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Tissue Culture (tissue + culture)
Kinds of Tissue Culture Terms modified by Tissue Culture Selected AbstractsSCHWANN CELL APOPTOSIS IN TISSUE CULTURE FOLLOWING THE ADMINISTRATION OF PRO-INFLAMMATORY CYTOKINESJOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 1 2000E. Scarpini In immune-mediated demyelination of the nervous system, glial cell apoptosis has been observed recently; however, the relevance of the phenomenon and the characterization of the involved molecules are still controversial. Cytokines are secreted by many cells, including inflammatory and glial cells, and appear to play a relevant role in the peripheral nervous system (PNS) immuno-mediated demyelination, being active in promoting the damage to Schwann cells, myelin, and axons. Even though the exact role of the different cytokines is at present uncertain, they have a sequential different expression in PNS immune-mediated demyelination and could induce apoptotic death of Schwann cells in the vicinity of the inflammatory reaction via the expression of CD95 (Apo1/Fas). This study has been designed to detect in rat primary Schwann cell tissue cultures whether the administration of IL-1B and IFN-y can induce cell death. Identification of apoptotic Schwann cell was performed by morphological, immunohistochemical, and electron-microscopy analysis. Our results show that Schwann cells stimulated by proinflammatory cytokines IL-1B and IFN-y show morphological evidence of nuclear chromatin condesation at the DAPI staining and are TUNEL positive. The same features of apoptotic cell death were observed by electron microscopy. These findings provide evidence to support the hypothesis that cytokines can directly damage Schwann cells in disorders of the PNS. [source] Development of Auxotrophic Agrobacterium tumefaciens for Gene Transfer in Plant Tissue CultureBIOTECHNOLOGY PROGRESS, Issue 3 2004Jason I. Collens Auxotrophic strains of Agrobacterium tumefaciens were generated for use in liquid co-culture with plant tissue for transient gene expression. Twenty-one auxotrophs were recovered from 1,900 tetracycline-resistant insertional mutants generated with a suicide vector transposon mutagenesis system. Twelve of these auxotrophs were characterized on a nutrient matrix. Isolates were screened for growth in plant cell and root culture, and three auxotrophs were identified that had limited growth: adenine (ade-24), leucine (leu-27), and cysteine (cys-32). Ade-24 displayed poor T-DNA delivery in a transient expression test delivering GUS from a binary vector, while cys-32 displayed the best ability to deliver DNA of these three auxotrophs. The growth yield of cys-32 on cysteine was assessed to provide a quantitative basis for co-culture nutrient supplementation. The utility of cys-32 for delivering T-DNA to plant tissues is demonstrated, where an 85-fold enhancement in GUS expression over wild-type A. tumefacienswas achieved. [source] Solitary embolic cutaneous aspergillosis in the immunocompromised patient with acute myelogenous leukemia , a propos another case caused by Aspergillus flavusINTERNATIONAL JOURNAL OF DERMATOLOGY, Issue 12 2003Aleksandar L. Krunic MD A 68-year-old male with acute myelogenous leukemia was admitted for consolidation chemotherapy. The in-hospital course was complicated by neutropenia, fever and nodular pulmonary opacities suggestive of multifocal pneumonia. The patient subsequently developed a single, solitary necrotic crusted nodule on the right cheek. Skin biopsy demonstrated embolic vascular invasion with septate hyphae, dichotomous branching and minimal inflammation. Tissue culture revealed Aspergillus flavus. Despite systemic antifungal therapy with amphotericin B and granulocyte transfusions, the patient developed respiratory failure and died of disseminated aspergillosis, sepsis and renal failure. The clinical presentation of disseminated infection with Aspergillus flavus as a solitary embolic cutaneous lesion is extremely rare. We have reviewed other cases described in the literature and suggest this pattern of cutaneous involvement as more typical of disseminated infection with Aspergillus flavus. [source] A preliminary examination of the role of NFAT 3 in human skin, cultured keratocytes and dermal fibroblastsJOURNAL OF CUTANEOUS PATHOLOGY, Issue 9 2010Wael I. Al-Daraji Background: Ciclosporin A (CsA) is widely utilized for the treatment of inflammatory skin diseases such as psoriasis. The therapeutic effects of CsA are thought to be mediated via its immunosuppressive action on infiltrating lymphocytes in skin lesions. CsA and tacrolimus block T cell activation by inhibiting the phosphatase calcineurin and preventing translocation from the cytoplasm to the nucleus of the transcription factor Nuclear Factor of Activated T cells (NFAT). Methods: RT-PCR and Western Analysis were used to investigate the presence of NFAT-3 mRNA and protein in human keratocytes. Tissue culture of human keratocytes and immunostaining of cells on coverslips and confocal microscopy were used to assess the degree of nuclear localisation of NFAT-3 in cultured cells. Keratome biopsies were taken from patients with psoriasis (lesional and non-lesional skin) and normal skin and immunohistochemistry was used to assess the NFAT-3 localisation in these biopsies using a well characterized anti-NFAT-3 antibody. Results: The NFAT-3 mRNA and protein expression was demonstrated using RT-PCR and Western blotting. The expression of NFAT-3 in human keratocytes and response to different agonists provides perhaps a unique opportunity to examine the regulation, subcellular localization and kinetics of translocation of different NFATs in primary cultured human cells. As with NFAT 1, NFAT 2 and recently NFAT 5, differentiation-promoting agents that increase intracellular calcium concentration induced nuclear translocation of NFAT-3 in cultured keratocytes but with different kinetics. Conclusion: These data provide the first evidence of that NFAT-3 is expressed in normal skin, psoriasis and that NFAT-3 functionally active in human keratocytes and that nuclear translocation of NFAT-3 in human skin cells has different kinetics than NFAT 1 suggesting that NFAT-3 may play an important role in regulation of keratocytes proliferation and differentiation at a different stage. Inhibition of this pathway in human epidermal keratocytes many account, in part for the therapeutic effects of CsA and tacrolimus in skin disorders such as psoriasis. Al-Daraji WI. A preliminary examination of the role of NFAT 3 in human skin, cultured keratocytes and dermal fibroblasts. [source] Necrotizing fasciitis: delay in diagnosis results in loss of limbINTERNATIONAL JOURNAL OF DERMATOLOGY, Issue 10 2006Rajat Varma MD A 58-year-old man presented to the Emergency Room with a 1-day history of severe pain in the left lower extremity preceded by several days of redness and swelling. He denied any history of trauma. He