Replicative senescence, telomere shortening and cell proliferation rate in Gaddi goat's skin fibroblast cell line

We assessed aging in continuous donor skin fibroblast cell line GGM5 up to the 25th passage by in vitro replicative senescence, telomere dynamics and chromosomal abnormalities. Cell proliferation rate increased from 0.84+/-0.26 (primary cells) to 1.20+/-0.17 (13-15 passage group) per day and reduced to 0.65+/-0.14 in 22-25 passages. Cell proliferation rate was reduced by 45.7% after 87.62 CPDs. Cell viability reduced from 100% to 97.4% up to the 25th passages. Frequency of beta gal(+) cells increased in successive passages and days in culture. The correlation coefficient between frequency of beta gal(+) cells and growth rate was -0.50 to -0.61. Loss of mean TRF length was 13.8 nucleotides (passage 15) to 95.4 nucleotides per cell division in later passages. All cells showed Robertsonian translocation in 22-25 passaged cells. The SCNT pre-implantation embryos production was highest (22.5%) in donor cells used from 10-15 passages as compared to early (相似文献   

Retrovirus-induced feline pure red cell aplasia: the kinetics of erythroid marrow failure

Cats viremic with feline leukemia virus subgroup C (FeLV-C) develop pure red cell aplasia (PRCA) characterized by the loss of detectable late erythroid progenitors (CFU-E) in marrow culture. Normal numbers of early erythroid progenitors (BFU-E) and granulocyte-macrophage progenitors (CFU-GM) remain, suggesting that the maturation of BFU-E to CFU-E is impaired in vivo. We have examined the cell cycle kinetics of BFU-E and their response to hematopoietic growth factor(s) to better characterize erythropoiesis as anemia develops. Within 3 weeks of FeLV-C infection, yet 6-42 weeks before anemia, the traction of BFU-E in DNA synthesis as determined by tritiated thymidine suicide increased to 43 +/- 4% (normal 23 +/- 2%) while there was no change in the cell cycle kinetics of CFU-GM. In additional studies, we evaluated the response of marrow to the hematopoietic growth factor(s) present in medium conditioned by FeLV-infected feline embryonic fibroblasts (FEA/FeLV CM). With cells from normal cats or cats viremic with FeLV-C but not anemic, a 4-fold increase in erythroid bursts was seen in cultures with 5% FEA/FeLV CM when compared to cultures without CM. However, just prior to the onset of anemia, when the numbers of detectable CFU-E decreased, BFU-E no longer responded to FEA/FeLV CM in vitro. BFU-E from anemic cats also required 10% cat or human serum for optimal in vitro growth. These altered kinetics and in vitro growth characteristics may relate to the in vivo block of BFU-E differentiation and PRCA. Finally, when marrow from cats with PRCA was placed in suspension culture for 2 to 4 days in the presence of cat serum and CM, the numbers of BFU-E increased 2- to 4-fold although no CFU-E were generated. By 4 to 7 days, CFU-E were detected, suggesting that conditions contributing to the block of erythroid maturation did not persist. The suspension culture technique provides an approach to study further the defect in erythroid differentiation characteristic of feline PRCA.     

Mouse Pancreas Tissue Slice Culture Facilitates Long-Term Studies of Exocrine and Endocrine Cell Physiology in situ

Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ.     

Thyroid hormones promote endocrine differentiation at expenses of exocrine tissue

Diabetes is caused by loss or dysfunction of pancreatic beta cells. Generation of beta cells in vitro is a promising strategy to develop a full-scale cell therapy against diabetes, and the development of methods without gene transfer may provide safer protocols for human therapy. Here we show that thyroid hormone receptors are expressed in embryonic murine pancreas. Addition of the thyroid hormone T3 in an ex vivo culture model of embryonic (E12.5) dorsal pancreas, mimicking embryonic pancreatic development, promoted an increase of ductal cell number at expenses of the acinar compartment. Double labeled cells expressing specific markers for ductal and acinar cells were observed, suggesting cell reprogramming. Increased mRNA levels of the pro-endocrine gene Ngn3 and an increased number of beta cells were detected in cultures treated previously with T3 suggesting that ductal cells promoted by T3 can subsequently differentiate into endocrine cells. So, indirectly, T3 induced endocrine differentiation. Moreover, T3 induced the expression of the pro-endocrine gene Ngn3 in the acinar 266-6 cell line. The pro-endocrine effect of T3 in the pancreatic explants and in the acinar cell line, was abrogated by the Akt inhibitor Ly294002 indicating the involvement of Akt signaling in this process. Altogether we show numerous evidences that define T3 as a promising candidate to generate endocrine cells from exocrine tissue, using ectopically gene expression free protocols, for cell therapy against diabetes.     

