T-type (aromatic) amino acid transporter. Monocarboxylate transporter 10 (MCT10) has been reported to transport aromatic amino acids but not iodothyronines. Within the MCT family, MCT10 is most homologous to MCT8, which is a very important iodothyronine transporter but does not transport amino acids. In view of this paradox, we decided to reinvestigate the possible transport of thyroid hormone by human (h) MCT10 in comparison with hMCT8. Transfection of COS1 cells with hMCT10 cDNA resulted in 1) the production of an approximately 55 kDa protein located to the plasma membrane as shown by immunoblotting and confocal microscopy, 2) a strong increase in the affinity labeling of intracellular type I deiodinase by N-bromoacetyl-[125I]T3, 3) a marked stimulation of cellular T4 and, particularly, T3 uptake, 4) a significant inhibition of T3 uptake by phenylalanine, tyrosine, and tryptophan of 12.5%, 22.2%, and 51.4%, respectively, and 5) a marked increase in the intracellular deiodination of T4 and T3 by different deiodinases. Cotransfection studies using the cytosolic thyroid hormone-binding protein μ-crystallin (CRYM) indicated that hMCT10 facilitates both cellular uptake and efflux of T4 and T3. In the absence of CRYM, hMCT10 and hMCT8 increased T3 uptake after 5 min incubation up to 4.0- and 1.9-fold, and in the presence of CRYM up to 6.9- and 5.8-fold, respectively. hMCT10 was less active toward T4 than hMCT8. These findings establish that hMCT10 is at least as active a thyroid hormone transporter as hMCT8, and that both transporters facilitate iodothyronine uptake as well as efflux. PMID:18337592

  2. Non-pathogenic pemphigus foliaceus (PF) IgG acts synergistically with a directly pathogenic PF IgG to increase blistering by p38MAPK-dependent desmoglein 1 clustering✩

    Science.gov (United States)

    Yoshida, Kenji; Ishii, Ken; Shimizu, Atsushi; Yokouchi, Mariko; Amagai, Masayuki; Shiraishi, Ken; Shirakata, Yuji; Stanley, John R.; Ishiko, Akira

    2017-01-01

    Background Pemphigus foliaceus (PF) is an autoimmune blistering disease caused by autoantibodies (Abs) against desmoglein 1 (Dsg1). PF sera contain polyclonal Abs which are heterogeneous mixture of both pathogenic and non-pathogenic Abs, as shown by isolation of monoclonal Abs (mAbs). Objective To investigate how pathogenic and non-pathogenic anti-Dsg1 Abs contribute to blister formation in PF. Methods Using organ-cultured human skin, we compared the effect of a single pathogenic anti-Dsg1 IgG mAb, a single non-pathogenic anti-Dsg1 IgG mAb, and their mixture on blister formation as analyzed by histology, subcellular localization of IgG deposits and desmosomal proteins by confocal microscopy, and desmosomal structure by electron microscopy. In addition, we measured keratinocyte adhesion by an in vitro dissociation assay. Results 24 h after injection, a single pathogenic anti-Dsg1 IgG caused a subcorneal blister with IgG and Dsg1 localized linearly on the cell surface of keratinocytes. A single non-pathogenic anti-Dsg1 IgG bound linearly on the keratinocytes but did not induce blisters. A pathogenic and a non-pathogenic IgG mAb injected together caused an aberrant granular pattern of IgG and Dsg1 in the lower epidermis with blister formation in the superficial epidermis. Electron microscopy demonstrated that the mixture of mAbs shortened desmosomal lengths more than a single mAb in the basal and spinous layers. Furthermore, although Dsg1 clustering required both cross-linking of Dsg1 molecules by the non-pathogenic IgG plus a pathogenic antibody, the latter could be in the form of a monovalent single chain variable fragment, suggesting that loss of trans-interaction of Dsg1 is required for clustering. Finally, a p38MAPK inhibitor blocked Dsg1 clustering. When pathogenic strength was measured by the dissociation assay, a mixture of pathogenic and non-pathogenic IgG mAbs disrupted keratinocyte adhesion more than a single pathogenic mAb. This pathogenic effect was only

  3. Non-pathogenic pemphigus foliaceus (PF) IgG acts synergistically with a directly pathogenic PF IgG to increase blistering by p38MAPK-dependent desmoglein 1 clustering.

    Science.gov (United States)

    Yoshida, Kenji; Ishii, Ken; Shimizu, Atsushi; Yokouchi, Mariko; Amagai, Masayuki; Shiraishi, Ken; Shirakata, Yuji; Stanley, John R; Ishiko, Akira

    2017-03-01

    Pemphigus foliaceus (PF) is an autoimmune blistering disease caused by autoantibodies (Abs) against desmoglein 1 (Dsg1). PF sera contain polyclonal Abs which are heterogeneous mixture of both pathogenic and non-pathogenic Abs, as shown by isolation of monoclonal Abs (mAbs). To investigate how pathogenic and non-pathogenic anti-Dsg1 Abs contribute to blister formation in PF. Using organ-cultured human skin, we compared the effect of a single pathogenic anti-Dsg1 IgG mAb, a single non-pathogenic anti-Dsg1 IgG mAb, and their mixture on blister formation as analyzed by histology, subcellular localization of IgG deposits and desmosomal proteins by confocal microscopy, and desmosomal structure by electron microscopy. In addition, we measured keratinocyte adhesion by an in vitro dissociation assay. 24h after injection, a single pathogenic anti-Dsg1 IgG caused a subcorneal blister with IgG and Dsg1 localized linearly on the cell surface of keratinocytes. A single non-pathogenic anti-Dsg1 IgG bound linearly on the keratinocytes but did not induce blisters. A pathogenic and a non-pathogenic IgG mAb injected together caused an aberrant granular pattern of IgG and Dsg1 in the lower epidermis with blister formation in the superficial epidermis. Electron microscopy demonstrated that the mixture of mAbs shortened desmosomal lengths more than a single mAb in the basal and spinous layers. Furthermore, although Dsg1 clustering required both cross-linking of Dsg1 molecules by the non-pathogenic IgG plus a pathogenic antibody, the latter could be in the form of a monovalent single chain variable fragment, suggesting that loss of trans-interaction of Dsg1 is required for clustering. Finally, a p38MAPK inhibitor blocked Dsg1 clustering. When pathogenic strength was measured by the dissociation assay, a mixture of pathogenic and non-pathogenic IgG mAbs disrupted keratinocyte adhesion more than a single pathogenic mAb. This pathogenic effect was only partially suppressed by the p38MAPK

  4. Interdependence of Gemcitabine Treatment, Transporter Expression, and Resistance in Human Pancreatic Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Wolfgang Hagmann

    2010-09-01

    Full Text Available Gemcitabine is widely used as first-line chemotherapeutic drug in the treatment of pancreatic cancer. Our previous experimental chemotherapy studies have shown that treatment of human pancreatic carcinoma cells with 5-fluorouracil (5-FU alters the cellular transporter expression profile and that modulation of the expression of multidrug resistance protein 5 (MRP5; ABCC5 influences the chemoresistance of these tumor cells. Here, we studied the influence of acute and chronic gemcitabine treatment on the expression of relevant uptake and export transporters in pancreatic carcinoma cells by reverse transcription-polymerase chain reaction (RT-PCR, quantitative RT-PCR, and immunoblot analyses. The specific role of MRP5 in cellular gemcitabine sensitivity was studied by cytotoxicity assays using MRP5-overexpressing and MRP5-silenced cells. Exposure to gemcitabine (12 nM for 3 days did not alter the messenger RNA (mRNA expression of MRP1, MRP3, MRP5, and equilibrative nucleoside transporter 1 (ENT1, whereas high dosages of the drug (20 µM for 1 hour elicited up-regulation of these transporters in most cell lines studied. In cells with acquired gemcitabine resistance (up to 160 nM gemcitabine, the mRNA or protein expression of the gemcitabine transporters MRP5 and ENT1 was upregulated in several cell lines. Combined treatment with 5-FU and gemcitabine caused a 5- to 40-fold increase in MRP5 and ENT1 expressions. Cytotoxicity assays using either MRP5-overexpressing (HEK and PANC-1 or MRP5-silenced (PANC1/shMRP5 cells indicated that MRP5 contributes to gemcitabine resistance. Thus, our novel data not only on drug-induced alterations of transporter expression relevant for gemcitabine uptake and export but also on the link between gemcitabine sensitivity and MRP5 expression may lead to improved strategies of future chemotherapy regimens using gemcitabine in pancreatic carcinoma patients.

  5. Adeno-associated virus type 6 is retrogradely transported in the non-human primate brain.

    Science.gov (United States)

    San Sebastian, W; Samaranch, L; Heller, G; Kells, A P; Bringas, J; Pivirotto, P; Forsayeth, J; Bankiewicz, K S

    2013-12-01

    We recently demonstrated that axonal transport of adeno-associated virus (AAV) is serotype-dependent. Thus, AAV serotype 2 (AAV2) is anterogradely transported (e.g., from cell bodies to nerve terminals) in both rat and non-human primate (NHP) brain. In contrast, AAV serotype 6 (AAV6) is retrogradely transported from terminals to neuronal cell bodies in the rat brain. However, the directionality of axonal transport of AAV6 in the NHP brain has not been determined. In this study, two Cynomolgus macaques received an infusion of AAV6 harboring green fluorescent protein (GFP) into the striatum (caudate and putamen) by magnetic resonance (MR)-guided convection-enhanced delivery. One month after infusion, immunohistochemical staining of brain sections revealed a striatal GFP expression that corresponded well with MR signal observed during gene delivery. As shown previously in rats, GFP expression was detected throughout the prefrontal, frontal and parietal cortex, as well as the substantia nigra pars compacta and thalamus, indicating retrograde transport of the vector in NHP. AAV6-GFP preferentially transduced neurons, although a few astrocytes were also transduced. Transduction of non-neuronal cells in the brain was associated with the upregulation of the major histocompatibility complex-II and lymphocytic infiltration as previously observed with AAV1 and AAV9. This contrasts with highly specific neuronal transduction in the rat brain. Retrograde axonal transport of AAV6 from a single striatal infusion permits efficient transduction of cortical neurons in significant tissue volumes that otherwise would be difficult to achieve.

  6. PURIFIED IGG F C-BINDING PROTEINS FROM M22 GROUP A STREPTOCOCCUS ARE ABLE TO INDUCE EXPERIMENTAL GLOMERULONEPHRITIS

    Directory of Open Access Journals (Sweden)

    L. A. Burova

    2012-01-01

    Full Text Available Abstract. Pathogenesis of acute post-streptococcal glomerulonephritis (APSGN, a major complication of group A streptococcal (GAS throat or skin disease, remains unclear. Over years, various theories were based on distinct streptococcal extracellular factors, as well as immunological mimicry of streptococci for renal tissue antigens was considered. Previously we reported that a lot of clinical GAS isolates with proven nephritogenic ability show a non-immune binding of monomeric or aggregated IgG. Moreover, using a rabbit model of APSGN, we obtained evidence for important causative role of streptococcal IgG Fc-binding proteins (IgG FcBPs belonging to the M family surface proteins. I.e., rabbits injected by whole IgG FcBP-positive streptococci showed induction of renal glomerular changes, with deposition of IgG and complement C3, resembling the picture recorded in human APSGN. These typical renal changes were always preceded by development of circulating anti-IgG antibodies. Present study was performed in the same rabbit model. Both purified IgG FcBPs isolated from type M22 GAS were found to elicit glomerular degenerative damage of renal glomeruli comparable to those caused by whole bacteria, as well as induce anti-IgG antibodies, deposition of IgG and C3 complement and production of proinflammatory cytokines (IL-1β, TNFα, IL-6 by glomerular mesangial and endothelial cells. By contrast, rabbits injected with proteins A or G, IgG FcBPs of S. aureus and group G streptococci, respectively, exhibited only low levels of circulating anti-IgG and reversible glomerular changes. In these settings, we have not observed any features of membranousproliferative glomerulonephritis (GN resembling morphological traits of acute post-streptococcal GN in humans. These data correlated with results obtained after injection of intact Staphylococcus aureus (Cowan 1 strain or group G streptococci (G148 strain. Both microbial types are known to harbor IgG Fc

  7. Cellular interactions of a lipid-based nanocarrier model with human keratinocytes: Unravelling transport mechanisms.

    Science.gov (United States)

    Silva, Elisabete; Barreiros, Luísa; Segundo, Marcela A; Costa Lima, Sofia A; Reis, Salette

    2017-04-15

    Knowledge of delivery system transport through epidermal cell monolayer is vital to improve skin permeation and bioavailability. Recently, nanostructured lipid carriers (NLCs) have gained great attention for transdermal delivery due to their biocompatibility, high drug payload, occlusive properties and skin hydration effect. However, the nanocarriers transport related mechanisms in epidermal epithelial cells are not yet understood. In this research, the internalization and transport pathways of the NLCs across the epidermal epithelial cell monolayer (HaCaT cells) were investigated. The 250nm sized witepsol/miglyol NLCs, prepared by hot homogenization had reduced cytotoxicity and no effect on the integrity of cell membrane in human HaCaT keratinocytes. The internalization was time-, concentration- and energy-dependent, and the uptake of NLCs was a vesicle-mediated process by macropinocytosis and clathrin-mediated pathways. 3% of NLCs were found at the apical membrane side of the HaCaT monolayer through exocytosis mechanism. Additionally, the endoplasmic reticulum, Golgi apparatus and microtubules played crucial roles in the transport of NLCs out of HaCaT cells. NLCs were transported intact across the human keratinocytes monolayer, without disturbing the tight junction's structure. From the transcytosis data only approximately 12% of the internalized NLCs were passed from the apical to the basolateral side. The transcytosis of NLCs throughout the HaCaT cell monolayer towards the basolateral membrane side requires the involvement of the endoplasmic reticulum, Golgi apparatus and microtubules. Our findings may contribute to a systematic understanding of NLCs transport across epidermal epithelial cell monolayers and their optimization for clinical transdermal application. Transdermal drug delivery is a challenging and growing area of clinical application. Lipid nanoparticles such as nanostructured lipid carriers (NLCs) have gained wide interest for transdermal drug

  8. Efficient Mucosal Delivery of Vaccine Using the FcRn-Mediated IgG Transfer Pathway

    Science.gov (United States)

    Ye, Lilin; Zeng, Rongyu; Bai, Yu; Roopenian, Derry C.; Zhu, Xiaoping

    2011-01-01

    Vaccine strategies to prevent invasive mucosal pathogens are being sought because 80–90% of infectious diseases are initiated at mucosal surfaces. However, our ability to deliver an intact vaccine antigen across a mucosal barrier for induction of effective immunity is limited. The neonatal Fc receptor (FcRn) mediates the transport of IgG across polarized epithelial cells lining mucosal surfaces. By mimicking IgG transfer at mucosal surfaces, intranasal immunization with a model antigen, herpes simplex virus type-2 (HSV-2) glycoprotein gD fused with an IgG Fc fragment, in combination with the adjuvant CpG, resulted in complete protection of wild type, but not FcRn knockout, mice that were intravaginally challenged with virulent HSV-2 186. This immunization strategy induced efficient mucosal and systemic antibody, B and T cell immune responses, including memory immune responses, which remained stable at least 6 months post-vaccination and conferred protection for a majority of animals. These results demonstrate that the FcRn-IgG transcellular transport pathway may represent a novel mucosal vaccine delivery route for a subunit vaccine against abundant mucosal pathogens. PMID:21240266

  9. Gene Expression Variability in Human Hepatic Drug Metabolizing Enzymes and Transporters

    Science.gov (United States)

    Yang, Lun; Price, Elvin T.; Chang, Ching-Wei; Li, Yan; Huang, Ying; Guo, Li-Wu; Guo, Yongli; Kaput, Jim; Shi, Leming; Ning, Baitang

    2013-01-01

    Interindividual variability in the expression of drug-metabolizing enzymes and transporters (DMETs) in human liver may contribute to interindividual differences in drug efficacy and adverse reactions. Published studies that analyzed variability in the expression of DMET genes were limited by sample sizes and the number of genes profiled. We systematically analyzed the expression of 374 DMETs from a microarray data set consisting of gene expression profiles derived from 427 human liver samples. The standard deviation of interindividual expression for DMET genes was much higher than that for non-DMET genes. The 20 DMET genes with the largest variability in the expression provided examples of the interindividual variation. Gene expression data were also analyzed using network analysis methods, which delineates the similarities of biological functionalities and regulation mechanisms for these highly variable DMET genes. Expression variability of human hepatic DMET genes may affect drug-gene interactions and disease susceptibility, with concomitant clinical implications. PMID:23637747

  10. Placental transfer of IgG antibodies specific to Klebsiella and Pseudomonas LPS and to group B Streptococcus in twin pregnancies.