also denied any systemic symptoms including fever and chills. His past medical history was significant for diabetes, hypertension, deep vein thrombosis, and Evans' syndrome, an autoimmune hemolytic anemia and thrombocytopenia, for which he was taking oral prednisone. Physical examination revealed a warm, tender, weeping, edematous, discolored left lower extremity. From the medial aspect of the ankle up to the calf, there was an indurated, dusky, violaceous plaque with focal areas of ulceration (Fig. 1). Figure 1. Grossly edematous lower extremity with well-demarcated, dusky, violaceous plaque with focal ulceration Laboratory data revealed a white blood cell count of 6.7 × 103/mm3[normal range, (4.5,10.8) × 103/mm3], hemoglobin of 11.5 g/dL (13.5,17.5 g/dL), and platelets of 119 × 103/mm3[(140,440) × 103/mm3]. Serum electrolytes were within normal limits. An ultrasound was negative for a deep vein thrombosis. After the initial evaluation, the Emergency Room physician consulted the orthopedic and dermatology services. Orthopedics did not detect compartment syndrome and did not pursue surgical intervention. Dermatology recommended a biopsy and urgent vascular surgery consultation to rule out embolic or thrombotic phenomena. Despite these recommendations, the patient was diagnosed with "cellulitis" and admitted to the medicine ward for intravenous nafcillin. Over the next 36 h, the "cellulitis" had advanced proximally to his inguinal region. His mental status also declined, and he showed signs of septic shock, including hypotension, tachycardia, and tachypnea. Vascular surgery was immediately consulted, and the patient underwent emergency surgical debridement. The diagnosis of necrotizing fasciitis was then made. Tissue pathology revealed full-thickness necrosis through the epidermis with subepidermal splitting. Dermal edema was also present with a diffuse neutrophilic infiltrate (Fig. 2). This infiltrate extended through the fat into the subcutaneous tissue and fascia. Tissue cultures sent at the time of surgery grew Escherichia coli. Initial blood cultures also came back positive for E. coli. Anaerobic cultures remained negative. Figure 2. Necrotic epidermis with subepidermal splitting. Marked dermal edema with mixed infiltrate and prominent neutrophils. Hematoxylin and eosin: original magnification, ×20 After surviving multiple additional debridements, the patient eventually required an above-the-knee amputation due to severe necrosis. [source] Potential and Bottlenecks of Bioreactors in 3D Cell Culture and Tissue ManufacturingADVANCED MATERIALS, Issue 32-33 2009David Wendt Abstract Over the last decade, we have witnessed an increased recognition of the importance of 3D culture models to study various aspects of cell physiology and pathology, as well as to engineer implantable tissues. As compared to well-established 2D cell-culture systems, cell/tissue culture within 3D porous biomaterials has introduced new scientific and technical challenges associated with complex transport phenomena, physical forces, and cell,microenvironment interactions. While bioreactor-based 3D model systems have begun to play a crucial role in addressing fundamental scientific questions, numerous hurdles currently impede the most efficient utilization of these systems. We describe how computational modeling and innovative sensor technologies, in conjunction with well-defined and controlled bioreactor-based 3D culture systems, will be key to gain further insight into cell behavior and the complexity of tissue development. These model systems will lay a solid foundation to further develop, optimize, and effectively streamline the essential bioprocesses to safely and reproducibly produce appropriately scaled tissue grafts for clinical studies. [source] Oxygen transport and consumption by suspended cells in microgravity: A multiphase analysisBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2008Ohwon Kwon Abstract A rotating bioreactor for the cell/tissue culture should be operated to obtain sufficient nutrient transfer and avoid damage to the culture materials. Thus, the objective of the present study is to determine the appropriate suspension conditions for the bead/cell distribution and evaluate oxygen transport in the rotating wall vessel (RWV) bioreactor. A numerical analysis of the RWV bioreactor is conducted by incorporating the Eulerian,Eulerian multiphase and oxygen transport equations. The bead size and rotating speed are the control variables in the calculations. The present results show that the rotating speed for appropriate suspensions needs to be increased as the size of the bead/cell increases: 10 rpm for 200 µm; 12 rpm for 300 µm; 14 rpm for 400 µm; 18 rpm for 600 µm. As the rotating speed and the bead size increase from 10 rpm/200 µm to 18 rpm/600 µm, the mean oxygen concentration in the 80% midzone of the vessel is increased by ,85% after 1-h rotation due to the high convective flow for 18 rpm/600 µm case as compared to 10 rpm/200 µm case. The present results may serve as criteria to set the operating parameters for a RWV bioreactor, such as the size of beads and the rotating speed, according to the growth of cell aggregates. In addition, it might provide a design parameter for an advanced suspension bioreactor for 3-D engineered cell and tissue cultures. Biotechnol. Bioeng. 2008;99: 99,107. © 2007 Wiley Periodicals, Inc. [source] Three-dimensional slice cultures from murine fetal gut for investigations of the enteric nervous systemDEVELOPMENTAL DYNAMICS, Issue 1 2007Marco Metzger Abstract Three-dimensional intestinal cultures offer new possibilities for the examination of growth potential, analysis of time specific gene expression, and spatial cellular arrangement of enteric nervous system in an organotypical environment. We present an easy to produce in vitro model of the enteric nervous system for analysis and manipulation of cellular differentiation processes. Slice cultures of murine fetal colon were cultured on membrane inserts for up to 2 weeks without loss of autonomous contractility. After slice preparation, cultured tissue reorganized within the first days in vitro. Afterward, the culture possessed more than 35 cell layers, including high prismatic epithelial cells, smooth muscle cells, glial cells, and neurons analyzed by immunohistochemistry. The contraction frequency of intestinal slice culture could be modulated by the neurotransmitter serotonin and the sodium channel blocker tetrodotoxin. Coculture experiments with cultured neurospheres isolated from enhanced green fluorescent protein (eGFP) transgenic mice demonstrated that differentiating eGFP-positive neurons were integrated into the intestinal tissue culture. This slice culture model of enteric nervous system proved to be useful for studying cell,cell interactions, cellular signaling, and cell differentiation processes in a three-dimensional cell arrangement. Developmental Dynamics 236:128,133, 2007. © 2006 