Cryopreservation of Parathyroid Glands

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Marlon A. Guerrero

2010-01-01

Full Text Available The risk of permanent hypoparathyroidism following thyroid and parathyroid surgery is around 1% in the hands of experienced endocrine surgeons. Although this complication is rare, rendering a patient permanently aparathyroid has significant consequences on the health and quality of life of the patient. Immediate autotransplantation of parathyroid glands that are injured or unintentionally removed offers the best possibility of graft viability and functionality. However, since the majority of cases of hypoparathyroidism are transient, immediate autotransplantation can complicate postoperative surveillance in certain patients, especially those with primary hyperparathyroidism. Cryopreservation of parathyroid tissue is an alternate technique that was developed to treat patients with permanent hypoparathyroidism. This method allows for parathyroid tissue to be stored and then autotransplanted in a delayed fashion once permanent hypoparathyroidism is confirmed. This article provides a contemporary review on cryopreservation of parathyroid tissue and its current role in thyroid and parathyroid surgery.

Cryopreservation of Human Mucosal Leukocytes.

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Sean M Hughes

Full Text Available Understanding how leukocytes in the cervicovaginal and colorectal mucosae respond to pathogens, and how medical interventions affect these responses, is important for developing better tools to prevent HIV and other sexually transmitted infections. An effective cryopreservation protocol for these cells following their isolation will make studying them more feasible.To find an optimal cryopreservation protocol for mucosal mononuclear leukocytes, we compared cryopreservation media and procedures using human vaginal leukocytes and confirmed our results with endocervical and colorectal leukocytes. Specifically, we measured the recovery of viable vaginal T cells and macrophages after cryopreservation with different cryopreservation media and handling procedures. We found several cryopreservation media that led to recoveries above 75%. Limiting the number and volume of washes increased the fraction of cells recovered by 10-15%, possibly due to the small cell numbers in mucosal samples. We confirmed that our cryopreservation protocol also works well for both endocervical and colorectal leukocytes. Cryopreserved leukocytes had slightly increased cytokine responses to antigenic stimulation relative to the same cells tested fresh. Additionally, we tested whether it is better to cryopreserve endocervical cells on the cytobrush or in suspension.Leukocytes from cervicovaginal and colorectal tissues can be cryopreserved with good recovery of functional, viable cells using several different cryopreservation media. The number and volume of washes has an experimentally meaningful effect on the percentage of cells recovered. We provide a detailed, step-by-step protocol with best practices for cryopreservation of mucosal leukocytes.

Cryopreservation of mutton snapper ( Lutjanus analis sperm

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EDUARDO G. SANCHES

2013-09-01

Full Text Available This study aimed to develop a protocol of semen cryopreservation of the mutton snapper Lutjanus analis. The interaction between three extenders ( pH 6.1; 7.8 and 8.2 , two concentrations of dimethyl sulfoxide ( DMSO, 5 and 10% and three cooling rates ( -90; -60 and -30°C.min−1 on the sperm motility rate and motility time were analyzed by a factorial experiment. A sample of 30 fishes ( 1,261 ± 449 g collected in the nature was kept in floating net cages. The semen was frozen by using cryogenic straws, in nitrogen vapour and transferred, later, to liquid nitrogen. Fertilization test was accomplished to evaluate the viability of the cryopreserved sperm. The highest sperm motility rate and motility time ( P < 0.05 was achieved by combining extender C ( pH 8.2 with DMSO ( 10% and cooling rate of -60°C.min−1 ( P < 0.05 . The use of cryopreserved sperm presented fertilization rates higher than 59% validating the present protocol for mutton snapper.

Cryopreservation of preimplantation embryos of cattle, sheep, and goats.

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Youngs, Curtis R

2011-08-05

Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage. Preimplantation embryos consist predominantly of water, and the avoidance of intracellular ice crystal formation during the cryopreservation process is of paramount importance to maintain embryo viability. Embryos are placed into a hypertonic solution (1.4 - 1.5 M) of a cryoprotective agent (CPA) such as ethylene glycol (EG) or glycerol (GLYC) to create an osmotic gradient that facilitates cellular dehydration. After embryos reach osmotic equilibrium in the CPA solution, they are individually loaded in the hypertonic CPA solution into 0.25 ml plastic straws for freezing. Embryos are placed into a controlled rate freezer at a temperature of -6°C. Ice crystal formation is induced in the CPA solution surrounding the embryo, and crystallization causes an increase in the concentration of CPA outside of the embryo, causing further cellular dehydration. Embryos are cooled at a rate of 0.5°C/min, enabling further dehydration, to a temperature of -34°C before being plunged into liquid nitrogen (-196°C). Cryopreserved embryos must be thawed prior to transfer to a recipient (surrogate) female. Straws containing the embryos are removed from the liquid nitrogen dewar, held in room temperature air for 3 to 5 sec, and placed into a 37°C water bath for 25 to 30 sec. Embryos cryopreserved in GLYC are placed into a 1 M solution of sucrose for 10 min for removal of the CPA before transfer to a recipient (surrogate) female. Embryos cryopreserved in EG, however, may be directly transferred to the uterus of a recipient.

Effect of cryopreservation and lyophilization on viability and growth of strict anaerobic human gut microbes.

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Bircher, Lea; Geirnaert, Annelies; Hammes, Frederik; Lacroix, Christophe; Schwab, Clarissa

2018-04-17

Strict anaerobic gut microbes have been suggested as 'next-generation probiotics' for treating several intestinal disorders. The development of preservation techniques is of major importance for therapeutic application. This study investigated cryopreservation (-80°C) and lyophilization survival and storage stability (4°C for 3 months) of the strict anaerobic gut microbes Bacteroides thetaiotaomicron, Faecalibacterium prausnitzii, Roseburia intestinalis, Anaerostipes caccae, Eubacterium hallii and Blautia obeum. To improve preservation survival, protectants sucrose and inulin (both 5% w/v) were added for lyophilization and were also combined with glycerol (15% v/v) for cryopreservation. Bacterial fitness, evaluated by maximum growth rate and lag phase, viability and membrane integrity were determined using a standardized growth assay and by flow cytometry as markers for preservation resistance. Lyophilization was more detrimental to viability and fitness than cryopreservation, but led to better storage stability. Adding sucrose and inulin enhanced viability and the proportion of intact cells during lyophilization of all strains. Viability of protectant-free B. thetaiotaomicron, A. caccae and F. prausnitzii was above 50% after cryopreservation and storage and increased to above 80% if protectants were present. The addition of glycerol, sucrose and inulin strongly enhanced the viability of B. obeum, E. hallii and R. intestinalis from 0.03-2% in protectant-free cultures to 11-37%. This is the first study that quantitatively compared the effect of cryopreservation and lyophilization and the addition of selected protectants on viability and fitness of six strict anaerobic gut microbes. Our results suggest that efficiency of protectants is process- and species-specific. © 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Proximate composition analysis posterior to the cryopreservation of Chaetoceros calcitrans

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Joan Salas-Leiva

2015-12-01

Full Text Available Objective. The effect of cryopreservation on the proximate composition of microalgae Chaetoceros calcitrans was evaluated. Materials and methods. C. calcitrans was cultured and cryopreserved using 5% (v/v dimethylsulfoxide as cryoprotectant. The freezing was controlled at a rate of 3°C/min, up to -60°C and then the microalgae were immersed in liquid nitrogen (-196°C. After storage in liquid nitrogen, microalgae were rapidly thawed out and subcultured. The percentage of proteins, lipids and carbohydrates was analyzed using absorption spectrophotometry and the organic matter was studied by gravimetric analysis. Results. There was no significant variation between the proximate composition of C. calcitrans cryopreserved and the controls (p>0.05. Conclusion. Our results show that, despite low cell recovery after the preservation of this organism at low temperatures, there is no apparent loss of nutritional characteristics caused by the storing process at low temperatures.

In vitro evaluation of encapsulated primary rat hepatocytes pre- and post-cryopreservation at -80°C and in liquid nitrogen.

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Durkut, Serap; Elçin, A Eser; Elçin, Y Murat

2015-02-01

Encapsulation techniques have the potential to protect hepatocytes from cryoinjury. In this study, we comparatively evaluated the viability and metabolic function of primary rat hepatocytes encapsulated in calcium alginate microbeads, in chitosan tripolyphosphate beads, and in three-layered alginate-chitosan-alginate (ACA) microcapsules, before and after cryopreservation at -80°C and in liquid nitrogen (LN2) for 1 and 3 months. Findings demonstrated that LN2 was atop of -80°C in regard to preservation of viability (> 90%) and hepatic functions. LN2-cryopreserved hepatocytes encapsulated in ACA microcapsules retained metabolic function post-thawing, with > 90% of the albumin, total protein and urea syntheses activities, and > 80% of oxidative function.

Cryopreservation of microencapsulated canine sperm.

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Shah, Shambhu; Otsuki, Tsubasa; Fujimura, Chika; Yamamoto, Naoki; Yamashita, Yasuhisa; Higaki, Shogo; Hishinuma, Mitsugu

2011-03-01

The objective was to develop a method for cryopreserving microencapsulated canine sperm. Pooled ejaculates from three beagle dogs were extended in egg yolk tris extender and encapsulated using alginate and poly-L-lysine at room temperature. The microcapsules were cooled at 4 °C, immersed in pre-cooled extender (equivalent in volume to the microcapsules) to reach final concentration of 7% (v/v) glycerol and 0.75% (v/v) Equex STM paste, and equilibrated for 5, 30 and 60 min at 4 °C. Thereafter, microcapsules were loaded into 0.5 mL plastic straws and frozen in liquid nitrogen. In Experiment 1, characteristics of microencapsulated canine sperm were evaluated after glycerol addition at 4 °C. Glycerol exposure for 5, 30 and 60 min did not significantly affect progressive motility, viability, or acrosomal integrity of microencapsulated sperm compared with pre-cooled unencapsulated sperm (control). In Experiment 2, characteristics of frozen-thawed canine microencapsulated sperm were evaluated at 0, 3, 6, and 9 h of culture at 38.5 °C. Pre-freeze glycerol exposure for 5, 30, and 60 min at 4 °C did not influence post-thaw quality in unencapsulated sperm. Post-thaw motility and acrosomal integrity of microencapsulated sperm decreased more than those of unencapsulated sperm (P < 0.05) following glycerol exposure for 5 min. However, motility, viability and acrosomal integrity of microencapsulated sperm after 30 and 60 min glycerol exposure were higher than unencapsulated sperm cultured for 6 or 9 h (P < 0.05). In conclusion, since microencapsulated canine sperm were successfully cryopreserved, this could be a viable alternative to convention sperm cryopreservation in this species. Copyright © 2011 Elsevier Inc. All rights reserved.

Cryopreservation of Arachis pintoi (leguminosae) somatic embryos.

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Rey, H Y; Faloci, M; Medina, R; Dolce, N; Engelmann, F; Mroginski, L

2013-01-01

In this study, we successfully cryopreserved cotyledonary somatic embryos of diploid and triploid Arachis pintoi cytotypes using the encapsulation-dehydration technique. The highest survival rates were obtained when somatic embryos were encapsulated in calcium alginate beads and precultured in agitated (80 rpm) liquid establishment medium (EM) with daily increasing sucrose concentration (0.50, 0.75, and 1.0 M). The encapsulated somatic embryos were then dehydrated with silica gel for 5 h to 20% moisture content (fresh weight basis) and cooled either rapidly (direct immersion in liquid nitrogen, LN) or slowly (1 degree C per min from 25 degree C to -30 degree C followed by immersion in LN). Beads were kept in LN for a minimum of 1 h and then were rapidly rewarmed in a 30 degree C water-bath for 2 min. Finally, encapsulated somatic embryos were post-cultured in agitated (80 rpm) liquid EM with daily decreasing sucrose concentration (0.75 and 0.5 M) and transferred to solidified EM. Using this protocol, we obtained 26% and 30% plant regeneration from cryopreserved somatic embryos of diploid and triploid cytotypes. No morphological abnormalities were observed in any of the plants regenerated from cryopreserved embryos and their genetic stability was confirmed with 10 isozyme systems and nine RAPD profiles.

Seedling development and evaluation of genetic stability of cryopreserved Dendrobium hybrid mature seeds.

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Galdiano, Renato Fernandes; de Macedo Lemos, Eliana Gertrudes; de Faria, Ricardo Tadeu; Vendrame, Wagner Aparecido

2014-03-01

Vitrification, a simple, fast, and recommended cryopreservation method for orchid germplasm conservation, was evaluated for Dendrobium hybrid "Dong Yai" mature seeds. The genetic stability of regenerated seedlings was also evaluated using flow cytometry. Mature seeds from this hybrid were submitted to plant vitrification solution (PVS2) for 0, 0.5, 1, 2, 3, 4, 5, or 6 h at 0 °C. Subsequently, they were plunged into liquid nitrogen (LN) at -196 °C for 1 h and recovered in half-strength Murashige and Skoog culture medium (1/2 MS), and seed germination was evaluated after 30 days. Seeds directly submitted to LN did not germinate after cryopreservation. Seeds treated with PVS2 between 1 and 3 h presented the best germination (between 51 and 58%), although longer exposure to PVS2 returned moderated germination (39%). Germinated seeds were further subcultured in P-723 culture medium and developed whole seedlings in vitro after 180 days, with no abnormal characteristics, diseases, or nutritional deficiencies. Seedlings were successfully acclimatized under greenhouse conditions with over 80% survival. Flow cytometry analysis revealed no chromosomal changes on vitrified seedlings, as well as seedlings germinated from the control treatment (direct exposure to LN). These findings indicate that vitrification is a feasible and safe germplasm cryopreservation method for commercial Dendrobium orchid hybrid conservation.

Cryopreservation, semen use and the likelihood of fatherhood in male Hodgkin lymphoma survivors: an EORTC-GELA Lymphoma Group cohort study.

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van der Kaaij, M A E; van Echten-Arends, J; Heutte, N; Meijnders, P; Abeilard-Lemoisson, E; Spina, M; Moser, E C; Allgeier, A; Meulemans, B; Lugtenburg, P J; Aleman, B M P; Noordijk, E M; Fermé, C; Thomas, J; Stamatoullas, A; Fruchart, C; Eghbali, H; Brice, P; Smit, W G J M; Sebban, C; Doorduijn, J K; Roesink, J M; Gaillard, I; Coiffier, B; Lybeert, M L M; Casasnovas, O; André, M; Raemaekers, J M M; Henry-Amar, M; Kluin-Nelemans, J C

2014-03-01

How does the successful cryopreservation of semen affect the odds of post-treatment fatherhood among Hodgkin lymphoma (HL) survivors? Among 334 survivors who wanted to have children, the availability of cryopreserved semen doubled the odds of post-treatment fatherhood. Cryopreservation of semen is the easiest, safest and most accessible way to safeguard fertility in male patients facing cancer treatment. Little is known about what proportion of patients achieve successful semen cryopreservation. To our knowledge, neither the factors which influence the occurrence of semen cryopreservation nor the rates of fatherhood after semen has been cryopreserved have been analysed before. This is a cohort study with nested case-control analyses of consecutive Hodgkin survivors treated between 1974 and 2004 in multi-centre randomized controlled trials. A written questionnaire was developed and sent to 1849 male survivors. Nine hundred and two survivors provided analysable answers. The median age at treatment was 31 years. The median follow-up after cryopreservation was 13 years (range 5-36). Three hundred and sixty-three out of 902 men (40%) cryopreserved semen before the start of potentially gonadotoxic treatment. The likelihood of semen cryopreservation was influenced by age, treatment period, disease stage, treatment modality and education level. Seventy eight of 363 men (21%) used their cryopreserved semen. Men treated between 1994 and 2004 had significantly lower odds of cryopreserved semen use compared with those treated earlier, whereas alkylating or second-line (chemo)therapy significantly increased the odds of use; no other influencing factors were identified. We found an adjusted odds ratio of 2.03 (95% confidence interval 1.11-3.73, P = 0.02) for post-treatment fatherhood if semen cryopreservation was performed. Forty-eight out of 258 men (19%) who had children after HL treatment became a father using cryopreserved semen. Data came from questionnaires and so this

ROLE OF MONOCYTE PHAGOCYTIC SYSTEM IN FORMATION OF ANTIVIRAL RESISTANCE IN MICE AFTER PRELIMINARY INJECTION OF CRYOPRESERVED CORD BLOOD

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Kozhina OYu

2013-03-01

Full Text Available Now the task of preventive maintenance and search of biologically active substances, capable to make active the nonspecific immune response, remains an actual during flu epidemic. It has been previously established, that cryopreserved leucoconcentrate of human cord blood (cLHCB can act as modulator of activity of immunity. In the given work there was estimated influence of preventive injection of cLHCB and its components on functional activity of monocyte phagocytic system cells (MPSC in mice in the conditions of the induced influenzal infection. Preliminary introduction of cLHCB and its components 6 months prior to infection by flu virus makes 2 times increase of functional activity of macrophages, preventing inhibition of a nonspecific link of immunity. Thus, cLHCB inhibit of secondary immune deficiency development. The found increase in phagocytic activity of peritoneal cavity cells and 3 times increasesing of CD11b-marker expression after preventive injection of cLHCB testifies to rise of adherence and protective potential of MPSC that is one of possible mechanisms of formation of resistance to a flu virus. It is shown, that intranasal cLHCB injection before development of viral infection it can be o recommended as the method of preventive maintenance of flu.

Cryopreservation of PLBs of Brassidium Fly Away Using Encapsulation-Dehydration Technique

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Arulvilee Rajasegar

2015-12-01

Full Text Available In vitro grown protocorm-like bodies (PLBs of Brassidium Fly Away orchid hybrid were cryopreserved using encapsulation- dehydration technique. The viability of the cryopreserved cells was determined by 2,3,5-triphenyltetrazolium chloride (TTC assay. For the preculture treatment, the PLBs were excised into two standard sizes of 1-2 and 4-5 mm and were precultured on half-strength Murashige and Skoog (MS semi solid medium supplemented with diff erent concentrations of sucrose (0, 0.2, 0.4, 0.6, 0.8 and 1.0M. The PLBs size 4-5 mm and 0.6 M sucrose concentration was selected based on highest viability obtained in TTC assay. The PLBs were encapsulated for 30 minutes using 3% (w/v liquid s odium alginate medium supplemented with 0.4M sucrose and 0.1M calcium chl oride and osmoprotected in 0.75M sucrose solution for 24 hours at 25°C. Th e beads were then dehydrated using 50g heat-sterilised silica gel for four hours , cryopreserved for 24 hours, thawed in a 40±2°C water bath for 90 seconds, and r egenerated in semi-solid half-strength. Biochemical analyses were conducted and th e cryopreserved PLBs had produced lower content of chlorophyll while the highest specifi c peroxidase activity was observed in cryopreserved PLBs

Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification.

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Sakai, A; Kobayashi, S; Oiyama, I

1990-06-01

The nucellar cells of navel orange(Citrus sinensis Osb. var. brasiliensis Tanaka) were successfully cryopreserved by vitrification. In this method, cells were sufficiently dehydrated with highly concentrated cryoprotective solution(PVS2) prior to direct plunge in liquid nitrogen. The PVS2 contains(w/v) 30% glycerol, 15% ethylene glycol and 15% DMSO in Murashige-Tucker medium(MT) containing 0.15 M sucrose. Cells were treated with 60% PVS2 at 25°C for 5 min and then chilled PVS2 at 0°C for 3 min. The cell suspension of about 0.1 ml was loaded in a 0.5 ml transparent plastic straw and directly plunged in liquid nitrogen for 30 min. After rapid warming, the cell suspension was expelled in 2 ml of MT medium containing 1.2 M sucrose. The average rate of survival was about 80%. The vitrified cells regenerated plantlets. This method is very simple and the time required for cryopreservation is only about 10 min.

Cryopreservation studies on the marine diatom Navicula subinflata Grun

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Redekar, P.D.; Wagh, A.B.

of cryoprotectant decreased the growth. These experiments after cryopreservation revealed that the maximum growth was recorded in Ethylene glycol treated cells for 0 day sample and 7 days and 30 days liquid nitrogen preserved samples. No growth was observed...

Impact of procedural steps and cryopreservation agents in the cryopreservation of chlorophyte microalgae.

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Tony V L Bui

Full Text Available The maintenance of traditional microalgae collections based on liquid and solid media is labour intensive, costly and subject to contamination and genetic drift. Cryopreservation is therefore the method of choice for the maintenance of microalgae culture collections, but success is limited for many species. Although the mechanisms underlying cryopreservation are understood in general, many technical variations are present in the literature and the impact of these are not always elaborated. This study describes two-step cryopreservation processes in which 3 microalgae strains representing different cell sizes were subjected to various experimental approaches to cryopreservation, the aim being to investigate mechanistic factors affecting cell viability. Sucrose and dimethyl sulfoxide (DMSO were used as cryoprotectants. They were found to have a synergistic effect in the recovery of cryopreserved samples of many algal strains, with 6.5% being the optimum DMSO concentration. The effect of sucrose was shown to be due to improved cell survival and recovery after thawing by comparing the effect of sucrose on cell viability before or after cryopreservation. Additional factors with a beneficial effect on recovery were the elimination of centrifugation steps (minimizing cell damage, the reduction of cell concentration (which is proposed to reduce the generation of toxic cell wall components and the use of low light levels during the recovery phase (proposed to reduce photooxidative damage. The use of the best conditions for each of these variables yielded an improved protocol which allowed the recovery and subsequent improved culture viability of a further 16 randomly chosen microalgae strains. These isolates included species from Chlorellaceae, Palmellaceae, Tetrasporaceae, Palmellopsis, Scenedesmaceae and Chlamydomonadaceae that differed greatly in cell diameter (3-50 µm, a variable that can affect cryopreservation success. The collective improvement

Viability of ectomycorrhizal fungi following cryopreservation.

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Crahay, Charlotte; Declerck, Stéphane; Colpaert, Jan V; Pigeon, Mathieu; Munaut, Françoise

2013-02-01

The use of ectomycorrhizal (ECM) fungi in biotechnological processes requires their maintenance over long periods under conditions that maintain their genetic, phenotypic, and physiological stability. Cryopreservation is considered as the most reliable method for long-term storage of most filamentous fungi. However, this technique is not widespread for ECM fungi since many do not survive or exhibit poor recovery after freezing. The aim of this study was to develop an efficient cryopreservation protocol for the long-term storage of ECM fungi. Two cryopreservation protocols were compared. The first protocol was the conventional straw protocol (SP). The mycelium of the ECM isolates was grown in Petri dishes on agar and subsequently collected by punching the mycelium into a sterile straw before cryopreservation. In the second protocol, the cryovial protocol (CP), the mycelium of the ECM isolates was grown directly in cryovials filled with agar and subsequently cryopreserved. The same cryoprotectant solution, freezing, and thawing process, and re-growth conditions were used in both protocols. The survival (positive when at least 60 % of the replicates showed re-growth) was evaluated before and immediately after freezing as well as after 1 week, 1 m, and 6 m of storage at -130 °C. Greater survival rate (80 % for the CP as compared to 25 % for the SP) and faster re-growth (within 10 d for the CP compared to the 4 weeks for the SP) were observed for most isolates with the CP suggesting that the preparation of the cultures prior to freezing had a significant impact on the isolates survival. The suitability of the CP for cryopreservation of ECM fungi was further confirmed on a set of 98 ECM isolates and displayed a survival rate of 88 % of the isolates. Only some isolates belonging to Suillus luteus, Hebeloma crustuliniforme, Paxillus involutus and Thelephora terrestris failed to survive. This suggested that the CP is an adequate method for the ultra-low cryopreservation of

Augmented dried versus cryopreserved amniotic membrane as an ocular surface dressing.

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Claire L Allen

Full Text Available Dried amniotic membrane (AM can be a useful therapeutic adjunct in ophthalmic surgery and possesses logistical advantages over cryopreserved AM. Differences in preservation techniques can significantly influence the biochemical composition and physical properties of AM, potentially affecting clinical efficacy. This study was established to investigate the biochemical and structural effects of drying AM in the absence and presence of saccharide lyoprotectants and its biocompatibility compared to cryopreserved material.AM was cryopreserved or dried with and without pre-treatment with trehalose or raffinose and the antioxidant epigallocatechin (EGCG. Structural and visual comparisons were assessed using electron microscopy. Localisation, expression and release of AM biological factors were determined using immunoassays and immunofluorescence. The biocompatibility of the AM preparations co-cultured with corneal epithelial cell (CEC or keratocyte monolayers were assessed using cell proliferation, cytotoxicity, apoptosis and migration assays.Drying devitalised AM epithelium, but less than cryopreservation and cellular damage was reduced in dried AM pre-treated with trehalose or raffinose. Dried AM alone, and with trehalose or raffinose showed greater factor retention efficiencies and bioavailability compared to cryopreserved AM and demonstrated a more sustained biochemical factor time release in vitro. Cellular health assays showed that dried AM with trehalose or raffinose are compatible and superior substrates compared to cryopreserved AM for primary CEC expansion, with increased proliferation and reduced LDH and caspase-3 levels. This concept was supported by improved wound healing in an immortalised human CEC line (hiCEC co-cultured with dried and trehalose or raffinose membranes, compared to cryopreserved and fresh AM.Our modified preservation process and our resultant optimised dried AM has enhanced structural properties and biochemical stability

Effects of some cryopreservation procedures on recalcitrant zygotic embryos of Ammocharis coranica.

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Nomali, Z; Ngobese; Sershen; Berjak, P; Pammenter, N W

2014-01-01

Cryopreservation, the most promising method for the long-term conservation of recalcitrant (desiccation-sensitive) seed germplasm, is often associated with high viability losses. Cryo-procedures involve a sequence of steps which must be optimised to reduce the impact of the stresses. This study reports on the effects of some of the steps of cryopreservation on the recalcitrant zygotic embryos of the amaryllid, Ammocharis coranica. Embryos were subjected to cryoprotection with glycerol and/or DMSO, rapid (flash) drying, and rapid (>100 degree C s(-1)) or slow (1 degree C s(-1)) cooling. Rapid dehydration (from c. 2.7 to 0.9 g g(-1) over 60 min) and cooling had a detrimental effect on the viability of the embryos, which was exacerbated when these steps were applied sequentially. After cooling, seedling production (30%) was obtained only from embryos that had been cryoprotected with glycerol prior to drying and rapid cooling, while 30% of non-treated embryos and 70% of those that had undergone cathodic protection during flash drying produced callus. Noting that no post-cryo survival of A. coranica embryos had previously been obtained, this study identified cryoprotection with glycerol and the incorporation of cathodic protection during flash drying as promising intervention points for future studies.

Cryopreservation of dermal fibroblasts and keratinocytes in hydroxyethyl starch-based cryoprotectants.

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Naaldijk, Yahaira; Johnson, Adiv A; Friedrich-Stöckigt, Annett; Stolzing, Alexandra

2016-12-01

Preservation of human skin fibroblasts and keratinocytes is essential for the creation of skin tissue banks. For successful cryopreservation of cells, selection of an appropriate cryoprotectant agent (CPA) is imperative. The aim of this study was to identify CPAs that minimize toxic effects and allow for the preservation of human fibroblasts and keratinocytes in suspension and in monolayers. We cryopreserved human fibroblasts and keratinocytes with different CPAs and compared them to fresh, unfrozen cells. Cells were frozen in the presence and absence of hydroxyethyl starch (HES) or dimethyl sulfoxide (DMSO), the latter of which is a commonly used CPA known to exert toxic effects on cells. Cell numbers were counted immediately post-thaw as well as three days after thawing. Cellular structures were analyzed and counted by labeling nuclei, mitochondria, and actin filaments. We found that successful cryopreservation of suspended or adherent keratinocytes can be accomplished with a 10% HES or a 5% HES, 5% DMSO solution. Cell viability of fibroblasts cryopreserved in suspension was maintained with 10% HES or 5% HES, 5% DMSO solutions. Adherent, cryopreserved fibroblasts were successfully maintained with a 5% HES, 5% DMSO solution. We conclude that skin tissue cells can be effectively cryopreserved by substituting all or a portion of DMSO with HES. Given that DMSO is the most commonly used CPA and is believed to be more toxic than HES, these findings are of clinical significance for tissue-based replacement therapies. Therapies that require the use of keratinocyte and fibroblast cells, such as those aimed at treating skin wounds or skin burns, may be optimized by substituting a portion or all of DMSO with HES during cryopreservation protocols.

Behavior of lateral buds of Hancornia speciosa after cryopreservation by encapsulation-vitrification

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Débora de Oliveira Prudente

2017-05-01

Full Text Available Hancornia speciosa is a fruitful species from Cerrado biome with high economic potential. However, the intense and disordered extractivism have caused a reduction of its population in its endemic area. In addition, seed recalcitrance negatively affects the conventional conservation of the species. Aiming to find alternatives that enable the long-term conservation of this species, the study’s objective was to assess the behavior of lateral bud’s regrowth after cryopreservation procedures by encapsulation-vitrification technique. Sodium alginate capsules containing lateral buds were pre-cultured in liquid WPM supplemented with 1.0 M glycerol, and subsequently exposed to different concentrations of sucrose (0.3; 0.75 and 1.0 M for 24 or 48 hours. The capsules were subjected to dehydration in silica gel or airflow hood for 0, 1, 2 and 3 hours before different incubation times in PVS2 (0, 15, 30, 60 and 120 minutes at 0°C. A high regeneration percentage of lateral buds was observed after cryopreservation of capsules treated with 0.75 M sucrose plus 1.0 M glycerol (24 hours, associated with dehydration in an airflow hood (1 hour and immersion in PVS2 (15 minutes. Encapsulation-vitrification allowed the long-term conservation, and provided high plant material survival rates after cryopreservation of Hancornia speciosa sensitive explants.

B Cells and B Cell Blasts Withstand Cryopreservation While Retaining Their Functionality for Producing Antibody.

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Fecher, Philipp; Caspell, Richard; Naeem, Villian; Karulin, Alexey Y; Kuerten, Stefanie; Lehmann, Paul V

2018-05-31

In individuals who have once developed humoral immunity to an infectious/foreign antigen, the antibodies present in their body can mediate instant protection when the antigen re-enters. Such antigen-specific antibodies can be readily detected in the serum. Long term humoral immunity is, however, also critically dependent on the ability of memory B cells to engage in a secondary antibody response upon re-exposure to the antigen. Antibody molecules in the body are short lived, having a half-life of weeks, while memory B cells have a life span of decades. Therefore, the presence of serum antibodies is not always a reliable indicator of B cell memory and comprehensive monitoring of humoral immunity requires that both serum antibodies and memory B cells be assessed. The prevailing view is that resting memory B cells and B cell blasts in peripheral blood mononuclear cells (PBMC) cannot be cryopreserved without losing their antibody secreting function, and regulated high throughput immune monitoring of B cell immunity is therefore confined to-and largely limited by-the need to test freshly isolated PBMC. Using optimized protocols for freezing and thawing of PBMC, and four color ImmunoSpot ® analysis for the simultaneous detection of all immunoglobulin classes/subclasses we show here that both resting memory B cells and B cell blasts retain their ability to secrete antibody after thawing, and thus demonstrate the feasibility of B cell immune monitoring using cryopreserved PBMC.

B Cells and B Cell Blasts Withstand Cryopreservation While Retaining Their Functionality for Producing Antibody

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Philipp Fecher

2018-05-01

Full Text Available In individuals who have once developed humoral immunity to an infectious/foreign antigen, the antibodies present in their body can mediate instant protection when the antigen re-enters. Such antigen-specific antibodies can be readily detected in the serum. Long term humoral immunity is, however, also critically dependent on the ability of memory B cells to engage in a secondary antibody response upon re-exposure to the antigen. Antibody molecules in the body are short lived, having a half-life of weeks, while memory B cells have a life span of decades. Therefore, the presence of serum antibodies is not always a reliable indicator of B cell memory and comprehensive monitoring of humoral immunity requires that both serum antibodies and memory B cells be assessed. The prevailing view is that resting memory B cells and B cell blasts in peripheral blood mononuclear cells (PBMC cannot be cryopreserved without losing their antibody secreting function, and regulated high throughput immune monitoring of B cell immunity is therefore confined to—and largely limited by—the need to test freshly isolated PBMC. Using optimized protocols for freezing and thawing of PBMC, and four color ImmunoSpot® analysis for the simultaneous detection of all immunoglobulin classes/subclasses we show here that both resting memory B cells and B cell blasts retain their ability to secrete antibody after thawing, and thus demonstrate the feasibility of B cell immune monitoring using cryopreserved PBMC.

Recovery and cryopreservation of epididymal sperm from agouti (Dasiprocta aguti) using powdered coconut water (ACP-109c) and Tris extenders.

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Silva, M A; Peixoto, G C X; Santos, E A A; Castelo, T S; Oliveira, M F; Silva, A R

2011-10-01

The objective was to compare the use of powdered coconut water (ACP-109c; ACP Biotecnologia, Fortaleza, CE, Brazil) and Tris extenders for recovery and cryopreservation of epididymal sperm from agouti. The caudae epididymus and proximal ductus deferens from 10 sexually mature agoutis were subjected to retrograde washing using ACP-109c (ACP Biotecnologia) or Tris. Epididymal sperm were evaluated for motility, vigor, sperm viability, membrane integrity, and morphology. Samples were centrifuged, and extended in the same diluents plus egg yolk (20%) and glycerol (6%), frozen in liquid nitrogen, and subsequently thawed at 37°C for 1 min, followed by re-evaluation of sperm characteristics. The two extenders were similarly efficient for epididymal recovery, with regard to the number and quality of sperm recovered. However, for both extenders, sperm quality decreased (P Biotecnologia) group, which was significantly better than 9.7 ± 2.6% motile sperm with 1.2 ± 0.3 vigor in Tris. In conclusion, agouti epididymal sperm were successfully recovered using either ACP-109c (ACP Biotecnologia) or Tris extenders; however, ACP-109c (ACP Biotecnologia) was a significantly better extender for processing and cryopreserving these sperm. Copyright © 2011 Elsevier Inc. All rights reserved.

Directional freezing for the cryopreservation of adherent mammalian cells on a substrate

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Braslavsky, Ido

2018-01-01

Successfully cryopreserving cells adhered to a substrate would facilitate the growth of a vital confluent cell culture after thawing while dramatically shortening the post-thaw culturing time. Herein we propose a controlled slow cooling method combining initial directional freezing followed by gradual cooling down to -80°C for robust preservation of cell monolayers adherent to a substrate. Using computer controlled cryostages we examined the effect of cooling rates and dimethylsulfoxide (DMSO) concentration on cell survival and established an optimal cryopreservation protocol. Experimental results show the highest post-thawing viability for directional ice growth at a speed of 30 μm/sec (equivalent to freezing rate of 3.8°C/min), followed by gradual cooling of the sample with decreasing rate of 0.5°C/min. Efficient cryopreservation of three widely used epithelial cell lines: IEC-18, HeLa, and Caco-2, provides proof-of-concept support for this new freezing protocol applied to adherent cells. This method is highly reproducible, significantly increases the post-thaw cell viability and can be readily applied for cryopreservation of cellular cultures in microfluidic devices. PMID:29447224

Data on antioxidant activity in grapevine (Vitis vinifera L.) following cryopreservation by vitrification

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María Fernanda Lazo-Javalera; Martín Ernesto Tiznado-Hernández; Irasema Vargas-Arispuro; Elisa Valenzuela-Soto; María del Carmen Rocha-Granados; Marcos Edel Martínez-Montero; Marisela Rivera-Domínguez

2015-01-01

Cryopreservation is used for the long-term conservation of plant genetic resources. This technique very often induces lethal injury or tissue damage. In this study, we measured indicators of viability and cell damage following cryopreservation and vitrification-cryopreservation in Vitis vinifera L. axillary buds cv. ?Flame seedless? stored in liquid nitrogen (LN) for: three seconds, one hour, one day, one week and one month; after LN thawed at 38??C for three minutes. The enzymatic activity o...

Experimental study on active specific immunotherapy utilizing the immune reaction of low-dose irradiated tumor tissue, 9

International Nuclear Information System (INIS)

Ogawa, Yasuhiro; Maeda, Tomoho; Yoshida, Shoji

1983-01-01

We have already reported the remarkable effect of an active specific immunotherapy using cryopreserved tumor cells and infiltrating mononuclear cells prepared from a low-dose irradiated tumor tissue after cytoreductive radiotherapy. In the present study, the effect of a biological response modifier, OK-432 combined with the active specific immunotherapy was investigated. Twelve-week-aged female C3H/He mice transplanted with MM 46 tumor cells were received local radiotherapy with a dose of 3,000 rads by high energy electron beams on the fifth day after inoculation. The tumor cells and infiltrating mononuclear cells cryopreserved for two months were thawed and treated with mitomycin-C at concentration of 20 μg/10 7 cells at 37 0 C for 30 min. Then, these cells were injected subcutaneously into the left hind paws as a mitomycin C-treated, cryopreserved active specific immunotherapy on the thirteenth day, and daily dose of 1 KE of OK-432 was injected intraperitoneally from the sixth to the tenth days. The inhibition of the tumor growth was similarly observed in the group which received this active specific immunotherapy combined with a biological response modifier, OK-432. (author)

Comparison of glycerolisation with cryopreservation methods on HIV-1 inactivation

International Nuclear Information System (INIS)

Van Baare, J.; Pagnon, J.; Cameron, P.; Vardaxis, N.; Middlekoop, E.; Crowe, S.

1999-01-01

Cryopreservation and glycerolisation are two successful long-term preservation methods for human cadaveric donor skin, which is used in the treatment of bum patients. High concentrations of glycerol has been shown to be antibacterial and virucidal. Because fear of possible transmission of HIV-1 following allograft transplantation, this study was undertaken to investigate whether HIV can be effectively eliminated from skin explants. HIV-1 Ba-L, which has been shown to infect monocytes in skin explants and also dendritic cells, was. For the experiments we used cell-free virus, exogenously HIV infected peripheral blood mononuclear cells (PBMCs) and exogenously HIV infected cadaver split skin. Different concentrations of glycerol at various temperatures and the glycerolisation procedure as used by the Euro Skin Bank were used to determine the effects on HIV-1 Ba-L infectivity. For the cryopreservation technique we used 10% DMSO and a controlled rate freezer. HIV-1 Ba-L transfer was determined by adding uninfected PBMCs to the infected material and reverse transcriptase was measured. Cell-free HIV-1 Ba-L was not inactivated by 50% glycerol but was effectively inactivated within 30 minutes by 70% and 85% glycerol at 4 degree C, room temperature and 37 degree C. In contrast, cell-free HIV-1 Ba-L was not inactivated by cryopreservation. Most importantly, we have shown that HIV-1 Ba-L present in split skin is inactivated by incubating skin in 70% glycerol for three hours at 37-C. HIV in exogenously infected skin was not inactivated by cryopreservation. High concentrations of glycerol effectively inactivates free HIV-1 Ba-L and intracellular HIV-1 Ba-L. Also the current glycerolisation procedure carried out by the Euro Skin Bank effectively inactivates infectious virus. However, the cryopreservation technique did not show any reduction in HIV-1 Ba-L infectivity

Effects of fenbendazole on routine immune response parameters of BALB/c mice.

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Cray, Carolyn; Villar, David; Zaias, Julia; Altman, Norman H

2008-11-01

Fenbendazole (FBZ) is an anthelmintic drug widely used to treat and prevent pinworm outbreaks in laboratory rodents. Although data in nonrodent species indicate possible effects of fenbendazole on the bone marrow and lymphocyte proliferation and function, little has been reported regarding possible effects on the rodent immune system. The purpose of the current study was to determine the effects of a therapeutic regimen of FBZ on immune parameters in BALB/c mice. Both 9-wk on-off and 5-wk continuous medicated feed protocols were assessed. No significant differences between normal and FBZ diet treated mice were observed in the following parameters: complete blood count, blood chemistry, quantitation of major T and B cell markers in spleen, quantitation of T cell markers in the thymus, spleen cell proliferation to T and B cell mitogens, bone marrow colony-forming cell assays, skin graft rejection, and primary and secondary humoral immune responses. These data indicate that FBZ treatment does not affect many standard broad measures of immune function.

Cryopreservation of somatic embryogenic cultures of Pinus pinaster: effects on regrowth and embryo maturation.

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Álvarez, José M; Cortizo, Millán; Ordás, Ricardo J

2012-01-01

Pinus pinaster is one of the most economically important conifers in the world. Somatic embryogenesis is a powerful tool in breeding programmes because it allows the generation of a great number of different clonal lines from seeds of superior genotypes. Unfortunately, embryogenic competence decreases with the age of cultures. Therefore, it is necessary to have a cryopreservation protocol that ensures a continuous supply of juvenile mass while allowing good maturation and conversion rates into vigorously growing plants. In this work we studied the influence of several cryopreservation parameters, such as cryoprotectant solution and pre-cooling temperature, on embryogenic culture regrowth and embryo maturation. Recovery of rewarmed samples after cryopreservation in a -150 degree C freezer depended on the cooling temperature reached prior to plunging the tubes into liquid nitrogen. As a result, we present an optimised cryopreservation protocol that ensures high recovery and embryo maturation rates. The protocol presented is a simple and fast alternative and enabled successful cryopreservation and recovery of 100 percent of the lines tested. Cryopreserved lines presented the same maturation rates as non-cryopreserved controls.

[Cryopreservation of teeth].

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Zimmerli, Melanie; Filippi, Andreas

2010-01-01

After tooth loss dental implants or fixed prosthetic restorations are not indicated in children and adolescents due to incomplete maxillary and mandibular development. Cryopreservation is a method for long-term storage of healthy teeth which were removed for orthodontic reasons or due to traumatic origin. These preserved teeth can be used as autogenous replants or transplants after tooth loss. During transport to and from the freezing facilities prior to freezing the teeth are stored in a cell culture medium. The tooth is transferred into a freezing tube containing cell culture medium and cryoprotectant DMSO. Teeth autotransplanted after cryopreservation show vitality of the PDL cells. Usually no enamel and/or dentinal cracks can be observed. After tooth loss transplantation of cryopreserved teeth could be an effective and biological therapy for tooth replacement.

Coconut (Cocos nucifera l.) pollen cryopreservation.

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Karun, A; Sjini, K K; Niral, V; Amarnth, C H; Remya, P; Rajesh, M K; Samsudeen, K; Jerard, B A; Engelmann, F

2014-01-01

Coconut genetic resources are threatened by pests and pathogens, natural hazards and human activities. Cryopreservation is the only method allowing the safe and cost-effective long-term conservation of recalcitrant seed species such as coconut. The objective of this work was to test the effect of cryopreservation and of cryostorage duration on coconut pollen germination and fertility. Pollen of two coconut varieties (West Coast Tall WWCTW and Chowghat Orange Dwarf CODC) was collected in March-May over three successive years, desiccated to 7.5 % moisture content (FW) and cryopreserved by direct immersion in liquid nitrogen. Germination and pollen tube length (PTL) of desiccated and cryopreserved pollen were not significantly different for both WCT and COD over the three harvest months of the three consecutive years of study. Pollen germination ranged from 24 to 32 % in desiccated pollen whereas it was between 26 and 29 % in cryopreserved COD pollen. In the case of WCT, germination ranged from 30 to 31 % in desiccated pollen, while it was between 28 and 32 % in cryopreserved pollen. PTL of cryopreserved pollen ranged between 224-390 nm and 226-396 mm for COD and WCT, respectively. Germination of COD pollen varied between 29.0 and 44.1 % after 4 years and 1.0/1.5 years cryostorage, respectively. Germination of WCT pollen did not change significantly between 0 and 6 years cryostorage, being comprised between 32 (24 h) and 40 % (1.5 years). Germination and vigour of cryopreserved pollen were generally higher compared to that of pollen dried in oven and non-cryopreserved. Normal seed set was observed in COD and WCT palms using pollen cryostored for 6 months and 4 years. Cryopreserved pollen of five Tall and five Dwarf accessions displayed 24-31 % and 25-49 % germination, respectively. These results show that it is now possible to establish pollen cryobanks to contribute to coconut germplasm long-term conservation.

Cryopreservation of Populus trichocarpa and Salix dormant buds with recovery by grafting or direct rooting.

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Bonnart, Remi; Waddell, John; Haiby, Kathy; Widrlenchner, Mark P; Volk, Gayle M

2014-01-01

Methods are needed for the conservation of clonally maintained trees of Populus and Salix. In this work, Populus trichocarpa and Salix genetic resources were cryopreserved using dormant scions as the source explant. We quantified the recovery of cryopreserved materials that originated from diverse field environments by using either direct sprouting or grafting. Scions (either at their original moisture content of 48 to 60% or dried to 30%) were slowly cooled to -35 degree C, transferred to the vapor phase of liquid nitrogen (LNV, -160 degree C), and warmed before determining survival. Dormant buds from P. trichocarpa clones from Westport and Boardman, OR had regrowth levels between 42 and 100%. Direct rooting of cryopreserved P. trichocarpa was also possible. Ten of 11 cryopreserved Salix accessions, representing 10 different species, exhibited at least 40% bud growth and rooting after 6 weeks when a bottom-heated rooting system was implemented. We demonstrate that dormant buds of P. trichocarpa and Salix accessions can be cryopreserved and successfully regenerated without the use of tissue culture.

Cryopreserved semen in ecotoxicological bioassays: sensitivity and reliability of cryopreserved Sparus aurata spermatozoa.

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Fabbrocini, Adele; D'Adamo, Raffaele; Del Prete, Francesco; Langellotti, Antonio Luca; Rinna, Francesca; Silvestri, Fausto; Sorrenti, Gerarda; Vitiello, Valentina; Sansone, Giovanni

2012-10-01

The aim of this study was to evaluate the feasibility of using cryopreserved S. aurata semen in spermiotoxicity tests. Cryopreservation is a biotechnology that can provide viable gametes and embryos on demand, rather than only in the spawning season, thus overcoming a limitation that has hindered the use of some species in ecotoxicological bioassays. Firstly, the sperm motility pattern of cryopreserved semen was evaluated after thawing by means of both visual and computer-assisted analyses. Motility parameters in the cryopreserved semen did not change significantly in the first hour after thawing, meaning that they were maintained for long enough to enable their use in spermiotoxicity tests. In the second phase of the research, bioassays were performed, using cadmium as the reference toxicant, in order to evaluate the sensitivity of cryopreserved S. aurata semen to ecotoxicological contamination. The sensitivity of the sperm motility parameters used as endpoints (motility percentages and velocities) proved to be comparable to what has been recorded for the fresh semen of other aquatic species (LOECs from 0.02 to 0.03 mg L(-1)). The test showed good reliability and was found to be rapid and easy to perform, requiring only a small volume of the sample. Moreover, cryopreserved semen is easy to store and transfer and makes it possible to perform bioassays in different sites or at different times with the same batch of semen. The proposed bioassay is therefore a promising starting point for the development of toxicity tests that are increasingly tailored to the needs of ecotoxicology and environmental quality evaluation strategies. Copyright © 2012 Elsevier Inc. All rights reserved.

Effect of Cryopreservation and Post-Cryopreservation Somatic Embryogenesis on the Epigenetic Fidelity of Cocoa (Theobroma cacao L.).

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Adu-Gyamfi, Raphael; Wetten, Andy; Marcelino Rodríguez López, Carlos

2016-01-01

While cocoa plants regenerated from cryopreserved somatic embryos can demonstrate high levels of phenotypic variability, little is known about the sources of the observed variability. Previous studies have shown that the encapsulation-dehydration cryopreservation methodology imposes no significant extra mutational load since embryos carrying high levels of genetic variability are selected against during protracted culture. Also, the use of secondary rather than primary somatic embryos has been shown to further reduce the incidence of genetic somaclonal variation. Here, the effect of in vitro conservation, cryopreservation and post-cryopreservation generation of somatic embryos on the appearance of epigenetic somaclonal variation were comparatively assessed. To achieve this we compared the epigenetic profiles, generated using Methylation Sensitive Amplified Polymorphisms, of leaves collected from the ortet tree and from cocoa somatic embryos derived from three in vitro conditions: somatic embryos, somatic embryos cryopreserved in liquid nitrogen and somatic embryos generated from cryoproserved somatic embryos. Somatic embryos accumulated epigenetic changes but these were less extensive than in those regenerated after storage in LN. Furthermore, the passage of cryopreserved embryos through another embryogenic stage led to further increase in variation. Interestingly, this detected variability appears to be in some measure reversible. The outcome of this study indicates that the cryopreservation induced phenotypic variability could be, at least partially, due to DNA methylation changes. Phenotypic variability observed in cryostored cocoa somatic-embryos is epigenetic in nature. This variability is partially reversible, not stochastic in nature but a directed response to the in-vitro culture and cryopreservation.

Evaluation of human platelet lysate and dimethyl sulfoxide as cryoprotectants for the cryopreservation of human adipose-derived stem cells.

Science.gov (United States)

Wang, Chuan; Xiao, Ran; Cao, Yi-Lin; Yin, Hong-Yu

2017-09-09

Cryopreservation provides an effective technique to maintain the functional properties of human adipose-derived stem cells (ASCs). Dimethylsulfoxide (DMSO) and fetal bovine serum (FBS) are frequently used as cryoprotectants for this purpose. However, the use of DMSO can result in adverse effects and toxic reactions and FBS can introduce risks of viral, prion, zoonose contaminations and evoke immune responses after injection. It is therefore crucial to reduce DMSO concentrations and use serum-free solution in the cryopreservation process. Human platelet lysate (PL) is a promising candidate for use as an alternative to DMSO and FBS. Therefore, in this study, with an aim to identify a cryoprotective agent for ASC cryopreservation, we determined the viability, proliferation potential, phenotype, and differentiation potential of fresh ASCs and ASCs cryopreserved using different combinations of three cryoprotective agents: fetal bovine serum (FBS), dimethylsulfoxide (DMSO), and human platelet lysate (PL). The viability of the ASCs cryopreserved with 90% FBS and 10% DMSO, 95% FBS and 5% DMSO, and 97% PL and 3% DMSO was >80%, and the proliferation potentials, cell phenotypes, and differentiation potentials of these groups were similar to those of fresh ASCs. Together, our findings suggest that a combination of 97% PL and 3% DMSO is an ideal cryoprotective agent for the efficient cryopreservation of human ASCs. Copyright © 2017 Elsevier Inc. All rights reserved.

Embryo cryopreservation and preeclampsia risk.

Science.gov (United States)

Sites, Cynthia K; Wilson, Donna; Barsky, Maya; Bernson, Dana; Bernstein, Ira M; Boulet, Sheree; Zhang, Yujia

2017-11-01

To determine whether assisted reproductive technology (ART) cycles involving cryopreserved-warmed embryos are associated with the development of preeclampsia. Retrospective cohort study. IVF clinics and hospitals. A total of 15,937 births from ART: 9,417 singleton and 6,520 twin. We used linked ART surveillance, birth certificate, and maternal hospitalization discharge data, considering resident singleton and twin births from autologous or donor eggs from 2005-2010. We compared the frequency of preeclampsia diagnosis for cryopreserved-warmed versus fresh ET and used multivariable logistic regression to adjust for confounders. Among pregnancies conceived with autologous eggs resulting in singletons, preeclampsia was greater after cryopreserved-warmed versus fresh ET (7.51% vs. 4.29%, adjusted odds ratio = 2.17 [95% CI 1.67-2.82]). Preeclampsia without and with severe features, preeclampsia with preterm delivery, and chronic hypertension with superimposed preeclampsia were more frequent after cryopreserved-warmed versus fresh ET (3.99% vs. 2.55%; 2.95% vs. 1.41%; 2.76 vs. 1.48%; and 0.95% vs. 0.43%, respectively). Among pregnancies from autologous eggs resulting in twins, the frequency of preeclampsia with severe features (9.26% vs. 5.70%) and preeclampsia with preterm delivery (14.81% vs. 11.74%) was higher after cryopreserved versus fresh transfers. Among donor egg pregnancies, rates of preeclampsia did not differ significantly between cryopreserved-warmed and fresh ET (10.78% vs. 12.13% for singletons and 28.0% vs. 25.15% for twins). Among ART pregnancies conceived using autologous eggs resulting in live births, those involving transfer of cryopreserved-warmed embryos, as compared with fresh ETs, had increased risk for preeclampsia with severe features and preeclampsia with preterm delivery. Copyright © 2017 American Society for Reproductive Medicine. All rights reserved.

Schistosoma mansoni: parasitology and immunology of baboons vaccinated with irradiated cryopreserved schistosomula

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Damian, R T; Powell, M R; Roberts, M L [Georgia Univ., Athens (USA). Dept. of Zoology; Clark, J D [Georgia Univ., Athens (USA). Lab. Animal Medicine; Stirewalt, M A; Lewis, F A [Biomedical Research Inst., Rockville, MD (USA)

1985-06-01

Young baboons (Papio cynocephalus) were vaccinated with ..gamma..-irradiated (500 Gy) cryopreserved Puerto Rican strain schistosomula of S. mansoni. Protection against heterologous, normal Kenyan Strain S. mansoni challenge infection was erratic and partial; and two putative correlates of immunity, reduced worm fecundity and change in worm location (anterior shift) were not observed. However, immunization of baboons with this vaccine resulted in a stimulated immune system. Both cellular and humoral anamnesis were demonstrable in vaccinated-challenged baboons. Schistosome infection-associated IgM hypergammaglobulinemia was also greatly reduced in vaccinated-challenged baboons. However IgG antibodies to adult, egg, and cercarial antigens were increased after challenge infection in preimmunized baboons. Vaccination appears to have resulted in a redirection of the immune system into anti-parasite channels, but this more specific immune response was insufficient to confer good protection against challenge infection in this experiment. The dampening effect of the vaccine on the hypergammaglobulinemia of schistosomiasis is another candidate for a possible ''anti-pathogenesis'' effect of irradiated schistosome larval vaccines.

Data on antioxidant activity in grapevine (Vitis vinifera L.) following cryopreservation by vitrification.

Science.gov (United States)

Lazo-Javalera, María Fernanda; Tiznado-Hernández, Martín Ernesto; Vargas-Arispuro, Irasema; Valenzuela-Soto, Elisa; Rocha-Granados, María Del Carmen; Martínez-Montero, Marcos Edel; Rivera-Domínguez, Marisela

2015-12-01

Cryopreservation is used for the long-term conservation of plant genetic resources. This technique very often induces lethal injury or tissue damage. In this study, we measured indicators of viability and cell damage following cryopreservation and vitrification-cryopreservation in Vitis vinifera L. axillary buds cv. "Flame seedless" stored in liquid nitrogen (LN) for: three seconds, one hour, one day, one week and one month; after LN thawed at 38 °C for three minutes. The enzymatic activity of catalase (CAT) and superoxide dismutase (SOD), as well as the amount of malondialdehyde (MDA), total protein and viability were assayed.

Data on antioxidant activity in grapevine (Vitis vinifera L. following cryopreservation by vitrification

Directory of Open Access Journals (Sweden)

María Fernanda Lazo-Javalera

2015-12-01

Full Text Available Cryopreservation is used for the long-term conservation of plant genetic resources. This technique very often induces lethal injury or tissue damage. In this study, we measured indicators of viability and cell damage following cryopreservation and vitrification-cryopreservation in Vitis vinifera L. axillary buds cv. “Flame seedless” stored in liquid nitrogen (LN for: three seconds, one hour, one day, one week and one month; after LN thawed at 38 °C for three minutes. The enzymatic activity of catalase (CAT and superoxide dismutase (SOD, as well as the amount of malondialdehyde (MDA, total protein and viability were assayed.

Effect of Antioxidants and Apoptosis Inhibitors on Cryopreservation of Murine Germ Cells Enriched for Spermatogonial Stem Cells.

Directory of Open Access Journals (Sweden)

Seung-Jung Ha

Full Text Available Spermatogonial stem cells (SSCs are germline stem cells that serve as the foundation of spermatogenesis to maintain fertility throughout a male's lifetime. To treat male infertility using stem cell banking systems and transplantation, it is important to be able to preserve SSCs for long periods of time. Therefore, this study was conducted to develop an optimal cryopreservation protocol for SSCs using antioxidants and apoptosis inhibitors in freezing medium. No differences were observed compared to controls when SSCs were cryopreserved in the presence of apoptosis inhibitors by themselves. However, mouse germ cells cryopreserved in basal medium containing the antioxidant hypotaurine (14 mM resulted in significantly greater proliferation potential and mitochondrial activity. Furthermore, treatment groups with combinations containing 200 mM trehalose and 14 mM hypotaurine showed higher proliferation rates compared to controls. In addition, several serum free conditions were evaluated for SSC cryopreservation. Treatment media containing 10% or 20% knockout serum replacement resulted in similar cryopreservation results compared to media containing FBS. SSC transplantation was also performed to confirm the functionality of SSCs frozen in 14 mM hypotaurine. Donor SSCs formed normal spermatogenic colonies and sperm in the recipient testis. These data indicate that inclusion of 14 mM hypotaurine in cryopreservation media is an effective way to efficiently cryopreserve germ cells enriched for SSCs and that knockout serum replacement can replace FBS in germ cell cryopreservation media.

Testicular tissue cryopreservation in prepubertal male children: an analysis of parental decision-making.

Science.gov (United States)

Ginsberg, Jill P; Li, Yimei; Carlson, Claire A; Gracia, Clarisa R; Hobbie, Wendy L; Miller, Victoria A; Mulhall, John; Shnorhavorian, Margarett; Brinster, Ralph L; Kolon, Thomas F

2014-09-01

Infertility is an unfortunate treatment-related consequence for some pediatric malignancies as well as some non-malignant conditions treated with stem cell transplant. Unlike pubertal males, prepubertal males cannot produce semen for cryopreservation. This manuscript reports on the acceptability and safety of a multi-institutional protocol for offering testicular tissue cryopreservation to families of prepubertal male children at highest risk for infertility. Data on decision influences, decision-making control, and emotional state when considering this option are described. Prepubertal males facing gonadotoxic therapy were offered testicular cryopreservation. Post-biopsy, patients were followed for acute side effects. In addition, parents and patients were asked to complete questionnaires, whether or not they chose to cryopreserve tissue. Seventy-four prepubertal male children were approached. Fifty-seven families (77%) consented to the testicular biopsy; 48 of 57 underwent the procedure. There was one post-operative side effect. Parents who agreed to testicular cryopreservation and those that did not felt in control of this decision. Parents who consented to the biopsy and refusers were not deterred by the experimental nature of the protocol. An important decision-making influence was the risk of the biopsy. Biopsy and cryopreservation of testicular tissue from prepubertal male children was performed successfully and safely at three institutions. Parents faced with this option at diagnosis can make an informed decision and weigh carefully the risks and benefits. Although asked to make a decision soon after they were given a difficult diagnosis, parents uniformly felt in control of this decision. © 2014 Wiley Periodicals, Inc.

Cryopreserved Dental Pulp Tissues of Exfoliated Deciduous Teeth Is a Feasible Stem Cell Resource for Regenerative Medicine

Science.gov (United States)

Yamaza, Haruyoshi; Akiyama, Kentaro; Hoshino, Yoshihiro; Song, Guangtai; Kukita, Toshio; Nonaka, Kazuaki; Shi, Songtao; Yamaza, Takayoshi

2012-01-01

Human exfoliated deciduous teeth have been considered to be a promising source for regenerative therapy because they contain unique postnatal stem cells from human exfoliated deciduous teeth (SHED) with self-renewal capacity, multipotency and immunomodulatory function. However preservation technique of deciduous teeth has not been developed. This study aimed to evaluate that cryopreserved dental pulp tissues of human exfoliated deciduous teeth is a retrievable and practical SHED source for cell-based therapy. SHED isolated from the cryopreserved deciduous pulp tissues for over 2 years (25–30 months) (SHED-Cryo) owned similar stem cell properties including clonogenicity, self-renew, stem cell marker expression, multipotency, in vivo tissue regenerative capacity and in vitro immunomodulatory function to SHED isolated from the fresh tissues (SHED-Fresh). To examine the therapeutic efficacy of SHED-Cryo on immune diseases, SHED-Cryo were intravenously transplanted into systemic lupus erythematosus (SLE) model MRL/lpr mice. Systemic SHED-Cryo-transplantation improved SLE-like disorders including short lifespan, elevated autoantibody levels and nephritis-like renal dysfunction. SHED-Cryo amended increased interleukin 17-secreting helper T cells in MRL/lpr mice systemically and locally. SHED-Cryo-transplantation was also able to recover osteoporosis bone reduction in long bones of MRL/lpr mice. Furthermore, SHED-Cryo-mediated tissue engineering induced bone regeneration in critical calvarial bone-defect sites of immunocompromised mice. The therapeutic efficacy of SHED-Cryo transplantation on immune and skeletal disorders was similar to that of SHED-Fresh. These data suggest that cryopreservation of dental pulp tissues of deciduous teeth provide a suitable and desirable approach for stem cell-based immune therapy and tissue engineering in regenerative medicine. PMID:23251621

Cryopreservation of roe deer abomasal nematodes for morphological identification.

Science.gov (United States)

Beraldo, Paola; Pascotto, Ernesto

2014-02-01

Conventional methods to preserve adult nematodes for taxonomic purposes involve the use of fixative or clearing solutions (alcohol, formaldehyde, AFA and lactophenol), which cause morphological alterations and are toxic. The aim of this study is to propose an alternative method based on glycerol-cryopreservation of nematodes for their subsequent identification. Adults of trichostrongylid nematodes from the abomasum of roe deer (Capreolus capreolus Linnaeus) were glycerol-cryopreserved and compared with those fixed in formaldehyde, fresh and frozen without cryoprotectans. Morphology, transparency and elasticity of the anterior and posterior portion of male nematodes were compared, especially the caudal cuticular bursa and genital accessories. The method presented is quick and easy to use, and the quality of nematode specimens is better than that of nematodes fixed by previously used fixatives. Moreover, glycerol cryopreserved nematodes can be stored for a long time at -20 degrees C in perfect condition and they could be suitable for further analyses, such as histological or ultrastructural examinations.

High-throughput optimization by statistical designs: example with rat liver slices cryopreservation.

Science.gov (United States)

Martin, H; Bournique, B; Blanchi, B; Lerche-Langrand, C

2003-08-01

The purpose of this study was to optimize cryopreservation conditions of rat liver slices in a high-throughput format, with focus on reproducibility. A statistical design of 32 experiments was performed and intracellular lactate dehydrogenase (LDHi) activity and antipyrine (AP) metabolism were evaluated as biomarkers. At freezing, modified University of Wisconsin solution was better than Williams'E medium, and pure dimethyl sulfoxide was better than a cryoprotectant mixture. The best cryoprotectant concentrations were 10% for LDHi and 20% for AP metabolism. Fetal calf serum could be used at 50 or 80%, and incubation of slices with the cryoprotectant could last 10 or 20 min. At thawing, 42 degrees C was better than 22 degrees C. After thawing, 1h was better than 3h of preculture. Cryopreservation increased the interslice variability of the biomarkers. After cryopreservation, LDHi and AP metabolism levels were up to 84 and 80% of fresh values. However, these high levels were not reproducibly achieved. Two factors involved in the day-to-day variability of LDHi were identified: the incubation time with the cryoprotectant and the preculture time. In conclusion, the statistical design was very efficient to quickly determine optimized conditions by simultaneously measuring the role of numerous factors. The cryopreservation procedure developed appears suitable for qualitative metabolic profiling studies.

Cryopreservation of crane semen

Science.gov (United States)

Gee, G.F.; Harris, James

1991-01-01

The method for the cryopreservation of crane semen at Patuxent Wildlife Research Center is described in detail. Cryopreservation is useful for the long-term storage of crane semen and for specialized propagation needs. A 50% fertility rate from most sandhill cranes, Grus canadensis, inseminated with frozen-thawed semen can be expected. Additional research should improve the fertility rate and determine how applicable the technique is to other crane species.

STABILITY IN REAL TIME OF SOME CRYOPRESERVED MICROBIAL STRAINS WITH REFERENCE TO GENETICALLY MODIFIED MICROORGANISMS

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DANIELA VINTILĂ

2007-05-01

Full Text Available The aim of this work is to analyze the viability of microorganisms from Collection of Industrial Microorganisms from Faculty of Animal Science and Biotechnology – Timisoara, during freezing and thawing as part of cryopreservation technique. The stability in real time of 19 strains cryopreserved in 16% glycerol was evaluated during a 6-months period. The strains studied were: Escherichia coli, Lactobacillus acidophilus, Rhizobium meliloti, Saccharomyces cerevisiae, Aspergillus oryzae, Aspergillus niger, Trichoderma viride, Bacillus globigii, Bacillus licheniformis, and 9 strains of Bacillus subtilis. The strains cryopreserved at -20oC and -70oC were activated using the fast thawing protocol. A better cell recovery was achieved with the -70oC protocol reaching an average viability for E. coli of 86,3%, comparing with 78,6% in -20oC protocol. The cell recovery percentages for the other strains were: 92,4% for L. acidophilus, 93,9% for A.niger, 89% for A. oryzae, 86,7% for T. viride, 94,2% for R. meliloti, 82,1% for S. cerevisiae, 89,9% for B. licheniformis. Regarding the viability of genetically modified microorganisms, the values shows a good recovering after freezing and thawing, even after 180 days of cryopreservation. With the -20oC protocol lower viability was observed due probably to the formation of eutectic mixtures and recrystalization processes.

CFU-C populations in blood and bone marrow of dogs after lethal irradiation and allogeneic transfusion with cryopreserved blood mononuclear cells

International Nuclear Information System (INIS)

Nothdurft, W.; Fliedner, T.M.; Calvo, W.; Flad, H.-D.; Huget, R.; Koerbling, M.; Krumbacher-von Loringen, K; Ross, W.M.; Schnappauf, H.-P.; Steinbach, I.

1978-01-01

Colony forming units in agar (CFU-C) were assayed in both bone marrow and peripheral blood of dogs during haemopoietic recovery after lethal total-body irradiation (1200 R) and allogeneic transfusion of blood mononuclear cells (MNC) from histocompatible donors. MNC had been collected from the peripheral blood by continuous-flow centrifugation leucapheris and cryopreserved at -196 deg C until transfusion. Two groups of dogs were studied. Group 1 dogs (n = 12) were given between 0.39 and 2.76 x 10 9 MNC per kg body wt. Group 2 dogs (n = 14) were transfused with a similar number of MNC, ranging from 0.51 to 1.87 x 10 9 per kg body wt., but in addition underwent immuno-suppressive therapy with methotrexate. In group 1 dogs, there was a rather good correlation between the number of CFU-C in the regenerating bone marrow and the recovery of the peripheral blood granulocyte values. The regeneration of the CPU-C population in the bone marrow of methotrexate-treated dogs showed a somewhat more heterogeneous picture than in dogs of group 1 and in dogs that, in a previous study, were transfused with autologous MNC. The minimum time interval required for the reconstitution of peripheral blood CFU-C to normal levels was 2-4 weeks but usually took from 4-14 weeks. (author)

INTERRELATION BETWEEN TYPE AND CULTIVAR OF BERRY-LIKE CROP AND CUTTINGS CRYOPRESERVATION RESULTS

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L. V. Gorbunov

2013-04-01

Full Text Available Optimal cryopreservation parameters were determined for the berry-like crop cuttings: blackcurrant (Dachnica, Yuviley, Sofiivska, Kitayskaya; redcurrant (Kitaivska, Djoker, Svyatkova; gooseberries (Krasen, Malachite, Kolobok; raspberries (Novost’ Kuz’mina, Struyka, Skromnica. This procedure allowed increasing viability of deconservation samples in 2–5 times compared to the existing methods of cryopreservation. Maximal values of cutting viability were obtained after their temperature adaptation at –10 °C during 14–60 days, stepwise cooling of the samples with 0.1–0.5 °C/hr rate down to –30 °C (exposure 3–7 days, direct plunging into liquid nitrogen, storage from 1 to 30 days and thawing rate of 70 °C/min. It was found that the in the region of allowable values for maximum viability of investigated birch (control cuttings humidity values correspond 32±40%, for blackcurrant are 40±50%, and raspberries 37±40 % in affirmative temperature span and 14±28 %, 30±40 %, 20±23 % in below zero. Using dispersion analysis it has been established that the development probability of frozenthawed plant cuttings significantly depends on the selected cultivar and cryopreservation method. It is noted that the force of influence η2А, due to the individual properties of the studied cultivars of berries, is comparable to that of the force of influence η2В of cryopreservation procedures. The differences of average indexes of viability of the studied cultivars were showed within a single species. It was found that these rates are different for black currant by 92%, redcurrant — 54%, gooseberries — 48%, raspberries — 70%. Using paired Student’s t-test has reduced to 3 times the error of representativeness of the sample that enabled to improve the reliability of the significance of differences compared to the values of the P ≥0,99. Selection of the cuttings’ cultivars and cryopreservation method significantly affect the

Cryopreservation of in vitro grown shoot tips and apical meristems of the forage legume Arachis pintoi.

Science.gov (United States)

Rey, Hebe Y; Faloci, Mirta; Medina, Ricardo; Dolce, Natalia; Mroginski, Luis; Engelmann, Florent

2009-01-01

A cryopreservation protocol using the encapsulation-dehydration procedure was established for shoot tips (2-3 mm in length) and meristems (0.3-0.5 mm) sampled from in vitro plantlets of diploid and triploid cytotypes of Arachis pintoi. The optimal protocol was the following: after dissection, explants were precultured for 24 h on establishment medium (EM), encapsulated in calcium alginate beads and pretreated in liquid EM medium with daily increasing sucrose concentration (0.5, 0.75, 1.0 M) and desiccated to 22-23 percent moisture content (fresh weight basis). Explants were frozen using slow cooling (1 C per min from 25C to -30C followed by direct immersion in liquid nitrogen), thawed rapidly and post-cultured in liquid EM medium enriched with daily decreasing sucrose concentrations (0.75, 0.50, 0.1 M). Explants were then transferred to solid EM medium in order to achieve shoot regeneration, then on Murashige and Skoog medium supplemented with 0.05 microM naphthalene acetic acid to induce rooting of shoots. With this procedure, 53 percent and 56 percent of cryopreserved shoot tips of the diploid and triploid cytotypes, respectively, survived and formed plants. However, only 16 percent of cryopreserved meristems of both cytotypes regenerated plants. Using ten isozyme systems and seven RAPD profiles, no modification induced by cryopreservation could be detected in plantlets regenerated from cryopreserved material.