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide new expandable sources of beta cells to alleviate the donor scarcity in human islet transplantation as therapy for diabetes. Although recent advances have been made towards this aim, mechanisms that regulate beta cell expansion and differentiation from a stem/progenitor cell remain to be characterized. Here, we describe a protocol for an injury model in the adult mouse pancreas that can function as a tool to study mechanisms of tissue remodeling and beta cell proliferation and differentiation. Partial duct ligation (PDL) is an experimentally induced injury of the rodent pancreas involving surgical ligation of the main pancreatic duct resulting in an obstruction of drainage of exocrine products out of the tail region of the pancreas. The inflicted damage induces acinar atrophy, immune cell infiltration and severe tissue remodeling. We have previously reported the activation of Neurogenin (Ngn) 3 expressing endogenous progenitor-like cells and an increase in beta cell proliferation after PDL. Therefore, PDL provides a basis to study signals involved in beta cell dynamics and the properties of an endocrine progenitor in adult pancreas. Since, it still remains largely unclear, which factors and pathways contribute to beta cell neogenesis and proliferation in PDL, a standardized protocol for PDL will allow for comparison across laboratories.     

Effects of sarcolectin (SCL) on human peripheral blood mononuclear cells

Sarcolectin (SCL) is a tissue growth factor found in various human or animal tissues, functioning in balance with interferons (IFNs) that can inhibit growth and affect cell differentiation. Like somatotropin, SCL is found in the pituitary gland. In humans, the SCL gene is located on chromosome 12 (q12-q13) and expressed as a 55 kDa protein consisting of 469 amino-acids. After a single activation of peripheral blood mononuclear cells (PBMC) obtained from more than 30 individuals, highly significant cell proliferation was found to peak after 7 days in culture. The presence of adherent cells was necessary for cell proliferation. SCL induced over-expression of alpha-IL-2 receptor (CD25) leading to proliferation of CD3+/CD4+/CD45RO+ T cells. Thus in PBMC, SCL induced CD4+ T cell growth and expression of inflammatory cytokine genes, including TNF-alpha, IL-1beta, IL-6 and IL-8. IFNs are also produced following activation as a feedback response which is maintained for about 20 days.     

Establishment of Besnoitia darlingi from opossums (Didelphis virginiana) in experimental intermediate and definitive hosts,propagation in cell culture,and description of ultrastructural and genetic characteristics

Besnoitia darlingi from naturally infected opossums (Didelphis virginiana) from Mississippi, USA, was propagated experimentally in mice, cats, and cell culture and was characterised according to ultrastructural, genetic, and life-history characteristics. Cats fed tissue cysts from opossums shed oocysts with a prepatent period of nine or 11 days. Oocysts, bradyzoites, or tachyzoites were infective to outbred and interferon-gamma gene knockout mice. Tachyzoites were successfully cultivated and maintained in vitro in bovine monocytes and African green monkey cells and revived after an 18-month storage in liquid nitrogen. Schizonts were seen in the small intestinal lamina propria of cats fed experimentally-infected mouse tissues. These schizonts measured up to 45 x 25 microm and contained many merozoites. A few schizonts were present in mesenteric lymph nodes and livers of cats fed tissue cysts. Ultrastructurally, tachyzoites and bradyzoites of B. darlingi were similar to other species of Besnoitia. A close relationship to B. besnoiti and an even closer relationship to B. jellisoni was indicated for B. darlingi on the basis of the small subunit and ITS-1 portions of nuclear ribosomal DNA.     

Ability of CD8(+) T cell anti-feline immunodeficiency virus activity correlated with peripheral CD4(+) T cell counts and plasma viremia

In the host defense mechanism against feline immunodeficiency virus (FIV) infection, CD8(+) T cells specifically attack virus-infected cells and suppress the replication of the virus in a non-cytolytic manner by secreting soluble factors. In this study, we measured CD8(+) T cell anti-FIV activity in 30 FIV-infected cats. We investigated its relationship with the number of peripheral blood lymphocytes, particularly the CD4(+) T cell and CD8(+) T cell counts, and the relationship between anti-FIV activity and the number of T cells of CD8alpha(+)beta(lo) and CD8alpha(+)beta(-) phenotypes. A clearly significant correlation was observed between anti-FIV activity and the number of CD4(+) T cells. A weaker anti-FIV activity was associated with a greater decrease in the number of CD4(+) T cells. However, there was no significant correlation between anti-FIV activity and the number of B or CD8(+) T cells. Compared with SPF cats, FIV-infected cats had significantly higher CD8alpha(+)beta(lo) T cell and CD8alpha(+)beta(-) T cell counts, but, no significant correlation was observed between these cell counts and anti-FIV activity. This anti-FIV activity significantly correlated with plasma viremia, which was detected in cats with a weak anti-FIV activity. These results suggest that the anti-FIV activity of CD8(+) T cells plays an important role in plasma viremia and the maintenance of CD4(+) T cells in the body. It is unlikely that CD8alpha(+)beta(lo) or CD8alpha(+)beta(-) T cells appearing after FIV infection represent a phenotype of CD8(+) cells with anti-FIV activity.     