Wiley-Liss, Inc. [source] Methanol exposure interferes with morphological cell movements in the Drosophila embryo and causes increased apoptosis in the CNSDEVELOPMENTAL NEUROBIOLOGY, Issue 3 2004Dervla M. Mellerick Abstract Despite the significant contributions of tissue culture and bacterial models to toxicology, whole animal models for developmental neurotoxins are limited in availability and ease of experimentation. Because Drosophila is a well understood model for embryonic development that is highly accessible, we asked whether it could be used to study methanol developmental neurotoxicity. In the presence of 4% methanol, approximately 35% of embryos die and methanol exposure leads to severe CNS defects in about half those embryos, where the longitudinal connectives are dorsally displaced and commissure formation is severely reduced. In addition, a range of morphological defects in other germ layers is seen, and cell movement is adversely affected by methanol exposure. Although we did not find any evidence to suggest that methanol exposure affects the capacity of neuroblasts to divide or induces inappropriate apoptosis in these cells, in the CNS of germ band retracted embryos, the number of apoptotic nuclei is significantly increased in methanol-exposed embryos in comparison to controls, particularly in and adjacent to the ventral midline. Apoptosis contributes significantly to methanol neurotoxicity because embryos lacking the cell death genes grim, hid, and reaper have milder CNS defects resulting from methanol exposure than wild-type embryos. Our data suggest that when neurons and glia are severely adversely affected by methanol exposure, the damaged cells are cleared by apoptosis, leading to embryonic death. Thus, the Drosophila embryo may prove useful in identifying and unraveling mechanistic aspects of developmental neurotoxicity, specifically in relation to methanol toxicity. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 308,318, 2004 [source] Selective Potentiometric Measurement of Physiologically Significant ThiolsELECTROANALYSIS, Issue 3 2005Svetlana Gracheva Abstract The reaction of benzoquinone with reduced thiol species has been investigated using potentiometric techniques. The specificity of this reaction for thiol functionality has been briefly assessed and the nature of the electrode response has been elucidated. The system has been characterized in terms of selectivity, sensitivity and the efficacy of using the system for the quantification of thiol containing pharmaceuticals has been demonstrated. The simplicity of the detection methodology is shown to markedly contrast alternative thiol detection strategies. The transfer of the technology to a mass production format through the adoption of screen print electrode formats has been achieved and the efficacy of the approach demonstrated. Recovery experiments using captopril and penicillamine in tissue culture as a model analyte system were performed. [source] Characteristics of strawberry plants propagated by in vitro bioreactor culture and ex vitro propagation methodENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 3 2009Samir C. Debnath Abstract Reproducible protocol for regeneration of complete plantlets from ,Bounty' strawberry (Fragaria ananassa Duch.), using a combination of gelled medium and bioreactor system, has been standardized. Sepals, leaf discs, and petiole halves produced multiple buds and shoots when cultured on semi solid-gelled medium containing 4 ,M thidiazuron (TDZ) for 4 wk followed by transferring in liquid medium containing 2,,M TDZ in a bioreactor system and cultured for another 4 wk. TDZ induced shoot proliferation at 0.1,,M in the bioreactor system but inhibited shoot elongation. TDZ-induced shoots were elongated and rooted in vitro on gelled medium containing 2,,M zeatin. Such bioreactor-derived tissue culture (BC) plantlets obtained from sepal explants were grown ex vitro and compared with those propagated by tissue culture on gelled medium (GC) and by conventional runner cuttings (RC), for growth, morphology, anthocyanin content, and antioxidant activity after three growth seasons. The BC and GC plants produced more crowns, runners, leaves, and berries than the RC plants although berry weight per plant did not differ significantly. BC and GC plants produced berries with more anthocyanin contents and antioxidant activities than those produced by the RC plants. However, intersimple sequence repeat (ISSR) marker assay produced a homogenous amplification profile in the tissue culture and donor control plants confirming the clonal fidelity of micropropagated plants. In vitro culture on TDZ and zeatin-containing nutrient media apparently induced the juvenile branching characteristics that favored enhanced vegetative growth with more crown, runners, leaf, and berry production. [source] Preclinical development of hybrid cell vaccines for multiple myelomaEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 1 2007Renata Walewska Abstract Immunotherapy may provide alternative or supplementary treatment of multiple myeloma (MM). We propose that hybrid cells, formed by fusing professional antigen-presenting cells with malignant plasma cells, would induce immune responses capable of mediating tumour regression. The human B-lymphoblastoid cell line, HMy2, was fused in vitro with CD138+ bead-separated myeloma plasma cells from five patients with MM. The hybrid cell lines generated in these studies grew stably in tissue culture, and maintained their phenotypic and functional characteristics, providing self-renewing cell lines with potential for therapeutic vaccination. The hybrid cells stimulated allogeneic and autologous T-cell proliferative responses in vitro to a considerably greater degree than their respective parent myeloma plasma cells, and directly activated both CD4+ and CD8+ T-cell responses. The enhanced T-cell stimulation correlated with expression of CD80 on the hybrid cells, and was inhibited by CTLA4-Ig fusion protein. The hybrid cell lines expressed several tumour-associated antigens known to be expressed in myeloma. These data show that self-replicating cell lines with enhanced immunostimulatory properties and potential for therapeutic vaccination can be generated by in vitro fusion of ex vivo myeloma cells and B-lymphoblastoid cell lines. [source] Calponin is expressed by fibroblasts and meningeal cells but not olfactory ensheathing cells in the adult peripheral olfactory systemGLIA, Issue 2 2007Chrystelle Ibanez Abstract Olfactory ensheathing cells (OECs), the principal glial cells of the peripheral olfactory system, have many phenotypic similarities with Schwann cells of the peripheral nervous system. This makes reliably distinguishing these two cells types difficult, especially following transplantation into areas of injury in the central nervous system. In an attempt to identify markers by which these two cells types can be distinguished, a recent proteomic analysis of fetal