Contradictory Immune Response in Post Liver Transplantation Hepatitis B and C

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Akinobu Takaki

2014-01-01

Full Text Available Hepatitis B and C often progress to decompensated liver cirrhosis requiring orthotopic liver transplantation (OLT. After OLT, hepatitis B recurrence is clinically controlled with a combination of hepatitis B immunoglobulin (HBIG and nucleos(tide analogues. Another approach is to induce self-producing anti-hepatitis B virus (HBV antibodies using a HBV envelope antigen vaccine. Patients who had not been HBV carriers such as acutely infected liver failure or who received liver from HBV self-limited donor are good candidate. For chronic HBV carrier patients, a successful response can only be achieved in selected patients such as those treated with experimentally reduced immunosuppression protocols or received an anti-HBV adaptive memory carrying donor liver. Hepatitis C virus (HCV reinfects transplanted livers at a rate of >90%. HCV reinfected patients show different severities of hepatitis, from mild and slowly progressing to severe and rapidly progressing, possibly resulting from different adaptive immune responses. More than half the patients require interferon treatment, although the success rate is low and carries risks for leukocytopenia and rejection. Managing the immune response has an important role in controlling recurrent hepatitis C. This study aimed to review the adaptive immune response in post-OLT hepatitis B and C.

Cryopreservation of Leishmania Species in Manisa Province.

Science.gov (United States)

Çavuş, İbrahim; Ocak, Fulya; Kaya, Tuğba; Özbilgin, Ahmet

2017-09-01

It was aimed to assess the success of the cryopreservation process which is carried out in order to preserve the genetic material and the virulence of the Leishmania species that are an important health problem in our region. Leishmania tropica, L. infantum, L. major, and L. donovani strains in Novy-MacNeal-Nicolle (NNN) medium in MCBU were used. Promastigotes cultured in the NNN medium were transferred to RPMI 1640 medium; promastigotes in the logarithmic phase were washed three times with PBS, and 15% dimethylsulfoxide (DMSO) was added. Leishmania species were transferred to 12 separate tubes. The tubes were stored at -86°C for one night by placing them in Coolcell boxes. The tubes were transferred into a liquid nitrogen tank. One cryotube per Leishmania strain is thawed monthly and cultured in NNN medium. For the duration of study it was observed that each Leishmania isolate preserved 60-65% of their viability and entered the logarithmic phase on the 7th day following the inoculation in the NNN medium. Abnormalities in the structures and movements of the promastigotes were not observed in microscopic examinations. The following conclusions were made: cryopreservation is important for studies planned related to leishmaniasis and cryopreservation with DMSO is successful.

Successful Oocyte Cryopreservation in Reproductive-Aged Cancer Survivors.

Science.gov (United States)

Druckenmiller, Sarah; Goldman, Kara N; Labella, Patty A; Fino, M Elizabeth; Bazzocchi, Antonia; Noyes, Nicole

2016-03-01

To demonstrate that oocyte cryopreservation is a feasible reproductive option for patients with cancer of childbearing age who require gonadotoxic therapies. This study is a university-based retrospective review of reproductive-aged cancer patient treatment cycles that included ovarian stimulation, transvaginal oocyte retrieval, oocyte cryopreservation, and, in some cases, subsequent oocyte thaw, in vitro fertilization, and embryo transfer. Outcome measures included ovarian stimulation response, number of oocytes retrieved, cryopreserved, and thawed, and pregnancy data. From 2005 to 2014, 176 reproductive-aged patients with cancer (median age 31 years, interquartile range 24-36) completed 182 oocyte cryopreservation cycles. Median time between consult request and oocyte retrieval was 12 days (interquartile range 10-14). Median peak stimulation estradiol was 1,446 pg/mL (interquartile range 730-2,687); 15 (interquartile range 9-23) oocytes were retrieved and 10 (interquartile range 5-18) metaphase II oocytes were cryopreserved per cycle. Ten patients (11 cycles) have returned to attempt pregnancy with their cryopreserved oocytes. Among thawed oocytes, the cryopreservation survival rate was 86% (confidence interval [CI] 78-94%). Nine of 11 thaw cycles resulted in embryos suitable for transfer. The embryo implantation rate was 27% (CI 8-46%) and the live birth rate was 44% (CI 12-77%) per embryo transfer. Chance for live birth with embryos created from cryopreserved oocytes was similar between the patients with cancer in this study and noncancer patients who underwent the same treatment at our center (44% [CI 12-77%] compared with 33% [CI 22-44%] per embryo transfer). Oocyte cryopreservation is now a feasible fertility preservation option for reproductive-aged patients with cancer who require gonadotoxic therapies.

Optimizing human semen cryopreservation by reducing test vial volume and repetitive test vial sampling

DEFF Research Database (Denmark)

Jensen, Christian F S; Ohl, Dana A; Parker, Walter R

2015-01-01

OBJECTIVE: To investigate optimal test vial (TV) volume, utility and reliability of TVs, intermediate temperature exposure (-88°C to -93°C) before cryostorage, cryostorage in nitrogen vapor (VN2) and liquid nitrogen (LN2), and long-term stability of VN2 cryostorage of human semen. DESIGN......: Prospective clinical laboratory study. SETTING: University assisted reproductive technology (ART) laboratory. PATIENT(S): A total of 594 patients undergoing semen analysis and cryopreservation. INTERVENTION(S): Semen analysis, cryopreservation with different intermediate steps and in different volumes (50......-1,000 μL), and long-term storage in LN2 or VN2. MAIN OUTCOME MEASURE(S): Optimal TV volume, prediction of cryosurvival (CS) in ART procedure vials (ARTVs) with pre-freeze semen parameters and TV CS, post-thaw motility after two- or three-step semen cryopreservation and cryostorage in VN2 and LN2. RESULT...

Schistosoma mansoni: parasitology and immunology of baboons vaccinated with irradiated cryopreserved schistosomula

Energy Technology Data Exchange (ETDEWEB)

Damian, R.T.; Powell, M.R.; Roberts, M.L. (Georgia Univ., Athens (USA). Dept. of Zoology); Clark, J.D. (Georgia Univ., Athens (USA). Lab. Animal Medicine); Stirewalt, M.A.; Lewis, F.A. (Biomedical Research Inst., Rockville, MD (USA))

1985-06-01

Young baboons (Papio cynocephalus) were vaccinated with ..gamma..-irradiated (500 Gy) cryopreserved Puerto Rican strain schistosomula of S. mansoni. Protection against heterologous, normal Kenyan Strain S. mansoni challenge infection was erratic and partial; and two putative correlates of immunity, reduced worm fecundity and change in worm location (anterior shift) were not observed. However, immunization of baboons with this vaccine resulted in a stimulated immune system. Both cellular and humoral anamnesis were demonstrable in vaccinated-challenged baboons. Schistosome infection-associated IgM hypergammaglobulinemia was also greatly reduced in vaccinated-challenged baboons. However IgG antibodies to adult, egg, and cercarial antigens were increased after challenge infection in preimmunized baboons. Vaccination appears to have resulted in a redirection of the immune system into anti-parasite channels, but this more specific immune response was insufficient to confer good protection against challenge infection in this experiment. The dampening effect of the vaccine on the hypergammaglobulinemia of schistosomiasis is another candidate for a possible ''anti-pathogenesis'' effect of irradiated schistosome larval vaccines.

Schistosoma mansoni: parasitology and immunology of baboons vaccinated with irradiated cryopreserved schistosomula

International Nuclear Information System (INIS)

Damian, R.T.; Powell, M.R.; Roberts, M.L.

1985-01-01

Young baboons (Papio cynocephalus) were vaccinated with γ-irradiated (500 Gy) cryopreserved Puerto Rican strain schistosomula of S. mansoni. Protection against heterologous, normal Kenyan Strain S. mansoni challenge infection was erratic and partial; and two putative correlates of immunity, reduced worm fecundity and change in worm location (anterior shift) were not observed. However, immunization of baboons with this vaccine resulted in a stimulated immune system. Both cellular and humoral anamnesis were demonstrable in vaccinated-challenged baboons. Schistosome infection-associated IgM hypergammaglobulinemia was also greatly reduced in vaccinated-challenged baboons. However IgG antibodies to adult, egg, and cercarial antigens were increased after challenge infection in preimmunized baboons. Vaccination appears to have resulted in a redirection of the immune system into anti-parasite channels, but this more specific immune response was insufficient to confer good protection against challenge infection in this experiment. The dampening effect of the vaccine on the hypergammaglobulinemia of schistosomiasis is another candidate for a possible ''anti-pathogenesis'' effect of irradiated schistosome larval vaccines. (author)

Comparison of the interleukin-1β-inducing potency of allergenic spores from higher fungi (Basidiomycetes) in a cryopreserved human whole blood system

Science.gov (United States)

Rivera-Mariani, Félix E.; Vysyaraju, Kranthi; Negherbon, Jesse; Levetin, Estelle; Horner, W. Elliot; Hartung, Thomas; Breysse, Patrick N.

2014-01-01

Background Spores from basidiomycete fungi (basidiospores) are highly prevalent in the atmosphere of urban and rural settings. Studies have confirmed their potential to affect human health as allergens. Less is known about their potential to serve as stimuli of the innate immune system and induce pro-inflammatory reactions. Methods In this study, we evaluated the pro-inflammatory potential of spores from 11 allergenic gilled (Pleurotus ostreatus, Oudemansiella radicata, Armillaria tabescens, Coprinus micaceus, Pluteus cervinus, Chlorophyllum molybdites) and non-gilled (Pisolithus arhizus, Merulius tremullosus, Calvatia cyathiformis, Lycoperdon pyriforme, Boletus bicolor) basidiomycetes fungi based on their potency to induce the release of the pro-inflammatory cytokine interleukin (IL)-1β in a cryopreserved human whole blood system. In addition, the role of morphological features of the spores (surface area, shape, and pigmentation) were examined for their role in the spores’ interleukin (IL)-1β-including potency. Peripheral blood from healthy volunteers was collected, pooled, and cryopreserved. After stimulating the cryopreserved pooled blood with 106 to 103 basidiospores/ml, the concentration of IL-1β in culture supernatants was determined with ELISA. Results Basidiospores manifested concentration-dependent IL-1β-inducing potency, which was more noteworthy among basidiospores from gilled basidiomycetes. At higher concentrations of basidiospores, the IL-1β-inducing potency was able to be differentiated in the cryopreserved human whole blood system. Morphological features did not correlate with the IL-1β-inducing potency of the basidiospores, suggesting that non-morphological properties modulate the IL-1β-inducing potency. Conclusion Our data provides evidence of the pro-inflammatory potential of basidiospores, and the utility of cryopreserved human whole blood as a human-based in-vitro system to study the immune reactivity of allergenic basidiospores. PMID

The Different Calcium+2 Intensity Profile and Quality of Oocyte and Goat Sperms after Cryopreservation

Science.gov (United States)

Ciptadi, G.; Rahayu, S.; Fatchiyah; Wahyuningsih, S.; Budiarto, A.; Nasich, M.; Putri, A. R. I.; Mudawamah, M.; Ihsan, M. N.

2018-02-01

This research aims were to study the effect of the oocyte and sperms cryopreservation of Indonesian local goat on the post-thawing quality and profile or characters of Calcium+2 intensity in relating with their fertility capacity. A study was conducted to test the freezing method and post-thawing viability both stock cells stored in the deep freezer and liquid nitrogen (-80°C of vs -196°C). A fertility test of sperms has been conducted through in vitro of sperm quality, while the oocytes cryopreserved test was done by in vitro maturation (IVM) rate (%). The profile of Calcium 2+ was performed and analysis by Confocal Laser Scanned Microscope (CLSM). The result showed that IVM rate of goat oocyte is considered lower when cryopreserved in -80°C than in -196°C. Meanwhile, sperm is considered having a good quality in 2 methods of cryopreservation with post-thawing motility > 40 % (SNI 2014). There is an important difference between Calcium intensity of fresh and post-thawing both for oocyte and spermatozoa. Calcium +2 profiles is varied individually on the peak of intensity, but it considered expressed the same profile of each fresh and post-thawing cell. In vitro fertilization test need to be performed to complete the viability and fertility competence of these sperm and oocyte freezing stocks.

Mature Oocyte Cryopreservation for Fertility Preservation.

Science.gov (United States)

Liang, Tina; Motan, Tarek

2016-01-01

In recent decades, advances in cancer treatment have led to a dramatic improvement in long term survival. This has led to an increasing focus on quality of life after surviving cancer treatment, with fertility being an important aspect. Given the known reproductive risks of cancer therapies, there has been a growing interest in the field of fertility preservation (also referred to as oncofertility). Mature oocyte cryopreservation is no longer considered experimental and has become a realistic option for reproductive aged women prior to undergoing cancer treatment. Additionally, as cryopreservation techniques improve, mature oocyte cryopreservation is increasing being marketed to healthy women without cancer wishing to delay child bearing, also termed "social egg freezing". This chapter provides a review of the current technology, use, and outcomes of mature oocyte cryopreservation. It also outlines the ethical debate surrounding social egg freezing and directions for future research in female fertility preservation.

Cryopreservation of adenovirus-transfected dendritic cells (DCs) for clinical use.

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Gülen, D; Maas, S; Julius, H; Warkentin, P; Britton, H; Younos, I; Senesac, J; Pirruccello, Samuel M; Talmadge, J E

2012-05-01

In this study, we examined the effects of cryoprotectant, freezing and thawing, and adenovirus (Adv) transduction on the viability, transgene expression, phenotype, and function of human dendritic cells (DCs). DCs were differentiated from cultured peripheral blood (PB) monocytes following Elutra isolation using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 6 days and then transduced using an Adv vector with an IL-12 transgene. Fresh, cryopreserved, and thawed transduced immature DCs were examined for their: 1) cellular concentration and viability; 2) antigenicity using an allogeneic mixed lymphocyte reaction (MLR); 3) phenotype (HLA-DR and CD11c) and activation (CD83); and 4) transgene expression based on IL-12 secretion. Stability studies revealed that transduced DCs could be held in cryoprotectant for as long as 75 min at 2-8°C prior to freezing with little effect on their viability and cellularity. Further, cryopreservation, storage, and thawing reduced the viability of the transduced DCs by an average of 7.7%; and had no significant impact on DC phenotype and activation. In summary, cryopreservation, storage, and thawing had no significant effect on DC viability, function, and transgene expression by Adv-transduced DCs. Copyright © 2012 Elsevier B.V. All rights reserved.

Safety and efficacy of cryopreserved autologous platelet concentrates in HLA-alloimmunized patients with hematologic malignancies.

Science.gov (United States)

Gerber, Bernhard; Alberio, Lorenzo; Rochat, Sophie; Stenner, Frank; Manz, Markus G; Buser, Andy; Schanz, Urs; Stussi, Georg

2016-10-01

Curative chemotherapy approaches in patients with malignancies and platelet (PLT) transfusion refractoriness due to alloimmunization may be hampered by the lack of suitable PLT donors. For these patients, transfusion of cryopreserved autologous PLTs is an option, but is time- and resource-consuming. We aimed at further simplifying this process. A retrospective single-center analysis was conducted on the transfusion of cryopreserved autologous PLTs in nine female alloimmunized, PLT transfusion-refractory patients treated for acute leukemia (n = 8) and non-Hodgkin's lymphoma (n = 1). No additional processing was used before transfusion, and most notably, washing and centrifugation steps were omitted. Clinical efficacy and safety, as well as a flow cytometric assessment of structural and functional PLT changes, were analyzed. A total of 40 autologous PLT concentrates were thawed at bedside and transfused a median of 32 (range, 9 to 994) days after cryopreservation. No major bleeds and no severe dimethyl sulfoxide toxicity were observed. The median PLT count increments did not differ 1 and 18 to 24 hours after transfusion and reached 6 × 10 9 /L (interquartile range [IQR], 3 × 10 9 -7.5 × 10 9 /L) and 6 × 10 9 /L (IQR, 2.5 × 10 9 -9.5 × 10 9 /L), respectively. Cryopreservation resulted in partial activation of one-third of the PLTs. In vitro stimulation with strong agonists induced additional full activation of cryopreserved PLTs: median, 55% (IQR, 42%-60%) after thrombin and 39% (IQR, 36%-39%) after convulxin. The transfusion of cryopreserved autologous PLTs is feasible and safe. Despite the cryopreservation process, PLT functionality is partially maintained. © 2016 AABB.

Rapid expansion of T cells: Effects of culture and cryopreservation and importance of short-term cell recovery.

Science.gov (United States)

Sadeghi, Arian; Ullenhag, Gustav; Wagenius, Gunnar; Tötterman, Thomas H; Eriksson, Fredrik

2013-06-01

Successful cell therapy relies on the identification and mass expansion of functional cells for infusion. Cryopreservation of cells is an inevitable step in most cell therapies which also entails consequences for the frozen cells. This study assessed the impact of cryopreservation and the widely used protocol for rapid expansion of T lymphocytes. The effects on cell viability, immunocompetence and the impact on apoptotic and immunosuppressive marker expression were analyzed using validated assays. Cryopreservation of lymphocytes during the rapid expansion protocol did not affect cell viability. Lymphocytes that underwent mass expansion or culture in high dose IL-2 were unable to respond to PHA stimulation by intracellular ATP production immediately after thawing (ATP = 16 ± 11 ng/ml). However, their reactivity to PHA was regained within 48 hours of recovery (ATP = 356 ± 61 ng/ml). Analysis of mRNA levels revealed downregulation of TGF-β and IL-10 at all time points. Culture in high dose IL-2 led to upregulation of p73 and BCL-2 mRNA levels while FoxP3 expression was elevated after culture in IL-2 and artificial TCR stimuli. FoxP3 levels decreased after short-term recovery without IL-2 or stimulation. Antigen specificity, as determined by IFNγ secretion, was unaffected by cryopreservation but was completely lost after addition of high dose IL-2 and artificial TCR stimuli. In conclusion, allowing short-time recovery of mass expanded and cryopreserved cells before reinfusion could enhance the outcome of adoptive cell therapy as the cells regain immune competence and specificity.

Partial dehydration and cryopreservation of Citrus seeds.

Science.gov (United States)

Graiver, Natalia; Califano, Alicia; Zaritzky, Noemí

2011-11-01

Three categories of seed storage behavior are generally recognized among plant species: orthodox, intermediate and recalcitrant. Intermediate seeds cannot be stored in liquid nitrogen (LN) without a previous partial dehydration process. The water content (WC) of the seeds at the moment of immersion in LN must be regarded as the most critical factor in cryopreservation. The purpose of this study was to investigate the basis of the optimal hydration status for cryopreservation of Citrus seeds: C. sinensis (sweet orange), C. paradisi (grapefruit), C. reticulata (mandarin) in LN. To study the tolerance to dehydration and LN exposure, seeds were desiccated by equilibration at relative humidities between 11 and 95%. Sorption isotherms were determined and modeled; lipid content of the seeds was measured. Seed desiccation sensitivity was quantified by the quantal response model. Differential scanning calorimetry (DSC) thermograms were determined on cotyledon tissue at different moisture contents to measure ice melting enthalpies and unfrozen WC. Samples of total seed lipid extract were also analyzed by DSC to identify lipid transitions in the thermograms. The limit of hydration for LN Citrus seeds treatment corresponded to the unfrozen WC in the tissue, confirming that seed survival strictly depended on avoidance of intracellular ice formation. Copyright © 2011 Society of Chemical Industry.

Hyperthermic treatment at 56 °C induces tumour-specific immune protection in a mouse model of prostate cancer in both prophylactic and therapeutic immunization regimens.

Science.gov (United States)

De Sanctis, Francesco; Sandri, Sara; Martini, Matteo; Mazzocco, Marta; Fiore, Alessandra; Trovato, Rosalinda; Garetto, Stefano; Brusa, Davide; Ugel, Stefano; Sartoris, Silvia

2018-06-14

Most active cancer immunotherapies able to induce a long-lasting protection against tumours are based on the activation of tumour-specific cytotoxic T lymphocytes (CTLs). Cell death by hyperthermia induces apoptosis followed by secondary necrosis, with the production of factors named "danger associated molecular pattern" (DAMP) molecules (DAMPs), that activate dendritic cells (DCs) to perform antigen uptake, processing and presentation, followed by CTLs cross priming. In many published studies, hyperthermia treatment of tumour cells is performed at 42-45 °C; these temperatures mainly promote cell surface expression of DAMPs. Treatment at 56 °C of tumour cells was shown to induce DAMPs secretion rather than their cell surface expression, improving DC activation and CTL cross priming in vitro. Thus we tested the relevance of this finding in vivo on the generation of a tumour-specific memory immune response, in the TRAMP-C2 mouse prostate carcinoma transplantable model. TRAMP-C2 tumour cells treated at 56 °C were able not only to activate DCs in vitro but also to trigger a tumour-specific CTL-dependent immune response in vivo. Prophylactic vaccination with 56 °C-treated TRAMP-C2 tumour cells alone provided protection against TRAMP-C2 tumour growth in vivo, whilst in the therapeutic regimen, control of tumour growth was achieved combining immunization with adjuvant chemotherapy. Copyright © 2018 Elsevier Ltd. All rights reserved.

Karyotype of cryopreserved bone marrow cells

Directory of Open Access Journals (Sweden)

M.L.L.F. Chauffaille

2003-07-01

Full Text Available The analysis of chromosomal abnormalities is important for the study of hematological neoplastic disorders since it facilitates classification of the disease. The ability to perform chromosome analysis of cryopreserved malignant marrow or peripheral blast cells is important for retrospective studies. In the present study, we compared the karyotype of fresh bone marrow cells (20 metaphases to that of cells stored with a simplified cryopreservation method, evaluated the effect of the use of granulocyte-macrophage colony-stimulating factor (GM-CSF as an in vitro mitotic index stimulator, and compared the cell viability and chromosome morphology of fresh and cryopreserved cells whenever possible (sufficient metaphases for analysis. Twenty-five bone marrow samples from 24 patients with hematological disorders such as acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, chronic myeloid leukemia, megaloblastic anemia and lymphoma (8, 3, 3, 8, 1, and 1 patients, respectively were selected at diagnosis, at relapse or during routine follow-up and one sample was obtained from a bone marrow donor after informed consent. Average cell viability before and after freezing was 98.8 and 78.5%, respectively (P < 0.05. Cytogenetic analysis was successful in 76% of fresh cell cultures, as opposed to 52% of cryopreserved samples (P < 0.05. GM-CSF had no proliferative effect before or after freezing. The morphological aspects of the chromosomes in fresh and cryopreserved cells were subjectively the same. The present study shows that cytogenetic analysis of cryopreserved bone marrow cells can be a reliable alternative when fresh cell analysis cannot be done, notwithstanding the reduced viability and lower percent of successful analysis that are associated with freezing.

Karyotype of cryopreserved bone marrow cells.

Science.gov (United States)

Chauffaille, M L L F; Pinheiro, R F; Stefano, J T; Kerbauy, J

2003-07-01

The analysis of chromosomal abnormalities is important for the study of hematological neoplastic disorders since it facilitates classification of the disease. The ability to perform chromosome analysis of cryopreserved malignant marrow or peripheral blast cells is important for retrospective studies. In the present study, we compared the karyotype of fresh bone marrow cells (20 metaphases) to that of cells stored with a simplified cryopreservation method, evaluated the effect of the use of granulocyte-macrophage colony-stimulating factor (GM-CSF) as an in vitro mitotic index stimulator, and compared the cell viability and chromosome morphology of fresh and cryopreserved cells whenever possible (sufficient metaphases for analysis). Twenty-five bone marrow samples from 24 patients with hematological disorders such as acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, chronic myeloid leukemia, megaloblastic anemia and lymphoma (8, 3, 3, 8, 1, and 1 patients, respectively) were selected at diagnosis, at relapse or during routine follow-up and one sample was obtained from a bone marrow donor after informed consent. Average cell viability before and after freezing was 98.8 and 78.5%, respectively (P < 0.05). Cytogenetic analysis was successful in 76% of fresh cell cultures, as opposed to 52% of cryopreserved samples (P < 0.05). GM-CSF had no proliferative effect before or after freezing. The morphological aspects of the chromosomes in fresh and cryopreserved cells were subjectively the same. The present study shows that cytogenetic analysis of cryopreserved bone marrow cells can be a reliable alternative when fresh cell analysis cannot be done, notwithstanding the reduced viability and lower percent of successful analysis that are associated with freezing.

Recent advances in the cryopreservation of shoot-derived germplasm of economically important fruit trees of Actinidia, Diospyros, Malus, Olea, Prunus, Pyrus and Vitis.

Science.gov (United States)

Benelli, Carla; De Carlo, Anna; Engelmann, Florent

2013-01-01

This paper presents the advances made over the last decade in cryopreservation of economically important vegetatively propagated fruit trees. Cryopreservation protocols have been established using both dormant buds sampled on field-grown plants and shoot tips sampled on in vitro plantlets. In the case of dormant buds, scions are partially dehydrated by storage at -5 °C, and then cooled slowly to -30 °C using low cooling rates (c.a. 1 °C/h) before immersion in liquid nitrogen. After slow rewarming and rehydration of samples, regrowth takes place either through grafting of buds on rootstocks or excision of apices and inoculation in vitro. In the case of shoot tips of in vitro plantlets, the cryopreservation techniques employed are the following: controlled rate cooling procedures involving slow prefreezing followed by immersion in liquid nitrogen or vitrification-based procedures including encapsulation-dehydration, vitrification, encapsulation-vitrification and droplet-vitrification. The current status of cryopreservation for a series of fruit tree species including Actinidia, Diospyros, Malus, Olea, Prunus, Pyrus and Vitis is presented. Routine application of cryopreservation for long-term germplasm storage in genebanks is currently limited to apple and pear, for which large cryopreserved collections have been established at NCGRP, Fort Collins (USA), using dormant buds and in vitro shoot tips, respectively. However, there are a growing number of examples of pilot scale testing experiments under way for different species in various countries. Progress in the further development and application of cryopreservation techniques will be made through a better understanding of the mechanisms involved in the induction of tolerance to dehydration and cryopreservation in frozen explants. Copyright © 2012 Elsevier Inc. All rights reserved.

Sperm cryopreservation in fish and shellfish.

Science.gov (United States)

Tiersch, Terrence R; Yang, Huiping; Jenkins, Jill A; Dong, Qiaoxiang

2007-01-01

Initial success in sperm cryopreservation came at about the same time for aquatic species and livestock. However, in the 50-plus years since then cryopreserved sperm of livestock has grown into a billion-dollar global industry, while despite work in some 200 species with well over 200 published reports, cryopreservation of aquatic species sperm remains essentially a research activity with little commercial application. Most research has focused on large-bodied culture and sport fishes, such as salmonids, carps, and catfishes, and mollusks such as commercially important oyster and abalone species. However, only a handful of studies have addressed sperm cryopreservation in small fishes, such as zebrafish, and in endangered species. Overall, this work has yielded techniques that are being applied with varying levels of success around the world. Barriers to expanded application include a diverse and widely distributed literature base, technical problems, small sperm volumes, variable results, a general lack of access to the technology, and most importantly, the lack of standardization in practices and reporting. The benefits of cryopreservation include at least five levels of improvements for existing industries and for creation of new industries. First, cryopreservation can be used to improve existing hatchery operations by providing sperm on demand and simplifying the timing of induced spawning. Second, frozen sperm can enhance efficient use of facilities and create new opportunities in the hatchery by eliminating the need to maintain live males, potentially freeing resources for use with females and larvae. Third, valuable genetic lineages such as endangered species, research models, or improved farmed strains can be protected by storage of frozen sperm. Fourth, cryopreservation opens the door for rapid genetic improvement. Frozen sperm can be used in breeding programs to create improved lines and shape the genetic resources available for aquaculture. Finally

Improved recovery of post-thaw motility and vitality of human spermatozoa cryopreserved in the presence of dithiothreitol.

Science.gov (United States)

Rao, B; David, G

1984-10-01

Semen was collected in the laboratory from nine healthy donors. The concentrations and the percentages of live and motile spermatozoa in all semen samples were within the normal range. Each sample was diluted with citrate-egg yolk-glycerol medium with and without 5 mM dithiothreitol (DTT). Samples were frozen in liquid nitrogen vapor (-70 degrees C) for 7 min and subsequently stored in liquid nitrogen. The effect of DTT in cryopreservation of sperm was determined by comparing percentage of motile and live spermatozoa between controls and DTT-treated post-thaw samples. Percentage of motile spermatozoa was determined by two techniques, laser Doppler velocimetry (LDV) and light microscopy. The percentage of live spermatozoa was measured by microscopic evaluation after staining with eosin-nigrosin. It was shown that the addition of DTT to the freezing medium significantly improved the recovery of motile and live spermatozoa in the post-thaw samples. The mean motility recovery, as measured by LDV, was 44.9% in the controls as compared to 73.9% in the DTT-treated samples. Similarly the mean recovery of live spermatozoa in the controls and DTT-treated samples was 66.5 and 86.6%, respectively. Based on these results, a new hypothesis implicating lipid peroxidation in cryoinjury is proposed. It is also suggested that the use of DTT in the freezing medium may offer an advantage over the commonly used techniques of human sperm cryopreservation.

Dehydration improves cryopreservation of coconut (Cocos nucifera L.).

Science.gov (United States)

Sisunandar; Sopade, Peter A; Samosir, Yohannes M S; Rival, Alain; Adkins, Steve W

2010-12-01

Cryopreservation of coconut can be used as a strategy to back up the establishment of living collections which are expensive to maintain and are under constant threat from biotic and abiotic factors. Unfortunately, cryopreservation protocols still need to be developed that are capable of producing a sizeable number of field-grown plants. Therefore, we report on the development of an improved cryopreservation protocol which can be used on a wide range of coconut cultivars. The cryopreservation of zygotic embryos and their recovery to soil-growing plants was achieved through the application of four optimised steps viz.: (i) rapid dehydration; (ii) rapid cooling; (iii) rapid warming and recovery in vitro and (iv) acclimatization and soil-supported growth. The thermal properties of water within the embryos were monitored using differential scanning calorimetry (DSC) in order to ensure that the freezable component was kept to a minimum. The feasibility of the protocol was assessed using the Malayan Yellow Dwarf (MYD) cultivar in Australia and then tested on a range of cultivars which were freshly harvested and studied in Indonesia. The most efficient protocol was one based on an 8-h rapid dehydration step followed by rapid cooling step. Best recovery percentages were obtained when a rapid warming step and an optimised in vitro culture step were used. Following this protocol, 20% (when cryopreserved 12 days after harvesting) and 40% (when cryopreserved at the time of harvest) of all MYD embryos cryopreserved could be returned to normal seedlings growing in soil. DSC showed that this protocol induced a drop in embryo fresh weight to 19% and significantly reduced the amount of water remaining that could produce ice crystals (0.1%). Of the 20 cultivars tested, 16 were found to produce between 10% and 40% normal seedlings while four cultivars generated between 0% and 10% normal seedlings after cryopreservation. This new protocol is applicable to a wide range of coconut

In vitro reactivity to concanavalin A of bovine lymphocytes after cryopreservation

NARCIS (Netherlands)

Kuil, H.

1984-01-01

Cryopreservation of bovine peripheral lymphocytes and its effect on the in vitro response to concanavalin A tested in a microculture system is described. Using DMSO as cryoprotectant in the medium, the cells were cooled to −30°C at 1.3°C/minute and further to −80°C at 6°C/minute and then rapidly to

Cryopreservation of zebrafish (Danio rerio) oocytes by vitrification.

Science.gov (United States)

Guan, M; Rawson, D M; Zhang, T

2010-01-01

Cryopreservation of fish oocytes is challenging because these oocytes have low membrane permeability to water and cryoprotectant and are highly chilling sensitive. Vitrification is considered to be a promising approach for their cryopreservation as it involves rapid freezing and thawing of the oocytes and therefore minimising the chilling injury. In the present study, vitrification properties and the toxicity of a range of vitrification solutions containing different concentrations of Me2SO, methanol, propylene glycol and ethylene glycol were investigated. Two different base media and vitrification methods were compared. The effect of different post-thaw dilution solutions together with incubation periods on oocyte viability were also investigated. Stage III zebrafish oocytes were equilibrated in increasing concentrations of cryoprotectants for 30 min in 3 steps. Oocytes were thawed rapidly in a water bath and cryoprotectants were removed in 4 steps. Oocyte viability was assessed using trypan blue staining. The results showed that vitrification solutions V3 and V4 in KCl buffer had low toxicity and vitrified well. The survivals of oocytes after stepwise dilution using solutions containing permeable cryoprotectants were significant higher than those diluted in 0.5M glucose, and the use of CVA65 vitrification system improved oocyte survival when compared with plastic straws after 30 min at 22 degrees C post-thawing. Cryopreservation of zebrafish oocytes by vitrification is reported here for the first time, although oocyte survivals after cryopreservation assessed by trypan blue staining were relatively high shortly after thawing, they became swollen and translucent after incubation in KCl buffer. Further studies are needed to optimise the post-thaw culturing conditions.

Efficacy of postal communication with patients who have cryopreserved pre-embryos.

Science.gov (United States)

Brzyski, R G

1998-11-01

To compare the characteristics of patients who did and did not respond to a request for information regarding their cryopreserved pre-embryos. Mail survey. Academic-assisted reproductive technology program. One hundred thirty-six patients with cryopreserved pre-embryos. Patients were surveyed by first-class mail regarding their plans for their cryopreserved pre-embryos and their interest in embryo donation. Age, number of stored pre-embryos, and duration of storage of responders and nonresponders at 6 weeks after mailing. Eighty-three patients (62%) did not respond to the survey. Compared with responders, nonresponders were significantly older at the time of embryo cryopreservation, had fewer pre-embryos cryopreserved, and had the pre-embryos cryopreserved for a longer duration. Five responders (9%) expressed an interest in embryo donation. Three patients requested disposal of pre-embryos. Sixteen surveys (12%) were returned as undeliverable. As a group, these patients had the fewest pre-embryos cryopreserved and had the longest duration of storage. A disturbing number of patients with cryopreserved pre-embryos ignored efforts by our program to maintain contact. Older patients with few cryopreserved pre-embryos may require special attention to avoid abandonment.

Seasonal and cryopreservation impacts on semen quality in boars

Science.gov (United States)

Seasonal boar infertility occurs worldwide and contributes to economic loss to the pork industry. The current study evaluated cooled vs cryopreserved semen quality of 11 Duroc boars collected in June (cool season) and August 2014 (warm season). Semen was cooled to 16°C (cooled) or frozen over liquid...

Effects of chilled storage and cryopreservation on sperm characteristics, antioxidant enzyme activities, and lipid peroxidation in Pacific cod Gadus microcephalus

Science.gov (United States)

Wang, Xueying; Shi, Xuehui; Liu, Yifan; Yu, Daode; Guan, Shuguang; Liu, Qinghua; Li, Jun

2016-07-01

The present study evaluated the effects of chilled storage and cryopreservation on sperm motion characteristics, antioxidant enzyme activities, and lipid peroxidation in the Pacific cod Gadus macrocephalus. Sperm motility and the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (Gr), and lipid peroxidation (measured via malondialdehyde (MDA) content) were determined after the milt was stored at 4°C for 12 h, cryopreserved without cryoprotectant in 12% propylene glycol (PG), cryopreserved in 12% PG+0.1 mol/L trehalose, or cryopreserved in 12% PG spermatozoa but centrifuged to decant the supernatant prior to cryopreservation (only sperm cells were cryopreserved). After chilled storage or cryopreservation, the SOD, CAT and GPx activities were reduced in sperm cells and increased in seminal plasma in almost all treatments; sperm motility parameters were also decreased. However, the addition of trehalose into the cryoprotectant could significantly improve the postthaw sperm quality as revealed by the sperm average path velocity. This improvement might be attributed to the function of trehalose in scavenging reactive oxygen species. Chilled storage and cryopreservation had significant effects on sperm motion characteristics, antioxidant enzyme activities, and lipid peroxidation in the Pacific cod.

Differential immune response of congenic mice to ultraviolet-treated major histocompatibility complex class II-incompatible skin grafts

International Nuclear Information System (INIS)

Vermeer, B.J.; Santerse, B.; Van De Kerckhove, B.A.; Schothorst, A.A.; Claas, F.H.

1988-01-01

The influence of ultraviolet (UVB) irradiation on the survival of H-2 class II-disparate skin grafts was studied in congenic mouse strains. Isolated skin was UVB irradiated in vitro at a dose of 40 mJ/cm 2 from both sides to remove Ia immunogenicity. Immediately after irradiation the skin was transplanted onto the flank of allogeneic mice. When B10.AQR grafts were transplanted onto B10.T(6R) recipients, a significant prolongation of the survival time was observed, while 50% of the UVB-treated grafts were not rejected at all. However, in the opposite direction--i.e., B10.T(6R) grafts onto B10.AQR recipients, no significant prolongation of the survival was observed. To test whether this effect was due to a difference in susceptibility of the donor skin to UVB irradiation or to a different immune response in the recipients, (B10.T(6R) x B10.AQR) grafts were transplanted onto the parent strains. Similar results were obtained, in that UVB-treated grafts did not show a prolonged survival in B10.AQR recipients, whereas a significant prolongation (50% of the grafts survived more than 100 days) was observed in B10.T(6R) recipients. UVB-treated (B10.T(6R) x B10.AQR)F1 grafts were also transplanted onto (B10.T(6R) x C57B1/10)F1, (B10.AQR x C57B1/10)F1, (B10.T(6R) x Balb/c)F1 and (B10.AQR x Balb/c)F1 recipients--but in none of these combinations was a prolonged survival time observed. These data suggest that, in contrast to all in vitro experiments, the abrogation of the immune response by UVB treatment of the stimulator cells is, in vivo, not a general phenomenon. The genetic constitution of the responder mice seems to play an important role in determining whether or not an immune response takes place

Potentials of short term and long term cryopreserved sperm of the ...

African Journals Online (AJOL)

To service the growing demand for male African giant catfish (Clarias gariepinus) broodstock for aquaculture in Nigeria, and to conserve valuable genetic resources, we improved both short-term (in deep freezer at -35°C) and long-term cryopreservation (in liquid nitrogen at -296°C) of catfish sperm. Catfish sperm ...

Heat and mass transfer during the cryopreservation of a bioartificial liver device: a computational model.

Science.gov (United States)

Balasubramanian, Saravana K; Coger, Robin N

2005-01-01

Bioartificial liver devices (BALs) have proven to be an effective bridge to transplantation for cases of acute liver failure. Enabling the long-term storage of these devices using a method such as cryopreservation will ensure their easy off the shelf availability. To date, cryopreservation of liver cells has been attempted for both single cells and sandwich cultures. This study presents the potential of using computational modeling to help develop a cryopreservation protocol for storing the three dimensional BAL: Hepatassist. The focus is upon determining the thermal and concentration profiles as the BAL is cooled from 37 degrees C-100 degrees C, and is completed in two steps: a cryoprotectant loading step and a phase change step. The results indicate that, for the loading step, mass transfer controls the duration of the protocol, whereas for the phase change step, when mass transfer is assumed negligible, the latent heat released during freezing is the control factor. The cryoprotocol that is ultimately proposed considers time, cooling rate, and the temperature gradients that the cellular space is exposed to during cooling. To our knowledge, this study is the first reported effort toward designing an effective protocol for the cryopreservation of a three-dimensional BAL device.

Cryopreservation of Embryos of the Mediterranean Fruit Fly Ceratitis capitata Vienna 8 Genetic Sexing Strain.

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Antonios A Augustinos

Full Text Available The Mediterranean fruit fly, Ceratitis capitata, is one of the most serious pests of fruit crops world-wide. During the last decades, area-wide pest management (AW-IPM approaches with a sterile insect technique (SIT component have been used to control populations of this pest in an effective and environment-friendly manner. The development of genetic sexing strains (GSS, such as the Vienna 8 strain, has been played a major role in increasing the efficacy and reducing the cost of SIT programs. However, mass rearing, extensive inbreeding, possible bottleneck phenomena and hitch-hiking effects might pose major risks for deterioration and loss of important genetic characteristics of domesticated insect. In the present study, we present a modified procedure to cryopreserve the embryos of the medfly Vienna 8 GSS based on vitrification and used this strain as insect model to assess the impact of the cryopreservation process on the genetic structure of the cryopreserved insects. Forty-eight hours old embryos, incubated at 24°C, were found to be the most suitable developmental stage for cryopreservation treatment for high production of acceptable hatch rate (38%. Our data suggest the absence of any negative impact of the cryopreservation process on egg hatch rate, pupation rates, adult emergence rates and stability of the temperature sensitive lethal (tsl character on two established cryopreserved lines (flies emerged from cryopreserved embryos, named V8-118 and V8-228. Taken together, our study provides an optimized procedure to cryopreserve the medfly Vienna 8 GSS and documents the absence of any negative impact on the genetic structure and quality of the strain. Benefits and sceneries for utilization of this technology to support operational SIT projects are discussed in this paper.

Cryopreservation of Embryos of the Mediterranean Fruit Fly Ceratitis capitata Vienna 8 Genetic Sexing Strain.

Science.gov (United States)

Augustinos, Antonios A; Rajamohan, Arun; Kyritsis, Georgios A; Zacharopoulou, Antigone; Haq, Ihsan Ul; Targovska, Asya; Caceres, Carlos; Bourtzis, Kostas; Abd-Alla, Adly M M

2016-01-01

The Mediterranean fruit fly, Ceratitis capitata, is one of the most serious pests of fruit crops world-wide. During the last decades, area-wide pest management (AW-IPM) approaches with a sterile insect technique (SIT) component have been used to control populations of this pest in an effective and environment-friendly manner. The development of genetic sexing strains (GSS), such as the Vienna 8 strain, has been played a major role in increasing the efficacy and reducing the cost of SIT programs. However, mass rearing, extensive inbreeding, possible bottleneck phenomena and hitch-hiking effects might pose major risks for deterioration and loss of important genetic characteristics of domesticated insect. In the present study, we present a modified procedure to cryopreserve the embryos of the medfly Vienna 8 GSS based on vitrification and used this strain as insect model to assess the impact of the cryopreservation process on the genetic structure of the cryopreserved insects. Forty-eight hours old embryos, incubated at 24°C, were found to be the most suitable developmental stage for cryopreservation treatment for high production of acceptable hatch rate (38%). Our data suggest the absence of any negative impact of the cryopreservation process on egg hatch rate, pupation rates, adult emergence rates and stability of the temperature sensitive lethal (tsl) character on two established cryopreserved lines (flies emerged from cryopreserved embryos), named V8-118 and V8-228. Taken together, our study provides an optimized procedure to cryopreserve the medfly Vienna 8 GSS and documents the absence of any negative impact on the genetic structure and quality of the strain. Benefits and sceneries for utilization of this technology to support operational SIT projects are discussed in this paper.

Wound-Healing Potential of Cultured Epidermal Sheets Is Unaltered after Lyophilization: A Preclinical Study in Comparison to Cryopreserved CES

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H. Jang

2013-01-01

Full Text Available Lyophilized Cultured Epidermal Sheets (L-CES have been reported to be as effective as the cryopreserved CES (F-CES in treating skin ulcers. However, unlike F-CES, no preclinical study assessing wound-healing effects has been conducted for L-CES. The present study was set out to investigate the microstructure, cytokine profile, and wound-healing effects of L-CES in comparison to those of F-CES. Keratinocytes were cultured to prepare CES, followed by cryopreservation at −70°C and lyophilization. Under microscopic observation, intact cells with apparent intracellular junctions were observed in L-CES. The L-CES, like fresh CES, consisted of three to four well-maintained epidermal layers, as shown by the expression of keratins, involucrin, and p63. There were no differences in the epidermal layer or protein expression between L-CES and F-CES, and both CES were comparable to fresh CES. TGF-α, EGF, VEGF, IL-1α, and MMPs were detected in L-CES at levels similar to those in F-CES. In a mouse study, wounds treated with L-CES or F-CES completely healed at least 4 days faster than untreated wounds. CES-treated wounds completely healed by day 10, while the untreated wounds did not heal by day 14. Masson’s trichrome staining showed that collagen deposition in the CES-treated wounds was highly increased in the dermis of the wound center compared to that in the control wounds. Thus, this study demonstrates that L-CES is as clinically effective as F-CES for wound treatment.

Cryopreservation of Embryos and Oocytes in Human Assisted Reproduction

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János Konc

2014-01-01

Full Text Available Both sperm and embryo cryopreservation have become routine procedures in human assisted reproduction and oocyte cryopreservation is being introduced into clinical practice and is getting more and more widely used. Embryo cryopreservation has decreased the number of fresh embryo transfers and maximized the effectiveness of the IVF cycle. The data shows that women who had transfers of fresh and frozen embryos obtained 8% additional births by using their cryopreserved embryos. Oocyte cryopreservation offers more advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation, and postponing childbirth, and eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In this review, the basic principles, methodology, and practical experiences as well as safety and other aspects concerning slow cooling and ultrarapid cooling (vitrification of human embryos and oocytes are summarized.

Cryopreservation of embryos and oocytes in human assisted reproduction.

Science.gov (United States)

Konc, János; Kanyó, Katalin; Kriston, Rita; Somoskői, Bence; Cseh, Sándor

2014-01-01

Both sperm and embryo cryopreservation have become routine procedures in human assisted reproduction and oocyte cryopreservation is being introduced into clinical practice and is getting more and more widely used. Embryo cryopreservation has decreased the number of fresh embryo transfers and maximized the effectiveness of the IVF cycle. The data shows that women who had transfers of fresh and frozen embryos obtained 8% additional births by using their cryopreserved embryos. Oocyte cryopreservation offers more advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation, and postponing childbirth, and eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In this review, the basic principles, methodology, and practical experiences as well as safety and other aspects concerning slow cooling and ultrarapid cooling (vitrification) of human embryos and oocytes are summarized.

First attempts to cryopreserve red abalone (Haliotis rufescens oocytes

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Ramírez Torrez, A.

2015-01-01

Full Text Available Overall, few advances in the cryopreservation of complex cells such as oocytes, embryo or tissue have been registered and in less quantity have been reported for aquatic species. Abalone has high economic interest worldwide and the conservation of abalone germplasm may help to enhance its culture and develop repopulation programs. In this work, we reported the cytotoxic effect of two concentration of trehalose (0.2 and 0.4 M on red abalone oocytes incubated for 10, 15 and 20 min. Also, we reported the cryopreservation of red abalone oocytes using a 3-steps cryopreservation protocol and 5 thawing protocols. Significant differences on cytotoxic effect were found (p<0.01. However, none of the cryoprotectant was optimum to cryopreserve red abalone oocyte. In conclusion, it is necessary to find an appropriate method to dehydrate or make the cryoprotectant penetrate on the abalone oocyte before proceeding to cryopreservation.

Strategies for commercialization of cryopreserved fish semen

Directory of Open Access Journals (Sweden)

Terrence R. Tiersch

2008-07-01

Full Text Available Initial success in sperm cryopreservation occurred at about the same time for aquatic species and livestock. However, in the 50 plus years since then cryopreserved sperm of livestock has grown into a billion-dollar global industry, while cryopreserved sperm of aquatic species remains a research activity with little commercial application despite work in more than 90 species and more than 200 published reports. Most research work has focused on large-bodied culture and sport fishes, such as salmon, trout, carp, and catfish, and mollusks such as commercially important oyster and abalone species. However, only a few studies have addressed sperm cryopreservation in small fishes such as zebrafish, or in endangered species. Overall, this work has yielded techniques that are being applied with varied levels of success around the world. Barriers to expanded application include a diverse and widely distributed literature base, technical problems, small sperm volumes, variable results, a general lack of access to the technology, and most importantly, a lack of standardization in practices and reporting. The benefits of cryopreservation include at least five levels of improvements for existing industries and for creation of new industries. First, cryopreservation can be used to improve existing hatchery operations by providing sperm on demand and simplifying the timing of induced spawning. Second, frozen sperm can enhance efficient use of facilities and create new opportunities in the hatchery by eliminating the need to maintain live males, potentially freeing resources for use with females and larvae. Third, valuable genetic lineages such as endangered species, research models or improved farmed strains can be protected by storage of frozen sperm. Fourth, cryopreservation opens the door for rapid genetic improvement. Frozen sperm can be used in breeding programs to create improved lines and shape the genetic resources available for aquaculture. Finally

Evaluation of amides and centrifugation temperature in boar semen cryopreservation.

Science.gov (United States)

Bianchi, I; Calderam, K; Maschio, E F; Madeira, E M; da Rosa Ulguim, R; Corcini, C D; Bongalhardo, D C; Corrêa, E K; Lucia, T; Deschamps, J C; Corrêa, M N

2008-03-15

Two experiments were conducted to evaluate the use of amides as cryoprotectants and two centrifugation temperatures (15 or 24 degrees C) in boar semen cryopreservation protocols. Semen was diluted in BTS, cooled centrifuged, added to cooling extenders, followed by the addition of various cryoprotectants. In experiment 1, mean (+/-S.E.M.) sperm motility for 5% dimethylformamide (DMF; 50.6+/-1.9%) and 5% dimethylacetamide (DMA; 53.8+/-1.7%) were superior (P0.05). In experiment 2, we tested MF, DMF, and DMA at 3, 5, and 7%. Sperm motility and membrane integrity were higher for 5% DMA (53.8+/-1.7 and 50.9+/-1.9%) and 5% DMF (50.6+/-1.9 and 47.9+/-2.1%), in comparison with 7% DMF and all MF concentrations (P0.05). In conclusion, boar semen was successfully cryopreserved by replacement of glycerol with amides (especially 5% DMA) and centrifugation at 15 degrees C, with benefits for post-thaw sperm motility and membrane integrity.

Immune Checkpoint Targets for Host-Directed Therapy to Prevent and Treat Leishmaniasis

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Rajiv Kumar

2017-11-01

Full Text Available Leishmaniasis encompasses a group of diseases caused by protozoan parasites belonging to the genus Leishmania. These diseases range from life threatening visceral forms to self-healing cutaneous lesions, and each disease manifestations can progress to complications involving dissemination of parasites to skin or mucosal tissue. A feature of leishmaniasis is the key role host immune responses play in disease outcome. T cells are critical for controlling parasite growth. However, they can also contribute to disease onset and progression. For example, potent regulatory T cell responses can develop that suppress antiparasitic immunity. Alternatively, hyperactivated CD4+ or CD8+ T cells can be generated that cause damage to host tissues. There is no licensed human vaccine and drug treatment options are often limited and problematic. Hence, there is an urgent need for new strategies to improve the efficacy of current vaccine candidates and/or enhance both antiparasitic drug effectiveness and subsequent immunity in treated individuals. Here, we describe our current understanding about host immune responses contributing to disease protection and progression in the various forms of leishmaniasis. We also discuss how this knowledge may be used to develop new strategies for host-directed immune therapy to prevent or treat leishmaniasis. Given the major advances made in immune therapy in the cancer and autoimmune fields in recent years, there are significant opportunities to ride on the back of these successes in the infectious disease domain. Conversely, the rapid progress in our understanding about host immune responses during leishmaniasis is also providing opportunities to develop novel immunotherapy strategies that could have broad applications in diseases characterized by inflammation or immune dysfunction.

Evaluation of the damage in fish spermatozoa cryopreservation

Science.gov (United States)

Li, Jun; Liu, Qinghua; Zhang, Shicui

2006-12-01

Cryodamages occur during sperm cryopreservation. Cryopreservation of fish sperm usually results in marked decrease in sperm quality, such as swelling or disruption of the plasma membrane, mitochondrial dysfunction, diminished sperm motility, impaired velocity, shorter motility period, denaturation, and release of some enzymes from spermatozoa. In this paper, damages in morphology, physiology, biochemistry and metabolism, and genetic integrity of fish semen after cryopreservation are discussed. New approaches in assessment of fish thawed sperm quality such as computer assisted sperm analysis, flow cytometic analysis combined with fluorescent probes and single cell gel electrophoresis are also briefly reviewed.

LONG-TERM PRECONDITIONING OF PLANTLETS: A PRACTICAL METHOD FOR ENHANCING SURVIVAL OF PINEAPPLE (Ananas comosus Merr.) SHOOT TIPS CRYOPRESERVED USING VITRIFICATION.

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Hu, W H; Liu, S F; Liaw, S I

2015-01-01

The purpose of this study was to develop an efficient cryopreservation protocol for pineapple (Ananas comosus Merr.) shoot tips. The optimal state of pineapple plantlets was investigated by using sucrose preconditioning to enhance survival after cryostorage. To achieve a suitable state of plantlets before cryopreservation, 0.2 M to 0.4 M sucrose concentrations combined with short- (0-7 days), medium- (15-30 days), and long-term (75-150 days) preconditioning periods were compared. The highest survival (100 %) was achieved using the following procedure: intact plantlets underwent long-term preconditioning with 0.2 M sucrose for 135 days, dissected shoot tips were treated with a loading solution containing 2.0 M glycerol + 0.4 M sucrose for 60 min at 25 degree and the shoot tips were dehydrated in PVS2 for 2h at 0 degree C before being plunged in liquid nitrogen. Rewarming was conducted in a water-bath for 30 s at 40 degree C and PVS2 was replaced with a 1.2 M sucrose solution for 30 min at 25 degree C. The shoot tips were transferred on semisolid medium and left in the dark for 1 week, then in dim light for 3 weeks.

In Vitro Propagarion and Cryopreservation of Important Grape Cultivars (Vitis Vinifera L. and Rootstocks

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F. CELEBI TOPRAK

2014-06-01

Full Text Available Grape (Vitis vinifera L. is among the most important species that is cultivated almost all around the world. There are over one thousand varieties that are grown for raisin, fresh consumption and wine making purposes. The grape germplasm resources are generally maintained as whole plants under field conditions. The traditional way of germplasm preservation is very risky due to natural uncertainties. In vitro technologies can help producing healthy propagation materials free from viroids, viruses, bacteria, phytoplasmas, fungi, and nematodes. When combined with cryopreservation technologies in vitro preservation systems can allow safe protection and propagation of valuable Vitis genetic resources. In this study, 12 commercial cultivar and two rootstock materials were tested for the applicability of long term preservation by in vitro clonal propagation and cryopreservation techniques. Axillary shoot tips collected from newly emerging shoots were placed in Magenda boxes containing 30 g/l sucrose on MS medium and cultured in a growth chamber adjusted to 16 h ligth/25o C and 8 h dark/17o C. All grape genotypes tested responded well to this application and produced healthy root and shoots. Shoot explants from these in vitro stocks were subcultured in every three months for one year. Apical dome explants excised from in vitro grape plants were stored in liquid nitrogen for cryopreservation. Genotypes varied in their responses to cryopservation treatment. Five genotypes showed shoot or callus formation. Regenerated shoots continued to grow and produced normal shoots and roots, but no plants could be developed from calli. Flow cytometry analysis of regenerants from continuous subculture and cryopreservation did not show any chromosome number abnormalities. In vitro micropropagation is an excellent choice for a long-term conservation of grape germplasm, which allows access to actively growing plant materials without seasonal restriction. Such cultures are

[Ultrastructural changes in the MP3 neuron of the mollusk Lymnaea stagnalis after cryopreservation of the isolated brain].

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Dmitrieva, E V; Moshkov, D A; Gakhova, E N

2006-01-01

Investigation of a possibility of long-term storage of frozen (-196 degrees C) viable neurons and nervous tissue is one of the central present day problems. In this study ultrastructural changes in neurons of frozen-thawed snail brain were examined as a function of time. We studied the influence of cryopreservation, cryoprotectant (Me2SO), cooling to 4-6 degrees C, and a prolonged incubation in physiological solution at 4-6 degrees C on dictyosomes of Golgi apparatus, endoplasmic reticulum (ER) cisternae and mitochondria. It has been found that responses of these intracellular structures of cryopreserved neurons to the above influences are similar: dissociation of Golgi dictyosomes, swelling of endoplasmic reticulum cisternae and mitochondrial cristae. Both freezing-thawing and cryoprotectant were seen to cause an increase in the number of lysosomes, liposomes, myelin-like structures, and to form large vacuoles. The structural changes in molluscan neurons caused by cryopreservation with Me2SO (2 M) were reversible.

Cryopreservation of ectomycorrhizal fungi has minor effects on root colonization of Pinus sylvestris plantlets and their subsequent nutrient uptake capacity.

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Crahay, Charlotte; Wevers, Jan; Munaut, Françoise; Colpaert, Jan V; Declerck, Stéphane

2013-08-01

The use of ectomycorrhizal (ECM) fungi for afforestation, bioremediation, and timber production requires their maintenance over long periods under conditions that preserve their genetic, phenotypic, and physiological stability. Cryopreservation is nowadays considered as the most suitable method to maintain the phenotypic and genetic stability of a large number of filamentous fungi including the ECM fungi. Here, we compared the ability of eight ECM fungal isolates to colonize Pinus sylvestris roots and to transport inorganic phosphate (Pi) and NH4 (+) from the substrate to the plant after cryopreservation for 6 months at -130 °C or after storage at 4 °C. Overall, the mode of preservation had no significant effect on the colonization rates of P. sylvestris, the concentrations of ergosterol in the roots and substrate, and the uptake of Pi and NH4 (+). Comparing the isolates, differences were sometimes observed with one or the other method of preservation. Suillus bovinus exhibited a reduced ability to form mycorrhizas and to take up Pi following cryopreservation, while one Suillus luteus isolate exhibited a decreased ability to take up NH4 (+). Conversely, Hebeloma crustuliniforme, Laccaria bicolor, Paxillus involutus, and Pisolithus tinctorius exhibited a reduced ability to form mycorrhizas after storage at 4 °C, although this did not result in a reduced uptake of Pi and NH4 (+). Cryopreservation appeared as a reliable method to maintain important phenotypic characteristics (i.e., root colonization and nutrient acquisition) of most of the ECM fungal isolates studied. For 50 % of the ECM fungal isolates, the colonization rate was even higher with the cultures cryopreserved at -130 °C as compared to those stored at 4 °C.

The effect of Curcuma longa extracted (curcumin) on the quality of cryopreserved boar semen.

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Chanapiwat, Panida; Kaeoket, Kampon

2015-09-01

The aim of this study was to determine the optimal concentration of curcumin needed for cryopreservation of boar semen. Semen samples (n = 9) were collected from nine Duroc boars which having proven fertility were used for routine artificial insemination. Semen samples were collected and divided into six groups (groups A-F) according to various concentrations of curcumin in freezing extender (i.e. 0, 0.125, 0.25, 0.50, 0.75 and 1.0 mmol/L, respectively). The semen was frozen by traditional liquid nitrogen vapor method and stored at -196°C in the liquid nitrogen tank. After storage, frozen semen samples were thawed at 50°C for 12 s and evaluated for progressive motility, viability and acrosome integrity. The present results indicated that the addition of curcumin at 0.25 (group C) or 0.50 mmol/L curcumin (group D) yielded the higher percentage of progressive motility (33.3 and 36.1%, respectively) (P curcumin during cryopreservation at a concentration of 0.25 or 0.50 mmol/L is the optimal concentration of curcumin for improving the quality (i.e. increased progressive motility and acrosome integrity) of cryopreserved boar semen. © 2015 Japanese Society of Animal Science.

Apoptosis in fresh and cryopreserved cardiac valves of pig samples.

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Rendal Vázquez, M Esther; Díaz Román, T M; Rodríguez Cabarcos, M; Zavanella Botta, C; Domenech García, N; González Cuesta, M; Sánchez Dopico, M J; Pértega Díaz, S; Andión Núñez, C

2008-06-01

To analyse the influence of cold ischemic time (CIT) (2-24 h) and of cryopreservation (liquid phase) on the viability of the valvular fibroblasts and in the presence of apoptosis. Cardiac valves from 10 pigs were evaluated by anatomo-pathological study of the wall, muscle and leaflet. At the same time, the presence of cellular death due to apoptosis was investigated in two ways; directly on tissue by Apodetec system and by two-colour flow cytometry assay analyzing a suspension of fibroblast from valve leaflets using Anexina V and propidium iodure (PI). We established three groups of samples to compare different experimental conditions: 2 h of ischemia (group 1), 24 h of ischemia (group 2), and a programme of cryopreservation (-1 degrees C/min) after 2 h of ischemia, followed by storage in liquid nitrogen during a week and thawing was performed (group 3). The analysis of viabilities showed slight differences between all three groups. The results indicated CIT of 24 h undergoing more structural affectation than CIT of 2 h. Flow cytometry analysis did not show important differences between groups; however cryopreserved samples (group 3) slightly less viability and a higher percentage of death by apoptosis than group 1 and 2 using flow cytometry. Apoptosis was confirmed on tissue from all valves but mainly in samples of group 2 and group 3. In summary, the viability of the valves in the case of ischemic times of 2 h, 24 h or after cryopreservation/thawing differs slightly. The death of the cells is mainly mediated by necrosis and not by apoptosis.

Chapter 1 Historical Background on Gamete and Embryo Cryopreservation.

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Ali, Jaffar; AlHarbi, Naif H; Ali, Nafisa

2017-01-01

This chapter describes the development of the science of cryopreservation of gametes and embryos of various species including human. It attempts to record in brief the main contributions of workers in their attempts to cryopreserve gametes and embryos. The initial difficulties faced and subsequent developments and triumphs leading to present-day state of the art are given in a concise manner. The main players and their contributions are mentioned and the authors' aim is to do justice to them. This work also attempts to ensure that credit is correctly attributed for significant advances in gamete and embryo cryopreservation. In general this chapter has tried to describe the historical development of the science of cryopreservation of gametes and embryos as accurately as possible without bias or partiality.

Alternative procedures for the cryopreservation of brown bear ejaculates depending on the flexibility of the "in cooling" period (5°C).

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López-Urueña, E; Alvarez, M; Gomes-Alves, S; Manrique, P; Anel-López, L; Chamorro, C A; Borragan, S; de Paz, P; Anel, L

2014-12-01

The adaptability of cryopreservation protocols for brown bear spermatozoa collected under field conditions and frozen in a nearby laboratory (transported for a few hours) or shipped to a reference laboratory for sex sorting (transported for a few days) was evaluated. Forty-nine electroejaculates from 15 mature brown bears were extended to 100×10(6) sperm/mL in a TES-Tris-Fructose based extender and cryopreserved (-20°C/min to -100°C and stored at -196°C). After thawing, the quality of the seminal samples was assessed for total (TM), progressive (PM) motility and kinetic parameters - by CASA -, and viability (VIAB), viable and non-apoptotic status (YOPRO-), high membrane mitochondrial potential (MIT) and intact acrosomes (iACR) - by flow cytometry -. In Experiment 1, we assessed different storage times (0, 0.5, 1 - control -, 4-5, 7-8 and 11-12 h) at 5°C from final dilution to freezing. After thawing, non-equilibrated samples (0 h) showed lower values of iACR, TM and PM. No significant differences were found for the different periods of equilibration tested. In Experiment 2, we evaluated three long-term storage times (24, 48 and 72 h) at 5°C before freezing using storage for 1h as control. The post-thawing quality of brown bear spermatozoa declined markedly after 48-72 h of pre-freezing. In conclusion, our findings suggest the possibility of extending the pre-freezing cooling period up to 24h post-collection without freezing. This knowledge should enable the adaptation of the freezing protocols for when a special handling conditions are required such as the shipment of seminal samples to technological centers for the pre-freezing application of enhancer spermatic biotechnologies. Copyright © 2014 Elsevier Inc. All rights reserved.

Cryopreservation of Mammalian Oocyte for Conservation of Animal Genetics

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Jennifer R. Prentice

2011-01-01

Full Text Available The preservation of the female portion of livestock genetics has become an international priority; however, in situ conservation strategies are extremely expensive. Therefore, efforts are increasingly focusing on the development of a reliable cryopreservation method for oocytes, in order to establish ova banks. Slow freezing, a common method for cryopreservation of oocytes, causes osmotic shock (solution effect and intracellular ice crystallization leading to cell damage. Vitrification is an alternative method for cryopreservation in which cells are exposed to a higher concentration of cryoprotectants and frozen with an ultra rapid freezing velocity, resulting in an ice crystal free, solid glass-like structure. Presently, vitrification is a popular method for cryopreservation of embryos. However, vitrification of oocytes is still challenging due to their complex structure and sensitivity to chilling.

Ocular Fluid As a Replacement for Serum in Cell Cryopreservation Media.

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Vivek Phani Varma

Full Text Available Cryostorage is of immense interest in biomedical research, especially for stem cell-based therapies and fertility preservation. Several protocols have been developed for efficient cryopreservation of cells and tissues, and a combination of dimethyl sulfoxide (DMSO and fetal bovine serum (FBS is commonly used. However, there is a need for an alternative to FBS because of ethical reasons, high cost, and risk of contamination with blood-borne diseases. The objective of the present study was to examine the possibility of using buffalo (Bubalus bubalis ocular fluid (BuOF to replace FBS in cryomedia. Frozen-thawed cells, which were cryopreserved in a cryomedia with BuOF, were assessed for viability, early and late apoptosis, and proliferation. Three cell lines (CHO, HEK, and C18-4, mouse embryonic stem (mES cells, and primary cells, such as mouse embryonic fibroblast (MEF cells, human peripheral blood mononuclear cells (hPBMCs, and mouse bone marrow cells (mBMCs, were cryopreserved in cryomedia containing 10% DMSO (D10 with 20% FBS (D10S20 or D10 with 20% BuOF (D10O20. For all three cell lines and mES cells cryopreserved in either D10S20 or D10O20, thawed cells showed no difference in cell viability or cell recovery. Western blot analysis of frozen-thawed-cultured cells revealed that the expression of Annexin V and proliferating cell nuclear antigen (PCNA proteins, and the ratio of BAX/BCL2 proteins were similar in all three cell lines, mES cells, and hPBMCs cryopreserved in D10S20 and D10O20. However, initial cell viability, cell recovery after culture, and PCNA expression were significantly lower in MEF cells, and the BAX/BCL2 protein ratio was elevated in mBMCs cryopreserved in D10O20. Biochemical and proteomic analysis of BuOF showed the presence of several components that may have roles in imparting the cryoprotective property of BuOF. These results encourage further research to develop an efficient serum-free cryomedia for several cell types

Effect of cryopreservation on the pre-hatching behavior in the Mexican fruit fly Anastrepha ludens Loew (Diptera, Tephritidae).

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Rajamohan, Arun; Rinehart, Joseph P; Leopold, Roger A

2018-02-01

In a sampling of untreated embryos of the economically important fruit pest species, Anastrepha ludens, the cumulative hatch percentage in the lab was noted to be ∼85%. Approximately 70% of the larvae had eclosed through the posterior pole of the egg. This process is effected by the act of Pole Reversal (PR) of the fully developed pre-hatch larva from the wider anterior to the narrower posterior pole of the egg. Investigation of the effects of cryopreservation and various pretreatments prior to cryostorage on the PR behavior was prompted by the observation of significantly lower proportion of cryopreserved embryos exhibiting the PR behavior. Pretreatments (dechorionation and permeabilization) followed by vitrification resulted in delayed hatching, reflecting a slower embryonic development rate of ∼10 h. A smaller proportion of the treated embryos either eclosed from the anterior end of the egg or did not eclose at all despite complete development and prehatch gnawing activity. In the untreated controls, 24.0% of the embryos eclosed from the anterior pole. After permeabilization and cryopreservation, 83% and 55% (adjusted hatch) of the embryos were noted to hatch this way, respectively. An analysis of the hatch count after the treatments shows that factors contributing to the embryos' inability to properly invert polarity is not solely due to cryopreservation but also due to the pretreatment procedures including dechorionation and permeabilization. In fact, the permeabilization pre-treatment contributed the highest to this phenomenon lending support to the view that chemical toxicity rather than physical effects of cryopreservation play a major role in post-cryopreservation effects. Published by Elsevier Inc.

Cryopreservation techniques and their application in vegetatively propagated crop plants in Finland

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A. NUKARI

2008-12-01

Full Text Available Cryopreservation protocols have been introduced as techniques for germplasm preservation of vegetatively propagated horticultural and staple food crops. In Finland, cryopreservation has been studied since 1990’s, beginning with cryopreservation of forest tree breeding material and since 2004 on cryopreservation of genetic resources of horticultural plants and potato. Priority was given to cryopreservation of raspberry (Rubus ideaus L., strawberry (Fragaria x ananassa Duch. and potato (Solanum tuberosum L. and the possibility to use cryotherapy in eradication of raspberry bushy dwarf virus (RBDV from in vitro cultures were studied on raspberry. Modified droplet vitrification cryopreservation protocols were designed for raspberry and strawberry and cryotherapy combined with thermotherapy was proven to be a successful application to eliminate RBDV from infected raspberries. Cryotherapy method can be applied for a large scale elimination of viruses from plant germplasm and from candidate nuclear stock in a certified plant production scheme. Routine use of cryotechniques in germplasm preservation of vegetatively propagated horticultural plants was started. Besides for long term germplasm preservation, cryopreservation techniques can be applied also for maintenance of mother stocks in certified plant production schemes and in commercial plant production. Cryopreservation of potato shoot tips needs additional detailed research to obtain sufficient recovery and regrowth rates.;

Cryopreserved irradiated tracheal homograft reconstruction for subglottic-tracheal stenosis

International Nuclear Information System (INIS)

Somyos Kunachak; Yongyudh Vajaradul; Boonchu Kulapaditharom

1999-01-01

Subglottic-tracheal stenosis is a common clinical entity. Handling on severe case is often problematic. Various tracheal replacement techniques have been used with varying degree of success and dispute. In this study we worked on cryopreserved irradiated tracheal homograft, of which its use in human has not been reported. The tracheas were harvested from donor cadavers within 24 hours of death in a sterile condition. After 1-2 weeks of preservation at -70 degree C, the grafts were irradiated at 25 kGy, then stored at -70 degree C until used. Four patients, 2 males and 2 females (aged 2-40 years, mean 16 years) with severe subglottic-tracheal stenosis underwent segmental tracheal graft reconstruction using this graft. Immunosuppressant was not given in any patient. The follow up period ranged from 11-1 5 months. Three patients were successfully decapulated, 1 patient developed local infection and dislodgement of intraluminal stent with subsequent restenosis. Postoperative tracheal lumen appeared near normal with histologic evidence of normal respiratory epithelium at the grafted site. In conclusion, cryopreserved irradiated tracheal homograft is a valuable alternative for tracheal transplant or reconstruction, without the need of immunosuppression

Ovarian tissue cryopreservation and transplantation among alternatives for fertility preservation in the Nordic countries

DEFF Research Database (Denmark)

Rodriguez-Wallberg, Kenny A; Tanbo, Tom; Tinkanen, Helena

2016-01-01

cryopreservation to be experimental. In Iceland, embryo cryopreservation is the only option for fertility preservation. Most centers use slow-freezing methods for ovarian tissue cryopreservation. Most patients selected for ovarian tissue cryopreservation were newly diagnosed with cancer and the tissue...