    Science.gov (United States)

    Stach, S C L; Brizot, M L; Liao, A W; Palmeira, P; Francisco, R P V; Carneiro-Sampaio, M M S; Zugaib, M

    2015-02-01

    Group B Streptococcus (GBS), Klebsiella spp. and Pseudomonas spp. are important aetiological agents of neonatal infections in Brazil. There is a lack of data in the literature regarding the specific transport of immunoglobulin G (IgG) against these pathogens in multiple pregnancies. Maternal (n = 55) and umbilical cord (n = 110) blood samples were prospectively collected at birth from 55 twin pregnancies. The factors associated with cord levels and transfer ratios of IgG against GBS, Klebsiella and Pseudomonas were examined. The IgG umbilical cord serum levels specific to GBS, Klebsiella LPS and Pseudomonas LPS were significantly associated with maternal-specific IgG concentrations and the presence of diabetes. The anti-Klebsiella IgG cord serum concentrations were also related to birthweight and the presence of hypertension. The transfer ratios against GBS and Pseudomonas LPS were associated with maternal-specific IgG concentrations. The transfer ratios for GBS and Pseudomonas LPS were associated with gestational age at delivery and the presence of diabetes, respectively. None of the examined parameters were related to Klebsiella LPS transfer ratios. We conclude that in twin pregnancies, specific maternal IgG serum concentrations and diabetes were the parameters associated with umbilical cord serum IgG concentrations reactive with the three pathogens investigated. All the other parameters investigated showed different associations with neonatal-specific IgG levels according to the antigen studied. There was no uniformity of the investigated parameters regarding association with placental IgG transfer ratios against the GBS, Pseudomonas LPS and Klebsiella LPS. © 2014 John Wiley & Sons Ltd.

  11. Energetic and molecular water permeation mechanisms of the human red blood cell urea transporter B.

    Science.gov (United States)

    Azouzi, Slim; Gueroult, Marc; Ripoche, Pierre; Genetet, Sandrine; Colin Aronovicz, Yves; Le Van Kim, Caroline; Etchebest, Catherine; Mouro-Chanteloup, Isabelle

    2013-01-01

    Urea transporter B (UT-B) is a passive membrane channel that facilitates highly efficient permeation of urea. In red blood cells (RBC), while the major function of UT-B is to transport urea, it is assumed that this protein is able to conduct water. Here, we have revisited this last issue by studying RBCs and ghosts from human variants with defects of aquaporin 1 (AQP1) or UT-B. We found that UT-B's osmotic water unit permeability (pfunit) is similar to that of AQP1. The determination of diffusional permeability coefficient (Pd) allowed the calculation of the Pf/Pd ratio, which is consistent with a single-file water transport. Molecular dynamic simulations of water conduction through human UT-B confirmed the experimental finding. From these results, we propose an atomistic description of water-protein interactions involved in this permeation. Inside the UT-B pore, five water molecules were found to form a single-file and move rapidly along a channel by hydrogen bond exchange involving two critical threonines. We further show that the energy barrier for water located in the central region coincides with a water dipole reorientation, which can be related to the proton exclusion observed experimentally. In conclusion, our results indicate that UT-B should be considered as a new member of the water channel family.

  12. Expression and purification of human and Saccharomyces cerevisiae equilibrative nucleoside transporters.

    Science.gov (United States)

    Boswell-Casteel, Rebba C; Johnson, Jennifer M; Roe-Žurž, Zygy; Duggan, Kelli D; Schmitz, Hannah; Hays, Franklin A

    2018-02-01

    Nucleosides play an essential role in the physiology of eukaryotes by acting as metabolic precursors in de novo nucleic acid synthesis and energy metabolism. Nucleosides also act as ligands for purinergic receptors. Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that aid in regulating plasmalemmal flux of purine and pyrimidine nucleosides and nucleobases. ENTs exhibit broad substrate selectivity across different isoforms and utilize diverse mechanisms to drive substrate flux across membranes. However, the molecular mechanisms and chemical determinants of ENT-mediated substrate recognition, binding, inhibition, and transport are poorly understood. To determine how ENT-mediated transport occurs at the molecular level, greater chemical insight and assays employing purified protein are essential. This article focuses on the expression and purification of human ENT1, human ENT2, and Saccharomyces cerevisiae ScENT1 using novel expression and purification strategies to isolate recombinant ENTs. ScENT1, hENT1, and hENT2 were expressed in W303 Saccharomyces cerevisiae cells and detergent solubilized from the membrane. After detergent extraction, these ENTs were further purified using immobilized metal affinity chromatography and size exclusion chromatography. This effort resulted in obtaining quantities of purified protein sufficient for future biophysical analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. A functional splice variant of the human Golgi CMP-sialic acid transporter.

    Science.gov (United States)

    Salinas-Marín, Roberta; Mollicone, Rosella; Martínez-Duncker, Iván

    2016-12-01

    The human Golgi Cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-Sia) transporter SLC35A1, a member of the nucleotide sugar transporter family, translocates CMP-Sia from the cytosol into the Golgi lumen where sialyltransferases use it as donor substrate for the synthesis of sialoglycoconjugates. In 2005, we reported a novel Congenital Disorder of Glycosylation (CDG) termed CDG-IIf or SLC35A1-CDG, characterized by macrothrombocytopenia, neutropenia and complete lack of the sialyl-Lex antigen (NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc-R) on polymorphonuclear cells. This disease was caused by the presence of inactive SLC35A1 alleles. It was also found that the SLC35A1 generates additional isoforms through alternative splicing. In this work, we demonstrate that one of the reported isoforms, the del177 with exon 6 skipping, is able to maintain sialylation in HepG2 cells submitted to wt knockdown and restore sialylation to normal levels in the Chinese Hamester Ovary (CHO) cell line Lec2 mutant deficient in CMP-Sia transport. The characteristics of the alternatively spliced protein are discussed as well as therapeutic implications of this finding in CDGs caused by mutations in nucleotide sugar transporters (NSTs).

  14. Functionally Relevant Residues of Cdr1p: A Multidrug ABC Transporter of Human Pathogenic Candida albicans

    Directory of Open Access Journals (Sweden)

    Rajendra Prasad

    2011-01-01

    Full Text Available Reduced intracellular accumulation of drugs (due to rapid efflux mediated by the efflux pump proteins belonging to ABC (ATP Binding Cassette and MFS (Major Facilitators superfamily is one of the most common strategies adopted by multidrug resistance (MDR pathogenic yeasts. To combat MDR, it is essential to understand the structure and function of these transporters so that inhibitors/modulators to these can be developed. The sequence alignments of the ABC transporters reveal selective divergence within much conserved domains of Nucleotide-Binding Domains (NBDs which is unique to all fungal transporters. Recently, the role of conserved but divergent residues of Candida Drug Resistance 1 (CDR1, an ABC drug transporter of human pathogenic Candida albicans, has been examined with regard to ATP binding and hydrolysis. In this paper, we focus on some of the recent advances on the relevance of divergent and conserved amino acids of CaCdr1p and also discuss as to how drug interacts with Trans Membrane Domains (TMDs residues for its extrusion from MDR cells.

  15. Passive transport pathways for Ca2+ and Co2+ in human red blood cells

    DEFF Research Database (Denmark)

    Simonsen, Lars Ole; Harbak, Henrik; Bennekou, Poul

    2011-01-01

    The passive transport of calcium and cobalt and their interference were studied in human red cells using (45)Ca and (57)Co as tracers. In ATP-depleted cells, with the ATP concentration reduced to about 1µM, the progress curve for (45)Ca uptake at 1mM rapidly levels off with time, consistent...... pathway in red cells, since it is not itself transported by the Ca-pump and, moreover, is effectively buffered in the cytosol by binding to hemoglobin, without interfering with Ca(2+) buffering. The molecular identity of the putative common transporter(s) remains to be defined....... with a residual Ca-pump activity building up at increasing [Ca(T)](c) to reach at [Ca(T)](c) about 5µmol(lcells)(-1) a maximal pump rate that nearly countermands the passive Ca influx, resulting in a linear net uptake at a low level. In ATP-depleted cells treated with vanadate, supposed to cause Ca-pump arrest...

  16. Modulation of cholesterol transport by maternal hypercholesterolemia in human full-term placenta.

    Directory of Open Access Journals (Sweden)

    Ran Zhang

    Full Text Available The significance of maternal cholesterol transporting to the fetus under normal as well as pathological circumstances is less understood. The objective of this study was to observe the effects of maternal hypercholesterolemia on placental cholesterol transportation. Human full-time placenta, maternal and venous cord blood were sampled at delivery from the pregnant women with serum total cholesterol (TC concentrations at third trimester higher than 7.25 mM (n = 19 and the pregnant women with normal TC concentrations (n = 19. Serum lipids and expression of genes related to cholesterol transportation were measured by western blot or real-time PCR. The results indicated that serum TC, high density lipoprotein cholesterol (HDL-C, and low density lipoprotein cholesterol (LDL-C levels were significantly increased, in pregnancies, but decreased in cord blood in hypercholesterolemic group compared to the matched control group. All the subjects were no-drinking, non-smoker, and gestational disease free. The mRNA expression of lipoprotein receptors, including LDLR and VLDLR were significantly increased, while the protein expression of PCSK9 was significantly increased in hypercholesterolemic placenta. In conclusion, maternal hypercholesterolemia might decrease the transportation of cholesterol from mother to fetus because of the high levels of PCSK9 protein expression.

  17. Lymphadenopathy of IgG4-related sclerosing disease.

    Science.gov (United States)

    Cheuk, Wah; Yuen, Hunter K L; Chu, Stephenie Y Y; Chiu, Edmond K W; Lam, L K; Chan, John K C

    2008-05-01

    IgG4-related sclerosing disease is a recently recognized syndrome characterized by mass-forming lesions in exocrine glands or extranodal tissues due to lymphoplasmacytic infiltrates and sclerosis, a raised serum IgG4 level and increased IgG4+ plasma cells in the involved tissues. We report the morphologic features of the enlarged regional (n=3) and nonregional lymph nodes (n=3) in patients with this syndrome. The patients presented with autoimmune pancreatitis, lymphoplasmacytic sclerosing cholangitis, chronic sclerosing dacryoadenitis, or chronic sclerosing sialadenitis. The histologic features of the lymph nodes could be categorized into 3 patterns: Castleman diseaselike, follicular hyperplasia, and interfollicular expansion by immunoblasts and plasma cells. The percentage of IgG4+/IgG+ plasma cells was markedly elevated (mean 62% vs. 9.9% in 54 control lymph nodes comprising a wide variety of reactive conditions). We also report 6 cases of primary lymphadenopathy characterized by increased IgG4+/IgG+plasma cells (mean 58%). These cases share many clinical and pathologic similarities with IgG4-related sclerosing disease. In fact, 2 of these patients developed lymphoplasmacytic sclerosing cholangitis or lacrimal and submandibular gland involvement during the clinical course. These cases therefore probably represent primary lymph node manifestation of the disease. The importance of recognition of the lymphadenopathic form of IgG4-related sclerosing disease lies in the remarkable response to steroid therapy, and the potential of mistaking the disease for lymphoma either clinically or histologically.

  18. Rubella IgG Antibody among Nigerian Pregnant Women without ...

    African Journals Online (AJOL)

    Rubella IgG Antibody among Nigerian Pregnant Women without Vaccination History. ... A total of 273 consenting rubella vaccine naïve antenatal clinic attendees aged 15-42 years (Median age = 28 years) were randomly selected and their sera analyzed for qualitative and quantitative anti-rubella IgG detection. Overall ...

  19. IgG glycosylation changes and MBL2 polymorphisms

    DEFF Research Database (Denmark)

    Troelsen, Lone N; Jacobsen, Søren; Abrahams, Jodie L

    2012-01-01

    To examine whether IgG glycosylation changes and MBL2 genotypes are associated with systemic inflammation and joint destruction in rheumatoid arthritis (RA).......To examine whether IgG glycosylation changes and MBL2 genotypes are associated with systemic inflammation and joint destruction in rheumatoid arthritis (RA)....

  20. IgG Fc Receptors - Role in phagocytosis & Staphyloccal evasion -

    NARCIS (Netherlands)

    Stemerding, A.M.

    2014-01-01

    IgG mediates protection against bacterial infection by activating further opsonization by complement, and via direct tagging of bacteria for destruction through zspecific interaction with phagocyte IgG receptors. The studies presented in this thesis focus on exploring features of IgG-FcR

  1. IgG4-Related Disease: A Multispecialty Condition

    Directory of Open Access Journals (Sweden)

    Iuri Usêda Santana

    2014-01-01

    Full Text Available IgG4-related disease (IgG4-RD is a recently recognized group of conditions, characterized by tumor-like swelling of involved organs, lymphoplasmacytic infiltrate rich in IgG4-positive plasma cells, variable degrees of fibrosis, and elevated serum IgG4 concentrations. Currently IgG4-RD is recognized as a systemic condition that can affect several organs and tissues. Herein we report the case of a 34-year-old male patient who was admitted to our hospital with diffuse abdominal pain, weight loss, and painful stiffness in his neck. He had a history of tumoral mass of the left maxillary region, right palpebral ptosis with protrusion of the eyeball, and chronic dry cough for about 6 years. Laboratory tests revealed polyclonal hypergammaglobulinemia and increased serum IgG4 levels. Immunohistochemical staining of the maxillary biopsy was compatible with IgG4-RD. He had an excellent response to corticosteroid therapy. This case highlights that IgG4-RD should be included in the differential diagnosis with multisystem diseases.

  2. Raman spectroscopy based screening of IgG positive and negative sera for dengue virus infection

    Science.gov (United States)

    Bilal, M.; Saleem, M.; Bial, Maria; Khan, Saranjam; Ullah, Rahat; Ali, Hina; Ahmed, M.; Ikram, Masroor

    2017-11-01

    A quantitative analysis for the screening of immunoglobulin-G (IgG) positive human sera samples is presented for the dengue virus infection. The regression model was developed using 79 samples while 20 samples were used to test the performance of the model. The R-square (r 2) value of 0.91 was found through a leave-one-sample-out cross validation method, which shows the validity of this model. This model incorporates the molecular changes associated with IgG. Molecular analysis based on regression coefficients revealed that myristic acid, coenzyme-A, alanine, arabinose, arginine, vitamin C, carotene, fumarate, galactosamine, glutamate, lactic acid, stearic acid, tryptophan and vaccenic acid are positively correlated with IgG; while amide III, collagen, proteins, fatty acids, phospholipids and fucose are negatively correlated. For blindly tested samples, an excellent agreement has been found between the model predicted, and the clinical values of IgG. The parameters, which include sensitivity, specificity, accuracy and the area under the receiver operator characteristic curve, are found to be 100%, 83.3%, 95% and 0.99, respectively, which confirms the high quality of the model.

  3. Anti-amyloidogenic activity of IgGs contained in normal plasma.

    Science.gov (United States)

    O'Nuallain, Brian; Williams, Angela D; McWilliams-Koeppen, Helen P; Acero, Luis; Weber, Alfred; Ehrlich, Hartmut; Schwarz, Hans P; Solomon, Alan

    2010-05-01

    We have previously shown that a subpopulation of naturally occurring human IgGs has therapeutic potential for the amyloid-associated disorders. These molecules cross-react with conformational epitopes on amyloidogenic assemblies, including amyloid beta (Abeta) protein fibrils that are a pathological hallmark of Alzheimer's disease. Using our europium-linked immunosorbant assay, we established that approximately 95% of 260 screened donor plasma samples had amyloid fibril-reactive IgGs and Abeta conformer-reactive IgGs with minimal binding to Abeta monomers. Anti-amyloidogenic reactivity was diverse and attributed to Abeta targeting multiple fibril-related binding sites and/or variations in multidentate binding. There was no correlation between anti-fibril and anti-oligomer reactivity and donor age (19 to 60 years old) or gender. These findings demonstrate the inherent but diverse anti-amyloidogenic activity of natural IgGs contained in normal plasma. Our studies provide support for investigating the clinical significance and physiological function of this novel class of antibodies.

  4. Self-reactive VH4-34-expressing IgG B cells recognize commensal bacteria.

    Science.gov (United States)

    Schickel, Jean-Nicolas; Glauzy, Salomé; Ng, Yen-Shing; Chamberlain, Nicolas; Massad, Christopher; Isnardi, Isabelle; Katz, Nathan; Uzel, Gulbu; Holland, Steven M; Picard, Capucine; Puel, Anne; Casanova, Jean-Laurent; Meffre, Eric

    2017-07-03

    The germline immunoglobulin (Ig) variable heavy chain 4-34 (VH4-34) gene segment encodes in humans intrinsically self-reactive antibodies that recognize I/i carbohydrates expressed by erythrocytes with a specific motif in their framework region 1 (FWR1). VH4-34-expressing clones are common in the naive B cell repertoire but are rarely found in IgG memory B cells from healthy individuals. In contrast, CD27(+)IgG(+) B cells from patients genetically deficient for IRAK4 or MYD88, which mediate the function of Toll-like receptors (TLRs) except TLR3, contained VH4-34-expressing clones and showed decreased somatic hypermutation frequencies. In addition, VH4-34-encoded IgGs from IRAK4- and MYD88-deficient patients often displayed an unmutated FWR1 motif, revealing that these antibodies still recognize I/i antigens, whereas their healthy donor counterparts harbored FWR1 mutations abolishing self-reactivity. However, this paradoxical self-reactivity correlated with these VH4-34-encoded IgG clones binding commensal bacteria antigens. Hence, B cells expressing germline-encoded self-reactive VH4-34 antibodies may represent an innate-like B cell population specialized in the containment of commensal bacteria when gut barriers are breached. © 2017 Schickel et al.