OECs and adult Schwann cells identified the actin-binding protein calponin as a potential marker expressed by OECs but not Schwann cells. Since many studies designed with the translational goal of autologous transplantation in mind have used adult OECs, this study examined the expression of calponin by adult OECs, both in vivo within the peripheral olfactory system and in vitro. Calponin colocalized with strongly fibronectin positive fibroblasts in the olfactory mucosa (OM) and meningeal cells in the olfactory bulb (OB) but not with S100, or neuropeptide-Y positive OECs. In tissue culture, calponin was strongly expressed by fibronectin-expressing fibroblasts from OM, sciatic nerve and skin and by meningeal cells from the OB, but not by p75NTR - and S100,-expressing OECs. These data, supported by Western blotting, indicate that calponin can not be used to distinguish adult OECs and Schwann cells. © 2006 Wiley-Liss, Inc. [source] Optimization of culture conditions for plant regeneration of Panicum spp. through somatic embryogenesisGRASSLAND SCIENCE, Issue 1 2010Mi-Suk Seo Abstract We developed a rapid and efficient shoot regeneration system for Panicum spp. by adjusting the regeneration medium and studying the responses of different genotypes and the influence of explant types (mature seed, immature embryo and shoot apex). We used Panicum meyerianum (Nees) and Panicum longijubatum (Stapf) which were shown to perform well, to select the optimal medium for shoot regeneration. The highest frequency of shoot regeneration was obtained on Murashige and Skoog medium supplemented with 30 g L,1 maltose and 1 mg L,1 N-phenyl-N,-[(1,2,3-thidiazol-5-yl) urea]. The callus formed green spots after 1 week of culture and showed primary green shoots after 2 weeks. In this system, the calli derived from mature seed of nine Panicum genotypes showed large variation in shoot regeneration ability: from 0 to 69.9% in the frequency of shoot formation and from 0 to 8.4 in the number of shoots per callus. Guineagrass (Panicum maximum Jacq.) showed no ability and switchgrass (Panicum virgatum L.) showed low ability to regenerate from mature seed-derived calli; however, both were able to be regenerated from immature embryos and calli derived from shoot apices. We developed an efficient protocol for high shoot regeneration of various Panicum genotypes which provides a foundation for efficient tissue culture and genetic improvement of Panicum. [source] Evaluation of tissue culture response from mature seeds of Panicum spp.GRASSLAND SCIENCE, Issue 3 2008Mi-Suk Seo Abstract The genus Panicum contains important warm-season forage grasses and species with potential as biomass crops. We selected Panicum genotypes with high response to tissue culture for genetic improvement. The highest frequency of callus induction from mature seed of Panicum maximum cultivar Natsukaze was obtained on MS medium containing 4.0 mg L,1 2,4-dichlorophenoxyacetic acid and solidified with 0.3% Gelrite. We compared germination frequencies and callus induction capacities among 24 genotypes of 11 Panicum species on this medium. Callus induction frequencies varied among genotypes. Those with high germination frequencies generally had high callus induction frequencies. On the other hand, especially in P. maximum, the callus induction ratio (callus induction frequency/germination frequency) depended on the reproductive mode and ploidy. The callus induction ratio of three sexual accessions of P. maximum were very low compared to apomictic accessions, and besides, a tetraploid sexual accession Noh PL1 had very low germination and callus induction frequencies. Callus induction and regeneration capacities were independent of each other. For shoot regeneration, we transferred callus derived from the 24 genotypes onto MS medium supplemented with 1.0 mg L,1 kinetin and 0.4% Gelrite. Six of the genotypes regenerated plantlets. Among them, Panicum meyerianum produced the highest shoot regeneration frequency of 61.6% and the maximum number of shoots callus,1 in the shortest time. The callus of P. meyerianum also showed vigorous proliferation. We thus selected high-response genotypes of P. meyerianum. [source] Plasticity in the adult rat pancreas: Transdifferentiation of exocrine to hepatocyte-like cells in primary cultureHEPATOLOGY, Issue 6 2004Jessy Lardon Under certain experimental conditions, hepatocytes can arise in the pancreas. It has been suggested that the pancreas retains a source of hepatocyte progenitor cells. However, such cells have not been yet identified in the adult pancreas. We describe here the transdifferentiation of primary rat pancreatic exocrine cells into hepatocyte-like cells during 5 days of tissue culture in the presence of dexamethasone (DX). Using reverse-transcription polymerase chain reaction and immunocytochemistry, it was observed that DX treatment induced albumin RNA and protein expression in the cells. Coexpression of albumin and amylase, and the absence of cell proliferation, demonstrated a direct transdifferentiation of acinar cells to hepatocytic cells. CCAAT enhancer-binding protein-ß protein, a liver-enriched transcription factor that is considered to be the master switch in pancreatohepatic transdifferentiation, and ,-fetoprotein were markedly upregulated in the cells after treatment with DX. We compared transcriptional profiles of freshly isolated exocrine cells and DX-treated cells using oligonucleotide microarrays and found that multiple liver-specific genes are induced along with albumin, and that certain pancreatic genes are downregulated in the DX-treated cells. In conclusion, these observations support the notion of plasticity in the adult pancreas and that exocrine cells can be reprogrammed to transdifferentiate into other cell types such as hepatocytes. (HEPATOLOGY 2004;39:1499,1507.) [source] Clinical islet transplant: current and future directions towards toleranceIMMUNOLOGICAL REVIEWS, Issue 1 2003A. M. James Shapiro Summary:, The ultimate goal of islet transplantation is to completely correct the diabetic state from an unlimited donor source, without the need for chronic immunosuppressive drug therapy. Although islet transplantation provides an opportunity to develop innovative strategies for tolerance in the clinic, both alloimmune and autoimmune barriers must be controlled, if stable graft function is to be maintained long-term. After islet extraction from the pancreas, the cellular graft may be stored in tissue culture or cryopreserved for banking, providing an opportunity not only to optimally condition the recipient but also to allow in vitro immunologic manipulation of the graft before transplantation, unlike solid organ grafts. As such, islets may be considered a ,special case.' Remarkable progress has occurred in the last three years, with dramatic improvements in outcomes after clinical islet transplantation. The introduction of a steroid-free, sirolimus-based, anti-rejection protocol and islets