Cryopreservation and revival of mesenchymal stromal cells

DEFF Research Database (Denmark)

Haack-Sørensen, Mandana; Kastrup, Jens

2011-01-01

initiated. As there has been a precedent for the use of bone marrow stem cells in the treatment of hematological malignancies and ischemic heart diseases through randomized clinical safety and efficacy trials, the development of new therapies based on culture-expanded human mesenchymal stromal cells (MSCs......Over the past few years, the pace of preclinical stem cell research is astonishing and adult stem cells have become the subject of intense research. Due to the presence of promising supporting preclinical data, human clinical trials for stem cell regenerative treatment of various diseases have been......) opens up new possibilities for cell therapy. To facilitate these applications, cryopreservation and long-term storage of MSCs becomes an absolute necessity. As a result, optimization of this cryopreservation protocol is absolutely critical. The major challenge during cellular cryopreservation...

Cryopreservation of human colorectal carcinomas prior to xenografting

International Nuclear Information System (INIS)

Linnebacher, Michael; Maletzki, Claudia; Ostwald, Christiane; Klier, Ulrike; Krohn, Mathias; Klar, Ernst; Prall, Friedrich

2010-01-01

Molecular heterogeneity of colorectal carcinoma (CRC) is well recognized, forming the rationale for molecular tests required before administration of some of the novel targeted therapies that now are rapidly entering the clinics. For clinical research at least, but possibly even for future individualized tumor treatment on a routine basis, propagation of patients' CRC tissue may be highly desirable for detailed molecular, biochemical or functional analyses. However, complex logistics requiring close liaison between surgery, pathology, laboratory researchers and animal care facilities are a major drawback in this. We here describe and evaluate a very simple cryopreservation procedure for colorectal carcinoma tissue prior to xenografting that will considerably reduce this logistic complexity. Fourty-eight CRC collected ad hoc were xenografted subcutaneously into immunodeficient mice either fresh from surgery (N = 23) or after cryopreservation (N = 31; up to 643 days). Take rates after cryopreservation were satisfactory (71%) though somewhat lower than with tumor tissues fresh from surgery (74%), but this difference was not statistically significant. Re-transplantation of cryopreserved established xenografts (N = 11) was always successful. Of note, in this series, all of the major molecular types of CRC were xenografted successfully, even after cryopreservation. Our procedure facilitates collection, long-time storage and propagation of clinical CRC specimens (even from different centres) for (pre)clinical studies of novel therapies or for basic research

Oocyte cryopreservation beyond cancer: tools for ethical reflection.

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Linkeviciute, Alma; Peccatori, Fedro A; Sanchini, Virginia; Boniolo, Giovanni

2015-08-01

This article offers physicians a tool for structured ethical reflection on challenging situations surrounding oocyte cryopreservation in young healthy women. A systematic literature review offers a comprehensive overview of the ethical debate surrounding the practice. Ethical Counseling Methodology (ECM) offers a practical approach for addressing ethical uncertainties. ECM consists of seven steps: (i) case presentation; (ii) analysis of possible implications; (iii) presentation of ethical question(s); (iv) explanation of ethical terms; (v) presentation of the ethical arguments in favor of and against the procedure; (vi) examination of the individual patient's beliefs and wishes; and (vii) conclusive summary. The most problematic aspects in the ethical debate include the distinction between medical and non-medical use of oocyte cryopreservation, safety and efficiency of the procedure, and marketing practices aimed at healthy women. Female empowerment and enhanced reproductive choices (granted oocyte cryopreservation is a safe and efficient technique) are presented as ethical arguments supporting the practice, while ethical reservations towards oocyte cryopreservation are based on concerns about maternal and fetal safety and wider societal implications. Oocyte cryopreservation is gaining popularity among healthy reproductive age women. However, despite promised benefits it also involves risks that are not always properly communicated in commercialized settings. ECM offers clinicians a tool for structured ethical analysis taking into consideration a wide range of implications, various ethical standpoints, and patients' perceptions and beliefs.

Cryopreservation of citrus seed via dehydration followed by immersion in liquid nitrogen

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An important method for plant germplasm conservation is offered by a biotechnology-based approach of cryopreservation. Cryopreservation refers to the storage of plant material at ultralow temperatures in liquid nitrogen. A procedure for cryopreservation of polyembryonic seeds was improved for select...

Effects of bestatin on the host immunity in patients treated for urogenital cancer

International Nuclear Information System (INIS)

Ozono, S.; Iwai, A.; Babaya, K.; Hiramatsu, T.; Yoshida, K.; Yamada, K.; Hirao, Y.; Aoyama, H.; Ohara, S.; Okajima, E.

1990-01-01

To examine effects of bestatin on the host immunity of patients with urogenital cancer, 54 patients were randomized into 2 groups: bestatin treated and controls. In each group, the patients were divided into 2 subgroups: one which received basic treatment expected to greatly affect host immunity ('invasive treatment') while the other one received other types of basic treatment ('non-invasive treatment'). Peripheral lymphocyte, OKT 4/8 ratio and purified protein derivative (PPD) skin reaction were used as immunological markers. There were significant differences in the 'invasive' treatment group between bestatin treated patients and controls concerning lymphocyte counts and PPD skin reactions and in the 'non-invasive' group concerning lymphocyte counts and OKT 4/8 ratios. These results suggest that bestatin may potentiate host immunity in patients with urogenital cancer. Further studies on larger materials are, however, needed before more definite conclusions can be drawn. (orig.)

Immature dendritic cells generated from cryopreserved human monocytes show impaired ability to respond to LPS and to induce allogeneic lymphocyte proliferation.

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Guilherme Ferreira Silveira

Full Text Available Dendritic cells play a key role in the immune system, in the sensing of foreign antigens and triggering of an adaptive immune response. Cryopreservation of human monocytes was investigated to understand its effect on differentiation into immature monocyte-derived dendritic cells (imdDCs, the response to inflammatory stimuli and the ability to induce allogeneic lymphocyte proliferation. Cryopreserved (crp-monocytes were able to differentiate into imdDCs, albeit to a lesser extent than freshly (frh-obtained monocytes. Furthermore, crp-imdDCs had lower rates of maturation and cytokine/chemokine secretion in response to LPS than frh-imdDCs. Lower expression of Toll-like receptor 4 (at 24 and 48 h and higher susceptibility to apoptosis in crp-imdDCs than in fresh cells would account for the impaired maturation and cytokine/chemokine secretion observed. A mixed leukocyte reaction showed that lymphocyte proliferation was lower with crp-imdDCs than with frh-imdDCs. These findings suggested that the source of monocytes used to generate human imdDCs could influence the accuracy of results observed in studies of the immune response to pathogens, lymphocyte activation, vaccination and antigen sensing. It is not always possible to work with freshly isolated monocytes but the possible effects of freezing/thawing on the biology and responsiveness of imdDCs should be taken into account.

Nucleons II: cryopreservation and metabolic activity.

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Reyes, R; Flores-Alonso, J C; Rodríguez-Hernández, H M; Merchant-Larios, H M; Delgado, N M

2001-01-01

The establishment of intracytoplasmatic sperm injection (ICSI) as a routine procedure in assisted fertilization has been used in the treatment of male infertility. The major technical problem that has arisen with the use of immotile sperm for ICSI has been differentiating between live and dead cells. Nucleons from human, pig, hamster, mouse, rat, and bull have been able to induce their chromatin decondensation by the action of heparin/GSH. Cryopreservation is deleterious to sperm function, killing more than 50% of the spermatozoa during the process. Nucleon cryostorage was performed at 5 and -5 degrees C and analyzed for total area (mu2), perimeter (mu), width (mu), and length (mu), using Metamorph Imaging System software. On the other hand, fluorescein diacetate (FDA) is hydrolyzed by intracellular estereases to produce fluorescein, which exhibits green fluorescence when excited by blue light. This fact is a striking result since the presence of this metabolic activity opens the possibility to select the nucleons for ICSI. In the present study, the authors decided to search for a suitable metabolic test, which might reflect the metabolism and viability of these chromatin structures. This is a simple cryostorage technique that after months of cryopreservation, allow the use of nucleons for ICSI with suitable fertilization and pregnancies rates.

Immunization with a DNA vaccine encoding Toxoplasma gondii Superoxide dismutase (TgSOD) induces partial immune protection against acute toxoplasmosis in BALB/c mice.

Science.gov (United States)

Liu, Yuan; Cao, Aiping; Li, Yawen; Li, Xun; Cong, Hua; He, Shenyi; Zhou, Huaiyu

2017-06-07

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that infects all warm-blooded animals including humans and causes toxoplasmosis. An effective vaccine could be an ideal choice for preventing and controlling toxoplasmosis. T. gondii Superoxide dismutase (TgSOD) might participate in affecting the intracellular growth of both bradyzoite and tachyzoite forms. In the present study, the TgSOD gene was used to construct a DNA vaccine (pEGFP-SOD). TgSOD gene was amplified and inserted into eukaryotic vector pEGFP-C1 and formed the DNA vaccine pEGFP-SOD. Then the BALB/c mice were immunized intramuscularly with the DNA vaccine and those injected with pEGFP-C1, PBS or nothing were treated as controls. Four weeks after the last immunization, all mouse groups followed by challenging intraperitoneally with tachyzoites of T. gondii ME49 strain. Results showed higher levels of total IgG, IgG2α in the sera and interferon gamma (IFN-γ) in the splenocytes from pEGFP-SOD inoculated mice than those unvaccinated, or inoculated with either empty plasmid vector or PBS. The proportions of CD4 + T cells and CD8 + T cells in the spleen from pEGFP-SOD inoculated mice were significantly (p < 0.05) increased compared to control groups. In addition, the survival time of mice immunized with pEGFP-SOD was significantly prolonged as compared to the controls (p < 0.05) although all the mice died. The present study revealed that the DNA vaccine triggered strong humoral and cellular immune responses, and aroused partial protective immunity against acute T. gondii infection in BALB/c mice. The collective data suggests the SOD may be a potential vaccine candidate for further development.

Cryopreservation of mammalian semen.

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Curry, Mark R

2007-01-01

Mammalian spermatozoa were among the very first cells to be successfully cryopreserved and over the last five decades the use of frozen-thawed semen for artificial insemination has come to play an important role in domestic livestock production. More recently, semen freezing has increasingly been utilized in the establishment of genetic resource banks for endangered species. Semen is collected, most commonly either by use of an artificial vagina or by electroejaculation of an anaesthetized animal, and basic sperm parameters assessed. Semen is extended using a TEST-egg yolk-glycerol diluent, packaged in 0.25-mL plastic straws and slowly cooled to 5 degrees C over a period of 1-2 h. Cooled straws are frozen by suspending within liquid nitrogen vapor above the liquid nitrogen surface before plunging into the liquid phase. Straws are thawed briefly in air before immersing in a 35 degrees C water bath for 15 s, and often are used directly for insemination without any further processing.

Cryopreservation of sperm bundles (spermatozeugmata) from endangered livebearing goodeids.

Science.gov (United States)

Liu, Yue; Torres, Leticia; Tiersch, Terrence R

2018-04-14

More than half of fishes in the family Goodeidae are considered to be endangered, threatened, or vulnerable. Sperm cryopreservation is an effective tool for conserving genetic resources of imperiled populations, but development of protocols with livebearing fishes faces numerous challenges including the natural packaging of sperm into bundles. In this study the cryopreservation of sperm bundles (spermatozeugmata) of three goodeids species was evaluated. Sperm quality was evaluated by activation with NaCl-NaOH solution (at 300 mOsmol/kg and pH 11.8), and analysis of dissociable bundles and dissociation duration. Using Redtail Splitfin (Xenotoca eiseni) as a model, the effects of cryoprotectants (dimethyl sulfoxide, methanol, and glycerol) with different concentrations (5-15% v/v %), equilibration exposure times (1-60 min), cooling rates (5-40 °C/min), concentrations (4 × 10 4 -4 × 10 6 bundles/ml), buffers (HBSS, PBS and NaCl), and buffer osmolalities (200-400 mOsmol/kg) were investigated. After cooling and thawing, sperm bundles maintained their packed form. A specific protocol was developed (10% dimethyl sulfoxide, 20-min equilibration, 10 °C/min cooling rate, 4 × 10 6 bundles/ml, and 300 mOsmol/kg HBSS). This protocol yielded 89 ± 5% of post-thaw dissociable bundles with 209 ± 10 s of dissociation duration for X. eiseni, 96 ± 9% with 814 ± 14 s for Blackfin Goodea (Goodea atripinni), and 66 ± 2% with 726 ± 25 s for Striped Goodeid (Ataeniobius toweri). This is the first study of cryopreservation of sperm within bundles for livebearing fishes and provides a basis for establishment of germplasm repositories for goodeids and other livebearers. Copyright © 2018 Elsevier Inc. All rights reserved.

Bactericidal active ingredient in cryopreserved plasma-treated water with the reduced-pH method for plasma disinfection

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Kitano, Katsuhisa; Ikawa, Satoshi; Nakashima, Yoichi; Tani, Atsushi; Yokoyama, Takashi; Ohshima, Tomoko

2016-09-01

For the plasma disinfection of human body, plasma sterilization in liquid is crucial. We found that the plasma-treated water (PTW) has strong bactericidal activity under low pH condition. Physicochemical properties of PTW is discussed based on chemical kinetics. Lower temperature brings longer half-life and the bactericidal activity of PTW can be kept by cryopreservation. High performance PTW, corresponding to the disinfection power of 22 log reduction (B. subtilis spore), can be obtained by special plasma system equipped with cooling device. This is equivalent to 65% H2O2, 14% sodium hypochlorite and 0.33% peracetic acid, which are deadly poison for human. But, it is deactivated soon at higher temperature (4 sec. at body temperature), and toxicity to human body seems low. For dental application, PTW was effective on infected models of human extracted tooth. Although PTW has many chemical components, respective chemical components in PTW were isolated by ion chromatography. In addition to peaks of H2O2, NO2- and NO3-, a specific peak was detected. and only this fraction had bactericidal activity. Purified active ingredient of PTW is the precursor of HOO, and further details will be discussed in the presentation. MEXT (15H03583, 23340176, 25108505). NCCE (23-A-15).

Cryopreservation of coconut (Cocos nucifera L.) zygotic embryos by vitrification.

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Sajini, K K; Karun, A; Amamath, C H; Engelmann, F

2011-01-01

The present study investigates the effect of preculture conditions, vitrification and unloading solutions on survival and regeneration of coconut zygotic embryos after cryopreservation. Among the seven plant vitrification solutions tested, PVS3 was found to be the most effective for regeneration of cryopreserved embryos. The optimal protocol involved preculture of embryos for 3 days on medium with 0.6 M sucrose, PVS3 treatment for 16 h, rapid cooling and rewarming and unloading in 1.2 M sucrose liquid medium for 1.5 h. Under these conditions, 70-80 survival (corresponding to size enlargement and weight gain) was observed with cryopreserved embryos and 20-25 percent of the plants regenerated (showing normal shoot and root growth) from cryopreserved embryos were established in pots.

Effects of combined cryopreservation and decellularization on the biomechanical, structural and biochemical properties of porcine pulmonary heart valves.

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Theodoridis, Karolina; Müller, Janina; Ramm, Robert; Findeisen, Katja; Andrée, Birgit; Korossis, Sotirios; Haverich, Axel; Hilfiker, Andres

2016-10-01

Non-fixed, decellularized allogeneic heart valve scaffolds seem to be the best choice for heart valve replacement, their availability, however, is quite limited. Cryopreservation could prolong their shelf-life, allowing for their ideal match to a recipient. In this study, porcine pulmonary valves were decellularized using detergents, either prior or after cryopreservation, and analyzed. Mechanical integrity was analyzed by uniaxial tensile testing, histoarchitecture by histological staining, and composition by DNA, collagen (hydroxyproline) and GAG (chondroitin sulfate) quantification. Residual sodium dodecyl sulfate (SDS) in the scaffold was quantified by applying a methylene blue activation assay (MBAS). Cryopreserved decellularized scaffolds (DC) and scaffolds that were decellularized after cryopreservation (CD) were compared to fresh valves (F), cryopreserved native valves (C), and decellularized only scaffolds (D). The E-modulus and tensile strength of decellularized (D) tissue showed no significant difference compared to DC and CD. The decellularization resulted in an overall reduction of DNA and GAG, with DC containing the lowest amount of GAGs. The DNA content in the valvular wall of the CD group was higher than in the D and DC groups. CD valves showed slightly more residual SDS than DC valves, which might be harmful to recipient cells. In conclusion, cryopreservation after decellularization was shown to be preferable over cryopreservation before decellularization. However, in vivo testing would be necessary to determine whether these differences are significant in biocompatibility or immunogenicity of the scaffolds. Absence of adverse effects on biomechanical stability of acellular heart valve grafts by cryopreservation, neither before nor after decellularization, allows the identification of best matching patients in a less time pressure dictated process, and therefore to an optimized use of a very limited, but best-suited heart valve prosthesis

Selecting Infants With Cryptorchidism and High Risk of Infertility for Optional Adjuvant Hormonal Therapy and Cryopreservation of Germ Cells: Experience From a Pilot Study

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Jorgen Thorup

2018-06-01

Full Text Available IntroductionOrchiopexy for congenital cryptorchid testes is recommended between ½ and 1 year of age to preserve testicular germ cell maturation. Early operation is not enough to preserve fertility in 22 and 36% of cases. Aim of this study was to set up a protocol for optional adjuvant hormonal therapy after orchiopexy and thereafter cryopreservation of testicular biopsies from infants with bilateral cryptorchidism and high infertility risk.Materials and methodsWe included 17 boys with bilateral cryptorchidism, normal FSH, and impaired germ cell number per tubular transverse section (G/T in testicular biopsies at orchiopexy, 7 months to 3½ years old. Postoperatively, optional adjuvant LHRH (kryptocur® 0.2 mg/0.1 mL 2× every second day in 16 weeks were offered. Ten boys were applicable for age matching according to parent’s choice of treatment regime and G/T. Five of them had kryptocur®, and five were controls. Repeat bilateral testicular biopsy evaluation and cryopreservation were offered to all boys 12 months after primary orchiopexy. For cryopreservation, tissue pieces were incubated with a cryoprotectant with a slow program freezing.ResultsTwo out of five kryptorcur®-treated boys normalized both the average G/T and the number of adult dark spermatogonia (Ad-S. Another kryptocur®-treated boy with initial low G/T and no Ad-S increased the G/T and achieved normal number of Ad-S at time of cryopreservation. In the control group, two patients reached only normal lower range regarding the G/T and the number of Ad-S. None of boys with less than average 0.2 G/T improved significantly, whether they were kryptocur®-treated or not.ConclusionBased on literature and the present results, we recommend adjuvant LHRH treatment to boys with cryptorchidism and insufficient genuine gonadotropin stimulation at time of surgery, as these patients have high infertility risk. Cryopreservation should be an option in case of treatment failure

Immunization with Paracoccidioides brasiliensis radioattenuated yeast cells induces Th1 immune response in Balb/C mice

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Martins, Estefania M.N.; Andrade, Antero S.R. [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN-CNEN/MG), Belo Horizonte, MG (Brazil)], e-mail: estefaniabio@yahoo.com.br, e-mail: antero@cdtn.br; Resende, Maria Aparecida de [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Microbiologia], e-mail: maresend@mono.icb.ufmg.br; Reis, Bernardo S.; Goes, Alfredo M. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Bioquimica e Imunologia], e-mail: goes@mono.icb.ufmg.br, e-mail: brsgarbi@mono.icb.ufmg.br

2009-07-01

Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent mycosis in Latin America. To date, there is no effective vaccine. In our laboratory yeast cells of P. brasiliensis were attenuated by gamma irradiation. We defined an absorbed dose in which the pathogen loses the reproductive ability, while retaining the morphology, the synthesis and secretion of proteins and the oxidative metabolism. The immunization with these cells was able to confer protection in BALB/c mice. The aim of the present work was evaluate the immune response pathway activated in mice immunized with P. brasiliensis radioattenuated yeast cells. The protector effect was evaluated in BALB/c mice groups immunized once or twice, respectively. Each group was divided in three sub groups that were challenge 30, 45 or 60 days after the immunization. These groups were called G1A, G1B and G1C in the group immunized once and G2A, G2B and G2C in the group immunized twice. Recovery of CFUs and cytokines determination (IFN - {gamma}, IL - 10 and IL IV 4) were performed three months post challenge. Quantitative RT-PCR was the method of choice used to quantify the expression of cytokines. The sera were collected weekly to evaluate the IgG antibody titers and the IgG1 and IgG2a pattern in the course of infection. A significant reduction in CFUs recovery was verified 90 days post challenge in mice submitted to one immunization: 73.0%, 96.0% and 76.3% for sub-groups G1A, G1B and G1C, respectively. In the group submitted to two immunizations, a remarkable increase in the protection was obtained. No CFUs was recovered from sub-groups G2B and G2C and very few CFUs (reduction of 98.6%) were recovered from the lungs of sub group G2A. In mice submitted to one immunization, Th1 and Th2 cytokines were simultaneously produced. In the group submitted to two immunizations, levels of IL-10 and IL-4 were very low, while IFN-{gamma} production was maintained indicating that a Th1 pattern was

Immunization with Paracoccidioides brasiliensis radioattenuated yeast cells induces Th1 immune response in Balb/C mice

International Nuclear Information System (INIS)

Martins, Estefania M.N.; Andrade, Antero S.R.; Resende, Maria Aparecida de; Reis, Bernardo S.; Goes, Alfredo M.

2009-01-01

Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent mycosis in Latin America. To date, there is no effective vaccine. In our laboratory yeast cells of P. brasiliensis were attenuated by gamma irradiation. We defined an absorbed dose in which the pathogen loses the reproductive ability, while retaining the morphology, the synthesis and secretion of proteins and the oxidative metabolism. The immunization with these cells was able to confer protection in BALB/c mice. The aim of the present work was evaluate the immune response pathway activated in mice immunized with P. brasiliensis radioattenuated yeast cells. The protector effect was evaluated in BALB/c mice groups immunized once or twice, respectively. Each group was divided in three sub groups that were challenge 30, 45 or 60 days after the immunization. These groups were called G1A, G1B and G1C in the group immunized once and G2A, G2B and G2C in the group immunized twice. Recovery of CFUs and cytokines determination (IFN - γ, IL - 10 and IL IV 4) were performed three months post challenge. Quantitative RT-PCR was the method of choice used to quantify the expression of cytokines. The sera were collected weekly to evaluate the IgG antibody titers and the IgG1 and IgG2a pattern in the course of infection. A significant reduction in CFUs recovery was verified 90 days post challenge in mice submitted to one immunization: 73.0%, 96.0% and 76.3% for sub-groups G1A, G1B and G1C, respectively. In the group submitted to two immunizations, a remarkable increase in the protection was obtained. No CFUs was recovered from sub-groups G2B and G2C and very few CFUs (reduction of 98.6%) were recovered from the lungs of sub group G2A. In mice submitted to one immunization, Th1 and Th2 cytokines were simultaneously produced. In the group submitted to two immunizations, levels of IL-10 and IL-4 were very low, while IFN-γ production was maintained indicating that a Th1 pattern was dominant. For

Egg yolk and glycerol requirements for freezing boar spermatozoa treated with methyl β-cyclodextrin or cholesterol-loaded cyclodextrin.

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Blanch, Eva; Tomás, Cristina; Hernández, Marta; Roca, Jordi; Martínez, Emilio A; Vázquez, Juan M; Mocé, Eva

2014-04-24

Egg yolk (EY) and glycerol are common constituents of extenders used for sperm cryopreservation. It has been demonstrated that using cholesterol-loaded cyclodextrins (CLC) improves sperm cryosurvival in several species. However, standard freezing extenders might not be the most appropriate for CLC-treated sperm. This study evaluated the EY and glycerol requirements for freezing CLC-treated boar spermatozoa. Semen samples from 34 ejaculates coming from 4 boars were used. Each ejaculate was split into three aliquots: one was used untreated (control), and the other two were treated with 1 mg of CLC or methyl-β-cyclodextrin/120 × 10(6) sperm for 15 min at 22 C prior to cryopreservation. Our results indicated that reducing the concentration of EY was detrimental for sperm viability after thawing (31.57 ± 2 vs. 19.89% ± 2 for 20 and 10% EY, respectively; P semen treated with CLC. On the other hand, it was observed that the traditional concentration of glycerol (3%) was not the appropriate for freezing CLC-treated sperm (61.10 ± 3 vs. 47.87% ± 3 viable sperm for control and CLC-treated sperm, respectively; P extenders for CLC-treated sperm. Nevertheless, additional studies will be needed to evaluate alternative cryoprotectants and to determine the effect of high glycerol concentrations on sperm functionality.

Social oocyte cryopreservation: a portrayal of Brazilian women.

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Santo, Elisangela V Espirito; Dieamant, Felipe; Petersen, Claudia G; Mauri, Ana L; Vagnini, Laura D; Renzi, Adriana; Zamara, Camila; Oliveira, João Batista A; Baruffi, Ricardo L R; Franco, José G

2017-06-01

This study aimed to determine what Brazilian childless women of reproductive age think about oocyte cryopreservation to postpone pregnancy and their reasons for performing or not performing this procedure. Women of reproductive age were randomly selected from the general population using different e-mail lists and were invited to participate in the study by completing an online web survey regarding social oocyte cryopreservation. The survey was also distributed through social media to women of reproductive age. Although most of the responders had a partner (86.9%) and had already planned the pregnancy of their first child (69.6%), 85.4% (379) considered the potential of social oocyte freezing to improve their chances of giving birth later in life. Those that had already planned pregnancy were two times more likely to intend to freeze their oocytes (p=0.03). The most important barrier for not undergoing oocyte cryopreservation was cost. The women who indicated that they could not currently undergo the procedure now because of cost were two times (p=0.03) more likely to intend to cryopreserve their oocytes than women who thought that they would not need to delay pregnancy. Brazilian women who think that they are not ready to have a family are discovering the option of oocyte cryopreservation. Most participants considered safeguarding their reproductive potential. Making the procedure more accessible could give women the opportunity to make proactive decisions about the future of their fertility.

Trehalose preincubation increases mesenchymal (CD271+ stem cells post-cryopreservation viability

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Indra Kusuma

2016-10-01

Full Text Available Background: Dimethyl sulfoxide (Me2SO is a common cryoprotective agent widely used in cell preservation system. Me2SO is currently known to cause epigenetic changes which are  critical in stem cells development and cellular differentiation. Therefore, it is imperative to develop cryopreservation techniques that protect cellular functions and avert Me2SO adverse effect. Trehalose was able to protect organism in extreme condition such as dehydration and cold. This study aimed to verify the protective effect of trehalose preincubation procedure in cryopreservation.Methods: The study was conducted using experimental design. Thawed mesenchymal (CD271+ stem cells from YARSI biorepository were used for the experiment. Trehalose preincubation was performed for 1 hour, internalized trehalose was confirmed by FTIR-ATR measurement. Three groups consisted of (1 cryopreserved without trehalose preincubation, (2 cryopreserved with trehalose preincubation, and (3 did not undergo cryopreservation were evaluated after 24 hours in LN2 for viability in culture. The absorbance from each group was measured at 450 nm. The analysis performed using paired student t test.Results: Viability of thawed mesenchymal (CD271+ stem cells that undergo trehalose preincubation prior cryopreservation was significantly higher (p<0.05 compared to group without trehalose preincubation. Higher viability observed between group with trehalose preincubation compared with controlled group suggests protection to trypsinization. Mesenchymal (CD271+ stem cells incubated for 1 hour in 100 mM trehalose supplemented medium  results in 15%  trehalose loading efficiency.Conclusion: These findings confirm the protective effect of trehalose preincubation in cryopreservation. Future research should be directed to elucidate the trehalose internalization mechanism and eventually the protective mechanism of trehalose in mammalian cell cryopreservation.

Cryopreservation of GABAergic Neuronal Precursors for Cell-Based Therapy.

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Daniel Rodríguez-Martínez

Full Text Available Cryopreservation protocols are essential for stem cells storage in order to apply them in the clinic. Here we describe a new standardized cryopreservation protocol for GABAergic neural precursors derived from the medial glanglionic eminence (MGE, a promising source of GABAergic neuronal progenitors for cell therapy against interneuron-related pathologies. We used 10% Me2SO as cryoprotectant and assessed the effects of cell culture amplification and cellular organization, as in toto explants, neurospheres, or individualized cells, on post-thaw cell viability and retrieval. We confirmed that in toto cryopreservation of MGE explants is an optimal preservation system to keep intact the interneuron precursor properties for cell transplantation, together with a high cell viability (>80% and yield (>70%. Post-thaw proliferation and self-renewal of the cryopreserved precursors were tested in vitro. In addition, their migration capacity, acquisition of mature neuronal morphology, and potency to differentiate into multiple interneuron subtypes were also confirmed in vivo after transplantation. The results show that the cryopreserved precursor features remained intact and were similar to those immediately transplanted after their dissection from the MGE. We hope this protocol will facilitate the generation of biobanks to obtain a permanent and reliable source of GABAergic precursors for clinical application in cell-based therapies against interneuronopathies.

CHARACTERISTICS AND FERTILITY OF SUMATRAN TIGER SPERMATOZOA CRYOPRESERVED WITH DIFFERENT SUGARS.

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Wayan Kurniani Karja, Ni; Fahrudin, Mokhamad; Setiadi, Mohamad Agus; Tumbelaka, Ligaya Ita; Sudarwati, Retno; Hastuti, Yohana Tri; Mulia, Bongot Huas; Widianti, Ardyta; Sultan, Keni; Terazono, Tsukasa; Namula, Zhao; Taniguchi, Masayasu; Tanihara, Fuminori; Takemoto, Tatsuya; Kikuchi, Kazuhiro; Sato, Yoko; Otoi, Takeshige

Cryopreservation of semen is one of the most important methods for the preservation of endangered tigers. This study evaluated the effects of sugar supplementation on the cryosurvival of spermatozoa from Sumatran tigers (Panthera tigris sumatrae). The post-thaw characteristics and fertility of spermatozoa cryopreserved with different sugars (glucose, lactose, and trehalose) were evaluated using heterologous in-vitro fertilisation with cat oocytes. All parameters of post-thaw spermatozoa significantly decreased as compared with those of fresh spermatozoa. The index of sperm motility for semen cryopreserved with lactose was significantly higher than that for semen cryopreserved with trehalose. The percentage of total fertilisation for tiger spermatozoa cryopreserved with trehalose was significantly lower than that for control cat spermatozoa. Our findings indicated that supplementation with lactose or glycerol as the main sugar in the egg yolk extender resulted in a better motility and fertility potential for post-thawed spermatozoa.

Evaluation of ebselen supplementation on cryopreservation medium in human semen.

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Khodayari Naeini, Zohreh; Hassani Bafrani, Hassan; Nikzad, Hossein

2014-04-01

An effect of cryopreservation on human sperm is sublethal cryodamage, in which cell viability post-thaw is lost more rapidly at later times than in fresh cells. This study examined whether the addition of an antioxidant to cryopreservation medium could improve the post-thaw parameters and evaluation of sperm chromatin quality of cryopreserved human spermatozoa from men with normal semen parameters. Semen samples (n=35) were collected by masturbation and assessed following WHO standards. Individual samples were classified as two portions. One portion (n=10) was for elucidate the concentration of ebselen.Then the samples(n=25) were divided in to 5groups.The first aliquot remained fresh.The second aliquots was mixed with cryopreservation medium.The third aliquots were mixed with cryopreservation medium containing solvent of ebselen.The forth and fifth aliquots were mixed with cryopreservation medium containing 1.25 and 2.5 µm of ebselen.Samples were frozen and thawed samples were assessed for sperm parameters.Three-way ANOVA Multivariate measures were used to assess. According to this assesment the differences are observed in existent groups in post-thaw count, motility index, vitality staining, and morphology and DNA fragmentation. After freezing the media containing of ebselen, DNA fragmentation is significantly different in comparison with control group. ebselen with 1.25 µm dose was significantly associated with post-thaw DNA fragmentation (p=0.047). Similarly ebselen with 2.5 µm dose was significantly associated with post-thaw DNA fragmentation (p=0.038). But other parameters were not altered. These results suggest that the addition of ebselen to cryopreservation medium doesnot improve post-thaw parameters and DNA fragmentation of sperm.

Harnessing what lies within: Programming immunity with biocompatible devices to treat human disease

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Roberts, Reid Austin

Advances in our mechanistic insight of cellular function and how this relates to host physiology have revealed a world which is intimately connected at the macro and micro level. Our increasing understanding of biology exemplifies this, where cells respond to environmental cues through interconnected networks of proteins which function as receptors and adaptors to elicit gene expression changes that drive appropriate cellular programs for a given stimulus. Consequently, our deeper molecular appreciation of host homeostasis implicates aberrations of these pathways in nearly all major human disease categories, including those of infectious, metabolic, neurologic, oncogenic, and autoimmune etiology. We have come to recognize the mammalian immune system as a common network hub among all these varied pathologies. As such, the major goal of this dissertation is to identify a platform to program immune responses in mammals so that we may enhance our ability to treat disease and improve health in the 21st century. Using advances in materials science, in particular a recently developed particle fabrication technology termed Particle Replication in Non-wetting Templates (PRINT), our studies systematically assess the murine and human immune response to precisely fabricated nano- and microscale particles composed of biodegradable and biocompatible materials. We then build on these findings and present particle design parameters to program a number of clinically attractive immune responses by targeting endogenous cellular signaling pathways. These include control of particle uptake through surface modification, design parameters that modulate the magnitude and kinetics of biological signaling dynamics that can be used to exacerbate or dampen inflammatory responses, as well as particle designs which may be of use in treating allergies and autoimmune disorders. In total, this dissertation provides evidence that rational design of biocompatible nano- and microparticles is a viable

Detection of tumor-associated cells in cryopreserved peripheral blood mononuclear cell samples for retrospective analysis.

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Zhu, Peixuan; Stanton, Melissa L; Castle, Erik P; Joseph, Richard W; Adams, Daniel L; Li, Shuhong; Amstutz, Platte; Tang, Cha-Mei; Ho, Thai H

2016-07-02

Cryopreserved peripheral blood mononuclear cells (PBMCs) are commonly collected in biobanks. However, little data exist regarding the preservation of tumor-associated cells in cryopreserved collections. The objective of this study was to determine the feasibility of using the CellSieve™ microfiltration assay for the isolation of circulating tumor cells (CTCs) and circulating cancer-associated macrophage-like cells (CAMLs) from cryopreserved PBMC samples. Blood samples spiked with breast (MCF-7), prostate (PC-3), and renal (786-O) cancer cell lines were used to establish analytical accuracy, efficiency, and reproducibility after cryopreservation. The spiked samples were processed through Ficoll separation, and cryopreservation was followed by thawing and microfiltration. MCF-7 cells were successfully retrieved with recovery efficiencies of 90.5 % without cryopreservation and 87.8 and 89.0 %, respectively, on day 7 and day 66 following cryopreservation. The corresponding recovery efficiencies of PC-3 cells were 83.3 % without cryopreservation and 85.3 and 84.7 %, respectively, after cryopreservation. Recovery efficiencies of 786-O cells were 92.7 % without cryopreservation, and 82.7 and 81.3 %, respectively, after cryopreservation. The recovered cells retained the morphologic characteristics and immunohistochemical markers that had been observed before freezing. The protocols were further validated by quantitation of CAMLs in blood samples from two patients with renal cell carcinoma (RCC). The recovery rates of CTCs and CAMLs from cryopreserved samples were not statistically significant different (P > 0.05) from matched fresh samples. To our knowledge, this is the first report that CAMLs could be cryopreserved and analyzed after thawing with microfiltration technology. The application of microfiltration technology to cryopreserved samples will enable much greater retrospective study of cancer patients in relation to long-term outcomes.

Immunization of baboons with attenuated schistosomula of Schistosoma haematobium: levels of protection induced by immunization with larvae irradiated with 20 and 60 krad

International Nuclear Information System (INIS)

Harrison, R.A.; Bickle, Q.D.; Sturrock, R.F.; Taylor, M.G.; Webbe, G.; Kiare, S.; James, E.R.; Andrews, B.J.

1990-01-01

The authors have demonstrated that baboons can be immunized with S. haemotobium schistosomula irradiated with 20 krad in a regimen that induces 90% protection. While this high level of protection has stimulated a discussion on the feasibility of a human volunteer trial (Von Lichtenberg, 1985), results of further studies particularly on (i) the pathogensis of immunization per se (Byram et al., 1989), (ii) the longevity of protection, and (iii) the protective efficacy of cryopreserved irradiated S. haemotobium schistosomula (R. Harrison et al., in preparation), prevent recommending this form of vaccination for human application. (author)

Impact of starting material (fresh versus cryopreserved marrow) on mesenchymal stem cell culture.

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Kaplan, Alesia; Sackett, Katie; Sumstad, Darin; Kadidlo, Dianne; McKenna, David H

2017-09-01

Mesenchymal stem cells (MSCs) continue to be investigated in multiple clinical trials as potential therapy for different disorders. There is ongoing controversy surrounding the clinical use of cryopreserved versus fresh MSCs. However, little is known about how cryopreservation affects marrow as starting material. The growth kinetics of MSC cultures derived from fresh versus cryopreserved marrow were compared. Data were reviewed on the growth kinetics of MSCs derived from fresh versus cryopreserved marrow of nine donors. Marrow harvested from each donor was separated into four aliquots (one fresh and three cryopreserved for culture). Data on the date of mononuclear cell cryopreservation/thaw, MSC counts at Passages 1 and 2, MSC doubling, MSC fold expansion, viability (of mononuclear cells and final MSCs), and on flow cytometry markers of mononuclear cells and final MSCs were analyzed for the fresh and cryopreserved marrow groups. In total, 21 MSC lots (seven fresh and 14 cryopreserved) were obtained. The average age of cryopreserved mononuclear cell product was 295 days (range, 18-1241 days). There were no significant differences between MSC numbers at Passage 1 (p = 0.1), final MSC numbers (p = 0.5), MSC doubling (p = 0.7), or MSC fold expansion (p = 0.7). A significant difference was observed in viability by flow cytometry for both mononuclear cells (p = 0.002) and final MSCs (p = 0.009), with higher viability in the fresh marrow group. This study demonstrates that MSCs derived from cryopreserved marrow have the same growth characteristics as fresh marrow-derived MSCs. Further studies are needed to explore potential differences in clinical efficacy. © 2017 AABB.

Hepatitis C, innate immunity and alcohol: friends or foes?

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Osna, Natalia A; Ganesan, Murali; Kharbanda, Kusum K

2015-02-05

Hepatitis C and alcohol are the most widespread causes of liver disease worldwide. Approximately 80% of patients with a history of hepatitis C and alcohol abuse develop chronic liver injury. Alcohol consumption in hepatitis C virus (HCV)-infected patients exacerbates liver disease leading to rapid progression of fibrosis, cirrhosis and even hepatocellular carcinoma. Hepatocytes are the main sites of HCV-infection and ethanol metabolism, both of which generate oxidative stress. Oxidative stress levels affect HCV replication and innate immunity, resulting in a greater susceptibility for HCV-infection and virus spread in the alcoholic patients. In this review paper, we analyze the effects of ethanol metabolism and other factors on HCV replication. In addition, we illustrate the mechanisms of how HCV hijacks innate immunity and how ethanol exposure regulates this process. We also clarify the effects of HCV and ethanol metabolism on interferon signaling-a crucial point for activation of anti-viral genes to protect cells from virus-and the role that HCV- and ethanol-induced impairments play in adaptive immunity which is necessary for recognition of virally-infected hepatocytes. In conclusion, ethanol exposure potentiates the suppressive effects of HCV on innate immunity, which activates viral spread in the liver and finally, leads to impairments in adaptive immunity. The dysregulation of immune response results in impaired elimination of HCV-infected cells, viral persistence, progressive liver damage and establishment of chronic infection that worsens the outcomes of chronic hepatitis C in alcoholic patients.

Hepatitis C, Innate Immunity and Alcohol: Friends or Foes?

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Natalia A. Osna

2015-02-01

Full Text Available Hepatitis C and alcohol are the most widespread causes of liver disease worldwide. Approximately 80% of patients with a history of hepatitis C and alcohol abuse develop chronic liver injury. Alcohol consumption in hepatitis C virus (HCV-infected patients exacerbates liver disease leading to rapid progression of fibrosis, cirrhosis and even hepatocellular carcinoma. Hepatocytes are the main sites of HCV-infection and ethanol metabolism, both of which generate oxidative stress. Oxidative stress levels affect HCV replication and innate immunity, resulting in a greater susceptibility for HCV-infection and virus spread in the alcoholic patients. In this review paper, we analyze the effects of ethanol metabolism and other factors on HCV replication. In addition, we illustrate the mechanisms of how HCV hijacks innate immunity and how ethanol exposure regulates this process. We also clarify the effects of HCV and ethanol metabolism on interferon signaling—a crucial point for activation of anti-viral genes to protect cells from virus—and the role that HCV- and ethanol-induced impairments play in adaptive immunity which is necessary for recognition of virally-infected hepatocytes. In conclusion, ethanol exposure potentiates the suppressive effects of HCV on innate immunity, which activates viral spread in the liver and finally, leads to impairments in adaptive immunity. The dysregulation of immune response results in impaired elimination of HCV-infected cells, viral persistence, progressive liver damage and establishment of chronic infection that worsens the outcomes of chronic hepatitis C in alcoholic patients.

Germination, carbohydrate composition and vigor of cryopreserved Caesalpinia echinata seeds

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Rafael Fonsêca Zanotti

2012-10-01

Full Text Available The present study investigated the germination and vigor of Caesalpinia echinata (Brazilwood seeds stored at negative temperatures. Recently harvested seeds were cryopreserved at -18º or -196ºC and periodically evaluated for germination, seed vigor and carbohydrate composition. The temperatures did not influence the germination percentages or vigor. The germination percentage decreased from 88% in recently harvested seeds to 60% after 730 days of storage. The different temperature and storage times tested did not affect the vigor seed germination as indicated by the measures of plant growth and survival. The different temperatures used did not cause changes in the carbohydrate composition. The tegument cell walls were rich in lignin, arabinose and xylose. The cytoplasm of the cotyledons and embryos had high levels of glucose, fructose, and sucrose. The cryopreservation technique here presented was effective in the conservation of Brazilwood seeds for the medium term.

Novel glyceryl glucoside is a low toxic alternative for cryopreservation agent

Energy Technology Data Exchange (ETDEWEB)

Su, Cathy; Allum, Allison J. [Department of Environmental & Radiological Health Sciences, Colorado State University, 1618 Campus Delivery, Fort Collins, CO 80523 (United States); Aizawa, Yasushi [Research and Development Group, Toyo Sugar Refining Co. Ltd., Tokyo 103-0046 (Japan); Kato, Takamitsu A., E-mail: Takamitsu.Kato@Colostate.edu [Department of Environmental & Radiological Health Sciences, Colorado State University, 1618 Campus Delivery, Fort Collins, CO 80523 (United States)

2016-08-05

Glyceryl glucoside (GG, α-D-glucosyglycerol) is a natural glycerol derivative found in alcoholic drinks. Recently GG has been used as an alternative for glycerol in cosmetic products. However, the safety of using GG is still unclear. Currently, dimethyl sulfoxide (DMSO) and glycerol are wildly used in cryopreservation. Despite GG being a derivative of glycerol, the ability of GG in cryopreservation is still unknown. By using a system of Chinese Hamster Ovary cells (CHO), A549 cells and AG1522 cells, the study examined the cryoprotective effects of DMSO, glycerol and GG. Cytotoxic and genotoxic responses induced by the three chemicals were also investigated with CHO to determine the safety of GG for cosmetic products. Our data suggests that GG has great cryopresearvation ability in the concentration of 30%–40% (v/v). For cytotoxic studies, DMSO showed the highest cytotoxicity above 3% (v/v) in cell doubling time delay among three chemicals. For the acute cytotoxicity with trypan blue dye exclusion assay, GG showed stronger cell killing effect within 24 h above 4% (v/v). For the continuous cytotoxicity with colony formation assay for 7 days, DMSO showed significantly reduced clonogenic ability above 2%. In genotoxicity studies, CHO treated with glycerol at 2% concentration induced three times higher frequencies of sister chromatid exchange (SCE) than background levels. GG did not induce significant amounts of SCE compared to background. Micronuclei formation was equally observed in the 2% and above concentrations of glycerol and GG. Our data showed that GG has significant effects on cryopreservation compared to DMSO. Glycerol and GG have similar cytotoxicity effects to CHO, but glycerol induced genotoxic responses in the same concentration. Therefore, we conclude that GG may be a safer alternative compound to glycerol in cosmetic products and safer alternative to DMSO in cryopreservation. -- Highlights: •Glyceryl Glucoside is low cytotoxicity and genotoxicity

Effects of three different types of antifreeze proteins on mouse ovarian tissue cryopreservation and transplantation.

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Jaewang Lee

Full Text Available Ovarian tissue (OT cryopreservation is effective in preserving fertility in cancer patients who have concerns about fertility loss due to cancer treatment. However, the damage incurred at different steps during the cryopreservation procedure may cause follicular depletion; hence, preventing chilling injury would help maintain ovarian function.This study was designed to investigate the beneficial effects of different antifreeze proteins (AFPs on mouse ovarian tissue cryopreservation and transplantation.Ovaries were obtained from 5-week-old B6D2F1 mice, and each ovary was cryopreserved using two-step vitrification and four-step warming procedures. In Experiment I, ovaries were randomly allocated into fresh, vitrification control, and nine experimental groups according to the AFP type (FfIBP, LeIBP, type III and concentration (0.1, 1, 10 mg/mL used. After vitrification and warming, 5,790 ovarian follicles were evaluated using histology and TUNEL assays, and immunofluorescence for τH2AX and Rad51 was used to detect DNA double-strand breaks (DSBs and repair (DDR, respectively. In Experiment II, 20 mice were randomly divided into two groups: one where the vitrification and warming media were supplemented with 10 mg/mL LeIBP, and the other where media alone were used (control. Ovaries were then autotransplanted under both kidney capsules 7 days after vitrification together with the addition of 10 mg/mL LeIBP in the vitrification-warming media. After transplantation, the ovarian follicles, the percentage of apoptotic follicles, the extent of the CD31-positive area, and the serum FSH levels of the transplanted groups were compared.In Experiment I, the percentage of total grade 1 follicles was significantly higher in the 10 mg/mL LeIBP group than in the vitrification control, while all AFP-treated groups had significantly improved grade 1 primordial follicle numbers compared with those of the vitrification control. The number of apoptotic (TUNEL

The S(c)ensory Immune System Theory.

Science.gov (United States)

Veiga-Fernandes, Henrique; Freitas, António A

2017-10-01

Viewpoints on the immune system have evolved across different paradigms, including the clonal selection theory, the idiotypic network, and the danger and tolerance models. Herein, we propose that in multicellular organisms, where panoplies of cells from different germ layers interact and immune cells are constantly generated, the behavior of the immune system is defined by the rules governing cell survival, systems physiology and organismic homeostasis. Initially, these rules were imprinted at the single cell-protist level, but supervened modifications in the transition to multicellular organisms. This context determined the emergence of the 'sensory immune system', which operates in a s(c)ensor mode to ensure systems physiology, organismic homeostasis, and perpetuation of its replicating molecules. Copyright © 2017 Elsevier Ltd. All rights reserved.

DNA sensor cGAS-mediated immune recognition

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Pengyan Xia

2016-09-01

Full Text Available Abstract The host takes use of pattern recognition receptors (PRRs to defend against pathogen invasion or cellular damage. Among microorganism-associated molecular patterns detected by host PRRs, nucleic acids derived from bacteria or viruses are tightly supervised, providing a fundamental mechanism of host defense. Pathogenic DNAs are supposed to be detected by DNA sensors that induce the activation of NFκB or TBK1-IRF3 pathway. DNA sensor cGAS is widely expressed in innate immune cells and is a key sensor of invading DNAs in several cell types. cGAS binds to DNA, followed by a conformational change that allows the synthesis of cyclic guanosine monophosphate–adenosine monophosphate (cGAMP from adenosine triphosphate and guanosine triphosphate. cGAMP is a strong activator of STING that can activate IRF3 and subsequent type I interferon production. Here we describe recent progresses in DNA sensors especially cGAS in the innate immune responses against pathogenic DNAs.

C3d enhanced DNA vaccination induced humoral immune response to glycoprotein C of pseudorabies virus

International Nuclear Information System (INIS)

Tong Tiezhu; Fan Huiying; Tan Yadi; Xiao Shaobo; Ling Jieyu; Chen Huanchun; Guo Aizhen

2006-01-01

Murine C3d were utilized to enhance immunogenicity of pseudorabies virus (PrV) gC DNA vaccination. Three copies of C3d and four copies of CR2-binding domain M28 4 were fused, respectively, to truncated gC gene encoding soluble glycoprotein C (sgC) in pcDNA3.1. BALB/c mice were, respectively, immunized with recombinant plasmids, blank vector, and inactivated vaccine. The antibody ELISA titer for sgC-C3d 3 DNA was 49-fold more than that for sgC DNA, and the neutralizing antibody obtained 8-fold rise. Protection of mice from death after lethal PrV (316 LD 5 ) challenge was augmented from 25% to 100%. Furthermore, C3d fusion increased Th2-biased immune response by inducing IL-4 production. The IL-4 level for sgC-C3d 3 DNA immunization approached that for the inactivated vaccine. Compared to C3d, M28 enhanced sgC DNA immunogenicity to a lesser extent. In conclusion, we demonstrated that murine C3d fusion significantly enhanced gC DNA immunity by directing Th1-biased to a balanced and more effective Th1/Th2 response

Optimization of Artificial Propagation in Piracanjuba Fish Brycon orbignyanus Using Cryopreserved Semen.

Science.gov (United States)

Felizardo, V O; Melo, C C V; Murgas, L D S; Andrade, E S; Navarro, R D; Ftreitas, T F

BACKGROUND: Cryopreserved semen could facilitate procedures during the artificial reproduction in fish. Factors affecting cryopreservation efficiency are important to define efficient protocols. This study investigated the application of cryoprotectants on the quality of piracanjuba fish semen, the sperm concentration required for oocyte fertilization and spermatic activation. We evaluated two intracellular cryoprotectant solutions (DMSO and methanol) and two extracellular cryoprotectant solutions (egg yolk and lactose) to cryopreserved piracanjuba semen. Sperm motility rate, motility duration and spermatic alterations were assessed. The protocol for piracanjuba semen cryopreservation can use solutions including either DMSO or methanol as intracellular cryoprotectant and egg yolk or lactose as extracellular cryoprotectants.

Phospholipase C-β in immune cells.

Science.gov (United States)

Kawakami, Toshiaki; Xiao, Wenbin

2013-09-01

Great progress has recently been made in structural and functional research of phospholipase C (PLC)-β. We now understand how PLC-β isoforms (β1-β4) are activated by GTP-bound Gαq downstream of G protein-coupled receptors. Numerous studies indicate that PLC-βs participate in the differentiation and activation of immune cells that control both the innate and adaptive immune systems. The PLC-β3 isoform also interplays with tyrosine kinase-based signaling pathways, to inhibit Stat5 activation by recruiting the protein-tyrosine phosphatase SHP-1, with which PLC-β3 and Stat5 form a multi-molecular signaling platform, named SPS complex. The SPS complex has important regulatory roles in tumorigenesis and immune cell activation. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effectiveness of human spermatozoa biomarkers as indicators of structural damage during cryopreservation.

Science.gov (United States)

Gómez-Torres, María José; Medrano, Llanos; Romero, Alejandro; Fernández-Colom, Pedro José; Aizpurúa, Jon

2017-10-01

Human spermatozoa cryopreservation techniques are used to maintain and protect male fertility in cases such as infertility and malignancy treatments. However, during cryopreservation, the spermatozoa's metabolic rate is reduced and they undergo dramatic functional and structural changes owing to exposure to cryoprotectants and freezing-thawing procedures. While the effects of cryopreservation on cells are documented, to date the induced cryodamage on structural and/or functional sperm biomarkers is not well established at multivariate scale. To address this question, we performed basic sperm analysis, sperm DNA fragmentation assessment, spontaneous acrosome reaction measurement, and cytoskeleton evaluation after thawing samples from subjects with normal and low-quality semen. A cryodamage rate was used to determine the effects of the freeze-thaw process on spermatozoa. In addition, a Principal Component Analysis (PCA) was used for data reduction and to evaluate sperm-specific patterns during the cryopreservation process. We found that the vitality, progressive motility and sperm count from low-quality samples after cryopreservation show higher damage rates (≥40%) than in normal sperm samples. However, cytoskeleton, DNA, tail and mid-piece and acrosome display the highest cryodamage rates (∼50-99%) and are equally susceptible to cryopreservation-induced damage in both low- and normal-quality semen samples. Overall, the evaluation of these parameters provides meaningful information about different aspects of sperm functionality after cryopreservation. Copyright © 2017 Elsevier Inc. All rights reserved.

USE OF DIFFERENT EXTENDERS TO CRYOPRESERVATION OF MANGALARGA MARCHADOR SPERM

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Jessica Neri Nascimento

2015-07-01

Full Text Available The techniques applied to animal reproduction such as artificial insemination, transfer and in vitro production of embryos, heat synchronization and induction, and gametes cryopreservation have been more utilized in veterinary practice each day. Nevertheless, some techniques have not achieved their full technical capacity within the equine reproduction field, such as semen cryopreservation. The aim of this study was to evaluate the characteristics of post-thawing semen (total motility, strength, plasmatic and acrosomal membrane integrity of Mangalarga Marchador breed stallions, using three different extenders (Botucrio, FR5 and FR6. Ejaculates from five stallions were collected and the gel-free semen was diluted in a 1:1 dilution in skim milk extender, and centrifuged at 600 g for 10 minutes. After the centrifugation the supernatant was removed and sperm pellet was divided and re-suspended using three different extenders to a concentration of 200 x 106 cells/mL. The samples were packed into 0.5 mL straws, placed in a stainless steel support and kept inside the refrigerator (5 oC for one hour. Subsequently, these straws were kept at a height of 6 cm from liquid nitrogen for 15 minutes in an isotherm box and, after that, plunged into liquid nitrogen (-196 oC and stored in a liquid nitrogen holding tank. There were no differences in the parameters evaluated when extenders using mixed amides and glycerol (Botucrio and FR6 were used (P > 0.05. All parameters evaluated were lower for the extender containing only glycerol (P<0.05. The use of cryoprotectants (methylformamide and dimethylformamide in association with glycerol concentrations around 1 to 2% is an alternative for semen cryopreservation of Mangalarga Marchador breed stallions.

Differential protective effects of immune lymphoid cells against transplanted line Ib leukemia and immune polioencephalomyelitis

International Nuclear Information System (INIS)

Duffey, P.S.; Lukasewycz, O.A.; Olson, D.S.; Murphy, W.H.

1978-01-01

The capacity of immune cells obtained from the major lymphoid compartments to protect C58 mice from transplanted line Ib leukemia, and from an age-dependent autoimmune CNS disease (immune polioencephalomyelitis = IPE) elicited by immunizing old C58 mice with inactivated Ib cells was quantified. Cells used for comparative adoptive protection tests were harvested from the major lymphoid compartments 14 to 15 days after young C58 mice were immunized with inactivated Ib cell preparations. Regression curves were plotted from survival data and the log 10 PD 50 values were determined. Immune spleen (ISC) and peritoneal cells (IPEC) were significantly more protective against transplanted Ib cells than immune lymph node (ILNC), thymic (ITC), and marrow cells (IMC). In contrast, IPEC and IMC were not protective against IPE and ITC were only marginally protective. ILNC afforded significant protection to transplantable leukemia but were only marginally protective to IPE. When ISC were treated with anti-thy 1.2 serum and complement, protection against transplanted leukemia and IPE was reduced > 99%. When donors of immune lymphoid cells were treated with 12.5 mg of cortisone acetate daily for 2 days before lymphoid cells were harvested, protection against transplanted Ib cells by ISC was reduced by approximately 90% whereas protection against IPE was totally eliminated. Considered together, these results indicate that the protective mechanisms to transplantable leukemia and IPE differ significantly in the same indicator mouse strain

Surface-enhanced Raman spectroscopy of genomic DNA from in vitro grown tomato (Lycopersicon esculentum Mill.) cultivars before and after plant cryopreservation.

Science.gov (United States)

Muntean, Cristina M; Leopold, Nicolae; Tripon, Carmen; Coste, Ana; Halmagyi, Adela

2015-06-05

In this work the surface-enhanced Raman scattering (SERS) spectra of five genomic DNAs from non-cryopreserved control tomato plants (Lycopersicon esculentum Mill. cultivars Siriana, Darsirius, Kristin, Pontica and Capriciu) respectively, have been analyzed in the wavenumber range 400-1800 cm(-1). Structural changes induced in genomic DNAs upon cryopreservation were discussed in detail for four of the above mentioned tomato cultivars. The surface-enhanced Raman vibrational modes for each of these cases, spectroscopic band assignments and structural interpretations of genomic DNAs are reported. We have found, that DNA isolated from Siriana cultivar leaf tissues suffers the weakest structural changes upon cryogenic storage of tomato shoot apices. On the contrary, genomic DNA extracted from Pontica cultivar is the most responsive system to cryopreservation process. Particularly, both C2'-endo-anti and C3'-endo-anti conformations have been detected. As a general observation, the wavenumber range 1511-1652 cm(-1), being due to dA, dG and dT residues seems to be influenced by cryopreservation process. These changes could reflect unstacking of DNA bases. However, not significant structural changes of genomic DNAs from Siriana, Darsirius and Kristin have been found upon cryopreservation process of tomato cultivars. Based on this work, specific plant DNA-ligand interactions or accurate local structure of DNA in the proximity of a metallic surface, might be further investigated using surface-enhanced Raman spectroscopy. Copyright © 2015 Elsevier B.V. All rights reserved.

The effect of the melatonin on cryopreserved mouse testicular cells

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Ghasem Saki

2016-01-01

Full Text Available Background: After improvements in various cancer treatments, life expectancy has been raised, but success in treatment causes loss of fertility in many of the survived young men. Cryopreservation of immature testicular tissues or cells introduced as the only way to preserve fertility. However, freezing has some harmful effects. Melatonin, a pineal gland hormone, has receptors in reproductive systems of different species. It is assumed that melatonin has free radical scavenger properties. Objective: The aim of this study was to evaluate the effects of melatonin on the cryopreserved testicular cells in mouse. Materials and Methods: Cells from 7- 10 days old NMRI mice testes were isolated using two step enzymatic digestion. The testicular cells were divided into two groups randomly and cryopreserved in two different freezing media with and without the addition of 100 μm melatonin. Finally, apoptosis of the cells was assayed by flow cytometry. Also, lactate dehydrogenase activity test was performed to assess the cytotoxicity. Results: The results of lactate dehydrogenase showed the nearly cytotoxic effect of melatonin. The results of flow cytometry showed increase in apoptosis in the cryopreserved cells in the media containing melatonin compared to the control group. Conclusion: The present study shows that melatonin has an apoptotic effect on cryopreserved mouse testicular cells.

Cholesterol addition aids the cryopreservation of dromedary camel (Camelus dromedarius) spermatozoa.

Science.gov (United States)

Crichton, Elizabeth G; Pukazhenthi, Budhan S; Billah, M; Skidmore, Julian A

2015-01-15

The cryopreservation of dromedary camel (Camelus dromedarius) sperm has proved challenging with little success reported. The routine application of artificial insemination with frozen semen would assist the flow of valuable genetic material nationally and internationally. The current study sought to examine the effects of cholesterol (cholesterol-loaded cyclodextrin [CLC]) preloading on camel sperm cryosurvival. Ejaculates (n = 3 males; 3 ejaculates per male) were collected using an artificial vagina during the breeding season and extended in HEPES-buffered Tyrode's albumin lactate pyruvate (TALP) and allowed to liquefy in the presence of papain (0.1 mg/mL) before removal of the seminal plasma by centrifugation. Sperm pellets were resuspended (120 million/mL) in fresh TALP and incubated (15 minutes; 37 °C) with 0, 1.5, or 4.5 mg CLC/mL. Sperm suspensions were then centrifuged and reconstituted in INRA-96 containing 20% (v:v) egg yolk and 2.5% (v:v) methylformamide, loaded in 0.5-mL plastic straws, sealed, and cooled for 20 minutes at 4 °C. Straws were frozen over liquid nitrogen (4 cm above liquid; 15 minutes), plunged, and stored. Sperm motility, forward progressive status, and acrosomal integrity were recorded at 0 and 3 hours after thawing and compared with these same parameters before freezing. Aliquots also were stained with chlortetracycline hydrochloride to assess spontaneous sperm capacitation status before freezing and post-thaw. Pretreatment with CLC (1.5 and 4.5 mg/mL) enhanced cryosurvival. Post-thaw sperm motility was highest (P < 0.05) in 1.5 mg CLC/mL immediately after thawing (44%) and after 3 hours incubation at room temperature (34%). Highest post-thaw sperm progressive status was also achieved in the presence of 1.5 CLC. Greater proportions of spermatozoa retained acrosomal membrane integrity when cryopreserved in the presence of CLC, but there was no difference between 1.5 and 4.5 CLC. Although thawed spermatozoa underwent spontaneous

Fibroproliferative activity in patients with immune thrombocytopenia (ITP) treated with thrombopoietic agents

DEFF Research Database (Denmark)

Ghanima, Waleed; Junker, Peter; Hasselbalch, Hans Carl

2011-01-01

This study assessed the grade of bone marrow (BM) fibrosis and its association with a seromarker for collagen-III formation and fibrosis-related cytokines in 25 immune thrombocytopenia (ITP) patients treated with thrombopoietin receptor agonists (Tpo-RA) who had at least one BM biopsy. Assessment...

Optimized cryopreservation of mixed microbial communities for conserved functionality and diversity.

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Frederiek-Maarten Kerckhof

Full Text Available The use of mixed microbial communities (microbiomes for biotechnological applications has steadily increased over the past decades. However, these microbiomes are not readily available from public culture collections, hampering their potential for widespread use. The main reason for this lack of availability is the lack of an effective cryopreservation protocol. Due to this critical need, we evaluated the functionality as well as the community structure of three different types of microbiomes before and after cryopreservation with two cryoprotective agents (CPA. Microbiomes were selected based upon relevance towards applications: (1 a methanotrophic co-culture (MOB, with potential for mitigation of greenhouse gas emissions, environmental pollutants removal and bioplastics production; (2 an oxygen limited autotrophic nitrification/denitrification (OLAND biofilm, with enhanced economic and ecological benefits for wastewater treatment, and (3 fecal material from a human donor, with potential applications for fecal transplants and pre/probiotics research. After three months of cryopreservation at -80 °C, we found that metabolic activity, in terms of the specific activity recovery of MOB, aerobic ammonium oxidizing bacteria (AerAOB and anaerobic AOB (AnAOB, anammox in the OLAND mixed culture, resumes sooner when one of our selected CPA [dimethyl sulfoxide (DMSO and DMSO plus trehalose and tryptic soy broth (DMSO+TT] was added. However, the activity of the fecal community was not influenced by the CPA addition, although the preservation of the community structure (as determined by 16S rRNA gene sequencing was enhanced by addition of CPA. In summary, we have evaluated a cryopreservation protocol that succeeded in preserving both community structure and functionality of value-added microbiomes. This will allow individual laboratories and culture collections to boost the use of microbiomes in biotechnological applications.

Transplantation of cryopreserved allogeneic bone marrow after its long-term storage to lethally irradiated dogs

International Nuclear Information System (INIS)

Novikova, N.N.; Fedotenkov, A.G.; Sukyasyan, G.V.; Timakova, L.A.

1982-01-01

The study of the dog bone marrow preserved at -196 deg C during 6-12 years has shown that in the body of lethally irradiated animals (8Gy), due to the antigenic difference in the tissues of the donor and the irradiated recipients, the cells of cryopreserved allogeneic bone marrow were differentiated by the lymphoid type similar to that observed in transplantation of freshly prepared myelocaryocytes. However, their proliferative activity in the period of active lymphocyte transformation was quantitatively less manifest than in freshly transplanted cells. The results of the study evidence that the bone marrow cells cryopreserved during 6-12 years retain their functional activity

Droplet vitrification technique for cryopreservation of different pineapple (Ananas comosus L. Merrill) accessions

Science.gov (United States)

Germplasm conservation of pineapple is crucial to secure the genetic variability of the genus for breeding programs and supporting new research. Long-term conservation is done through cryopreservation, by storing cells or tissues at ultra-low temperature in liquid nitrogen (LN; -196°C) or in the LN ...

Ovarian tissue cryopreserved for fertility preservation from patients with Ewing or other sarcomas appear to have no tumour cell contamination

DEFF Research Database (Denmark)

Greve, Tine; Wielenga, Vera Timmermans; Grauslund, Morten

2013-01-01

The chemotherapy required to treat patients with sarcoma may as a side-effect induce infertility in girls and young women. If these patients have ovarian cortical tissue cryopreserved prior to chemotherapy, they may, if necessary, have the tissue transplanted and restore their fertility. The aim...

Two-piece cryopreserved tracheal allotransplantation: an experimental study.

Science.gov (United States)

Iyikesici, Tuncel; Tuncozgur, Bulent; Sanli, Maruf; Isik, Ahmet Feridun; Meteroglu, Fatih; Elbeyli, Levent

2009-10-01

For successful reconstruction with tracheal allotransplants following long tracheal resections, problems related to the preservation and vascularisation of the tracheal graft have to be solved. In this study, instead of using a long-segment single-piece graft, we used a graft that has been split into two. The aim was to use this graft after cryopreservation in order to ease neo-vascularisation and to maintain tracheal integrity by transplanting it to two separate regions of the dog cervical trachea. This experimental study was conducted in animal laboratories of the medical school on 11 half-blood dogs. The trachea obtained from the first dog was 8 cm in length; it was split into two pieces of 4 cm each and stored in the preservation solution at -80 degrees C for 4 weeks. Following this, the dog was sacrificed. Two 2 cm portions of cervical trachea were excised from the second dog. These parts were then reconstructed with two tracheal grafts of the same length as the cryopreserved ones. Ten dogs that were grouped into five groups of two dogs each underwent the same procedure. The subjects had a bronchoscopic evaluation on the third postoperative week. Anastomosis regions of the test tracheas were resected to be examined histopathologically. Seven subjects were found to have third-degree obstructions during bronchoscopy; two had close to fourth-degree obstructions. In the histopathological examination, contrary to the findings of the bronchoscopies, 75% of the anastomoses had intact epithelium. The cartilage was seen to have well-preserved structural characteristics in all the anastomoses. Twelve anastomoses had moderate, seven mild and one had severe inflammation. All anastomoses had either good or very good level of vascularisation. The integrity of the tracheal epithelium can be maintained with cryopreservation and split anastomosis technique. The cartilage preserves its structural characteristics despite losing its viability, thereby offering an advantage to

Cryopreservation of in vitro-grown shoot tips of apricot (Prunus ...

African Journals Online (AJOL)

akpobome

2013-03-20

Mar 20, 2013 ... Thus, they can be preserved in such a state for a long period (Sakai, 1995). Many new cryopreservation ..... formation of intracellular ice crystals during rapid cooling in LN (Wilkinson et al., 1998). Effect preculture and ..... Cryopreservation of protocorm-like bodies. (PLBs) of Phalaenopsis bellina. (Rchb.f.) ...

A new strategy and system for the ex vivo ovary perfusion and cryopreservation: An innovation.

Science.gov (United States)

Ali Mohamed, Mohamed Shehata

2017-06-01

Children and young adults, who suffer from cancer, receive gonadotoxic therapy, which destroys their fertile abilities after survival. Ovarian cryopreservation and transplantation provide the promising solution to this problem, where the ovary can be removed before the gonadotoxic therapy and reimplanted after patient's survival, where the ovary is to be cryopreserved during the period of the therapy. However, cryopreservation of the whole ovary is still facing great obstacles, namely the ischemic reperfusion injury and the defective cryopreservation related to the defective ability to universally deliver the cryopreservation/warming solutions through the ovarian vascular bed. Meanwhile, the currently applied technique of ovarian tissue cryopreservation provides limited follicular recovery because many follicles are lost until the development of revascularization post-transplantation. To solve the problems, an innovative system has been developed to insure immediate and universal delivery of the cryopreservation/warming solutions to the graft, in addition to keeping the graft under continuous perfusion before and after cryopreservation, minimizing any chance for microthrombi formation or ischemia-reperfusion. This innovative system can be applied in the following surgical and clinical interventions: 1) Allogeneic ovarian transplantation; 2) Preservation of fertility after systemic chemotherapy or bone marrow transplantation in young females, where the ovaries could be removed before the therapy and exposed to the adequate cryopreservation provided by the system till re-implantation after the patient's survival; 3) The system is also suitable for the corresponding applications on the testicles.