In vitro growth of oocyte-granulosa cell complexes isolated from cryopreserved ovine tissue.

A culture system has been designed in which enzymatically isolated oocyte-granulosa cell complexes from fresh and frozen-thawed ovine ovarian tissue can be grown to antral size in vitro. Oocyte-granulosa complexes ranging from 100 to 240 microns in diameter were dissected from stromal tissue and grown individually in serum-free medium for 30 days. Complexes < 190 microns generally excluded their oocytes or lost three-dimensional structure early in the culture period. In contrast, complexes isolated from fresh or frozen-thawed tissue and measuring 190-240 microns on the day of isolation formed antral cavities in 25 +/- 9% and 18 +/- 6% (mean +/- SEM) of cases, respectively. The effect of gonadotrophin supplementation to the culture medium was tested on frozen-thawed oocyte-granulosa cell complexes only. In cultures supplemented with both FSH and LH or FSH alone, there was no significant difference in the number of oocyte-granulosa cell complexes that formed antral cavities (18 +/- 7%). However, antrum formation was significantly less frequent in cultures lacking gonadotrophin stimulation (7 +/- 4%). All oocyte-granulosa cell complexes maintained a three-dimensional structure throughout culture and developed a functional P450 aromatase enzyme complex, as revealed by the induction of oestradiol production during 8 days of culture after antrum formation in serum-free medium containing testosterone. Oocytes recovered after 30 days of culture were viable and had increased in diameter from 78 +/- 2 microns on the day of isolation, to 131 +/- 3 microns at the end of culture. These results show that oocyte-granulosa cell complexes isolated from cryopreserved ovarian tissue can be grown to antral size in vitro with similar efficiency to those isolated from fresh tissue.     

IL-9 enhances growth of ICC, maintains network structure and strengthens rhythmicity of contraction in culture

Interstitial cells of Cajal (ICC) play a critical role in the control of gastrointestinal motility as pacemaker cells and as regulators of enteric innervation. ICC are one of the first cell types that are injured during an inflammatory process and maintenance of ICC health or promotion of growth and development maybe crucial in recovery after injury. The aim of this study was to evaluate the role of IL-9 in the growth, development and maintenance of ICC in culture. IL-9 in concentrations from 0.02 to 1 microg/ml promoted individual ICC growth and maintenance of the ICC network structure inside tissue explants under culture conditions. The number of ICC grown out of the explants increased significantly at day 4 of culture in the presence of 0.02, 0.5 and 1 microg/ml IL-9. In the presence of 0.5 microg/ml IL-9, explants in culture maintained a higher frequency and stabilized the frequency of spontaneous contractile activity. The ultrastructure of the ICC after 4 days in culture was similar to that in situ. Our data indicate that IL-9 promotes ICC growth in culture and it can be hypothesized that IL-9 is a critical factor in the maintenance of ICC health and ICC repair after injury.     

Metabolic effects of radiation induced rat insulinoma at pancreatic, hepatic and subscapular transplantation sites

Metabolic effects of 3 different sites of transplantation of cultured tumour cells from a radiation induced insulinoma (28 X 10(6) viable cells per rat) were examined in 15-18 weeks old male NEDH rats. Subscapular implantation consistently produced a highly vascularised encapsulated tumour associated with hyperinsulinaemia, hyperphagia and hypoglycaemia by 21 days, which progressed to fatal neuroglycopaenic coma at 37 +/- 3 days (mean +/- SEM). Implantation of tumour cells into the hepatic portal vein resulted in a multilobular hepatic tumour in two out of nine rats, with hyperinsulinaemia and fatal hypoglycaemia by 49-54 days. Irregularities of glucose homeostasis were observed in a further three rats by 62 days. Intrapancreatic implantation consistently produced a similar tumour to that observed at the subscapular site. Implantation into the pancreas produced the most rapid onset of hyperinsulinaemia, hyperphagia and hypoglycaemia, with survival for only 28 +/- 3 days. The results demonstrate an important effect of transplantation site on the function and metabolic consequences of the NEDH rat insulinoma.     