prepared from two (or rarely three) donors led to high rates of insulin independence. The ,Edmonton Protocol' has been successfully replicated by other centers in an international multicenter trial. A number of key refinements in pancreas transportation, processing, purification on non-ficoll-based media, storage of islets in culture for two days and newer immunological conditioning and induction therapies have led to continued advancement through extensive collaboration between key centers. This review outlines the historical development of islet transplantation over the past 30 years, provides an update on current clinical outcomes, and summarizes a series of unique opportunities for development and early testing of tolerance protocols in patients. [source] Visualizing thymocyte motility using 2-photon microscopyIMMUNOLOGICAL REVIEWS, Issue 1 2003Ellen A. Robey Summary:, Our view of a thymocyte based on its behavior in tissue culture and appearance in fixed tissue sections was of a round sessile cell. Its travel through the thymus might occur slowly, perhaps even passively, leaving it in contact with the support cells that happened to be in its immediate environment. However, when we got our first look at the behavior of thymocytes in a 3D cellular stromal cell environment, that picture changed dramatically. Instead we found that thymocytes are actively crawling, allowing them to explore their environment over relatively long distances and interact with peptide-major histocompatibility complex (pMHC)-bearing thymic stromal cells in both dynamic and stable modes. In this review, we discuss the implications of thymocyte motility for T-cell repertoire selection and for the mechanisms that determine the spatial organization of thymocyte subsets within the thymus. [source] Hyaluronan-binding peptide can inhibit tumor growth by interacting with Bcl-2INTERNATIONAL JOURNAL OF CANCER, Issue 1 2004Ninfei Liu Abstract Previous studies have indicated that proteins that bind hyaluronan can also inhibit the growth of tumor cells. To determine if synthetic peptides also possessed these properties, we tested a series of polypeptides containing structural motifs from different proteins for their ability to bind [3H]hyaluronan, and identified one compound termed P4 that had a particularly strong interaction. Further studies revealed that P4 also inhibited the growth of tumor cells in tissue culture as well as on the chorioallantoic membranes of chicken embryos. In addition, expression vectors for P4 caused tumor cells to grow slower in nude mice and reduced their vascularization. The P4 peptide also inhibited VEGF-induced angiogenesis in the chorioallantoic membranes of chicken embryos. Studies on cultured cells indicated that P4 induced apoptosis, which was blocked by a pan-caspase inhibitor. Confocal microscopy revealed that shortly after its uptake, P4 became associated with mitochondria. Immunoprecipitation indicated that P4 could bind to Bcl-2 and Bcl-xL, which are associated with mitochondria and regulate apoptosis. This was also supported by the fact that P4 induced the release of cytochrome c from preparations of mitochondria. Taken together, these results suggest that P4 binds to Bcl-2 and related proteins and this activates the apoptotic cascade. © 2003 Wiley-Liss, Inc. [source] Erythema induratum with pulmonary tuberculosis: histopathologic features resembling true vasculitisINTERNATIONAL JOURNAL OF DERMATOLOGY, Issue 3 2001Yong Suk Lee MD A 22-year-old South Korean woman presented with a 4-month history of several nodules on both legs. She looked healthy, but suffered from tenderness and swelling of the legs. Physical examination showed multiple, nonulcerating, erythematous nodules occurring on the calves, knee joints, and thighs (Fig. 1). A biopsy specimen of the skin revealed necrotizing vasculitis of medium-sized arteries with fibrinoid necrosis at the border between the dermis and the subcutis. Dense cellular infiltrates, including numerous neutrophils and lymphocytes, presented within and around the vessel walls as in polyarteritis nodosa, with some eosinophils (Fig. 2A,B). There were no other generalized symptoms. She was diagnosed with cutaneous polyarteritis nodosa and was initially treated with systemic steroids. She was given an intravenous injection of Solu-Cortef, 60 mg/6 h for 7 days. This was replaced with oral prednisolone for 2 weeks. The skin lesions and symptoms improved. Figure 1. Small, nut-sized, erythematous, brown-colored nodules and patches on the lower extremities, even above the knee joints Figure 2. (A) Dense infiltration within and around artery (× 40). (B) Slightly expanded lobular panniculitis with vasculitis (× 100) Six months later, she complained of general weakness and recurrent skin lesions. Purified protein derivative (PPD) test gave a moderate positive reaction and chest X-ray examination showed the features of pulmonary tuberculosis: radio-opaque infiltrations in the right lower lung field. A repeated biopsy revealed mild vasculitis with more diffuse lobular infiltrations of the subcutaneous tissue compared with the former specimen. Polymerase chain reaction (PCR) and tissue culture for Mycobacterium tuberculosis were performed from a biopsy specimen. DNA was extracted from skin tissue with an AplisystemTM DNA/RNA detection kit using the resin-mediated boiling method (Stargene, Seoul, South Korea). The primers were designed on the basis of the M. tuberculosis gene IS6110 target (sense primer, 5,-CCA GAT GCA CCG TCG AAC GGC TGA T-3, antisense primer, 5,-CGC TCG CTG AAC CGG ATC GAT GTG T-3,). The amplification was performed with uracil- N -glycosylase (UNG), to prevent carry-over contamination, and internal control primers, to correct for false-negative reaction (Kox LF, Rhienthong D, Miranda AM et al. A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples. J Clin Microbiol 1994; 32: 672,678; Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene 1990; 93: 125,128). According to the manufacturer's instructions, amplification was carried out for 40 cycles with denaturation at 94 °C for 40 s, annealing at 70 °C for 1 min, and extension at 72 °C for 1 min in a thermal cycler (Perkin,Elmer Cetus, Norwalk, CT, USA). The results of PCR and tissue culture for M. tuberculosis using the biopsy specimen were all negative (Fig. 3). Figure 3. Negative result in PCR for M. tuberculosis (negative control is not shown; M, marker; P, positive control; I, internal control; S, specimen) The patient was finally diagnosed with erythema induratum with pulmonary tuberculosis and was started on antituberculosis medication (isoniazid 400 mg, rifampicin 600 mg, ethambutol 800 mg, and pyrazinamide 1500 mg daily). She showed prompt improvement after 2 weeks of medication. After 9 months of antituberculosis therapy, her skin lesions and chest X-ray had cleared. She was followed up for 4 months with no recurrence of skin and pulmonary lesions. [source] Lack of oxygen in