The use of cryopreserved sea urchin embryos (Paracentrotus lividus) in marine quality assessment.

Science.gov (United States)

Paredes, E; Bellas, J

2015-06-01

We have established for first time an ecotoxicological bioassay using cryopreserved sea urchin embryos (Paracentotus lividus) and provided a comparison to the already standardized sea urchin embryo-larval bioassay, using selected (organic and inorganic) pollutants and sediment elutriates from 4 different locations from Ria de Vigo harbour (Galicia, NW Iberian Peninsula). A cryopreservation protocol was designed in order to enable the successful cryopreservation and cryobanking of gametes and embryos to be used for marine quality assessment and ensure the accessibility to high quality reproductive material all year round, as an option to conditioning adults for out of season reproduction. The calculated EC50 using the cryopreserved blastula was 53.7 μg L(-1) for copper, 81.0 μg L(-1) for lead, 300.6 μg L(-1) for BP-3 and 300.6 μg L(-1) for 4-MBC. The sensitivity of the classic sea urchin embryo-larval bioassay was compared with the bioassay conducted with cryopreserved blastula. The results showed that the use of cryopreserved blastula bioassay allows detecting lower concentrations of pollutants in comparison with the classic bioassay. Copyright © 2015 Elsevier Ltd. All rights reserved.

Optimization of a protocol for cryopreservation of mouse spermatozoa using cryotubes.

Science.gov (United States)

Hasegawa, Ayumi; Yonezawa, Kazuya; Ohta, Akihiko; Mochida, Keiji; Ogura, Atsuo

2012-01-01

The rapid increase in the number of genetically modified mouse strains has produced a high demand for their frozen spermatozoa from laboratories and mouse banking facilities. Historically, plastic straws have been used preferentially as containers for frozen mammalian spermatozoa because spermatozoa frozen in plastic straws have a high survival rate after thawing. However, plastic straws are more fragile and are used less often than the cryotubes used for conventional cell freezing. In this study, we sought to develop a new protocol for sperm freezing using cryotubes as the container to increase the accessibility of mouse sperm cryopreservation. Epididymal spermatozoa were collected from mature ICR or C57BL/6J (B6) males and were suspended in 18% raffinose and 3% skim milk solution. We then optimized the following conditions using the sperm survival rate as an index: 1) distance of cryotubes from the surface of the liquid nitrogen at freezing, 2) volume of the sperm suspension in the cryotube and 3) temperature of warming sperm during thawing. The best result was obtained when cryotubes containing 10 µl of sperm suspension were immersed 1 cm below the surface of the liquid nitrogen and then thawed at 50 C. The fertilization rates using spermatozoa frozen and thawed using this method were 63.1% in ICR mice and 28.2% in B6 mice. The latter rate was increased to 62.3% by adding reduced glutathione to the fertilization medium. After embryo transfer, 68% and 62% of the fertilized oocytes developed into normal offspring in the ICR and B6 strains, respectively. These results show that cryotubes can be used for cryopreservation of mouse spermatozoa under optimized conditions. This protocol is easy and reproducible, and it may be used in laboratories that do not specialize in sperm cryopreservation.

Immunization of C57BL/6 Mice with GRA2 Combined with MPL Conferred Partial Immune Protection against Toxoplasma gondii

Science.gov (United States)

Babaie, Jalal; Amiri, Samira; Homayoun, Robab; Azimi, Ebrahim; Mohabati, Reyhaneh; Berizi, Mahboobe; Sadaie, M. Reza; Golkar, Majid

2018-01-01

We have previously reported that immunization with GRA2 antigen of Toxoplasma gondii induces protective immunity in CBA/J (H2k) and BALB/c mice (H2d). We aimed to examine whether immunization of a distinct strain of rodent with recombinant dense granule antigens (GRA2) combined with monophosphorryl lipid A (MPL) adjuvant elicits protective immune response against T. gondii. C57BL/6 (H2b haplotype) mice were immunized with GRA2, formulated in MPL adjuvant. Strong humoral response, predominantly of IgG1 subclass and cellular response, IFN-γ, was detected at three weeks post immunization. Mice immunized with GRA2 had significantly (p < 0.01) fewer brain cysts than those in the adjuvant group, upon challenge infection. Despite the production of a strong antibody response, IFN-γ production and brain cyst reduction were not significant when the immunized mice were infected four months after the immunization. We can conclude that GRA2 immunization partially protects against T. gondii infection in C57BL/6 mice, though the potency and longevity of this antigen as a standalone vaccine may vary in distinct genetic backgrounds. This observation further emphasizes the utility of GRA2 for incorporation into a multi-antigenic vaccine against T. gondii.

DMPD: C-type lectin receptors in antifungal immunity. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 18160296 C-type lectin receptors in antifungal immunity. Willment JA, Brown GD. Tre...nds Microbiol. 2008 Jan;16(1):27-32. Epub 2007 Dec 21. (.png) (.svg) (.html) (.csml) Show C-type lectin receptors in antifun...gal immunity. PubmedID 18160296 Title C-type lectin receptors in antifungal immunity. Author

Generation of Platelet Microparticles after Cryopreservation of Apheresis Platelet Concentrates Contributes to Hemostatic Activity

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İbrahim Eker

2017-03-01

Full Text Available Objective: In the last decade, substantial evidence has accumulated about the use of cryopreserved platelet concentrates, especially in trauma. However, little reference has been made in these studies to the morphological and functional changes of platelets. Recently platelets have been shown to be activated by cryopreservation processes and to undergo procoagulant membrane changes resulting in the generation of platelet-derived microparticles (PMPs, platelet degranulation, and release of platelet-derived growth factors (PDGFs. We assessed the viabilities and the PMP and PDGF levels of cryopreserved platelets, and their relation with thrombin generation. Materials and Methods: Apheresis platelet concentrates (APCs from 20 donors were stored for 1 day and cryopreserved with 6% dimethyl sulfoxide. Cryopreserved APCs were kept at -80 °C for 1 day. Thawed APCs (100 mL were diluted with 20 mL of autologous plasma and specimens were analyzed for viabilities and PMPs by flow cytometry, for thrombin generation by calibrated automated thrombogram, and for PDGFs by enzyme-linked immunosorbent assay testing. Results: The mean PMP and PDGF levels in freeze-thawed APCs were significantly higher (2763±399.4/μL vs. 319.9±80.5/μL, p<0.001 and 550.9±73.6 pg/mL vs. 96.5±49 pg/mL, p<0.001, respectively, but the viability rates were significantly lower (68.2±13.7% vs. 94±7.5%, p<0.001 than those of fresh APCs. The mean endogenous thrombin potential (ETP of freeze-thawed APCs was significantly higher than that of the fresh APCs (3406.1±430.4 nM.min vs. 2757.6±485.7 nM.min, p<0.001. Moreover, there was a significant positive poor correlation between ETP levels and PMP levels (r=0.192, p=0.014. Conclusion: Our results showed that, after cryopreservation, while levels of PMPs were increasing, significantly higher and earlier thrombin formation was occurring in the samples analyzed despite the significant decrease in viability. Considering the damage caused

Cryopreservation of yamú (Brycon amazonicus) sperm for large scale fertilization

DEFF Research Database (Denmark)

Velasco-Santamaría, Yohana M.; Medina-Robles, Mauricio; Cruz-Casallas, Pablo E.

2006-01-01

  To determine the effect of straw size and thawing temperature on cryopreserved sperm quality of yamú (Brycon amazonicus), ovulation and spermiation were induced in sexually mature broodstock using Carp Pituitary Extract. Sperm quality was evaluated by motility, activation time and fertility...... assays consisted of 40 g eggs inseminated with approximately 5.0 mL (ca. 75,000 motile spermatozoa/egg) of cryopreserved sperm in large straws thawed at 35 °C. The fertilization rate was estimated 6 h post-insemination. In all straws, postthaw motility was significantly lower than for fresh sperm (pb0.......05) to sperm frozen in 0.5-mL straws (48±2%, 51±2%, 52±2% and 54±3%, respectively). In large scale fertilization trials, fresh sperm showed a higher (pb0.05) fertilization rate (83±1%) than frozen-thawed sperm (68±1%). Although the fertility percentage with fresh sperm was significantly higher than with frozen...

The effects of X-irradiation on ex vivo expansion of cryopreserved human hematopoietic stem/progenitor cells

International Nuclear Information System (INIS)

Hayashi, Naoki; Takahashi, Kenji; Kashiwakura, Ikuo

2010-01-01

In our previous study (Life Sciences 84: 598, 2009), we demonstrated that placental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells have the effect to support the regeneration of freshly prepared X-irradiated hematopoietic stem/progenitor cells (HSPCs). Generally, HSPCs are supplied from companies, institutions, and cell banks that cryopreserve them for clinical and experimental use. In this study, the influence of cryopreservation on the responses of HSPCs to irradiation and co-culture with stromal cells is assessed. After cryopreservation with the optimal procedure, 2 Gy-irradiated HSPCs were cultured with or without stromal cells supplemented with combination of interleukin-3, stem cell factor, and thrombopoietin. The population of relatively immature CD34 + /CD38 - cells in cryopreserved cells was significantly higher than in fresh cells prior to cryopreservation; furthermore, the hematopoietic progenitor populations of CD34 + /CD45RA + cells and CD34 + /CD117 + cells in cryopreserved cells were significantly lower than that in fresh cells. However, the rate of expansion in the cryopreserved HSPCs was lower than in the fresh HSPCs. In the culture of cryopreserved cells irradiated with 2 Gy, the growth rates of CD34 + cells, CD34 + /CD38 - cells, and hematopoietic progenitors were greater than growth rates of their counter parts in the culture of fresh cells. Surprisingly, the effect to support the hematopoiesis in co-culture with stromal cells was never observed in the X-irradiated HSPCs after cryopreservation. The present results demonstrated that cryopreserving process increased the rate of immature and radio-resistant HSPCs but decreased the effects to support the hematopoiesis by stromal cells, thus suggesting that cryopreservation changes the character of HSPCs. (author)

Delicate regulation of the cGAS-MITA-mediated innate immune response.

Science.gov (United States)

Luo, Wei-Wei; Shu, Hong-Bing

2018-02-19

Although it has long been demonstrated that cytosolic DNA is a potent immune stimulant, it is only in recent years that the molecular mechanisms of DNA-stimulated innate immune responses have emerged. Studies have established critical roles for the DNA sensor cyclic GMP-AMP synthase (cGAS) and the adapter protein MITA/STING in the innate immune response to cytosolic DNA or DNA viruses. Although the regulation of cGAS-MITA/STING-mediated signaling remains to be fully investigated, understanding the processes involved may help to explain the mechanisms of innate immune signaling events and perhaps autoinflammatory diseases and to provide potential therapeutic targets for drug intervention. In this review, we summarize recent progress on the regulation of the cGAS-MITA/STING-mediated innate immune response to DNA viruses at the organelle-trafficking, post-translational and transcriptional levels.Cellular & Molecular Immunology advance online publication, 19 February 2018; doi:10.1038/cmi.2016.51.

Cryopreservation of canine sperm using egg yolk and soy bean based extenders.

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Sánchez-Calabuig, María Jesús; Maillo, Verónica; Beltrán-Breña, Paula; de la Fuente Martínez, Julio; Galera-Carrillo, Silvestre; Pérez-Gutiérrez, José Félix; Pérez-Cerezales, Serafín

2017-09-01

Animal protein-based extenders are widely used despite being a potential source of bacterial or mycoplasma contamination. Its replacement with vegetal protein-based extenders could represent an interesting alternative for dog sperm cryopreservation. This technique could be further improved by the addition of Tris-Glucose-Citric acid (TGC) that could physically protect the spermatozoa and improve its homeostasis. The aim of this study was to evaluate a cryopreservation protocol for dog spermatozoa using a soybean-based extender (LP1 ℗ ) as well as the effects of the addition of (TGC) immediately after the semen collection. Eleven ejaculates from purebred adult dogs were collected, centrifuged in the absence or presence of TGC and processed as fresh or cryopreserved spermatozoa with: egg yolk-based extender (CaniPRO) or LP1 ℗ . Freezing the spermatozoa in LP1 ℗ reduced the amplitude of the lateral head displacement, the percentage of spermatozoa that showed the intact acrosome and the mitochondrial function (P<0.05). These samples also showed a trend towards increased percentage of apoptotic spermatozoa (P<0.05). The addition of TGC before centrifugation did not improve the seminal parameters and adversely affected motility (P<0.05) in the spermatozoa cryopreserved in CaniPRO. However, TGC did not affect motility and increased (P<0.05) the percentage of intact acrosomes in the spermatozoa cryopreserved in LP1 ℗ , reaching similar values than those cryopreserved in CaniPRO. In conclusion, LP1 ® plus TGC provide the same level of protection to dog spermatozoa cryopreservation than the egg yolk based extender CaniPRO when comparing standard post-thaw sperm quality parameters. Copyright © 2017. Published by Elsevier Urban & Partner Sp. z o.o.

Cryopreservation of spermatozoa of black marlin, Makaira indica (Teleostei: Istiophoridae).

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van der Straten, K M; Leung, L K-P; Rossini, R; Johnston, S D

2006-01-01

As a first step towards the development of a method for the cryopreservation of black marlin spermatozoa, this study investigated the effect of dimethylsulfoxide (DMSO) concentration and pellet size on post-thaw spermatozoal motility. Spermatozoa were recovered from the spermatic duct of testes retrieved post-mortem from four adult black marlin caught in the Coral Sea spawning grounds of Australia. Undiluted spermatozoa were stored on ice for 4 to 10 hours during transport to shore, then evaluated for motility after activation in seawater (1:10 v:v). Spermatozoa were prepared for cryopreservation in pellets by extension (1:3 v:v) in a defined fish Ringer's solution to give two final DMSO concentrations of 2.5% or 5.0%. Diluted spermatozoa were frozen directly on a dry ice block in pellet sizes of either 0.25 ml or 0.50 ml. Frozen pellets were thawed in a water bath at 40 degrees C for 60 seconds and assessed for post-thaw motility following activation in seawater. Spermatozoa recovered within 50 minutes of death and chilled on ice for 4 to 10 hours showed a mean (+/- SEM) motility immediately following activation of 91.6 +/- 7.9%. 50% of the spermatozoa remained motile for approximately 4 to 5 minutes. Following cryopreservation, mean motility declined significantly across all cryoprotectant and pellet size combinations (P < 0.001) but spermatozoa frozen in 2.5% DMSO showed higher motility than those frozen in 5.0% DMSO (P = 0.014). Pellet size had no effect on post-thaw motility (P = 0.179).

Sperm harvesting and cryopreservation during vasectomy reversal is not cost effective.

Science.gov (United States)

Boyle, Karen E; Thomas, Anthony J; Marmar, Joel L; Hirshberg, Steven; Belker, Arnold M; Jarow, Jonathan P

2006-04-01

To determine whether sperm harvesting and cryopreservation at the time of vasectomy reversal is cost-effective. Model of actual costs and results at five institutions. Multicenter study comprising five centers, including university hospitals and private practices. Men undergoing vasectomy reversal. We established two models for vasectomy reversal. The first model was sperm harvesting and cryopreservation at the time of vasectomy reversal. The second model was sperm harvesting at the time of IVF only if the patient remained azoospermic after vasectomy reversal. Vasectomy reversal procedures modeled included bilateral vasovasostomy and bilateral epididymovasostomy. The costs for each procedure at the five institutions were collated and median costs determined. Median cost of procedure and calculated financial comparisons. The median cost of testicular sperm extraction/cryopreservation performed at the time of bilateral vasovasostomy was $1,765 (range, $1,025-$2,800). The median cost of microsurgical epididymal sperm aspiration or testicular sperm extraction with cryopreservation performed at the time of epididymovasostomy was $1,209 (range, $905-$2,488). The average of the median costs for percutaneous sperm aspiration or testicular sperm aspiration for those patients with a failed vasectomy reversal was $725 (range, $400-$1,455). Sperm retrieval with cryopreservation at the time of vasectomy reversal is not a cost-effective management strategy.

Humoral and cellular immune responses in BALB/c and C57BL/6 mice immunized with cytoplasmic (CRA) and flagellar (FRA) recombinant repetitive antigens, in acute experimental Trypanosoma cruzi infection.

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Pereira, Valéria R A; Lorena, Virginia M B; Nakazawa, Mineo; Luna, Carlos F; Silva, Edimilson D; Ferreira, Antonio G P; Krieger, Marco Aurélio; Goldenberg, Samuel; Soares, Milena B P; Coutinho, Eridan M; Correa-Oliveira, Rodrigo; Gomes, Yara M

2005-06-01

In previous studies, cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) proteins induced specific humoral and cellular immune responses in susceptible and resistant mice in the absence of Trypanosoma cruzi infection with a significant induction of the Interferon-gamma (IFN-gamma) production in those animals. In this follow-up paper, the immunostimulatory and protective effects of these proteins were evaluated by immunizing with CRA or FRA antigens, BALB/c and C57BL/6 mice and challenging with a T. cruzi (Y strain). Both proteins induced humoral response with high levels of IgG isotypes as well as cellular immunity with high levels of IFN-gamma when compared to controls. However, the lymphocyte proliferative response was minimal. The survival rate at 30 days post-infection was significant in CRA (60%) or FRA (50%)--immunized BALB/c mice and CRA (83.3%)--immunized C57BL/6 mice. Taken as a whole these findings indicate that CRA and FRA are immunogenic and potentially important for protective immunity.

Cryopreservation of artificial gut microbiota produced with in vitro fermentation technology.

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Bircher, Lea; Schwab, Clarissa; Geirnaert, Annelies; Lacroix, Christophe

2018-01-01

Interest in faecal microbiota transplantation (FMT) has increased as therapy for intestinal diseases, but safety issues limit its widespread use. Intestinal fermentation technology (IFT) can produce controlled, diverse and metabolically active 'artificial' colonic microbiota as potential alternative to common FMT. However, suitable processing technology to store this artificial microbiota is lacking. In this study, we evaluated the impact of the two cryoprotectives, glycerol (15% v/v) and inulin (5% w/v) alone and in combination, in preserving short-chain fatty acid formation and recovery of major butyrate-producing bacteria in three artificial microbiota during cryopreservation for 3 months at -80°C. After 24 h anaerobic fermentation of the preserved microbiota, butyrate and propionate production were maintained when glycerol was used as cryoprotectant, while acetate and butyrate were formed more rapidly with glycerol in combination with inulin. Glycerol supported cryopreservation of the Roseburia spp./Eubacterium rectale group, while inulin improved the recovery of Faecalibacterium prausnitzii. Eubacterium hallii growth was affected minimally by cryopreservation. Our data indicate that butyrate producers, which are key organisms for gut health, can be well preserved with glycerol and inulin during frozen storage. This is of high importance if artificially produced colonic microbiota is considered for therapeutic purposes. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Poly (I:C) enhances the anti-tumor activity of canine parvovirus NS1 protein by inducing a potent anti-tumor immune response.

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Gupta, Shishir Kumar; Yadav, Pavan Kumar; Tiwari, A K; Gandham, Ravi Kumar; Sahoo, A P

2016-09-01

The canine parvovirus NS1 (CPV2.NS1) protein selectively induces apoptosis in the malignant cells. However, for an effective in vivo tumor treatment strategy, an oncolytic agent also needs to induce a potent anti-tumor immune response. In the present study, we used poly (I:C), a TLR3 ligand, as an adjuvant along with CPV2.NS1 to find out if the combination can enhance the oncolytic activity by inducing a potent anti-tumor immune response. The 4T1 mammary carcinoma cells were used to induce mammary tumor in Balb/c mice. The results suggested that poly (I:C), when given along with CPV2.NS1, not only significantly reduced the tumor growth but also augmented the immune response against tumor antigen(s) as indicated by the increase in blood CD4+ and CD8+ counts and infiltration of immune cells in the tumor tissue. Further, blood serum analysis of the cytokines revealed that Th1 cytokines (IFN-γ and IL-2) were significantly upregulated in the treatment group indicating activation of cell-mediated immune response. The present study reports the efficacy of CPV2.NS1 along with poly (I:C) not only in inhibiting the mammary tumor growth but also in generating an active anti-tumor immune response without any visible toxicity. The results of our study may help in developing CPV2.NS1 and poly (I: C) combination as a cancer therapeutic regime to treat various malignancies.

Survival, growth and reproduction of cryopreserved larvae from a marine invertebrate, the Pacific oyster (Crassostrea gigas.

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Marc Suquet

Full Text Available This study is the first demonstration of successful post-thawing development to reproduction stage of diploid cryopreserved larvae in an aquatic invertebrate. Survival, growth and reproductive performances were studied in juvenile and adult Pacific oysters grown from cryopreserved embryos. Cryopreservation was performed at three early stages: trochophore (13±2 hours post fertilization: hpf, early D-larvae (24±2 hpf and late D-larvae (43±2 hpf. From the beginning (88 days at the end of the ongrowing phase (195 days, no mortality was recorded and mean body weights did not differ between the thawed oysters and the control. At the end of the growing-out phase (982 days, survival of the oysters cryopreserved at 13±2 hpf and at 43±2 hpf was significantly higher (P<0.001 than those of the control (non cryopreserved larvae. Only the batches cryopreserved at 24±2 hpf showed lower survival than the control. Reproductive integrity of the mature oysters, formely cryopreserved at 13±2 hpf and 24±2 hpf, was estimated by the sperm movement and the larval development of their offspring in 13 crosses gamete pools (five males and five females in each pool. In all but two crosses out of 13 tested (P<0.001, development rates of the offspring were not significantly different between frozen and unfrozen parents. In all, the growth and reproductive performances of oysters formerly cryopreserved at larval stages are close to those of controls. Furthermore, these performances did not differ between the three initial larval stages of cryopreservation. The utility of larvae cryopreservation is discussed and compared with the cryopreservation of gametes as a technique for selection programs and shellfish cryobanking.

Utilization and quality of cryopreserved red blood cells in transfusion medicine

NARCIS (Netherlands)

Henkelman, S.; Noorman, F.; Badloe, J. F.; Lagerberg, J. W. M.

Cryopreserved (frozen) red blood cells have been used in transfusion medicine since the Vietnam war. The main method to freeze the red blood cells is by usage of glycerol. Although the usage of cryopreserved red blood cells was promising due to the prolonged storage time and the limited cellular

Differential protective effects of immune lymphoid cells against transplanted line Ib leukemia and immune polioencephalomyelitis. [X radiation, mice

Energy Technology Data Exchange (ETDEWEB)

Duffey, P.S.; Lukasewycz, O.A.; Olson, D.S.; Murphy, W.H.

1978-12-01

The capacity of immune cells obtained from the major lymphoid compartments to protect C58 mice from transplanted line Ib leukemia, and from an age-dependent autoimmune CNS disease (immune polioencephalomyelitis = IPE) elicited by immunizing old C58 mice with inactivated Ib cells was quantified. Cells used for comparative adoptive protection tests were harvested from the major lymphoid compartments 14 to 15 days after young C58 mice were immunized with inactivated Ib cell preparations. Regression curves were plotted from survival data and the log/sub 10/PD/sub 50/ values were determined. Immune spleen (ISC) and peritoneal cells (IPEC) were significantly more protective against transplanted Ib cells than immune lymph node (ILNC), thymic (ITC), and marrow cells (IMC). In contrast, IPEC and IMC were not protective against IPE and ITC were only marginally protective. ILNC afforded significant protection to transplantable leukemia but were only marginally protective to IPE. When ISC were treated with anti-thy 1.2 serum and complement, protection against transplanted leukemia and IPE was reduced > 99%. When donors of immune lymphoid cells were treated with 12.5 mg of cortisone acetate daily for 2 days before lymphoid cells were harvested, protection against transplanted Ib cells by ISC was reduced by approximately 90% whereas protection against IPE was totally eliminated. Considered together, these results indicate that the protective mechanisms to transplantable leukemia and IPE differ significantly in the same indicator mouse strain.

Hatchery-scale trials using cryopreserved spermatozoa of black-lip pearl oyster, Pinctada margaritifera

OpenAIRE

Hui, Belinda; Vonau, Vincent; Moriceau, Jacques; Tetumu, Roger; Vanaa, Vincent; Demoy-schneider, Marina; Suquet, Marc; Le Moullac, Gilles

2011-01-01

Cryopreservation is a valuable tool for genetic improvement programs. Several bivalve mollusc species have already been the subject of such programs and the Tahitian black pearl oyster industry is now planning the development of selective breeding for desirable traits in Pinctada margaritifera. The ability to cryopreserve spermatozoa would, therefore, offer significant benefits to the cultured black pearl industry. Spermatozoa were cryopreserved with CPA 0.7 M trehalose in 0.8 M Me2SO and...

Determination of metabolic stability using cryopreserved hepatocytes from rainbow trout (Oncorhynchus mykiss)

Science.gov (United States)

Standard protocols for isolating, cryopreserving, and thawing rainbow trout hepatocytes are described, along with procedures for using fresh or cryopreserved hepatocytes to assess chemical metabolic stability in fish by means of a substrate depletion approach. Variations on thes...

Improved recovery of cryotherapy-treated shoot tips following thermotherapy of in vitro-grown stock shoots of raspberry (Rubus idaeus L.).

Science.gov (United States)

Wang, Qiaochun; Valkonen, Jari P T

2009-01-01

Raspberry bushy dwarf virus (RBDV) can be efficiently eradicated from raspberry plants (Rubus idaeus) by a procedure combining thermotherapy and cryotherapy. However, the bottleneck of this procedure is that, following thermotherapy, cryopreserved shoot tips become chlorotic during regrowth and eventually die after several subcultures. In addition, survival of heat-treated stock shoots and recovery of cryopreserved shoot tips following thermotherapy are low. The present study focused towards improving regrowth of cryopreserved raspberry shoot tips following thermotherapy. Results showed that preconditioning stock shoots with salicylic acid (SA; 0.01-0.1 mM) markedly increased survival of stock shoots after 4 weeks of thermotherapy. Regrowth of cryopreserved shoot tips following thermotherapy was also significantly enhanced when SA (0.05-0.1 mM) was used for preconditioning stock shoots. Addition of either Fe-ethylenediaminetetracetic acid (Fe-EDTA, 50 mg per L) or Fe-ethylenediaminedi(o)hydroxyphenylacetic acid (Fe-EDDHA, 50 mg per L) to post-culture medium strongly promoted regrowth and totally prevented chlorosis of shoots regenerated from cryopreserved shoot tips following thermotherapy. Using the parameters optimized in the present study, about 80 percent survival of heat-treated stock shoots and about 33 percent regrowth of cryopreserved shoot tips following thermotherapy were obtained. Morphology of plants regenerated from cryopreserved shoot tips following thermotherapy was identical to that of control plants, based on observations of leaf shape and size, internode length and plant height. Optimization of the thermotherapy procedure followed by cryotherapy will facilitate the wider application of this technique to eliminate viruses which can invade meristems.

Cryopreservation of Tetraclinis articulata (vahl.) Masters.

Science.gov (United States)

Serrano-Martinez, Francisco; Casas, José Luis

2011-01-01

Tetraclinis articulata shoot tips excised from in vitro grown shoots were cryopreserved using a modified PVS2-based vitrification protocol. Preliminary experiments with non-cryostored shoot tips showed that the high concentrations of sucrose in loading (LS), vitrification (PVS2) and unloading (US) solutions employed in the protocol were very toxic for the explants. Replacement of sucrose by sorbitol in equal molar concentration in all these solutions enhanced survival of shoot tips after all treatments. However, cold-hardening of donor shoots before shoot tip excision was strictly required to obtain post-rewarming survival. Therefore, the protocol was outlined as follows: pre-conditioning of explants at 4 degree C for 3 weeks in the dark; excision of 1 mm long shoot tips; loading for 20 min in modified LS at room temperature; dehydration in modified PVS2 at 0 degree C for 60 min; immersion in liquid nitrogen (LN); rewarming at 40 degree C for 2 min and subsequent transfer of shoot tips in modified US for 20 min.

Effects of alternative cryoprotectants, diluents, straw size and cholesterol addition on cryopreserved rooster sperm

Science.gov (United States)

Rooster sperm do not cryopreserve well. This is due in part to osmotic changes that sperm undergo during addition and removal of cryoprotectant (CPA), as well as membrane damage during the membrane phase transition from fluid at 37 degrees C to gel at low temperatures. When a CPA is removed from spe...

Paradoxical aging in HIV: immune senescence of B Cells is most prominent in young age.

Science.gov (United States)

Rinaldi, Stefano; Pallikkuth, Suresh; George, Varghese K; de Armas, Lesley R; Pahwa, Rajendra; Sanchez, Celeste M; Pallin, Maria Fernanda; Pan, Li; Cotugno, Nicola; Dickinson, Gordon; Rodriguez, Allan; Fischl, Margaret; Alcaide, Maria; Gonzalez, Louis; Palma, Paolo; Pahwa, Savita

2017-04-01

Combination antiretroviral therapies (cART)can lead to normal life expectancy in HIV-infected persons, and people aged >50 yrs represent the fastest growing HIV group. Although HIV and aging are independently associated with impaired humoral immunity, immune status in people aging with HIV is relatively unexplored. In this study influenza vaccination was used to probe age associated perturbations in the B cell compartment of HIV-negative "healthy controls" (HC) and virologically controlled HIV-infected participants on cART (HIV) (n=124), grouped by age as young (aged (40-59yrs) or old ( > 60 yrs). H1N1 antibody response at d21 post-vaccination correlated inversely with age in both HC and HIV. Immunophenotyping of cryopreserved PBMC demonstrated increased frequencies of double negative B cells and decreased plasmablasts in old compared to young HC. Remarkably, young HIV were different from young HC but similar to old HC in B cell phenotype, influenza specific spontaneous (d7) or memory (d21) antibody secreting cells. We conclude that B cell immune senescence is a prominent phenomenon in young HIV in comparison to young HC, but distinctions between old HIV and old HC are less evident though both groups manifest age-associated B cell dysfunction.

Paradoxical aging in HIV: immune senescence of B Cells is most prominent in young age

Science.gov (United States)

George, Varghese K.; de Armas, Lesley R.; Pahwa, Rajendra; Sanchez, Celeste M.; Pallin, Maria Fernanda; Pan, Li; Cotugno, Nicola; Dickinson, Gordon; Rodriguez, Allan; Fischl, Margaret; Alcaide, Maria; Gonzalez, Louis; Palma, Paolo; Pahwa, Savita

2017-01-01

Combination antiretroviral therapies (cART) can lead to normal life expectancy in HIV-infected persons, and people aged >50 yrs represent the fastest growing HIV group. Although HIV and aging are independently associated with impaired humoral immunity, immune status in people aging with HIV is relatively unexplored. In this study influenza vaccination was used to probe age associated perturbations in the B cell compartment of HIV-negative “healthy controls” (HC) and virologically controlled HIV-infected participants on cART (HIV) (n=124), grouped by age as young (<40 yrs), middle-aged (40-59yrs) or old (≥60 yrs). H1N1 antibody response at d21 post-vaccination correlated inversely with age in both HC and HIV. Immunophenotyping of cryopreserved PBMC demonstrated increased frequencies of double negative B cells and decreased plasmablasts in old compared to young HC. Remarkably, young HIV were different from young HC but similar to old HC in B cell phenotype, influenza specific spontaneous (d7) or memory (d21) antibody secreting cells. We conclude that B cell immune senescence is a prominent phenomenon in young HIV in comparison to young HC, but distinctions between old HIV and old HC are less evident though both groups manifest age-associated B cell dysfunction. PMID:28448963

Fertility preservation for girls and young women with cancer: population-based validation of criteria for ovarian tissue cryopreservation.

Science.gov (United States)

Wallace, W Hamish B; Smith, Alice Grove; Kelsey, Thomas W; Edgar, Angela E; Anderson, Richard A

2014-09-01

Ovarian tissue cryopreservation with later reimplantation has been shown to preserve fertility in adult women, but this approach remains unproven and experimental in children and adolescents. We aimed to assess the use of the Edinburgh selection criteria for ovarian tissue cryopreservation in girls and young women with cancer to determine whether we are offering this invasive procedure to the patients who are most at risk of premature ovarian insufficiency. Cryopreservation of ovarian tissue has been selectively offered to girls and young women with cancer who met the Edinburgh selection criteria since 1996. Between Jan 1, 1996, and June 30, 2012, 410 female patients younger than 18 years at diagnosis were treated for cancer (including leukaemia and brain tumours) at the Edinburgh Children's Cancer Centre, which serves the whole South East of Scotland region. We determined the ovarian status of these patients from review of clinical records and classified them as having premature ovarian insufficiency or not, or as unable to be determined. Patients younger than 12 years at time of data cutoff (Jan 31, 2013) were excluded from the analysis. 34 (8%) of the 410 patients met the Edinburgh selection criteria and were offered ovarian tissue cryopreservation before starting cancer treatment. 13 patients declined the procedure and 21 consented, and the procedure was completed successfully in 20 patients. Of the 20 patients who had ovarian tissue successfully cryopreserved, 14 were available for assessment of ovarian function. Of the 13 patients who had declined the procedure, six were available for assessment of ovarian function. Median age at the time of follow-up for the 20 assessable patients was 16·9 years (IQR 15·5-21·8). Of the 14 assessable patients who had successfully undergone ovarian cryopreservation, six had developed premature ovarian insufficiency at a median age of 13·4 years (IQR 12·5-14·6), one of whom also had a natural pregnancy. Of the six

Proteomic identification of the related immune-enhancing proteins in shrimp Litopenaeus vannamei stimulated with vitamin C and Chinese herbs.

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Qiao, Jie; Du, Zhiheng; Zhang, Yueling; Du, Hong; Guo, Lingling; Zhong, Mingqi; Cao, Jingsong; Wang, Xiuying

2011-12-01

Recently, strong interest has been focused on immunostimulants to reducing the diseases in shrimp aquaculture. However, information regarding to the related immune-enhancing proteins in shrimps is not available yet. In this study, vitamin C (Vc), Chinese herbs (CH), and the mixture of vitamin C and Chinese herbs (Mix) were tested for their enhancement on shrimp's immune activity. Compared with those in the control group, values of phenoloxidase (PO), superoxide dismutase (SOD) and antibacterial (Ua) activity in the Mix-treated group were improved significantly 12 or 24 days after the treatment. The cumulative mortality was also lower in the Mix-treated group after infection with Vibrio parahemolyticus. Furthermore, comparative proteomic approach was used to assess the protein expression profile in shrimps. Approximately 220-290 and 300-400 protein spots were observed in the 2-DE gels. Among them, 29 and 28 altered proteins from hemocytes and hepatopancreas, respectively, were subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) analysis. The results revealed that the main altered proteins showed high homologies with Litopenaeus vannamei hemocyanin, hemolymph clottable protein, hemoglobin beta, cytosolic MnSOD, trypsin, cathepsin I(L) and zinc proteinase Mpc1. Together, these studies found Vc and CH were suitable immunostimulants to shrimp L. vannamei, and 7 altered proteins could be involved in the enhanced immune activities. Copyright © 2011 Elsevier Ltd. All rights reserved.

Characterization of proacrosin/acrosin system after liquid storage and cryopreservation of turkey semen (Meleagris gallopavo).

Science.gov (United States)

Słowińska, M; Liszewska, E; Dietrich, G J; Ciereszko, A

2012-09-15

This study was designed to identify the effect of liquid storage at 4 °C for 48 h and cryopreservation on the proacrosin/acrosin system of turkey spermatozoa. Anti-acrosin I antibodies were produced and used to demonstrate Western blot analysis profile of the proacrosin/acrosin system of sperm and seminal plasma and possible changes in the proacrosin/acrosin system of turkey sperm stored for 2.5, 24, and 48 h or cryopreserved. At the same time acrosin-like activity was examined by the measurement of amidase activity of sperm extracts, sperm suspension, and seminal plasma of turkey semen. A computer-assisted sperm analysis system was used to monitor the sperm motility characteristics of turkey sperm stored for 48 h or cryopreserved. Different profiles of the sperm proacrosin/acrosin system were observed regarding the presence or absence of inhibitors (p-nitrophenyl-p'-guanidine benzoate [NPGB] and Kazal family inhibitor) during the extraction process. When NPGB was present three main bands were observed with the molecular weight ranging from 66 to 35 kDa. Bands corresponding to acrosin I and II were not observed. In sperm extract without NPGB, three or four bands were observed with the molecular weight ranging from 41 to 30 kDa. The bands corresponding to acrosin I and II were observed. During liquid storage a decrease in sperm motility and an increase in sperm-extracted amidase activity were observed. After 24 and 48 h of storage, extracted amidase activity was higher than at 2.5 h by 24% and 31%, respectively. However, no changes in the Western blot analysis profiles of sperm extract and seminal plasma were visible during liquid storage. After cryopreservation a decrease in sperm motility and all sperm motility parameters were observed. In contrast to liquid storage, cryopreservation did not increase extracted amidase activity. However, changes in Western blot analysis profiles were visible in sperm extract and seminal plasma after cryopreservation. After

Replacement of serum with ocular fluid for cryopreservation of immature testes.

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Pothana, Lavanya; Devi, Lalitha; Venna, Naresh Kumar; Pentakota, Niharika; Varma, Vivek Phani; Jose, Jedy; Goel, Sandeep

2016-12-01

Cryopreservation of immature testis is a feasible approach for germplasm preservation of male animals. Combinations of dimethyl sulfoxide (DMSO) and foetal bovine serum (FBS) are used for testis cryopreservation. However, an alternative to FBS is needed, because FBS is expensive. Buffalo ocular fluid (BuOF), a slaughter house by-product, could be an economical option. The objective of the present study was to assess whether BuOF can replace FBS for cryopreservation of immature mouse (Mus musculus), rat (Rattus norvegicus), and buffalo (Bubalus bubalis) testes. Results showed that rodent and buffalo testes frozen in DMSO (10% for rodents and 20% for buffalo) with 20% FBS or BuOF had similar numbers of viable and DNA-damaged cells (P > 0.05). The expression of cell proliferation- (PCNA) and apoptosis-specific proteins (Annexin V and BAX/BCL2 ratio) were also comparable in mouse and buffalo testes frozen in DMSO with FBS or BuOF (P > 0.05). Interestingly, rat testis frozen in DMSO with BuOF had lower expression of Annexin V protein than testis frozen in DMSO with FBS (P  0.05). These findings provide evidence that BuOF has potential to replace FBS for cryopreservation of immature rodent and buffalo testis. Further investigation is needed to explore whether BuOF can replace FBS for testis cryopreservation of other species. Copyright © 2016 Elsevier Inc. All rights reserved.

C/EBPβ Promotes Immunity to Oral Candidiasis through Regulation of β-Defensins.

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Simpson-Abelson, Michelle R; Childs, Erin E; Ferreira, M Carolina; Bishu, Shrinivas; Conti, Heather R; Gaffen, Sarah L

2015-01-01

Humans or mice subjected to immunosuppression, such as corticosteroids or anti-cytokine biologic therapies, are susceptible to mucosal infections by the commensal fungus Candida albicans. Recently it has become evident that the Th17/IL-17 axis is essential for immunity to candidiasis, but the downstream events that control immunity to this fungus are poorly understood. The CCAAT/Enhancer Binding Protein-β (C/EBPβ) transcription factor is important for signaling by multiple inflammatory stimuli, including IL-17. C/EBPβ is regulated in a variety of ways by IL-17, and controls several downstream IL-17 target genes. However, the role of C/EBPβ in vivo is poorly understood, in part because C/EBPβ-deficient mice are challenging to breed and work with. In this study, we sought to understand the role of C/EBPβ in the context of an IL-17-dependent immune response, using C. albicans infection as a model system. Confirming prior findings, we found that C/EBPβ is required for immunity to systemic candidiasis. In contrast, C/EBPβ(-/-) mice were resistant to oropharyngeal candidiasis (OPC), in a manner indistinguishable from immunocompetent WT mice. However, C/EBPβ(-/-) mice experienced more severe OPC than WT mice in the context of cortisone-induced immunosuppression. Expression of the antimicrobial peptide β-defensin (BD)-3 correlated strongly with susceptibility in C/EBPβ(-/-) mice, but no other IL-17-dependent genes were associated with susceptibility. Therefore, C/EBPβ contributes to immunity to mucosal candidiasis during cortisone immunosuppression in a manner linked to β-defensin 3 expression, but is apparently dispensable for the IL-17-dependent response.

Efficacy and prospects for cryopreserved thyroid gland transplantation as a method for treatment of different forms of primary hypothyroidism including postradiation

International Nuclear Information System (INIS)

Karachentsev, Yu.I.; Lyakh, I.A.; Khasiev, V.V.

1999-01-01

It was established that auto-and allo transplantation of cryopreserved thyroid is a physiologically well grounded technique of treatment and can be an alternative to substitution hormonotherapy of hypothyroidism and can be a main method of treatment in cases when hormone treatment is not effective or is contraindicated.Talking into account the efficiency of cryopreserved thyroid transplantation in patients with primary hypothyroidism,it is possible to predict its positive effect for c postradiation hypothyroidism treatment,if substitution hormonotherapy is not effective

Midterm Results of Aortic Valve Replacement with Cryopreserved Homografts

Directory of Open Access Journals (Sweden)

Emre Özker

2012-06-01

Full Text Available Objective: The aim of this study was to analyze the midterm clinical results of aortic valve replacement with cryopreserved homografts.Materials and Methods: Aortic valve replacement was performed in 40 patients with cryopreserved homograft. The indications were aortic valve endocarditis in 20 patients (50%, truncus arteriosus in 6 patients (15%, and re-stenosis or regurtitation after aortic valve reconstruction in 14 (35% patients. The valve sizes ranged from 10 to 27mm. A full root replacement technique was used for homograft replacement in all patients.Results: The 30-day postoperative mortality rate was 12.5% (5 patients. There were four late deaths. Only one of them was related to cardiac events. Overall mortality was 22.5%. Thirty-three patients were followed up for 67±26 months. Two patients needed reoperation due to aortic aneurysm caused by endocarditis. The mean transvalvular gradient significantly decreased after valve replacement (p<0.003. The last follow up showed that the 27 (82% patients had a normal left ventricular function.Conclusion: Cryopreserved homografts are safe alternatives to mechanical valves that can be used when there are proper indications. Although it has a high perioperative mortality rate, cryopreserved homograft implantation is an alternative for valve replacement, particularly in younger patients and for complex surgical problems such as endocarditis that must be minimalized.

Comparison of two different extenders for cryopreservation of epididymal dog sperm.

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Martins, Mim; Justino, R C; Sant'anna, M C; Trautwein, L G C; Souza, F F

2012-12-01

The collection of epididymal sperm is an option for preservation of germplasm of genetically superior animals that need to be orchiectomized or have died. The extender type used to freeze sperm is important to avoid spermatozoal membrane damage and to preserve semen quality after cryopreservation. The objective of this study was to verify the effects of a commercial bovine extender (Bovimix(®); Nutricell, Campinas) and a traditional TRIS-citric acid-glucose-egg yolk-7% glycerol extender on cryopreservation of canine epididymal sperm. The testes of 13 adult dogs were kept at 5 °C for 24 h in saline solution, and epididymal sperm was recovered in Ringers solution without lactate and were evaluated for motility. Samples with ≥ 80% motility were pooled and then divided before dilution and packaging in 0.5 ml plastic straws, equilibration at 4 °C for 1 h, freezing in nitrogen vapour for 20 min and storing at -196 °C. The straws were thawed at 56 °C for 10 s and were evaluated for motility by computer assisted analysis (CASA). The semen parameters, sperm movement index, linearity, total motility and rapid progressive motility were statistically higher in Bovimix(®) than TRIS. In contrast, amplitude of lateral head displacement, slow sperm and static sperm were lower in Bovimix(®). Despite the high percentage of sperm defects in epididymal cells, regardless of the extender, we concluded that Bovimix(®) is a viable alternative for the freezing of canine epididymal sperm. © 2012 Blackwell Verlag GmbH.

Oxygenated thawing and rewarming alleviate rewarming injury of cryopreserved pancreatic islets.

Science.gov (United States)

Komatsu, Hirotake; Barriga, Alyssa; Medrano, Leonard; Omori, Keiko; Kandeel, Fouad; Mullen, Yoko

2017-05-06

Pancreatic islet transplantation is an effective treatment for Type 1 diabetic patients to eliminate insulin injections; however, a shortage of donor organs hinders the widespread use. Although long-term islet storage, such as cryopreservation, is considered one of the key solutions, transplantation of cryopreserved islets is still not practical due to the extensive loss during the cryopreservation-rewarming process. We have previously reported that culturing islets in a hyperoxic environment is an effective treatment to prevent islet death from the hypoxic injury during culture. In this study, we explored the effectiveness of thawing and rewarming cryopreserved islets in a hyperoxic environment. Following cryopreservation of isolated human islets, the thawing solution and culture media were prepared with or without pre-equilibration to 50% oxygen. Thawing/rewarming and the pursuant two-day culture were performed with or without oxygenation. Short-term recovery rate, defined as the volume change during cryopreservation and thawing/rewarming, was assessed. Ischemia-associated and inflammation-associated gene expressions were examined using qPCR after the initial rewarming period. Long-term recovery rate, defined as the volume change during the two-day culture after the thawing/rewarming, was also examined. Islet metabolism and function were assessed by basal oxygen consumption rate and glucose stimulated insulin secretion after long-term recovery. Oxygenated thawing/rewarming did not alter the short-term recovery rate. Inflammation-associated gene expressions were elevated by the conventional thawing/rewarming method and suppressed by the oxygenated thawing/rewarming, whereas ischemia-associated gene expressions did not change between the thawing/rewarming methods. Long-term recovery rate experiments revealed that only the combination therapy of oxygenated thawing/rewarming and oxygenated culture alleviated islet volume loss. These islets showed higher metabolism

Sperm Cryopreservation before Testicular Cancer Treatment and Its Subsequent Utilization for the Treatment of Infertility

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Jana Žáková

2014-01-01

Full Text Available Aims. In this study we report our results with storage of cryopreserved semen intended for preservation and subsequent infertility treatment in men with testicular cancer during the last 18 years. Methods. Cryopreserved semen of 523 men with testicular cancer was collected between October 1995 and the end of December 2012. Semen of 34 men (6.5% was used for fertilization of their partners. They underwent 57 treatment cycles with cryopreserved, fresh, and/or donor sperm. Results. A total of 557 men have decided to freeze their semen before cancer treatment. Azoospermia was diagnosed in 34 men (6.1%, and semen was cryopreserved in 532 patients. Seminoma was diagnosed in 283 men (54.1% and nonseminomatous germ cell tumors in 240 men (45.9%. 34 patients who returned for infertility treatment underwent 46 treatment cycles with cryopreserved sperm. Totally 16 pregnancies were achieved, that is, 34.8% pregnancy rate. Conclusion. The testicular cancer survivors have a good chance of fathering a child by using sperm cryopreserved prior to the oncology treatment, even when it contains only limited number of spermatozoa.

Cryopreservation of tissue engineered constructs for bone.

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Kofron, Michelle D; Opsitnick, Natalie C; Attawia, Mohamed A; Laurencin, Cato T

2003-11-01

The large-scale clinical use of tissue engineered constructs will require provisions for its mass availability and accessibility. Therefore, it is imperative to understand the effects of low temperature (-196 degrees C) on the tissue engineered biological system. Initial studies used samples of the osteoblast-like cell line (SaOS-2) adhered to a two-dimensional poly(lactide-co-glycolide) thin film (2D-PLAGA) or a three-dimensional poly(lactide-co-glycolide) sintered microsphere matrix (3D-PLAGA) designed for bone tissue engineering. Experimental samples were tested for their ability to maintain cell viability, following low temperature banking for one week, in solutions of the penetrating cryoprotective agents, dimethylsulfoxide (DMSO), ethylene glycol, and glycerol. Results indicated the DMSO solution yielded the greatest percent cell survival for SaOS-2 cells adhered to both the 2D- and 3D-PLAGA scaffolds; therefore, DMSO was used to cryopreserve mineralizing primary rabbit osteoblasts cells adhered to 2D-PLAGA matrices for 35 days. Results indicated retention of the extracellular matrix architecture as no statistically significant difference in the pre- and post-thaw mineralized structures was measured. Percent cell viability of the mineralized constructs following low temperature storage was approximately 50%. These are the first studies to address the issue of preservation techniques for tissue engineered constructs. The ability to successfully cryopreserve mineralized tissue engineered matrices for bone may offer an unlimited and readily available source of bone-like materials for orthopaedic applications.

Cancer patients treated with sunitinib or sorafenib have sufficient antibody and cellular immune responses to warrant influenza vaccination.

Science.gov (United States)

Mulder, Sasja F; Jacobs, Joannes F M; Olde Nordkamp, Michel A M; Galama, Joep M D; Desar, Ingrid M E; Torensma, Ruurd; Teerenstra, Steven; Mulders, Peter F A; Vissers, Kris C P; Punt, Cornelis J A; de Vries, I Jolanda M; van Herpen, Carla M L

2011-07-01

The tyrosine kinase inhibitors sorafenib and sunitinib have efficacy in several types of cancer. Recent studies indicate that these agents affect the immune system. The way it affects the immune response to influenza vaccination is unknown. The aim of this study was to elucidate the specific immune response to seasonal flu vaccination in cancer patients treated with sunitinib or sorafenib. Sunitinib- or sorafenib-treated cancer patients were vaccinated against seasonal influenza with an inactivated vaccine. Healthy controls and patients with metastatic renal cell cancer (mRCC) without systemic treatment (nontreated mRCC controls) were included for comparison. Antibody responses were measured at baseline, day 8, and day 22 by a standard hemagglutination inhibition assay and cellular T-cell responses at baseline and day 8 by proliferation assay and secretion of cytokines. Forty subjects were enrolled: 16 patients treated with sunitinib, 6 patients with sorafenib, 7 nontreated mRCC controls, and 11 healthy controls. All patients treated with sunitinib and sorafenib developed seroprotection rates comparable with controls. Functional T-cell reactivity was observed in all groups, except for patients treated with sorafenib who showed a decreased proliferation rate and IFN-γ/IL-2 production and increased IL-10 compared with healthy controls. We conclude that influenza vaccination should be recommended to cancer patients treated with sunitinib or sorafenib.

Grafting with Cryopreserved Amniotic Membrane versus Conservative Wound Care in Treatment of Pressure Ulcers: A Randomized Clinical Trial.

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Dehghani, Mehdi; Azarpira, Negar; Mohammad Karimi, Vahid; Mossayebi, Hamid; Esfandiari, Elaheh

2017-10-01

To compare the healing process of pressure ulcers treated with cryopreserved human amniotic membrane allograft and routine pressure ulcer care in our hospital. From January 2012 to December 2013, in a prospective randomized clinical trial (IRCT201612041335N2), 24 patients with second and third stage of pressure ulcers were enrolled in this study. All patients needed split-thickness skin grafts for pressure ulcer-wound coverage. Selected patients had symmetric ulcers on both upper and lower extremities. The patients were randomly divided into two groups: amnion and control. In the amnion group, the ulcer was covered with cryopreserved amniotic membrane and in the control group it was treated with local Dilantin powder application. The duration and success rate of complete healing was compared between the two groups. The study group was composed of 24 pressure ulcers in 24 patients (19 males and 5 females) with a mean age of 44±12.70 years. The demographic characteristics, ulcer area, and underlying diseases were similar in both groups. The early sign of response, such as decrease in wound discharge, was detected 12-14 days after biological dressing. Complete pressure ulcer healing occurred only in the amnion group ( p pressure ulcers.

Establishment of colloid gold immunity chromatography assay for cardiac troponin I (cTnI)

International Nuclear Information System (INIS)

Wang Dezhi; Chen Jiying; Qin Lili; Zhao Baojian; Zhang Chunming

2006-01-01

Objective: To establish the colloid gold Immunity chromatography assay for cardiac troponin I. Methods: To purify cTnI from human cardiac muscle and immunize rabbit with it. cTnI antibody of rabbit anti-human cardiac muscle has been prepared and colloid gold immunity chromatography assay was established by using immunity chromatography technology. Results: Anti-serum titles of cTnI were 1:100000, Ka=2.38 x 10 9 L/mol; Methodological index: Sensitivity: 5 ng/ml; Specificity: cTnI is no cross-reaction with cTnT, cTnC and CK-MB. conclusion: The assay is highly specific, quick and simple. It can be widely used for the early diagnosis of AMI and scientific research. (authors)

Challenges in cryopreservation of regulatory T cells (Tregs) for clinical therapeutic applications.

Science.gov (United States)

Golab, Karolina; Leveson-Gower, Dennis; Wang, Xiao-Jun; Grzanka, Jakub; Marek-Trzonkowska, Natalia; Krzystyniak, Adam; Millis, J Michael; Trzonkowski, Piotr; Witkowski, Piotr

2013-07-01

Promising results of initial studies applying ex-vivo expanded regulatory T cell (Treg) as a clinical intervention have increased interest in this type of the cellular therapy and several new clinical trials involving Tregs are currently on the way. Methods of isolation and expansion of Tregs have been studied and optimized to the extent that such therapy is feasible, and allows obtaining sufficient numbers of Tregs in the laboratory following Good Manufacturing Practice (GMP) guidelines. Nevertheless, Treg therapy could even more rapidly evolve if Tregs could be efficiently cryopreserved and stored for future infusion or expansions rather than utilization of only freshly isolated and expanded cells as it is preferred now. Currently, our knowledge regarding the impact of cryopreservation on Treg recovery, viability, and functionality is still limited. Based on experience with cryopreserved peripheral blood mononuclear cells (PBMCs), cryopreservation may have a detrimental effect on Tregs, can decrease Treg viability, cause abnormal cytokine secretion, and compromise expression of surface markers essential for proper Treg function and processing. Therefore, optimal strategies and conditions for Treg cryopreservation in conjunction with cell culture, expansion, and processing for clinical application still need to be investigated and defined. Copyright © 2013 Elsevier B.V. All rights reserved.

Ovarian tissue cryopreservation by stepped vitrification and monitored by X-ray computed tomography.

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Corral, Ariadna; Clavero, Macarena; Gallardo, Miguel; Balcerzyk, Marcin; Amorim, Christiani A; Parrado-Gallego, Ángel; Dolmans, Marie-Madeleine; Paulini, Fernanda; Morris, John; Risco, Ramón

2018-04-01

Ovarian tissue cryopreservation is, in most cases, the only fertility preservation option available for female patients soon to undergo gonadotoxic treatment. To date, cryopreservation of ovarian tissue has been carried out by both traditional slow freezing method and vitrification, but even with the best techniques, there is still a considerable loss of follicle viability. In this report, we investigated a stepped cryopreservation procedure which combines features of slow cooling and vitrification (hereafter called stepped vitrification). Bovine ovarian tissue was used as a tissue model. Stepwise increments of the Me 2 SO concentration coupled with stepwise drops-in temperature in a device specifically designed for this purpose and X-ray computed tomography were combined to investigate loading times at each step, by monitoring the attenuation of the radiation proportional to Me 2 SO permeation. Viability analysis was performed in warmed tissues by immunohistochemistry. Although further viability tests should be conducted after transplantation, preliminary results are very promising. Four protocols were explored. Two of them showed a poor permeation of the vitrification solution (P1 and P2). The other two (P3 and P4), with higher permeation, were studied in deeper detail. Out of these two protocols, P4, with a longer permeation time at -40 °C, showed the same histological integrity after warming as fresh controls. Copyright © 2018 Elsevier Inc. All rights reserved.

Ejaculate traits and sperm cryopreservation in the endangered Baird's tapir (Tapirus bairdii).

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Pukazhenthi, Budhan S; Togna, Gina Della; Padilla, Luis; Smith, Diorene; Sanchez, Carlos; Pelican, Katey; Sanjur, Oris I

2011-01-01

There is little information on the reproductive biology of the male Baird's tapir (Tapirus bairdii). In this study, we characterized the ejaculate traits and evaluated the efficacy of 2 cryodiluents on sperm cryosurvival. Ejaculates were assessed for volume, pH, sperm motility, forward progression, osmolality, sperm concentration, sperm morphology, and acrosomal integrity. For cryopreservation, ejaculates with >50% total sperm motility were washed, and sperm pellets were resuspended in either Botu-Crio (CryoVital, Grandau, Germany) or INRA 96 containing 2% egg yolk and 2.5% each of methyl- and dimethylformamide (INRA 96), and they were cryopreserved over liquid nitrogen vapor. Thawed samples were incubated in vitro (25 °C) and evaluated for percent total sperm motility, forward progression, and acrosomal integrity at hourly intervals for 4 hours. Spermic ejaculates were obtained from all males, and the mean seminal volume, sperm concentration per milliliter, percent sperm motility, progressive status, and percent morphologically normal cells were 20.4 ± 4.3 mL, 101.2 ± 24.0 × 10(6)/mL, 46.1% ± 5.0%, 2.9 ± 0.1, and 6.9% ± 1.4%, respectively. There was a positive significant correlation between percent normal sperm and animal age (r = 0.66; P tapir; demonstrate that tapir spermatozoa can be cryopreserved in diluents containing amides alone or in combination with glycerol; and provide fundamental information critical for development of assisted reproductive technologies for the Baird's tapir.

Dynamics of adaptive and innate immunity in patients treated during primary human immunodeficiency virus infection: results from Maraviroc in HIV Acute Infection (MAIN) randomized clinical trial.

Science.gov (United States)

Ripa, M; Pogliaghi, M; Chiappetta, S; Galli, L; Pensieroso, S; Cavarelli, M; Scarlatti, G; De Biasi, S; Cossarizza, A; De Battista, D; Malnati, M; Lazzarin, A; Nozza, S; Tambussi, G

2015-09-01

We evaluated the dynamics of innate and adaptive immunity in patients treated with combined antiretroviral therapy (cART) during primary human immunodeficiency virus infection (PHI), enrolled in a prospective randomized trial (MAIN, EUDRACT 2008-007004-29). After 48 weeks of cART, we documented a reduction in activated B cells and CD8(+) T cells. Natural killer cell and dendritic cell frequencies were measured and a decrease in CD16(+) CD56(dim) with a reciprocal rise in CD56(high) natural killer cells and an increase in myeloid and plasmacytoid dendritic cells were recorded. In conclusion, 48 weeks of cART during PHI showed significant benefits for both innate and adaptive immunity. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

In vitro propagation and cryopreservation of Aerides odorata Lour. (Orchidaceae).

Science.gov (United States)

Hongthongkham, J; Bunnag, S

2014-05-01

An efficient method for in vitro propagation and cryopreservation of Aerides odorata was established. Leaf segments were cultured on New Dogashima (ND) mediums supplemented with various concentrations of Benzyladenine (BA) (0-5 mg L(-1)) combined with Naphthaleneacetic Acid (NAA) (0-2 mg L(-1)). The optimal treatment for inducing Protocorm-like Bodies (PLBs) from leaf segments was obtained from the combination of 1 or 3 mg L(-1) BA and 0.5 or 1 mg L(-1) NAA; whereas, the addition of BA or NAA alone induced shoot and/or root initiation rather than PLB or callus formation. Shoots rapidly developed on ND mediums containing 5 mg L(-1) BA. Cryopreservation of leaf segment-derived PLBs was successful using the encapsulation-dehydration method. The maximum survival percentage of Cryopreserved (Cryp) PLBs was achieved by encapsulating PLBs with 2% Na-alginate combined with 2 M glycerol and 0.4 M sucrose. The encapsulated PLBs were then precultured in 0.75 M sucrose for 24 h and dehydrated for 6 h before plunging into liquid nitrogen. Genetic stability of Cryp PLBs after regrowth was assessed by flow cytometry. The findings showed no different patterns of ploidy levels and morphology between Cryp and non-cryopreserved (Ncryp) control plantlets.

Eggs on Ice. Imaginaries on Eggs and Cryopreservation in Denmark

DEFF Research Database (Denmark)

Herrmann, Janne Rothmar; Kroløkke, Charlotte

2018-01-01

While Denmark is widely known as a global exporter of cryopreserved sperm, Danish women’s eggs follow very different trajectories. This paper combines legal and rhetorical analyses with the concept of sociotechnical imaginaries (Jasanoff, 2015). In establishing the genealogy of the sociotechnical...... imaginaries that shaped the Danish regulation on the cryopreservation of eggs, we analyze the relevant Acts, Bills, preparatory work and readings in Parliament along with the concurrent public and ethical debates that in time relaxed the legal limit for the cryopreservation of eggs to the current 5 years...... and today continue to ignite discussions on elective egg freezing. We rely on welfare state perspectives to discuss why reproduction, in the Danish context, is seen as a legitimate and appropriate sphere to regulate and we turn to feminist theorizing to discuss their gendered implications captured...

Recent Advances in Boar Sperm Cryopreservation: State of the Art and Current Perspectives.

Science.gov (United States)

Yeste, M

2015-07-01

While sperm cryopreservation is the best technology to store boar semen for long-term periods, only 1% of all artificial inseminations (AI) conducted worldwide are made using frozen-thawed boar sperm. With the emergence of long-term extenders for liquid storage, the use of cryopreserved sperm in routine AI is less required. However, banks of boar semen contain cryopreserved sperm and planning inseminations in AI centres may benefit from the use of frozen-thawed semen. Therefore, there is an interest in the use of this technology to preserve boar sperm. In this regard, although the first attempts to cryopreserve boar semen date back to the seventies and this technology is still considered as optimal, some relevant improvements have been made in the last decade. After giving a general picture about boar sperm cryodamage, the present review seeks to shed light on these recent cryopreservation advances. These contributions regard to protein markers for predicting ejaculate freezability, sperm selection prior to start cryopreservation procedures, additives to freezing and thawing extenders, relevance of the AI-technique and insemination-to-ovulation interval. In conclusion, most of these progresses have allowed counteracting better boar sperm cryodamage and are thus considered as forward steps for this storage method. It is also worth noting that, despite being lower than fresh/extended semen, reproductive performance outcomes following AI with frozen-thawed boar sperm are currently acceptable. © 2015 Blackwell Verlag GmbH.

Milt cryopreservation for rheophilic fish threatened by extinction in the Rio Grande, Brazil.

Science.gov (United States)

de Andrade, Estefânia Souza; Paula, Daniella Aparecida de Jesus; Felizardo, Viviane de Oliveira; Murgas, Luis David Solis; Veras, Galileu Crovatto; Vieira e Rosa, Priscila

2014-01-01

Specific protocols for milt cryopreservation have been established for some freshwater fish species. However, cryopreservation reduces sperm quality, giving unsatisfactory results in reproduction. The objective of this work was to evaluate the effect of different cryoprotectants on the quality of Prochilodus lineatus, Brycon orbignyanus and Piaractus mesopotamicus milt after cryopreservation. The milt was diluted in different cryoprotectant solutions containing 10% methanol, dimethyl sulfoxide, glycerol, propylene glycol or ethylene glycol combined with the Beltsville Thawing Solution extender (5%), then placed in the vapour of a liquid nitrogen (LN) storage tank for 24 h, after which they were immersed in LN. After rewarming, the rate (%) and duration (s) of milt motility and abnormal morphology were evaluated. All of cryoprotectant solutions tested used maintained the viability of P. lineatus and P. mesopotamicus milt. However, in P. lineatus, glycerol ensured a lower percentage of abnormal morphology. In case of P. mesopotamicus, all of the cryoprotectant solutions tested may be used in the cryopreservation process, with the exception of those containing glycerol. For B. orbignyanus, cryoprotectant solutions containing methanol and ethylene glycol are recommended for use in the cryopreservation process, although they reduced the quality of sperm post-rewarming.

Efficacy Testing of H56 cDNA Tattoo Immunization against Tuberculosis in a Mouse Model.

Science.gov (United States)

Platteel, Anouk C M; Nieuwenhuizen, Natalie E; Domaszewska, Teresa; Schürer, Stefanie; Zedler, Ulrike; Brinkmann, Volker; Sijts, Alice J A M; Kaufmann, Stefan H E

2017-01-01

Tuberculosis (TB), caused by Mycobacterium tuberculosis ( Mtb ), remains a global threat. The only approved vaccine against TB, Mycobacterium bovis bacillus Calmette-Guérin (BCG), provides insufficient protection and, being a live vaccine, can cause disseminated disease in immunocompromised individuals. Previously, we found that intradermal cDNA tattoo immunization with cDNA of tetanus toxoid fragment C domain 1 fused to cDNA of the fusion protein H56, comprising the Mtb antigens Ag85B, ESAT-6, and Rv2660c, induced antigen-specific CD8 + T cell responses in vivo . As cDNA tattoo immunization would be safer than a live vaccine in immunocompromised patients, we tested the protective efficacy of intradermal tattoo immunization against TB with H56 cDNA, as well as with H56_E, a construct optimized for epitope processing in a mouse model. As Mtb antigens can be used in combination with BCG to boost immune responses, we also tested the protective efficacy of heterologous prime-boost, using dermal tattoo immunization with H56_E cDNA to boost BCG immunization in mice. Dermal H56 and H56_E cDNA immunization induced H56-specific CD4 + and CD8 + T cell responses and Ag85B-specific IgG antibodies, but did not reduce bacterial loads, although immunization with H56_E ameliorated lung pathology. Both subcutaneous and intradermal immunization with BCG resulted in broad cellular immune responses, with increased frequencies of CD4 + T effector memory cells, T follicular helper cells, and germinal center B cells, and resulted in reduced bacterial loads and lung pathology. Heterologous vaccination with BCG/H56_E cDNA induced increased H56-specific CD4 + and CD8 + T cell cytokine responses compared to vaccination with BCG alone, and lung pathology was significantly decreased in BCG/H56_E cDNA immunized mice compared to unvaccinated controls. However, bacterial loads were not decreased after heterologous vaccination compared to BCG alone. CD4 + T cells responding to Ag85B- and ESAT-6

Comparison of cryopreserved human sperm in vapor and liquid phases of liquid nitrogen: effect on motility parameters, morphology, and sperm function.

Science.gov (United States)

Punyatanasakchai, Piyaphan; Sophonsritsuk, Areephan; Weerakiet, Sawaek; Wansumrit, Surapee; Chompurat, Deonthip

2008-11-01

To compare the effects of cryopreserved sperm in vapor and liquid phases of liquid nitrogen on sperm motility, morphology, and sperm function. Experimental study. Andrology laboratory at Ramathibodi Hospital, Thailand. Thirty-eight semen samples with normal motility and sperm count were collected from 38 men who were either patients of an infertility clinic or had donated sperm for research. Each semen sample was divided into two aliquots. Samples were frozen with static-phase vapor cooling. One aliquot was plunged into liquid nitrogen (-196 degrees C), and the other was stored in vapor-phase nitrogen (-179 degrees C) for 3 days. Thawing was performed at room temperature. Motility was determined by using computer-assisted semen analysis, sperm morphology was determined by using eosin-methylene blue staining, and sperm function was determined by using a hemizona binding test. Most of the motility parameters of sperm stored in the vapor phase were not significantly different from those stored in the liquid phase of liquid nitrogen, except in amplitude of lateral head displacement. The percentages of normal sperm morphology in both vapor and liquid phases also were not significantly different. There was no significant difference in the number of bound sperm in hemizona between sperm cryopreserved in both vapor and liquid phases of liquid nitrogen. Cryopreservation of human sperm in a vapor phase of liquid nitrogen was comparable to cryopreservation in a liquid phase of liquid nitrogen.

Circulating angiogenic cells can be derived from cryopreserved peripheral blood mononuclear cells.

Directory of Open Access Journals (Sweden)

Tanja Sofrenovic

Full Text Available Cell transplantation for regenerative medicine has become an appealing therapeutic method; however, stem and progenitor cells are not always freshly available. Cryopreservation offers a way to freeze cells as they are generated, for storage and transport until required for therapy. This study was performed to assess the feasibility of cryopreserving peripheral blood mononuclear cells (PBMCs for the subsequent in vitro generation of their derived therapeutic population, circulating angiogenic cells (CACs.PBMCs were isolated from healthy human donors. Freshly isolated cells were either analyzed immediately or cryopreserved in media containing 6% plasma serum and 5% dimethyl sulfoxide. PBMCs were thawed after being frozen for 1 (early thaw or 28 (late thaw days and analyzed, or cultured for 4 days to generate CACs. Analysis of the cells consisted of flow cytometry for viability and phenotype, as well as functional assays for their adhesion and migration potential, cytokine secretion, and in vivo angiogenic potential.The viability of PBMCs and CACs as well as their adhesion and migration properties did not differ greatly after cryopreservation. Phenotypic changes did occur in PBMCs and to a lesser extent in CACs after freezing; however the potent CD34(+VEGFR2(+CD133(+ population remained unaffected. The derived CACs, while exhibiting changes in inflammatory cytokine secretion, showed no changes in the secretion of important regenerative and chemotactic cytokines, nor in their ability to restore perfusion in ischemic muscle.Overall, it appears that changes do occur in cryopreserved PBMCs and their generated CACs; however, the CD34(+VEGFR2(+CD133(+ progenitor population, the secretion of pro-vasculogenic factors, and the in vivo angiogenic potential of CACs remain unaffected by cryopreservation.

Effect of cryopreservation and in vitro culture of bovine fibroblasts on histone acetylation levels and in vitro development of hand-made cloned embryos

Science.gov (United States)

Chacon, L.; Gomez, M.C.; Jenkins, J.A.; Leibo, S.P.; Wirtu, G.; Dresser, B.L.; Pope, C.E.