  5. Transport of amino acids and GABA analogues via the human proton-coupled amino acid transporter, hPAT1

    DEFF Research Database (Denmark)

    Larsen, Mie; Larsen, Birger Brodin; Frølund, Bente

    2008-01-01

    The objective of this study was to investigate transepithelial amino acid transport as a function of Caco-2 cell culture time. Furthermore, the objective was to investigate apical uptake characteristics of hPAT1-mediated transport under various experimental conditions. Apical amino acid uptake......, which has been shown to function as a carboxylic acid bioisostere for substrates of the GABA receptor and transport systems....

  6. Riedel's thyroiditis association with IgG4-related disease.

    Science.gov (United States)

    Stan, Marius N; Sonawane, Vikram; Sebo, Thomas J; Thapa, Prabin; Bahn, Rebecca S

    2017-03-01

    IgG4-positive (+) plasma cells have been reported in both Riedel's thyroiditis (RT) and Hashimoto's thyroiditis (HT). These cells are the hallmark of IgG4-related disease (IgG4-RD). We sought to determine whether RT is part of IgG4-RD spectrum. This was a case-control study performed at a tertiary medical centre. We included RT cases from the period 1958 to 2008 that had sufficient paraffin-embedded tissue for IgG4 immunostaining. Controls were patients with HT, age and gender matched, with similar pathology criteria. The main outcome measures were the intensity of the IgG4 staining and the clinical and histological correlates with IgG4-RD. Six pairs of RT and HT were analysed. The mean age was 44·7 years. In both groups, 5/6 cases had positive IgG4 staining. The mean number of IgG4 + cells/ HPF, normalized to the degree of inflammation, was 3·2 ± 3·0 SD (RT) vs 0·9 ± 0·7 (HT), P = 0·15, for fibrotic areas and 2·1 ± 2·3 SD vs 1·0 ± 0·8 (P = 0·39) for areas with lymphoid aggregates. We found the number of IgG4 +  cells in RT to be inversely correlated with the duration of disease (P = 0·046). Three RT cases had associated comorbidities from the IgG4-RD spectrum while none of the HT cases had such conditions. Riedel's thyroiditis is a component of IgG4-RD with the density of the IgG4 +  lymphocytic infiltrate being time dependent. In this small study, we did not identify differences in IgG4 infiltration between RT and HT, minimizing the utility of this marker in RT diagnosis. © 2016 John Wiley & Sons Ltd.

  7. IgG4-Associated Cholangitis--A Mimic of PSC

    NARCIS (Netherlands)

    Beuers, Ulrich; Hubers, Lowiek M.; Doorenspleet, Marieke; Maillette de Buy Wenniger, Lucas; Klarenbeek, Paul L.; Boonstra, Kirsten; Ponsioen, Cyriel; Rauws, Erik; de Vries, Niek

    2015-01-01

    IgG4-associated cholangitis (IAC) is an inflammatory disorder of the biliary tract representing a major manifestation of IgG4-related disease (IgG4-RD) often with elevation of serum IgG4 levels, infiltration of IgG4+ plasma cells in the affected tissue and good response to immunosuppressive

  8. IgG trafficking in the adult pig small intestine

    DEFF Research Database (Denmark)

    Möller, Rebecca; Hansen, Gert H; Danielsen, E Michael

    2017-01-01

    immunoglobulins, including IgG made by plasma cells in the lamina propria, are secreted via the brush border to the lumen as part of the mucosal defense. Here, IgG has been proposed to perform a luminal immune surveillance which eventually includes a reuptake through the brush border as pathogen-containing immune......Immunoglobulin G (IgG) transfer in opposite directions across the small intestinal brush border serves different purposes in early life and in adulthood. In the neonate, maternal IgG is taken up from the gut lumen into the blood, conferring passive immunity to the offspring, whereas in the adult...... complexes. In the present work, we studied luminal uptake of FITC-conjugated and gold-conjugated IgG in cultured pig jejunal mucosal explants. After 1 h, binding to the brush border was seen in upper crypts and lower parts of the villi. However, no endocytotic uptake into EEA-1-positive compartments...

  9. Genetically encoded photocrosslinkers locate the high-affinity binding site of antidepressant drugs in the human serotonin transporter

    DEFF Research Database (Denmark)

    Rannversson, Hafsteinn; Andersen, Jacob; Hall, Lena Sørensen

    2016-01-01

    Despite the well-established role of the human serotonin transporter (hSERT) in the treatment of depression, the molecular details of antidepressant drug binding are still not fully understood. Here we utilize amber codon suppression in a membrane-bound transporter protein to encode...

  10. The breast cancer resistance protein transporter ABCG2 is expressed in the human kidney proximal tubule apical membrane.

    NARCIS (Netherlands)

    Huls, M.; Brown, C.D.; Windass, A.S.; Sayer, R.; Heuvel, J.J.M.W. van den; Heemskerk, S.; Russel, F.G.M.; Masereeuw, R.

    2008-01-01

    The Breast Cancer Resistance Protein (BCRP/ABCG2) is a transporter restricting absorption and enhancing excretion of many compounds including anticancer drugs. This transporter is highly expressed in many tissues; however, in human kidney, only the mRNA was found in contrast to the mouse kidney,

  11. Enhanced human service transportation models : joint demonstration. Phase 1, System planning and design process evaluation : baseline analysis

    Science.gov (United States)

    2007-11-13

    This document presents the findings from the baseline phase of the evaluation of the process being used by eight sites to develop a design for a Travel Management Coordination Center (TMCC) for improved coordination of human service transportation wi...

  12. Human engineering analysis for the high speed civil transport flight deck

    Science.gov (United States)

    Regal, David M.; Alter, Keith W.

    1993-01-01

    The Boeing Company is investigating the feasibility of building a second generation supersonic transport. If current studies support its viability, this airplane, known as the High Speed Civil Transport (HSCT), could be launched early in the next century. The HSCT will cruise at Mach 2.4, be over 300 feet long, have an initial range of between 5000 and 6000 NM, and carry approximately 300 passengers. We are presently involved in developing an advanced flight deck for the HSCT. As part of this effort we are undertaking a human engineering analysis that involves a top-down, mission driven approach that will allow a systematic determination of flight deck functional and information requirements. The present paper describes this work.

  13. Genetic Polymorphisms in Organic Cation Transporter 1 Attenuates Hepatic Metformin Exposure in Humans

    DEFF Research Database (Denmark)

    Sundelin, Elias; Gormsen, Lars Christian; Jensen, Jonas Brorson

    2017-01-01

    Metformin has been used successfully to treat type 2 diabetes for decades. However, the efficacy of the drug varies considerably from patient to patient and this may in part be due to its pharmacokinetic properties. The aim of this study was to examine if common polymorphisms in SLC22A1, encoding...... the transporter protein OCT1, affect the hepatic distribution of metformin in humans. We performed noninvasive 11C-metformin positron emission tomography (PET)/computed tomography (CT) to determine hepatic exposure in 12 subjects genotyped for variants in SLC22A1. Hepatic distribution of metformin...... was significantly reduced after oral intake in carriers of M420del and R61C variants in SLC22A1 without being associated with changes in circulating levels of metformin. Our data show that genetic polymorphisms in transporter proteins cause variation in hepatic exposure to metformin, and it demonstrates...

  14. Rhythmic potassium transport regulates the circadian clock in human red blood cells.

    Science.gov (United States)

    Henslee, Erin A; Crosby, Priya; Kitcatt, Stephen J; Parry, Jack S W; Bernardini, Andrea; Abdallat, Rula G; Braun, Gabriella; Fatoyinbo, Henry O; Harrison, Esther J; Edgar, Rachel S; Hoettges, Kai F; Reddy, Akhilesh B; Jabr, Rita I; von Schantz, Malcolm; O'Neill, John S; Labeed, Fatima H

    2017-12-07

    Circadian rhythms organize many aspects of cell biology and physiology to a daily temporal program that depends on clock gene expression cycles in most mammalian cell types. However, circadian rhythms are also observed in isolated mammalian red blood cells (RBCs), which lack nuclei, suggesting the existence of post-translational cellular clock mechanisms in these cells. Here we show using electrophysiological and pharmacological approaches that human RBCs display circadian regulation of membrane conductance and cytoplasmic conductivity that depends on the cycling of cytoplasmic K+ levels. Using pharmacological intervention and ion replacement, we show that inhibition of K+ transport abolishes RBC electrophysiological rhythms. Our results suggest that in the absence of conventional transcription cycles, RBCs maintain a circadian rhythm in membrane electrophysiology through dynamic regulation of K+ transport.

  15. Unbiased simulations reveal the inward-facing conformation of the human serotonin transporter and Na+ ion release

    DEFF Research Database (Denmark)

    Koldsø, Heidi; Noer, Pernille Rimmer; Grouleff, Julie

    2011-01-01

    Monoamine transporters are responsible for termination of synaptic signaling and are involved in depression, control of appetite, and anxiety amongst other neurological processes. Despite extensive efforts, the structures of the monoamine transporters and the transport mechanism of ions and subst......Monoamine transporters are responsible for termination of synaptic signaling and are involved in depression, control of appetite, and anxiety amongst other neurological processes. Despite extensive efforts, the structures of the monoamine transporters and the transport mechanism of ions...... and substrates are still largely unknown. Structural knowledge of the human serotonin transporter (hSERT) is much awaited for understanding the mechanistic details of substrate translocation and binding of antidepressants and drugs of abuse. The publication of the crystal structure of the homologous leucine...

  16. Abnormal mitochondrial transport and morphology as early pathological changes in human models of spinal muscular atrophy

    Directory of Open Access Journals (Sweden)

    Chong-Chong Xu

    2016-01-01

    Full Text Available Spinal muscular atrophy (SMA, characterized by specific degeneration of spinal motor neurons, is caused by mutations in the survival of motor neuron 1, telomeric (SMN1 gene and subsequent decreased levels of functional SMN. How the deficiency of SMN, a ubiquitously expressed protein, leads to spinal motor neuron-specific degeneration in individuals affected by SMA remains unknown. In this study, we examined the role of SMN in mitochondrial axonal transport and morphology in human motor neurons by generating SMA type 1 patient-specific induced pluripotent stem cells (iPSCs and differentiating these cells into spinal motor neurons. The initial specification of spinal motor neurons was not affected, but these SMA spinal motor neurons specifically degenerated following long-term culture. Moreover, at an early stage in SMA spinal motor neurons, but not in SMA forebrain neurons, the number of mitochondria, mitochondrial area and mitochondrial transport were significantly reduced in axons. Knocking down of SMN expression led to similar mitochondrial defects in spinal motor neurons derived from human embryonic stem cells, confirming that SMN deficiency results in impaired mitochondrial dynamics. Finally, the application of N-acetylcysteine (NAC mitigated the impairment in mitochondrial transport and morphology and rescued motor neuron degeneration in SMA long-term cultures. Furthermore, NAC ameliorated the reduction in mitochondrial membrane potential in SMA spinal motor neurons, suggesting that NAC might rescue apoptosis and motor neuron degeneration by improving mitochondrial health. Overall, our data demonstrate that SMN deficiency results in abnormal mitochondrial transport and morphology and a subsequent reduction in mitochondrial health, which are implicated in the specific degeneration of spinal motor neurons in SMA.

  17. Binding of the multimodal antidepressant drug vortioxetine to the human serotonin transporter.

    Science.gov (United States)

    Andersen, Jacob; Ladefoged, Lucy Kate; Wang, Danyang; Kristensen, Trine N Bjerre; Bang-Andersen, Benny; Kristensen, Anders S; Schiøtt, Birgit; Strømgaard, Kristian

    2015-11-18

    Selective inhibitors of the human serotonin transporter (hSERT) have been first-line treatment against depression for several decades. Recently, vortioxetine was approved as a new therapeutic option for the treatment of depression. Vortioxetine represents a new class of antidepressant drugs with a multimodal pharmacological profile that in addition to potent inhibition of hSERT include agonistic or antagonistic effects at different serotonin receptors. We used a combination of computational, chemical, and biological methods to decipher the molecular basis for high affinity binding of vortioxetine in hSERT. X-ray crystal structures of the bacterial amino acid transporter LeuT and the Drosophila melanogaster dopamine transporter were used to build homology models of hSERT. Comparative modeling and ligand docking suggest that vortioxetine can adopt several distinct binding modes within the central binding site of hSERT. To distinguish between the identified binding modes, we determined the effect of 57 functional hSERT point mutants on vortioxetine potency and characterized seven structurally related analogs of vortioxetine in a subset of the point mutants. This allowed us to determine the orientation of vortioxetine within the central binding site and showed that only one of the proposed binding modes is functionally relevant. The findings provide important new insight about the molecular basis for high affinity recognition of vortioxetine in hSERT, which is essential for future structure-based drug discovery of novel multimodal drugs with fine-tuned selectivity across different transporter and receptor proteins in the human brain.

  18. SLC2A9 is a high-capacity urate transporter in humans.

    Directory of Open Access Journals (Sweden)

    Mark J Caulfield

    2008-10-01

    Full Text Available Serum uric acid levels in humans are influenced by diet, cellular breakdown, and renal elimination, and correlate with blood pressure, metabolic syndrome, diabetes, gout, and cardiovascular disease. Recent genome-wide association scans have found common genetic variants of SLC2A9 to be associated with increased serum urate level and gout. The SLC2A9 gene encodes a facilitative glucose transporter, and it has two splice variants that are highly expressed in the proximal nephron, a key site for urate handling in the kidney. We investigated whether SLC2A9 is a functional urate transporter that contributes to the longstanding association between urate and blood pressure in man.We expressed both SLC2A9 splice variants in Xenopus laevis oocytes and found both isoforms mediate rapid urate fluxes at concentration ranges similar to physiological serum levels (200-500 microM. Because SLC2A9 is a known facilitative glucose transporter, we also tested whether glucose or fructose influenced urate transport. We found that urate is transported by SLC2A9 at rates 45- to 60-fold faster than glucose, and demonstrated that SLC2A9-mediated urate transport is facilitated by glucose and, to a lesser extent, fructose. In addition, transport is inhibited by the uricosuric benzbromarone in a dose-dependent manner (Ki = 27 microM. Furthermore, we found urate uptake was at least 2-fold greater in human embryonic kidney (HEK cells overexpressing SLC2A9 splice variants than nontransfected kidney cells. To confirm that our findings were due to SLC2A9, and not another urate transporter, we showed that urate transport was diminished by SLC2A9-targeted siRNA in a second mammalian cell line. In a cohort of men we showed that genetic variants of SLC2A9 are associated with reduced urinary urate clearance, which fits with common variation at SLC2A9 leading to increased serum urate. We found no evidence of association with hypertension (odds ratio 0.98, 95% confidence interval [CI

  19. Expression and functional activity of nucleoside transporters in human choroid plexus

    Directory of Open Access Journals (Sweden)

    Grujicic Danica

    2010-01-01

    Full Text Available Abstract Background Human equilibrative nucleoside transporters (hENTs 1-3 and human concentrative nucleoside transporters (hCNTs 1-3 in the human choroid plexus (hCP play a role in the homeostasis of adenosine and other naturally occurring nucleosides in the brain; in addition, hENT1, hENT2 and hCNT3 mediate membrane transport of nucleoside reverse transcriptase inhibitors that could be used to treat HIV infection, 3'-azido-3'-deoxythymidine, 2'3'-dideoxycytidine and 2'3'-dideoxyinosine. This study aimed to explore the expression levels and functional activities of hENTs 1-3 and hCNTs 1-3 in human choroid plexus. Methods Freshly-isolated pieces of lateral ventricle hCP, removed for various clinical reasons during neurosurgery, were obtained under Local Ethics Committee approval. Quantification of mRNAs that encoded hENTs and hCNTs was performed by the hydrolysis probes-based reverse transcription real time-polymerase chain reaction (RT-qPCR; for each gene of interest and for 18 S ribosomal RNA, which was an endogenous control, the efficiency of PCR reaction (E and the quantification cycle (Cq were calculated. The uptake of [3H]inosine by the choroid plexus pieces was investigated to explore the functional activity of hENTs and hCNTs in the hCP. Results RT-qPCR revealed that the mRNA encoding the intracellularly located transporter hENT3 was the most abundant, with E-Cq value being only about 40 fold less that the E-Cq value for 18 S ribosomal RNA; mRNAs encoding hENT1, hENT2 and hCNT3 were much less abundant than mRNA for the hENT3, while mRNAs encoding hCNT1 and hCNT2 were of very low abundance and not detectable. Uptake of [3H]inosine by the CP samples was linear and consisted of an Na+-dependent component, which was probably mediated by hCNT3, and Na+-independent component, mediated by hENTs. The latter component was not sensitive to inhibition by S-(4-nitrobenzyl-6-thioinosine (NBMPR, when used at a concentration of 0.5 μM, a finding that

  20. L-arginine transport in the human coronary and peripheral circulation.

    Science.gov (United States)

    Miner, S E S; Al-Hesayen, A; Kelly, S; Benson, T; Thiessen, J J; Young, V R; Parker, J D

    2004-03-16

    Nitric oxide synthase (NOS) uses arginine for the production of nitric oxide (NO). High intracellular concentrations of arginine suggest that NOS activity should be independent of plasma arginine supply. However, under certain conditions, increased plasma arginine concentrations appear to be associated with increased NOS activity. The purpose of this study was to explore arginine transport within the human coronary and peripheral circulation Mass-labeled 15N2-arginine was infused to steady state before cardiac catheterization in 31 patients. After diagnostic angiography, a catheter was placed in the coronary sinus. The transcardiac concentration gradient (aorta-coronary sinus) of 15N2-arginine was used as a measure of arginine transport at baseline and during infusions of acetylcholine and N(G)-monomethyl-L-arginine (L-NMMA). No gradient was detected at rest. During the infusion of acetylcholine, a significant gradient was detected (2.5+/-1.2 micromol/L, P=0.01) corresponding to a fractional extraction of 11.7+/-7.5%. This is consistent with in vitro studies that suggest that stimulation of NOS induces arginine transport. During the infusion of L-NMMA, the concentration of 15N2-arginine increased in the coronary sinus, producing a gradient of -3.9+/-1.3 micromol/L (P=0.0002), corresponding to a fractional production of 20.5+/-5.0%. This is consistent with in vitro studies that suggest that L-NMMA induces the efflux of arginine from the cell to the extracellular space via transporter-mediated transstimulation. The use of steady-state 15N2-arginine to examine transorgan L-arginine gradients represents a novel tool for the study of L-arginine transport and the mechanisms of endothelial and NOS dysfunction.