articular cartilage: consequences for chondrocyte biologyINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 2 2010Jérôme E. Lafont Summary Controlling the chondrocytes phenotype remains a major issue for cartilage repair strategies. These cells are crucial for the biomechanical properties and cartilage integrity because they are responsible of the secretion of a specific matrix. But chondrocyte dedifferentiation is frequently observed in cartilage pathology as well as in tissue culture, making their study more difficult. Given that normal articular cartilage is hypoxic, chondrocytes have a specific and adapted response to low oxygen environment. While huge progress has been performed on deciphering intracellular hypoxia signalling the last few years, nothing was known about the particular case of the chondrocyte biology in response to hypoxia. Recent findings in this growing field showed crucial influence of the hypoxia signalling on chondrocytes physiology and raised new potential targets to repair cartilage and maintain tissue integrity. This review will thus focus on describing hypoxia-mediated chondrocyte function in the native articular cartilage. [source] An in vitro examination of an extracellular matrix scaffold for use in wound healingINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 5 2002Denis E. Solomon Summary. This paper describes evidence that an extracellular matrix (ECM) secreted by human umbilical vein endothelial cells (HUVECs) assembled on gelatin coated plates overlaid by a mixed matrix secreted by human dermal microvascular endothelial cells (HDMECs) and human dermal fibroblasts provides a viable acellular scaffold for use in wound healing. Trypsinized epidermal keratinocytes or colonies from Dispase-digested fresh and cadaver skin tissue adhered and proliferated on either HUVECs ECM/gelatin or mixed matrix overlaid on HUVECs ECM/gelatin. An epithelial,mesenchymal interaction, previously thought to be tissue-specific, was exposed as well as concomitant integrin versatility. Furthermore, heterologous HDMECs and dermal fibroblasts attached and proliferated on the mixed matrix as well as HUVECs ECM. The conditioned medium from HUVECs (HUVECs CM) was found to neutralize the lingering after effects of Dispase, and could be used for the tissue culture of epidermal keratinocytes, HDMECs and dermal fibroblasts, which share related extracellular secretions. Taken together, these results indicate that cultured epithelial autografts can be redesigned to include both epithelial and dermal elements, and advances the acellular ,sandwich' ECM scaffold as a possible structural replacement for the lamina densa and lamina lucida, damaged or completely missing in some wounds and burns. [source] Commercial considerations in tissue engineeringJOURNAL OF ANATOMY, Issue 4 2006Jonathan Mansbridge Abstract Tissue engineering is a field with immense promise. Using the example of an early tissue-engineered skin implant, Dermagraft, factors involved in the successful commercial development of devices of this type are explored. Tissue engineering has to strike a balance between tissue culture, which is a resource-intensive activity, and business considerations that are concerned with minimizing cost and maximizing customer convenience. Bioreactor design takes place in a highly regulated environment, so factors to be incorporated into the concept include not only tissue culture considerations but also matters related to asepsis, scaleup, automation and ease of use by the final customer. Dermagraft is an allogeneic tissue. Stasis preservation, in this case cryopreservation, is essential in allogeneic tissue engineering, allowing sterility testing, inventory control and, in the case of Dermagraft, a cellular stress that may be important for hormesis following implantation. Although the use of allogeneic cells provides advantages in manufacturing under suitable conditions, it raises the spectre of immunological rejection. Such rejection has not been experienced with Dermagraft. Possible reasons for this and the vision of further application of allogeneic tissues are important considerations in future tissue-engineered cellular devices. This review illustrates approaches that indicate some of the criteria that may provide a basis for further developments. Marketing is a further requirement for success, which entails understanding of the mechanism of action of the procedure, and is illustrated for Dermagraft. The success of a tissue-engineered product is dependent on many interacting operations, some discussed here, each of which must be performed simultaneously and well. [source] Genetically Manipulated Human Skeletal Myoblast Cells for Cardiac TransplantationJOURNAL OF CARDIAC SURGERY, Issue 6 2002Kh H Haider Aim: Considering the promise of skeletal myoblast cell transplantation to improve cardiac function in myocardial myopathies, we aim in the present study to investigate the potential of human skeletal myoblast cells (HSMC) as a carrier for therapeutic genes for the heart muscle. Methods: Skeletal muscle sample is obtained from rectus femoris of the donor and is processed in the tissue culture to generate HSMC by a patented process of Cell Therapy Inc. The HSMC are grown in large 225 mm2 tissue culture flasks coated with collagen for enhanced cell adherence, using patented Super Medium (Cell Therapy Inc., Singapore) containing 10% fetal calf serum, to 80% confluence. The HSMC are passaged at regular time intervals of 48-72 hours to prevent in vitro differentiation. The HSMC thus obtained are transduced three times with retroviral vector carrying Lac-Z reporter gene before transplantation. The Lac-Z transduced HSMC are harvested by trypsinization, washed and re-suspended in serum free Super Medium. Ischemic Porcine model is created by clamping ameroid ring around left circumflex coronary artery in Yorkshire swine, four weeks prior to cell transplantation. For cell transplantation, the animal is anaesthetized, ventilated and heart is exposed by left thoracotomy. Fifteen injections (0.25 ml each) containing 300 million cells are injected in to the left ventricle endocardially under direct vision. For control animal, only culture medium without cells is injected. The animal is euthanized at pre-determined time, heart is explanted and processed for histological examination. The cryosectioning of the tissue and subsequent staining for Lac-Z expression and Hematoxylin-Eosin staining is carried out by standard methods. Results: The skeletal muscle samples processed by the patented method of Cell Therapy yield 85-90% pure HSMC. The preliminary data shows that repeated transductions of myoblast cells with retrovirus carrying Lac-Z yield highly efficient 70-75% Lac-Z positive HSMC population (Figure 1). Dye exclusion test using Trypan blue reveals >95% cell viability at the time of injection. Gross sections of the cardiac tissue stained positive for Lac-Z expression (Figure 2). Histological