2011-01-01

In this study, the relative acetylation levels of histone 3 in lysine 9 (H3K9ac) in cultured and cryopreserved bovine fibroblasts was measured and we determined the influence of the epigenetic status of three cultured (C1, C2 and C3) donor cell lines on the in vitro development of reconstructed bovine embryos. Results showed that cryopreservation did not alter the overall acetylation levels of H3K9 in bovine fibroblasts analysed immediately after thawing (frozen/thawed) compared with fibroblasts cultured for a period of time after thawing. However, reduced cleavage rates were noted in embryos reconstructed with fibroblasts used immediately after thawing. Cell passage affects the levels of H3K9ac in bovine fibroblasts, decreasing after P1 and donor cells with lower H3K9ac produced a greater frequency of embryo development to the blastocyst stage. Cryopreservation did not influence the total cell and ICM numbers, or the ICM/TPD ratios of reconstructed embryos. However, the genetic source of donor cells did influence the total number of cells and the trophectoderm cell numbers, and the cell passage influenced the total ICM cell numbers. ?? Copyright Cambridge University Press 2010.

Humoral immune response of C57Bl/6j and BALB/c mice immunized with irradiated tachyzoites of Toxoplasma gondii RH strain and oral challenge with ME-49 strain

Energy Technology Data Exchange (ETDEWEB)

Jimenez Galisteo Junior, Andres [Instituto de Pesquisas Energeticas e Nucleares IPEN/CNEN-SP, Sao Paulo, SP (Brazil). Centro de Biotecnologia; Instituto de Medicina Tropical de Sao Paulo, Sao Paulo, SP (Brazil). Lab. de Protozoologia; E-mail: galisteo@usp.br; Zorgi, Nahiara Esteves; Andrade Junior, Heitor Franco de [Instituto de Medicina Tropical de Sao Paulo, Sao Paulo, SP (Brazil). Lab. de Protozoologia; Alves, Janaina Baptista; Nascimento, Nanci do [Instituto de Pesquisas Energeticas e Nucleares IPEN/CNEN-SP, Sao Paulo, SP (Brazil). Centro de Biotecnologia; Hiramoto, Roberto Mitsuyoshi [instituto Adolfo Lutz, Sao Paulo, SP (Brazil)

2007-07-01

Toxoplasmosis, a prevalent widespread infection in man and animals, is mainly transmitted by oral route, through ingestion of oocysts from water and food contaminated with cat feces or infected animal tissue cysts in undercooked meat. Vaccine development implies in effective intestinal immunity, the first site of parasite entry. Radiation (255 Gy/{sup 60}Co) sterilized T. gondii RH strain tachyzoites (RST) induced significant protection when parentally administered, similar to chronically infected and acute disease protected animal. We study the humoral immune response in C57Bl/6j and BALB/c mice immunized with 10{sup 7} RST, by oral (with aluminium hydroxide 3%) or parenteral 3 biweekly administrations. T. gondii antigens specific ELISA for IgG, IgA, IgG1, IgG2a and IgG2b detection was performed in weekly blood samples during immunization. Also we evaluate of the intestinal epithelial of immunized mice the integrity of the radiated parasites by electronic microscopy. After 2 weeks, immunized and control animals were challenged with 10 cysts of ME-49 strain p.o. Protection was determined at the 30th day by brain cyst counting. As it was possible to observe in the intestinal mucosal, the aluminium hydroxide seems to maintain unchanged the parasite morphology and its mechanisms of invasion, probably due to keeping it safe from extreme pH condition of stomach. All immunized groups presented significant protection when challenged with ME-49; however, BALB/c mice showed better protection levels, with only one positive animal on brain microscopic analysis. IgG production in the serum of the animals was higher in groups immunized by i.p route, however, IgA and IgG1 levels were higher in BALB/c mice immunized by oral route. This higher protection found in BALB/c group could probably also be related to the Th2 response, demonstrated by higher IgG1 levels. All these data provide insights in oral immunization schedules for toxoplasmosis prevention, suggesting that oral

Humoral immune response of C57Bl/6j and BALB/c mice immunized with irradiated tachyzoites of Toxoplasma gondii RH strain and oral challenge with ME-49 strain

International Nuclear Information System (INIS)

Jimenez Galisteo Junior, Andres

2007-01-01

Toxoplasmosis, a prevalent widespread infection in man and animals, is mainly transmitted by oral route, through ingestion of oocysts from water and food contaminated with cat feces or infected animal tissue cysts in undercooked meat. Vaccine development implies in effective intestinal immunity, the first site of parasite entry. Radiation (255 Gy/ 60 Co) sterilized T. gondii RH strain tachyzoites (RST) induced significant protection when parentally administered, similar to chronically infected and acute disease protected animal. We study the humoral immune response in C57Bl/6j and BALB/c mice immunized with 10 7 RST, by oral (with aluminium hydroxide 3%) or parenteral 3 biweekly administrations. T. gondii antigens specific ELISA for IgG, IgA, IgG1, IgG2a and IgG2b detection was performed in weekly blood samples during immunization. Also we evaluate of the intestinal epithelial of immunized mice the integrity of the radiated parasites by electronic microscopy. After 2 weeks, immunized and control animals were challenged with 10 cysts of ME-49 strain p.o. Protection was determined at the 30th day by brain cyst counting. As it was possible to observe in the intestinal mucosal, the aluminium hydroxide seems to maintain unchanged the parasite morphology and its mechanisms of invasion, probably due to keeping it safe from extreme pH condition of stomach. All immunized groups presented significant protection when challenged with ME-49; however, BALB/c mice showed better protection levels, with only one positive animal on brain microscopic analysis. IgG production in the serum of the animals was higher in groups immunized by i.p route, however, IgA and IgG1 levels were higher in BALB/c mice immunized by oral route. This higher protection found in BALB/c group could probably also be related to the Th2 response, demonstrated by higher IgG1 levels. All these data provide insights in oral immunization schedules for toxoplasmosis prevention, suggesting that oral vaccines could

Cryopreservation of human embryos and its contribution to in vitro fertilization success rates

NARCIS (Netherlands)

Wong, Kai Mee; Mastenbroek, Sebastiaan; Repping, Sjoerd

2014-01-01

Cryopreservation of human embryos is now a routine procedure in assisted reproductive technologies laboratories. There is no consensus on the superiority of any protocol, and substantial differences exist among centers in day of embryo cryopreservation, freezing method, selection criteria for which

Parental desire and acceptability of spermatogonial stem cell cryopreservation in boys with cancer

NARCIS (Netherlands)

van den Berg, H.; Repping, S.; van der Veen, F.

2007-01-01

BACKGROUND: In the near future, a substantial proportion of adults will be childhood cancer survivors. The cryopreservation and transplantation of spermatogonial stem cells (SSCs) is currently successful in animals; application in humans seems likely in the near future. Cryopreserving SSCs might

Preliminary investigation of cryopreservation by encapsulation ...

African Journals Online (AJOL)

Protocorm-like bodies (PLBs) of Brassidium Shooting Star, a new commercial ornamental orchid hybrid, were cryopreserved by an encapsulation-dehydration technique. The effects of PLB size, various sucrose concentrations in preculture media and sodium alginate concentration for encapsulation were the main ...

A quality assurance initiative for commercial-scale production in high-throughput cryopreservation of blue catfish sperm.

Science.gov (United States)

Hu, E; Liao, T W; Tiersch, T R

2013-10-01

Cryopreservation of fish sperm has been studied for decades at a laboratory (research) scale. However, high-throughput cryopreservation of fish sperm has recently been developed to enable industrial-scale production. This study treated blue catfish (Ictalurus furcatus) sperm high-throughput cryopreservation as a manufacturing production line and initiated quality assurance plan development. The main objectives were to identify: (1) the main production quality characteristics; (2) the process features for quality assurance; (3) the internal quality characteristics and their specification designs; (4) the quality control and process capability evaluation methods, and (5) the directions for further improvements and applications. The essential product quality characteristics were identified as fertility-related characteristics. Specification design which established the tolerance levels according to demand and process constraints was performed based on these quality characteristics. Meanwhile, to ensure integrity throughout the process, internal quality characteristics (characteristics at each quality control point within process) that could affect fertility-related quality characteristics were defined with specifications. Due to the process feature of 100% inspection (quality inspection of every fish), a specific calculation method, use of cumulative sum (CUSUM) control charts, was applied to monitor each quality characteristic. An index of overall process evaluation, process capacity, was analyzed based on in-control process and the designed specifications, which further integrates the quality assurance plan. With the established quality assurance plan, the process could operate stably and quality of products would be reliable. Copyright © 2013 Elsevier Inc. All rights reserved.

Optimization of conditions for long-term prefreezing storage of brown bear sperm before cryopreservation.

Science.gov (United States)

López-Urueña, E; Alvarez, M; Gomes-Alves, S; Anel-López, L; Martínez-Rodríguez, C; Manrique, P; Borragan, S; Anel, L; de Paz, P

2015-10-15

Brown bear ejaculates are usually collected in field conditions and may need to be shipped to a laboratory for the application of reproductive biotechnologies before cryopreservation. The aim of this study was to extend the prefreezing step to 48 hours (1 hour vs. long-term storage [LS] to 24 and 48 hours) to enable the sample to be transported. The effects of storage temperature (experiment 1), glycerol concentration (experiment 2), and dilution rate (experiment 3) on sperm were evaluated. Electroejaculates from brown bears were stored under different experimental conditions and cryopreserved. The sperm motility and viability, apoptotic status, and acrosomal status of sperm were assessed before freezing (prefreezing), after thawing, and after 2-hour incubation at 37 °C (thermal stress test). In all experiments, one control sample was frozen using a standard protocol (control). In experiment 1, three temperatures during LS with 6% glycerol were tested: 5 °C (T5), 15 °C (T15), and room temperature (RT). The LS-T5 sample yielded the highest postthawing results for viability (42.4%), progressive motility (15.6%), and intact acrosome (83.1%) after 24 hours in comparison with the other temperatures (P bear electroejaculates are at 5 °C, at a concentration of 100 × 10(6) sperm/mL, and with 6% glycerol. Copyright © 2015 Elsevier Inc. All rights reserved.

Effect on post-cryopreserved semen characteristics of Holstein bulls of adding combinations of vitamin C and either catalase or reduced glutathione to Tris extender.

Science.gov (United States)

Eidan, Sajeda M

2016-04-01

This study was undertaken to investigate the influence of adding combinations of vitamin C to Tris extender with either catalase or reduced glutathione on post-cryopreserved semen characteristics of Holstein bulls for different preservation periods (cooling at 5°C, 48 h, 1, 2 and 3 months post cryopreservation, PC). Seven Holstein bulls of 2.5-3 years of age were used in this experiment. Semen was collected via artificial vagina in one ejaculate per bull per week for the 7 week experimental period. Pooled semen was equally divided into three treatments using Tris extender. Combinations of vitamin C (2.5mM) were added with either catalase (100 IU/ml, T2) or reduced glutathione (2mM, T3) to Tris extender and comparisons in response were made with the control group (Tris extender, T1). Individual sperm motility (IM), viability (V), plasma membrane integrity (PMI), and acrosome integrity (AI) were assessed during all periods of the study along with Malondialdehyde (MDA) concentrations and freezing ability. The IM was greater (P ≤ 0.01) in the T2 as compared with the T1 group at all periods of the study. Furthermore, the IM were greater (P ≤ 0.01) in the T3 as compared with the T1 group at the 48 h time period and at 3 months PC. The V, PMI and AI were greater (P ≤ 0.01) in T2 and T3 as compared with the T1 group at all the experimental periods. The MDA was greater (P ≤ 0.01) in the T2 as compared with the T1 group at 3 months PC. In conclusion, there was improved semen quality if semen of Holstein bulls was collected and stored in combinations of vitamin C with either catalase (T2) or reduced glutathione (T3) being added to Tris extender. Copyright © 2016 Elsevier B.V. All rights reserved.

Cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cGAMP.

Science.gov (United States)

Ablasser, Andrea; Schmid-Burgk, Jonathan L; Hemmerling, Inga; Horvath, Gabor L; Schmidt, Tobias; Latz, Eicke; Hornung, Veit

2013-11-28

The innate immune defence of multicellular organisms against microbial pathogens requires cellular collaboration. Information exchange allowing immune cells to collaborate is generally attributed to soluble protein factors secreted by pathogen-sensing cells. Cytokines, such as type I interferons (IFNs), serve to alert non-infected cells to the possibility of pathogen challenge. Moreover, in conjunction with chemokines they can instruct specialized immune cells to contain and eradicate microbial infection. Several receptors and signalling pathways exist that couple pathogen sensing to the induction of cytokines, whereas cytosolic recognition of nucleic acids seems to be exquisitely important for the activation of type I IFNs, master regulators of antiviral immunity. Cytosolic DNA is sensed by the receptor cyclic GMP-AMP (cGAMP) synthase (cGAS), which catalyses the synthesis of the second messenger cGAMP(2'-5'). This molecule in turn activates the endoplasmic reticulum (ER)-resident receptor STING, thereby inducing an antiviral state and the secretion of type I IFNs. Here we find in murine and human cells that cGAS-synthesized cGAMP(2'-5') is transferred from producing cells to neighbouring cells through gap junctions, where it promotes STING activation and thus antiviral immunity independently of type I IFN signalling. In line with the limited cargo specificity of connexins, the proteins that assemble gap junction channels, most connexins tested were able to confer this bystander immunity, thus indicating a broad physiological relevance of this local immune collaboration. Collectively, these observations identify cGAS-triggered cGAMP(2'-5') transfer as a novel host strategy that serves to rapidly convey antiviral immunity in a transcription-independent, horizontal manner.

Development of a vitrification-based cryopreservation protocol for the storage of saltcedar (Tamarix boveana Bunge).

Science.gov (United States)

Cano-Castillo, M; Casas, J L

2012-01-01

We cryopreserved in vitro shoot tips of saltcedar (Tamarix boveana Bunge) using the vitrification technique. The success of the cryopreservation protocol was strongly affected by preculture, loading duration, dehydration duration in plant vitrification solution 2 (PVS2), and medium composition during post-warming regrowth. The highest explant regrowth (50 percent) occurred when the following conditions were employed: preculture in 0.4 M glycerol; treatment with a loading solution (LS) consisting of 2 M glycerol + 0.4 M sucrose in culture medium for 40 min at room temperature; and dehydration in PVS2 at 0 degree C for 45 min before rapid immersion in liquid nitrogen (LN). Rewarming was performed in a water-bath at 40 degree C for 2 min. Explants were then immersed in unloading solution for 10 min before plating on recovery medium supplemented with 0.01 mg per liter thidiazuron (TDZ). TDZ was progressively eliminated from the medium over a period of 6 weeks. Plantlets were transferred to a double-layer medium to enhance rooting. This protocol was successfully applied to three individuals of T. boveana harvested from the wild.

Cross-immunity among mammary carcinomas in C3H/HE mice immunized with gamma-irradiated tumor cells

International Nuclear Information System (INIS)

Waga, Takashi

1980-01-01

By immunization with gamma-irradiated (13,000 rad) tumor cells, cross-immunity between ascites mammary carcinomas and among solid mammary carcinomas in C3H/He mice was studied. The results were as follows: (1) Two ascites mammary carcinomas designated MM 46 (high vitality) and MM 48 (intermediate vitality) were used in this experiment. The immunization with the tumor of high vitality (MM 46) induced strong cross-immunity against the challenge of the tumor of intermediate vitality (MM 48). The immunization with the tumor of intermediate vitality (MM 48) induced weak cross-immunity against the challenge of the tumor of high vitality (MM 46). (2) Three solid mammary carcinomas designated MT 10 (intermediate vitality), MT 7 (high vitality) and MT X (the highest vitality) were used in this experiment. The immunization with the tumor of high vitality (MT 7) induced strong cross-immunity against the challenge of the tumor of intermediate vitality (MT 10), and induced moderate cross-immunity against the challenge of the tumor of the highest vitality (MT X). The immunization with the tumor of intermediate vitality (MT 10) induced moderate cross-immunity against the challenge of the tumor of high vitality (MT 7), but could not induce any cross-immunity against the challenge of the tumor of the highest vitality (MT X). (author)

Effects of Droplet-Vitrification Cryopreservation Based on Physiological and Antioxidant Enzyme Activities of Brassidium Shooting Star Orchid

Directory of Open Access Journals (Sweden)

Safrina Rahmah

2015-01-01

Full Text Available Protocorm-like bodies (PLBs of Brassidium Shooting Star orchid were successfully cryopreserved using droplet-vitrification method. Vitrification based cryopreservation protocol is comprised of preculture, osmoprotection, cryoprotection, cooling, rewarming, and growth recovery and each and every step contributes to the achievement of successful cryopreservation. In order to reveal the lethal and nonlethal damage produced by cryopreservation, histological observation, scanning electron microscopy (SEM, and biochemical analysis were carried out in both cryopreserved and noncryopreserved PLBs of Brassidium Shooting Star orchid comparing with the control PLBs stock culture. Histological and scanning electron microscopy analyses displayed structural changes in cryopreserved PLBs due to the impact of cryoinjury during exposure to liquid nitrogen. Total soluble protein significantly increased throughout the dehydration process and the highest value was achieved when PLBs were stored in liquid nitrogen. Ascorbate peroxidase (APX and catalase (CAT showed the highest enzyme activities in both dehydration and cryostorage treatments indicating that stress level of PLBs was high during these stages.

Highly efficient vitrification method for cryopreservation of human oocytes.

Science.gov (United States)

Kuwayama, Masashige; Vajta, Gábor; Kato, Osamu; Leibo, Stanley P

2005-09-01

Two experiments were performed to develop a method to cryopreserve MII human oocytes. In the first experiment, three vitrification methods were compared using bovine MII oocytes with regard to their developmental competence after cryopreservation: (i) vitrification within 0.25-ml plastic straws followed by in-straw dilution after warming (ISD method); (ii) vitrification in open-pulled straws (OPS method); and (iii) vitrification in plastic handle (Cryotop method). In the second experiment, the Cryotop method, which had yielded the best results, was used to vitrify human oocytes. Out of 64 vitrified oocytes, 58 (91%) exhibited normal morphology after warming. After intracytoplasmic sperm injection, 52 became fertilized, and 32 (50%) developed to the blastocyst stage in vitro. Analysis by fluorescence in-situ hybridization of five blastocysts showed that all were normal diploid embryos. Twenty-nine embryo transfers with a mean number of 2.2 embryos per transfer on days 2 and 5 resulted in 12 initial pregnancies, seven healthy babies and three ongoing pregnancies. The results suggest that vitrification using the Cryotop is the most efficient method for human oocyte cryopreservation.

Human oocyte cryopreservation and the fate of cortical granules.

Science.gov (United States)

Ghetler, Yehudith; Skutelsky, Ehud; Ben Nun, Isaac; Ben Dor, Liah; Amihai, Dina; Shalgi, Ruth

2006-07-01

To examine the effect of the commonly used oocyte cryopreservation protocol on the cortical granules (CGs) of human immature germinal vesicle (GV) and mature metaphase II (MII) oocytes. Laboratory study. IVF unit. Unfertilized, intracytoplasmic sperm injected (ICSI) oocytes, and immature oocytes were cryopreserved using a slow freezing-rapid thawing program with 1,2-propanediol (PROH) as a cryoprotectant. Cortical granule exocytosis (CGE) was assessed by either confocal microscopy or transmission electron microscopy (TEM). The survival rates of frozen-thawed oocytes (mature and immature) were significantly lower compared with zygotes. Both mature and immature oocytes exhibited increased fluorescence after cryopreservation, indicating the occurrence of CGE. Mere exposure of oocytes to cryoprotectants induced CGE of 70% the value of control zygotes. The TEM revealed a drastic reduction in the amount of CGs at the cortex of frozen-thawed GV and MII oocytes, as well as appearance of vesicles in the ooplasm. The commonly used PROH freezing protocol for human oocytes resulted in extensive CGE. This finding explains why ICSI is needed to achieve fertilization of frozen-thawed human oocytes.

Development of a sperm cryopreservation protocol for the Argentine black and white tegu (Tupinambis merianae).

Science.gov (United States)

Young, Carly; Ravida, Nicole; Curtis, Michelle; Mazzotti, Frank; Durrant, Barbara

2017-01-01

Of the 934 lizard species evaluated by the International Union for the Conservation of Nature (IUCN), at least one-third is threatened with extinction. However, there are no reports of semen cryopreservation efforts for lizards. Invasive Argentine black and white tegus were captured in the Florida Everglades, and sperm was collected postmortem. Initial motility score (IMS; % motile × speed of progression 2 × 100), plasma membrane integrity (IPL), and acrosome integrity (IAC) were recorded. Sperm was diluted in TEST-yolk buffer with a final glycerol or dimethylsulfoxide (DMSO)concentration of 8%, 12%, or 16%, and frozen at 0.3 °C, 1.0 °C, or 6.3 °C/min. At thaw, all variables were expressed as the percentage of initial (%IMS, %IPL, and %IAC). The 0.3 °C freeze rate was more successful than 1.0 °C and 6.3 °C/min in preserving %IMS and %IPL. DMSO preserved %IMS, %IPL, and %IAC better than glycerol. To determine the best overall cryopreservation protocol, a sperm quality index was calculated, giving equal weight to each of the three indicators of cryosurvival. Because there were significant interactions between freeze rate and cryoprotectant concentration, each treatment was compared with all others. The sperm quality index analysis revealed that tegu sperm frozen at 0.3 °C/min with 12% DMSO exhibited the highest postthaw viability compared with all other treatments. Copyright © 2016 Elsevier Inc. All rights reserved.

Protectants used in the cryopreservation of microorganisms

Czech Academy of Sciences Publication Activity Database

Hubálek, Zdeněk

2003-01-01

Roč. 46, č. 3 (2003), s. 205-229 ISSN 0011-2240 Institutional research plan: CEZ:AV0Z6093917 Keywords : cryopreservation * cryoprotectants Subject RIV: EE - Microbiology, Virology Impact factor: 1.445, year: 2003

Semen cryopreservation in fish: effects on sperm motility and fertility

International Nuclear Information System (INIS)

Martinez, Jose Gregorio; Pardo Carrasco, Sandra

2010-01-01

The cryopreservation of semen in fish, as in many species even shows effects that decrease sperm quality and directly engage cell ability to successfully participate in the processes of fertilization and embryonic development. the characteristics such as mobility and fertilizing capacity of fertilization of sperm are considered to be quality criteria that allow to measure the success or failure of the process, since they are considered integrative variables, being indicators that depend not on a single factor, but on the stability and welfare of all structures, enzymes and subcellular functional compounds that give place to these spermatic characteristics. membrane damage (Adenylate cyclase, ion channels, grouping of other proteins, among others) and their implication in the route of signaling pathway leading to spermatic activation, ATP degradation and fragmentation of nuclear and mitochondrial DNA (genome), degradation of kinase enzymes and other cytosolic proteins (proteome) are considered today, as some of the molecular factors that most affect during cryopreservation and markedly decreasing the fertilizing capacity and mobility of sperm in fish. Proposals on the molecular mechanisms, by which these subcellular factors interact and act as consequence of cryopreservation, are some of the topics covered in this review. Understanding the principles and factors that are involved in the origin of such damages, will allow to improved cryopreservation processes, making them less harmful and more efficient.

Sperm cryopreservation before cancer treatment: a 15-year monocentric experience.

Science.gov (United States)

Bizet, P; Saias-Magnan, J; Jouve, E; Grillo, J M; Karsenty, G; Metzler-Guillemain, C; Perrin, J

2012-03-01

Sperm banking is an important procedure to preserve fertility before cancer therapy. The aim of this study was to comprehensively analyse cryopreservation activity retrospectively for 1080 patients referred to the sperm bank for sperm cryopreservation before cancer treatment. This study included 1007 patients diagnosed with testicular cancer (TC) (41.7%), lymphoma (26%), other haematological cancers (9.4%) or other types of cancer (22.8%); of these, 29 patients did not produce any semen sample and cryopreservation was impossible for 67 patients. Semen characteristics before treatment were within normal ranges, except moderate asthenospermia. Sperm concentration was significantly lower in TC than in non-TC. Straws from 57 patients (6.3%) were used in assisted reproductive technologies, which led to a 46.8% cumulative birth rate. Straws were destroyed for 170 patients (18.7%) and 140 patients performed semen analyses after cancer therapy. After an average delay of 22.5 months after the end of therapy, 43 patients (30.7%) exhibited azoospermia. This study of a large population of cancer patients revealed a high level of successful sperm storage. Utilization of cryopreserved spermatozoa led to good chances of fatherhood. Nevertheless, sperm banks should be aware of the low rates of straw use and straw destruction by cancer patients. Copyright © 2011 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Effectiveness and safety of traditional Chinese medicine in treating acquired immune deficiency syndrome: 2004-2014.

Science.gov (United States)

Liu, Zhi-Bin; Yang, Ji-Ping; Xu, Li-Ran

2015-12-23

Substantial progress has been made in China in using traditional Chinese medicine (TCM) to treat acquired immune deficiency syndrome (AIDS). Our objective was to review the latest developments in TCM treatment of AIDS in China between 2004 and 2014. We reviewed the content of original articles investigating the efficacy and safety of TCM for treating AIDS published in Chinese and English language journals. Relevant references from 2004 to 2014 were found using PubMed and the China National Knowledge Infrastructure Database. We found that TCM has been widely used for treating AIDS and its complications in China. The number of TCM studies has increased, which indicates efficacy and safety. Measures of efficacy in the reviewed articles included the alleviation of human immunodeficiency virus (HIV)-related signs and symptoms, improvements in quality of life, improvements in long-term survival, counteraction of the adverse side effects of antiviral drugs, promotion of immune reconstitution, and improvement of laboratory results. In sum, the literature indicates that TCM is safe. TCM plays an important role in the treatment of AIDS. Some studies have attempted to measure the efficacy and safety of TCM for treating AIDS, but more evidence is needed. Therefore, more research on this topic is required in the future.

Cryopreservation of mouse embryos by ethylene glycol-based vitrification.

Science.gov (United States)

Mochida, Keiji; Hasegawa, Ayumi; Taguma, Kyuichi; Yoshiki, Atsushi; Ogura, Atsuo

2011-11-18

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic modifications and the number is consistently increasing year by year. Its technical development started with slow freezing methods in the 1970s(1), then followed by vitrification methods developed in the late 1980s(2). Generally, the latter technique is advantageous in its quickness, simplicity, and high survivability of recovered embryos. However, the cryoprotectants contained are highly toxic and may affect subsequent embryo development. Therefore, the technique was not applicable to certain strains of mice, even when the solutions are cooled to 4°C to mitigate the toxic effect during embryo handling. At the RIKEN BioResource Center, more than 5000 mouse strains with different genetic backgrounds and phenotypes are maintained(3), and therefore we have optimized a vitrification technique with which we can cryopreserve embryos from many different strains of mice, with the benefits of high embryo survival after vitrifying and thawing (or liquefying, more precisely) at the ambient temperature(4). Here, we present a vitrification method for mouse embryos that has been successfully used at our center. The cryopreservation solution contains ethylene glycol instead of DMSO to minimize the toxicity to embryos(5). It also contains Ficoll and sucrose for prevention of devitrification and osmotic adjustment, respectively. Embryos can be handled at room temperature and transferred into liquid nitrogen within 5 min. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. We also describe the procedure of thawing vitrified embryos in detail because it is a critical step for efficient recovery of live mice. These methodologies would be helpful to researchers and

Fertility in cancer patients after cryopreservation of one ovary

DEFF Research Database (Denmark)

Schmidt, K T; Andersen, Anders Nyboe; Greve, T

2013-01-01

This questionnaire study describes the fertility and ovarian function in 143 adult female cancer survivors with only one ovary due to cryopreservation of the other. The women were asked about their ovarian function (as defined by the presence of a spontaneous menstrual cycle), pregnancies...... and their outcome. The mean follow-up time was 58months after cryopreservation (range 24-129months). The risk of premature ovarian failure was high in the group of patients with leukaemia (13/15; 87%) but low in the breast cancer group (5/54; 9%). Fifty-seven women had actively tried to become pregnant after end...

Cryopreservation of cocoa (Theobroma cacao L.) somatic embryos by vitrification.

Science.gov (United States)

Adu-Gyamfi, Raphael; Wetten, Andy

2012-01-01

Losses of cultivated cocoa (Theobroma cacao L.) due to diseases and continued depletion of forests that harbour the wild progenitors of the crop make ex situ conservation of cocoa germplasm of paramount importance. In order to enhance security of in situ germplasm collections, 2-3 mm floral-derived secondary somatic embryos were cryopreserved by vitrification. This work demonstrates the most uncomplicated clonal cocoa cryopreservation. Optimal post-cryostorage survival (74.5 percent) was achieved by 5 d preculture of SSEs on 0.5 M sucrose medium followed by 60 min dehydration in cold PVS2. To minimise free radical related cryo-injury, cation sources were removed from the embryo development solution and/or the recovery medium, the former treatment resulting in a significant benefit. After optimisation with cocoa genotype AMAZ 15, the same protocol was effective across all five additional cocoa genotypes tested. For the multiplication of clones, embryos regenerated following cryopreservation were used as explant sources, and vitrification was found to maintain their embryogenic potential.

Haemoglobin C and S role in acquired immunity against Plasmodium falciparum malaria.

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Federica Verra

2007-10-01

Full Text Available A recently proposed mechanism of protection for haemoglobin C (HbC; beta6Glu-->Lys links an abnormal display of PfEMP1, an antigen involved in malaria pathogenesis, on the surface of HbC infected erythrocytes together with the observation of reduced cytoadhesion of parasitized erythrocytes and impaired rosetting in vitro. We investigated the impact of this hypothesis on the development of acquired immunity against Plasmodium falciparum variant surface antigens (VSA encoding PfEMP1 in HbC in comparison with HbA and HbS carriers of Burkina Faso. We measured: i total IgG against a single VSA, A4U, and against a panel of VSA from severe malaria cases in human sera from urban and rural areas of Burkina Faso of different haemoglobin genotypes (CC, AC, AS, SC, SS; ii total IgG against recombinant proteins of P. falciparum asexual sporozoite, blood stage antigens, and parasite schizont extract; iii total IgG against tetanus toxoid. Results showed that the reported abnormal cell-surface display of PfEMP1 on HbC infected erythrocytes observed in vitro is not associated to lower anti- PfEMP1 response in vivo. Higher immune response against the VSA panel and malaria antigens were observed in all adaptive genotypes containing at least one allelic variant HbC or HbS in the low transmission urban area whereas no differences were detected in the high transmission rural area. In both contexts the response against tetanus toxoid was not influenced by the beta-globin genotype. These findings suggest that both HbC and HbS affect the early development of naturally acquired immunity against malaria. The enhanced immune reactivity in both HbC and HbS carriers supports the hypothesis that the protection against malaria of these adaptive genotypes might be at least partially mediated by acquired immunity against malaria.

Low-density Lipoprotein Improves Motility and Plasma Membrane Integrity of Cryopreserved Canine Epididymal Spermatozoa.

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Prapaiwan, N; Tharasanit, T; Punjachaipornpol, S; Yamtang, D; Roongsitthichai, A; Moonarmart, W; Kaeoket, K; Manee-In, S

2016-05-01

Cryopreservation of caudal epididymal spermatozoa is an effective technique to conserve genetic potentials of superior dogs when it is not possible to collect ejaculated spermatozoa. Although hen egg yolk is commonly supplemented into the semen extender, active substances within the egg yolk which protect sperm against cryoinjury remain to be discovered. Among its compositions, low-density lipoprotein (LDL) has been reported to have a cryoprotective property for sperm cryopreservation. However, the effects of LDL on dog epididymal spermatozoa during cryopreservation have not yet been investigated. This study aimed to investigate the effects of LDL on epididymal spermatozoa quality following cryopreservation and thawing. After routine castration of 12 dogs, caudal epididymides from individuals were separated from the testes and cut into a few pieces in a Tris-buffer. Spermatozoa recovered from each sample were examined at once for sperm quality and divided into six groups of extender: no LDL, 20% egg yolk, 4%, 8%, 16%, and 24% LDL, before cryopreservation. The sperm aliquots were then equilibrated and conventionally frozen. After thawing, sperm motility, morphology, plasma membrane integrity, and acrosome integrity were evaluated. The results revealed that 4% LDL and 20% egg yolk yielded significantly higher sperm motility (57.69% and 52.69%, respectively, p<0.05) than other LDLs. In addition, 4% LDL yielded the significantly highest plasma membrane integrity (70.54%, p<0.05). In conclusion, the supplementation of 4% LDL in Tris-glucose extender could be applied for cryopreservation of canine epididymal spermatozoa.

Low-density Lipoprotein Improves Motility and Plasma Membrane Integrity of Cryopreserved Canine Epididymal Spermatozoa

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N. Prapaiwan

2016-05-01

Full Text Available Cryopreservation of caudal epididymal spermatozoa is an effective technique to conserve genetic potentials of superior dogs when it is not possible to collect ejaculated spermatozoa. Although hen egg yolk is commonly supplemented into the semen extender, active substances within the egg yolk which protect sperm against cryoinjury remain to be discovered. Among its compositions, low-density lipoprotein (LDL has been reported to have a cryoprotective property for sperm cryopreservation. However, the effects of LDL on dog epididymal spermatozoa during cryopreservation have not yet been investigated. This study aimed to investigate the effects of LDL on epididymal spermatozoa quality following cryopreservation and thawing. After routine castration of 12 dogs, caudal epididymides from individuals were separated from the testes and cut into a few pieces in a Tris-buffer. Spermatozoa recovered from each sample were examined at once for sperm quality and divided into six groups of extender: no LDL, 20% egg yolk, 4%, 8%, 16%, and 24% LDL, before cryopreservation. The sperm aliquots were then equilibrated and conventionally frozen. After thawing, sperm motility, morphology, plasma membrane integrity, and acrosome integrity were evaluated. The results revealed that 4% LDL and 20% egg yolk yielded significantly higher sperm motility (57.69% and 52.69%, respectively, p<0.05 than other LDLs. In addition, 4% LDL yielded the significantly highest plasma membrane integrity (70.54%, p<0.05. In conclusion, the supplementation of 4% LDL in Tris-glucose extender could be applied for cryopreservation of canine epididymal spermatozoa.

Liposome encapsulated soy lecithin and cholesterol can efficiently replace chicken egg yolk in human semen cryopreservation medium.

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Mutalik, Srinivas; Salian, Sujith Raj; Avadhani, Kiran; Menon, Jyothsna; Joshi, Haritima; Hegde, Aswathi Raju; Kumar, Pratap; Kalthur, Guruprasad; Adiga, Satish Kumar

2014-06-01

Cryopreservation of spermatozoa plays a significant role in reproductive medicine and fertility preservation. Chicken egg yolk is used as an extender in cryopreservation of human spermatozoa using glycerol egg yolk citrate (GEYC) buffered medium. Even though 50% survival of spermatozoa is generally achieved with this method, the risk of high levels of endotoxins and transmission pathogens from chicken egg yolk is a matter of concern. In the present study we attempted to establish a chemically defined cryopreservation medium which can replace the chicken egg yolk without affecting sperm survival. Ejaculates from 28 men were cryopreserved with GEYC based freezing medium or liposome encapsulated soy lecithin-cholesterol based freezing medium (LFM). The semen samples were subjected to rapid thawing after 14 days of storage in liquid nitrogen. Post-thaw analysis indicated significantly higher post-thaw motility and sperm survival in spermatozoa cryopreserved with LFM compared to conventional GEYC freezing medium. The soy lecithin and cholesterol at the ratio of 80:20 with sucrose showed the highest percentage of post-thaw motility and survival compared to the other compositions. In conclusion, chemically defined cryopreservation medium with liposome encapsulated soy lecithin and cholesterol can effectively replace the chicken egg yolk from human semen cryopreservation medium without compromising post-thaw outcome.

[Therapeutic effect of double fill nine tastes soup in treating recurrent respiratory infection (RRI) and change of immune function in children].

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Wang, Youcheng; Zhang, Lijuan; Hu, Guohua; Wang, Menghe; Tang, Xiaoyuan; Guo, Hui; Shi, Yimei; Chen, Shufang; Shi, Changchun

2012-04-01

To investigate the therapeutic effect of double fill nine tastes soup in treating children recurrent respiratory infection (RRTI) and the change of immune function. 77 RRTI patients were randomly selected into observation and control groups. The observation group was treated with Chinese medicine- double fill nine tastes soup,water frying points 2 times oral. The control was treated with transfer factor oral liquid,every 10 mL,2 times daily oral. Treatment periods were both two months. IgA, IgG, IgM and IL-12, TNF-alpha, INF-gamma were detected before and after treatment to assess the clinical effects and the changes of immune factors, meanwhile, a health group was established. Before treatment, compared with the health group, the serum IgA, IgG, IgM, IgE, IL-12, TNF-alpha, IFN-gamma in both groups were significantly different (P soup has significant effects in treating recurrent respiratory infection (RRI) and enhance the immune function in children.

Effect of cryopreservation on mitochondrial activity in buffalo sperm Bubalus bubalis

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O. Kandil

2010-02-01

Full Text Available Sperm mitochondrial activity is investigated and used as “in vitro” spermatozoa vitality indicator and about quality effectiveness of different sperm diluents. It was studied the cytochemically activity of NADPH-diaphorase and LDH-C4 in cryopreserved buffalo sperm. Low intensity of the enzyme reaction was established in all examined sperm samples in both enzymes, regardless from the used cryoprotectors. The main part of the enzyme reaction was localized in mitochondrial sheath and in a very small degree in the head base of spermatozoa. No increase of the enzymes activities or the spermatozoa motility has been found after the incubating with Sp-TALP medium although the established caffeine stimulating effect on the glycolysis and fresh spermatozoa motility. Established by us low sperm motility after cryopreservation may be due to low LDH and NADPH-diaphorase activity due to glycolisis disturbances and ATP synthesis. This method allows to estimate quality of buffalo semen and to find some different disturbances in mitochondrial sheath, which could not be found by routine morphological studies and could be used in practice ejaculates with high number of metabolic active sperm cells.

Immune regulation in chronic hepatitis C virus infection

DEFF Research Database (Denmark)

Hartling, Hans Jakob; Ballegaard, Vibe Cecilie; Nielsen, Nick Schou

2016-01-01

The immunological result of infection with Hepatitis C virus (HCV) depends on the delicate balance between a vigorous immune response that may clear the infection, but with a risk of unspecific inflammation and, or a less inflammatory response that leads to chronic infection. In general, exhaustion...... and impairment of cytotoxic function of HCV-specific T cells and NK cells are found in patients with chronic HCV infection. In contrast, an increase in immune regulatory functions is found primarily in form of increased IL-10 production possibly due to increased level and function of anti-inflammatory Tregs...

Structural and functional analyses of DNA-sensing and immune activation by human cGAS.

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Kato, Kazuki; Ishii, Ryohei; Goto, Eiji; Ishitani, Ryuichiro; Tokunaga, Fuminori; Nureki, Osamu

2013-01-01

The detection of cytosolic DNA, derived from pathogens or host cells, by cytosolic receptors is essential for appropriate host immune responses. Cyclic GMP-AMP synthase (cGAS) is a newly identified cytosolic DNA receptor that produces cyclic GMP-AMP, which activates stimulator of interferon genes (STING), resulting in TBK1-IRF3 pathway activation followed by the production of type I interferons. Here we report the crystal structure of human cGAS. The structure revealed that a cluster of lysine and arginine residues forms the positively charged DNA binding surface of human cGAS, which is important for the STING-dependent immune activation. A structural comparison with other previously determined cGASs and our functional analyses suggested that a conserved zinc finger motif and a leucine residue on the DNA binding surface are crucial for the DNA-specific immune response of human cGAS, consistent with previous work. These structural features properly orient the DNA binding to cGAS, which is critical for DNA-induced cGAS activation and STING-dependent immune activation. Furthermore, we showed that the cGAS-induced activation of STING also involves the activation of the NF-κB and IRF3 pathways. Our results indicated that cGAS is a DNA sensor that efficiently activates the host immune system by inducing two distinct pathways.

Structural and functional analyses of DNA-sensing and immune activation by human cGAS.

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Kazuki Kato

Full Text Available The detection of cytosolic DNA, derived from pathogens or host cells, by cytosolic receptors is essential for appropriate host immune responses. Cyclic GMP-AMP synthase (cGAS is a newly identified cytosolic DNA receptor that produces cyclic GMP-AMP, which activates stimulator of interferon genes (STING, resulting in TBK1-IRF3 pathway activation followed by the production of type I interferons. Here we report the crystal structure of human cGAS. The structure revealed that a cluster of lysine and arginine residues forms the positively charged DNA binding surface of human cGAS, which is important for the STING-dependent immune activation. A structural comparison with other previously determined cGASs and our functional analyses suggested that a conserved zinc finger motif and a leucine residue on the DNA binding surface are crucial for the DNA-specific immune response of human cGAS, consistent with previous work. These structural features properly orient the DNA binding to cGAS, which is critical for DNA-induced cGAS activation and STING-dependent immune activation. Furthermore, we showed that the cGAS-induced activation of STING also involves the activation of the NF-κB and IRF3 pathways. Our results indicated that cGAS is a DNA sensor that efficiently activates the host immune system by inducing two distinct pathways.

CRYOPRESERVATION OF REPRODUCTIVE PRODUCTS AS AN EFFECTIVE METHOD FOR PRESERVING THE BIODIVERSITY OF STURGEON FISH SPECIES (REVIEW

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I. Kononenko

2016-06-01

Full Text Available Purpose. In recent years, cryopreservation of reproductive products has widely been used as one of the accessible and in some cases the only ways of preserving and supporting the number of endangered fish species including sturgeon fish species. Currently, the method of cryopreservation is represented by various techniques and ways using individual and species approaches. Numerous publications on the issue of fish sperm cryopreservation mostly contain inconsistent results and ambiguous data. Thus, the analysis of the existing information about principles and methods of fish sperm cryopreservation is an important issue for further studies. Moreover, summarizing the existing information will enable us to plan the experiment more efficiently and reasonably and to get the desired outcomes with higher reliability. Findings. The study presents main principles of widely used methods of fish sperm cryopreservation, the analysis of main factors of influence on outcomes of freezing or unfreezing as well as the analysis of results received when using various ways and methods of cryopreservation. Besides, the paper shows the importance of forming and full functioning of fish sperm cryobanks. Originality. The paper summarizes the existing information on the issue of fish sperm low temperature cryopreservation. The information is given in the form of successive presentation of the research outcomes received at each point of freezing or unfreezing when using different techniques as well as results of different factors influence on it. Moreover, a review of achievements in the field of cryopreservation and main principles of forming fish sperm cryobanks are given. Practical value. The presented review of traditional and modern literature data in the issue of cryopreservation can be used when planning, redesigning and experimenting fish sperm freezing or unfreezing.

Modification of trout sperm membranes associated with activation and cryopreservation. Implications for fertilizing potential

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Abstract We investigated the effects of two trout sperm activation solutions on sperm physiology and membrane organization prior to and following cryopreservation using flow cytometry and investigated their impact on in vitro fertility. Cryopreservation caused greater phospholipid disorder (high pl...

Development of cryopreservation for Loxocarya cinerea---an endemic Australian plant species important for post-mining restoration.

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Kaczmarczyk, Anja; Funnekotter, Bryn; Turner, Shane R; Bunn, Eric; Bryant, Gary; Hunt, Taavi E; Mancera, Ricardo L

2013-01-01

We report the development of a cryopreservation protocol for the endemic Western Australian plant species Loxocarya cinerea (Restionaceae). Shoot tips from two genotypes, SXH404 and SXH804, were cryopreserved using the droplet-vitrification technique. Control explants, which were cryoprotected, but not cooled, showed regeneration for both genotypes (SXH404, 22.1 +/- 5.9%; SXH804, 67.7 +/- 9.6%). Extension of incubation in PVS2 from 30 to 60 min did not lead to survival after cryopreservation. Thermal analysis using differential scanning calorimetry confirmed the beneficial effect of a loading phase but also revealed no or very little ice formation after cryoprotection of shoot tips in other treatments. Regeneration following cryopreservation was obtained for genotype SXH804 (4.3 +/- 2.1%) but not for SXH404. Regenerated explants of L. cinerea SXH804 were morphologically identical to tissue-cultured plants. As an alternative to shoot tips, callus tissues of clone SXH404 were successfully cryopreserved (> 66.7% post LN survival) using the same protocol.

The role of splenic irradiation in treating HIV-associated immune thrombocytopenia

International Nuclear Information System (INIS)

Blauth, Jeanette; Fisher, Scot; Henry, David; Nichini, Franco

1999-01-01

Purpose: To evaluate our experience in treating patients with HIV-associated thrombocytopenia using splenic irradiation. Methods and Materials: From 1993 to 1998, 10 patients with HIV-related immune thrombocytopenia (ITP) were treated in our department with low-dose splenic irradiation. All patients had either failed more conventional treatment modalities or possessed some contraindication to them. Results: Nine of 10 patients had at least a small, transient rise in their platelet counts, but only two received a substantial therapeutic benefit. Of these two, one died shortly after completing his course of radiation therapy while the other maintained near normal platelet counts up to approximately 3((1)/(2)) years following treatment. There were no treatment-related morbidities and one patient was treated twice. Conclusion: While most patients with HIV-associated ITP may initially respond favorably to splenic irradiation with small rises in platelet count, few responses are likely to be sustained or provide clinically significant outcomes. Our results support those previously reported by others treating this same condition. What remains to be investigated is whether there are any prognostic indicators to help identify those patients most likely to respond to this treatment, thus enabling us to reserve splenic irradiation for those who might derive a substantial benefit from it

Triplet pregnancy after intracytoplasmic sperm injection of cryopreserved oocytes: case report.

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Young, E; Kenny, A; Puigdomenech, E; Van Thillo, G; Tiverón, M; Piazza, A

1998-08-01

To report a triplet pregnancy that occurred after intracytoplasmic injection of sperm into cryopreserved oocytes. Case report. Instituto de Ginecología y Fertilidad (IFER), Buenos Aires, Argentina. A 36-year-old infertile patient with premature ovarian failure and a previous term pregnancy with fresh donated oocytes. We administered leuprolide acetate for pituitary down-regulation followed by E2 valerianate in incremental doses until an endometrial lining of >8 mm was observed by ultrasound. Thawing of frozen donated oocytes, intracytoplasmic sperm injection (ICSI), and translaparoscopic fallopian tube ET also were performed. Natural micronized progesterone was administered intravaginally (600 mg/d) before ET. Ultrasound at the 8th week of gestation revealed a triplet pregnancy with active fetal heartbeats. A triple intrauterine gestation was achieved with the use of microinjection into cryopreserved oocytes. This case illustrates the feasibility of oocyte cryopreservation for clinical use in the era of ICSI.

Semen quality before cryopreservation and after thawing in 543 patients with testicular cancer

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MacKenna, Antonio; Crosby, Javier; Huidobro, Cristián; Correa, Eduardo; Duque, Gonzalo

2017-01-01

Objective The main objective of this study was to assess semen characteristics of patients with testicular cancer before cryopreservation and after thawing, to evaluate the consequences of this technique on sperm quality in patients with testicular cancer. Methods Five hundred eighty-nine samples from 543 patients with testicular cancer were cryopreserved between 1995 and 2015, one aliquot per patient was used for a thawing test to assess the impact of cryopreservation on sperm motility; semen analysis was performed before cryo preservation and after thawing, the result interpretation was carried out using the 2010 World Health Organization (WHO) Laboratory Manual, and consent forms were signed by the patients for freezing and when sperm was used for reproductive purposes. Results Hypospermia was observed in 28.7% of samples, the median sperm concentration was 18 million/mL with 35% oligozoospermia; twenty-two patients (4.1%) had azoospermia and 12.7% had severe oligozoospermia, the median sperm count was 31.3 million and 261 semen samples (44.3%) were normal in all parameters according to the WHO; total motile sperm count before cryopreservation and after thawing was 12 (0-412.2) and 7 (0-303.9) million sperm, respectively (p < 0.00001, 95% CI 5.48-14.91), which represents a 32% reduction; concerning the utilization of cryopreserved semen samples, only twelve patients (2.2%) used their frozen sperm for reproductive purposes. Conclusions An impairment in semen quality was found in almost half of the samples from patients with testicular cancer, only few patients had azoospermia or severe oligozoospermia; sperm cryopreservation significantly reduces sperm motility and total motile sperm count and very few patients use their frozen sperm for reproductive purposes. PMID:28333030

Sperm DNA fragmentation induced by cryopreservation: new insights and effect of a natural extract from Opuntia ficus-indica.

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Meamar, Mehrdad; Zribi, Nassira; Cambi, Marta; Tamburrino, Lara; Marchiani, Sara; Filimberti, Erminio; Fino, Maria Grazia; Biggeri, Annibale; Menezo, Yves; Forti, Gianni; Baldi, Elisabetta; Muratori, Monica

2012-08-01

To analyze the effect of cryopreservation on sperm DNA fragmentation (SDF) in two cytometric sperm populations, PI(brighter) and PI(dimmer), and to test the effects of Opuntia ficus-indica (OFI) extracts, which contain antioxidants and flavanoids, and of resveratrol on cryopreservation of human semen. In vitro prospective study. Institutional study. Twenty-one normozoospermic men undergoing semen analysis for couple infertility. Cryopreservation using the routine method in the presence of OFI extracts or resveratrol. Measurement of SDF by TUNEL/PI flow cytometric method to evaluate sperm motility (by automated motion analysis, CASA system) and viability (by eosin/nigrosin staining) in the two populations of sperm PI(br) and PI(dim). Cryopreservation induced an increase of SDF only in the PI(br) sperm population. The increase was negatively dependent on the basal values of SDF in the same population. Addition of OFI extracts and resveratrol to the cryopreservation medium slightly but statistically significantly reduced SDF in the PI(br) population without affecting the deleterious effect of cryopreservation on sperm motion parameters or viability. The increase of SDF in the PI(br) population, which is unrelated to semen quality, suggests that caution must be taken in using cryopreserved semen, as morphologically normal and motile sperm may be damaged. The addition of substances with multifunctional properties such as OFI extracts to cryopreservation medium is only slightly effective in preventing the dramatic effects on SDF. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Early investigation on cryopreservation of Dendrobium sonia-28 ...

African Journals Online (AJOL)

User

2011-05-02

May 2, 2011 ... This study was conducted to determine the potential of cryostoring and regenerating Dendrobium ... Key words: Orchid, protocorm-like bodies, cryopreservation. ... technology is now taken as an ideal alternative propa-.

Trichinella spiralis Calreticulin Binds Human Complement C1q As an Immune Evasion Strategy.

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Zhao, Limei; Shao, Shuai; Chen, Yi; Sun, Ximeng; Sun, Ran; Huang, Jingjing; Zhan, Bin; Zhu, Xinping

2017-01-01

As a multicellular parasitic nematode, Trichinella spiralis regulates host immune responses by producing a variety of immunomodulatory molecules to escape from host immune attack, but the mechanisms underlying the immune evasion are not well understood. Here, we identified that T. spiralis calreticulin ( Ts -CRT), a Ca 2+ -binding protein, facilitated T. spiralis immune evasion by interacting with the first component of human classical complement pathway, C1q. In the present study, Ts -CRT was found to be expressed on the surface of different developmental stages of T. spiralis as well as in the secreted products of adult and muscle larval worms. Functional analysis identified that Ts -CRT was able to bind to human C1q, resulting in the inhibition of C1q-initiated complement classical activation pathway reflected by reduced C4/C3 generation and C1q-dependent lysis of antibody-sensitized sheep erythrocytes. Moreover, recombinant Ts -CRT (r Ts -CRT) binding to C1q suppressed C1q-induced THP-1-derived macrophages chemotaxis and reduced monocyte-macrophages release of reactive oxygen intermediates (ROIs). Blocking Ts -CRT on the surface of newborn larvae (NBL) of T. spiralis with anti- Ts -CRT antibody increased the C1q-mediated adherence of monocyte-macrophages to larvae and impaired larval infectivity. All of these results suggest that T. spiralis -expressed Ts -CRT plays crucial roles in T. spiralis immune evasion and survival in host mostly by directly binding to host complement C1q, which not only reduces C1q-mediated activation of classical complement pathway but also inhibits the C1q-induced non-complement activation of macrophages.

Effects of cryopreservation on excretory function, cellular adhesion molecules and vessel lumen formation in human umbilical vein endothelial cells.

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Cai, Guoping; Lai, Binbin; Hong, Huaxing; Lin, Peng; Chen, Weifu; Zhu, Zhong; Chen, Haixiao

2017-07-01

Cryopreservation is widely used in regenerative medicine for tissue preservation. In the present study, the effects of cryopreservation on excretory function, cellular adhesion molecules and vessel lumen formation in human umbilical vein endothelial cells (HUVECs) were investigated. After 0, 4, 8, 12 or 24 weeks of cryopreservation in liquid nitrogen, the HUVECs were thawed. The excretory functions markers (endothelin‑1, prostaglandin E1, von Willebrand factor and nitric oxide) of HUVECs were measured by ELISA assay. The expression of intercellular adhesion molecule‑1 (ICAM‑1) in HUVECs was analyzed using flow cytometry. An angiogenesis assay was used to determine the angiogeneic capabilities of the thawed HUVECs. The results demonstrated that cryopreserved/thawed and recultivated HUVECs were unsuitable for tissue‑engineered microvascular construction. Specifically, the excretory function of the cells was significantly decreased in the post‑cryopreserved HUVECs at 24 weeks. In addition, the level of ICAM‑1 in HUVECs was significantly upregulated from the fourth week of cryopreservation. Furthermore, the tube‑like structure‑forming potential was weakened with increasing cryopreservation duration, and the numbers of lumen and the length of the pipeline were decreased in the thawed HUVECs, in a time‑dependent manner. In conclusion, the results of the present study revealed that prolonged cryopreservation may lead to HUVEC dysfunction and did not create stable cell lines for tissue‑engineered microvascular construction.

Application of Pectin From Rauvolfia serpentina (L.) Benth to the Cryopreservation of Human Leucocyte Cell Suspensions.

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Zaitseva, O O; PoleZhaeva, T V; Khudvakov, A N; Solomina, O N; Golovchenko, V V

　　BACKGROUND: Due to their valuable medicinal properties and high physiological activity, plant polysaccharides are currently being extensively studied. The present study aims to investigate rauwolfian (pectin for Rauvolfia serpentina) supplementation on human leukocytes cryopreservation. We determined the сharacteristics of leukocytes undergoing freezing with pectin at different temperatures. Donor leukocytes were frozen under the protection of comprehensive cryoprotectant solution and stored in electrical freezers (-20C, -40C, -80C). A regular decrease of all values starting from a higher temperature (-20С) through to the lower temperature (-80С) was identified. The study showed that pectin rauwolfian stimulated both the oxygen-independent and the oxygen-dependent killer response. We also found that the oxygen-dependent neutrophil killer effect was reduced as the storage temperature was lowered. It was determined that the LPO levels in the cells with added pectin-containing solutions remained the same before freezing, while their antioxidant activity positively increased, which is beneficial for neutrophils for their further freezing to -20C, -40C and -80C. The results of the study make it possible to assume that rauwolfian, a pectin extracted from Rauvolfia serpentina, has an exocellular protectant effect as part of cryopreservative solution on human white blood cells stored at different low temperatures. The versatility of the substance is probably due to the degree of the macromolecule branching, in particular, the structure of carbohydrate side chains, which contain a large number of hydroxyl groups.

Exploring the Possibility of Cryopreservation of Feline and Canine Erythrocytes by Rapid Freezing with Penetrating and Non-Penetrating Cryoprotectants.

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Denys Pogozhykh

Full Text Available Efficient application of veterinary blood transfusion approaches for small companion animals requires readily available supply of the donor material. This can be achieved by developing of effective biobanking technologies allowing long-term storage of donor blood components via cryopreservation. Transfusion of an erythrocyte concentrate allows the successful correction of various hematological pathologies, severe bleeding, and etc. While in the past there were several approaches to cryopreserve red blood cells of dogs, to our knowledge there is virtually no data on cryopreservation of feline erythrocytes. In this paper, we performed a comprehensive parameter optimization for low temperature storage of RBCs of both species. Here, the efficiency of single-component and multicomponent cryoprotective media as well as necessary time of pre-incubation with penetrating and non-penetrating cryoprotectants prior to rapid freezing is analyzed. This study showed that glycerol was not sufficient for cryopreservation of red blood cells of the studied species under the investigated conditions. Application of 10% (v/v ME2SO allowed for a significant reduction of canine and feline erythrocytes hemolysis after thawing. 17.5% hydroxyethyl starch demonstrated the highest cryoprotective activity for both species. It was found that dog RBCs should be incubated in cryoprotective media for 30 min at 22°C prior to freezing, while for cat RBCs 20 min is sufficient. Combination of CPAs was less effective. Presented data may be considered in further studies in veterinary transfusion and blood banking optimization.

Cryopreservation and distribution of radiation-attenuated helminth larvae and the use of radioisotopes to monitor their survival. Coordinated programme on preparation of irradiated vaccines against some human diseases

International Nuclear Information System (INIS)

James, E.

1982-06-01

Techniques for the cryopreservation of schistosomula are described, from the methanol/two-step cooling technique, through a technique which uses 40% methanol and rapid cooling to the current technique which employs a two-step addition of ethanediol and rapid cooling. Levels of survival with these techniques have improved from 0.3% to 5.9% and now to 47% of control values. The 40% methanol/rapid cooling technique is described in detail as this forms the basis for understanding the role of cryoprotective additives and cooling and warming rates in the cryopreservation of schistosomula. The toxicity of 12 different potentially cryoprotective compounds is described. Cryopreservation of S.japonicum and S.bovis is described. The effect of the age of the schistosomula and their cryopreservability is related to the development of water sensitivity and the permeation and damage produced by glycerol and it is postulated that morphological changes occurring in the tegument during transformation from a cercaria to schistosomulum may account for these observations. Studies with 14 C-ethanediol are described which attempt to provide an understanding of permeability of cryoprtectants to schistosomula of different ages and at different temperatures. Vaccination studies with cryopreserved and radiation-attenuated schistosomula are also reported. Radiation-attenuated schistosomula are also reported. Radiation-attenuated cercariae and schistosomula produced high (64% to 89%) levels of protection in baboons, and cryopreserved schistosomula produced comparable levels of protection in vaccinated mice to normal schistosomula. Cryopreserved radiation-attenuated schistosomula produced a significant level of protection (49% reduction) in sheep although the numbers of normally motile organisms injected was low (1,000 per dose, 2 doses). It is concluded that normally-motile cryopreserved radiation-attenuated schistosomula are as immunogenic as fresh organisms

Complement C3 gene: Expression characterization and innate immune response in razor clam Sinonovacula constricta.

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Peng, Maoxiao; Niu, Donghong; Wang, Fei; Chen, Zhiyi; Li, Jiale

2016-08-01

Complement component 3 (C3) is central to the complement system, playing an important role in immune defense, immune regulation and immune pathology. Several C3 genes have been characterized in invertebrates but very few in shellfish. The C3 gene was identified from the razor clam Sinonovacula constricta, referred to here as Sc-C3. It was found to be highly homologous with the C3 gene of Ruditapes decussatus. All eight model motifs of the C3 gene were found to be included in the thiolester bond and the C345C region. Sc-C3 was widely expressed in all healthy tissues with expression being highest in hemolymph. A significant difference in expression was revealed at the umbo larvae development stage. The expression of Sc-C3 was highly regulated in the hemolymph and liver, with a distinct response pattern being noted after a challenge with Micrococcus lysodeikticus and Vibrio parahemolyticus. It is therefore suggested that a complicated and unique response pathway may be present in S. constricta. Further, serum of S. constricta containing Sc-C3 was extracted. This was activated by LPS or bacterium for verification for function. The more obvious immune function of Sc-C3 was described as an effective membrane rupture in hemocyte cells of rabbit, V. parahemolyticus and Vibrio anguillarum. Thus, Sc-C3 plays an essential role in the immune defense of S. constricta. Copyright © 2016 Elsevier Ltd. All rights reserved.

Slow cryopreservation is not superior to vitrification in human spermatozoa; an experimental controlled study

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Mohamed Shehata Ali Mohamed

2015-10-01

Full Text Available Background: Spermatozoa cryopreservation is used for the management of infertility and some other medical conditions. The routinely applied cryopreservation technique depends on permeating cryoprotectants, whose toxic effects have raised the attention towards permeating cryoprotectants-free vitrification technique. Objective: To compare between the application of slow cryopreservation and vitrification on human spermatozoa. Materials and Methods: This was an experimental controlled study involving 33 human semen samples, where each sample was divided into three equal parts; fresh control, conventional slow freezing, and permeating cryoprotectants-free vitrification. Viability and mitochondrial membrane potential (MMP of control and post-thawing spermatozoa were assessed with the sperm viability kit and the JC-1 kit, respectively, using fluorescence-activated cell sorting analysis. Results: Significant reduction of the progressive motility, viability and MMP was observed by the procedure of freezing and thawing, while there was not any significant difference between both cryopreservation techniques. Cryopreservation resulted in 48% reduction of the percentage of viable spermatozoa and 54.5% rise in the percentage of dead spermatozoa. In addition, high MMP was reduced by 24% and low MMP was increased by 34.75% in response to freezing and thawing. Progressive motility of spermatozoa was correlated significantly positive with high MMP and significantly negative with low MMP in control as well as post-thawing specimens (r=0.8881/ -0.8412, 0.7461/ -0.7510 and 0.7603/ -0.7839 for control, slow and vitrification respectively, p=0.0001. Conclusion: Although both cryopreservation techniques have similar results, vitrification is faster, easier and associated with less toxicity and costs. Thus, vitrification is recommended for the clinical application.

Changing rooster sperm membranes to facilitate cryopreservation

Science.gov (United States)

Cryopreservation damages rooster sperm membranes. Part of this damage is due to membrane transitioning from the fluid to the gel state as temperature is reduced. This damage may be prevented by increasing membrane fluidity at low temperatures by incorporating cholesterol or unsaturated lipids into t...

Improved quality of cryopreserved cheetah (Acinonyx jubatus) spermatozoa after centrifugation through Accudenz.

Science.gov (United States)

Crosier, Adrienne E; Henghali, Josephine N; Howard, Jogayle; Pukazhenthi, Budhan S; Terrell, Kimberly A; Marker, Laurie L; Wildt, David E

2009-01-01

Sperm cryopreservation, in combination with assisted reproductive techniques, is a valuable tool for the genetic management of endangered felids. However, the acrosome of the cheetah spermatozoon is especially sensitive to cryopreservation, with approximately 40% of spermatozoa experiencing acrosomal damage immediately after thawing and then another approximately 15% loss during the next 4 hours in vitro. Additionally, thawing causes a reduction in sperm motility by approximately 20% with another decrease of approximately 12% during subsequent incubation in vitro. We hypothesized that slow removal of glycerol from cryopreserved cheetah spermatozoa using an Accudenz gradient would improve acrosomal integrity, sperm motility longevity, and structural morphology. Accudenz was compared with traditional cheetah sperm processing methods for glycerol removal that involves washing, multistep resuspension, and swim-up processing. Electroejaculates (n = 21 total from 8 males) were washed in Ham F10 medium, and sperm pellets were resuspended in TEST-yolk buffer with 0% glycerol. Samples were cryopreserved in straws in 4% final glycerol, thawed, and assessed for percent intact acrosomes (% IA), percent motility (% M), and forward progressive status (FPS; scale, 0-5). Sperm motility index (SMI) was calculated as (% M + [FPS x 20]) / 2. In study 1, glycerol removal by centrifugation through an Accudenz gradient (4%, 10%) was compared with traditional sperm washing (control) and multistep resuspension protocols. At each time after centrifugation (hourly for 4 hours), % IA was improved (P cheetah sperm mitigates the significant loss in sperm quality that occurs after freeze-thawing. This alleviation of cellular damage resulting from cryopreservation contributes to a more than 10% improvement in overall sperm motility and, more importantly, allows retention of 40% or more of sperm with intact acrosomes.

An evaluation of soybean lecithin as an alternative to avian egg yolk in the cryopreservation of fish sperm.

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Yildiz, Cengiz; Bozkurt, Yusuf; Yavas, Ilker

2013-08-01

Plant-derived lecithin has been used as a more sanitary alternative to avian egg yolk in livestock sperm cryopreservation protocols but its efficacy for cryopreserving fish sperm has not previously been tested comparatively. Here various concentrations of soybean lecithin were evaluated for the cryopreservation of carp (Cyprinus carpio) sperm. Sexually mature fish were induced to spermiation and ovulation with ovopel. The extenders were prepared by using 300 mM glucose, 10% DMSO, supplemented with different ratios of lecithin (5%, 10%, 15%, and 20%) and 10% egg yolk (control I). Negative control was made without egg yolk and soybean lecithin (control II). The pooled semen was diluted separately at ratio of 1:3 (v/v) by using egg yolk and soybean-based extenders. Diluted semen placed into 0.25 ml straws were equilibrated at 4 °C for 15 min and frozen in liquid nitrogen vapor. Fertilization was conducted using a ratio of 1 × 10(5)spermatozoa/egg. Supplementation of 10% lecithin to extender showed the best cryoprotective effect for sperm motility and duration of motility against freezing damage compared to 15%, 20% and control II groups (plecithin provided a greater result in terms of fertilization success when compared to extenders containing 20% lecithin or control II (plecithin has a similar cryoprotective actions with conventional egg yolk-based extender against freezing damages and fertilization. Therefore, soybean lecithin is a suitable alternative to avian egg yolk for the cryopreservation of fish sperm. Copyright © 2013 Elsevier Inc. All rights reserved.

Immunization with Tc52 or its amino terminal domain adjuvanted with c-di-AMP induces Th17+Th1 specific immune responses and confers protection against Trypanosoma cruzi.

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Marina N Matos

2017-02-01

Full Text Available The development of new adjuvants enables fine modulation of the elicited immune responses. Ideally, the use of one or more adjuvants should result in the induction of a protective immune response against the specific pathogen. We have evaluated the immune response and protection against Trypanosoma cruzi infection in mice vaccinated with recombinant Tc52 or its N- and C-terminal domains (NTc52 and CTc52 adjuvanted either with the STING (Stimulator of Interferon Genes agonist cyclic di-AMP (c-di-AMP, a pegylated derivative of α-galactosylceramide (αGC-PEG, or oligodeoxynucleotides containing unmethylated CpG motifs (ODN-CpG. All groups immunized with the recombinant proteins plus adjuvant: Tc52+c-di-AMP, NTc52+c-di-AMP, CTc52+c-di-AMP, NTc52+c-di-AMP+αGC-PEG, NTc52+CpG, developed significantly higher anti-Tc52 IgG titers than controls. Groups immunized with c-di-AMP and Tc52, NTc52 or CTc52 showed the highest Tc52-specific IgA titers in nasal lavages. All groups immunized with the recombinant proteins plus adjuvant developed a strong specific cellular immune response in splenocytes and lymph node cells with significant differences for groups immunized with c-di-AMP and Tc52, NTc52 or CTc52. These groups also showed high levels of Tc52-specific IL-17 and IFN-γ producing cells, while NTc52+CpG group only showed significant difference with control in IFN-γ producing cells. Groups immunized with c-di-AMP and Tc52, NTc52 or CTc52 developed predominantly a Th17 and Th1immune response, whereas for NTc52+CpG it was a dominant Th1 response. It was previously described that αGC-PEG inhibits Th17 differentiation by activating NKT cells. Thus, in this work we have also included a group immunized with both adjuvants (NTc52+c-di-AMP+αGC-PEG with the aim to modulate the Th17 response induced by c-di-AMP. This group showed a significant reduction in the number of Tc52-specific IL-17 producing splenocytes, as compared to the group NTc52+c-di-AMP, which has

Development of methods for cryopreservation of rooster sperm from the endangered breed "Gallina Valenciana de Chulilla" using low glycerol concentrations.

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Blanch, E; Tomás, C; Casares, L; Gómez, E A; Sansano, S; Giménez, I; Mocé, E

2014-06-01

Glycerol (11%; v:v) is the cryoprotectant most often used for the cryopreservation of rooster sperm. However, chicken breeds differ in the resistance of their sperm to the cryopreservation process and endangered or local breeds usually present low fertilizing ability when conventional sperm cryopreservation protocols are used. The objective of this study was to optimize the protocol for the cryopreservation of the sperm from the endangered breed "Gallina Valenciana de Chulilla". For this purpose, 10 pools of semen from 43 roosters of this breed were cryopreserved using 8%, 7%, 6%, or 4% glycerol, and the sperm quality was determined immediately after thawing and in the insemination doses. Lohmann Brown Classic laying hens (n = 40) were used for the insemination trials. The sperm quality after cryopreservation progressively decreased as the glycerol concentration was reduced (P roosters frozen using 4% glycerol exhibited lower sperm quality but similar fertilizing ability compared with samples processed using higher glycerol concentrations. These results may provide useful information for developing cryopreservation protocols for other breeds. Copyright © 2014 Elsevier Inc. All rights reserved.

Cryopreserved Cadaveric Arterial Allograft for Arterial Reconstruction in Patients with Prosthetic Infection.

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Lejay, Anne; Delay, Charline; Girsowicz, Elie; Chenesseau, Bettina; Bonnin, Emilie; Ghariani, Mohamed-Zied; Thaveau, Fabien; Georg, Yannick; Geny, Bernard; Chakfe, Nabil

2017-11-01

The aim of this study was to report outcomes of cryopreserved arterial allografts used as a vascular substitute in the setting of prosthetic material infection. A retrospective analysis of prospectively collected data was conducted including all consecutive interventions performed with cryopreserved arterial allografts used for vascular reconstruction in the setting of prosthetic material infection between January 2005 and December 2014. Five year outcomes included allograft related re-interventions, survival, primary patency, and limb salvage rates. Fifty-three procedures were performed using cryopreserved allografts for vascular prosthetic infection: 25 procedures (47%) were performed at aorto-iliac level (Group 1) and 28 procedures (53%) at peripheral level (Group 2). The mean follow-up was 52 months. Five year allograft related re-intervention was 55% in Group 1 (6 allograft ruptures and 5 allograft aneurysm degenerations) and 33% in Group 2 (2 allograft ruptures and 7 allograft aneurysm degenerations). Five year survival was 40% and 68%, primary patency was 89% and 59% and limb salvage was 100% and 89% for Group 1 and 2 respectively. Use of cryopreserved arterial allografts provides acceptable results but is tempered by suboptimal 5 year outcomes with high re-intervention rates. Copyright © 2017 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.