  1. A species difference in the transport activities of H2 receptor antagonists by rat and human renal organic anion and cation transporters.

    Science.gov (United States)

    Tahara, Harunobu; Kusuhara, Hiroyuki; Endou, Hitoshi; Koepsell, Hermann; Imaoka, Tomoki; Fuse, Eiichi; Sugiyama, Yuichi

    2005-10-01

    A clinical drug-drug interaction between famotidine (a H2 receptor antagonist) and probenecid has not been reproduced in rats. The present study hypothesized that the species-dependent probenecid sensitivity is due to a species difference in the contribution of renal organic anion and cation transporters. The transport activities of the H2 receptor antagonists (cimetidine, famotidine, and ranitidine) by rat and human basolateral organic anion and cation transporters [human organic anion transporter (hOAT) 1, hOAT2, r/hOAT3, rat organic cation transporter (rOct) 1, and r/hOCT2] were compared using their cDNA transfectants. The transport activities (Vmax/Km) of famotidine (Km, 345 microM) by rOat3 were 8- and 15-fold lower than those of cimetidine (Km, 91 microM) and ranitidine (Km, 155 microM), respectively, whereas the activity by hOAT3 (Km, 124 microM) was 3-fold lower than that of cimetidine (Km, 149 microM) but similar to that of ranitidine (Km, 234 microM). Comparison of the relative transport activity with regard to that of cimetidine suggests that famotidine was more efficiently transported by hOAT3 than rOat3, and vice versa, for ranitidine. Only ranitidine was efficiently transported by hOAT2 (Km, 396 microM). rOct1 accepts all of the H2 receptor antagonists with a similar activity, whereas the transport activities of ranitidine and famotidine (Km, 61/56 microM) by r/hOCT2 were markedly lower than that of cimetidine (Km, 69/73 microM). Probenecid was a potent inhibitor of r/OAT3 (Ki, 2.6-5.8 microM), whereas it did not interact with OCTs. These results suggest that, in addition to the absence of OCT1 in human kidney, a species difference in the transport activity by hOAT3 and rOat3 accounts, at least in part, for the species difference in the drug-drug interaction between famotidine and probenecid.

  2. Involvement of monocarboxylate transporter 1 (SLC16A1) in the uptake of l-lactate in human astrocytes.

    Science.gov (United States)

    Ideno, Masaya; Kobayashi, Masaki; Sasaki, Shotaro; Futagi, Yuya; Narumi, Katsuya; Furugen, Ayako; Iseki, Ken

    2018-01-01

    Astrocytes, the most abundant glial cells in the central nervous system (CNS), help neurons survive. Monocarboxylate transporters (MCTs) are reported to transport l-lactate, which is important for CNS physiology and cognitive function. However, it remains unclear which MCT isoform is functionally expressed by human astrocytes. The aim of this study was to establish the contribution of each MCT isoform to l-lactate transport in human astrocytes. The function of l-lactate transport was studied using NHA cells as a human astrocyte model and radiolabeled l-lactate. The expression of MCT in human astrocytes was detected by immunohistochemistry staining. The cellular uptake of l-lactate was found to be pH- and concentration-dependent with a Km value for l-lactate uptake of 0.64mM. This Km was similar to what has been previously established for MCT1-mediated l-lactate uptake. α-Cyano-4- hydroxycinnamate (CHC) and 5-oxoproline, which are both MCT1 inhibitors, were found to significantly inhibit the uptake of l-lactate, suggesting MCT1 is primarily responsible for l-lactate transport. Moreover, MCT1 protein was expressed in human astrocytes. pH-dependent l-lactate transport is mediated by MCT1 in human astrocytes. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Intracellular loop 5 is important for the transport mechanism and molecular pharmacology of the human serotonin transporter

    DEFF Research Database (Denmark)

    Said, Saida; Neubauer, Henrik Amtoft; Müller, Heidi Kaastrup

    2015-01-01

    The serotonin transporter (SERT) belongs to a family of transport proteins called the neurotransmitter:sodium symporters. The specialized members of this family transport different neurotransmitters across the cell membrane, thereby regulating signaling between neurons. Most of these transporters...... are important drug targets in treating i.e. affective disorders such as depression and anxiety, and for drugs of abuse such as ecstasy and cocaine. The normal function of the SERT relies on large conformational changes and its inhibition by antidepressants represents a conformational lock. Understanding...... the molecular mechanism of inhibition and which structural elements are involved in inhibitor binding and conformational changes of the transporter will provide clues for the development of improved drugs for the treatment of depression. Guided by our previous studies, we combined different biochemical methods...

  4. Binding of the Multimodal Antidepressant Drug Vortioxetine to the Human Serotonin Transporter

    DEFF Research Database (Denmark)

    Andersen, Jacob; Ladefoged, Lucy Kate; Wang, Danyang

    2015-01-01

    Selective inhibitors of the human serotonin transporter (hSERT) have been first-line treatment against depression for several decades. Recently, vortioxetine was approved as a new therapeutic option for the treatment of depression. Vortioxetine represents a new class of antidepressant drugs...... with a multimodal pharmacological profile that in addition to potent inhibition of hSERT include agonistic or antagonistic effects at different serotonin receptors. We used a combination of computational, chemical, and biological methods to decipher the molecular basis for high affinity binding of vortioxetine in h...

  5. BDNF val66met association with serotonin transporter binding in healthy humans

    DEFF Research Database (Denmark)

    Fisher, P. M.; Ozenne, B.; Svarer, C.

    2017-01-01

    The serotonin transporter (5-HTT) is a key feature of the serotonin system, which is involved in behavior, cognition and personality and implicated in neuropsychiatric illnesses including depression. The brain-derived neurotrophic factor (BDNF) val66met and 5-HTTLPR polymorphisms have predicted......-carriers have increased subcortical 5-HTT binding. The small difference suggests limited statistical power may explain previously reported null effects. Our finding adds to emerging evidence that BDNF val66met contributes to differences in the human brain serotonin system, informing how variability in the 5-HTT...

  6. Movement of NH3 through the human urea transporter B: a new gas channel

    OpenAIRE

    Geyer, R. Ryan; Musa-Aziz, Raif; Enkavi, Giray; Mahinthichaichan, P.; Tajkhorshid, Emad; Boron, Walter F.

    2013-01-01

    Aquaporins and Rh proteins can function as gas (CO2 and NH3) channels. The present study explores the urea, H2O, CO2, and NH3 permeability of the human urea transporter B (UT-B) (SLC14A1), expressed in Xenopus oocytes. We monitored urea uptake using [14C]urea and measured osmotic water permeability (Pf) using video microscopy. To obtain a semiquantitative measure of gas permeability, we used microelectrodes to record the maximum transient change in surface pH (ΔpHS) caused by exposing oocytes...

  7. Pituitary adenylate cyclase-activating polypeptide: a potent activator of human intestinal ion transport.

    Science.gov (United States)

    Fuchs, M; Adermann, K; Raab, H R; Forssmann, W G; Kuhn, M

    1996-12-26

    To investigate the effects of PACAP-27 on electrolyte transport across the isolated human intestinal mucosa, changes in short-circuit current (Isc) were measured in Ussing chamber experiments. Serosally added PACAP-27 increased Isc in a concentration-dependent manner, eliciting a similar maximal effect in both the jejunal and the colonic mucosa. Bumetanide inhibited Isc responses, indicating stimulation of Cl- secretion. The potency and efficacy of PACAP-27 were comparable to those of VIP, suggesting that both peptides activate intestinal secretion by way of a common receptor located in the basolateral membrane of the intestinal epithelium.

  8. Human Enteroids as a Model of Upper Small Intestinal Ion Transport Physiology and Pathophysiology.

    Science.gov (United States)

    Foulke-Abel, Jennifer; In, Julie; Yin, Jianyi; Zachos, Nicholas C; Kovbasnjuk, Olga; Estes, Mary K; de Jonge, Hugo; Donowitz, Mark

    2016-03-01

    Human intestinal crypt-derived enteroids are a model of intestinal ion transport that require validation by comparison with cell culture and animal models. We used human small intestinal enteroids to study neutral Na(+) absorption and stimulated fluid and anion secretion under basal and regulated conditions in undifferentiated and differentiated cultures to show their functional relevance to ion transport physiology and pathophysiology. Human intestinal tissue specimens were obtained from an endoscopic biopsy or surgical resections performed at Johns Hopkins Hospital. Crypts were isolated, enteroids were propagated in culture, induced to undergo differentiation, and transduced with lentiviral vectors. Crypt markers, surface cell enzymes, and membrane ion transporters were characterized using quantitative reverse-transcription polymerase chain reaction, immunoblot, or immunofluorescence analyses. We used multiphoton and time-lapse confocal microscopy to monitor intracellular pH and luminal dilatation in enteroids under basal and regulated conditions. Enteroids differentiated upon withdrawal of WNT3A, yielding decreased crypt markers and increased villus-like characteristics. Na(+)/H(+) exchanger 3 activity was similar in undifferentiated and differentiated enteroids, and was affected by known inhibitors, second messengers, and bacterial enterotoxins. Forskolin-induced swelling was completely dependent on cystic fibrosis transmembrane conductance regulator and partially dependent on Na(+)/H(+) exchanger 3 and Na(+)/K(+)/2Cl(-) cotransporter 1 inhibition in undifferentiated and differentiated enteroids. Increases in cyclic adenosine monophosphate with forskolin caused enteroid intracellular acidification in HCO3(-)-free buffer. Cyclic adenosine monophosphate-induced enteroid intracellular pH acidification as part of duodenal HCO3(-) secretion appears to require cystic fibrosis transmembrane conductance regulator and electrogenic Na(+)/HCO3(-) cotransporter 1

  9. Evaluation of the Serotonin Transporter Ligand 123I-ADAM for SPECT Studies on Humans

    DEFF Research Database (Denmark)

    Frokjaer, V.G.; Pinborg, Lars Hageman; Madsen, J.

    2008-01-01

    Imaging serotonin transporters in the living human brain is important in several fields, such as normal psychophysiology, mood disorders, eating disorders, and neurodegenerative disorders. The aim of this study was to compare different kinetic and semiquantitative methods for assessing serotonin......; range, 24-43 y), and metabolite-corrected arterial input was measured. Tissue reference models (simplified reference tissue model, Logan reference tissue model, and ratio method) were validated against the outcome of a 1-tissue-compartment model, and performance with decreasing scan length was evaluated...

  10. Characterization of intracellular regions in the human serotonin transporter for phosphorylation sites

    DEFF Research Database (Denmark)

    Sørensen, Lena; Strømgaard, Kristian; Kristensen, Anders S

    2014-01-01

    /dephosphorylation during transporter regulation by multiple pathways. In particular, activation and/or inhibition of kinases including PKC, PKG, p38MAPK, and CaMKII modulate SERT function and trafficking. The molecular mechanisms by which kinase activity is linked to SERT regulation are poorly understood, including...... the identity of specific phosphorylated residues. To elucidate SERT phosphorylation sites, we have generated peptides corresponding to the entire intracellular region of human SERT and performed in vitro phosphorylation assays with a panel of kinases suggested to be involved in SERT regulation or for which...

  11. Pathomorphological characteristic of IgG4-related diseases

    Directory of Open Access Journals (Sweden)

    O. O. Dyadyk

    2016-08-01

    Full Text Available IgG4-related diseases are a relatively new group of diseases of unknown etiology which are characterized by the development of fibrosis of organs with the presence of big amounts of IgG4-positive plasma-cells in the area of the lesions and increased levels of IgG4 in serum. The organs that may be affected are pancreas, salivary gland, and others, clinical cases of kidney damage are described as well. Renal involvement in IgG4-related diseases most often occurs on the type of tubulointerstitial nephritis, with the further development of acute or chronic kidney injury. The clinic may be represented by the pseudotumor of kidney, renal tissue heterogeneity on the results of CT-studies; acute or chronic renal disease; combination with other organ damage (autoimmune pancreatitis, sclerosing cholangitis, sclerosing lymphoplasmacytic cholecystitis, colitis, sialadenitis, retroperitoneal fibrosis, etc.. Laboratory findings include an increased level of IgG4 in the blood serum, hypocomplementemia, eosinophilia. Histologically, there is interstitial inflammation with many plasma cells, interstitial fibrosis, tubular atrophy, thickening of the tubular basement membrane, some cases are a type of membranous glomerulonephritis. The aim of the study is to identify the patients with IgG4-related diseases with renal impairment and widening the pathological database of such patients with renal impairment to determine the classification criteria of this pathological condition. Materials and methods will include the deceased kidney screening, screening of patients with autoimmune and allergic diseases, nephrological patients screening with the lifetime biopsy (in some cases – repeat biopsy with chronic or acute kidney impairment. There will be clinical and pathological comparison in kidney damage and other diseases with the development of criteria for the classification of lesions in the presence of IgG4-positive substrates and further development of practical

  12. Does the Maternal Serum IgG Level during Pregnancy in Primary Antibody Deficiency Influence the IgG Level in the Newborn?

    OpenAIRE

    Vasantha Nagendran; Noel Emmanuel; Bansal, Amolak S.

    2015-01-01

    Purpose. To find out if the serum IgG level in the newborn baby was affected by low maternal serum IgG during pregnancy in two newly diagnosed primary antibody deficient patients. Method. Infant cord blood IgG level was compared with maternal IgG level in 2 mothers with newly diagnosed primary antibody deficiency, who declined replacement IgG treatment during pregnancy. Results. Both mothers delivered healthy babies with normal IgG levels at birth. Conclusions. The normal IgG levels and sound...

  13. Insights into molecular properties of the human monocarboxylate transporter 8 by combining functional with structural information

    Directory of Open Access Journals (Sweden)

    Kleinau Gunnar

    2011-08-01

    Full Text Available Abstract Background The monocarboxylate transporter 8 (MCT8 is a member of the major facilitator superfamily (MFS and transports specificly iodothyronines. MCT8 mutations are the underlying cause of a syndrome of severe X-linked psychomotor retardation known as the Allan-Herndon-Dudley syndrome. This syndrome is characterized by abnormally high T3, low/normal T4 serum levels and slightly elevated serum TSH. To date, more than 25 pathogenic mutations in hMCT8 are known and they are valuable indicators of important regions for structural and functional MCT8 properties. Methods We designed a structural human MCT8 model and studied reported pathogenic missense mutations with focus on the estimation of those amino acid positions which are probably sensitive for substrate transport. Furthermore, assuming similarities between determinants of T3 binding observed in the published crystal structure of the thyroid hormone receptor beta occupied by its ligand T3 and the structural MCT8 model, we explore potential T3 binding sites in the MCT8 substrate channel cavity. Results We found that all known pathogenic missense mutations are located exclusively in the transmembrane helices and to a high degree at conserved residues among the MCT family. Furthermore, mutations either of or to prolines/glycines are located mainly at helices 9-12 and are expected to cause steric clashes or structural misfolding. In contrast, several other mutations are close to the potential substrate channel and affected amino acids are likely involved in the switching mechanism between different transporter conformations. Finally, three potential substrate binding sites are predicted for MCT8. Conclusions Naturally occurring mutations of MCT8 provide molecular insights into protein regions important for protein folding, substrate binding and the switching mechanism during substrate transport. Future studies guided by this information should help to clarify structure

  14. The influence of foot posture on the cost of transport in humans.

    Science.gov (United States)

    Cunningham, C B; Schilling, N; Anders, C; Carrier, D R

    2010-03-01

    Although humans appear to be specialized for endurance running, the plantigrade posture of our feet, in which the heel contacts the substrate at the beginning of a step, seems incompatible with economical running. In this study, we tested the hypothesis that plantigrade foot posture reduces the energetic cost of transport (COT) during walking in humans. When human subjects walked with their heels slightly elevated in a 'low-digitigrade' posture, COT increased by 53% above that of normal plantigrade walking. By contrast, there was no difference in COT when subjects ran with digitigrade versus plantigrade foot posture. Stride frequency increased and stride length decreased when subjects switched to digitigrade walking; however, this change did not influence the COT. Additionally, we found that possible reductions in postural stability appear not to have caused the elevated cost of digitigrade walking. Digitigrade walking, however, did (1) increase the external mechanical work performed by the limbs; (2) reduce the pendular exchange of kinetic and potential energy of the center of mass; (3) increase the average ground reaction force moment at the ankle joint; and (4) increase the recruitment of major extensor muscles of the ankle, knee, hip and back. These observations suggest that plantigrade foot posture improves the economy of walking. Relative to other mammals, humans are economical walkers, but not economical runners. Given the great distances hunter-gatherers travel, it is not surprising that humans retained a foot posture, inherited from our more arboreal great ape ancestors, that facilitates economical walking.