Dickkopf-3 is expressed in a subset of adult human pancreatic beta cells

The Dickkopf (Dkk) gene family of secretory modulators of canonical Wnt/beta catenin signals is involved in the control of stem cell proliferation, homeostasis and differentiation. Bioinformatic data on dkk-1/3 gene expression, indicating high expression levels in the human pancreas, led us to analyze these two proteins in adult human pancreatic tissue. Dkk-1/3 mRNA levels and protein distribution were analyzed in isolated human islets vs. the exocrine/ductal pancreatic cells and in paraffin sections of adult human pancreata. Using real time PCR only lowest amounts of dkk-1 mRNA were detectable in the endocrine fractions. Immunohistochemistry did not reveal any Dkk-1 protein in adult human pancreatic tissue. Interestingly, Dkk-3 mRNA and protein were clearly present in adult human pancreatic islets. Messenger RNA levels for Dkk-3 were significantly higher in isolated islets as compared to the exocrine/ductal fraction. Co-staining with an antibody against insulin identified the beta cells of the pancreas as the Dkk-3-positive cells. Notably, only a subset of beta cells contained Dkk-3. As shown by western blot analysis Dkk-3 seems to be proteolytically processed in beta cells. To our knowledge, this is the first study describing a molecule with which the pool of pancreatic beta cells can be further subdivided. Future studies will show whether this sub-classification of beta cells translates into functional differences.     

Rapid purification of human ductal cells from human pancreatic fractions with surface antibody CA19-9

Generating human insulin-secreting cells for cell therapy of diabetes represents a highly competitive world challenge. Human ductal cells can give rise to islets in vivo and in vitro. The goal of this study was to devise a rapid sorting method to highly purify human ductal cells from pancreatic tissue using a pan-ductal membrane antibody carbohydrate antigen 19-9 (CA19-9). Human pancreatic sections confirmed antibody specificity. The human exocrine fraction (30% ductal cells) was sorted with magnetic bead technology or by FACS. Immunocytochemistry post-sorting determined ductal cell content. The manual magnetic bead technique resulted in 74%+/-2 (n = 4) CA19 positive cells. Whereas the automated AutoMACS technique (n = 5) yielded 92.6%+/-0.5 CA19-9 positive cells with only a minor beta cell contamination (0.2%+/-0.03); cell yield post-sorting was 12.9%+/-2.5 (1.69+/-0.41 x 10(6) cells) with 51.7%+6.5 (n = 5) viability post-sorting. The FACS (n = 6) resulted in 97.1%+/-0.82 CA19-9 positive cells, a cell yield of 25.5%+/-5.6 (5.03+/-1.0 x 10(6)), with 72.1%+/-6.1 viability post-sorting.     

Non-enzymatic,Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO₂ incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.     

Factors controlling pancreatic islet neogenesis.

We have established a model in which cellophane wrapping induces reiteration of the normal ontogeny of beta-cell differentiation from ductal tissue. The secretion of insulin is physiologic and coordinated to the needs of the animal. Streptozotocin-induced diabetes in hamsters can be "cured" at least half the time. There appears to be activation of growth factor(s) within the pancreas, acting in an autocrine, paracrine, or juxtacrine manner to induce ductal cell proliferation and differentiation into functioning beta cells. Given the results of our studies to date, it does not seem premature to envisage new approaches to the treatment of diabetes mellitus. Identification of the factor(s) regulating islet-cell proliferation and differentiation in our model may permit islets to be grown in culture. This concept could be extended to induce endocrine cell differentiation in vitro as well. Furthermore, islet-cell growth factors could be used to provide "trophic support" to islet transplants as a means of maintaining graft viability. There may also be greater scope for gene therapy when the growth factor(s) have been isolated, purified, sequenced, and cloned.     

Maintenance of fetal human pancreatic beta cells in tissue culture

Large quantities of viable human islet tissue (beta cells) are required for transplant and for investigations of the autoimmune basis of Type I diabetes. Fetal pancreas offers a potential advantage over other possible sources of beta cells in that it retains some capacity for growth in vitro. We have cultured a total of 45 human pancreata from fetuses of gestational ages from 18 to 23 weeks. Each pancreas was obtained within minutes after delivery and usually cultured within 30 minutes. Pancreata were dispersed and cultured for up to 32 days. Maintenance and growth of the beta cells was assessed by the content of insulin in extracts of cultured tissue. As has been reported by others, fetal human beta cells survived in vitro for over 4 weeks. In three experiments in which a direct comparison was made, collagenase digestion of the fetal pancreas resulted in a significantly greater loss of insulin content compared to minced tissue cultured without digestion. Storage of three pancreata in medium overnight at 4 degrees C significantly reduced the insulin content of the pancreas compared to pancreata cultured immediately. During culture, the majority of the beta cells (based on insulin content) were found in small, macroscopic clumps attached to the surface of the culture dish, and surrounded by a nearly confluent monolayer of fibroblastoid cells. There was a marked decrease in the insulin content of the tissue during culture, most of it (to less than 25% of the original) occurring over the first 4-6 days of culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth factor and somatic cell regulation of mouse gonocyte-derived colony formation in vitro