Effectiveness analyses may underestimate protection of infants after group C meningococcal immunization.

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Vu, David M; Kelly, Dominic; Heath, Paul T; McCarthy, Noel D; Pollard, Andrew J; Granoff, Dan M

2006-07-15

Group C meningococcal conjugate-vaccine effectiveness in the United Kingdom declines from ~90% in the first year to 0% between 1 and 4 years after immunization in infants immunized at 2, 3, and 4 months of age and to 61% in toddlers given a single dose. Confidence intervals are wide, and the extent of protection is uncertain. Serum samples were obtained from children 3-5 years of age who were participants in a preschool booster-vaccine trial. Serum bactericidal activity was measured with human complement. Group C anticapsular antibody concentrations were measured by a radioantigen binding assay. Passive protection was analyzed in an infant rat bacteremia model. Serum samples from UK children who had been immunized 2-3 years earlier as infants or toddlers had higher levels of radioantigen binding, bactericidal activity, and passive protection than did historical control serum samples from unimmunized children (P or =1 : 4 (considered to be protective) than those immunized as toddlers (61% vs. 24%; Pprotection (50% and 41%, respectively; P=.4). We found no evidence of lower immunity in children immunized as infants than as toddlers. On the basis of serum bactericidal activity and/or passive protection, 40%-50% of both age groups are protected at 2-3 years after immunization, which was significantly greater than in unimmunized historical controls (<5%).

Immune responses to transgene and retroviral vector in patients treated with ex vivo-engineered T cells

NARCIS (Netherlands)

Lamers, C.H.; Willemsen, R.; Elzakker, P. van; Steenbergen-Langeveld, S. van; Broertjes, M.; Oosterwijk-Wakka, J.C.; Oosterwijk, E.; Sleijfer, S.; Debets, R.; Gratama, J.W.

2011-01-01

Adoptive transfer of immune effector cells that are gene modified by retroviral transduction to express tumor-specific receptors constitutes an attractive approach to treat cancer. In patients with metastatic renal cell carcinoma, we performed a study with autologous T cells genetically retargeted

Development of a sperm cryopreservation protocol for redside dace, Clinostomus elongatus: implications for genome resource banking

DEFF Research Database (Denmark)

Butts, Ian A.E.; Mokdad, A.; Trippel, E.A.

2013-01-01

of extirpation.We developed cryopreservation protocols by testing the effects of diluent (buffered sperm motility-inhibiting saline solution [BSMIS]; BSMIS + glycine; sucrose; and Hanks’ balanced salt solution [HBSS]), cryoprotectant (dimethyl sulfoxide [DMSO]; propylene glycol [PG]; N,N-dimethylacetamide [DMA...... rate was not significant for BSMIS + glycine or for HBSS, but an effect was detected for sucrose, with sperm frozen at 5◦C/min or 10◦C/min having higher motility than sperm frozen at 1◦C/min. The effect of extender was not significant at 1◦C/min or 5◦C/min, but an effect was detected at 10◦C...

Cryopreservation of collared peccary (Pecari tajacu) semen using different freezing curves, straw sizes, and thawing rates.

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Silva, M A; Peixoto, G C X; Castelo, T S; Lima, G L; Silva, A M; Oliveira, M F; Silva, A R

2013-08-01

The objective of this study was to verify the effect of different freezing curves, straw sizes, and thawing rates on the cryopreservation of collared peccary semen. Twelve ejaculates were obtained from captive adult males by electroejaculation, and evaluated for sperm motility, kinetic rating, viability, morphology, and functional membrane integrity. The ejaculates were diluted in a coconut water extender (ACP-116c) with egg yolk and glycerol, packaged into 0.25 mL or 0.50 mL plastic straws and cryopreserved in liquid nitrogen following a slow (-10 °C/min) or a fast (-40 °C/min) freezing curve. After one week, samples were thawed at 37 °C/1 min or 70 °C/8s and evaluated as reported for fresh semen, and also for kinematic parameters (computerized analysis). A significant decrease in sperm motility and kinetic rating was observed after glycerol addition at 5 °C and also after thawing for all the treatments (Pstraw size and thawing rate were taken as reference (P>0.05). In general, values for sperm characteristics found after thawing at 37 °C were better preserved than at 70 °C (Pstraws, which were similar for semen packaging (P>0.05). The evaluation of the kinematic parameters of sperm motility confirmed these results at values varying from 20% to 30% motile sperm for the samples tha wed at 37 °C, and values fewer than 12% motile sperm for samples thawed at 70 °C (Pstraws, but the thawing should be conducted at 37 °C/1 min. Copyright © 2013 Elsevier Inc. All rights reserved.

Cryopreservation of medicinal plants: role of melatonin

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Many useful plant species found in Canada are of conservation concern. In vitro storage and cryopreservation techniques guarantees safety of these species and have potential applications which may result in sustainable agriculture. Shoot tips of in vitro-grown plantlets of American elm, St John’s Wo...

Macrophage Inducible C-Type Lectin As a Multifunctional Player in Immunity

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Emmanuel C. Patin

2017-07-01

Full Text Available The macrophage-inducible C-type lectin (Mincle is an innate immune receptor on myeloid cells sensing diverse entities including pathogens and damaged cells. Mincle was first described as a receptor for the mycobacterial cell wall glycolipid, trehalose-6,6′-dimycolate, or cord factor, and the mammalian necrotic cell-derived alarmin histone deacetylase complex unit Sin3-associated protein 130. Upon engagement by its ligands, Mincle induces secretion of innate cytokines and other immune mediators modulating inflammation and immunity. Since its discovery more than 25 years ago, the understanding of Mincle’s immune function has made significant advances in recent years. In addition to mediating immune responses to infectious agents, Mincle has been linked to promote tumor progression, autoimmunity, and sterile inflammation; however, further studies are required to completely unravel the complex role of Mincle in these distinct host responses. In this review, we discuss recent findings on Mincle’s biology with an emphasis on its diverse functions in immunity.

Cryopreservation of Mycobacterium bovis isolates

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Cássia Yumi Ikuta

2016-11-01

Full Text Available Research, development of new biotechnological methods, diagnostic tests, confirmation of results, and reinvestigations are possible because of the availability of well-preserved living organisms maintained without any changes. Cryopreservation is a simpler, more reliable and long-term stable method for culture maintenance. Storage temperature and composition of the suspending vehicle are factors that affect the viability of mycobacterial strains. Three vehicles and three storage temperatures were evaluated to define a suitable cryoprotective medium for the preservation of Mycobacterium bovis strains. Colonies of sixteen M. bovis isolates were used to prepare the suspensions, which were then added to three vehicles: sterile 0.85% saline solution (SS, Middlebrook 7H9 broth (7H9, and Middlebrook 7H9 broth with sodium pyruvate (7H9p replacing glycerol. Aliquots of these suspensions were frozen by three different methods, directly in the -20°C freezer, directly in the -80°C freezer, and at -196°C by immersion in liquid nitrogen (LN. The frozen aliquots were thawed at room temperature after 45, 90 and 120 days. Mycobacterial viability was assessed by counting the living cells on plates of Stonebrink medium before and after the freezing procedure. Storage at -20°C exhibited a lower recovery of M. bovis compared to storage at -80°C (Dunn’s test, p=0.0018 and LN (Dunn’s test, p=0.0352. There was no statistically significant difference between storage at -80°C and in LN (Dunn’s test, p=0.1403, yet -80°C showed better results than LN. All three suspending vehicles showed no statistically significant difference in terms of viability (Friedman’s test, p=0.7765. Given the low loss proportion of 5% during storage at -20°C and the high cost equipment required for storage at -80°C and LN, we recommend storage at -20°C or -80°C, when this is available, for preservation of M. bovis field strains.

Cryopreservation and xenografting of human ovarian fragments: medulla decreases the phosphatidylserine translocation rate

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Vladimir Isachenko

2016-11-01

Full Text Available Abstract Background Phosphatidylserine is the phospholipid component which plays a key role in cell cycle signaling, specifically in regards to necrosis and apoptosis. When a cell affected by some negative factors, phosphatidylserine is no longer restricted to the intracellular side of membrane and can be translocated to the extracellular surface of the cell. Cryopreservation can induce translocation of phosphatidylserine in response to hypoxia, increasing intracellular Ca2+, osmotic disruption of cellular membranes, generation of reactive oxygen species and lipid peroxidation. As such the aim of this study was to test the level of phosphatidylserine translocation in frozen human medulla-contained and medulla-free ovarian tissue fragments. Methods Ovarian fragments from twelve patients were divided into small pieces of two types, medulla-free cortex (Group 1, n = 42, 1.5–3.0 × 1.5–3.0 × 0.5–0.8 mm and cortex with medulla (Group 2, n = 42, 1.5–3.0 × 1.5–3.0 × 1.5–2.0 mm, pre-cooled after operative removal to 5 °C for 24 h and then conventionally frozen with 6 % dimethyl sulfoxide, 6 % ethylene glycol and 0.15 M sucrose in standard 5-ml cryo-vials. After thawing at +100 °C and step-wise removal of cryoprotectants in 0.5 M sucrose, ovarian pieces were xenografted to SCID mice for 45 days. The efficacy of tissues cryopreservation, taking into account the presence or absence of medulla, was evaluated by the development of follicles (histology with hematoxylin-eosin and through the intensity of translocation of phosphatidylserine (FACS with FITC-Annexin V and Propidium Iodide. Results For Groups 1 and 2, the mean densities of follicles per 1 mm3 were 9.8, and 9.0, respectively. In these groups, 90 and 90 % preantral follicles appeared morphologically normal. However, FACS analysis showed a significantly decreased intensity of translocation of phosphatidylserine (FITC-Annexin V positive after

Cryopreservation of spores of Dicksonia sellowiana: an endangered tree fern indigenous to South and Central America.

Science.gov (United States)

Rogge, G D; Viana, A M; Randi, A M

2000-01-01

Spores of Dicksonia sellowiana (Presl.) Hook., an endangered tree fern, were stored in liquid nitrogen. Surface sterilized spores were placed in 1 ml sterile polypropylene cryotubes and were plunged into liquid nitrogen cryo-cans for 15 minutes, 15 days, 1 month and 3 months. In all, of the treatments the percentage of germination was higher than the control (fresh spores). Germination in Dyer and MS media supplement with 10 (-7) M and 5 x 10(-7) M BA was also promoted as comparing to control. There was no difference between the germination of spores thawed rapidly in a water bath at 45 degree C during 5 minutes or slowly at room temperature. Cryopreservation seems to promote germination of some dormant spores of D. sellowiana. The pre-treatment in cryoprotective solution of dimethyl sulphoxide 15%(v/v) in 1 M glycerol inhibited the germination of cryopreserved spores

EFFECT OF DIFFERENT CRYOPROTECTIVE AGENTS ON SKIM MILK AND DIMITROPOULUS EXTENDER FOR STALLION SEMEN CRYOPRESERVATION

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R.I. Arifiantini

2014-10-01

Full Text Available s to assess different CPAs on stallion semen cryopreservation. Skim milk (SM and Dimitropoulos(DV were the extenders used in this study; each was added by glycerol (Gly, combination of ethyleneglycol-glycerol (EG+Gly or dimethilformamide (DMF. Each semen sample was evaluated and dividedequally into six tubes; semen in the three tubes was diluted 1:1 with (SM, while in the remaining tubesthe semen was diluted 1:1 by DV. After being diluted, all tubes were centrifuged at 1006xg for 10minutes. The supernatan discarded, the pellet was rediluted by SM trehalosa or DV trehalose, and addedby G, EG+Gly, or DMF to reach the final sperm concentration of 200x106/ml. The extended semen wasindividually packed in 0.3 ml minitube, equilibrated at 4oC for 2 hours, frozen in liquid nitrogen vaporfor 10 minutes, and then was stored in liquid nitrogen container at -196 oC. After 24 hours, the semenwas thawed at 37 oC for 30 second. There were no significantly different (p>0.05 on the percentages ofmotile and viable sperm in SMT (21.7% and 43.4%, respectively compared with those extended withDV T extender (26.9% and 50.8%, respectively. DMF demonstrated better results as CPA compared tothe others; and DVTDMF combination had the best protection during cryopreservation in this study.

Immunization alters body odor.

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Kimball, Bruce A; Opiekun, Maryanne; Yamazaki, Kunio; Beauchamp, Gary K

2014-04-10

Infections have been shown to alter body odor. Because immune activation accompanies both infection and immunization, we tested the hypothesis that classical immunization might similarly result in the alteration of body odors detectable by trained biosensor mice. Using a Y-maze, we trained biosensor mice to distinguish between urine odors from rabies-vaccinated (RV) and unvaccinated control mice. RV-trained mice generalized this training to mice immunized with the equine West Nile virus (WNV) vaccine compared with urine of corresponding controls. These results suggest that there are similarities between body odors of mice immunized with these two vaccines. This conclusion was reinforced when mice could not be trained to directly discriminate between urine odors of RV- versus WNV-treated mice. Next, we trained biosensor mice to discriminate the urine odors of mice treated with lipopolysaccharide (LPS; a general elicitor of innate immunological responses) from the urine of control mice. These LPS-trained biosensors could distinguish between the odors of LPS-treated mouse urine and RV-treated mouse urine. Finally, biosensor mice trained to distinguish between the odors of RV-treated mouse urine and control mouse urine did not generalize this training to discriminate between the odors of LPS-treated mouse urine and control mouse urine. From these experiments, we conclude that: (1) immunization alters urine odor in similar ways for RV and WNV immunizations; and (2) immune activation with LPS also alters urine odor but in ways different from those of RV and WNV. Published by Elsevier Inc.

Serum-Free Cryopreservation of Five Mammalian Cell Lines in Either a Pelleted or Suspended State

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Corsini Joe

2004-01-01

Full Text Available Herein we have explored two practical aspects of cryopreserving cultured mammalian cells during routine laboratory maintenance. First, we have examined the possibility of using a serum-free, hence more affordable, cryopreservative. Using five mammalian lines (Crandell Feline Kidney, MCF7, A72, WI 38 and NB324K, we found that the serum-free alternative preserves nearly as efficiently as the serum-containing preservatives. Second, we compared cryostorage of those cells in suspended versus a pellet form using both aforementioned cryopreservatives. Under our conditions, cells were in general recovered equally well in a suspended versus a pellet form.

Requirement for Innate Immunity and CD90+ NK1.1− Lymphocytes to Treat Established Melanoma with Chemo-Immunotherapy

Science.gov (United States)

Moskalenko, Marina; Pan, Michael; Fu, Yichun; de Moll, Ellen H.; Hashimoto, Daigo; Mortha, Arthur; Leboeuf, Marylene; Jayaraman, Padmini; Bernardo, Sebastian; Sikora, Andrew G.; Wolchok, Jedd; Bhardwaj, Nina; Merad, Miriam; Saenger, Yvonne

2015-01-01

We sought to define cellular immune mechanisms of synergy between tumor-antigen–targeted monoclonal antibodies and chemotherapy. Established B16 melanoma in mice was treated with cytotoxic doses of cyclophosphamide in combination with an antibody targeting tyrosinase-related protein 1 (αTRP1), a native melanoma differentiation antigen. We find that Fcγ receptors are required for efficacy, showing that antitumor activity of combination therapy is immune mediated. Rag1−/− mice deficient in adaptive immunity are able to clear tumors, and thus innate immunity is sufficient for efficacy. Furthermore, previously treated wild-type mice are not significantly protected against tumor reinduction, as compared with mice inoculated with irradiated B16 alone, consistent with a primarily innate immune mechanism of action of chemo-immunotherapy. In contrast, mice deficient in both classical natural killer (NK) lymphocytes and nonclassical innate lymphocytes (ILC) due to deletion of the IL2 receptor common gamma chain IL2γc−/−) are refractory to chemo-immunotherapy. Classical NK lymphocytes are not critical for treatment, as depletion of NK1.1+ cells does not impair antitumor effect. Depletion of CD90+NK1.1− lymphocytes, however, both diminishes therapeutic benefit and decreases accumulation of macrophages within the tumor. Tumor clearance during combination chemo-immunotherapy with monoclonal antibodies against native antigen is mediated by the innate immune system. We highlight a novel potential role for CD90+NK1.1− ILCs in chemo-immunotherapy. PMID:25600438

Cryopreservation of adult unrelated donor products in hematopoietic cell transplantation: the OneMatch experience and systematic review of the literature.

Science.gov (United States)

Aziz, Joseph; Morris, Gail; Rizk, Mina; Shorr, Risa; Mercer, Dena; Young, Kimberly; Allan, David

2017-11-01

The frequency of cryopreserving blood stem or progenitor products from unrelated donors is not known and the underlying reasons are poorly documented. Greater insight is needed to develop policies on cryopreservation that balance donor safety with patient needs. Cryopreservation requests between January 1, 2014, and May 31, 2016, at the OneMatch Stem Cell and Marrow Network at Canadian Blood Services were reviewed and a systematic review of the literature was performed. Thirty products of 719 (4.2%) unrelated donor collections facilitated by OneMatch were cryopreserved. Patient-related reasons were most common and included the need to delay transplant for continued antimicrobial treatment (six patients), patient too deconditioned to proceed with scheduled transplant (five patients), and/or need for more treatment for relapsed disease (three patients). Donor-related issues leading to cryopreservation requests were less common (five cases), mainly due to lack of donor availability after attempting to reschedule. Cryopreservation of a product that was never infused occurred infrequently (two cases, 7%). In our systematic review of the literature, 993 cases were identified in 32 published reports. Both patient-related and donor-related reasons were cited but not specifically reported, precluding quantitative insight regarding the relative frequency of causes. The impact of cryopreservation on hematopoietic engraftment appears negligible when compared to controls in a subset of studies; however, reporting of outcomes was inconsistent. Future studies with standard outcome measures are needed to clarify the impact of cryopreservation on engraftment and other transplant outcomes. International guidelines that consider the ethical framework surrounding requests for donor product cryopreservation are needed. © 2017 AABB.

Effect of traditional Chinese medicine for treating human immunodeficiency virus infections and acquired immune deficiency syndrome: Boosting immune and alleviating symptoms.

Science.gov (United States)

Zou, Wen; Wang, Jian; Liu, Ying

2016-01-01

To respond to the human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) epidemic in China, the integration of antiretroviral therapy (ART) and traditional Chinese medicine (TCM) has important implications in health outcomes, especially in China where the use of TCM is widespread. The National Free TCM Pilot Program for HIV Infected People began in 5 provinces (Henan, Hebei, Anhui, Hubei, and Guangdong) in 2004, and quickly scaled up to 19 provinces, autonomous regions, and municipalities in China including some places with high prevalence, 26,276 adults have been treated thus far. Usually, people with HIV infection seek TCM for four main reasons: to enhance immune function, to treat symptoms, to improve quality of life, and to reduce side effects related to medications. Evidences from randomized controlled clinical trials suggested some beneficial effects of use of traditional Chinese herbal medicine for HIV infections and AIDS. More proofs from large, well-designed, rigorous trials is needed to give firm support. Challenges include interaction between herbs and antiretroviral drugs, stigma and discrimination. The Free TCM Program has made considerable progress in providing the necessary alternative care and treatment for HIV-infected people in China, and has strong government support for continued improvement and expansion, establishing and improving a work mechanism integrating Chinese and Western medicines.

Generation of juvenile rainbow trout derived from cryopreserved whole ovaries by intraperitoneal transplantation of ovarian germ cells.

Science.gov (United States)

Lee, Seungki; Katayama, Naoto; Yoshizaki, Goro

2016-09-23

Cryopreservation of fish sperm offers the practical applications in the selective breeding and biodiversity conservation. However, because of the lack of cryopreservation methods for fish eggs and embryos, maternally inherited cytoplasmic compartments cannot be successfully preserved. We previously developed an alternative method to derive functional eggs and sperm from cryopreserved whole testis by transplanting testicular cells into female and male recipients. However, if target fish had ovaries, the previous method employing male-derived germ cells would be ineffective. Here, we aimed to generate functional gametes from cryopreserved whole ovaries by transplanting ovarian germ cells into peritoneal cavity of sterile hatchlings. Cryopreservation conditions for rainbow trout ovaries (1.0 M DMSO, 0.1 M trehalose, and 10% egg yolk) were optimized by testing several different cryoprotective agents. Ovarian germ cells from thawed ovaries were intraperitoneally transplanted into allogeneic triploid hatchlings. Transplanted germ cells migrated toward and were incorporated into recipient gonads, where they underwent gametogenesis. Transplantation efficiency of ovarian germ cells remained stable after cryopreservation period up to 1185 days. Although all triploid recipients that did not undergo transplantation were functionally sterile, 5 of 25 female recipients and 7 of 25 male recipients reached sexual maturity at 2.5 years post-transplantation. Inseminating the resultant eggs and sperm generated viable offspring displaying the donor characteristics of orange body color, green fluorescence, and chromosome numbers. This method is thus a breakthrough tool for the conservation of endangered fish species that are crucial to cryopreserve the genetic resources of female fish. Copyright © 2016 Elsevier Inc. All rights reserved.

Protection against avian metapneumovirus subtype C in turkeys immunized via the respiratory tract with inactivated virus.

Science.gov (United States)

Cha, Ra Mi; Khatri, Mahesh; Sharma, Jagdev M

2011-01-10

Avian metapneumovirus subtype C (aMPV/C) causes a severe upper respiratory tract (URT) infection in turkeys. Turkeys were inoculated oculonasally with inactivated aMPV/C adjuvanted with synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid (Poly IC). Immunized turkeys had elevated numbers of mucosal IgA+ cells in the URT and increased levels of virus-specific IgG and IgA in the lachrymal fluid and IgG in the serum. After 7 or 21 days post immunization, turkeys were challenged oculonasally with pathogenic aMPV/C. Immunized groups were protected against respiratory lesions induced by the challenge virus. Further, the viral copy number of the challenge virus in the URT were significantly lower in the immunized turkeys than in the unimmunized turkeys (P<0.05). These results showed that inactivated aMPV/C administered by the respiratory route induced protective immunity against pathogenic virus challenge. Copyright © 2010 Elsevier Ltd. All rights reserved.

Distinctive cellular immunity in genetically susceptible BALB/c mice recovered from Leishmania major infection or after subcutaneous immunization with killed parasites

International Nuclear Information System (INIS)

Liew, F.Y.; Dhaliwal, J.S.

1987-01-01

Genetically susceptible BALB/c mice are refractory to further infection after recovery from Leishmania major infection after a sublethal dose of gamma-irradiation. In contrast, mice immunized with killed promastigotes s.c. develop exacerbated lesions after infection. Both groups of mice produce only a low level of specific antibody and no detectable cytotoxic T cells, but do have a strong antigen-specific DTH, which is adoptively transferable with Lyt-1+2-, L3T4+ T cells. Kinetic and histological studies revealed that mice immunized s.c. developed Jones-Mote hypersensitivity, peaking at 15 hr. with little mononuclear cell infiltration at the site of antigen administration; whereas mice that had recovered from infection developed tuberculin-type of reactivity, peaking at 24 to 48 hr, with intense mononuclear cell infiltration. Splenic T cells from recovered mice, when injected into the footpads of normal recipients together with live promastigotes, were able to retard lesion development; whereas T cells from s.c. immunized mice, when similarly transferred, accelerated disease progression. Antigen-specific culture supernatant of spleen cells from recovered mice also activated normal resident peritoneal macrophages to kill intracellular L. major amastigotes and tumor cells. Culture supernatants of spleen cells from s.c. immunized or normal mice were devoid of such activities. Part of the macrophage-activating potential can be inhibited by antibody specific for IFN-gamma. These results therefore demonstrate that whereas the Jones-Mote reaction is correlated with disease exacerbation, the tuberculin-type of DTH may be protective. Furthermore, in vivo immunity is directly related to the capacity of T cells to produce macrophage-activating factor

Fertilization and Embryo Development of Fresh and Cryopreserved Sibling Oocytes

Directory of Open Access Journals (Sweden)

Robert F. Casper

2010-01-01

Full Text Available Background: Oocyte cryopreservation is potentially the best way to preserve female fertility forunmarried women or young girls at risk of losing ovarian function. The aim of this study was tocompare fertilization and embryo development in frozen-thawed oocytes to their fresh siblings inwomen undergoing in vitro fertilization (IVF and embryo transfer (ET.Materials and Methods: Eleven infertile women undergoing infertility treatment, between theages of 24 to 37 years (mean ± SD = 31.6 ± 3.5, were included in this study. Mature oocytesfrom each patient were randomized into cryopreserved and fresh groups prior to intracytoplasmicsperm injection (ICSI. One hundred and thirty nine oocytes were retrieved, of which 105 were atmetaphase II (MII. Forty- five fresh MII oocytes were kept in culture whereas their sibling 60 MIIoocytes were cryopreserved using a slow cooling protocol. The frozen oocytes remained in LN2for 2 hours before thawing. ICSI was performed 1-2 hours after thawing for frozen oocytes and 4-5hours after retrieval for fresh oocytes. Fertilization and embryo development were compared.Results: Following thawing, 31 oocytes (51.6 % survived and 22 fertilized (79% while 32 freshoocytes fertilized upon ICSI (71%. The mean ± SE scores for embryos developing from frozenthawedoocytes were significantly lower at 48 and 72 hours post-ICSI than for embryos resultingfrom fresh oocytes (p<0.05.Conclusion: Our data demonstrated that oocyte freezing resulted in acceptable survival ratesfollowing cryopreservation, and similar fertilization rates following ICSI as compared to the freshsibling oocytes. However the number of blastomeres and the embryo quality on day three wassuperior in embryos from fresh oocytes when compared to the frozen oocytes.

[Prospects for Application of Gases and Gas Hydrates to Cryopreservation].

Science.gov (United States)

Shishova, N V; Fesenko, E E

2015-01-01

In the present review, we tried to evaluate the known properties of gas hydrates and gases participating in the formation of gas hydrates from the point of view of the mechanisms of cryoinjury and cryoprotection, to consider the papers on freezing biological materials in the presence of inert gases, and to analyze the perspectives for the development of this direction. For the purpose, we searched for the information on the physical properties of gases and gas hydrates, compared processes occured during the formation of gas hydrates and water ice, analyzed the influence of the formation and growth of gas hydrates on the structure of biological objects. We prepared a short review on the biological effects of xenon, krypton, argon, carbon dioxide, hydrogen sulfide, and carbon monoxide especially on hypothermal conditions and probable application of these properties in cryopreservation technologies. The description of the existing experiments on cryopreservation of biological objects with the use of gases was analyzed. On the basis of the information we found, the most perspective directions of work in the field of cryopreservation of biological objects with the use of gases were outlined. An attempt was made to forecast the potential problems in this field.

Immune response in the lungs following oral immunization with bacterial lysates of respiratory pathogens.

Science.gov (United States)

Ruedl, C; Frühwirth, M; Wick, G; Wolf, H

1994-03-01

We have investigated the local immune response of the BALB/c mouse respiratory tract after oral immunization with a bacterial lysate of seven common respiratory pathogens. After two immunization on five consecutive days, we examined the immunoglobulin (immunoglobulin G [IgG], IgM, and IgA) secretion rates of cells isolated from the lungs and compared them with those of spleen cells of orally immunized and nonimmunized animals by using a new test system based on time-resolved fluorescence. The procedure followed the principle of the classical ELISPOT test with nitrocellulose-bottomed microtiter plates, but europium (Eu3+)-linked streptavidin rather than enzyme-conjugated streptavidin was used, with the advantage of quantifying secreted immunoglobulins instead of detecting single antibody-secreting cells. Lymphocytes isolated from the lungs of treated animals revealed significant increases in total and antigen-specific IgA synthesis compared with the rates of the controls, whereas IgG and IgM production rates showed no remarkable differences. In addition, the sera of treated mice revealed higher antigen-specific IgA titers but not increased IgM and IgG levels. We conclude that priming the gut-associated lymphoid tissue with bacterial antigens of pneumotropic microorganisms can elicit an enhanced IgA response in a distant mucosal effector site, such as the respiratory tract, according to the concept of a common mucosa-associated immune system.

Signalling through C-type lectin receptors: shaping immune responses

NARCIS (Netherlands)

Geijtenbeek, Teunis B. H.; Gringhuis, Sonja I.

2009-01-01

C-type lectin receptors (CLRs) expressed by dendritic cells are crucial for tailoring immune responses to pathogens. Following pathogen binding, CLRs trigger distinct signalling pathways that induce the expression of specific cytokines which determine T cell polarization fates. Some CLRs can induce

Isolation and Characterization of Human Dental Pulp Stem Cells from Cryopreserved Pulp Tissues Obtained from Teeth with Irreversible Pulpitis.

Science.gov (United States)

Malekfar, Azin; Valli, Kusum S; Kanafi, Mohammad Mahboob; Bhonde, Ramesh R

2016-01-01

Human dental pulp stem cells (DPSCs) are becoming an attractive target for therapeutic purposes because of their neural crest origin and propensity. Although DPSCs can be successfully cryopreserved, there are hardly any reports on cryopreservation of dental pulp tissues obtained from teeth diagnosed with symptomatic irreversible pulpitis during endodontic treatment and isolation and characterization of DPSCs from such cryopreserved pulp. The aim of this study was to cryopreserve the said pulp tissues to propagate and characterize isolated DPSCs. A medium consisting of 90% fetal bovine serum and 10% dimethyl sulfoxide was used for cryopreservation of pulp tissues. DPSCs were isolated from fresh and cryopreserved pulp tissues using an enzymatic method. Cell viability and proliferation were determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. DPSC migration and interaction were analyzed with the wound healing assay. Mesenchymal characteristics of DPSCs were verified by flow cytometric analysis of cell surface CD markers. The osteogenic and adipogenic potential of DPSCs was shown by von Kossa and oil red O staining methods, respectively, and the polymerase chain reaction method. We found no significant difference in CD marker expression and osteogenic and adipogenic differentiation potential of DPSCs obtained from fresh and cryopreserved dental pulp tissue. Our study shows that dental pulp can be successfully cryopreserved without losing normal characteristics and differentiation potential of their DPSCs, thus making them suitable for dental banking and future therapeutic purposes. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Cryopreservation of coconut (Cocos nucifera L.) zygotic embryos does not induce morphological, cytological or molecular changes in recovered seedlings.

Science.gov (United States)

Sisunandar; Rival, Alain; Turquay, Patricia; Samosir, Yohannes; Adkins, Steve W

2010-07-01

The present study aimed at exploring the fidelity of coconut (Cocos nucifera L.) plants recovered from cryopreservation. Zygotic embryos from various different cultivars were cryopreserved following four successive steps, namely: rapid dehydration, rapid freezing, rapid thawing and in vitro recovery followed by acclimatization. At the end of the acclimatization period, the seedlings were compared to counterparts of the same age, which were produced from non-cryopreserved embryos. Both series were submitted to morphological, cytological and molecular comparisons. No significant differences in terms of growth rates could be measured. In addition, no morphological variation could be detected through the measurement of shoot elongation rates, production of opened leaves, and the number and total length of primary roots. Karyotype analysis revealed the same chromosome number (2n = 32) in all studied cultivars independently of cryopreservation. No significant differences could be observed between control and cryopreserved material concerning the type of chromosomes, the length of the long and short arms, the arm length ratio and the centromeric index. However, idiogram analysis did show a greater number of black banding on chromosomes isolated from cryopreserved material. Genetic and epigenetic fidelity was assessed through microsatellite (SSR) analysis and global DNA methylation rates; no significant differences would be observed between genomic DNAs isolated from seedlings originating from cryopreserved embryos and respective controls. In conclusion, our results suggest that the method of cryopreservation under study did not induce gross morphological, genetic or epigenetic changes, thus suggesting that it is an appropriate method to efficiently preserve coconut germplasm.

Embryo transfer using cryopreserved Boer goat blastocysts ...

African Journals Online (AJOL)

The aim of this trial was to evaluate the effect of embryo cryopreservation techniques on the survivability of embryos and fertility following transfer to Boer goat does. The oestrous cycles of 27 mature recipients Boer goat does were synchronised using controlled internal drug release dispensers (CIDR's) for 16 days. At CIDR ...

Cryopreservation of human skeletal muscle impairs mitochondrial function

DEFF Research Database (Denmark)

Larsen, Steen; Wright-Paradis, C; Gnaiger, E

2012-01-01

functionality after long term cryopreservation (1 year). Skeletal muscle samples were preserved in dimethyl sulfoxide (DMSO) for later analysis. Human skeletal muscle fibres were thawed and permeabilised with saponin, and mitochondrial respiration was measured by high-resolution respirometry. The capacity...

ATP Release from Chemotherapy-Treated Dying Leukemia Cells Elicits an Immune Suppressive Effect by Increasing Regulatory T Cells and Tolerogenic Dendritic Cells.

Science.gov (United States)

Lecciso, Mariangela; Ocadlikova, Darina; Sangaletti, Sabina; Trabanelli, Sara; De Marchi, Elena; Orioli, Elisa; Pegoraro, Anna; Portararo, Paola; Jandus, Camilla; Bontadini, Andrea; Redavid, Annarita; Salvestrini, Valentina; Romero, Pedro; Colombo, Mario P; Di Virgilio, Francesco; Cavo, Michele; Adinolfi, Elena; Curti, Antonio

2017-01-01

Chemotherapy-induced immunogenic cell death can favor dendritic cell (DC) cross-priming of tumor-associated antigens for T cell activation thanks to the release of damage-associated molecular patterns, including ATP. Here, we tested the hypothesis that in acute myeloid leukemia (AML), ATP release, along with its well-known immune stimulatory effect, may also contribute to the generation of an immune suppressive microenvironment. In a cohort of AML patients, undergoing combined daunorubicin and cytarabine chemotherapy, a population of T regulatory cells (Tregs) with suppressive phenotype, expressing the immune checkpoint programmed cell death protein 1 (PD-1), was significantly increased. Moving from these results, initial in vitro data showed that daunorubicin was more effective than cytarabine in modulating DC function toward Tregs induction and such difference was correlated with the higher capacity of daunorubicin to induce ATP release from treated AML cells. DCs cultured with daunorubicin-treated AML cells upregulated indoleamine 2,3-dioxygenase 1 (IDO1), which induced anti-leukemia Tregs. These data were confirmed in vivo as daunorubicin-treated mice show an increase in extracellular ATP levels with increased number of Tregs, expressing PD-1 and IDO1 + CD39 + DCs. Notably, daunorubicin failed to induce Tregs and tolerogenic DCs in mice lacking the ATP receptor P2X7. Our data indicate that ATP release from chemotherapy-treated dying cells contributes to create an immune suppressive microenvironment in AML.

Aging of oxygen and hydrogen plasma discharge treated a-C:H and ta-C coatings

Energy Technology Data Exchange (ETDEWEB)

Bachmann, Svenja [Physics of Surfaces, Institute of Materials Science, Technische Universität Darmstadt, Alarich-Weiss-Str. 16, 64287 Darmstadt (Germany); BMW Group, Hufelandstraße 4, 80788 Munich (Germany); Schulze, Marcus [Physics of Surfaces, Institute of Materials Science, Technische Universität Darmstadt, Alarich-Weiss-Str. 16, 64287 Darmstadt (Germany); Center of Smart Interfaces, Technische Universität Darmstadt, Alarich-Weiss-Str. 10, 64287 Darmstadt (Germany); Morasch, Jan [Institute of Materials Science, Technische Universität Darmstadt, Surface Science Division, Jovanka-Bonschits-Straße 2, 64287 Darmstadt (Germany); Hesse, Sabine [Physics of Surfaces, Institute of Materials Science, Technische Universität Darmstadt, Alarich-Weiss-Str. 16, 64287 Darmstadt (Germany); Center of Smart Interfaces, Technische Universität Darmstadt, Alarich-Weiss-Str. 10, 64287 Darmstadt (Germany); Hussein, Laith [Eduard-Zintl-Institut, Department of Chemistry, Technische Universität Darmstadt, Alarich-Weiss-Str. 12, 64287, Darmstadt (Germany); Krell, Lisa; Schnagl, Johann [BMW Group, Hufelandstraße 4, 80788 Munich (Germany); Stark, Robert W. [Physics of Surfaces, Institute of Materials Science, Technische Universität Darmstadt, Alarich-Weiss-Str. 16, 64287 Darmstadt (Germany); Center of Smart Interfaces, Technische Universität Darmstadt, Alarich-Weiss-Str. 10, 64287 Darmstadt (Germany); and others

2016-05-15

Highlights: • The water CA of O{sub 2} and H{sub 2} plasma treated a-C:H and ta-C changes from hydrophillic to hydrophobic on aging. • XPS study indicates that the decrease in surface energy of plasma treated a-C:H and ta-C could be due to adsorption of organic component from air. • The COFLFM of O{sub 2} and H{sub 2} plasma treated a-C:H and ta-C decreased upon aging. • The COF of glycerol lubricated ta-C showed no sign of change upon aging. - Abstract: Surface modification with gas plasma is an efficient and easy way to improve the surface energy and the tribological behavior of diamond-like carbon (DLC) coatings, e.g., in biomedical implants or as protective coatings. However, the long-term performance of the plasma treated DLC coatings is not fully clear. We thus studied the long-term stability of two kinds of DLC coatings, namely (a) hydrogenated amorphous carbon (a-C:H) and (b) tetrahedral amorphous carbon (ta-C) treated at different radio frequency (RF) power and time of oxygen (O{sub 2}) and hydrogen (H{sub 2}) plasma. Their surface properties, e.g. surface wettability, structure and tribological behavior, were studied at regular intervals for a period of two months using contact angle goniometer, Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), lateral force microscopy (LFM) and ball on disc apparatus. The surface energy of both the coatings decreased upon aging. The higher the RF power and time of treatment, the higher was the hydrophobicity upon aging. XPS analysis showed that the increase in hydrophobicity could be due to adsorption of unavoidable volatile organic components in the atmosphere. The H{sub 2} plasma treated ta-C was capable of rearranging its structural bonds upon aging. The nano-friction measurements by LFM showed that the coefficient of friction of plasma treated a-C:H and ta-C decreased upon aging. The results indicate that the surface properties of plasma treated a‐C:H and ta‐C are not stable on long-term and are

Outlining adequate protocols for Lidia bull epididymal storage and sperm cryopreservation: use of glycerol, dimethylformamide and N-acetylcysteine

Directory of Open Access Journals (Sweden)

Elvira Matilla

2017-12-01

Full Text Available The Lidia bovine breed is an important hallmark of the Spanish cattle industry. Bulls are selected based upon aggressiveness and epididymal sperm cryopreservation is the way to obtain and store their genetics. There are not specifically designed protocols yet to perform Lidia bull sperm cryopreservation. The present study aimed to determine if a tris-fructose-citrate-egg yolk (20% v/v; TFY extender supplemented with 7% glycerol (TFY1 or 3.5% glycerol plus 3.5% dimethylformamide (DMF; TFY2 are suitable media for cryopreservation of epididymal Lidia bull sperm. Moreover, the effect of N-acetylcysteine (NAC, a potent antioxidant, was evaluated. The epididymis were stored at 4°C for 24, 48, 72 or 96 h, and both freezing media were tested as such or supplemented with 1 or 2.5 mM of NAC. Our data demonstrated that post-thaw viability was well maintained (TFY1: 50.8% ± 1.9 at 24 h and 52.4% ± 0.8 at 96 h and TFY2: 52.6% ± 1.6 at 24 h and 56.1% ± 1.8 at 96 h; mean % ± SEM; p>0.05 as also were total and progressive sperm motility, high mitochondrial membrane potential, ROS production, DNA status and acrosomal intactness of Lidia bull sperm up to 96 h of epididymal storage, all extender variations being similar (p>0.05. In conclusion, the use of TFY medium supplemented either with 7% glycerol alone or the combination of 3.5% glycerol and 3.5% DMF were equally safe choices for epididymal Lidia bull sperm cryopreservation, and NAC addition did not significantly improve sperm post-thaw quality.

Anti-oxidant supplementation improves boar sperm characteristics and fertility after cryopreservation: comparison between cysteine and rosemary (Rosmarinus officinalis).

Science.gov (United States)

Malo, C; Gil, L; Gonzalez, N; Martínez, F; Cano, R; de Blas, I; Espinosa, E

2010-08-01

Anti-oxidants partially ameliorated the detrimental effects of reactive oxidative substances produced during cryopreservation. The objective of the study was to determine the effect of anti-oxidant addition to the freezing extender on boar semen qualities and fertility capacity. Ejaculates were collected from a previously selected boar and semen samples were processed using the straw freezing procedure. In experiment 1, semen samples were cryopreserved in lactose-egg yolk solution supplemented with various concentrations of cysteine (0, 5 and 10mM) to determinate a cysteine concentration capable of producing a protective effect during cryopreservation. Semen quality (total motility, progressive motility, viability, acrosome integrity and hypoosmotic swelling test) was evaluated after freezing and thawing and then every hour for 3h. In experiment 2, ejaculates were cryopreserved with lactose-egg yolk extender with or without the following anti-oxidants: cysteine, rosemary (Rosmarinus officinalis) and cysteine plus rosemary. Semen quality was evaluated. In the experiment 3, fertility capacity of semen frozen in anti-oxidant supplementation extenders was examined in vitro. A total of 2232 oocytes were in vitro matured and inseminated with frozen-thawed sperm. In summary: (i) the effective concentration of cysteine in freezing extender was 10mM; (ii) the addition of exogenous rosemary or cysteine to the freezing extender positively affected post-thawed viability and acrosome integrity. Only rosemary supplementation improved total motility at 3h and progressive motility at any time; (iii) the inclusion of rosemary into the extender was effective in penetration and cleavage rate and also in the efficiency of the fertilization system. (c) 2010 Elsevier Inc. All rights reserved.

Kinetics of antibody responses after primary immunization with meningococcal serogroup C conjugate vaccine or secondary immunization with either conjugate or polysaccharide vaccine in adults

NARCIS (Netherlands)

de Voer, Richarda M.; van der Klis, Fiona R. M.; Engels, Carla W. A. M.; Schepp, Rutger M.; van de Kassteele, Jan; Sanders, Elisabeth A. M.; Rijkers, Ger T.; Berbers, Guy A. M.

2009-01-01

In the Netherlands the meningococcal serogroup C conjugate (MenCC) vaccine is administered as a single dose at 14 months. We evaluated the kinetics of isotype-specific antibodies in adults (n = 21) after primary immunization with MenCC or secondary immunization with MenCC or plain MenC

Spermatic Viability of Cryopreserved Semen of Piau swine breed analyzed by Thermo Resistant Test

Directory of Open Access Journals (Sweden)

Maurício Hoshino da Costa Barros

2014-06-01

Full Text Available ABSTRACT. Barros M.H.C., Shiomi H.H., Amorim L.S., Guimarães S.E.F., Lopes P.S., Siqueira J.B., Pinho R.O. & Guimarães J.D. [Spermatic Viability of Cryopreserved Semen of Piau swine breed analyzed by Thermo Resistant Test.] Viabilidade espermática de sêmen congelado de suínos da raça Piau avaliada pelo Teste de Termorresistência. Revista Brasileira de Medicina Veterinária 36(2:131-136, 2014. Departamento de Veterinária, Universidade Federal de Viçosa, Av. Peter Henry Rolfs, s/n, Viçosa, MG 36571-000, Brasil. E-mail: hugoshiomi@hotmail.com The objective of this study was to verify three protocols of semen cryopreservation on spermatic viability after thawing from Piau swine breed (Sus scrofa, by thermo resistant test (TTR. Twenty two ejaculates from 5 Piau mature boars were collected by glove hand technique. To freezing, the ejaculates was split and submitted to three protocols (P: (P1 freezing method proposed by Furst et al. (2005, altered by diluent media, (P2 freezing method proposed by Furst et al. (2005, altered by cooled curve; and (P3 freezing method proposed by Ohata et al. (2001. After thawing, semen was submitted to TTR, been incubated at 37o C by 2 hours, motility sperm (MOT and vigor (VIG was analyzed at 30 minutes of interval. MOT and VIG after thawing was 20.9±12.4, 29.5±10.9 and 49.5±12.1%; 2.5±0.5, 2.9±0.4 and 3.4±0.4, respectively to P1, P2 and P3. The TTR results show gradually decrease of motility and vigor along 2 hours of test procedure utilized, with best average to protocol 3 at all time of analyze. The protocol 3 tested by Piau boars shows highest values in cellular semen cryopreservation.

Cryopreservation of South African indigenous goat semen

African Journals Online (AJOL)

use

2011-12-05

Dec 5, 2011 ... sperm cell motility rate of 83.1%, progressive sperm cell motility of 49.3% ... cells can be stored and used after a long period of time. ... artificial insemination to enhance improvement of .... Effect of cryopreservation on semen quality (mean ± S.E) of South African .... Successful pregnancies with directional.

TRANSPLANTATION OF CRYOPRESERVED FETAL LIVER CELLS SEEDED INTO MACROPOROUS ALGINATE-GELATIN SCAFFOLDS IN RATS WITH LIVER FAILURE

Directory of Open Access Journals (Sweden)

D. V. Grizay

2015-01-01

Full Text Available Aim. To study the therapeutic potential of cryopreserved fetal liver cells seeded into macroporous alginategelatin scaffolds after implantation to omentum of rats with hepatic failure.Materials and methods.Hepatic failure was simulated by administration of 2-acetyl aminofl uorene followed partial hepatectomy. Macroporous alginate-gelatin scaffolds, seeded with allogenic cryopreserved fetal liver cells (FLCs were implanted into rat omentum. To prevent from colonization of host cells scaffolds were coated with alginate gel shell. Serum transaminase activity, levels of albumin and bilirubin as markers of hepatic function were determined during 4 weeks after failure model formation and scaffold implantation. Morphology of liver and scaffolds after implantation were examined histologically. Results. Macroporous alginate-gelatin scaffolds after implantation to healthy rats were colonized by host cells. Additional formation of alginate gel shell around scaffolds prevented the colonization. Implantation of macroporous scaffolds seeded with cryopreserved rat FLCs and additionally coated with alginate gel shell into omentum of rats with hepatic failure resulted in signifi cant improvement of hepatospecifi c parameters of the blood serum and positive changes of liver morphology. The presence of cells with their extracellular matrix within the scaffolds was confi rmed after 4 weeks post implantation.Conclusion. The data above indicate that macroporous alginate-gelatin scaffolds coated with alginate gel shell are promising cell carriers for the development of bioengineered liver equivalents.

Cell banking for regulatory T cell-based therapy: strategies to overcome the impact of cryopreservation on the Treg viability and phenotype.

Science.gov (United States)

Gołąb, Karolina; Grose, Randall; Placencia, Veronica; Wickrema, Amittha; Solomina, Julia; Tibudan, Martin; Konsur, Evelyn; Ciepły, Kamil; Marek-Trzonkowska, Natalia; Trzonkowski, Piotr; Millis, J Michael; Fung, John; Witkowski, Piotr

2018-02-09

The first clinical trials with adoptive Treg therapy have shown safety and potential efficacy. Feasibility of such therapy could be improved if cells are cryopreserved and stored until optimal timing for infusion. Herein, we report the evaluation of two cell-banking strategies for Treg therapy: 1) cryopreservation of CD4 + cells for subsequent Treg isolation/expansion and 2) cryopreservation of ex-vivo expanded Tregs (CD4 + CD25 hi CD127 lo/- cells). First, we checked how cryopreservation affects cell viability and Treg markers expression. Then, we performed Treg isolation/expansion with the final products release testing. We observed substantial decrease in cell number recovery after thawing and overnight culture. This observation might be explained by the high percentage of necrotic and apoptotic cells found just after thawing. Furthermore, we noticed fluctuations in percentage of CD4 + CD25 hi CD127 - and CD4 + FoxP3 + cells obtained from cryopreserved CD4 + as well as Treg cells. However, after re-stimulation Tregs expanded well, presented a stable phenotype and fulfilled the release criteria at the end of expansions. Cryopreservation of CD4 + cells for subsequent Treg isolation/expansion and cryopreservation of expanded Tregs with re-stimulation and expansion after thawing, are promising solutions to overcome detrimental effects of cryopreservation. Both of these cell-banking strategies for Treg therapy can be applied when designing new clinical trials.

Oocyte cryopreservation for donor egg banking.

Science.gov (United States)

Cobo, Ana; Remohí, José; Chang, Ching-Chien; Nagy, Zsolt Peter

2011-09-01

Oocyte donation is an efficient alternative to using own oocytes in IVF treatment for different indications. Unfortunately, 'traditional' (fresh) egg donations are challenged with inefficiency, difficulties of synchronization, very long waiting periods and lack of quarantine measures. Given the recent improvements in the efficiency of oocyte cryopreservation, it is reasonable to examine if egg donation through oocyte cryopreservation has merits. The objective of the current manuscript is to review existing literature on this topic and to report on the most recent outcomes from two established donor cryobank centres. Reports on egg donation using slow freezing are scarce and though results are encouraging, outcomes are not yet comparable to a fresh egg donation treatment. Vitrification on the other hand appears to provide high survival rates (90%) of donor oocytes and comparable fertilization, embryo development, implantation and pregnancy rates to traditional (fresh) egg donation. Besides the excellent outcomes, the ease of use for both donors and recipients, higher efficiency, lower cost and avoiding the problem of synchronization are all features associated with the benefit of a donor egg cryobank and makes it likely that this approach becomes the future standard of care. Oocyte donation is one of the last resorts in IVF treatment for couples challenged with infertility problems. However, traditional (fresh) egg donation, as it is performed today, is not very efficient, as typically all eggs from one donor are given to only one recipient, it is arduous as it requires an excellent synchronization between the donor and recipient and there are months or years of waiting time. Because of the development of an efficient oocyte cryopreservation technique, it is now possible to cryo-store donor (as well as non-donor) eggs, maintaining their viability and allowing their use whenever there is demand. Therefore, creating a donor oocyte cryobank would carry many advantages

A validation study of new cryopreservation bags for implementation in a blood and marrow transplant laboratory.

Science.gov (United States)

Pomper, Gregory J; Wilson, Emily; Isom, Scott; Hurd, David D

2011-06-01

A new cryopreservation bag for hematopoietic cell transplantation requires validation as a safe alternative to the bag currently being used in the laboratory. The new bag was validated using both laboratory and clinical criteria. Laboratory validation proceeded using paired samples of mononuclear cells processed using standard procedures. Cells cryopreserved in the new and old bags were compared for viability, cell counts, CD34 enumeration, colony-forming unit assays, and bag integrity. After completion of laboratory investigations, engraftment with the new bags was followed and compared to historical engraftment using the old bags. There were no significant differences between the old and new bags detected using laboratory studies. Bag integrity was equivalent. The validation data suggested impaired cell function after cryopreservation in the new bags, but there were no significant differences in engraftment potential using either material. Days to engraftment was longer using the new bags, but statistical analysis revealed an association with CD34 dose and not with cryopreservation bag type. The new bags were noninferior to the old bags. A change in cryopreservation bag type may appear to affect cell function and potentially affect engraftment. Multiple analyses may be needed to understand the effect of cell processing changes. © 2010 American Association of Blood Banks.

Reactivation of Herpesvirus in Patients With Hepatitis C Treated With Direct-Acting Antiviral Agents.

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Perelló M, Christie; Fernández-Carrillo, Carlos; Londoño, María-Carlota; Arias-Loste, Teresa; Hernández-Conde, Marta; Llerena, Susana; Crespo, Javier; Forns, Xavier; Calleja, José Luis

2016-11-01

We performed a case-series analysis of reactivation of herpesvirus in patients with hepatitis C virus (HCV) infection treated with direct-acting antiviral (DAA) agents. We collected data from 576 patients with HCV infection treated with DAA combinations at 3 hospitals in Spain, from November 2014 through November 2015. We also collected data from a control population (230 HCV-infected patients, matched for sex and age; 23 untreated and 213 treated with interferon-based regimens). Herpesvirus was reactivated in 10 patients who received DAA therapy (7 patients had cirrhosis and 3 patients had received liver transplants), a median of 8 weeks after the therapy was initiated. None of the controls had herpesvirus reactivation. Patients with herpesvirus reactivation were receiving the DAA agents sofosbuvir with ledipasvir (with or without ribavirin, 7/10), ombitasvir with paritaprevir and ritonavir plus dasabuvir (with or without ribavirin, 2/10), or sofosbuvir with simeprevir plus ribavirin (1/10). Two of the 10 patients developed postherpetic neuralgia and 1 patient developed kerato-uveitis. All 10 patients with herpesvirus reactivation achieved a sustained virologic response. Immune changes that follow clearance of HCV might lead to reactivation of other viruses, such as herpesvirus. Patients with HCV infection suspected of having herpesvirus infection should be treated immediately. Some groups also might be screened for herpesvirus infection. Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.

Helminthic therapy: using worms to treat immune-mediated disease.

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Elliott, David E; Weinstock, Joel V

2009-01-01

There is an epidemic of immune-mediated disease in highly-developed industrialized countries. Such diseases, like inflammatory bowel disease, multiple sclerosis and asthma increase in prevalence as populations adopt modern hygienic practices. These practices prevent exposure to parasitic worms (helminths). Epidemiologic studies suggest that people who carry helminths have less immune-mediated disease. Mice colonized with helminths are protected from disease in models of colitis, encephalitis, Type 1 diabetes and asthma. Clinical trials show that exposure to helminths reduce disease activity in patients with ulcerative colitis or Crohn's disease. This chapter reviews some of the work showing that colonization with helminths alters immune responses, against dysregulated inflammation. These helminth-host immune interactions have potentially important implications for the treatment of immune-mediated diseases.

Aspectos histológicos do ovário de coelhas após criopreservação Histological aspects of rabbit ovarian tissue after cryopreservation

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Beatriz Angélica Charlotte Thomaz

2005-11-01

e presença de PCNA positivo em todos os folículos.PURPOSE: to evaluate follicular preservation and histologic characteristics of the cryopreserved ovarian tissue and to compare with the fresh one, in rabbits. METHODS: ten adult female white rabbits were submitted to right oophorectomy. The dried ovary was dissected and the cortex was maintained with approximately 1.5 millimeter thickness. The tissue was fractionated into small sections, some reserved for control histologic study and others destined for cryopreservation. Six weeks later the ovarian tissue was thawed and evaluated histologically. After histologic processing, the control and the experimental samples were stained with hematoxylin and eosin and treated immunohistochemically by the PCNA technique for evaluation of DNA preservation. Histologic alterations present in the fresh and in the cryopreserved tissues were identified, and cryopreserved tissue viability was evaluated. RESULTS: in the cryopreserved tissue only primordial follicles persisted. Reversible alterations were identified: cytoplasmatic vacuolation (p=0,039, stromal lysis (p=0.648 and oocytes with irregular contours (p=0.007. Irreversible alterations: (hyalin degeneration and pyknosis were found, but not at significant levels (p=0.210. The immunohistochemical analysis showed PCNA staining of follicles at different stages of development in the fresh tissue and primordial follicles in the cryopreserved tissue, indicating the presence of active DNA in both tissues. CONCLUSION: in the cryopreserved ovarian tissue the following were observed: survival of only primordial follicles; significant reversible histologic alterations (cytoplasmic vacuolation, stromal lysis and oocytes with irregular contours; irreversible alterations (hyalin degeneration and pyknosis, and PCNA staining of all follicles.

Between Scylla and Charybdis: the role of the human immune system in the pathogenesis of hepatitis C.

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Spengler, Ulrich; Nischalke, Hans Dieter; Nattermann, Jacob; Strassburg, Christian P

2013-11-28

Hepatitis C virus (HCV) frequently elicits only mild immune responses so that it can often establish chronic infection. In this case HCV antigens persist and continue to stimulate the immune system. Antigen persistence then leads to profound changes in the infected host's immune responsiveness, and eventually contributes to the pathology of chronic hepatitis. This topic highlight summarizes changes associated with chronic hepatitis C concerning innate immunity (interferons, natural killer cells), adaptive immune responses (immunoglobulins, T cells, and mechanisms of immune regulation (regulatory T cells). Our overview clarifies that a strong anti-HCV immune response is frequently associated with acute severe tissue damage. In chronic hepatitis C, however, the effector arms of the immune system either become refractory to activation or take over regulatory functions. Taken together these changes in immunity may lead to persistent liver damage and cirrhosis. Consequently, effector arms of the immune system will not only be considered with respect to antiviral defence but also as pivotal mechanisms of inflammation, necrosis and progression to cirrhosis. Thus, avoiding Scylla - a strong, sustained antiviral immune response with inital tissue damage - takes the infected host to virus-triggered immunopathology, which ultimately leads to cirrhosis and liver cancer - the realm of Charybdis.

Effects of micro-encapsulation on morphology and endocrine function of cryopreserved neonatal porcine islet-like cell clusters.

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Murakami, M; Satou, H; Kimura, T; Kobayashi, T; Yamaguchi, A; Nakagawara, G; Iwata, H

2000-10-27

For the success of clinical islets transplantation, the development of a long-term storage method is necessary. However, the structure of digested islets is scanty for culture and cryopreservation. In this study, the effect of micro-encapsulation to cryopreserved porcine islet-like cell clusters (ICCs) was investigated. The ICCs prepared from neonatal pigs by collagenase digestion and culture technique were cryopreserved and micro-encapsulated in 5% agarose membranes. After cryopreservation, ICC cultured without encapsulation (group A) and cultured with encapsulation (group B) were assessed by comparison with no cryopreserved ICC (control) both in vitro by static incubation test and in vivo in a xenotransplantation study. Micro-encapsulation was able to maintain the fine morphology and the number of ICCs of group B after 7 days of culture. There were not significant differences in insulin secretion of group B and control on day 1 and 7 of culture (1 day:11+/-0.99, 7 days: 5.30+/-1.08 microU/ICC/hr NS versus control). On day 7 of culture, the retrieval rate of group B (105.2+/-9.8%) is obviously higher compared with group A (63.0+/-6.3%). In the xenotransplatation model, the ICCs of group B showed long survival time (7.9+/-0.4 weeks) and good transplantation effect. Our study suggests that micro-encapsulation is one of the useful method for cryopreserved ICC to maintain the fine morphology and effectively recover the endocrine function.

Value of lymphocyte cryo-preservation after a radiological or nuclear accident

International Nuclear Information System (INIS)

Laroche, P.; Lataillade, J.J.; Chambrette, V.; Voisin, Ph.

1997-01-01

The conventional cytogenetic method in biological dosimetry is most useful for the estimation of the received radiation dose. It shows resulting unstable chromosomal aberrations (dicentrics, centric rings and fragments) in peripheral blood lymphocytes. This method has been used over the past 30 years and is used in forensic medicine. Nevertheless, it is long and fastidious. Accordingly, the number of simultaneous analyses of blood samples is limited and depends on the capacity of specialized laboratories. This capacity may be insufficient in the case of large scale radiological or nuclear accidents. Cryo-preservation is the usual method to store cells before analysis or use, for instance for biological dosimetry purposes. Some investigations have shown that thawing following freezing may induce cell injury but few studies have been made on the effect of cryo-preservation on cells containing radiation-induced unstable chromosomal aberrations. In this work, lymphocytes were irradiated with 1 to 4 Gy gamma rays and stored in liquid nitrogen. The dicentric and centric ring yields were analysed after storage periods of 1 week, 1 month, 3 months, and 1 year. No difference in aberration frequency from control, unfrozen samples was observed over this period. Lymphocytes stored at -196 deg C for up to least 1 year may therefore be used for chromosome aberration scoring when overexposure to ionizing radiation is suspected. (author)

Antioxidant activity of aqueous extract of noni in dilutent for ram semen cryopreservation

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Ana Lauren Costa Nascimento

2016-03-01

Full Text Available Noni (Morinda citrifolia L. is a fruit consumed worldwide because of its nutritional and therapeutic properties resulting from the large amount of phenolic compounds, which has aroused interest of the scientific community. In order to identify new natural sources of antioxidants, the objective of this study was to evaluate the performance of noni in diluent for ram semen cryopreservation. A completely randomized design consisting of four treatments and three repetitions per treatment was used. The treatments differed in terms of the concentration of the aqueous extract of noni added to the diluent: control, no addition of the extract, and three concentrations (24, 72, and 120 µg/mL. The physical and chemical variables of the mature fruit were evaluated: total acidity (8.78, pH (4.12, and soluble solids (8.18%. The vitamin C content was 309.42 mg per 100 g fresh matter. The aqueous extract of noni was also evaluated regarding the quantity of total phenolic compounds, antioxidant activity, and lipid peroxidation inhibition capacity. The aqueous extract contained a moderate amount of phenolic compounds (47.96 ± 1.95 mg gallic acid equivalent/100 g extract. The concentrations of the aqueous extract of 72 and 120 µg/mL in diluent used for semen cryopreservation inhibited lipid peroxidation by 21.75% and 51.32%, respectively. There was no positive effect of the lowest concentration (24 µg/mL. The antioxidant activity index of noni was 33.33, corresponding to very strong antioxidant activity. The aqueous extract of noni exhibits very strong antioxidant activity and its addition to the diluent for semen cryopreservation at a concentration of 72 µg/mL is able to inhibit lipid peroxidation.

Development of soya milk extender for semen cryopreservation of Karan Fries (crossbreed cattle).

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Singh, V K; Singh, A K; Kumar, R; Atreja, S K

2013-01-01

Egg yolk based semen extenders are used widely, with the potential risk of xenobiotic contamination. This study was designed to develop a soya milk based extender to substitute egg yolk based extender for bovine semen cryopreservation. In the first experiment soya milk was prepared from fresh soya bean (Glycine max). Concentration of soya milk in tris based extender was standardized based on quality parameters of spermatozoa during liquid preservation at 5°C up to 72 h and compared with egg yolk tris (EYT) extender. Sperm in soya milk tris (SMT) extender with 25 percent soya milk showed no significant (P > 0.05) differences in all the quality parameters like motility, viability, membrane integrity and acrosome integrity, as compared to sperm in EYT extender up to 72h in liquid dilution. In the second experiment the Karan Fries semen was cryopreserved in SMT extender with 25 percent soya milk (selected from the first experiment) using different concentration of glycerol, as cryoprotectant, ranging from 6-7 percent with a difference of 0.2 percent to standardize optimum concentration based on post thaw motility of spermatozoa. Glycerol at a final concentration of 6.4 percent was found to be the best among all. Further, semen samples were split and cryopreserved in newly developed SMT extender containing 6.4 percent glycerol and compared with conventional EYT extender for post thaw sperm quality parameters and degree of cryocapacitation. There were no significant (P > 0.05) differences between sperm in EYT extender and SMT extender for post thaw motility, viability, membrane integrity, acrosome integrity and cryocapacitation. In conclusion, the newly developed SMT extender maintained comparable semen quality as compared to EYT extender hence it can.

ATP Release from Chemotherapy-Treated Dying Leukemia Cells Elicits an Immune Suppressive Effect by Increasing Regulatory T Cells and Tolerogenic Dendritic Cells

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Mariangela Lecciso

2017-12-01

Full Text Available Chemotherapy-induced immunogenic cell death can favor dendritic cell (DC cross-priming of tumor-associated antigens for T cell activation thanks to the release of damage-associated molecular patterns, including ATP. Here, we tested the hypothesis that in acute myeloid leukemia (AML, ATP release, along with its well-known immune stimulatory effect, may also contribute to the generation of an immune suppressive microenvironment. In a cohort of AML patients, undergoing combined daunorubicin and cytarabine chemotherapy, a population of T regulatory cells (Tregs with suppressive phenotype, expressing the immune checkpoint programmed cell death protein 1 (PD-1, was significantly increased. Moving from these results, initial in vitro data showed that daunorubicin was more effective than cytarabine in modulating DC function toward Tregs induction and such difference was correlated with the higher capacity of daunorubicin to induce ATP release from treated AML cells. DCs cultured with daunorubicin-treated AML cells upregulated indoleamine 2,3-dioxygenase 1 (IDO1, which induced anti-leukemia Tregs. These data were confirmed in vivo as daunorubicin-treated mice show an increase in extracellular ATP levels with increased number of Tregs, expressing PD-1 and IDO1+CD39+ DCs. Notably, daunorubicin failed to induce Tregs and tolerogenic DCs in mice lacking the ATP receptor P2X7. Our data indicate that ATP release from chemotherapy-treated dying cells contributes to create an immune suppressive microenvironment in AML.

Comparative proteome analysis of cryopreserved flagella and head plasma membrane proteins from sea bream spermatozoa: effect of antifreeze proteins.

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Zilli, Loredana; Beirão, José; Schiavone, Roberta; Herraez, Maria Paz; Gnoni, Antonio; Vilella, Sebastiano

2014-01-01

Cryopreservation induces injuries to fish spermatozoa that in turn affect sperm quality in terms of fertilization ability, motility, DNA and protein integrity and larval survival. To reduce the loss of sperm quality due to freezing-thawing, it is necessary to improve these procedures. In the present study we investigated the ability of two antifreeze proteins (AFPI and AFPIII) to reduce the loss of quality of sea bream spermatozoa due to cryopreservation. To do so, we compared viability, motility, straight-line velocity and curvilinear velocity of fresh and (AFPs)-cryopreserved spermatozoa. AFPIII addition to cryopreservation medium improved viability, motility and straight-line velocity with respect to DMSO or DMSO plus AFPI. To clarify the molecular mechanism(s) underlying these findings, the protein profile of two different cryopreserved sperm domains, flagella and head plasma membranes, was analysed. The protein profiles differed between fresh and frozen-thawed semen and results of the image analysis demonstrated that, after cryopreservation, out of 270 proteins 12 were decreased and 7 were increased in isolated flagella, and out of 150 proteins 6 showed a significant decrease and 4 showed a significant increase in head membranes. Mass spectrometry analysis identified 6 proteins (4 from isolated flagella and 2 present both in flagella and head plasma membranes) within the protein spots affected by the freezing-thawing procedure. 3 out of 4 proteins from isolated flagella were involved in the sperm bioenergetic system. Our results indicate that the ability of AFPIII to protect sea bream sperm quality can be, at least in part, ascribed to reducing changes in the sperm protein profile occurring during the freezing-thawing procedure. Our results clearly demonstrated that AFPIII addition to cryopreservation medium improved the protection against freezing respect to DMSO or DMSO plus AFPI. In addition we propose specific proteins of spermatozoa as markers related to

Cryopreservation and other assisted reproductive technologies for the conservation of threatened amphibians and reptiles: bringing the ARTs up to speed.

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Clulow, John; Clulow, Simon

2016-06-01

Amphibians and reptiles are experiencing serious declines, with the number of threatened species and extinctions growing rapidly as the modern biodiversity crisis unfolds. For amphibians, the panzootic of chytridiomycosis is a major driver. For reptiles, habitat loss and harvesting from the wild are key threats. Cryopreservation and other assisted reproductive technologies (ARTs) could play a role in slowing the loss of amphibian and reptile biodiversity and managing threatened populations through genome storage and the production of live animals from stored material. These vertebrate classes are at different stages of development in cryopreservation and other ARTs, and each class faces different technical challenges arising from the separate evolutionary end-points of their reproductive biology. For amphibians, the generation of live offspring from cryopreserved spermatozoa has been achieved, but the cryopreservation of oocytes and embryos remains elusive. With reptiles, spermatozoa have been cryopreserved in a few species, but no offspring from cryopreserved spermatozoa have been reported, and the generation of live young from AI has only occurred in a small number of species. Cryopreservation and ARTs are more developed and advanced for amphibians than reptiles. Future work on both groups needs to concentrate on achieving proof of concept examples that demonstrate the use of genome storage and ARTs in successfully recovering threatened species to increase awareness and support for this approach to conservation.

Assessment of basic seminal characteristics, sperm cryopreservation and heterologous in vitro fertilisation in the fishing cat (Prionailurus viverrinus).

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Thiangtum, Khongsak; Swanson, William F; Howard, JoGayle; Tunwattana, Wanchai; Tongthainan, Dakara; Wichasilpa, Wisid; Patumrattanathan, Pornchai; Pinyopoommintr, Tanu

2006-01-01

Conservation of the fishing cat, a threatened south-east Asian felid, could benefit from effective ex situ genetic management and breeding programmes, including the use of assisted reproduction. The aims of the present study were to: (1) characterise basal seminal traits of fishing cats in Thailand zoos; and (2) investigate the effect of cryopreservation on sperm motility, acrosomal integrity and in vitro function. Seminal traits were evaluated in electroejaculates collected from eight males. Spermatozoa were diluted in n-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid Tris (TEST)-yolk buffer (TYB) without glycerol, then diluted further with TYB with glycerol (4% final concentration) at either 25 degrees C or after slow cooling to 5 degrees C and frozen in straws over liquid nitrogen vapour. After thawing, sperm function was assessed by insemination of viable domestic cat oocytes. Fishing cat ejaculates averaged (+/- s.e.m.) 43.6 +/- 14.2 x 10(6) motile spermatozoa with 33.5 +/- 6.8% normal sperm morphology. Semen processing had a negligible effect (P > 0.05) on sperm motility and acrosomal integrity, but values were reduced (P cat oocytes, with 62.1% (36/58) of mature oocytes cleaving. Glycerol addition at 5 degrees C resulted in higher (P fishing cats and their spermatozoa exhibit adequate function after cryopreservation for in vitro fertilisation procedures.