examination showed the presence of grafted myoblast cells expressing Lac-Z gene in the cardiac tissue (Figure 3). Conclusion: In the light of our preliminary results, we conclude that HSMC may prove to be excellent carriers of transgene for cardiac muscle cells which otherwise are refractory to ordinary gene transfection methods. The use of HSMC mediated gene delivery to cardiac muscle is safer as compared to direct injection of viral vectors in to the heart muscle. Furthermore, the grafted myoblast cells will additionally serve to strengthen the weakened heart muscle. Figure 1.Human Skeletal myoblasts transduced with Lac-Z carrying retrovirus and stained with x-gal. Figure 2.Gross sections of heart muscle stained for Lac-Z expression. Figure 3.X-gal stained porcine heart muscle counter-stained with Eosin. The heart was explanted 6 weeks after transplantation of Lac-Z stained human myoblasts. The arrow shows Lac-Z expressing myoblast cells. [source] A role for the transcription factor HEY1 in glioblastomaJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 1 2009Esther Hulleman Abstract Glioblastoma multiforme (GBM), the highest-grade glioma, is the most frequent tumour of the brain with a very poor prognosis and limited therapeutic options. Although little is known about the molecular mechanisms that underlie glioblastoma formation, a number of signal transduction routes, such as the Notch and Ras signalling pathways, seem to play an important role in the formation of GBM. In the present study, we show by in situ hybridization on primary tumour material that the transcription factor HEY1, a target of the Notch signalling pathway, is specifically up-regulated in glioma and that expression of HEY1 in GBM correlates with tumour-grade and survival. In addition, we show by chromatin immunoprecipitations, luciferase assays and Northern blot experiments that HEY1 is a bona fide target of the E2F family of transcription factors, connecting the Ras and Notch signalling pathways. Finally, we show that ectopic expression of HEY1 induces cell proliferation in neural stem cells, while depletion of HEY1 by RNA interference reduces proliferation of glioblastoma cells in tissue culture. Together, these data imply a role for HEY1 in the progression of GBM, and therefore we propose that HEY1 may be a therapeutic target for glioblastoma patients. Moreover, HEY1 may represent a molecular marker to distinguish GBM patients with a longer survival prognosis from those at high risk. [source] Decorin synthesized by arterial smooth muscle cells is retained in fibrin gels and modulates fibrin contractionJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2007Pamela Y. Johnson Abstract Fibrin serves as a provisional extracellular matrix (ECM) for arterial smooth muscle cells (ASMC) after vascular injury, yet little is known about the effect of fibrin on ECM remodeling by these cells. To address this question, monkey ASMC were grown on fibrin gels and tissue culture (TC) plastic, and proteoglycan synthesis and accumulation were assessed by radiolabeling. Initial rates of 35S-sulfate incorporation into proteoglycans were identical for both groups, but increased proteoglycan accumulation was observed in cultures grown for 48 h on fibrin. This increased accumulation on fibrin was due to reduced proteoglycan turnover and retention within the fibrin gel. Decorin and biglycan constituted 40 and 14% of the total proteoglycan in the fibrin gels, whereas their combined contribution was only 12% in control matrices. To explore whether the retention of decorin in fibrin had any influence on the properties of the fibrin gel, ASMC-mediated fibrin contraction assays were performed. Both de novo synthesis of decorin as well as decorin added during polymerization inhibited the ability of the cells to contract fibrin. In contrast, decorin added exogenously to mature fibrin matrices had no effect on fibrin gel contraction. This study illustrates that decorin derived from ASMC selectively accumulates in fibrin and modifies fibrin architecture and mechanical properties. Such an accumulation may influence wound healing and the thrombotic properties of this provisional pro-atherosclerotic ECM. J. Cell. Biochem. 101: 281,294, 2007. © 2007 Wiley-Liss, Inc. [source] Enamel matrix derivative exhibits angiogenic effect in vitro and in a murine modelJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 8 2003Kuo Yuan Abstract Objectives: Angiogenesis is one of the most critical events in the wound healing process. Any increase in angiogenesis could result in more rapid and complete healing. A recent study found that enamel matrix derivative (EMD) could accelerate early periodontal wound healing. We wanted to clarify whether EMD caused an angiogenic effect and, thus, possibly enhanced wound healing. Methods: We performed in vitro proliferation and chemotaxis assays on human umbilical vein endothelial cell (HUVEC) cultures, and a tissue culture assay using blood vessel fragments in fibrin gel. Collagen membranes soaked with EMD were implanted subcutaneously in mice to test the in vivo angiogenic effect. Results: While there were no significant differences between the negative control and EMD groups in the proliferation assay, EMD treatment did exhibit a significantly greater dose-dependent chemotactic effect on HUVEC than control group treatments. The tissue culture in fibrin gel showed new blood vessel outgrowths in the EMD groups, but none in the negative control group. In the animal studies, significantly more endothelial cells were detected in the EMD group of mice. Conclusions: Our findings show that EMD does exhibit some angiogenic effects. However, the underlying molecules and mechanisms are still unidentified. We discuss several possibilities. Zusammenfassung Ziele: Die Angiogenese gehört zu den kritischsten Ereignissen bei der Wundheilung. Eine Erhöhung der Angiogenese könnte zu einer rascheren und kompletteren Wundheilung führen. Kürzlich zeigte eine Studie, dass Schmelzmatrixderivate (EMD) die frühe parodontale Wundheilung beschleunigen könnte. Wir wollten klären, ob EMD einen angiogenetischen Effekt verursacht und dies möglicherweise die Wundheilung verbessert. Methoden: Wir führten in vitro Proliferations- und Chemotaxis-Assays an menschlichen Umbilicalvenen-Endothelzellen (HUVEC)Kulturen durch und studierten eine Gewebekultur unter Nutzung von Blutgefäßfragmenten in Fibringel. Kollagenmembranen mit EMD getränkt wurden subkutan in Mäuse implantiert, um den angiogenetischen Effekt in vivo zu testen. Ergebnisse: Während es keine signifikanten Differenzen zwischen den negativen Kontrollen und den EMD Gruppen in dem Proliferationsassay gab, zeigte die EMD Behandlung einen signifikant größeren, dosisabhängigen chemotaktischen Effekt auf HUVEC verglichen mit den Kontrollen. Die Gewebekultur im Fibringel zeigte neue Blutgefäßbildungen in den EMD-Gruppen, aber keine bei den Negativkontrollen. Bei den Tierstudien wurden signifikant