  15. Evaluation of the new architect cytomegalovirus immunoglobulin M (IgM), IgG, and IgG avidity assays.

    Science.gov (United States)

    Lagrou, K; Bodeus, M; Van Ranst, M; Goubau, P

    2009-06-01

    A panel of new cytomegalovirus (CMV) assays for use on the Architect instrument has been developed, including a CMV avidity assay based on a new technology. The purpose of this study was to compare the performance characteristics of the fully automated CMV immunoglobulin M (IgM), IgG, and IgG avidity tests on the Architect instrument with those of other available assays. A total of 503 consecutive fresh patient serum specimens (routine serum specimens) and 96 serum specimens from 33 pregnant women with a recent CMV primary infection (seroconversion serum specimens) were tested for CMV IgM and IgG by the Architect (Abbott), Vidas (BioMérieux), and Enzygnost (Siemens) assays. The seroconversion sera and 100 preselected serum specimens IgM negative and IgG positive by the AxSYM assay were also tested by the IgG avidity tests on the Architect and Vidas instruments. The relative agreements for CMV IgM determination with routine sera between the Architect assay and the Vidas, Enzygnost, and AxSYM assays were 97%, 94%, and 93%, respectively, for the CMV IgM tests and 99%, 98%, and 98%, respectively, for the CMV IgG tests. The specificities of the CMV IgG avidity test were 98% for the Architect assay and 76% for the Vidas assay. No high CMV IgG avidity test results were found within the first 3 months after seroconversion by either of those assays. The correlation between the results of the newly developed CMV IgM and IgG tests on the Architect instrument with the Vidas and Enzygnost assays was excellent (> or = 94%). The CMV IgG avidity test reliably excluded patients with recent infections and showed an excellent specificity (98%).

  16. Evaluation of the New Architect Cytomegalovirus Immunoglobulin M (IgM), IgG, and IgG Avidity Assays▿

    Science.gov (United States)

    Lagrou, K.; Bodeus, M.; Van Ranst, M.; Goubau, P.

    2009-01-01

    A panel of new cytomegalovirus (CMV) assays for use on the Architect instrument has been developed, including a CMV avidity assay based on a new technology. The purpose of this study was to compare the performance characteristics of the fully automated CMV immunoglobulin M (IgM), IgG, and IgG avidity tests on the Architect instrument with those of other available assays. A total of 503 consecutive fresh patient serum specimens (routine serum specimens) and 96 serum specimens from 33 pregnant women with a recent CMV primary infection (seroconversion serum specimens) were tested for CMV IgM and IgG by the Architect (Abbott), Vidas (BioMérieux), and Enzygnost (Siemens) assays. The seroconversion sera and 100 preselected serum specimens IgM negative and IgG positive by the AxSYM assay were also tested by the IgG avidity tests on the Architect and Vidas instruments. The relative agreements for CMV IgM determination with routine sera between the Architect assay and the Vidas, Enzygnost, and AxSYM assays were 97%, 94%, and 93%, respectively, for the CMV IgM tests and 99%, 98%, and 98%, respectively, for the CMV IgG tests. The specificities of the CMV IgG avidity test were 98% for the Architect assay and 76% for the Vidas assay. No high CMV IgG avidity test results were found within the first 3 months after seroconversion by either of those assays. The correlation between the results of the newly developed CMV IgM and IgG tests on the Architect instrument with the Vidas and Enzygnost assays was excellent (≥94%). The CMV IgG avidity test reliably excluded patients with recent infections and showed an excellent specificity (98%). PMID:19339470

  17. Molecular Dynamics Simulation of the Crystallizable Fragment of IgG1—Insights for the Design of Fcabs

    Science.gov (United States)

    Lai, Balder; Hasenhindl, Christoph; Obinger, Christian; Oostenbrink, Chris

    2014-01-01

    An interesting format in the development of therapeutic monoclonal antibodies uses the crystallizable fragment of IgG1 as starting scaffold. Engineering of its structural loops allows generation of an antigen binding site. However, this might impair the molecule’s conformational stability, which can be overcome by introducing stabilizing point mutations in the CH3 domains. These point mutations often affect the stability and unfolding behavior of both the CH2 and CH3 domains. In order to understand this cross-talk, molecular dynamics simulations of the domains of the Fc fragment of human IgG1 are reported. The structure of human IgG1-Fc obtained from X-ray crystallography is used as a starting point for simulations of the wild-type protein at two different pH values. The stabilizing effect of a single point mutation in the CH3 domain as well as the impact of the hinge region and the glycan tree structure connected to the CH2 domains is investigated. Regions of high local flexibility were identified as potential sites for engineering antigen binding sites. Obtained data are discussed with respect to the available X-ray structure of IgG1-Fc, directed evolution approaches that screen for stability and use of the scaffold IgG1-Fc in the design of antigen binding Fc proteins. PMID:24451126

  18. Protein G, Protein A and Protein-A-Derived Peptides Inhibit the Agitation Induced Aggregation of IgG

    Science.gov (United States)

    Zhang, Jun; Topp, Elizabeth M.

    2012-01-01

    Controlling and preventing aggregation is critical to the development of safe and effective antibody drug products. The studies presented here test the hypothesis that Protein A and Protein G inhibit the agitation-induced aggregation of IgG. The hypothesis is motivated by the enhanced conformational stability of proteins upon ligand binding and the specific binding affinity of Protein A and Protein G to the Fc region of IgG. The aggregation of mixed human IgG from pooled human plasma was induced by agitation alone or in the presence of: (i) Protein A, (ii) Protein G or (iii) a library of 24 peptides derived from the IgG-binding domain of Protein A. Aggregation was assessed by UV spectroscopy, SDS-PAGE, high performance size-exclusion chromatography (HP-SEC), dynamic light scattering (DLS) and fluorescence spectroscopy. Additional information on IgG-ligand interactions was obtained using differential scanning fluorimetry (DSF) and competitive binding studies. The results demonstrate that Protein A provides near-complete inhibition of agitation-induced aggregation, while Protein G and two peptides from the peptide library show partial inhibition. The findings indicate that the IgG Protein A binding site is involved in the agitation-induced aggregation of IgG, and suggest a dominant role of colloidal interactions. PMID:22304418

  19. BDNF val66met association with serotonin transporter binding in healthy humans

    DEFF Research Database (Denmark)

    Fisher, P. M.; Ozenne, B.; Svarer, C.

    2017-01-01

    The serotonin transporter (5-HTT) is a key feature of the serotonin system, which is involved in behavior, cognition and personality and implicated in neuropsychiatric illnesses including depression. The brain-derived neurotrophic factor (BDNF) val66met and 5-HTTLPR polymorphisms have predicted...... differences in 5-HTT levels in humans but with equivocal results, possibly due to limited sample sizes. Within the current study we evaluated these genetic predictors of 5-HTT binding with [11C]DASB positron emission tomography (PET) in a comparatively large cohort of 144 healthy individuals. We used a latent......-carriers have increased subcortical 5-HTT binding. The small difference suggests limited statistical power may explain previously reported null effects. Our finding adds to emerging evidence that BDNF val66met contributes to differences in the human brain serotonin system, informing how variability in the 5-HTT...

  20. Treatment approaches to IgG4-related systemic disease.

    Science.gov (United States)

    Khosroshahi, Arezou; Stone, John H

    2011-01-01

    IgG4-related systemic disease (IgG4-RSD) is a systemic fibroinflammatory condition that can affect any organ system. Prompt recognition and management of this disease process are necessary to prevent sclerosis and permanent organ damage. Here, we review the advances in treatment approaches to IgG4-RSD. Most information regarding treatment is derived from retrospective case series of patients with autoimmune pancreatitis (AIP), and follow-up periods have generally been short. A variety of IgG4-RSD presentations respond rapidly to glucocorticoid treatment. Glucocorticoids have become a standard therapy for AIP, but the indications requiring treatment as well as the appropriate starting dose and duration of therapy remain controversial. The importance of maintenance of glucocorticoids following remission induction is debatable. As our knowledge grows regarding other organ manifestations of IgG4-RSD with longer follow-ups, the necessity of steroid-sparing agents to manage frequent relapses becomes clear. The natural history and long-term prognosis of IgG4-RSD are not well understood. Large prospective studies and randomized controlled trials of patients with wide spectrum manifestations of IgG4-RSD are required to support better approaches to treatment.

  1. Unique recognition of a low molecular weight Onchocerca volvulus antigen by IgG3 antibodies in chronic hyper-reactive oncho-dermatitis (Sowda).

    Science.gov (United States)

    Cabrera, Z; Buttner, D W; Parkhouse, R M

    1988-01-01

    Individual human Ig class responses to Onchocerca volvulus antigens have been evaluated by Western blotting using sera from cases of generalized onchocerciasis and chronic hyper-reactive onchocerciasis (Sowda). in all cases except IgG3 the patterns of recognition by human antibody classes were similar in Sowda and generalized onchocerciasis. Weak or undetectable responses were seen with IgG1, IgG2 and IgM. The total profiles of antigens recognized by the other Ig classes were different, although in some cases certain bands were commonly identified. The result with IgG3, however, was striking. Here, two major antigens (9 kD and 72kD) were recognized by IgG3 antibodies in Sowda sera but not generalized onchocerciasis sera. Furthermore, these two antigens were not recognised by any other Ig class, either in generalized or Sowda onchocerciasis, nor were they detected by antibodies of any class present in a collection of sera representative of other nematode infections. This difference in the IgG3 response was so pronounced that Sowda sera could be distinguished from generalized onchocerciasis sera by an IgG3-specific ELISA assay with a PBS parasite extract as the antigen. Thus, a correlation has been established between one particular clinical condition of onchocerciasis (Sowda) and a serological response, defined in terms of both the parasite antigens and an immunoglobulin class restricted antibody response. Images Fig. 1 Fig. 2 Fig. 5 PMID:3224441

  2. Nitric oxide transport in normal human thoracic aorta: effects of hemodynamics and nitric oxide scavengers.

    Directory of Open Access Journals (Sweden)

    Xiao Liu

    Full Text Available Despite the crucial role of nitric oxide (NO in the homeostasis of the vasculature, little quantitative information exists concerning NO transport and distribution in medium and large-sized arteries where atherosclerosis and aneurysm occur and hemodynamics is complex. We hypothesized that local hemodynamics in arteries may govern NO transport and affect the distribution of NO in the arteries, hence playing an important role in the localization of vascular diseases. To substantiate this hypothesis, we presented a lumen/wall model of the human aorta based on its MRI images to simulate the production, transport and consumption of NO in the arterial lumen and within the aortic wall. The results demonstrated that the distribution of NO in the aorta was quite uneven with remarkably reduced NO bioavailability in regions of disturbed flow, and local hemodynamics could affect NO distribution mainly via flow dependent NO production rate of endothelium. In addition, erythrocytes in the blood could moderately modulate NO concentration in the aorta, especially at the endothelial surface. However, the reaction of NO within the wall could only slightly affect NO concentration on the luminal surface, but strongly reduce NO concentration within the aortic wall. A strong positive correlation was revealed between wall shear stress and NO concentration, which was affected by local hemodynamics and NO reaction rate. In conclusion, the distribution of NO in the aorta may be determined by local hemodynamics and modulated differently by NO scavengers in the lumen and within the wall.

  3. Coupling Protein Dynamics with Proton Transport in Human Carbonic Anhydrase II.

    Science.gov (United States)

    Taraphder, Srabani; Maupin, C Mark; Swanson, Jessica M J; Voth, Gregory A

    2016-08-25

    The role of protein dynamics in enzyme catalysis is one of the most highly debated topics in enzymology. The main controversy centers around what may be defined as functionally significant conformational fluctuations and how, if at all, these fluctuations couple to enzyme catalyzed events. To shed light on this debate, the conformational dynamics along the transition path surmounting the highest free energy barrier have been herein investigated for the rate limiting proton transport event in human carbonic anhydrase (HCA) II. Special attention has been placed on whether the motion of an excess proton is correlated with fluctuations in the surrounding protein and solvent matrix, which may be rare on the picosecond and subpicosecond time scales of molecular motions. It is found that several active site residues, which do not directly participate in the proton transport event, have a significant impact on the dynamics of the excess proton. These secondary participants are shown to strongly influence the active site environment, resulting in the creation of water clusters that are conducive to fast, moderately slow, or slow proton transport events. The identification and characterization of these secondary participants illuminates the role of protein dynamics in the catalytic efficiency of HCA II.

  4. Arsenic-glutathione conjugate transport by the human multidrug resistance proteins (MRPs/ABCCs).

    Science.gov (United States)

    Leslie, Elaine M

    2012-03-01

    Millions of people world-wide are chronically exposed to inorganic forms of the environmental toxicant arsenic in drinking water. This has led to a public health crisis because arsenic is a human carcinogen, and causes a myriad of other adverse health effects. In order to prevent and treat arsenic-induced toxicity it is critical to understand the cellular handling of this metalloid. A large body of literature describes the importance of the cellular tripeptide glutathione (γ-Glu-Cys-Gly,GSH/GS) in the excretion of arsenic. The triglutathione conjugate of arsenite [As(III)(GS)(3)] and the diglutathione conjugate of monomethylarsonous acid [MMA(III)(GS)(2)] have been isolated from rat bile and mouse urine, and account for the majority of excreted arsenic, suggesting these are important transportable forms. The ATP-binding cassette (ABC) transporter proteins, multidrug resistance protein 1 (MRP1/ABCC1) and the related protein MRP2 (ABCC2), are thought to play an important role in arsenic detoxification through the cellular efflux of arsenic-GSH conjugates. Current knowledge on the cellular handling of arsenic with a special emphasis on the transport pathways of the arsenic-GSH conjugates As(III)(GS)(3), MMA(III)(GS)(2), and dimethylarsenic glutathione DMA(III)(GS), as well as, the seleno-bis(S-glutathionyl) arsinium ion [(GS)(2)AsSe](-) are reviewed. Copyright © 2011 Elsevier Inc. All rights reserved.

  5. Regulation of Na,K-pump-mediated transport by prolactin in cultured human prostate epithelial cells.

    Science.gov (United States)

    Duran, M J; Chosseler, M; Pressley, TA

    2004-11-01

    The prostate gland is unique in its ability to secrete large amounts of zinc and citrate, suggesting that it employs unusual transport mechanisms. Intracellular ionic homeostasis in prostate is likely to be mediated by the Na,K-pump, yet there have been few studies of its regulation in this tissue. Accordingly, we explored the expression of the Na,K-pump in PC3 cells, an established cell line of human prostate epithelial cells. Total RNA from confluent monolayers of PC3 cells was isolated, reverse transcribed, and the resulting complementary DNA was amplified by polymerase chain reaction using primers specific for each of the pump's constituent subunits. The amplification revealed a complex pattern of Na,K-pump expression, with detection of mRNAs encoding the alpha1-, alpha3-, alpha4-, betal-, beta2- and beta3-isoforms. We next examined the effect on pump activity of prolactin, an important mediator of cell proliferation in prostate cancer. Monolayers exposed to 10 nM prolactin for 24 hr revealed an inhibition of 40% in ouabain-sensitive 86Rb+ uptake, a sensitive measure of pump-mediated transport. These experiments suggest that the unique transport properties of prostate may depend, at least in part, on a complicated pattern of Na,K-pump expression and regulation.

  6. Volume-dependent K+ transport in rabbit red blood cells comparison with oxygenated human SS cells

    Energy Technology Data Exchange (ETDEWEB)

    Al-Rohil, N.; Jennings, M.L.