Sperm subpopulational dinamycs during the cryopreservation procedure in caprine (Capra aegagrus hircus ejaculates

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Barbas JP

2017-08-01

Full Text Available The objective of the present research was to determine specific sperm subpopulational dynamics in different processing steps during cryopreservation process by using objective functional sperm kinematic descriptors in goat ejaculates. Fresh ejaculates (n=40 collected from eight bucks were analised for volume, concentration, sperm viability, acrosome integrity, and sperm motility using computer-assisted sperm analysis (CASA system. Eight sperm kinematic descriptors (VCL, VSL, VAP, LIN, STR, BCF, ALH, and WOB were assessed using CASA system after five different handling step (1st: fresh semen collection (F; 2nd: 1st washing/centrifugation step (1WC; 3rd: 2nd washing / centrifugation step (2WC; 4th: cooling step at 4ºC (CL; and 5th: post-thawing step at 37ºC (PT during a standard cryopreservation protocol for goat semen. The results obtained from the kinematic parameters were analysed by using Principal Component Analysis (PCA and multivariate clustering procedures to identify specific kinematic subpopulations and establish the relationship between the distribution of the subpopulations found and the functional sperm motility in each step. Except for the 1st (SbpF1-SbpF3 and 4th (SbpCL1-SbpCL3 intervals, four sperm kinematic subpopulations (Sbp1LC1-Sbp1LC4, Sbp2LC1-Sbp2LC4 and SbpPD1-SbpPD4 were observed. Based on kinematic velocity parameters and the subpopulation disclosed, rapid, slow, vigorous, passive, non-progressive and progressive sperm were discerned. Moreover, based on kinematic linearity parameters and depending on the subpopulation uncovered, curvilinear, regular-linear, parabolic and erratic-non-linear trajectories were detected. Subpopulations remained varible throughout handling steps and multiple significant differences among the sperm kinematic parameters were observed (p<0.001 as well as in the frequency of distribution of kinematic subpopulations among steps (p<0.05. In conclusion, this study confirms the variability and

Sperm quality and cryopreservation of Brazilian freshwater fish species: a review.

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Viveiros, A T M; Godinho, H P

2009-03-01

The Brazilian freshwater fish diversity is the richest in the world. Only 0.7% of all Brazilian species have had any aspect of their sperm biology addressed up to this date. The majority of the fish species described in this review migrate during the spawning season (a phenomenon known as piracema). Urbanization, pollution, hydroelectric dams and deforestation are some of the causes of stock depletion or even local extinction of some of these species. The knowledge concerning sperm quality and minimum sperm:egg ratio is important to maximize the use of males without reducing hatching rates. Furthermore, sperm cryopreservation and gene banking can guarantee the conservation of genetic diversity and development of adequate breeding programs of native fish species. In this review, we present and evaluate the existing information on Brazilian fish species that have been subject to sperm quality and cryopreservation studies. The following parameters were evaluated: volume of extractable sperm, sperm motility, sperm concentration, freezing media, freezing methods, and post-thaw sperm quality. Although the existing protocols yield relatively high post-thaw motility and fertilization rates, the use of cryopreserved sperm in routine hatchery production is still limited in Brazil.

Cryopreservation of Boer goat spermatozoa: Comparison of two freezing extenders based on post-thaw sperm quality and fertility rates

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Fitra Aji Pamungkas

2014-06-01

Full Text Available Boer goat have recently been popularly used for cross breeding with local goats. However, it is currently considered a breed at very limited number with relatively high prices . In this context, the cryopreservation of spermatozoa is important because it could be conserved for a very long period of time. Egg yolk extenders are most commonly used for cryopreservation of goat sperm. The aim of this study was to compare the ability of two extenders to maintain sperm viability after cryopreservation. Semen from three male Boer goat aged about 2-3 years old was collected using artificial vagina and frozen with Tris and Triladyl extender. The results showed that percentage of motility, viability and membrane integrity of spermatozoa with Tris and Triladyl extenders at every stage of cryopreservation showed not significantly difference (P>0.05, except the percentage of sperm motility post thawing of Triladyl was higher than Tris extender (52.00±4.47% vs 47.50±2.74%, P<0.05. Cryopreserved semen in Tris extender provided the same fertility rates after cervical insemination compared to Triladyl (62.50% vs 60.00%. In conclusion, the Tris extender has the same capabilities to Triladyl in cryopreservation of Boer goat spermatozoa as to maintain sperm quality and fertility rates.

Low-density Lipoprotein Improves Motility and Plasma Membrane Integrity of Cryopreserved Canine Epididymal Spermatozoa

OpenAIRE

N. Prapaiwan; T. Tharasanit; S. Punjachaipornpol; D. Yamtang; A. Roongsitthichai; W. Moonarmart; K. Kaeoket; S. Manee-in

2016-01-01

Cryopreservation of caudal epididymal spermatozoa is an effective technique to conserve genetic potentials of superior dogs when it is not possible to collect ejaculated spermatozoa. Although hen egg yolk is commonly supplemented into the semen extender, active substances within the egg yolk which protect sperm against cryoinjury remain to be discovered. Among its compositions, low-density lipoprotein (LDL) has been reported to have a cryoprotective property for sperm cryopreservation. Howeve...

Protein Kinase C Enzymes in the Hematopoietic and Immune Systems.

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Altman, Amnon; Kong, Kok-Fai

2016-05-20

The protein kinase C (PKC) family, discovered in the late 1970s, is composed of at least 10 serine/threonine kinases, divided into three groups based on their molecular architecture and cofactor requirements. PKC enzymes have been conserved throughout evolution and are expressed in virtually all cell types; they represent critical signal transducers regulating cell activation, differentiation, proliferation, death, and effector functions. PKC family members play important roles in a diverse array of hematopoietic and immune responses. This review covers the discovery and history of this enzyme family, discusses the roles of PKC enzymes in the development and effector functions of major hematopoietic and immune cell types, and points out gaps in our knowledge, which should ignite interest and further exploration, ultimately leading to better understanding of this enzyme family and, above all, its role in the many facets of the immune system.

Platelet activating factor receptor binding plays a critical role in jet fuel-induced immune suppression

International Nuclear Information System (INIS)

Ramos, Gerardo; Kazimi, Nasser; Nghiem, Dat X.; Walterscheid, Jeffrey P.; Ullrich, Stephen E.

2004-01-01

Applying military jet fuel (JP-8) or commercial jet fuel (Jet-A) to the skin of mice suppresses the immune response in a dose-dependant manner. The release of biological response modifiers, particularly prostaglandin E 2 (PGE 2 ), is a critical step in activating immune suppression. Previous studies have shown that injecting selective cyclooxygenase-2 inhibitors into jet fuel-treated mice blocks immune suppression. Because the inflammatory phospholipid mediator, platelet-activating factor (PAF), up-regulates cyclooxygenase-2 production and PGE 2 synthesis by keratinocytes, we tested the hypothesis that PAF-receptor binding plays a role in jet fuel-induced immune suppression. Treating keratinocyte cultures with PAF and/or jet fuel (JP-8 and Jet-A) stimulates PGE 2 secretion. Jet fuel-induced PGE 2 production was suppressed by treating the keratinocytes with specific PAF-receptor antagonists. Injecting mice with PAF, or treating the skin of the mice with JP-8, or Jet-A, induced immune suppression. Jet fuel-induced immune suppression was blocked when the jet fuel-treated mice were injected with PAF-receptor antagonists before treatment. Jet fuel treatment has been reported to activate oxidative stress and treating the mice with anti-oxidants (Vitamins C, or E or beta-hydroxy toluene), before jet fuel application, interfered with immune suppression. These findings confirm previous studies showing that PAF-receptor binding can modulate immune function. Furthermore, they suggest that PAF-receptor binding may be an early event in the induction of immune suppression by immunotoxic environmental agents that target the skin

Vitrification versus slow freezing for women undergoing oocyte cryopreservation

NARCIS (Netherlands)

Glujovsky, Demián; Riestra, Barbara; Sueldo, Carlos; Fiszbajn, Gabriel; Repping, Sjoerd; Nodar, Florencia; Papier, Sergio; Ciapponi, Agustín

2014-01-01

Oocyte cryopreservation is a technique with considerable potential in reproductive medicine, including fertility preservation, as a way of delaying childbearing and as part of oocyte donation programs. Although the technique was relatively ineffective at first more recently numerous modifications

Transmission of innate immune signaling by packaging of cGAMP in viral particles.

Science.gov (United States)

Gentili, Matteo; Kowal, Joanna; Tkach, Mercedes; Satoh, Takeshi; Lahaye, Xavier; Conrad, Cécile; Boyron, Marilyn; Lombard, Bérangère; Durand, Sylvère; Kroemer, Guido; Loew, Damarys; Dalod, Marc; Théry, Clotilde; Manel, Nicolas

2015-09-11

Infected cells detect viruses through a variety of receptors that initiate cell-intrinsic innate defense responses. Cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) is a cytosolic sensor for many DNA viruses and HIV-1. In response to cytosolic viral DNA, cGAS synthesizes the second messenger 2'3'-cyclic GMP-AMP (cGAMP), which activates antiviral signaling pathways. We show that in cells producing virus, cGAS-synthesized cGAMP can be packaged in viral particles and extracellular vesicles. Viral particles efficiently delivered cGAMP to target cells. cGAMP transfer by viral particles to dendritic cells activated innate immunity and antiviral defenses. Finally, we show that cell-free murine cytomegalovirus and Modified Vaccinia Ankara virus contained cGAMP. Thus, transfer of cGAMP by viruses may represent a defense mechanism to propagate immune responses to uninfected target cells. Copyright © 2015, American Association for the Advancement of Science.

Crosstalk between Substrates and Rho-Associated Kinase Inhibitors in Cryopreservation of Tissue-Engineered Constructs

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Arindam Bit

2017-01-01

Full Text Available It is documented that human mesenchymal stem cells (hMSCs can be differentiated into various types of cells to present a tool for tissue engineering and regenerative medicine. Thus, the preservation of stem cells is a crucial factor for their effective long-term storage that further facilitates their continuous supply and transportation for application in regenerative medicine. Cryopreservation is the most important, practicable, and the only established mechanism for long-term preservation of cells, tissues, and organs, and engineered tissues; thus, it is the key step for the improvement of tissue engineering. A significant portion of MSCs loses cellular viability while freeze-thawing, which represents an important technical limitation to achieving sufficient viable cell numbers for maximum efficacy. Several natural and synthetic materials are extensively used as substrates for tissue engineering constructs and cryopreservation because they promote cell attachment and proliferation. Rho-associated kinase (ROCK inhibitors can improve the physiological function and postthaw viability of cryopreserved MSCs. This review proposes a crosstalk between substrate topology and interaction of cells with ROCK inhibitors. It is shown that incorporation of ionic nanoparticles in the presence of an external electrical field improves the generation of ROCK inhibitors to safeguard cellular viability for the enhanced cryopreservation of engineered tissues.

Viral (hepatitis C virus, hepatitis B virus, HIV) persistence and immune homeostasis

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Zhou, Yun; Zhang, Ying; Moorman, Jonathan P; Yao, Zhi Q; Jia, Zhan S

2014-01-01

Immune homeostasis is a host characteristic that maintains biological balance within a host. Humans have evolved many host defence mechanisms that ensure the survival of individuals upon encountering a pathogenic infection, with recovery or persistence from a viral infection being determined by both viral factors and host immunity. Chronic viral infections, such as hepatitis B virus, hepatitis C virus and HIV, often result in chronic fluctuating viraemia in the face of host cellular and humoral immune responses, which are dysregulated by multi-faceted mechanisms that are incompletely understood. This review attempts to illuminate the mechanisms involved in this process, focusing on immune homeostasis in the setting of persistent viral infection from the aspects of host defence mechanism, including interferon-stimulated genes, apolipoprotein B mRNA editing enzyme catalytic polypeptide 3 (APOBEC3), autophagy and interactions of various immune cells, cytokines and regulatory molecules. PMID:24965611

Cryopreservation of in vitro grown nodal segments of Rauvolfia serpentina by PVS2 vitrification.

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Ray, Avik; Bhattacharya, Sabita

2008-01-01

This paper describes the cryopreservation by PVS2 vitrification of Rauvolfia serpentina (L.) Benth ex kurz, an important tropical medicinal plant. The effects of type and size of explants, sucrose preculture (duration and concentration) and vitrification treatment were tested. Preliminary experiments with PVS1, 2 and 3 produced shoot growth only for PVS2. When optimizing the PVS2 vitrification of nodal segments, those of 0.31 - 0.39 cm in size were better than other nodal sizes and or apices. Sucrose preculture had a positive role in survival and subsequent regrowth of the cryopreserved explants. Seven days on 0.5 M sucrose solution significantly improved the viability of nodal segments. PVS2 incubation for 45 minutes combined with a 7-day preculture gave the optimum result of 66 percent. Plantlets derived after cryopreservation resumed growth and regenerated normally.

Morphological Findings in Trophozoites during Amoebic Abscess Development in Misoprostol-Treated BALB/c Mice

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Andrés Aceves-Cano

2015-01-01

Full Text Available During amoebic liver abscess (ALA formation in susceptible animals, immune response is regulated by prostaglandin E2 (PGE2 dependent mechanisms. The aim of this study was to analyze the effect of misoprostol (MPL, a PGE1 analogue, on ALA formation in BALB/c mice. Male mice from BALB/c strain were intrahepatically infected with 7.5×105 trophozoites of E. histolytica strain HM1:IMSS and treated with 10−4 M of MPL daily until sacrifice at 2, 4, and 7 days postinfection (p.i.. ALA formation was evaluated at 2, 4, and 7 days postinfection; trophozoite morphology was analyzed using immunohistochemistry and image analysis. Results showed an increase in frequency of ALA formation in infected and MPL-treated mice only at 2 days p.i. (P=0.03. A significant diminution in the size of trophozoites was detected in abscesses from mice independently of MPL treatment (from 5.8±1.1 µm at 2 days p.i. to 2.7±1.9 µm at 7 days p.i. compared with trophozoites dimensions observed in susceptible hamsters (9.6±2.7 µm (P<0.01. These results suggest that MPL treatment may modify the adequate control of inflammatory process to allow the persistence of trophozoites in the liver; however, natural resistance mechanisms cannot be discarded.

Heterologous humoral immune response in patients treated with human growth hormone from different sources

International Nuclear Information System (INIS)

Cardoso, A.I.; Llera, A.S.; Iacono, R.F.

1993-01-01

The existence of homologous anti-human growth hormone (anti-hGH) and heterologous anti-bovine growth hormone (anti-bGH) humoral immune responses in hypopituitary patients under hGH therapy has been reported previously. In order to study the influence of the hormone source, both responses were compared by radiobinding assays performed with [ 125 I]hGH or [ 125 I]bGH as tracers. 57 hypopituitary patients treated with extractive hGH, recombinant methionyl hGH or authentic recombinant hGH were studied. A very low incidence of heterologous antibodies was found in patients under recombinant hGH therapy, contrary to the high incidence observed in patients treated with extractive hGH preparations. In addition, immunochemical studies performed with a synthetic peptide (hGH 44-128) indicated that this peptide exhibited, in the anti-bGH/[ 125 I]bGH radioimmunoassay system, higher reactivity than the native hGH, suggesting that such fragment resembled an altered conformation of the hormone. The high heterologous response elicited only by the extractive hGH along with the behaviour of the hGH 44-128 fragment supports the fact that the extraction and purification procedures in extractive preparations may alter slightly the structure of the hGH molecule and trigger a heterologous immune response. 16 refs., 4 figs., 1 tab

Heterologous humoral immune response in patients treated with human growth hormone from different sources

Energy Technology Data Exchange (ETDEWEB)

Cardoso, A.I.; Llera, A.S.; Iacono, R.F. (and others) (Inst. de Estudios de la Inmunidad Humoral, Buenos Aires (Argentina))

1993-07-01

The existence of homologous anti-human growth hormone (anti-hGH) and heterologous anti-bovine growth hormone (anti-bGH) humoral immune responses in hypopituitary patients under hGH therapy has been reported previously. In order to study the influence of the hormone source, both responses were compared by radiobinding assays performed with [[sup 125]I]hGH or [[sup 125]I]bGH as tracers. 57 hypopituitary patients treated with extractive hGH, recombinant methionyl hGH or authentic recombinant hGH were studied. A very low incidence of heterologous antibodies was found in patients under recombinant hGH therapy, contrary to the high incidence observed in patients treated with extractive hGH preparations. In addition, immunochemical studies performed with a synthetic peptide (hGH 44-128) indicated that this peptide exhibited, in the anti-bGH/[[sup 125]I]bGH radioimmunoassay system, higher reactivity than the native hGH, suggesting that such fragment resembled an altered conformation of the hormone. The high heterologous response elicited only by the extractive hGH along with the behaviour of the hGH 44-128 fragment supports the fact that the extraction and purification procedures in extractive preparations may alter slightly the structure of the hGH molecule and trigger a heterologous immune response. 16 refs., 4 figs., 1 tab.

Cryopreservation Maintains Functionality of Human iPSC Dopamine Neurons and Rescues Parkinsonian Phenotypes In Vivo

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Dustin R. Wakeman

2017-07-01

Full Text Available A major challenge for clinical application of pluripotent stem cell therapy for Parkinson's disease (PD is large-scale manufacturing and cryopreservation of neurons that can be efficiently prepared with minimal manipulation. To address this obstacle, midbrain dopamine neurons were derived from human induced pluripotent stem cells (iPSC-mDA and cryopreserved in large production lots for biochemical and transplantation studies. Cryopreserved, post-mitotic iPSC-mDA neurons retained high viability with gene, protein, and electrophysiological signatures consistent with midbrain floor-plate lineage. To test therapeutic efficacy, cryopreserved iPSC-mDA neurons were transplanted without subculturing into the 6-OHDA-lesioned rat and MPTP-lesioned non-human-primate models of PD. Grafted neurons retained midbrain lineage with extensive fiber innervation in both rodents and monkeys. Behavioral assessment in 6-OHDA-lesioned rats demonstrated significant reversal in functional deficits up to 6 months post transplantation with reinnervation of the host striatum and no aberrant growth, supporting the translational development of pluripotent cell-based therapies in PD.

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

Science.gov (United States)

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

2013-08-01

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

Assessment of some critical factors in the freezing technique for the cryopreservation of precision-cut rat liver slices

NARCIS (Netherlands)

Maas, W.J.M.; Graaf, I.A.M. de; Schoen, E.D.; Koster, H.J.; Sandt, J.J.M. van de; Groten, J.P.

2000-01-01

A number of studies on the cryopreservation of precision-cut liver slices using various techniques have been reported. However, the identification of important factors that determine cell viability following cryopreservation is difficult because of large differences between the various methods

Stress preconditioning of rooster semen before cryopreservation improves fertility potential of thawed sperm.

Science.gov (United States)

Feyzi, S; Sharafi, M; Rahimi, S

2018-03-22

Avian semen cryopreservation is not as successful as that seen in mammals. This failure is mostly attributed to unique physiological characteristics of poultry semen that make it susceptible to cryo-damages. Utilization of sublethal oxidative stress for preconditioning of sperm, as an innovative approach, improves the cryo-survival of sperm in certain mammalian species. The purpose of this study was to investigate the effects of preconditioning of rooster semen with sublethal oxidative stress [very low concentrations of nitric oxide (NO)] before cryopreservation on the quality and fertility potential of thawed sperm. Semen samples were collected from 20 roosters, twice a wk, and different concentrations of NO [0 (NO-0), 0.01 (NO-0.01), 0.1 (NO-0.1), 1 (NO-1), 10 (NO-10), and 100 μM (NO-100)] were used to investigate the effects of controlled induction of sublethal stress before semen cryopreservation on the thawed sperm performance. A significantly higher (P 0.05) affected by different concentrations of NO. Sperm exposed to NO-1 produced the highest percentage of viable spermatozoa (Annexin-/PI-), which was significantly different from the other samples. Finally, rate of fertility after artificial insemination was significantly higher (P < 0.05) following treatment with NO-1 compared to NO-0 and NO-0.1. Application of 1 μM NO as a sublethal oxidative stress before cryopreservation of sperm efficiently increased numerous quality indices of thawed sperm as well as its fertility potential.

The 5'-AMP-Activated Protein Kinase (AMPK Is Involved in the Augmentation of Antioxidant Defenses in Cryopreserved Chicken Sperm.

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Thi Mong Diep Nguyen

Full Text Available Semen cryopreservation is a unique tool for the management of animal genetic diversity. However, the freeze-thaw process causes biochemical and physical alterations which make difficult the restoration of sperm energy-dependent functions needed for fertilization. 5'-AMP activated protein kinase (AMPK is a key sensor and regulator of intracellular energy metabolism. Mitochondria functions are known to be severely affected during sperm cryopreservation with deleterious oxidative and peroxidative effects leading to cell integrity and functions damages. The aim of this study was thus to examine the role of AMPK on the peroxidation/antioxidant enzymes defense system in frozen-thawed sperm and its consequences on sperm functions. Chicken semen was diluted in media supplemented with or without AMPK activators (AICAR or Metformin [MET] or inhibitor (Compound C [CC] and then cryopreserved. AMPKα phosphorylation, antioxidant enzymes activities, mitochondrial potential, ATP, citrate, viability, acrosome reaction ability (AR and various motility parameters were negatively affected by the freeze-thaw process while reactive oxygen species (ROS production, lipid peroxidation (LPO and lactate concentration were dramatically increased. AICAR partially restored superoxide dismutase (SOD, Glutathione Peroxidase (GPx and Glutathione Reductase (GR, increased ATP, citrate, and lactate concentration and subsequently decreased the ROS and LPO (malondialdehyde in frozen-thawed semen. Motility parameters were increased (i.e., + 23% for motility, + 34% for rapid sperm as well as AR (+ 100%. MET had similar effects as AICAR except that catalase activity was restored and that ATP and mitochondrial potential were further decreased. CC showed effects opposite to AICAR on SOD, ROS, LPO and AR and motility parameters. Taken together, our results strongly suggest that, upon freeze-thaw process, AMPK stimulated intracellular anti-oxidative defense enzymes through ATP regulation

A simple improvement of the conventional cryopreservation for human ES and iPS cells

OpenAIRE

sprotocols

2014-01-01

Authors: Midori Ozawa, Yutaka Ozawa, Masashi Iemura, Arihiro Kohara, Kana Yanagihara & Miho K Furue ### Abstract In this study, a simple method for the cryopreservation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells is proposed. It is based on the conventional slow-freezing method with 10% DMSO and modified mainly in a thawing protocol without specific equipment or reagents. Recovery rate of the cells cryopreserved by this method was equally high, which is compa...

Maternal immune activation during pregnancy in rats impairs working memory capacity of the offspring.

Science.gov (United States)

Murray, Brendan G; Davies, Don A; Molder, Joel J; Howland, John G

2017-05-01

Maternal immune activation during pregnancy is an environmental risk factor for psychiatric illnesses such as schizophrenia in the offspring. Patients with schizophrenia display an array of cognitive symptoms, including impaired working memory capacity. Rodent models have been developed to understand the relationship between maternal immune activation and the cognitive symptoms of schizophrenia. The present experiment was designed to test whether maternal immune activation with the viral mimetic polyinosinic:polycytidylic acid (polyI:C) during pregnancy affects working memory capacity of the offspring. Pregnant Long Evans rats were treated with either saline or polyI:C (4mg/kg; i.v.) on gestational day 15. Male offspring of the litters (2-3months of age) were subsequently trained on a nonmatching-to-sample task with odors. After a criterion was met, the rats were tested on the odor span task, which requires rats to remember an increasing span of different odors to receive food reward. Rats were tested using delays of approximately 40s during the acquisition of the task. Importantly, polyI:C- and saline-treated offspring did not differ in performance of the nonmatching-to-sample task suggesting that both groups could perform a relatively simple working memory task. In contrast, polyI:C-treated offspring had reduced span capacity in the middle and late phases of odor span task acquisition. After task acquisition, the rats were tested using the 40s delay and a 10min delay. Both groups showed a delay-dependent decrease in span, although the polyI:C-treated offspring had significantly lower spans regardless of delay. Our results support the validity of the maternal immune activation model for studying the cognitive symptoms of neurodevelopmental disorders such as schizophrenia. Copyright © 2017 Elsevier Inc. All rights reserved.

Cryopreservation: a cold look at technology for fertility preservation.

Science.gov (United States)

Gosden, Roger

2011-08-01

To outline the history of cryopreservation technology and its contributions to reproductive medicine, including fertility preservation. A search of the relevant literature using Medline and other online tools. Research and laboratory protocol development. The biology of preserving cells at low temperatures is complex and still being unraveled. Principles were first established more than half a century ago, with progress being driven empirically and often by trial and error. The protocols vary widely, and practice is still heavily dependent on operator skill, accounting for wide differences in the success rates between centers. No single protocol fits all specimen types, and differential vulnerability to cryoinjury remains a major obstacle. Nevertheless, semen cryopreservation has long been established, embryo banking is now highly effective, and vitrification appears to overcome problems with oocytes. Protocols in the future, although specific to the cell type and tissue, are likely to evolve toward generally acknowledged standards. But heterogeneity between patients and even within samples implies that each cell may have its own peculiar optimum for minimizing cryoinjury; because protocols are therefore compromises, "perfect" preservation may be unattainable. Cryopreservation has become a mainstay in the assisted reproduction laboratory and underpins fertility preservation for patients with cancer and other conditions. The practice is currently evolving from slow freezing methods toward more vitrification, and future technology is likely to reduce dependence on operator skill, which should raise success rates to higher, more uniform levels. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Autotransplantation of cryopreserved ovarian tissue in 12 women with chemotherapy-induced premature ovarian failure: the Danish experience

DEFF Research Database (Denmark)

Schmidt, Kirsten Tryde; Rosendahl, Mikkel; Ernst, Erik

2011-01-01

To describe a cohort of 12 Danish women who received autotransplantation of frozen-thawed cryopreserved ovarian tissue because of premature ovarian failure after cancer treatment.......To describe a cohort of 12 Danish women who received autotransplantation of frozen-thawed cryopreserved ovarian tissue because of premature ovarian failure after cancer treatment....

Survival, growth performance and immune capacity of the juvenile lined seahorse Hippocampus erectus fed with rifampicin-treated copepods.

Science.gov (United States)

Lin, Tingting; Wang, Changbo; Liu, Xin; Gao, Fen; Xiao, Dongxue; Zhang, Dong; Zhu, Xialian

2017-06-19

Gastrointestinal disease is one of the most serious diseases in cultured seahorse juveniles. Treatment with antimicrobials of live food (i.e. copepods and Artemia) that is used to feed the juveniles may be a promising measure to alleviate the occurrence of gastrointestinal disease. However, relevant investigations are rare. In the present study, we first investigated the antimicrobial efficacies on bacteria within copepods that were treated with 4 antimicrobials, including 3 antibiotics (i.e. enrofloxacin hydrochloride, oxytetracycline and rifampicin [RFP]) that are approved for use in aquaculture and 1 disinfectant (i.e. povidone iodine). We then assessed the effects of copepods treated with the antimicrobial that had the best antimicrobial efficacy on survival, growth performance and immune capacity of juvenile lined seahorses Hippocampus erectus. The results showed that RFP had the best antimicrobial efficacy on both Pseudoalteromonas spp. and Vibrio spp., 2 dominant bacteria with potential pathogenicity within the copepods; the proper concentration of RFP was 6 mg l-1. Moreover, H. erectus juveniles fed with RFP-treated copepods demonstrated an improved survivorship and immune capacity and had a lower abundance of pathogenic bacteria within their gastrointestinal tracts compared to juveniles fed with untreated copepods. These results suggest that treating live food with RFP is a potential measure for reducing the incidence of gastrointestinal disease in seahorse juveniles.

cGAS/STING Pathway in Cancer: Jekyll and Hyde Story of Cancer Immune Response.

Science.gov (United States)

Bose, Debojit

2017-11-18

The last two decades have witnessed enormous growth in the field of cancer immunity. Mechanistic insights of cancer immunoediting have not only enhanced our understanding but also paved the way to target and/or harness the innate immune system to combat cancer, called cancer immunotherapy. Cyclic GMP-AMP synthase (cGAS)/Stimulator of interferon genes(STING) pathway has recently emerged as nodal player in cancer immunity and is currently being explored as potential therapeutic target. Although therapeutic activation of this pathway has shown promising anti-tumor effects in vivo, evidence also indicates the role of this pathway in inflammation mediated carcinogenesis. This review highlights our current understanding of cGAS/STING pathway in cancer, its therapeutic targeting and potential alternate approaches to target this pathway. Optimal therapeutic targeting and artificial tunability of this pathway still demand in depth understanding of cGAS/STING pathway regulation and homeostasis.

cGAS/STING Pathway in Cancer: Jekyll and Hyde Story of Cancer Immune Response

Directory of Open Access Journals (Sweden)

Debojit Bose

2017-11-01

Full Text Available The last two decades have witnessed enormous growth in the field of cancer immunity. Mechanistic insights of cancer immunoediting have not only enhanced our understanding but also paved the way to target and/or harness the innate immune system to combat cancer, called cancer immunotherapy. Cyclic GMP-AMP synthase (cGAS/Stimulator of interferon genes(STING pathway has recently emerged as nodal player in cancer immunity and is currently being explored as potential therapeutic target. Although therapeutic activation of this pathway has shown promising anti-tumor effects in vivo, evidence also indicates the role of this pathway in inflammation mediated carcinogenesis. This review highlights our current understanding of cGAS/STING pathway in cancer, its therapeutic targeting and potential alternate approaches to target this pathway. Optimal therapeutic targeting and artificial tunability of this pathway still demand in depth understanding of cGAS/STING pathway regulation and homeostasis.

Effect of season on fresh and cryopreserved stallion semen

Science.gov (United States)

The objective of this study was to determine the effect of season on sperm quality parameters, expression of the fertility-related protein SP22 and selected mRNA transcripts infresh and cryopreserved stallion sperm. Four stallions were collected in each of the four seasons: summ...

Fertility rate of daily collected and cryopreserved fowl semen

NARCIS (Netherlands)

Voorst, van A.; Leenstra, F.R.

1995-01-01

Semen was collected for 4 consecutive d individually from experimental broiler breeder males that had not been massaged for 7 d. The semen was mixed and diluted with the Beltsville Poultry Semen Extender with dimethylsulfoxide (DMSO) as cryoprotectant and cryopreserved. After thawing of the semen,

Complement-mediated solubilization of immune complexes and their interaction with complement C3 receptors

DEFF Research Database (Denmark)

Petersen, Ivan; Baatrup, Gunnar; Jepsen, H H

1985-01-01

Some of the molecular events in the complement (C)-mediated solubilization of immune complexes (IC) have been clarified in recent years. The solubilization is primarily mediated by alternative C pathway proteins whereas factors in the classical pathway accelerate the process. Components of the me......Some of the molecular events in the complement (C)-mediated solubilization of immune complexes (IC) have been clarified in recent years. The solubilization is primarily mediated by alternative C pathway proteins whereas factors in the classical pathway accelerate the process. Components...... of the cellular localization, expression and structure of the C3 receptors, especially the C3b (CR1) receptor, has been considerably extended in the last few years, whereas our understanding of the physiological role of these receptors is still fragmentary. However, it is becoming increasingly evident...

Protective features of resveratrol on human spermatozoa cryopreservation may be mediated through 5' AMP-activated protein kinase activation.

Science.gov (United States)

Shabani Nashtaei, M; Amidi, F; Sedighi Gilani, M A; Aleyasin, A; Bakhshalizadeh, Sh; Naji, M; Nekoonam, S

2017-03-01

Biochemical and physical modifications during the freeze-thaw process adversely influence the restoration of energy-dependent sperm functions required for fertilization. Resveratrol, a phytoalexin, has been introduced to activate 5' AMP-activated protein kinase which is a cell energy sensor and a cell metabolism regulator. The cryoprotection of resveratrol on sperm cryoinjury via activation of AMP-activated protein kinase also remains to be elucidated. Our aim, thus, was to investigate: (i) the presence and intracellular localization of AMP-activated protein kinase protein; (ii) whether resveratrol may exert a protective effect on certain functional properties of fresh and post-thaw human spermatozoa through modulation of AMP-activated protein kinase. Spermatozoa from normozoospermic men were incubated with or without different concentrations of Compound C as an AMP-activated protein kinase inhibitor or resveratrol as an AMP-activated protein kinase activator for different lengths of time and were then cryopreserved. AMP-activated protein kinase is expressed essentially in the entire flagellum and the post-equatorial region. Viability of fresh spermatozoa was not significantly affected by the presence of Compound C or resveratrol. However, although Compound C caused a potent inhibition of spermatozoa motility parameters, resveratrol did not induce negative effect, except a significant reduction in motility at 25 μm for 1 h. Furthermore, resveratrol significantly increased AMP-activated protein kinase phosphorylation and mitochondrial membrane potential and decreased reactive oxygen species and apoptosis-like changes in frozen-thawed spermatozoa. Nevertheless, it was not able to compensate decreased sperm viability and motility parameters following cryopreservation. In contrast, Compound C showed opposite effects to resveratrol on AMP-activated protein kinase phosphorylation, reactive oxygen species, apoptosis-like changes, mitochondrial membrane potential, and

Aging of oxygen and hydrogen plasma discharge treated a-C:H and ta-C coatings

Science.gov (United States)

Bachmann, Svenja; Schulze, Marcus; Morasch, Jan; Hesse, Sabine; Hussein, Laith; Krell, Lisa; Schnagl, Johann; Stark, Robert W.; Narayan, Suman

2016-05-01

Surface modification with gas plasma is an efficient and easy way to improve the surface energy and the tribological behavior of diamond-like carbon (DLC) coatings, e.g., in biomedical implants or as protective coatings. However, the long-term performance of the plasma treated DLC coatings is not fully clear. We thus studied the long-term stability of two kinds of DLC coatings, namely (a) hydrogenated amorphous carbon (a-C:H) and (b) tetrahedral amorphous carbon (ta-C) treated at different radio frequency (RF) power and time of oxygen (O2) and hydrogen (H2) plasma. Their surface properties, e.g. surface wettability, structure and tribological behavior, were studied at regular intervals for a period of two months using contact angle goniometer, Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), lateral force microscopy (LFM) and ball on disc apparatus. The surface energy of both the coatings decreased upon aging. The higher the RF power and time of treatment, the higher was the hydrophobicity upon aging. XPS analysis showed that the increase in hydrophobicity could be due to adsorption of unavoidable volatile organic components in the atmosphere. The H2 plasma treated ta-C was capable of rearranging its structural bonds upon aging. The nano-friction measurements by LFM showed that the coefficient of friction of plasma treated a-C:H and ta-C decreased upon aging. The results indicate that the surface properties of plasma treated a-C:H and ta-C are not stable on long-term and are influenced by the environmental conditions.

Second-harmonic generation microscopy used to evaluate the effect of the dimethyl sulfoxide in the cryopreservation process in collagen fibers of differentiated chondrocytes

Science.gov (United States)

Andreoli-Risso, M. F.; Duarte, A. S. S.; Ribeiro, T. B.; Bordeaux-Rego, P.; Luzo, A.; Baratti, M. O.; Adur, J.; de Thomaz, A. A.; Pelegati, V. B.; Carvalho, H. F.; Cesar, C. L.; Kharmadayan, P.; Costa, F. F.; Olalla-Saad, S. T.

2012-03-01

Cartilaginous lesions are a significant public health problem and the use of adult stem cells represents a promising therapy for this condition. Cryopreservation confers many advantages for practitioners engaged in cell-based therapies. However, conventional slow freezing has always been associated with damage and mortality due to intracellular ice formation, cryoprotectant toxicity, and dehydration. The aim of this work is to observe the effect of the usual Dimethyl Sulfoxide (DMSO) cryopreservation process on the architecture of the collagen fiber network of chondrogenic cells from mesenchymal stem cells by Second Harmonic Generation (SHG) microscopy. To perform this study we used Mesenchymal Stem Cells (MSC) derived from adipose tissue which presents the capacity to differentiate into other lineages such as osteogenic, adipogenic and chondrogenic lineages. Mesenchymal stem cells obtained after liposuction were isolated digested by collagenase type I and characterization was carried out by differentiation of mesodermic lineages, and flow cytometry using specific markers. The isolated MSCs were cryopreserved by the DMSO technique and the chondrogenic differentiation was carried out using the micromass technique. We then compared the cryopreserved vs non-cryopreserved collagen fibers which are naturally formed during the differentiation process. We observed that noncryopreserved MSCs presented a directional trend in the collagen fibers formed which was absent in the cryopreserved MSCs. We confirmed this trend quantitatively by the aspect ratio obtained by Fast Fourier Transform which was 0.76 for cryopreserved and 0.52 for non-cryopreserved MSCs, a statistical significant difference.

Recovery patterns, histological observations and genetic integrity in Malus shoot tips cryopreserved using droplet vitrification and encapsulation-dehydration procedures

Science.gov (United States)

A droplet-vitrification procedure is described for cryopreservation of Malus shoot tips. Survival patterns, recovery types, histological observations, and genetic integrity were compared for Malus shoot tips cryopreserved using this droplet-vitrification procedure and an encapsulation-dehydration pr...

Individual differences in maternal response to immune challenge predict offspring behavior: Contribution of environmental factors

Science.gov (United States)

Bronson, Stefanie L.; Ahlbrand, Rebecca; Horn, Paul S.; Kern, Joseph R.; Richtand, Neil M.

2011-01-01

Maternal infection during pregnancy elevates risk for schizophrenia and related disorders in offspring. Converging evidence suggests the maternal inflammatory response mediates the interaction between maternal infection, altered brain development, and behavioral outcome. The extent to which individual differences in the maternal response to immune challenge influence the development of these abnormalities is unknown. The present study investigated the impact of individual differences in maternal response to the viral mimic polyinosinic:polycytidylic acid (poly I:C) on offspring behavior. We observed significant variability in body weight alterations of pregnant rats induced by administration of poly I:C on gestational day 14. Furthermore, the presence or absence of maternal weight loss predicted MK-801 and amphetamine stimulated locomotor abnormalities in offspring. MK-801 stimulated locomotion was altered in offspring of all poly I:C treated dams; however, the presence or absence of maternal weight loss resulted in decreased and modestly increased locomotion, respectively. Adult offspring of poly I:C treated dams that lost weight exhibited significantly decreased amphetamine stimulated locomotion, while offspring of poly I:C treated dams without weight loss performed similarly to vehicle controls. Social isolation and increased maternal age predicted weight loss in response to poly I:C but not vehicle injection. In combination, these data identify environmental factors associated with the maternal response to immune challenge and functional outcome of offspring exposed to maternal immune activation. PMID:21255612

Advances in Stallion Semen Cryopreservation.

Science.gov (United States)

Alvarenga, Marco Antonio; Papa, Frederico Ozanam; Ramires Neto, Carlos

2016-12-01

The use of stallion frozen semen minimizes the spread of disease, eliminates geographic barriers, and preserves the genetic material of the animal for an unlimited time. Significant progress on the frozen thawed stallion semen process and consequently fertility has been achieved over the last decade. These improvements not only increased fertility rates but also allowed cryopreservation of semen from "poor freezers." This article reviews traditional steps and new strategies for stallion semen handling and processing that are performed to overcome the deleterious effects of semen preservation and consequently improve frozen semen quality and fertility. Copyright © 2016 Elsevier Inc. All rights reserved.

Development of a cryopreservation protocol for type A spermatogonia

NARCIS (Netherlands)

Izadyar, Fariborz; Matthijs-Rijsenbilt, J. J.; den Ouden, Krista; Creemers, Laura B.; Woelders, Henri; de Rooij, Dirk G.

2002-01-01

The aim of this study was to develop a cryopreservation protocol for type A spermatogonia. Testes from 5- to 7-month-old calves were collected, and type A spermatogonia were isolated using two-step enzymatic digestion and Percoll separation. Cells were resuspended in minimum essential medium (MEM)

Whole ovary cryopreservation with vascular transplantation – A future development in female oncofertility

Directory of Open Access Journals (Sweden)

Mats Brännström

2010-07-01

Full Text Available Preservation of female fertility in female cancer victims is gaining more importance in the light of the excellent survival rates today after treatment for the types of cancer, which are common in women during childhood and fertile age. In this article we discuss the risks of infertility after various cancer treatments and in what patient categories fertility preservation should be discussed. The modes of female fertility preservation used today as clinically established methods or as experimental methods that are used in the human female are reviewed. The advantages and disadvantages of the methods are also identified. Whole ovary cryopreservation has been suggested as a possible future more efficient method of fertility preservation. The research on this method in different animal models is discussed in detail and unsolved questions are identified. To date there has not been any attempt to transplant a cryopreserved whole human ovary but human ovaries have already been cryopreserved for future fertility preservation purposes, when cryobiological and microsurgical techniques have been further refined.

Neural differentiation of novel multipotent progenitor cells from cryopreserved human umbilical cord blood

International Nuclear Information System (INIS)

Lee, Myoung Woo; Moon, Young Joon; Yang, Mal Sook; Kim, Sun Kyung; Jang, In Keun; Eom, Young-woo; Park, Joon Seong; Kim, Hugh C.; Song, Kye Yong; Park, Soon Cheol; Lim, Hwan Sub; Kim, Young Jin

2007-01-01

Umbilical cord blood (UCB) is a rich source of hematopoietic stem cells, with practical and ethical advantages. To date, the presence of other stem cells in UCB remains to be established. We investigated whether other stem cells are present in cryopreserved UCB. Seeded mononuclear cells formed adherent colonized cells in optimized culture conditions. Over a 4- to 6-week culture period, colonized cells gradually developed into adherent mono-layer cells, which exhibited homogeneous fibroblast-like morphology and immunophenotypes, and were highly proliferative. Isolated cells were designated 'multipotent progenitor cells (MPCs)'. Under appropriate conditions for 2 weeks, MPCs differentiated into neural tissue-specific cell types, including neuron, astrocyte, and oligodendrocyte. Differentiated cells presented their respective markers, specifically, NF-L and NSE for neurons, GFAP for astrocytes, and myelin/oligodendrocyte for oligodendrocytes. In this study, we successfully isolated MPCs from cryopreserved UCB, which differentiated into the neural tissue-specific cell types. These findings suggest that cryopreserved human UCB is a useful alternative source of neural progenitor cells, such as MPCs, for experimental and therapeutic applications

Modulation of immune cells and Th1/Th2 cytokines in insulin-treated ...

African Journals Online (AJOL)

and Th2 cytokines and the frequencies of innate and adaptive immunity cells .... As inclusion criteria, all participants were non- .... Ranges of normal values: Fasting glucose: 3.88-6.10 mM (0.7-1.10 g/L); .... of HbA1c, reflecting a poor control of diabetes37 and a .... rophage recruitment and adipose tissue inflammation in.

Effects of seed cryopreservation, stratification and scarification on germination for five rare species of pitcher plants.

Science.gov (United States)

Khanna, Sruti; Jenkins, Heather; Bucalo, Kylie; Determann, Ron O; Cruse-Sanders, Jennifer M; Pullman, Gerald S

2014-01-01

Habitat loss and over collection have caused North American pitcher plants to become rare, including U.S. federally endangered Sarracenia alabamensis and S. oreophila, and S. leucophylla, S. psittacina and S. purpurea spp. venosa, endangered in several states. To develop reliable seed cryopreservation protocols for endangered Sarracenia species enabling similar germination percentages before and after storage in liquid nitrogen (LN) either in vivo or using in vitro tools. Seed germination pre- and post-cryopreservation were compared following seed drying with germination in soil, aseptic environment with wet filter paper or enriched medium, and using scarification or stratification for dormancy removal. After cryostorage, germination in vitro (1/6- or 1/3-strength MS medium) increased compared to germination on peat moss. Germination pre- and post-cryopreservation was similar for S. alabamensis and S. oreophila when seeds were stratified and grown in vitro. S. leucophylla and S. psittacina also showed high germination after cryopreservation when germinated on medium following stratification. Rapid liquid nitrogen exposure and rewarming induced seed coat cracking that damaged seeds, likely allowing internal damage during acid scarification and microbial entry during germination in non-sterile environments.

Cryopreservation of chayote (Sechium edule JACQ.SW.) zygotic embryos and shoot-tips from in vitroplantlets

OpenAIRE

Abdelnour-Esquivel, Ana; Engelmann, Florent

2002-01-01

This paper presents the development of cryopreservation protocols for zygotic embryos and apices of chayote (Sechium edule Jacq. Sw.), a tropical plant species with recalcitrant seeds. Zygotic embryos of two cultivars, Ccocro negro (CN) and Claudio (Cl) could withstand cryopreservation, with survival percentages of 10 and 30 %, after desiccation to 23 and 19 % moisture content (fresh weight basis), respectively. Apices sampled on in vitro plantlets of cultivars Cl, 13 and JM we...

Structure of Human cGAS Reveals a Conserved Family of Second-Messenger Enzymes in Innate Immunity

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Philip J. Kranzusch

2013-05-01

Full Text Available Innate immune recognition of foreign nucleic acids induces protective interferon responses. Detection of cytosolic DNA triggers downstream immune signaling through activation of cyclic GMP-AMP synthase (cGAS. We report here the crystal structure of human cGAS, revealing an unanticipated zinc-ribbon DNA-binding domain appended to a core enzymatic nucleotidyltransferase scaffold. The catalytic core of cGAS is structurally homologous to the RNA-sensing enzyme, 2′-5′ oligo-adenylate synthase (OAS, and divergent C-terminal domains account for specific ligand-activation requirements of each enzyme. We show that the cGAS zinc ribbon is essential for STING-dependent induction of the interferon response and that conserved amino acids displayed within the intervening loops are required for efficient cytosolic DNA recognition. These results demonstrate that cGAS and OAS define a family of innate immunity sensors and that structural divergence from a core nucleotidyltransferase enables second-messenger responses to distinct foreign nucleic acids.

On the Effects of Thermal History on the Development and Relaxation of Thermo-Mechanical Stress in Cryopreservation.

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Eisenberg, David P; Steif, Paul S; Rabin, Yoed

2014-01-01

This study investigates the effects of the thermal protocol on the development and relaxation of thermo-mechanical stress in cryopreservation by means of glass formation, also known as vitrification. The cryopreserved medium is modeled as a homogeneous viscoelastic domain, constrained within either a stiff cylindrical container or a highly compliant bag. Annealing effects during the cooling phase of the cryopreservation protocol are analyzed. Results demonstrate that an intermediate temperature-hold period can significantly reduce the maximum tensile stress, thereby decreasing the potential for structural damage. It is also demonstrated that annealing at temperatures close to glass transition significantly weakens the dependency of thermo-mechanical stress on the cooling rate. Furthermore, a slower initial rewarming rate after cryogenic storage may drastically reduce the maximum tensile stress in the material, which supports previous experimental observations on the likelihood of fracture at this stage. This study discusses the dependency of the various stress components on the storage temperature. Finally, it is demonstrated that the stiffness of the container wall can affect the location of maximum stress, with implications on the development of cryopreservation protocols.

Endocrine disruption screening by protein and gene expression of vitellogenin in freshly isolated and cryopreserved rainbow trout hepatocytes.

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Markell, Lauren K; Mingoia, Robert T; Peterson, Heather M; Yao, Jianhong; Waters, Stephanie M; Finn, James P; Nabb, Diane L; Han, Xing

2014-08-18

Xenobiotics may activate the estrogen receptor, resulting in alteration of normal endocrine functions in animals and humans. Consequently, this necessitates development of assay end points capable of identifying estrogenic xenobiotics. In the present study, we screened the potential estrogenicity of chemicals via their ability to induce vitellogenin (VTG) expression in cultured primary hepatocytes from male trout. A routine method for VTG detection measures the secretion of the protein by enzyme-linked immunosorbent assay (ELISA) in freshly isolated trout hepatocytes. However, this lengthy (6 days) culturing procedure requires that hepatocyte isolation is performed each time the assay is run. We optimized this methodology by investigating the utility of cryopreserved hepatocytes, shortening the incubation time, performing a quantitative real-time PCR (qPCR) method for VTG quantification, and verifying the model system with reference chemicals 17β-estradiol, estrone, diethylstilbestrol, hexestrol, genistein, and a negative control, corticosterone. To test the performance of both freshly isolated and cryopreserved hepatocytes, mRNA was collected from hepatocytes following 24 h treatment for VTG gene expression analysis, whereas cell culture media was collected for a VTG ELISA 96 h post-treatment. EC50 values were obtained for each reference chemical except for corticosterone, which exhibited no induction of VTG gene or protein level. Our results show linear concordance between ELISA and qPCR detection methods. Although there was approximately 50% reduction in VTG inducibility following cryopreservation, linear concordance of EC50 values was found between freshly isolated and cryopreserved hepatocytes, indicating that cryopreservation does not alter the functional assessment of estrogen receptor activation and therefore VTG expression. These studies demonstrate that qPCR is a sensitive and specific method for detecting VTG gene expression that can be used together

[In vitro activity of human bone marrow cells after cryopreservation in liquid nitrogen for 21 - 25 years].

Science.gov (United States)

Huang, You-Zhang; Shen, Jian-Liang; Gong, Li-Zhong; Zheng, Pei-Hao; Liu, Yi; Yin, Wen-Jie; Cen, Jian; Wang, Ning; Zhao, De-Feng

2010-02-01

The aim of this study was to investigate the best method to preserve human bone marrow cells and the effectiveness of long term cryopreservation at -80 degrees C. The human bone marrow cells in 20 samples were firstly frozen by a programmed freezer or -80 degrees C refrigerator, and then were preserved in liquid nitrogen with DMSO-AuP (10% dimethylsulfonamide, 10% autologous plasma) or DMSO-HES-HuA (5% dimethylsulfonamide, 6% hydroxyethyl starch, 4% human serum albumin) as cryoprotectant for 21 to 25 years. They were thawed in 38 degrees C. The cell sample frozen in -80 degrees C refrigerator was frozen at a low frozen speed of 1 degrees C/min which was the same as the programmed freezer before -30 degrees C. Before detection the bone marrow cells were taken from liquid nitrogen and were thawed in 38 degrees C, then the suspension of bone marrow cells was prepared for detection. The cell morphology and recovery rate of erythrocytes, nucleocytes and platelets; the recovery rate of hematopoietic stem progenitors cells, as well as mesenchymal stem cells were determined. The results showed that the protective effectiveness of DMSO-HES-HuA was better than DMSO-AuP. The mature erythrocytes were destroyed lightly [(3.5 +/- 1.5)% versus (12.6 +/- 4.8)%], the hemolysis rate was lower [(3.3 +/- 1.6)% versus (23.1 +/- 5.1)%]. Osmotic fragility of erythrocytes in the former was not changed, but was dropped in the latter. The recovery rates of red cell, platelet, granulocyte-macrophage colony forming units and long term culture-initiating cells were higher in the former than that in the latter [(96.1 +/- 1.8)%, (70.0 +/- 9.5)%, (49.2 +/- 10.9)%, (54.2 +/- 13.8)% versus (76.3 +/- 5.6)%, (52.7 +/- 8.1)%, (43.5 +/- 12.3)%, (47.2 +/- 13.6)% respectively]. With each kind of cryoprotectant or frozen method, the frozen MSC could keep the original growth properties. With the same cryoprotectant and different frozen method, the cryopreservative effectiveness was not different. The

Cryopreservation of turkey semen: effect of breeding line and freezing method on post-thaw sperm quality, fertilization, and hatching

Science.gov (United States)

Cryopreservation methods for poultry semen are not reliable for germplasm preservation, especially for turkeys, where fertility rates from frozen/thawed semen are particularly low. The objective was to evaluate cryopreservation methods for effectiveness in promoting cryosurvival and post-thaw funct...

Cryopreservation of Indian red jungle fowl (Gallus gallus murghi) semen.

Science.gov (United States)

Rakha, B A; Ansari, M S; Akhter, S; Hussain, I; Blesbois, E

2016-11-01

The population of red jungle fowl is declining and needs special attention for its conservation with suitable approaches. For ex situ in vitro conservation of Indian red jungle fowl, establishment of semen cryobank is an appropriate option, for which an extender with adequate retrieval capacity for functional spermatozoa is required. Therefore, studies were designed to evaluate a wide range of extenders for cryopreservation of Indian red jungle fowl (Gallus gallus murghi) sperm to achieve maximal post-thawed semen quality and fertility. For this purpose, semen from eight mature cocks were collected, initially evaluated (percent sperm motility, volume and concentration), pooled, assessed for motility, plasma membrane integrity, viability and acrosome integrity, and divided into six aliquots for dilution (1:5; 37°C) in Beltsville poultry, red fowl extender, Lake, EK, Tselutin poultry and chicken semen extenders. Diluted semen was cooled from 37°C to 4°C @ -0.275°C/min. Glycerol (20%) was added to chilled semen, equilibrated for 10min, filled in 0.5mL French straws, kept over LN 2 vapours for 10min and plunged into LN 2 and stored at -196°C. Percentages of motility, plasma membrane integrity, viability and acrosome integrity were higher (Psemen extender. Copyright © 2016 Elsevier B.V. All rights reserved.

Recent advances in the field of ovarian tissue cryopreservation and opportunities for research.

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Ladanyi, Camille; Mor, Amir; Christianson, Mindy S; Dhillon, Namisha; Segars, James H

2017-06-01

The purpose of this study was to summarize the latest advances and successes in the field of ovarian tissue cryopreservation while identifying gaps in current knowledge that suggest opportunities for future research. A systematic review was performed according to PRISMA guidelines for all relevant full-text articles in PubMed published in English that reviewed or studied historical or current advancements in ovarian tissue cryopreservation and auto-transplantation techniques. Ovarian tissue auto-transplantation in post-pubertal women is capable of restoring fertility with over 80 live births currently reported with a corresponding pregnancy rate of 23 to 37%. The recently reported successes of live births from transplants, both in orthotopic and heterotopic locations, as well as the emerging methods of in vitro maturation (IVM), in vitro culture of primordial follicles, and possibility of in vitro activation (IVA) suggest new fertility options for many women and girls. Vitrification, as an ovarian tissue cryopreservation technique, has also demonstrated successful live births and may be a more cost-effective method to freezing with less tissue injury. Further, transplantation via the artificial ovary with an extracellular tissue matrix (ECTM) scaffolding as well as the effects of sphingosine-1-phosphate (SIP) and fibrin modified with heparin-binding peptide (HBP), heparin, and a vascular endothelial growth factor (VEGF) have demonstrated important advancements in fertility preservation. As a fertility preservation method, ovarian tissue cryopreservation and auto-transplantation are currently considered experimental, but future research may pave the way for these modalities to become a standard of care for women facing the prospect of sterility from ovarian damage.

Endogenous ligands for C-type lectin receptors: the true regulators of immune homeostasis.

Science.gov (United States)

García-Vallejo, Juan J; van Kooyk, Yvette

2009-07-01

C-type lectin receptors (CLRs) have long been known as pattern-recognition receptors implicated in the recognition of pathogens by the innate immune system. However, evidence is accumulating that many CLRs are also able to recognize endogenous 'self' ligands and that this recognition event often plays an important role in immune homeostasis. In the present review, we focus on the human and mouse CLRs for which endogenous ligands have been described. Special attention is given to the signaling events initiated upon recognition of the self ligand and the regulation of glycosylation as a switch modulating CLR recognition, and therefore, immune homeostasis.

Recent Progress in Cryopreservation of Bovine Oocytes

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In-Sul Hwang

2014-01-01

Full Text Available Principle of oocyte cryoinjury is first overviewed and then research history of cryopreservation using bovine oocytes is summarized for the last two decades with a few special references to recent progresses. Various types of cryodevices have been developed to accelerate the cooling rate and applied to the oocytes from large domestic species enriched with cytoplasmic lipid droplets. Two recent approaches include the qualitative improvement of IVM oocytes prior to the vitrification and the short-term recovery culture of vitrified-warmed oocytes prior to the subsequent IVF. Supplementation of L-carnitine to IVM medium of bovine oocytes has been reported to reduce the amount of cytoplasmic lipid droplets and improve the cryotolerance of the oocytes, but it is still controversial whether the positive effect of L-carnitine is reproducible. Incidence of multiple aster formation, a possible cause for low developmental potential of vitrified-warmed bovine oocytes, was inhibited by a short-term culture of the postwarm oocytes in the presence of Rho-associated coiled-coil kinase (ROCK inhibitor. Use of an antioxidant α-tocopherol, instead of the ROCK inhibitor, also supported the revivability of the postwarm bovine oocytes. Further improvements of the vitrification procedure, combined with pre- and postvitrification chemical treatment, would overcome the high sensitivity of bovine oocytes to cryopreservation.

Blocking junctional adhesion molecule C enhances dendritic cell migration and boosts the immune responses against Leishmania major.

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Romain Ballet

2014-12-01

Full Text Available The recruitment of dendritic cells to sites of infections and their migration to lymph nodes is fundamental for antigen processing and presentation to T cells. In the present study, we showed that antibody blockade of junctional adhesion molecule C (JAM-C on endothelial cells removed JAM-C away from junctions and increased vascular permeability after L. major infection. This has multiple consequences on the output of the immune response. In resistant C57BL/6 and susceptible BALB/c mice, we found higher numbers of innate immune cells migrating from blood to the site of infection. The subsequent migration of dendritic cells (DCs from the skin to the draining lymph node was also improved, thereby boosting the induction of the adaptive immune response. In C57BL/6 mice, JAM-C blockade after L. major injection led to an enhanced IFN-γ dominated T helper 1 (Th1 response with reduced skin lesions and parasite burden. Conversely, anti JAM-C treatment increased the IL-4-driven T helper 2 (Th2 response in BALB/c mice with disease exacerbation. Overall, our results show that JAM-C blockade can finely-tune the innate cell migration and accelerate the consequent immune response to L. major without changing the type of the T helper cell response.

Long-term cryopreservation of non-spore-forming fungi in Microbank™ beads for plant pathological investigations.

Science.gov (United States)

Lakshman, Dilip K; Singh, Vimla; Camacho, Manuel E

2018-05-01

Long-term preservation of experimental fungi without genetic, morphological, and pathogenic changes is of paramount importance in mycological and plant pathological investigations. Several cryogenic and non-cryogenic methods are available for the preservation of fungi, but the methods can be cumbersome, hazardous, expensive, and often not suitable for long-term storage of non-spore-forming (sterile) fungi. A method of preservation of spore-forming fungi in commercially available porous beads (Micrbank™) under cryogenic condition was successfully tested for three non-spore-forming basidiomycetes genera: Rhizoctonia solani (teleomorph: Thanatephorus cucumeris) (n = 19), Ceratobasidium species (n = 1), and Waitea circinata (n = 3), and a non-spore forming ascomycetes, Sclerotinia sclerotiorum (n = 1). For comparison, spore-forming ascomycetous fungi, Alternaria alternata (n = 1), Bauveria basiana (n = 2), Botrytis cinerea (n = 1), Fusarium oxysporum f.sp. gladiolii (n = 1), Trichoderma spp. (n = 3), and Thielaviopsis basicola (n = 2) were also cryopreserved in Microbank beads. Viable fungal isolates of all test species were retrieved after five years of storage at -80 °C, which was longer than the viabilities of the corresponding isolates cryopreserved in agar plugs or colonized wheat seeds. Fungi revived from the Microbank beads maintained identical morphology and cultural characteristics of the parent isolates. Randomly selected Rhizoctonia isolates revived from the Microbank beads maintained respective pathological properties of the parent isolates; also, no mutation was detected in the internal transcribed spacer (ITS) ribosomal DNA when compared with respective cultures maintained at ambient temperature. This finding demonstrated the utility of cryopreservation in Microbank beads as a convenient alternative to conventional long-term preservation of a wide group of fungal cultures for plant pathological investigations

Rabbit haemorrhagic disease: advantages of cELISA in assessing immunity in wild rabbits (Oryctolagus cuniculus).

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Zheng, Tao; Parkes, John P

2011-12-15

Rabbit haemorrhagic disease (RHD) is an acute fatal disease of domestic and wild European rabbits (Oryctolagus cuniculus) caused by RHD virus (RHDV). Accurate assessment of immunity is of great importance for the conservation and control of wild rabbits. We evaluated a competitive ELISA (cELISA) against isotype ELISAs for assessing the protective immunity against the disease by challenging 50 wild-caught rabbits with a lethal dose of RHDV. Death or survival to the challenge was used as a criterion to determine the performance characteristics of the assay for the assessment of immunity in rabbits. At 1:10 dilution, a serum exhibiting ≥ 25% inhibition (1:10(25)) was regarded as the presence of RHDV-specific antibodies. Eleven of 16 (68.8%) rabbits with antibodies at 1:10(25) (<1:40) died of RHD. When the cut-off was moved from 25% to 50% inhibition (1:10(50)) at 1:10 serum dilution, the assay sensitivity, specificity and accuracy for the protective immunity were improved from 84%, 54.2% and 69.4% to 84%, 100% and 91.8%, respectively. We also demonstrated at the epitope amino acid sequence level why the presence of the RHDV-cross reactive benign rabbit calicivirus, which interfered with isotype ELISAs, had little impact on the specificity of the cELISA for the diagnosis of RHDV infection. The presence of RHDV-specific antibody at 1:10(50) by the cELISA is a reliable indicator for the protective immunity. In contrast to isotype ELISAs, the cELISA is a valuable specific tool for monitoring the herd immunity to RHD for the conservation and management of wild rabbits in the field. Copyright © 2011 Elsevier B.V. All rights reserved.

Cryopreservation of human oocytes, zygotes, embryos and blastocysts: A comparison study between slow freezing and ultra rapid (vitrification methods

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Tahani Al-Azawi

2013-12-01

Full Text Available Preservation of female genetics is currently done primarily by means of oocyte and embryo cryopreservation. The field has seen much progress during its four-decade history, progress driven predominantly by research in humans. It can also be done by preservation of ovarian tissue or entire ovary for transplantation, followed by oocyte harvesting or natural fertilization. Two basic cryopreservation techniques rule the field, slow-rate freezing, the first to be developed and vitrification which in recent years, has gained a foothold. The slow-rate freezing method previously reported had low survival and pregnancy rates, along with the high cost of cryopreservation. Although there are some recent data indicating better survival rates, cryopreservation by the slow freezing method has started to discontinue. Vitrification of human embryos, especially at early stages, became a more popular alternative to the slow rate freezing method due to reported comparable clinical and laboratory outcomes. In addition, vitrification is relatively simple, requires no expensive programmable freezing equipment, and uses a small amount of liquid nitrogen for freezing. Moreover, oocyte cryopreservation using vitrification has been proposed as a solution to maintain women’s fertility by serving and freezing their oocytes at the optimal time. The aim of this research is to compare slow freezing and vitrification in cryopreservation of oocytes, zygotes, embryos and blastocysts during the last twelve years. Therefore, due to a lot of controversies in this regard, we tried to achieve an exact idea about the subject and the best technique used.

A role in immunity for Arabidopsis cysteine protease RD21, the ortholog of the tomato immune protease C14.

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Takayuki Shindo

Full Text Available Secreted papain-like Cys proteases are important players in plant immunity. We previously reported that the C14 protease of tomato is targeted by cystatin-like EPIC proteins that are secreted by the oomycete pathogen Phytophthora infestans (Pinf during infection. C14 has been under diversifying selection in wild potato species coevolving with Pinf and reduced C14 levels result in enhanced susceptibility for Pinf. Here, we investigated the role C14-EPIC-like interactions in the natural pathosystem of Arabidopsis with the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa. In contrast to the Pinf-solanaceae pathosystem, the C14 orthologous protease of Arabidopsis, RD21, does not evolve under diversifying selection in Arabidopsis, and rd21 null mutants do not show phenotypes upon compatible and incompatible Hpa interactions, despite the evident lack of a major leaf protease. Hpa isolates express highly conserved EPIC-like proteins during infections, but it is unknown if these HpaEPICs can inhibit RD21 and one of these HpaEPICs even lacks the canonical cystatin motifs. The rd21 mutants are unaffected in compatible and incompatible interactions with Pseudomonas syringae pv. tomato, but are significantly more susceptible for the necrotrophic fungal pathogen Botrytis cinerea, demonstrating that RD21 provides immunity to a necrotrophic pathogen.

Neonatal immune activation during early and late postnatal brain development differently influences depression-related behaviors in adolescent and adult C57BL/6 mice

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Jafar Majidi-Zolbanin

2014-06-01

Full Text Available Aim: Immune challenge during early and late neonatal periods can induce robust alterations in physiological and behavioral functions, resulting in greater risk for the development of neuropsychiatric disorders, such as anxiety and depression, later in life. In addition, previous studies concluded that increasing age correlates with increased depression behaviors in humans and rodents. This study aimed to investigate for the first time whether immune challenge with a viral mimic, synthetic double-stranded ribonucleic acid (Poly I: C during different neonatal periods can differently affect depression-related behaviors in adolescent and adult mice. Methods: Male C57BL/6 mice were treated with either saline or Poly I:C (1 mg/kg and 4 mg/kg on postnatal days (PND 3-5 (early neonatal phase or PND 14-16 (late neonatal phase, and then subjected to behavioral tests, including tail suspension test and forced swimming test, during adolescence (PND 35 or 40 and adulthood (PND 85 or 90. Results: The results demonstrated that early neonatal immune activation increases depression-related behaviors in both adolescent and adult mice, but late neonatal immune activation only increases depression in adult mice. In other words, these findings indicated that the nature of the offspring's neuropathology can depend on the severity of the insult, the pup's age at the time of the insult, and offspring age at the time of behavioral testing. Conclusion: These findings suggest that dose and timing of neonatal insult and offspring age may be important factors for evaluating neuropsychiatric disorders in adults who experienced early life infection.

Cryopreservation of ovarian tissue for a decade in Denmark: a view of the technique

DEFF Research Database (Denmark)

Rosendahl, Mikkel; Schmidt, Kirsten Louise Tryde; Ernst, Erik

2011-01-01

evaluation of mouse and human ovarian tissue after freezing with four different combinations of cryoprotectants. Viability was confirmed by transplantation of frozen-thawed human ovarian tissue (n = 49) to oophorectomized Nude mice. Viability after transport of fresh tissue 4-5 h prior to freezing had...... previously been validated. Overnight transport of fresh ovarian tissue prior to cryopreservation was evaluated when human ovarian tissue was kept on ice for 20 h and then cryopreserved. The thawed ovarian tissue was transplanted to an oophorectomized Nude mouse and histology confirmed viability. In Denmark...

[The destiny of cryopreserved embryos].

Science.gov (United States)

Karpel, L; Achour-Frydman, N; Frydman, R; Flis-Trèves, M

2007-12-01

To know the psychological motivations of couples who keep their embryos so long (five years and more) and do not make a decision about them. We studied 84 couples refrained from making a decision on their cryopreserved embryos for at least five years. They were invited to fill out a questionnaire focusing on three points: the reasons of the indecision, their own representation of the cryopreserved embryos and their choice for the future: donation to another couple, to research, pregnancy or no solution for the moment. Mean (S.D.) women's and men's age were respectively, 38.8 (2.5)- and 41.3 (2.5)-years old. On average, three (1-9) embryos are preserved since 7.5 (5-12) years. Most of couples are parents. Four major reasons explain their attitudes: feeling of being too aged (25%), fear of a multiple pregnancy (45%), disagreement between members of couple (20%) and fear of failure (42.5%). Multiple choices were given to the future of the embryos: 25% wanted a pregnancy, 8% wanted to give them to infertile couples, 20% to research and 27.5% did not find any solution. Twenty percent were hesitating. The representation of those embryos is more symbolic than material. Most of the time, they see them like a potential child, a hope for the future or a brother or sister of their alive children. Those embryos are symbolized. They are a proof of fertility, a hope for another child. So, whatever the legal statement, couples will be in a dilemma because it is never easy for an infertile person to renounce to embryos, and the hope for children.

Bioengineering Strategies to Treat Female Infertility.

Science.gov (United States)

Kuo, Che-Ying; Baker, Hannah; Fries, Melissa H; Yoo, James J; Kim, Peter C W; Fisher, John P

2017-06-01

Bioengineering strategies have demonstrated enormous potential to treat female infertility as a result of chemotherapy, uterine injuries, fallopian tube occlusion, massive intrauterine adhesions, congenital uterine malformations, and hysterectomy. These strategies can be classified into two broad categories as follows: (i) Transplantation of fresh or cryopreserved organs into the host and (ii) tissue engineering approaches that utilize a combination of cells, growth factors, and biomaterials that leverages the body's inherent ability to regenerate/repair reproductive organs. While whole organ transplant has demonstrated success, the source of the organ and the immunogenic effects of allografts remain challenging. Even though tissue engineering strategies can avoid these issues, their feasibilities of creating whole organ constructs are yet to be demonstrated. In this article we summarize the recent advancements in the applications of bioengineering to treat female infertility.

Collection, analysis and cryopreservation of semen from Malayan gaur (Bos gaurus hubbacki: A preliminary study

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M.S. Khairiah

2012-10-01

Full Text Available The Malayan gaur (Bos gaurus hubbacki or Seladang is classified as vulnerable by the International Union for Conservation of Nature and Natural Resources (IUCN. The Malayan gaur is mainly distributed in the tropical woodlands of Peninsular Malaysia and Southern Thailand. The aim of this study was to collect, analyze and cryopreserve the semen of wild Malayan gaur. Transrectal massage (TM and electroejaculation (EEJ technique was applied in semen collection of the Malayan gaur. The semen was then cryopreserved in liquid nitrogen using slow freezing technique. Makler counting chamber was used to evaluate sperm concentration and motility, while the sperm viability and morphology of fresh and post-thaw sperm was determined using eosin-nigrosin staining protocol. As a result, we have successfully collected the Malayan gaur semen using EEJ technique. Sperm motility, viability and morphological changes of the post-thaw semen of Malayan gaur were found undesirable due to the complication of the cryopreservation process. On the basis of current study it can be concluded that Malayan gaur bulls semen can be obtain by EEJ with no evidence of rectal trauma. Optimization of the process of cryopreservation for Malayan gaur sperm is needed to maintain the cryoviability of the good sperm quality. The data generated in this study would be useful in conservation of genetic diversity program for Malayan gaur.

The effect of Tribulus terrestris extract on motility and viability of human sperms after cryopreservation.