mehr Endothelzellen in den EMD Mäusegruppen entdeckt. Schlussfolgerungen: Unsere Ergebnisse zeigen, dass EMD einige angiogenetische Effekte zeigt. Jedoch sind die zugrunde liegenden Moleküle und die Mechanismen noch nicht geklärt. Wir diskutieren verschiedene Möglichkeiten. Résumé Objectifs: L'Angiogenèse est un des plus critiques éléments lors du processus de cicatrisation. La moindre augmentation de l'angiogenèse peut entraîner une cicatrisation plus rapide et plus complète. Une récente étude a montré que les dérivés de la matrice amellaire (EMD) pouvait accélérer plus tôt la cicatrisation parodontale. Nous voulions clarifier la possible responsabilité de l'EMD dans l'angiogenèse et si oui, l'amélioration de la cicatrisation. Méthodes: Nous avons réalisé in vitro la prolifération et un essai de chimiotactisme sur des cultures de cellules endothéliales de la veine ombilicale humaine (HUVEC), et un essai de culture tissulaire en utilisant des fragments de vaisseaux sanguins dans un gel de fibrine. Des membranes de collagène gorgées d'EMD furent implantées en sous-cutanée chez des souris pour tester l'effet angiogénique in vivo. Résultats: Bien qu'il n'y eut pas de différences significatives entre le contrôle négatif et le groupe EMD pour le test de prolifération, le traitement par EMD présentait un effet chimiotactique dose- dépendant significativement plus élevé sur les HUVEC. La culture tissulaire sur gel de fibrine présentait une surcroissance de nouveaux vaisseaux sanguins pour le groupe EMD, mais pas dans le groupe contrôle. Plus de cellules endothéliales furent en outre détectées lors de l'étude animale, pour le groupe de souris traitées par EMD. Conclusions: Nos données montrent que l'EMD présente quelques effets angiogéniques. Cependant, les molécules et les mécanismes responsables ne sont toujours pas identifiés. Nous discutons quelques possibilités. [source] Treatment development for systemic Tetrahymena sp. infection in guppies, Poecilia reticulata PetersJOURNAL OF FISH DISEASES, Issue 6 2010M Pimenta Leibowitz Abstract Antibacterial and antiparasitic agents and a cysteine protease inhibitor (E-64) were tested against Tetrahymena infection, a serious problem in guppy production worldwide. Chemicals were tested in vitro by a colorimetric assay for Tetrahymena survival. The most effective were niclosamide, albendazole and chloroquine, with 23%, 35% and 60% survival, respectively, following 2-h exposure to 100 ppm. Longer incubation periods resulted in greater reductions in survival. Niclosamide was further studied in vivo at different dosages, administered orally to Tetrahymena -infected guppies. Mortality rates were significantly lower in all treatment groups; in trial I, 30% and 33% mortality in 5 and 40 mg kg,1 niclosamide-fed fish vs. 59% mortality in controls; in trial II, 35%, 13% and 10% in 50, 100 and 200 mg kg,1 niclosamide-fed fish vs. 64% in controls. The effect of the cysteine protease inhibitor E64 was tested in tissue culture, by measuring histolytic activity of the parasite (Tet-NI) on a guppy-fin cell line, based on cell depletion. Tet-NI feeding activity was significantly reduced following pretreatment with E-64 relative to non-treated Tet-NI. E-64-pretreated Tet-NI was injected i.p. into guppies: recorded mortality rates were significantly lower (35%) than that in non-treated Tet-NI (60%), suggesting inhibition of the parasite's cysteine protease as a possible therapeutic approach. [source] Rapid and sensitive detection of mumps virus RNA directly from clinical samples by real-time PCRJOURNAL OF MEDICAL VIROLOGY, Issue 3 2005Kazue Uchida Abstract A rapid, sensitive, and specific assay to detect mumps virus RNA directly from clinical specimens using a real-time PCR assay was developed. The assay was capable of detecting five copies of standard plasmid containing cDNA from the mumps virus F gene. No cross-reactions were observed with other members of Paramyxoviridae, or with viruses or bacteria known to be meningitis pathogens. Seventy-three clinical samples consisting of throat swabs collected from patients with parotitis, and cerebrospinal fluid (CSF) collected from patients with aseptic meningitis, were examined with a real-time PCR assay developed by the authors, reverse-transcription nested-PCR (RT-n-PCR), and virus isolation using cell culture. Like the RT-n-PCR assay, the real-time PCR assay could detect mumps virus RNA in approximately 70% of both throat swabs and CSF samples, while, by tissue culture, mumps virus was isolated from only approximately 20% of CSF and 50% of throat swab samples. In addition, the real-time PCR assay could be developed easily into a quantitative assay for clinical specimens containing more than 1,800 copies of mumps virus RNA/ml by using serial dilutions of the standard plasmid. The results suggest that the real-time PCR assay is useful for identification of mumps virus infections, not only in typical cases, but also in suspected cases, which show only symptoms of meningitis or encephalitis. J. Med. Virol. 75:470,474, 2005. © 2005 Wiley-Liss, Inc. [source] Experimental hepatitis A virus infection in guinea pigsJOURNAL OF MEDICAL VIROLOGY, Issue 4 2001Britt Hornei Abstract Although many of the properties of hepatitis A virus (HAV) are known, several aspects of HAV pathogenesis are still not understood, such as the mechanism underlying the hepatotropism or HAV replication in extrahepatic sites. Detailed studies of these aspects were hampered mostly by the lack of accessible animal models, since only nonhuman primates are susceptible to experimental infections. An alternative animal model would also be of interest to assess the primary replication site and for the evaluation of the safety and efficacy of vaccines. A study was undertaken to determine whether HAV can infect guinea pigs and whether they are useful as a model for studying aspects of HAV pathogenesis and for the evaluation of vaccines. HAV variants adapted to primate or guinea pig tissue culture were used to inoculate guinea pigs intraperitoneally and by the oral route. The animals were observed for clinical disease, shedding of HAV in stools, viremia, seroconversion, evidence for liver damage by biochemical liver function tests, virus presence in the liver, development of hepatic histopathological changes, and occurrence of HAV in extrahepatic organs. The animals developed an active, clinically inapparent infection with specific histopathological changes in the liver. Although virus replication occurred, as shown by RT-PCR and isolation of infectious virus from feces and serum, it seems unlikely that guinea pigs are suitable for studying the clinical features of hepatitis A, because the clinical and laboratory parameters remained normal. However, guinea pigs appear useful for studying some aspects of HAV pathogenesis and for testing the safety of vaccines. J. Med. Virol. 64:402,409, 2001. © 2001 Wiley-Liss, Inc. [source] | |||||||||||||||||||||||||||||||||||||