    1989-07-01

    In this study the volume-dependent or N-ethylmaleimide (NEM)-stimulated, ouabain-insensitive K+ influx and efflux were measured with the tracer 86Rb+ in rabbit red blood cells. The purpose of the work was to examine the rabbit as a potential model for cell volume regulation in human SS red blood cells and also to investigate the relationship between the NEM-reactive sulfhydryl group(s) and the signal by which cell swelling activates the transport. Ouabain-resistant K+ efflux and influx increase nearly threefold in cells swollen hypotonically by 15%. Pretreatment with 2 mM NEM stimulates efflux 5-fold and influx 10-fold (each measured in an isotonic medium). The ouabain-resistant K+ efflux was dependent on the major anion in the medium. The anion dependence of K+ efflux in swollen or NEM-stimulated cells was as follows: Br- greater than Cl- much greater than NO3- = acetate. The magnitudes of both the swelling- and the NEM-stimulated fluxes are much higher in young cells (density separated but excluding reticulocytes) than in older cells. Swelling- or NEM-stimulated K+ efflux in rabbit red blood cells was inhibited 50% by 1 mM furosemide, and the inhibitory potency of furosemide was enhanced by extracellular K+, as is known to be true for human AA and low-K+ sheep red blood cells. The swelling-stimulated flux in both rabbit and human SS cells has a pH optimum at approximately 7.4. We conclude that rabbit red blood cells are a good model for swelling-stimulated K+ transport in human SS cells.

  7. IgG Glycan Hydrolysis Attenuates ANCA-Mediated Glomerulonephritis

    Science.gov (United States)

    van Timmeren, Mirjan M.; van der Veen, Betty S.; Stegeman, Coen A.; Petersen, Arjen H.; Hellmark, Thomas; Collin, Mattias

    2010-01-01

    Anti-neutrophil cytoplasmic autoantibodies (ANCA) directed against myeloperoxidase (MPO) and proteinase 3 (Pr3) are considered pathogenic in ANCA-associated necrotizing and crescentic glomerulonephritis (NCGN) and vasculitis. Modulation of ANCA IgG glycosylation may potentially reduce its pathogenicity by abolishing Fc receptor–mediated activation of leukocytes and complement. Here, we investigated whether IgG hydrolysis by the bacterial enzyme endoglycosidase S (EndoS) attenuates ANCA-mediated NCGN. In vitro, treatment of ANCA IgG with EndoS significantly attenuated ANCA-mediated neutrophil activation without affecting antigen-binding capacity. In a mouse model of anti-MPO IgG/LPS-induced NCGN, we induced disease with either unmodified or EndoS-treated (deglycosylated) anti-MPO IgG. In separate experiments, we administered EndoS systemically after disease induction with unmodified anti-MPO IgG. Pretreatment of anti-MPO IgG with EndoS reduced hematuria, leukocyturia, and albuminuria and attenuated both neutrophil influx and formation of glomerular crescents. After inducing disease with unmodified anti-MPO IgG, systemic treatment with EndoS reduced albuminuria and glomerular crescent formation when initiated after 3 but not 24 hours. In conclusion, IgG glycan hydrolysis by EndoS attenuates ANCA-induced neutrophil activation in vitro and prevents induction of anti-MPO IgG/LPS-mediated NCGN in vivo. Systemic treatment with EndoS early after disease induction attenuates the development of disease. Thus, modulation of IgG glycosylation is a promising strategy to interfere with ANCA-mediated inflammatory processes. PMID:20448018

  8. Multimodel estimates of premature human mortality due to intercontinental transport of air pollution

    Science.gov (United States)

    Liang, C.; Silva, R.; West, J. J.; Sudo, K.; Lund, M. T.; Emmons, L. K.; Takemura, T.; Bian, H.

    2015-12-01

    Numerous modeling studies indicate that emissions from one continent influence air quality over others. Reducing air pollutant emissions from one continent can therefore benefit air quality and health on multiple continents. Here, we estimate the impacts of the intercontinental transport of ozone (O3) and fine particulate matter (PM2.5) on premature human mortality by using an ensemble of global chemical transport models coordinated by the Task Force on Hemispheric Transport of Air Pollution (TF HTAP). We use simulations of 20% reductions of all anthropogenic emissions from 13 regions (North America, Central America, South America, Europe, Northern Africa, Sub-Saharan Africa, Former Soviet Union, Middle East, East Asia, South Asia, South East Asia, Central Asia, and Australia) to calculate their impact on premature mortality within each region and elsewhere in the world. To better understand the impact of potential control strategies, we also analyze premature mortality for global 20% perturbations from five sectors individually: power and industry, ground transport, forest and savannah fires, residential, and others (shipping, aviation, and agriculture). Following previous studies, premature human mortality resulting from each perturbation scenario is calculated using a health impact function based on a log-linear model for O3 and an integrated exposure response model for PM2.5 to estimate relative risk. The spatial distribution of the exposed population (adults aged 25 and over) is obtained from the LandScan 2011 Global Population Dataset. Baseline mortality rates for chronic respiratory disease, ischemic heart disease, cerebrovascular disease, chronic obstructive pulmonary disease, and lung cancer are estimated from the GBD 2010 country-level mortality dataset for the exposed population. Model results are regridded from each model's original grid to a common 0.5°x0.5° grid used to estimate mortality. We perform uncertainty analysis and evaluate the sensitivity

  9. Genetic Polymorphisms in Organic Cation Transporter 1 Attenuates Hepatic Metformin Exposure in Humans.

    Science.gov (United States)

    Sundelin, Eio; Gormsen, L C; Jensen, J B; Vendelbo, M H; Jakobsen, S; Munk, O L; Christensen, Mmh; Brøsen, K; Frøkiaer, J; Jessen, N

    2017-11-01

    Metformin has been used successfully to treat type 2 diabetes for decades. However, the efficacy of the drug varies considerably from patient to patient and this may in part be due to its pharmacokinetic properties. The aim of this study was to examine if common polymorphisms in SLC22A1, encoding the transporter protein OCT1, affect the hepatic distribution of metformin in humans. We performed noninvasive 11 C-metformin positron emission tomography (PET)/computed tomography (CT) to determine hepatic exposure in 12 subjects genotyped for variants in SLC22A1. Hepatic distribution of metformin was significantly reduced after oral intake in carriers of M420del and R61C variants in SLC22A1 without being associated with changes in circulating levels of metformin. Our data show that genetic polymorphisms in transporter proteins cause variation in hepatic exposure to metformin, and it demonstrates the application of novel imaging techniques to investigate pharmacogenetic properties in humans. © 2017 American Society for Clinical Pharmacology and Therapeutics.

  10. A comparison of linaclotide and lubiprostone dosing regimens on ion transport responses in human colonic mucosa.

    Science.gov (United States)

    Kang, Sang Bum; Marchelletta, Ronald R; Penrose, Harrison; Docherty, Michael J; McCole, Declan F

    2015-03-01

    Linaclotide, a synthetic guanylyl cyclase C (GC-C) agonist, and the prostone analog, Lubiprostone, are approved to manage chronic idiopathic constipation and constipation-predominant irritable bowel syndrome. Lubiprostone also protects intestinal mucosal barrier function in ischemia. GC-C signaling regulates local fluid balance and other components of intestinal mucosal homeostasis including epithelial barrier function. The aim of this study was to compare if select dosing regimens differentially affect linaclotide and lubiprostone modulation of ion transport and barrier properties of normal human colonic mucosa. Normal sigmoid colon biopsies from healthy subjects were mounted in Ussing chambers. Tissues were treated with linaclotide, lubiprostone, or vehicle to determine effects on short-circuit current (I sc). Subsequent I sc responses to the cAMP agonist, forskolin, and the calcium agonist, carbachol, were also measured to assess if either drug caused desensitization. Barrier properties were assessed by measuring transepithelial electrical resistance. I sc responses to linaclotide and lubiprostone were significantly higher than vehicle control when administered bilaterally or to the mucosal side only. Single versus cumulative concentrations of linaclotide showed differences in efficacy while cumulative but not single dosing caused desensitization to forskolin. Lubiprostone reduced forskolin responses under all conditions. Linaclotide and lubiprostone exerted a positive effect on TER that was dependent on the dosing regimen. Linaclotide and lubiprostone increase ion transport responses across normal human colon but linaclotide displays increased sensitivity to the dosing regimen used. These findings may have implications for dosing protocols of these agents in patients with constipation.

  11. Canakinumab (ACZ885, a fully human IgG1 anti-IL-1β mAb) induces sustained remission in pediatric patients with cryopyrin-associated periodic syndrome (CAPS).

    Science.gov (United States)

    Kuemmerle-Deschner, Jasmin B; Ramos, Eduardo; Blank, Norbert; Roesler, Joachim; Felix, Sandra D; Jung, Thomas; Stricker, Kirstin; Chakraborty, Abhijit; Tannenbaum, Stacey; Wright, Andrew M; Rordorf, Christiane

    2011-02-28

    Cryopyrin-associated periodic syndrome (CAPS) represents a spectrum of three auto-inflammatory syndromes, familial cold auto-inflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal-onset multisystem inflammatory disease/chronic infantile neurological cutaneous and articular syndrome (NOMID/CINCA) with etiology linked to mutations in the NLRP3 gene resulting in elevated interleukin-1β (IL-1β) release. CAPS is a rare hereditary auto-inflammatory disease, which may start early in childhood and requires a life-long treatment. Canakinumab, a fully human anti-IL-1β antibody, produces sustained selective inhibition of IL-1β. This study was conducted to assess the efficacy, safety, and pharmacokinetics of canakinumab in the treatment of pediatric CAPS patients. Seven pediatric patients (five children and two adolescents) with CAPS were enrolled in a phase II, open-label study of canakinumab in patients with CAPS. Canakinumab was administered at a dose of 2 mg/kg subcutaneously (s.c.) (for patients with body weight ≤ 40 kg) or 150 mg s.c. (for patients with body weight > 40 kg) with re-dosing upon each relapse. The primary efficacy variable was time to relapse following achievement of a complete response (defined as a global assessment of no or minimal disease activity and no or minimal rash and values for serum C-reactive protein (CRP) and/or serum amyloid A (SAA) within the normal range, CAPS. ClinicalTrials.gov: NCT00487708.

  12. Quantitative Prediction of Human Renal Clearance and Drug-Drug Interactions of Organic Anion Transporter Substrates Using In Vitro Transport Data: A Relative Activity Factor Approach.

    Science.gov (United States)

    Mathialagan, Sumathy; Piotrowski, Mary A; Tess, David A; Feng, Bo; Litchfield, John; Varma, Manthena V

    2017-04-01

    Organic anion transporters (OATs) are important in the renal secretion, and thus, the clearance, of many drugs; and their functional change can result in pharmacokinetic variability. In this study, we applied transport rates measured in vitro using OAT-transfected human embryonic kidney cells to predict human renal secretory and total renal clearance of 31 diverse drugs. Selective substrates to OAT1 (tenofovir), OAT2 (acyclovir and ganciclovir), and OAT3 (benzylpenicillin, oseltamivir acid) were used to obtain relative activity factors (RAFs) for these individual transporters by relating in vitro transport clearance (after physiologic scaling) to in vivo secretory clearance. Using the estimated RAFs (0.64, 7.3, and 4.1, respectively, for OAT1, OAT2, and OAT3, respectively) and the in vitro active clearances, renal secretory clearance and total renal clearance were predicted with average fold errors (AFEs) of 1.89 and 1.40, respectively. The results show that OAT3-mediated transport play a predominant role in renal secretion for 22 of the 31 drugs evaluated. This mechanistic static approach was further applied to quantitatively predict renal drug-drug interactions (AFE ∼1.6) of the substrate drugs with probenecid, a clinical probe OAT inhibitor. In conclusion, the proposed in vitro-in vivo extrapolation approach is the first comprehensive attempt toward mechanistic modeling of renal secretory clearance based on routinely employed in vitro cell models. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  13. Transport of Aflatoxin M1 in human intestinal Caco-2/TC7 cells

    Directory of Open Access Journals (Sweden)

    Francesca eCaloni

    2012-06-01

    Full Text Available Aflatoxin M1 (AFM1 is a hydroxylated metabolite of aflatoxin B1 (AFB1. After it is formed, it is secreted in the milk of mammals.Despite the potential risk of human exposure to AFM1, data reported in literature on the metabolism, toxicity and bioavailability of this molecule are limited and out of date. The aim of the present research was to study the absorption profile of AFM1 and possible damage to tight junctions of the intestinal Caco-2/TC7 clone grown on microporous filter supports. These inserts allowed for the separation of the apical and basolateral compartments which correspond to the in vivo lumen and the interstitial space/vascular systems of intestinal mucosa respectively.In this study, the Caco-2/TC7 cells were treated with different AFM1 concentrations (10-10,000 ng/kg for short (40 minutes and long periods of time (48 hours. The AFM1 influx/efflux transport and effects on tight junctions were evaluated by measuring trans-epithelial electrical resistance and observing tight junction protein (Zonula occludens-1 and occludin localization.The results showed that: i when introduced to the apical and basolateral compartments, AFM1 was poorly absorbed by the Caco-2/TC7 cells but its transport across the cell monolayer occurred very quickly (Papp value of 105.10 ± 7.98 cm/s x 10-6. ii The integrity of tight junctions was not permanently compromised after exposure to the mycotoxin. Viability impairment or barrier damage did not occur either.The present results contribute to the evaluation of human risk exposure to AFM1, although the AFM1 transport mechanism need to be clarified.

  14. Cellular distribution of glucose and monocarboxylate transporters in human brain white matter and multiple sclerosis lesions.

    Science.gov (United States)

    Nijland, Philip G; Michailidou, Iliana; Witte, Maarten E; Mizee, Mark R; van der Pol, Susanne M A; van Het Hof, Bert; Reijerkerk, Arie; Pellerin, Luc; van der Valk, Paul; de Vries, Helga E; van Horssen, Jack

    2014-07-01

    To ensure efficient energy supply to the high demanding brain, nutrients are transported into brain cells via specific glucose (GLUT) and monocarboxylate transporters (MCT). Mitochondrial dysfunction and altered glucose metabolism are thought to play an important role in the progression of neurodegenerative diseases, including multiple sclerosis (MS). Here, we investigated the cellular localization of key GLUT and MCT proteins in human brain tissue of non-neurological controls and MS patients. We show that in control brain tissue GLUT and MCT proteins were abundantly expressed in a variety of central nervous system cells, particularly in microglia and endothelial cells. In active MS lesions, GLUTs and MCTs were highly expressed in infiltrating leukocytes and reactive astrocytes. Astrocytes manifest increased MCT1 staining and maintain GLUT expression in inactive lesions, whereas demyelinated axons exhibit significantly reduced GLUT3 and MCT2 immunoreactivity in inactive lesions. Finally, we demonstrated that the co-transcription factor peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α), an important protein involved in energy metabolism, is highly expressed in reactive astrocytes in active MS lesions. Overexpression of PGC-1α in astrocyte-like cells resulted in increased production of several GLUT and MCT proteins. In conclusion, we provide for the first time a comprehensive overview of key nutrient transporters in white matter brain samples. Moreover, our data demonstrate an altered expression of these nutrient transporters in MS brain tissue, including a marked reduction of axonal GLUT3 and MCT2 expression in chronic lesions, which may impede efficient nutrient supply to the hypoxic demyelinated axons thereby contributing to the ongoing neurodegeneration in MS. Copyright © 2014 Wiley Periodicals, Inc.

  15. In vitro approaches to evaluate placental drug transport by using differentiating JEG-3 human choriocarcinoma cells.

    Science.gov (United States)

    Ikeda, Kenji; Utoguchi, Naoki; Tsutsui, Hidenobu; Yamaue, Satoko; Homemoto, Manami; Nakao, Erina; Hukunaga, Yumi; Yamasaki, Kyohei; Myotoku, Michiaki; Hirotani, Yoshihiko

    2011-02-01

    Human choriocarcinoma cells have been used as models for studying transcellular drug transport through placental trophoblasts. However, these models allow the transport of low-molecular-weight drugs through intercellular gap junctions. This study aimed at investigating the differentiation patterns of JEG-3 choriocarcinoma cells under different culture conditions and establishing the appropriate model of in vitro syncytiotrophoblast drug transport. Paracellular permeability was estimated by measuring the transepithelial electrical resistance (TEER) across JEG-3 cell layers. The mRNA expression levels of non-expressed in choriocarcinoma clone 1 (NECC1) and breast cancer resistance protein (BCRP), and those of E-cadherin (ECAD) and cadherin-11 (CDH11), which are adherens junction-associated proteins related to fusogenic ability of syncytiotrophoblasts differentiated from cytotrophoblasts, protein expression levels were considered as the differentiation signals. The highest TEER values were obtained in the JEG-3 cells cultured in the Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 (1:1) mixed medium (CS-C(®) ; Dainippon Sumitomo Pharma Co. Ltd., Osaka, Japan). By comparing the TEER values and the differentiation signals, the authors identified at least five JEG-3 cell-differentiation patterns. The differentiation pattern of JEG-3 cultured in CS-C resembled the syncytiotrophoblast-like differentiation signal characterizations in vivo. In conclusion, the syncytiotrophoblast-like models of differentiating JEG-3 cells cultured in CS-C might be appropriate for evaluating drug transport across the placental trophoblast. © 2010 The Authors. Basic & Clinical Pharmacology & Toxicology © 2010 Nordic Pharmacological Society.