At birth, the mouse gonocyte does not resume mitotic activity for several days in vivo but, in an in vitro clonogenic system, cell division commences soon after culture. Somatic testis cell underlays had potent inhibitory activity on gonocyte-derived colony formation (23 +/- 15% compared with 84 +/- 1% in controls; P = 0.0001) when added to cultures of gonocytes in vitro. A Sertoli cell line, TM4B, had an even more pronounced effect on gonocyte clonogenic capacity, with 1 +/- 1% compared with 72 +/- 17% colony formation in controls (P = 0.0003). Testis cells appeared to have a direct inhibitory effect since testis-conditioned medium did not show a significant reduction in the number of colonies. The observed reduction in colony formation with the testis cell underlay was not accounted for by decreased attachment of gonocytes as simultaneous addition of a single cell suspension of testis cells was still effective in significantly reducing colony number when compared with controls (P = 0.01). Therefore, the observed inhibition exerted by testis cells appears to be a consequence of decreased proliferation of gonocytes. Growth factors belonging to the transforming growth factor beta superfamily which are known to be expressed in testis, such as transforming growth factor beta and epidermal growth factor, did not exert any inhibitory action on gonocyte-derived colony formation when added together or alone. However, a shift to a smaller colony size occurred in the presence of transforming growth factor beta and transforming growth factor beta plus epidermal growth factor, indicating a reduction in colony cell proliferation. Evidence for the expression of the Müllerian inhibiting substance receptor on newborn gonocytes using in situ hybridization was inconclusive. This finding was in agreement with the lack of a direct action of Müllerian inhibiting substance on the formation of gonocyte-derived colonies in vitro. Leukaemia inhibitory factor, alone or in combination with forskolin, had neither an inhibitory nor an enhancing effect on gonocyte-derived colony formation. An in vitro clonogenic method to assay for the proliferation of gonocytes in the presence of specific growth factors, cell lines, testis cell underlays and cell suspensions was used to identify a somatic cell-mediated inhibitor which may be responsible for the inhibitory action on gonocyte proliferation in vivo shortly after birth.     

T cell subpopulations mediating inhibition of feline immunodeficiency virus replication in mucosally infected cats

Feline immunodeficiency virus (FIV) infection induces an increase in two subpopulations (CD8alpha(+)beta(low) and CD8alpha(+)beta(-)) within CD8(+) peripheral blood lymphocytes (PBLs) of cats. It is known that depletion of CD8(+) cells often results in augmentation of FIV proliferation in PBL culture, similarly to the case of human immunodeficiency virus. In this study, we attempted to define PBL subpopulations mediating antiviral activity in five cats intravaginally infected with a molecularly cloned FIV isolate. Several subpopulations (CD8alpha(+)beta(+), CD8alpha(+)beta(-), and CD4(+) cells) were shown to participate in inhibition of the FIV replication, at least in part, in a major histocompatibility complex-unrestricted manner. Moreover, the subpopulations showing anti-FIV activity were different among the individual cats.     

Prolongation of canine pancreas allograft survival with cyclosporin A: preliminary report.

Studies were conducted on dogs to test the efficacy of cyclosporin A (CyA) in prolonging normoglycaemia and graft survival after whole-organ pancreas allograft transplantation. Five dogs subjected to pancreatectomy alone served as controls. Withholding immunosuppression after transplantation (five animals) resulted in the same median duration of survival as occurred in the controls (13 days). Azathioprine and steroids (seven animals) produced median durations of normoglycaemia and survival of 9 and 23 days respectively. Animals given CyA 18 mg/kg/day (five) and 25 mg/kg/day (10), however, showed median durations of normoglycaemia of 18 and 55 days (p less than 0.05 and p less than 0.02) respectively and median survival times of 36 and 85 days (NS and p less than 0.02). If CyA proved effective in controlling rejection of pancreas allografts in man it would offer unstable diabetics in renal failure a more hopeful outlook than conventional immunosuppression.