Science.gov (United States)

Asadmobini, Atefeh; Bakhtiari, Mitra; Khaleghi, Sara; Esmaeili, Farzaneh; Mostafaei, Ali

2017-04-01

Semen cryopreservation produces significant amounts of reactive oxygen species (ROS), which may lead to impairment of sperm morphology, function, and ultimately, male fertility. Since Tribulus terrestris has antioxidant and free-radical-scavenging properties, this study aims to reveal the effect of the Tribulus terrestris extract on motility and vitality of human sperms after cryopreservation. Semen specimens from 80 healthy volunteers were divided into eight groups: fresh control (group I), freeze control (group II), groups III, IV, and V, which had 20, 40, and 50 μg/mL doses of Tribulus terrestris extract added before cryopreservation, and groups VI, VII, and VIII, which were supplemented by these extract doses after the freeze-thaw process. To evaluate the effects of the Tribulus terrestris extract, the semen samples were incubated with the extract and evaluated with a light microscope for motility and viability. After cryopreservation, a significant improvement in spermatozoa viability was observed in group VII. In groups VII and VIII, motility, according to World Health Organization (WHO) criteria, increased considerably (p Tribulus terrestris, which improves human sperm motility and viability, may be due to its antioxidant properties. On the basis of the results, the researchers concluded that Tribulus terrestris can be used as a safe therapeutic alternative to current modalities for the management of motility dysfunction in males. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of ozone, ultraviolet and peracetic acid disinfection of a primary-treated municipal effluent on the immune system of rainbow trout (Oncorhynchus mykiss).

Science.gov (United States)

Hébert, N; Gagné, F; Cejka, P; Bouchard, B; Hausler, R; Cyr, D G; Blaise, C; Fournier, M

2008-08-01

Municipal sewage effluents are complex mixtures that are known to compromise the health condition of aquatic organisms. The aim of this study was to evaluate the impacts of various wastewater disinfection processes on the immune system of juvenile rainbow trout (Oncorhynchus mykiss). The trout were exposed to a primary-treated effluent for 28 days before and after one of each of the following treatments: ultraviolet (UV) radiation, ozonation and peracetic acid. Immune function was characterized in leucocytes from the anterior head kidney by the following three parameters: phagocytosis activity, natural cytotoxic cells (NCC) function and lymphocyte (B and T) proliferation assays. The results show that the fish mass to length ratio was significantly decreased for the primary-treated and all three disinfection processes. Exposure to the primary-treated effluent led to a significant increase in macrophage-related phagocytosis; the addition of a disinfection step was effective in removing this effect. Both unstimulated and mitogen-stimulated T lymphocyte proliferation in fish decreased dramatically in fish exposed to the ozonated effluent compared to fish exposed to either the primary-treated effluent or to aquarium water. Stimulation of T lymphocytes proliferation was observed with the peracetic acid treatment group. In conclusion, the disinfection strategy used can modify the immune system in fish at the level of T lymphocyte proliferation but was effective to remove the effects on phagocytosis activity.

Fertility and flow cytometry study of frozen-thawed sperm in cryopreservation medium supplemented with soybean lecithin.

Science.gov (United States)

Masoudi, R; Sharafi, M; Zareh Shahneh, A; Towhidi, A; Kohram, H; Esmaeili, V; Shahverdi, A; Davachi, N Dadashpour

2016-08-01

Semen cryopreservation can provide genetic resources for a large number of females from a small number of superior males. Optimization of cryopreservation media to achieve the highest quality of post-thaw semen is crucial. Soybean lecithin has evaluated as a plant-based cryoprotectant for substitution of egg yolk in ram semen extender. Flow cytometric and fertility assessments were applied following cryopreservation procedure in two experimental groups (SL group: extender containing 1% w/v soybean lecithin and EY group: extender containing 20% v/v egg yolk). The higher percentage of live sperm and the lower percentage of dead sperm were obtained in SL (47.66 ± 1.38, 52.33 ± 1.69, respectively) extender compared to EY (41.16 ± 1.38, 58.83 ± 1.69). For motion characteristics, plasma membrane integrity, acrosome integrity and mitochondria activity, no significant difference was observed between SL and EY extenders. In artificial insemination experiment, there was no significant difference in pregnancy rate, lambing rate and twining rate between SL and EY extenders. It can be concluded that SL extender can be an efficient alternative extender to preserve ram sperm during cryopreservation procedure without adverse effects. Copyright © 2016 Elsevier Inc. All rights reserved.

Cryopreserving turkey semen in straws and nitrogen vapour using DMSO or DMA: effects of cryoprotectant concentration, freezing rate and thawing rate on post-thaw semen quality.

Science.gov (United States)

Iaffaldano, N; Di Iorio, M; Miranda, M; Zaniboni, L; Manchisi, A; Cerolini, S

2016-04-01

1. This study was designed to identify a suitable protocol for freezing turkey semen in straws exposed to nitrogen vapour by examining the effects of dimethylacetamide (DMA) or dimethylsulfoxide (DMSO) as cryoprotectant (CPA), CPA concentration, freezing rate and thawing rate on in vitro post-thaw semen quality. 2. Pooled semen samples were diluted 1:1 (v:v) with a freezing extender composed of Tselutin diluent containing DMA or DMSO to give final concentrations of 8% or 18% DMA and 4% or 10% DMSO. The semen was packaged in 0.25 ml plastic straws and frozen at different heights above the liquid nitrogen (LN2) surface (1, 5 and 10 cm) for 10 min. Semen samples were thawed at 4°C for 5 min or at 50°C for 10 s. After thawing, sperm motility, viability and osmotic tolerance were determined. 3. Cryosurvival of turkey sperm was affected by DMSO concentration. Freezing rate affected the motility of sperm cryopreserved using both CPAs, while thawing rates showed an effect on the motility of sperm cryopreserved using DMA and on the viability of sperm cryopreserved using DMSO. Significant interactions between freezing rate × thawing rate on sperm viability in the DMA protocol were found. 4. The most effective freezing protocol was the use of 18% DMA or 10% DMSO with freezing 10 cm above the LN2 surface and a thawing temperature of 50°C. An efficient protocol for turkey semen would improve prospects for sperm cryobanks and the commercial use of frozen turkey semen.

Consequences of metaphase II oocyte cryopreservation on mRNA content.

Science.gov (United States)

Chamayou, S; Bonaventura, G; Alecci, C; Tibullo, D; Di Raimondo, F; Guglielmino, A; Barcellona, M L

2011-04-01

We studied the consequences of freezing/thawing processes on mRNA contents in MII oocytes after slow-freezing/rapid thawing (SF/RT) and vitrification/warming (V/W) protocols, and compared the results to fresh MII oocytes. We quantified the nuclear transcript mRNA responsible for the translation of proteins belonging either to trans-regulatory protein family or to functional structural proteins such as proteins involved in DNA structural organization (NAP1L1, TOP1, H1F0H1), chromosomal structure maintenance (SMC, SCC3, RAD21, SMC1A, SMC1B, STAG3, REC8), mitochondrial energetic pathways (ATP5GJ, SDHC), cell cycle regulation and processes (CLTA, MAPK6, CKS2) and staminal cell potency-development competence stage (DPPA3, OCT4, FOXJ2). Surplus MII oocytes were donated from patients in IVF cycles and divided in three groups of 15 oocytes. Group 1 was comprised of non-cryopreserved oocytes and Groups 2 and 3 underwent SF/RT and V/W procedures, respectively. There was an overall decrease of mRNA extracted from cryopreserved oocytes compared to control group. Only 39.4% of mRNA content were preserved after SF/RT while 63.3% of mRNA content were maintained after V/W. Oocyte cryopreservation is associated with molecular injury associated with the decrease of stored mRNA. However the V/W protocol is more conservative than SF/RT resulting in a level of mRNA sufficient to maintain biologic functions in the subsequent fertilized oocyte. Copyright © 2011 Elsevier Inc. All rights reserved.

Carbon Nanostructure of Kraft Lignin Thermally Treated at 500 to 1000 °C.

Science.gov (United States)

Zhang, Xuefeng; Yan, Qiangu; Leng, Weiqi; Li, Jinghao; Zhang, Jilei; Cai, Zhiyong; Hassan, El Barbary

2017-08-21

Kraft lignin (KL) was thermally treated at 500 to 1000 °C in an inert atmosphere. Carbon nanostructure parameters of thermally treated KL in terms of amorphous carbon fraction, aromaticity, and carbon nanocrystallites lateral size ( L a ), thickness ( L c ), and interlayer space ( d 002 ) were analyzed quantitatively using X-ray diffraction, Raman spectroscopy, and high-resolution transmission electron microscopy. Experimental results indicated that increasing temperature reduced amorphous carbon but increased aromaticity in thermally treated KL materials. The L c value of thermally treated KL materials averaged 0.85 nm and did not change with temperature. The d 002 value decreased from 3.56 Å at 500 °C to 3.49 Å at 1000 °C. The L a value increased from 0.7 to 1.4 nm as temperature increased from 500 to 1000 °C. A nanostructure model was proposed to describe thermally treated KL under 1000 °C. The thermal stability of heat treated KL increased with temperature rising from 500 to 800 °C.

Cryopreservation does not alter main characteristics of Good Manufacturing Process-grade human multipotent mesenchymal stromal cells including immunomodulating potential and lack of malignant transformation.

Science.gov (United States)

Luetzkendorf, Jana; Nerger, Katrin; Hering, Julian; Moegel, Angelika; Hoffmann, Katrin; Hoefers, Christiane; Mueller-Tidow, Carsten; Mueller, Lutz P

2015-02-01

The immunomodulating capacity of multipotent mesenchymal stromal cells (MSCs) qualifies them as a therapeutic tool in several diseases. However, repeated transplantation with products of reproducible characteristics may be required. This could be achieved with cryopreserved aliquots of Good Manufacturing Practice (GMP)-grade MSCs. However, the impact of cryopreservation on the characteristics of GMP-MSCs is ill defined. We produced fresh and cryopreserved MSCs from human donors with a xenogen-free GMP protocol. Immunogenicity and immunomodulating capacity were tested in co-culture with putative recipient-specific peripheral blood mononuclear cells (PBMCs). Risk of malignant transformation was assessed in vitro and in vivo. Cryopreservation had no impact on viability and consensus criteria of MSCs. In co-culture with PBMCs, MSCs showed low immunogenicity and suppressed mitogen-stimulated proliferation of PBMC irrespective of cryopreservation. Cytogenetic aberrations were not observed consistently in fresh and cryopreserved products, and no signs of malignant transformation occurred in functional assays. MSC products from an elderly pretreated donor showed reduced functional quality, but imminent failure of functional criteria could be detected by an increased population doubling time in early passages. This study is the first systematic analysis on cryopreservation of xenogen-free human bone marrow-derived GMP-MSCs. The data support that cryopreservation does not alter the characteristics of the cells and thus may allow the generation of products for serial transplantation. In addition, the protocol allowed early detection of MSC products with low functional capacity. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

c-Myb is required for plasma cell migration to bone marrow after immunization or infection

Science.gov (United States)

O’Donnell, Kristy; Belz, Gabrielle T.; Nutt, Stephen L.

2015-01-01

Plasma cell migration is crucial to immunity, but little is known about the molecular regulators of their migratory programs. Here, we detail the critical role of the transcription factor c-Myb in determining plasma cell location. In the absence of c-Myb, no IgG+ antigen-specific plasma cells were detected in the bone marrow after immunization or virus infection. This was correlated with a dramatic reduction of plasma cells in peripheral blood, mislocalization in spleen, and an inability of c-Myb–deficient plasma cells to migrate along a CXCL12 gradient. Therefore, c-Myb plays an essential, novel role in establishing the long-lived plasma cell population in the BM via responsiveness to chemokine migration cues. PMID:26077717

Magnetic induction heating of superparamagnetic nanoparticles during rewarming augments the recovery of hUCM-MSCs cryopreserved by vitrification.

Science.gov (United States)

Wang, Jianye; Zhao, Gang; Zhang, Zhengliang; Xu, Xiaoliang; He, Xiaoming

2016-03-01

Cryopreservation by vitrification has been recognized as a promising strategy for long-term banking of living cells. However, the difficulty to generate a fast enough heating rate to minimize devitrification and recrystallization-induced intracellular ice formation during rewarming is one of the major obstacles to successful vitrification. We propose to overcome this hurdle by utilizing magnetic induction heating (MIH) of magnetic nanoparticles to enhance rewarming. In this study, superparamagnetic (SPM) Fe3O4 nanoparticles were synthesized by a chemical coprecipitation method. We successfully applied the MIH of Fe3O4 nanoparticles for rewarming human umbilical cord matrix mesenchymal stem cells (hUCM-MSCs) cryopreserved by vitrification. Our results show that extracellular Fe3O4 nanoparticles with MIH may efficiently suppress devitrification and/or recrystallization during rewarming and significantly improve the survival of vitrified cells. We further optimized the concentration of Fe3O4 nanoparticles and the current of an alternating current (AC) magnetic field for generating the MIH to maximize cell viability. Our results indicate that MIH in an AC magnetic field with 0.05% (w/v) Fe3O4 nanoparticles significantly facilitates rewarming and improves the cryopreservation outcome of hUCM-MSCs by vitrification. The application of MIH of SPM nanoparticles to achieve rapid and spatially homogeneous heating is a promising strategy for enhanced cryopreservation of stem cells by vitrification. Here we report the successful synthesis and application of Fe3O4 nanoparticles for magnetic induction heating (MIH) to enhance rewarming of vitrification-cryopreserved human umbilical cord matrix mesenchymal stem cells (hUCM-MSCs). We found that MIH-enhanced rewarming greatly improves the survival of vitrification-cryopreserved hUCM-MSCs. Moreover, the hUCM-MSCs retain their intact stemness and multilineage potential of differentiation post cryopreservation by vitrification with the

Effects of treated sewage effluent on immune function in rainbow trout (Oncorhynchus mykiss)

Energy Technology Data Exchange (ETDEWEB)

Hoeger, Birgit [Environmental Toxicology, University of Konstanz, P.O. Box X918, D-78457 Constance (Germany); Heuvel, Michael R. van den [Forest Research, Private Bag 3020, Sala St., Rotorua (New Zealand); Hitzfeld, Bettina C. [Swiss Agency for the Environment, Forests and Landscape (SAEFL), Substances, Soil, Biotechnology Division, Section Substances, 3003 Bern (Switzerland); Dietrich, Daniel R. [Environmental Toxicology, University of Konstanz, P.O. Box X918, D-78457 Constance (Germany)]. E-mail: daniel.dietrich@uni-konstanz.de

2004-12-20

In this study, the immune reactions of rainbow trout (Oncorhynchus mykiss) were examined, after exposure to 10, 30 and 70% of tertiary-treated municipal sewage effluent for 27 days. Exposures were conducted concurrently with and without an immune challenge using intraperitoneal injections of inactivated Aeromonas salmonicida salmonicida. Due to the time required to prepare and analyse samples, fish sampling was conducted over two consecutive days. There was no trout mortality for any of the experimental treatments. The exposure to effluent increased in vitro lymphocyte proliferation, decreased circulating lymphocytes and increased degrading erythrocytes in peripheral blood samples. Circulating lymphocytes were only decreased in the sham-injected, but not in the A. salmonicida-injected group. In addition to effluent effects, circulating lymphocytes and lymphocyte proliferation were decreased on day 2 of sampling as compared to day 1. Concentration-dependent degradation of erythrocytes was only observed on day 2 of sampling. Capture and removal of trout on day 1 of sampling presumably caused low-level stress that affected some results on day 2. Oxidative burst, phagocytosis, lysozyme, leucocyte populations other than lymphocytes and A. salmonicida-specific IgM production were not affected by exposure to effluent, and of these parameters, only oxidative burst and total leucocytes showed sampling day effects. From these results it can be observed, that with the exception of oxidative burst, those variables affected by effluent exposure were also significantly changed by the low-level sampling stress imposed by staggered sampling. Elevated liver mixed-function oxygenase activity as measured by 7-ethoxyresorufin-O-deethylase activity, and increased bile polycyclic aromatic hydrocarbon (PAH) metabolites were observed in response to sewage effluent exposure. As both PAHs and stress are known immune suppressors, it is difficult to conclude whether or not changes in immune

Effects of treated sewage effluent on immune function in rainbow trout (Oncorhynchus mykiss)

International Nuclear Information System (INIS)

Hoeger, Birgit; Heuvel, Michael R. van den; Hitzfeld, Bettina C.; Dietrich, Daniel R.

2004-01-01

In this study, the immune reactions of rainbow trout (Oncorhynchus mykiss) were examined, after exposure to 10, 30 and 70% of tertiary-treated municipal sewage effluent for 27 days. Exposures were conducted concurrently with and without an immune challenge using intraperitoneal injections of inactivated Aeromonas salmonicida salmonicida. Due to the time required to prepare and analyse samples, fish sampling was conducted over two consecutive days. There was no trout mortality for any of the experimental treatments. The exposure to effluent increased in vitro lymphocyte proliferation, decreased circulating lymphocytes and increased degrading erythrocytes in peripheral blood samples. Circulating lymphocytes were only decreased in the sham-injected, but not in the A. salmonicida-injected group. In addition to effluent effects, circulating lymphocytes and lymphocyte proliferation were decreased on day 2 of sampling as compared to day 1. Concentration-dependent degradation of erythrocytes was only observed on day 2 of sampling. Capture and removal of trout on day 1 of sampling presumably caused low-level stress that affected some results on day 2. Oxidative burst, phagocytosis, lysozyme, leucocyte populations other than lymphocytes and A. salmonicida-specific IgM production were not affected by exposure to effluent, and of these parameters, only oxidative burst and total leucocytes showed sampling day effects. From these results it can be observed, that with the exception of oxidative burst, those variables affected by effluent exposure were also significantly changed by the low-level sampling stress imposed by staggered sampling. Elevated liver mixed-function oxygenase activity as measured by 7-ethoxyresorufin-O-deethylase activity, and increased bile polycyclic aromatic hydrocarbon (PAH) metabolites were observed in response to sewage effluent exposure. As both PAHs and stress are known immune suppressors, it is difficult to conclude whether or not changes in immune

Thermo-mechanical stress analysis of cryopreservation in cryobags and the potential benefit of nanowarming.

Science.gov (United States)

Solanki, Prem K; Bischof, John C; Rabin, Yoed

2017-06-01

Cryopreservation by vitrification is the only promising solution for long-term organ preservation which can save tens of thousands of lives across the world every year. One of the challenges in cryopreservation of large-size tissues and organs is to prevent fracture formation due to the tendency of the material to contract with temperature. The current study focuses on a pillow-like shape of a cryobag, while exploring various strategies to reduce thermo-mechanical stress during the rewarming phase of the cryopreservation protocol, where maximum stresses are typically found. It is demonstrated in this study that while the level of stress may generally increase with the increasing amount of CPA filled in the cryobag, the ratio between width and length of the cryobag play a significant role. Counterintuitively, the overall maximum stress is not found when the bag is filled to its maximum capacity (when the filled cryobag resembles a sphere). Parametric investigation suggests that reducing the initial rewarming rate between the storage temperature and the glass transition temperature may dramatically decrease the thermo-mechanical stress. Adding a temperature hold during rewarming at the glass transition temperature may reduce the thermo-mechanical stress in some cases, but may have an adverse effect in other cases. Finally, it is demonstrated that careful incorporation of volumetric heating by means on nanoparticles in an alternating magnetic field, or nanowarming, can dramatically reduce the resulting thermo-mechanical stress. These observations display the potential benefit of a thermo-mechanical design of the cryopreservation protocols in order to prevent structural damage. Copyright © 2017 Elsevier Inc. All rights reserved.

C-type lectins: their network and roles in pathogen recognition and immunity.

Science.gov (United States)

Mayer, Sabine; Raulf, Marie-Kristin; Lepenies, Bernd

2017-02-01

C-type lectins (CTLs) represent the most complex family of animal/human lectins that comprises 17 different groups. During evolution, CTLs have developed by diversification to cover a broad range of glycan ligands. However, ligand binding by CTLs is not necessarily restricted to glycans as some CTLs also bind to proteins, lipids, inorganic molecules, or ice crystals. CTLs share a common fold that harbors a Ca 2+ for contact to the sugar and about 18 invariant residues in a phylogenetically conserved pattern. In vertebrates, CTLs have numerous functions, including serum glycoprotein homeostasis, pathogen sensing, and the initiation of immune responses. Myeloid CTLs in innate immunity are mainly expressed by antigen-presenting cells and play a prominent role in the recognition of a variety of pathogens such as fungi, bacteria, viruses, and parasites. However, myeloid CTLs such as the macrophage inducible CTL (Mincle) or Clec-9a may also bind to self-antigens and thus contribute to immune homeostasis. While some CTLs induce pro-inflammatory responses and thereby lead to activation of adaptive immune responses, other CTLs act as inhibitory receptors and dampen cellular functions. Since CTLs are key players in pathogen recognition and innate immunity, targeting CTLs may be a promising strategy for cell-specific delivery of drugs or vaccine antigens and to modulate immune responses.

Cryopreservation of in vitro -grown shoot tips of apricot ( Prunus ...

African Journals Online (AJOL)

In vitro grown apricot (Prunus armeniaca L.) cv. El-Hamawey shoot tips were successfully cryopreserved using an encapsulation-dehydration procedure. Shoot tips were encapsulated in calcium-alginate beads before preculture on woody plant (WP) medium supplemented with different sucrose concentrations; 0.1, 0.3, 0.5, ...

Ice Recrystallization Inhibiting Polymers Enable Glycerol-Free Cryopreservation of Micro-organisms.

Science.gov (United States)

Hasan, Muhammad; Fayter, Alice E R; Gibson, Matthew I

2018-06-22

All modern molecular biology and microbiology is underpinned not only by the tools to handle and manipulate microorganisms, but also those to store, bank and transport them. Glycerol is the current gold-standard cryoprotectant but it is intrinsically toxic to most micro-organisms: only a fraction of cells survive freezing and the presence of glycerol can impact down-stream applications and assays. Extremophile organisms survive repeated freeze/thaw cycles by producing antifreeze proteins which are potent ice recrystallization inhibitors. Here we introduce a new concept for the storage/transport of micro-organisms by using ice recrystallization inhibiting poly(vinyl alcohol) in tandem with poly(ethylene glycol). This cryopreserving formulation is shown to result in a 4-fold increase in E. coli yield post-thaw, compared to glycerol, utilizing lower concentrations, with successful cryopreservation at just 1.1 weight percent of additive. The mechanism of protection is demonstrated to be linked to inhibiting ice recrystallization (by comparison to a recombinant antifreeze protein) but also to the significantly lower toxicity of the polymers compared to glycerol. Optimized formulations are presented and shown to be broadly applicable to the cryopreservation of a panel of Gram negative, Gram positive and Mycobacteria strains. This represents a step-change in how micro-organisms will be stored by the design of new macromolecular ice growth inhibitors; it should enable a transition from traditional solvent-based to macromolecular microbiology storage methods.

Oral immune therapy: targeting the systemic immune system via the gut immune system for the treatment of inflammatory bowel disease.

Science.gov (United States)

Ilan, Yaron

2016-01-01

Inflammatory bowel diseases (IBD) are associated with an altered systemic immune response leading to inflammation-mediated damage to the gut and other organs. Oral immune therapy is a method of systemic immune modulation via alteration of the gut immune system. It uses the inherit ability of the innate system of the gut to redirect the systemic innate and adaptive immune responses. Oral immune therapy is an attractive clinical approach to treat autoimmune and inflammatory disorders. It can induce immune modulation without immune suppression, has minimal toxicity and is easily administered. Targeting the systemic immune system via the gut immune system can serve as an attractive novel therapeutic method for IBD. This review summarizes the current data and discusses several examples of oral immune therapeutic methods for using the gut immune system to generate signals to reset systemic immunity as a treatment for IBD.

Cryopreserved Ultra-Thick Human Amniotic Membrane for Conjunctival Surface Reconstruction After Excision of Conjunctival Tumors.

Science.gov (United States)

Tanaka, Thais S; Demirci, Hakan

2016-04-01

Cryopreserved ultra-thick human amniotic membrane (AM) is used for glaucoma surgery. We evaluated the use of cryopreserved ultra-thick human AM for conjunctival surface reconstruction after excision of a conjunctival tumor. We retrospectively reviewed the medical records of 28 patients who underwent conjunctival surface reconstruction with cryopreserved ultra-thick human AM after excision of the tumor. The AM was secured to the surrounding conjunctiva and underlying sclera with interrupted 8-0 Vicryl sutures. Clinical data regarding demographics, diagnosis, size and location of conjunctival tumors, patient outcome, and complications were gathered. Of 28 patients, 6 (21.4%) had malignant melanoma, 4 (14.3%) had squamous cell carcinoma, 6 (21.4%) had conjunctival intraepithelial neoplasia, 1 (3.6%) had sebaceous carcinoma, 1 (3.6%) had mucoepidermoid carcinoma, 1 (3.6%) had conjunctival intraepithelial dysplasia, 5 (17.9%) had pterygium, 2 (7.1%) had compound nevus, 1 (3.6%) had a large epithelial inclusion cyst, and 1 (3.6%) patient had a granuloma. The mean area of graft size was 156 ± 120 mm2. Postoperatively, the graft was well tolerated with no failure, discomfort, or dehiscence. During the 17-month mean follow-up, symblepharon, which was clinically nonsignificant, developed in 3 (11%) patients and partial stem cell deficiency was noted in 5 (18%) patients. Cryopreserved ultra-thick human AM is a well-tolerated, effective graft material that is easy to handle. It is a viable alternative for conjunctival surface reconstruction after excision of a conjunctival tumor.

Studies on the regeneration of the CFU-C population in blood and bone marrow or lethally irradiated dogs after autologous transfusion of cryopreserved mononuclear blood cells

International Nuclear Information System (INIS)

Nothdurft, W.; Bruch, C.; Fliedner, T.M.; Rueber, E.

1977-01-01

In a group of 8 lethally irradiated (1200 R) dogs, that were transfused autologously with cryopreserved mononuclear cells (MNC) derived from the peripheral blood by leucapheresis the concentration of colony-forming units in agar (CFU-C) in bone marrow and peripheral blood was estimated at regular intervals after irradiation and transfusion of MNC. The numbers of MNC transfused per kg body weight ranged from 0.32 x 10 9 to 1.63 x 10 9 with an incidence of CFU-C between 0.02 x 10 5 and 1.38 x 10 5 . In 6 dogs the CFU-C levels in the bone marrow reached the normal preirradiation values between days 15 and 20. But in 2 dogs that had received the lowest CFU-C numbers the regeneration of the bone marrow CFU-C was markedly delayed. In general the time course of the bone marrow repopulation by CFU-C for single dogs was reflected by a corresponding regeneration pattern of the blood CFU-C. The time course of the curves for the blood CFU-C levels on the other hand was of the same kind as for the granulocyte values in the peripheral blood, that reached the normal levels mainly around day 30 and thereafter. Considerable fluctuations were seen in the blood CFU-C levels of single dogs before irradiation and after mononuclear leucocyte transfusion. Despite of such limitations the blood CFU-C content appeared to be a useful indicator of haematopoietic regeneration of the bone marrow. (author)

Construction and application of a bovine immune-endocrine cDNA microarray.

Science.gov (United States)

Tao, Wenjing; Mallard, Bonnie; Karrow, Niel; Bridle, Byram

2004-09-01

A variety of commercial DNA arrays specific for humans and rodents are widely available; however, microarrays containing well-characterized genes to study pathway-specific gene expression are not as accessible for domestic animals, such as cattle, sheep and pigs. Therefore, a small-scale application-targeted bovine immune-endocrine cDNA array was developed to evaluate genetic pathways involved in the immune-endocrine axis of cattle during periods of altered homeostasis provoked by physiological or environmental stressors, such as infection, vaccination or disease. For this purpose, 167 cDNA sequences corresponding to immune, endocrine and inflammatory response genes were collected and categorized. Positive controls included 5 housekeeping genes (glyceraldehydes-3-phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase, ribosomal protein L19, beta-actin, beta2-microglobulin) and bovine genomic DNA. Negative controls were a bacterial gene (Rhodococcus equi 17-kDa virulence-associated protein) and a partial sequence of the plasmid pACYC177. In addition, RNA extracted from un-stimulated, as well as superantigen (Staphylococcus aureus enterotoxin-A, S. aureus Cowan Pansorbin Cells) and mitogen-stimulated (LPS, ConA) bovine blood leukocytes was mixed, reverse transcribed and PCR amplified using gene-specific primers. The endocrine-associated genes were amplified from cDNA derived from un-stimulated bovine hypothalamus, pituitary, adrenal and thyroid gland tissues. The array was constructed in 4 repeating grids of 180 duplicated spots by coupling the PCR amplified 213-630 bp gene fragments onto poly-l-lysine coated glass slides. The bovine immune-endocrine arrays were standardized and preliminary gene expression profiles generated using Cy3 and Cy5 labelled cDNA from un-stimulated and ConA (5 microg/ml) stimulated PBMC of 4 healthy Holstein cows (2-4 replicate arrays/cow) in a time course study. Mononuclear cell-derived cytokine and chemokine (IL-2, IL-1alpha

Studies on a transplantable C57BL/6 mouse lymphoma

International Nuclear Information System (INIS)

Kendall, C.E.

1977-01-01

A C57BL/6 mouse lymphoma was demonstrated to be of T cell origin by treating the lymphoma cells with anti-Thy 1.2 antisera in a complement-dependent cytotoxicity test. The lymphoma's growth pattern was described using flow microfluorometric determinations and spleen weight progression. C-type particles were identified in electron micrographs of the lymphoma. C57BL/6 mice were immunized against the lymphoma by injecting x-ray inactivated lymphoma cells into the mice. Protection of immunized mice against live lymphoma cells demonstrated tumor antigens on the lymphoma cells. The success of immunization was found to depend on: route of injection, antigen dosage, state of the antigen, number of injections and the vaccination-challenge interval. Attempts were made to passively transfer immunity from immunized C57BL/6 mice which had survived lymphoma challenge to non-treated, syngeneic mice. The route of immunization in the donors influenced the success of passively transferred immunity in the recipients. Serum from days 1 to 3 and days 11 to death (day 17) had an enhancing effect on lymphoma growth. However, sera from days 5 to 9 retarded lymphoma growth. The C57BL/6 lymphoma cells were injected into rabbits and other strains of mice to demonstrate tumor specificity. The lymphoma did not grow in rabbits and only grew in one mouse strain. This strain had the same major histocompatibility loci as C57BL/6 mice. Crosses were made between C57BL/6 mice and a resistant strain of mice (DBA/2 mice). The F 1 hybrids were found to be less susceptible to the lymphoma than the C57BL/6 strain. Sublethal x-irradiation of the F 1 mice decreased its ability to resist the C57BL/6 lymphoma. Immunization with x-ray inactivated C57BL/6 lymphoma cells increased survival after challenge with lymphoma in the F 1 mice

Efficient cryopreservation of human pluripotent stem cells by surface-based vitrification

NARCIS (Netherlands)

Neubauer, Julia C; Beier, Axel F; Geijsen, Niels; Zimmermann, Heiko

2015-01-01

Efficient cryopreservation of human stem cells is crucial for guaranteeing a permanent supply of high-quality cell material for drug discovery or regenerative medicine. Conventionally used protocols usually employing slow freezing rates, however, result in low recovery rates for human pluripotent

Zona pellucida damage to human embryos after cryopreservation and the consequences for their blastomere survival and in-vitro viability.

Science.gov (United States)

Van Den Abbeel, E; Van Steirteghem, A

2000-02-01

The study objective was to quantify zona pellucida (ZP) damage in cryopreserved human embryos. The influence of two different freezing containers was investigated, and the influence of freezing damage on the survival and viability of the embryos evaluated. ZP damage did not differ according to whether embryos originated from in-vitro fertilization (IVF) cycles or from IVF cycles in association with intracytoplasmic sperm injection (ICSI). The freezing container, however, significantly influenced the occurrence of ZP damage after cryopreservation. More damage was observed when the embryos were frozen-thawed using plastic cryovials than using plastic mini-straws (16.6% versus 2.3%; P plastic mini-straws. The further cleavage of frozen-thawed embryos suitable for transfer was not different whether there was ZP damage or not; however, it was higher when there was 100% blastomere survival as compared with when some blastomeres were damaged (79.0% versus 43.7%; P plastic mini-straws. In conclusion, the aim of a cryopreservation programme should be to have as many fully intact embryos as possible after thawing. Increased ZP damage might indicate a suboptimal cryopreservation procedure.

Effects of DHA-enriched hen egg yolk and L-cysteine supplementation on quality of cryopreserved boar semen.

Science.gov (United States)

Chanapiwat, Panida; Kaeoket, Kampon; Tummaruk, Padet

2009-09-01

The objective of the present study was to determine the effects of docosahexaenoic acid (DHA)-enriched hen egg yolks and L-cysteine supplementation on the qualities of the cryopreserved boar semen. A total of 15 ejaculates from 5 Pietrain boars were divided into 4 groups according to the compositions of the freezing extenders used, that is, normal hen egg yolk (group I), DHA-enriched hen egg yolk (group II), normal hen egg yolk with 5 mmol L(-1) of cysteine supplementation (group III) and DHA-enriched hen egg yolk with 5 mmol L(-1) of cysteine supplementation (group IV). The semen was cryopreserved using controlled rate freezer and was thawed at 50 degrees C for 12 s. Progressive motility, sperm viability, acrosome integrity and functional integrity of sperm plasma membrane of the post-thawed semen were evaluated. The supplementation of L-cysteine in the freezing extender alone (group III) improved progressive motility (P semen qualities (P > 0.05). In conclusion, the supplementation of antioxidant L-cysteine alone or in combination with DHA-enriched hen egg yolk significantly improved the post-thawed semen qualities, especially progressive motility and acrosome integrity.

Immune Evasion Strategies during Chronic Hepatitis B and C Virus Infection

Science.gov (United States)

Ortega-Prieto, Ana Maria; Dorner, Marcus

2017-01-01

Both hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are a major global healthcare problem with more than 240 million and 70 million infected, respectively. Both viruses persist within the liver and result in progressive liver disease, resulting in liver fibrosis, cirrhosis and hepatocellular carcinoma. Strikingly, this pathogenesis is largely driven by immune responses, unable to clear an established infection, rather than by the viral pathogens themselves. Even though disease progression is very similar in both infections, HBV and HCV have evolved distinct mechanisms, by which they ensure persistence within the host. Whereas HCV utilizes a cloak-and-dagger approach, disguising itself as a lipid-like particle and immediately crippling essential pattern-recognition pathways, HBV has long been considered a “stealth” virus, due to the complete absence of innate immune responses during infection. Recent developments and access to improved model systems, however, revealed that even though it is among the smallest human-tropic viruses, HBV may, in addition to evading host responses, employ subtle immune evasion mechanisms directed at ensuring viral persistence in the absence of host responses. In this review, we compare the different strategies of both viruses to ensure viral persistence by actively interfering with viral recognition and innate immune responses. PMID:28862649

Thixotropic injectable hydrogel using a polyampholyte and nanosilicate prepared directly after cryopreservation

Energy Technology Data Exchange (ETDEWEB)

Jain, Minkle; Matsumura, Kazuaki, E-mail: mkazuaki@jaist.ac.jp

2016-12-01

Success of tissue engineering applications in regenerative medicine requires the preservation of tissue-engineered products at a low temperature. This can be successfully achieved by the use of cryoprotective agent (CPA). In this study, we formulated a unique injectable hydrogel for the purpose of cell delivery after cryopreservation by using polyampholyte CPA. The polyampholyte showed excellent post-thaw cell survival, and after thawing, the polymeric CPA did not have to be removed because of its low cytotoxicity. The polyampholyte could be transformed into a hydrogel by mixing with nanosilicates. Previously, nanosilicates were used to improve mechanical properties, but this is the first report of the use of a nanosilicate together with CPA to formulate hydrogels. Inclusion of the nanosilicate led to the formation of thixotropic hydrogels, which can be injected using fine needles. These gels with tunable mechanical properties can be injected into defect sites to form scaffolds for cell growth and tissue repair, and they do not require any separate seeding of cells before injection, thus eliminating the need for cell harvesting and cell maintenance. This is a distinct system in which cells can be cryopreserved until before usage; when required, the cells in the polyampholyte can be revived to their original state and the thixotropic hydrogel can be formed. The combination of thixotropy and cytocompatibility of the gels could enable a wide range of biomedical applications such as cell delivery and orthopedic repair. - Graphical abstract: A novel thixotropic, cytocompatible and injectable nanocomposite hydrogel with tunable mechanical properties directly after cryopreservation was formulated using an efficient polymer cryoprotectant and laponite. This is an efficient system in which cells remain viable and can proliferate even after one week. The combined use of thixotropy and cytocompatibility enables this system to be used for cell delivery applications

Systemic Immune-Inflammation Index and Circulating T-Cell Immune Index Predict Outcomes in High-Risk Acral Melanoma Patients Treated with High-Dose Interferon

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Jiayi Yu

2017-10-01

Full Text Available High-dose interferon alfa-2b (IFN-α-2b improves the survival of patients with high-risk melanoma. We aimed to identify baseline peripheral blood biomarkers to predict the outcome of acral melanoma patients treated with IFN-α-2b. Pretreatment baseline parameters and clinical data were assessed in 226 patients with acral melanoma. Relapse-free survival (RFS and overall survival (OS were assessed using the Kaplan-Meier method, and multivariate Cox regression analyses were applied after adjusting for stage, lactate dehydrogenase (LDH, and ulceration. Univariate analysis showed that neutrophil-to-lymphocyte ratio ≥2.35, platelet-to-lymphocyte ratio ≥129, systemic immune-inflammation index (SII ≥615 × 109/l, and elevated LDH were significantly associated with poor RFS and OS. The SII is calculated as follows: platelet count × neutrophil count/lymphocyte count. On multivariate analysis, the SII was associated with RFS [hazard ratio (HR=1.661, 95% confidence interval (CI: 1.066-2.586, P=.025] and OS (HR=2.071, 95% CI: 1.204-3.564, P=.009. Additionally, we developed a novel circulating T-cell immune index (CTII calculated as follows: cytotoxic T lymphocytes/(CD4+ regulatory T cells × CD8+ regulatory T cells. On univariate analysis, the CTII was associated with OS (HR=1.73, 95% CI: 1.01-2.94, P=.044. The SII and CTII might serve as prognostic indicators in acral melanoma patients treated with IFN-α-2b. The indexes are easily obtainable via routine tests in clinical practice.

Ovarian cryopreservation after laparoscopic ovariectomy using the Endo-GIA stapling device and LAPRO-clip absorbable ligating clip in a woman: a case report

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Messner Alexandra

2011-02-01

Full Text Available Abstract Introduction Several options are available for preserving fertility before cytotoxic treatment, including ovarian tissue cryopreservation. Most reported surgical techniques include electrocoagulation. Our hypothesis is that avoidance of electrocoagulation may decrease ovarian cortex injury during cryopreservation procedures. Case presentation We report a laparoscopic technique of whole-ovary removal without coagulation using Endo-GIA forceps and clips. Laparoscopic ovariectomy was performed for cryopreservation in a 37-year-old Caucasian woman with breast cancer and for whom chemotherapy was planned. The procedure was completed quickly and without complication. This Endo-GIA procedure was of short duration with a short period of ischemia before freezing. Conclusion Laparoscopic ovariectomy using the Endo-GIA stapling device procedure without coagulation may diminish ovary injury before ovarian cryopreservation.

Ovarian cryopreservation after laparoscopic ovariectomy using the Endo-GIA stapling device and LAPRO-clip absorbable ligating clip in a woman: a case report

Science.gov (United States)

2011-01-01

Introduction Several options are available for preserving fertility before cytotoxic treatment, including ovarian tissue cryopreservation. Most reported surgical techniques include electrocoagulation. Our hypothesis is that avoidance of electrocoagulation may decrease ovarian cortex injury during cryopreservation procedures. Case presentation We report a laparoscopic technique of whole-ovary removal without coagulation using Endo-GIA forceps and clips. Laparoscopic ovariectomy was performed for cryopreservation in a 37-year-old Caucasian woman with breast cancer and for whom chemotherapy was planned. The procedure was completed quickly and without complication. This Endo-GIA procedure was of short duration with a short period of ischemia before freezing. Conclusion Laparoscopic ovariectomy using the Endo-GIA stapling device procedure without coagulation may diminish ovary injury before ovarian cryopreservation. PMID:21291518

Cryopreservation of canine ovarian and testicular fibroblasts.

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Yu, Il-Jeoung; Leibo, S P; Songsasen, Nucharin; Dresser, Betsy L; Kim, In-Shik

2009-01-01

To derive a practical procedure to store canine somatic cells, fibroblasts isolated from testicular or ovarian tissues were cryopreserved in 1.2 M ethylene glycol or in 1.2 M dimethylsulfoxide prepared in Dulbecco's Modified Eagle Medium as cryoprotectants, and were frozen either in plastic straws or vials. Thawed cells were cultured for 24 hr at 38.5 degree C in a humidified atmosphere of 5 percent CO2 95 percent air, and then their membrane integrity was assayed with a double fluorescent stain, Fertilight. In addition, frozen-thawed fibroblasts were cultured for 4 days, and then their functional survival was measured after staining small colonies with trypan blue. After freezing and thawing, membrane integrity of testicular fibroblasts was 55-70 percent and functional survival ranged from 20-40 percent. With frozen-thawed ovarian cells, the average membrane integrity was 55-75 percent and the average functional survival was 35-40 percent. When frozen in ethylene glycol, functional survival of ovarian fibroblasts was significantly higher than that of testicular cells (P less than 0.05). These methods should prove useful to preserve cells collected from canids in the wild.

Effect of different monosaccharides and disaccharides on boar sperm quality after cryopreservation.

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Gómez-Fernández, José; Gómez-Izquierdo, Emilio; Tomás, Cristina; Mocé, Eva; de Mercado, Eduardo

2012-07-01

The aim of the present study was to evaluate the cryoprotectant effect of different non-permeating sugars for boar sperm. Pooled semen from three boars was used for the experiments. In the first experiment, the sperm quality of boar sperm cryopreserved with an egg-yolk based extender supplemented with different monosaccharides (glucose, galactose or fructose) was compared to a control cryopreserved in lactose-egg yolk extender. In the second experiment, the effect of five disaccharides (lactose, sucrose, lactulose, trehalose or melibiose) on boar sperm cryosurvival was studied. Several sperm quality parameters were assessed by flow cytometry in samples incubated for 30 and 150 min at 37°C after thawing: percentages of sperm with intact plasma membrane (SIPM), sperm presenting high plasma membrane fluidity (HPMF), sperm with intracellular reactive oxygen substances production (IROSP) and apoptotic sperm (AS). In addition, the percentages of total motile (TMS) and progressively motile sperm (PMS) were assessed at the same incubation times with a computer-assisted sperm analysis system. Freezing extenders supplemented with each of the monosaccharide presented smaller cryoprotective effect than the control extender supplemented with lactose (Pextender supplemented with lactulose exhibited in general the lowest sperm quality, except for the percentage of capacitated sperm, which was highest (Pextender. Our results suggest that disaccharides have higher cryoprotective effect than monosaccharides, although the monosaccharide composition of the disaccharides is also important, since the best results were obtained with those disaccharides presenting glucose in their composition. Copyright © 2012 Elsevier B.V. All rights reserved.

Skin immunization by microneedle patch overcomes statin-induced suppression of immune responses to influenza vaccine.

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Vassilieva, Elena V; Wang, Shelly; Li, Song; Prausnitz, Mark R; Compans, Richard W

2017-12-19

Recent studies indicated that in elderly individuals, statin therapy is associated with a reduced response to influenza vaccination. The present study was designed to determine effects on the immune response to influenza vaccination induced by statin administration in a mouse model, and investigate potential approaches to improve the outcome of vaccination on the background of statin therapy. We fed middle aged BALB/c mice a high fat "western" diet (WD) alone or supplemented with atorvastatin (AT) for 14 weeks, and control mice were fed with the regular rodent diet. Mice were immunized with a single dose of subunit A/Brisbane/59/07 (H1N1) vaccine, either systemically or with dissolving microneedle patches (MNPs). We observed that a greater age-dependent decline in the hemagglutinin inhibition titers occurred in systemically-immunized mice than in MNP- immunized mice. AT dampened the antibody response in the animals vaccinated by either route of vaccine delivery. However, the MNP-vaccinated AT-treated animals had ~20 times higher total antibody levels to the influenza vaccine than the systemically vaccinated group one month postvaccination. We propose that microneedle vaccination against influenza provides an approach to ameliorate the immunosuppressive effect of statin therapy observed with systemic immunization.

Skin effector memory T cells do not recirculate and provide immune protection in alemtuzumab-treated CTCL patients.

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Clark, Rachael A; Watanabe, Rei; Teague, Jessica E; Schlapbach, Christoph; Tawa, Marianne C; Adams, Natalie; Dorosario, Andrew A; Chaney, Keri S; Cutler, Corey S; Leboeuf, Nicole R; Carter, Joi B; Fisher, David C; Kupper, Thomas S

2012-01-18

Cutaneous T cell lymphoma (CTCL) is a cancer of skin-homing T cells with variants that include leukemic CTCL (L-CTCL), a malignancy of central memory T cells (T(CM)), and mycosis fungoides (MF), a malignancy of skin resident effector memory T cells (T(EM)). We report that low-dose alemtuzumab (αCD52) effectively treated patients with refractory L-CTCL but not MF. Alemtuzumab depleted all T cells in blood and depleted both benign and malignant T(CM) from skin, but a diverse population of skin resident T(EM) remained in skin after therapy. T cell depletion with alemtuzumab required the presence of neutrophils, a cell type frequent in blood but rare in normal skin. These data suggest that T(CM) were depleted because they recirculate between the blood and the skin, whereas skin resident T(EM) were spared because they are sessile and non-recirculating. After alemtuzumab treatment, skin T cells produced lower amounts of interleukin-4 and higher amounts of interferon-γ. Moreover, there was a marked lack of infections in alemtuzumab-treated L-CTCL patients despite the complete absence of T cells in the blood, suggesting that skin resident T(EM) can protect the skin from pathogens even in the absence of T cell recruitment from the circulation. Together, these data suggest that alemtuzumab may treat refractory L-CTCL without severely compromising the immune response to infection by depleting circulating T(CM) but sparing the skin resident T(EM) that provide local immune protection of the skin.

Partial protection of baboons against Schistosoma mansoni using radiation-attenuated cryopreserved schistosomula

International Nuclear Information System (INIS)

James, E.R.; Dobinson, A.R.; Otieno, M.; Monorei, J.; Else, J.G.

1986-01-01

Three groups of five baboons were vaccinated in Kenya using three doses of 10,000 viable cryopreserved schistosomula attenuated with either 10, 20 or 60 krad 60 co-irradiation. The results from perfusion indicated reductions in worm burdens in the 10, 20 and 60 krad vaccinated groups of 18%, 23% and 20% respectively, none of which was statistically significant. No stunting of adult worms could be demonstrated in any of the groups. Mean tissue egg burdens were higher in all vaccinated groups and consequently egg production per worm pair was also higher than in the challenge controls. The logistics of preparing and delivering a cryopreserved radiation-attenuated vaccine were amply demonstrated; however, in this study the levels of protection achieved were not statistically significant; possible reasons for this are discussed. (author)

A rapid and simple method for cryopreservation of human liver slices

NARCIS (Netherlands)

de Kanter, R; Olinga, Peter; Hof, I.H; de Jager, M.H; Verwillegen, W.A; Slooff, M.JH; Meijer, D.K F; Groothuis, Geny; Koster, H

1. Precision-cut liver slices represent a suitable and convenient in vitro preparation for studying metabolism and toxicity mechanisms of drugs and toxic chemicals. Particularly in the case of human liver slices, cryopreservation would enable more efficient utilization of this scarce and irregularly

Supplemental effect of varying L-cysteine concentrations on the quality of cryopreserved boar semen

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Kaeoket, Kampon; Chanapiwat, Panida; Tummaruk, Padet; Techakumphu, Mongkol

2010-01-01

Cryopreservation is associated with the production of reactive oxygen species, which leads to lipid peroxidation of the sperm membrane and consequently a reduction in sperm motility and decreased fertility potential. The aim of this study was to determine the optimal concentration of L-cysteine needed for cryopreservation of boar semen. Twelve boars provided semen of proven motility and morphology for this study. The semen was divided into four portions in which the lactose-egg yolk (LEY) extender used to resuspend the centrifuged sperm pellet was supplemented with various concentrations of L-cysteine to reach 0 mmol L−1 (group I, control), 5 mmol L−1 (group II), 10 mmol L−1 (group III) and 15 mmol L−1 (group IV). Semen suspensions were loaded in straws (0.5 mL) and placed in a controlled-rate freezer. After cryopreservation, frozen semen samples were thawed and investigated for progressive motility, viability using SYBR-14/EthD-1 staining and acrosome integrity using FITC-PNA/EthD-1 staining. There was a significantly higher (P extender for improving the quality of frozen–thawed boar semen. PMID:20601963

Mechanisms underlying UV-induced immune suppression

International Nuclear Information System (INIS)

Ullrich, Stephen E.

2005-01-01

Skin cancer is the most prevalent form of human neoplasia. Estimates suggest that in excess of one million new cases of skin cancer will be diagnosed this year alone in the United States (www.cancer.org/statistics). Fortunately, because of their highly visible location, skin cancers are more rapidly diagnosed and more easily treated than other types of cancer. Be that as it may, approximately 10,000 Americans a year die from skin cancer. The cost of treating non-melanoma skin cancer is estimated to be in excess of US$ 650 million a year [J.G. Chen, A.B. Fleischer, E.D. Smith, C. Kancler, N.D. Goldman, P.M. Williford, S.R. Feldman, Cost of non-melanoma skin cancer treatment in the United States, Dermatol. Surg. 27 (2001) 1035-1038], and when melanoma is included, the estimated cost of treating skin cancer in the United States is estimated to rise to US$ 2.9 billion annually (www.cancer.org/statistics). Because the morbidity and mortality associated with skin cancer is a major public health problem, it is important to understand the mechanisms underlying skin cancer development. The primary cause of skin cancer is the ultraviolet (UV) radiation found in sunlight. In addition to its carcinogenic potential, UV radiation is also immune suppressive. In fact, data from studies with both experimental animals and biopsy proven skin cancer patients suggest that there is an association between the immune suppressive effects of UV radiation and its carcinogenic potential. The focus of this manuscript will be to review the mechanisms underlying the induction of immune suppression following UV exposure. Particular attention will be directed to the role of soluble mediators in activating immune suppression

Soybean lecithin-based extender preserves spermatozoa membrane integrity and fertilizing potential during goat semen cryopreservation.

Science.gov (United States)

Chelucci, Sara; Pasciu, Valeria; Succu, Sara; Addis, Daniela; Leoni, Giovanni G; Manca, Maria E; Naitana, Salvatore; Berlinguer, Fiammetta

2015-04-01

Soybean lecithin may represent a suitable alternative to egg yolk for semen cryopreservation in livestock species. However, additional studies are needed to elucidate its effects on spermatozoa functional properties. Semen collected from five Sarda bucks was cryopreserved in Tris-based extender and glycerol (4% v:v) with different supplementations. In a preliminary experiment, different soybean lecithin concentrations were tested (1%-6% wt/vol) and results in terms of viability, percentages of progressive motile and rapid spermatozoa, and DNA integrity after thawing showed that the most effective concentration was 1%. In the second experiment, semen was frozen in a Tris-based extender with no supplementation (EXT), with 1% lecithin (EXT LC), and 20% egg yolk (EXT EY). The effectiveness of these extenders was also compared with a commercial extender. The EXT EY led to the highest viability and motility parameters after freezing and thawing (P lecithin can be considered as a suitable alternative to egg yolk in goat semen cryopreservation, because it ensures higher fertilization rates and a better protection from membrane damage by cold shock. Copyright © 2015 Elsevier Inc. All rights reserved.

Evaluation of glycerol removal techniques, cryoprotectants, and insemination methods for cryopreserving rooster sperm with implications of regeneration of breed or line or both.

Science.gov (United States)

Purdy, P H; Song, Y; Silversides, F G; Blackburn, H D

2009-10-01

A series of experiments was designed to evaluate the quality of cryopreserved rooster sperm and its fertility so that programs needing to bank germplasm and recreate animals can do so utilizing a minimal amount of cryopreserved semen. In experiment 1, rooster semen from the National Animal Germplasm Program genebank was thawed and glycerol was removed using a discontinuous Accudenz column or by stepwise dilution. The postthaw sperm motilities, plasma membrane integrity, and concentration were determined before and after deglycerolization. Line differences in postthaw sperm concentration and progressive motility were observed before deglycerolization (Prooster semen was cryopreserved using Lake's diluent containing either dimethyl acetamide (DMA) or glycerol as the cryoprotectants. Postthaw analysis revealed that the samples cryopreserved with glycerol survived freezing better, determined by total motility (47.8 and 15.1% glycerol and DMA samples, respectively; P0.05). These results indicate that reasonable postthaw sperm quality and fertility can be derived using cryopreserved rooster semen. By utilizing this information, estimations can be made for storing sufficient material for line or breed, or both, recreation programs.

Towards gene banking amphibian maternal germ lines: short-term incubation, cryoprotectant tolerance and cryopreservation of embryonic cells of the frog, Limnodynastes peronii.

Science.gov (United States)

Lawson, Bianca; Clulow, Simon; Mahony, Michael J; Clulow, John

2013-01-01

Gene banking is arguably the best method available to prevent the loss of genetic diversity caused by declines in wild populations, when the causes of decline cannot be halted or reversed. For one of the most impacted vertebrate groups, the amphibians, gene banking technologies have advanced considerably, and gametes from the male line can be banked successfully for many species. However, cryopreserving the female germ line remains challenging, with attempts at cryopreserving oocytes unsuccessful due to their large size and yolk content. One possible solution is to target cryopreservation of early embryos that contain the maternal germ line, but consist of smaller cells. Here, we investigate the short term incubation, cryoprotectant tolerance, and cryopreservation of dissociated early embryonic cells from gastrulae and neurulae of the Striped Marsh Frog, Limnodynastes peronii. Embryos were dissociated and cells were incubated for up to 24 hours in various media. Viability of both gastrula and neurula cells remained high (means up to 40-60%) over 24 hours of incubation in all media, although viability was maintained at a higher level in Ca(2+)-free Simplified Amphibian Ringer; low speed centrifugation did not reduce cell viability. Tolerance of dissociated embryonic cells was tested for two cryoprotectants, glycerol and dimethyl sulphoxide; dissociated cells of both gastrulae and neurulae were highly tolerant to both-indeed, cell viability over 24 hours was higher in media containing low-to-medium concentrations than in equivalent cryoprotectant-free media. Viability over 24 hours was lower in concentrations of cryoprotectant higher than 10%. Live cells were recovered following cryopreservation of both gastrula and neurula cells, but only at low rates. Optimal cryodiluents were identified for gastrula and neurula cells. This is the first report of a slow cooling protocol for cryopreservation of amphibian embryonic cells, and sets future research directions for

Towards gene banking amphibian maternal germ lines: short-term incubation, cryoprotectant tolerance and cryopreservation of embryonic cells of the frog, Limnodynastes peronii.

Directory of Open Access Journals (Sweden)

Bianca Lawson

Full Text Available Gene banking is arguably the best method available to prevent the loss of genetic diversity caused by declines in wild populations, when the causes of decline cannot be halted or reversed. For one of the most impacted vertebrate groups, the amphibians, gene banking technologies have advanced considerably, and gametes from the male line can be banked successfully for many species. However, cryopreserving the female germ line remains challenging, with attempts at cryopreserving oocytes unsuccessful due to their large size and yolk content. One possible solution is to target cryopreservation of early embryos that contain the maternal germ line, but consist of smaller cells. Here, we investigate the short term incubation, cryoprotectant tolerance, and cryopreservation of dissociated early embryonic cells from gastrulae and neurulae of the Striped Marsh Frog, Limnodynastes peronii. Embryos were dissociated and cells were incubated for up to 24 hours in various media. Viability of both gastrula and neurula cells remained high (means up to 40-60% over 24 hours of incubation in all media, although viability was maintained at a higher level in Ca(2+-free Simplified Amphibian Ringer; low speed centrifugation did not reduce cell viability. Tolerance of dissociated embryonic cells was tested for two cryoprotectants, glycerol and dimethyl sulphoxide; dissociated cells of both gastrulae and neurulae were highly tolerant to both-indeed, cell viability over 24 hours was higher in media containing low-to-medium concentrations than in equivalent cryoprotectant-free media. Viability over 24 hours was lower in concentrations of cryoprotectant higher than 10%. Live cells were recovered following cryopreservation of both gastrula and neurula cells, but only at low rates. Optimal cryodiluents were identified for gastrula and neurula cells. This is the first report of a slow cooling protocol for cryopreservation of amphibian embryonic cells, and sets future research

Knowledge, attitudes, and intentions toward fertility awareness and oocyte cryopreservation among obstetrics and gynecology resident physicians.

Science.gov (United States)

Yu, L; Peterson, B; Inhorn, M C; Boehm, J K; Patrizio, P

2016-02-01

What knowledge, attitudes and intentions do US obstetrics and gynecology (OB/GYN) residents have toward discussing age-related fertility decline and oocyte cryopreservation with their patients? Most OB/GYN residents believe that age-related fertility decline, but not oocyte cryopreservation, should be discussed during well-woman annual exams; furthermore, nearly half of residents overestimated the age at which female fertility markedly declines. Oocyte cryopreservation can be utilized to preserve fertility potential. Currently, no studies of US OB/GYN residents exist that question their knowledge, attitudes, and intentions toward discussing age-related fertility decline and oocyte cryopreservation with patients. A cross-sectional online survey was conducted during the fall of 2014 among residents in American Council for Graduate (ACOG) Medical Education-approved OB/GYN residency programs. Program directors were emailed via the ACOG Council on Resident Education in Obstetrics and Gynecology server listing and asked to solicit resident participation. Participants included 238 residents evenly distributed between post-graduate years 1-4 with varied post-residency plans; 90% of residents were women and 75% were 26-30 years old. The survey was divided into three sections: demographics, fertility awareness, and attitudes toward discussing fertility preservation options with patients. Descriptive and inferential statistics were conducted. A strong majority of residents (83%) believed an OB/GYN should initiate discussions about age-related fertility decline with patients (mean patient age 31.8), and 73% percent believed these discussions should be part of an annual exam. One third of residents overestimated the age at which there is a slight decline in female fertility, while nearly half of residents overestimated the age at which female fertility markedly declines. Over three-quarters of residents (78.4%) also overestimated the likelihood of success using assisted

Attenuation of phosphamidon-induced oxidative stress and immune dysfunction in rats treated with N-acetylcysteine

Directory of Open Access Journals (Sweden)

S.G. Suke

2008-09-01

Full Text Available The effect of N-acetylcysteine, a thiolic antioxidant, on attenuation of phosphamidon-induced oxidative stress and immune dysfunction was evaluated in adult male Wistar rats weighing 200-250 g. Rats were divided into four groups, 8 animals/group, and treated with phosphamidon, N-acetylcysteine or the combination of both for 28 days. Oral administration of phosphamidon (1.74 mg/kg, an organophosphate insecticide, increased serum malondialdehyde (3.83 ± 0.18 vs 2.91 ± 0.24 nmol/mL; P < 0.05 and decreased erythrocyte superoxide dismutase (567.8 ± 24.36 vs 749.16 ± 102.61 U/gHb; P < 0.05, catalase activity (1.86 ± 0.18 vs 2.43 ± 0.08 U/gHb; P < 0.05 and whole blood glutathione levels (1.25 ± 0.21 vs 2.28 ± 0.08 mg/gHb; P < 0.05 showing phosphamidon-induced oxidative stress. Phosphamidon exposure markedly suppressed humoral immune response as assessed by antibody titer to ovalbumin (4.71 ± 0.51 vs 8.00 ± 0.12 -log2; P < 0.05, and cell-mediated immune response as assessed by leukocyte migration inhibition (25.24 ± 1.04 vs 70.8 ± 1.09%; P < 0.05 and macrophage migration inhibition (20.38 ± 0.99 vs 67.16 ± 5.30%; P < 0.05 response. Phosphamidon exposure decreased IFN-у levels (40.7 ± 3.21 vs 55.84 ± 3.02 pg/mL; P < 0.05 suggesting a profound effect of phosphamidon on cell-mediated immune response. A phosphamidon-induced increase in TNF-α level (64.19 ± 6.0 vs 23.16 ± 4.0 pg/mL; P < 0.05 suggests a contributory role of immunocytes in oxidative stress. Co-administration of N-acetylcysteine (3.5 mmol/kg, orally with phosphamidon attenuated the adverse effects of phosphamidon. These findings suggest that oral N-acetylcysteine treatment exerts protective effect and attenuates free radical injury and immune dysfunction caused by subchronic phosphamidon exposure.

Improved cryopreservability of stallion sperm using a sorbitol-based freezing extender.

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Pojprasath, T; Lohachit, C; Techakumphu, M; Stout, T; Tharasanit, T

2011-06-01

Cryopreservation of stallion semen is often associated with poor post-thaw sperm quality. Sugars are among the important components of a freezing extender and act as non-permeating cryoprotectants. This study aimed to compare the quality of stallion sperm frozen with glucose, fructose or sorbitol-containing freezing extenders. Semen was collected from six stallions of proven fertility and cryopreserved using a freezing extender containing different types of monosaccharide sugars (glucose, fructose or sorbitol). After thawing, the semen was examined for sperm motility, viability, acrosome integrity, plasma membrane functionality and sperm longevity. The fertility of semen frozen in the presence of sorbitol was also tested by artificial insemination. Sperm quality was significantly decreased following freezing and thawing (P sorbitol and glucose (P sorbitol-based extender when examined at 2 and 4 h post-thaw, all of these parameters plus plasma membrane functionality were improved for sperm frozen in the sorbitol extender than in the glucose extender when examined 10 min post-thaw. Two of four mares (50%) inseminated with semen frozen with a sorbitol-containing freezing extender became pregnant. It is concluded that different sugars have different abilities to protect against cryoinjury during freezing and thawing of stallion sperm. This study demonstrated that an extender containing sorbitol as primary sugar can be used to successfully cryopreserve equine sperm; moreover, the quality of frozen-thawed sperm appeared to be better than when glucose or fructose was the principle sugar in the freezing extender. Copyright © 2011 Elsevier Inc. All rights reserved.

Th1 immune response to Plasmodium falciparum recombinant thrombospondin-related adhesive protein (TRAP) antigen is enhanced by TLR3-specific adjuvant, poly(I:C) in BALB/c mice.

Science.gov (United States)

Mehrizi, A A; Ameri Torzani, M; Zakeri, S; Jafary Zadeh, A; Babaeekhou, L

2018-07-01

Sporozoite-based malaria vaccines have provided a gold standard for malaria vaccine development, and thrombospondin-related adhesive protein (TRAP) serves as the main vaccine candidate antigen on sporozoites. As recombinant malaria vaccine candidate antigens are poorly immunogenic, additional appropriate immunostimulants, such as an efficient adjuvant, are highly essential to modulate Th1-cell predominance and also to induce a protective and long-lived immune response. In this study, polyinosinic:polycytidylic acid [poly(I:C)], the ligand of TLR3, was considered as the potential adjuvant for vaccines targeting stronger Th1-based immune responses. For this purpose, BALB/c mice were immunized with rPfTRAP delivered in putative poly(I:C) adjuvant, and humoural and cellular immune responses were determined in different immunized mouse groups. Delivery of rPfTRAP with poly(I:C) induced high levels and titres of persisted and also high-avidity anti-rPfTRAP IgG antibodies comparable to complete Freund's adjuvant (CFA)/incomplete Freund's adjuvant (IFA) adjuvant after the second boost. In addition, rPfTRAP formulated with poly(I:C) elicited a higher ratio of IFN-γ/IL-5, IgG2a/IgG1, and IgG2b/IgG1 than with CFA/IFA, indicating that poly(I:C) supports the induction of a stronger Th1-based immune response. This is a first time study which reveals the potential of rPfTRAP delivery in poly(I:C) to increase the level, avidity and durability of both anti-PfTRAP cytophilic antibodies and Th1 cytokines. © 2018 John Wiley & Sons Ltd.

Cryopreservation of Sambar deer semen in Thailand.

Science.gov (United States)

Vongpralub, Thevin; Chinchiyanond, Wittaya; Hongkuntod, Pornchai; Sanchaisuriya, Pitcharat; Liangpaiboon, Sanan; Thongprayoon, Areeya; Somphol, Noppadon

2015-01-01

Little is known of the different freezing and thawing techniques for post-thaw survival of spermatozoa in Sambar deer. So, this study determined the effect of seminal plasma, egg yolk and glycerol extenders and their concentrations, plus cooling, freezing, and thawing protocols on the post-thaw quality of their semen. Semen samples were collected by electro-ejaculation from four Thai Sambar deer stags (Cervus unicolor equinus). As evaluated by post-thaw progressive motility and acrosome integrity removal of seminal plasma was beneficial; Tris-egg yolk was the most efficient extender; a 20% egg yolk concentration was better than the 0%, 10%, or 30%; and a 3% glycerol concentration was better than 5%, 7%, or 9%. Using the optimum dilution techniques, semen was loaded in 0.5 ml plastic straws. Cooling times from ambient temperature to 5°C in 3 hr resulted in higher post-thaw progressive motility and acrosome integrity than 1, 2, or 4 hr. Suspending the straws 4 cm above the surface for 15 min before plunging into liquid nitrogen was better than suspending at 2 or 6 cm. For thawing frozen semen, an intermediate thawing (50°C, 8 sec) protocol was more effective than the slower (37°C, 10 sec) or faster (70°C, 5 sec) thawing rates. Timed insemination following estrus synchronization of 10 hinds resulted in six confirmed pregnancies at 60 days. Five hinds delivered live fawns. This study provides an effective approach for semen cryopreservation and artificial insemination (AI), which should be valuable to scientists for genetics and reproductive management of Sambar deer in developing countries. © 2015 Wiley Periodicals, Inc.

HI-6 modulates immunization efficacy in a BALB/c mouse model.

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Pohanka, Miroslav

2013-11-01

HI-6 is used as an antidote to nerve agents. It can also act as an antagonist to acetylcholine receptors (AChRs) including the nicotinic receptor, α 7 nAChR which is involved in regulating the immune response through macrophages. This experiment investigated the efficacy of HI-6 to regulate the immune response. Laboratory BALB/c mice received HI-6 and/or keyhole limpet hemocyanin (KLH) as an antigen. Antibody production was investigated after either 21 or 65 days when either single or repeated dose of antigen was applied. We confirmed that HI-6 significantly improved vaccination efficacy when KLH was given in a dose of 1mg/kg. The effect was dose dependent. A combination of HI-6 and KLH produced a vaccination of almost the same efficacy as that for Freund's complete adjuvant. The findings point at the suitability of HI-6 for improving vaccination efficacy at the level of immunity regulation by the nervous system. Copyright © 2013 Elsevier B.V. All rights reserved.

Evaluation of Glycerol Removal Techniques, Cryoprotectants, and Insemination Methods for Cryopreserving Rooster Sperm with Implications for Breed and/or Line Regeneration

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A series of experiments was designed to evaluate the quality of cryopreserved rooster sperm and its fertility so that programs needing to bank germplasm and recreate animals can do so utilizing a minimal amount of cryopreserved semen. In Experiment 1, semen from roosters was collected and cryoprese...

Predictors of sperm recovery after cryopreservation in testicular cancer

Directory of Open Access Journals (Sweden)

James M Hotaling

2016-01-01

Full Text Available Our objective was to identify predictors of improved postthaw semen quality in men with testicular cancer banking sperm for fertility preservation. We reviewed 173 individual semen samples provided by 67 men with testicular germ cell tumor (TGCT who cryopreserved sperm before gonadotoxic treatment between 1994 and 2010 at our tertiary university medical center. Our main outcomes measures were independent predictors for the greater postthaw total motile count (TMC in men with TGCT. Men with NSGCT were more likely to be younger (P median fresh TMC each had increased odds of a postthaw TMC greater than median postthaw TMC. Interestingly, age, advanced cancer stage (II or III, rapid freezing protocol, and motility enhancer did not show increased odds of improved postthaw TMC in our models. In conclusion, men with TGCT or poor fresh TMC should consider preserving additional vials (at least 15 vials before oncologic treatment. Density gradient purification should be routinely used to optimize postthaw TMC in men with TGCT. Larger, randomized studies evaluating cancer stage and various cryopreservation techniques are needed to assist in counseling men with TGCT regarding fertility preservation and optimizing cryosurvival.

Vitality of oligozoospermic semen samples is improved by both swim-up and density gradient centrifugation before cryopreservation.

Science.gov (United States)

Counsel, Madeleine; Bellinge, Rhys; Burton, Peter

2004-05-01

To ascertain whether washing sperm from oligozoospermic and normozoospermic samples before cryopreservation improves post-thaw vitality. Normozoospermic (n = 18) and oligozoospermic (n = 16) samples were divided into three aliquots. The first aliquot remained untreated and the second and third aliquots were subjected to the swim-up and discontinuous density gradient sperm washing techniques respectively. Vitality staining was performed, samples mixed with cryopreservation media and frozen. Spermatozoa were thawed, stained, and vitality quantified and expressed as the percentage of live spermatozoa present. Post-thaw vitality in untreated aliquots from normozoospermic samples (24.9% +/- 2.3; mean +/- SEM) was significantly higher (unpaired t-tests; P vitality was significantly higher after swim-up in normozoospermic samples (35.6% +/- 2.1; P vitality in oligozoospermic (22.4% +/- 1.0; P vitality in cryopreserved oligozoospermic samples was improved by both the swim-up and density gradient centrifugation washing techniques prior to freezing.