  16. A transportation network for human ovarian tissue is indispensable to success for fertility preservation.

    Science.gov (United States)

    Kyono, K; Hashimoto, T; Toya, M; Koizumi, M; Sasaki, C; Shibasaki, S; Aono, N; Nakamura, Y; Obata, R; Okuyama, N; Ogura, Y; Igarashi, H

    2017-09-02

    The purpose of this study was to examine the efficacy of an ovarian tissue transportation network for fertility preservation (FP) for cancer patients in Japan. PubMed was searched for papers on transportation of human ovarian tissue for FP. We analyzed population, area, number of cancer patients for ovarian tissue cryopreservation (OTC), quality control/assessment and safety, cost of a cryopreservation center for the building for 30 years, and medical fees of cancer patients (operation, cryopreservation, and storage of ovarian tissue). More than twenty babies have been born in Denmark and Germany through a transportation system. Up to 400 new patients a year need OTC. The fees for removal, cryopreservation, and storage for 5 years, and transplantation of ovarian tissue are around €5,000, €4,000, and €5,000, respectively. It costs more than €5 million to establish and maintain one cryopreservation center for 30 years. If we have a few cryopreservation centers in Japan, we can cryopreserve 400 patients' ovarian tissue per year by safer slow freezing and maintain quality control/assessment. We need to lighten the patients' burden for easy to use FP by a government subsidy and medical insurance coverage. This model has been termed the Danish model ("the woman stays - the tissue moves"). This is truly patient-centered medicine. We can have maximum effects with the minimum burden. A transportation network like those of Denmark and Germany is the best strategy for FP in Japan. It may be the best system for cancer patients, medical staff, and the Ministry of Health, Labor, and Welfare.

  17. Assignment of the human organic anion transporting polypeptide (OATP) gene to chromosome 12p12 by fluorescence in situ hybridization

    NARCIS (Netherlands)

    Kullak-Ublick, G. A.; Beuers, U.; Meier, P. J.; Domdey, H.; Paumgartner, G.

    1996-01-01

    The organic anion transporting polypeptide (OATP) of human liver mediates the basolateral hepatocellular uptake of numerous cholephilic anions and steroidal compounds. The aim of this study was to clone the human OATP gene and to map its chromosomal localization by fluorescence in situ

  18. Xenobiotic-Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes Modulated by Toxcast Chemicals

    Science.gov (United States)

    Primary human hepatocyte cultures are useful in vitro model systems of human liver because when cultured under appropriate conditions the hepatocytes retain liver-like functionality such as metabolism, transport, and cell signaling. This model system was used to characterize the ...

  19. Membrane transporters for the special amino acid glutamine: Structure/function relationships and relevance to human health.

    Science.gov (United States)

    Pochini, Lorena; Scalise, Mariafrancesca; Galluccio, Michele; Indiveri, Cesare

    2014-08-01

    Glutamine together with glucose is essential for body’s homeostasis. It is the most abundant amino acid and is involved in many biosynthetic, regulatory and energy production processes. Several membrane transporters which differ in transport modes, ensure glutamine homeostasis by coordinating its absorption, reabsorption and delivery to tissues. These transporters belong to different protein families, are redundant and ubiquitous. Their classification, originally based on functional properties, has recently been associated with the SLC nomenclature. Function of glutamine transporters is studied in cells over-expressing the transporters or, more recently in proteoliposomes harboring the proteins extracted from animal tissues or over-expressed in microorganisms. The role of the glutamine transporters is linked to their transport modes and coupling with Na+ and H+. Most transporters share specificity for other neutral or cationic amino acids. Na+-dependent co-transporters efficiently accumulate glutamine while antiporters regulate the pools of glutamine and other amino acids. The most acknowledged glutamine transporters belong to the SLC1, 6, 7 and 38 families. The members involved in the homeostasis are the co-transporters B0AT1 and the SNAT members 1, 2, 3, 5 and 7; the antiporters ASCT2, LAT1 and 2. The last two are associated to the ancillary CD98 protein. Some information on regulation of the glutamine transporters exist, which, however, need to be deepened. No information at all is available on structures, besides some homology models obtained using similar bacterial transporters as templates. Some models of rat and human glutamine transporters highlight very similar structures between the orthologues. Moreover the presence of glycosylation and/or phosphorylation sites located at the extracellular or intracellular faces has been predicted. ASCT2 and LAT1 are over-expressed in several cancers, thus representing potential targets for pharmacological intervention.

  20. Membrane transporters for the special amino acid glutamine: Structure/function relationships and relevance to human health.

    Directory of Open Access Journals (Sweden)

    Lorena ePochini

    2014-08-01

    Full Text Available Glutamine together with glucose is essential for body’s homeostasis. It is the most abundant amino acid and is involved in many biosynthetic, regulatory and energy production processes. Several membrane transporters which differ in transport modes, ensure glutamine homeostasis by coordinating its absorption, reabsorption and delivery to tissues. These transporters belong to different protein families, are redundant and ubiquitous. Their classification, originally based on functional properties, has recently been associated with the SLC nomenclature. Function of glutamine transporters is studied in cells over-expressing the transporters or, more recently in proteoliposomes harboring the proteins extracted from animal tissues or over-expressed in microorganisms. The role of the glutamine transporters is linked to their transport modes and coupling with Na+ and H+. Most transporters share specificity for other neutral or cationic amino acids. Na+-dependent co-transporters efficiently accumulate glutamine while antiporters regulate the pools of glutamine and other amino acids. The most acknowledged glutamine transporters belong to the SLC1, 6, 7 and 38 families. The members involved in the homeostasis are the co-transporters B0AT1 and the SNAT members 1, 2, 3, 5 and 7; the antiporters ASCT2, LAT1 and 2. The last two are associated to the ancillary CD98 protein. Some information on regulation of the glutamine transporters exist, which, however, need to be deepened. No information at all is available on structures, besides some homology models obtained using similar bacterial transporters as templates. Some models of rat and human glutamine transporters highlight very similar structures between the orthologues. Moreover the presence of glycosylation and/or phosphorylation sites located at the extracellular or intracellular faces has been predicted. ASCT2 and LAT1 are over-expressed in several cancers, thus representing potential targets for

  1. Serotonin (5-HT) transport in human platelets is modulated by Src-catalysed Tyr-phosphorylation of the plasma membrane transporter SERT.

    Science.gov (United States)

    Zarpellon, Alessandro; Donella-Deana, Arianna; Folda, Alessandra; Turetta, Loris; Pavanetto, Martina; Deana, Renzo

    2008-01-01

    platelets possess tightly regulated systems for serotonin (5-HT) transport. This study analysed whether the 5-HT transport mediated by the plasma-membrane transporter SERT is regulated by its Tyr-phosphorylation. 5-HT transport was determined by filtration techniques, while immunoblotting procedures were adopted for detecting the Tyr-phosphorylation of SERT in human platelet fractions. 5-HT accumulation in platelets pre-treated with reserpine, which prevents the neurotransmitter transport into the dense granules, decreased upon cellular exposure to PP2 and SU6656, two structurally unrelated inhibitors of Src-kinases. By contrast, the protein Tyr-phosphatase inhibitor pervanadate increased the 5-HT accumulation. Anti-SERT immunostaining of the platelet fractions showed a major band displaying an apparent molecular mass of 50 kappaDa, indicating that, during the analytical procedure, SERT underwent proteolysis, which was counteracted by addition of 4 M urea in the cellular disrupting medium. The Tyr-phosphorylation degree of SERT immunoprecipitated from membrane extracts decreased by platelet treatment with SU6656 or PP2, and enhanced upon pervanadate treatment. The anti-SERT immunoprecipitates displayed anti-Src immunostaining and in vitro kinase activity towards a Src-specific peptide-substrate. Platelet treatment with PP2 or SU6656 also caused a decrease in the imipramine binding to platelets. It was concluded that the Src-mediated SERT Tyr-phosphorylation regulates the 5-HT transport by affecting the neurotransmitter binding sites.

  2. Mapping of the taurine transporter gene to mouse chromosome 6 and to the short arm of human chromosome 3

    Energy Technology Data Exchange (ETDEWEB)

    Patel, A.; Uhl, G.R.; Gregor, P. [National Inst. of Health, Baltimore, MD (United States)] [and others

    1995-01-01

    Transport proteins have essential functions in the uptake of neurotransmitters and neuromodulators. We have mapped the gene encoding the taurine transporter, Taut, to the central region of mouse chromosome 6. Analysis of a cross segregating the neurological mutant mnd2 excluded Taut as a candidate gene for this closely linked mutation. To map the human taurine transporter gene, TAUT, a sequence-tagged site (STS) corresponding to the 3{prime} untranslated region of the human cDNA was developed. TAUT was assigned to human chromosome 3 by typing this STS on a panel of somatic cell hybrids. Further analysis of a hybrid panel containing defined deletions of chromosome 3 suggested that TAUT maps to 3p21-p25. These data extend a conserved linkage group on mouse chromosome 6 and human chromosome 3p. Deletion of TAUT might contribute to some phenotypic features of the 3p{sup -} syndrome. 32 refs., 3 figs.

  3. Piracetam and TRH analogues antagonise inhibition by barbiturates, diazepam, melatonin and galanin of human erythrocyte D-glucose transport

    Science.gov (United States)

    Naftalin, Richard J; Cunningham, Philip; Afzal-Ahmed, Iram

    2004-01-01

    Nootropic drugs increase glucose uptake into anaesthetised brain and into Alzheimer's diseased brain. Thyrotropin-releasing hormone, TRH, which has a chemical structure similar to nootropics increases cerebellar uptake of glucose in murine rolling ataxia. This paper shows that nootropic drugs like piracetam (2-oxo 1 pyrrolidine acetamide) and levetiracetam and neuropeptides like TRH antagonise the inhibition of glucose transport by barbiturates, diazepam, melatonin and endogenous neuropeptide galanin in human erythrocytes in vitro. The potencies of nootropic drugs in opposing scopolamine-induced memory loss correlate with their potencies in antagonising pentobarbital inhibition of erythrocyte glucose transport in vitro (Pnootropics, D-levetiracetam and D-pyroglutamate, have higher antagonist Ki's against pentobarbital inhibition of glucose transport than more potent L-stereoisomers (Pnootropics, like aniracetam and levetiracetam, while antagonising pentobarbital action, also inhibit glucose transport. Analeptics like bemigride and methamphetamine are more potent inhibitors of glucose transport than antagonists of hypnotic action on glucose transport. There are similarities between amino-acid sequences in human glucose transport protein isoform 1 (GLUT1) and the benzodiazepine-binding domains of GABAA (gamma amino butyric acid) receptor subunits. Mapped on a 3D template of GLUT1, these homologies suggest that the site of diazepam and piracetam interaction is a pocket outside the central hydrophilic pore region. Nootropic pyrrolidone antagonism of hypnotic drug inhibition of glucose transport in vitro may be an analogue of TRH antagonism of galanin-induced narcosis. PMID:15148255

  4. IgG Glycan Hydrolysis Attenuates ANCA-Mediated Glomerulonephritis

    NARCIS (Netherlands)

    van Timmeren, Mirjan M.; van der Veen, Betty S.; Stegeman, Coen A.; Petersen, Arjen H.; Hellmark, Thomas; Collin, Mattias; Heeringa, Peter

    Anti-neutrophil cytoplasmic autoantibodies (ANCA) directed against myeloperoxidase (MPO) and proteinase 3 (Pr3) are considered pathogenic in ANCA-associated necrotizing and crescentic glomerulonephritis (NCGN) and vasculitis. Modulation of ANCA IgG glycosylation may potentially reduce its

  5. Increased IgG4-Positive Plasma Cells in Granulomatosis with Polyangiitis: A Diagnostic Pitfall of IgG4-Related Disease

    Directory of Open Access Journals (Sweden)

    Sing Yun Chang

    2012-01-01

    Full Text Available Granulomatosis with polyangiitis (Wegener’s (GPA may mimic IgG4-related disease (IgG4-RD on histologic examination of some biopsies, especially those from head and neck sites. IgG4 immunostain is often performed in this context for differential diagnosis with IgG4-RD. However, the prevalence of IgG4+ cells in GPA has not been explored. We examined the IgG4+ cells in 26 cases confirmed as GPA by a thorough clinical and pathologic assessment. Twenty-six biopsies consisted of 14 sinonasal/oral cavity/nasopharynx, 7 orbit/periorbital, 3 lung/pleura, 1 iliac fossa/kidney, and 1 dura specimens. Eight of 26 (31% biopsies revealed increased IgG4+ cells (>30/HPF and >40% in IgG4+/IgG+ ratio. The IgG4+ cells and IgG4+/IgG+ ratio ranged 37–137/hpf and 44–83%, respectively. Eight biopsies with increased IgG4+ cells were from sinonasal (n=4 or orbital/periorbital (n=4 sites. In conclusion, increased IgG4+ cells are not uncommonly seen in sinonasal or orbital/periorbital biopsies of GPA, which could pose as a diagnostic pitfall.

  6. Characterization of Chicken B-Locus (Igg) Allotypes

    Science.gov (United States)

    David, C. S.

    1972-01-01

    Chicken allotypes b1 and b2 are controlled by allelic genes and associated with the IgG class of immunoglobulins. The determinants cannot be detected on either isolated heavy and light polypeptide chains or the enzymatic fragments. The results suggest that either the intact IgG molecule is required for their expression or the conditions normally used for isolation of the subunits destroy or modify the antigenic specificities in chickens. PMID:4626653

  7. Functional expression of choline transporter like-protein 1 (CTL1) and CTL2 in human brain microvascular endothelial cells.

    Science.gov (United States)

    Iwao, Beniko; Yara, Miki; Hara, Naomi; Kawai, Yuiko; Yamanaka, Tsuyoshi; Nishihara, Hiroshi; Inoue, Takeshi; Inazu, Masato

    2016-02-01

    In this study, we examined the molecular and functional characterization of choline transporter in human brain microvascular endothelial cells (hBMECs). Choline uptake into hBMECs was a saturable process that was mediated by a Na(+)-independent, membrane potential and pH-dependent transport system. The cells have two different [(3)H]choline transport systems with Km values of 35.0 ± 4.9 μM and 54.1 ± 8.1 μM, respectively. Choline uptake was inhibited by choline, acetylcholine (ACh) and the choline analog hemicholinium-3 (HC-3). Various organic cations also interacted with the choline transport system. Choline transporter-like protein 1 (CTL1) and CTL2 mRNA were highly expressed, while mRNA for high-affinity choline transporter 1 (CHT1) and organic cation transporters (OCTs) were not expressed in hBMECs. CTL1 and CTL2 proteins were localized to brain microvascular endothelial cells in human brain cortical sections. Both CTL1 and CTL2 proteins were expressed on the plasma membrane and mitochondria. CTL1 and CTL2 proteins are mainly expressed in plasma membrane and mitochondria, respectively. We conclude that choline is mainly transported via an intermediate-affinity choline transport system, CTL1 and CTL2, in hBMECs. These transporters are responsible for the uptake of extracellular choline and organic cations. CTL2 participate in choline transport mainly in mitochondria, and may be the major site for the control of choline oxidation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Isolation and characterization of a monoclonal anti CK-2 alpha subunit antibody of the IgG1 subclass

    DEFF Research Database (Denmark)

    Schmidt-Spaniol, I; Boldyreff, B; Issinger, O G

    1992-01-01

    A monoclonal antibody was produced against the recombinant human alpha subunit of CK-2. The antibody was of the IgG1 subclass and it was isolated from serum-free cell culture media and purified by affinity chromatography on Protein G Sepharose. The antibody can be used to detect specifically the ...

  9. Isotype Diversification of IgG Antibodies to HIV Gag Proteins as a Therapeutic Vaccination Strategy for HIV Infection

    Directory of Open Access Journals (Sweden)

    Sonia Fernandez

    2013-08-01

    Full Text Available The development of vaccines to treat and prevent human immunodeficiency virus (HIV infection has been hampered by an incomplete understanding of “protective” immune responses against HIV. Natural control of HIV-1 infection is associated with T-cell responses against HIV-1 Gag proteins, particularly CD8+ T-cell responses restricted by “protective” HLA-B alleles, but other immune responses also contribute to immune control. These immune responses appear to include IgG antibodies to HIV-1 Gag proteins, interferon-a-dependant natural killer (NK cell responses and plasmacytoid dendritic cell (pDC responses. Here, it is proposed that isotype diversification of IgG antibodies against HIV-1 Gag proteins, to include IgG2, as well as IgG3 and IgG1 antibodies, will broaden the function of the antibody response and facilitate accessory cell responses against HIV-1 by NK cells and pDCs. We suggest that this should be investigated as a vaccination strategy for HIV-1 infection.

  10. Micropollutants in groundwater from septic systems: Transformations, transport mechanisms, and human health risk assessment.

    Science.gov (United States)

    Yang, Yun-Ya; Toor, Gurpal S; Wilson, P Chris; Williams, Clinton F

    2017-10-15

    Septic systems may contribute micropollutants to shallow groundwater and surface water. We constructed two in situ conventional drainfields (drip dispersal and gravel trench) and an advanced drainfield of septic systems to investigate the fate and transport of micropollutants to shallow groundwater. Unsaturated soil-water and groundwater samples were collected, over 32 sampling events (January 2013 to June 2014), from the drainfields (0.31-1.07 m deep) and piezometers (3.1-3.4 m deep). In addition to soil-water and groundwater, effluent samples collected from the septic tank were also analyzed for 20 selected micropollutants, including wastewater markers, hormones, pharmaceuticals and personal care products (PPCPs), a plasticizer, and their transformation products. The removal efficiencies of micropollutants from septic tank effluent to groundwater were similar among three septic systems and were 51-89% for sucralose and 53->99% for other micropollutants. Even with high removal rates within the drainfields, six PPCPs and sucralose with concentrations ranging from groundwater, respectively. The human health risk assessment showed that the risk to human health due to consumption of groundwater is negligible for the micropollutants monitored in the study. A better understanding of ecotoxicological effects of micropollutant mixtures from septic systems to ecosystem and human health is warranted for the long-term sustainability of septic systems. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Fate and Transport of Mercury in Environmental Media and Human Exposure

    Science.gov (United States)

    Kim, Moon-Kyung

    2012-01-01

    Mercury is emitted to the atmosphere from various natural and anthropogenic sources, and degrades with difficulty in the environment. Mercury exists as various species, mainly elemental (Hg0) and divalent (Hg2+) mercury depending on its oxidation states in air and water. Mercury emitted to the atmosphere can be deposited into aqueous environments by wet and dry depositions, and some can be re-emitted into the atmosphere. The deposited mercury species, mainly Hg2+, can react with various organic compounds in water and sediment by biotic reactions mediated by sulfur-reducing bacteria, and abiotic reactions mediated by sunlight photolysis, resulting in conversion into organic mercury such as methylmercury (MeHg). MeHg can be bioaccumulated through the food web in the ecosystem, finally exposing humans who consume fish. For a better understanding of how humans are exposed to mercury in the environment, this review paper summarizes the mechanisms of emission, fate and transport, speciation chemistry, bioaccumulation, levels of contamination in environmental media, and finally exposure assessment of humans. PMID:23230463

  12. Reduced expression of glutamate transporter EAAT2 and impaired glutamate transport in human primary astrocytes exposed to HIV-1 or gp120.

    Science.gov (United States)

    Wang, Zhuying; Pekarskaya, Olga; Bencheikh, Meryem; Chao, Wei; Gelbard, Harris A; Ghorpade, Anuja; Rothstein, Jeffrey D; Volsky, David J

    2003-07-20

    L-Glutamate is the major excitatory neurotransmitter in the brain. Astrocytes maintain low levels of synaptic glutamate by high-affinity uptake and defects in this function may lead to neuronal cell death by excitotoxicity. We tested the effects of HIV-1 and its envelope glycoprotein gp120 upon glutamate uptake and expression of glutamate transporters EAAT1 and EAAT2 in fetal human astrocytes in vitro. Astrocytes isolated from fetal tissues between 16 and 19 weeks of gestation expressed EAAT1 and EAAT2 RNA and proteins as detected by Northern blot analysis and immunoblotting, respectively, and the cells were capable of specific glutamate uptake. Exposure of astrocytes to HIV-1 or gp120 significantly impaired glutamate uptake by the cells, with maximum inhibition within 6 h, followed by gradual decline during 3 days of observation. HIV-1-infected cells showed a 59% reduction in V(max) for glutamate transport, indicating a reduction in the number of active transporter sites on the cell surface. Impaired glutamate transport after HIV-1 infection or gp120 exposure correlated with a 40-70% decline in steady-state levels of EAAT2 RNA and protein. EAAT1 RNA and protein levels were less affected. Treatment of astrocytes with tumor necrosis factor-alpha (TNF-alpha) decreased the expression of both EAAT1 and EAAT2, but neither HIV-1 nor gp120 were found to induce TNF-alpha production by astrocytes. These findings demonstrate that HIV-1 and gp120 induce transcriptional downmodulation of the EAAT2 transporter gene in human astrocytes and coordinately attenuate glutamate transport by the cells. Reduction of the ability of HIV-1-infected astrocytes to take up glutamate may contribute to the development of neurological disease.

  13. Stabilization of IgG by supercritical fluid drying: optimization of formulation and process parameters.

    Science.gov (United States)

    Jovanović, Natasa; Bouchard, Andréanne; Hofland, Gerard W; Witkamp, Geert-Jan; Crommelin, Daan J A; Jiskoot, Wim

    2008-02-01

    The aim of this study was to stabilize human serum immunoglobulin G (IgG) by a supercritical fluid (SCF) drying process. Solutions containing IgG (20mg/ml) and trehalose or hydroxypropyl-beta-cyclodextrin in a 1:4 (w/w) ratio were sprayed into a SCF phase consisting of CO(2) and ethanol at 100bar and 37 degrees C. Initially, a set of drying conditions previously developed to successfully stabilize lysozyme and myogobin formulations was used [N. Jovanović, A. Bouchard, G.W. Hofland, G.J. Witkamp, D.J.A. Crommelin, W. Jiskoot, Eur. J. Pharm. Sci. 27 (2006) 336-345]. Dried formulations were analyzed by Karl Fisher titration, scanning electron microscopy, X-ray powder diffraction, and modulated DSC. Protein structure in the solid-state was studied by FTIR and after reconstitution by UV/Vis, circular dichroism and fluorescence spectroscopy, GPC and SDS-PAGE. When IgG was dried under the above-mentioned conditions, substantial amounts of insoluble aggregates were formed. Addition of buffer helped to reduce the fraction of insoluble material but not of soluble aggregates. Full stabilization could be achieved by adjusting the process conditions: drying without ethanol while keeping the other conditions the same, or drying with ethanol at a temperature below the critical point (20 degrees C). In conclusion, it is possible to stabilize human IgG by SCF drying provided that the formulation and process conditions are tailored to meet the specific requirements of the protein.

  14. Transporter Classification Database (TCDB)

    Data.gov (United States)

    U.S. Department of Health & Human Services — The Transporter Classification Database details a comprehensive classification system for membrane transport proteins known as the Transporter Classification (TC)...

  15. Evidence that forskolin binds to the glucose transporter of human erythrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Lavis, V.R.; Lee, D.P.; Shenolikar, S.

    1987-10-25

    Binding of (4-/sup 3/H)cytochalasin B and (12-/sup 3/H)forskolin to human erythrocyte membranes was measured by a centrifugation method. Glucose-displaceable binding of cytochalasin B was saturable, with KD = 0.11 microM, and maximum binding approximately 550 pmol/mg of protein. Forskolin inhibited the glucose-displaceable binding of cytochalasin B in an apparently competitive manner, with K1 = 3 microM. Glucose-displaceable binding of (12-/sup 3/H)forskolin was also saturable, with KD = 2.6 microM and maximum binding approximately equal to 400 pmol/mg of protein. The following compounds inhibited binding of (12-/sup 3/H)forskolin and (4-/sup 3/H)cytochalasin B equivalently, with relative potencies parallel to their reported affinities for the glucose transport system: cytochalasins A and D, dihydrocytochalasin B, L-rhamnose, L-glucose, D-galactose, D-mannose, D-glucose, 2-deoxy-D-glucose, 3-O-methyl-D-glucose, phloretin, and phlorizin. A water-soluble derivative of forskolin, 7-hemisuccinyl-7-desacetylforskolin, displaced equivalent amounts of (4-/sup 3/H)cytochalasin B or (12-/sup 3/H)forskolin. Rabbit erythrocyte membranes, which are deficient in glucose transporter, did not bind either (4-/sup 3/H)cytochalasin B or (12-/sup 3/H)forskolin in a glucose-displaceable manner. These results indicate that forskolin, in concentrations routinely employed for stimulation of adenylate cyclase, binds to the glucose transporter. Endogenous ligands with similar specificities could be important modulators of cellular metabolism.

  16. Elucidation of the proton transport mechanism in human carbonic anhydrase II.

    Science.gov (United States)

    Maupin, C Mark; McKenna, Robert; Silverman, David N; Voth, Gregory A

    2009-06-10

    Human carbonic anhydrase II (HCA II) is one of the fastest known enzymes, which utilizes a rate-limiting proton transport (PT) step in its enzymatic reaction. To evaluate the PT event at an atomistic level, the multistate empirical valence bond (MS-EVB) method has been utilized in this work. It is observed that the PT event in HCA II exploits a transient active site water cluster to transport the excess proton between the catalytic zinc-bound water/hydroxide and the proton shuttling residue, His64. This PT event is found to be dependent on the enzyme's ability to form and stabilize the active site water cluster in addition to its ability to orient His64 in a favorable conformation. Evaluation of the PT free energy barrier for different orientations of His64 reveals this residue's vital role as a proton transporter and elucidates its direct effect on the barrier to PT through the active site water. It is suggested that the rate-limiting step oscillates between the active site water PT event to His64 and the de/protonation of His64 depending on the exogenous buffer concentration and the orientation of His64. In the absence of a PT acceptor/donor at position 64, it is found that the excess proton will utilize one of three distinct paths to enter/leave the active site. This latter result not only allows for an increased understanding of how enzymes capitalize on the protein/solvent interface to guide excess protons to/from areas of interest, it also provides valuable insight into the chemical rescue experiments on HCA II mutants.

  17. Interaction between rhein acyl glucuronide and methotrexate based on human organic anion transporters.

    Science.gov (United States)

    Yuan, Yuan; Yang, Hua; Kong, Linghua; Li, Yuan; Li, Ping; Zhang, Hongjian; Ruan, Jianqing

    2017-11-01

    Rhein, a major bioactive compound of many medicinal herbs and the prodrug of diacerein, is often used with low dose of methotrexate as drug combination to treat rheumatoid arthritis. In this study, potential drug-drug interaction between methotrexate and rhein was investigated based on organic anion transporters (OAT). Our study demonstrated that rhein acyl glucuronide (RAG), the major metabolite of rhein in the human blood circulation, significantly inhibited the uptake of p-aminohippurate in hOAT1 transfected cells with IC50 value of 691 nM and estrone sulfate uptake in hOAT3 transfected cells with IC50 value of 78.5 nM. As the substrate of both hOAT1 and hOAT3, the methotrexate transport was significantly inhibited by RAG in hOAT1 transfected cells at 50 μM and hOAT3 transfected cells at 1 μM by 69% and 87%, respectively. Further in vivo study showed that after co-administrated with RAG in rats the AUC0-24 values of methotrexate increased from 3109 to 5370 ng/mL*hr and the t1/2 was prolonged by 40.5% (from 7.4 to 10.4 h), demonstrating the inhibitory effect of RAG on methotrexate excretion. In conclusion, rhein acyl glucuronide could significantly decrease the transport of methotrexate by both hOAT1 and hOAT3. The combination use of rhein, diacerein or other rhein-containing herbs with methotrexate may cause obvious drug-drug interaction and require close monitoring for potential drug interaction in clinical practice. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Duodenal fatty acid sensor and transporter expression following acute fat exposure in healthy lean humans.

    Science.gov (United States)

    Cvijanovic, Nada; Isaacs, Nicole J; Rayner, Christopher K; Feinle-Bisset, Christine; Young, Richard L; Little, Tanya J

    2017-04-01

    Free fatty acids (FFAs) and their derivatives are detected by G-protein coupled receptors (GPRs) on enteroendocrine cells, with specific transporters on enterocytes. It is unknown whether acute fat exposure affects FFA sensors/transporters, and whether this relates to hormone secretion and habitual fat intake. We studied 20 healthy participants (10M, 10F; BMI: 22 ± 1 kg/m 2 ; age: 28 ± 2 years), after an overnight fast, on 2 separate days. On the first day, duodenal biopsies were collected endoscopically before, and after, a 30-min intraduodenal (ID) infusion of 10% Intralipid ® , and relative transcript expression of FFA receptor 1 (FFAR1), FFA receptor 4 (FFAR4), GPR119 and the FFA transporter, cluster of differentiation-36 (CD36) was quantified from biopsies. On the second day, ID Intralipid ® was infused for 120-min, and plasma concentrations of cholecystokinin (CCK) and glucagon-like peptide-1 (GLP-1) evaluated. Habitual dietary intake was assessed using food frequency questionnaires (FFQs). ID Intralipid ® increased expression of GPR119, but not FFAR1, FFAR4 and CD36, and stimulated CCK and GLP-1 secretion. Habitual polyunsaturated fatty acid (PUFA) consumption was negatively associated with basal GPR119 expression. GPR119 is an early transcriptional responder to duodenal lipid in lean humans, although this response appeared reduced in individuals with high PUFA intake. These observations may have implications for downstream regulation of gut hormone secretion and appetite. This study was registered as a clinical trial with the Australia and New Zealand Clinical Trial Registry (Trial number: ACTRN12612000376842). Copyright © 2016 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

  19. Multidrug ABC transporter Cdr1 of Candida albicans harbors specific and overlapping binding sites for human steroid hormones transport.

    Science.gov (United States)

    Baghel, Pratima; Rawal, Manpreet Kaur; Khan, Mohammad Firoz; Sen, Sobhan; Siddiqui, Mohammed Haris; Chaptal, Vincent; Falson, Pierre; Prasad, Rajendra

    2017-10-01

    The present study examines the kinetics of steroids efflux mediated by the Candida drug resistance protein 1 (Cdr1p) and evaluates their interaction with the protein. We exploited our in-house mutant library for targeting the 252 residues forming the twelve transmembrane helices (TMHs) of Cdr1p. The screening revealed 65 and 58 residues critical for β-estradiol and corticosterone transport, respectively. Notably, up to 83% critical residues for corticosterone face the lipid interface compared to 54% for β-estradiol. Molecular docking identified a possible peripheral corticosterone-binding site made of 8/14 critical/non-critical residues between TMHs 3, 4 and 6. β-estradiol transport was severely hampered by alanine replacements of Cdr1p core residues involving TMHs 2, 5 and 8, in a binding site made of 10/14 critical residues mainly shared with rhodamine 6G with which it competes. By contrast, TMH11 was poorly impacted, although being part of the core domain. Finally, we observed the presence of several contiguous stretches of 3-5 critical residues in TMHs 2, 5 and 10 that points to a rotation motion of these helices during the substrate transport cycle. The selective structural arrangement of the steroid-binding pockets in the core region and at the lipid-TMD interface, which was never reported before, together with the possible rotation of some TMHs may be the structural basis of the drug-transport mechanism achieved by these type II ABC transporters. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Stereoselective transport of hydrophilic quaternary drugs by human MDR1 and rat Mdr1b P-glycoproteins

    OpenAIRE

    Guido J E J Hooiveld; Heegsma, Janette; van Montfoort, Jessica E; Jansen, Peter L M; Meijer, Dirk K.F; Müller, Michael

    2002-01-01

    The present study was performed to evaluate and compare the ability of human MDR1-, and rat Mdr1b- and Mdr2-P-glycoproteins to transport hydrophilic monoquaternary drugs. Transport studies were performed with plasma membrane vesicles isolated from MDR1-, Mdr1b-, or Mdr2-overexpressing insect cells.As model substrates we used the N-methylated derivatives of the diastereomers quinidine and quinine, the monoquaternary compounds N-methylquinidine and N-methylquinine. Vincristine, an established M...

  1. [From the discovery of microbial Mep-Amt ammonium transporters to human Rhesus factors].

    Science.gov (United States)

    Boeckstaens, Mélanie

    2016-04-01

    Ammonium, ubiquitous on Earth, plays major and distinct roles in most organisms. While it can be a nitrogen source for many microorganisms and plants, it is a cytotoxic metabolic product actively detoxified by the liver in animals. Furthermore, in the latter, ammonium synthesis in the kidney is involved in acid/base homeostasis. Ammonium transport is ensured by a family of proteins, called Mep-Amt-Rh. This family is conserved in all domains of life and comprises the human Rh factors, notably known in transfusional medicine. While the study of bacterial, fungal and vegetal Mep-Amt transporters reveals a fine-tuned and rapid regulation of these proteins in function of environmental changes, the regulation of animal Rh proteins has been poorly addressed. This review notably highlights the importance of the yeast model in the study of the regulation of these proteins as well as in the functional characterization of Mep-Amt-Rh members of diverse origins. © 2016 médecine/sciences – Inserm.

  2. The presence and near-shore transport of human fecal pollution in Lake Michigan beaches

    Science.gov (United States)

    Molloy, S.L.; Liu, L.B.; Phanikumar, M.S.; Jenkins, T.M.; Wong, M.V.; Rose, J.B.; Whitman, R.L.; Shively, D.A.; Nevers, M.B.

    2005-01-01

    The Great Lakes are a source of water for municipal, agricultural and industrial use, and support significant recreation, commercial and sport fishing industries. Every year millions of people visit the 500 plus recreational beaches in the Great Lakes. An increasing public health risk has been suggested with increased evidence of fecal contamination at the shoreline. To investigate the transport and fate of fecal pollution at Great Lakes beaches and the health risk associated with swimming at these beaches, the near-shore waters of Mt Baldy Beach, Lake Michigan and Trail Creek, a tributary discharging into the lake were examined for fecal pollution indicators. A model of surf zone hydrodynamics coupled with a transport model with first-order inactivation of pollutant was used to understand the relative importance of different processes operating in the surf zone (e.g. physical versus biological processes). The Enterococcus human fecal pollution marker, which targets a putative virulence factor, the enterococcal surface protein (esp) in Enterococcus faecium, was detected in 2/28 samples (7%) from the tributaries draining into Lake Michigan and in 6/30 samples (20%) from Lake Michigan beaches. Preliminary analysis sugg