Decidual mesenchymal stem/stromal cells from preeclamptic patients secrete endoglin, which at high levels inhibits endothelial cell attachment invitro.

INTRODUCTION: In preeclampsia (PE), inadequate remodelling of spiral arterioles in the decidua basalis causes oxidative stress and subsequent increased release of antiangiogenic soluble endoglin (sENG) into the maternal circulation. Decidual mesenchymal stem/stromal cells (DMSCs) reside adjacent to endothelial cells in this vascular niche. Surprisingly, DMSCs express membrane-bound ENG (CD105). PE-affected DMSCs (PE-DMSCs) are abnormal and due to reduced extravillous invasion, more of them are present, but the significance of this is not known. METHODS: DMSCs were isolated and characterised from normotensive control and severe-PE placentae. Extracellular vesicle (EV) types, shed microvesicles (sMV) and exosomes, were isolated from DMSC conditioned media (DMSCCM), respectively. Secretion of ENG by DMSCs was assessed by ELISA of DMSCCM, with and without EV depletion. The effects of reducing ENG concentration, by blocking antibody, on human umbilical vein endothelial cell (HUVEC) attachment were assessed by xCELLigence real-time functional assays. RESULTS: ENG was detected in DMSCCM and these levels significantly decreased when depleted of exosomes and sMV. There was no significant difference in the amount of ENG secreted by control DMSCs and PE-DMSCs. Blocking ENG in concentrated DMSCCM, used to treat HUVECs, improved endothelial cell attachment. DISCUSSION: In normotensive pregnancies, DMSC secretion of ENG likely has a beneficial effect on endothelial cells. However, in PE pregnancies, shallow invasion of the spiral arterioles exposes more PE-DMSC derived sources of ENG (soluble and EV). The presence of these PE-DMSCs in the vascular niche contributes to endothelial cell dysfunction.

Postpartum IGF-I and IGFBP-2 levels are prospectively associated with the development of type 2 diabetes in women with previous gestational diabetes mellitus.

AIMS: Women with previous gestational diabetes mellitus (GDM) are at greater risk of developing type 2 diabetes. In the general population, the insulin-like growth factor (IGF) system has been implicated in the development of type 2 diabetes. The aim of this study was to determine if circulating IGF-I, IGF-II, IGFBP-1 and IGFBP-2 levels 12weeks following a GDM pregnancy are associated with an increased risk of developing type 2 diabetes. METHODS: IGF-I, IGF-II, IGFBP-1 and IGFBP-2 levels were measured in 98 normal glucose tolerant women, 12weeks following an index GDM pregnancy using enzyme immunoassay. Women were assessed for up to 10years for the development of overt type 2 diabetes. RESULTS: Among the 98 women with previous GDM, 21 (21%) developed diabetes during the median follow-up period of 8.5years. After adjusting for age and BMI, IGF-I and IGFBP-2 were significantly associated with the development of type 2 diabetes. In a clinical model of prediction of type 2 diabetes that included age, BMI, pregnancy fasting glucose and postnatal fasting glucose, the addition of IGF-I and IGFBP-2 resulted in an improvement in the net reclassification index of 17.8%. CONCLUSIONS: High postpartum IGF-I and low postpartum IGFBP-2 levels are a significant risk factor for the development of type 2 diabetes in women with a previous history of GDM. This is the first report that identifies IGF-I and IGFBP-2 as a potential biomarker for the prediction of type 2 diabetes in women with a history of GDM.

New biomarkers for the prediction of spontaneous preterm labour in symptomatic pregnant women: a comparison with fetal fibronectin.

OBJECTIVE: To identify cervicovaginal fluid (CVF) biomarkers predictive of spontaneous preterm birth in women with symptoms of preterm labour. DESIGN: Retrospective cohort study. SETTING: Melbourne, Australia. POPULATION: Women with a singleton pregnancy admitted to the Emergency Department between 22 and 36 weeks of gestation presenting with symptoms of preterm labour. METHODS: Two-dimensional electrophoresis was used to analyse the CVF proteome. Validation of putative biomarkers was performed using enzyme-linked immunosorbent assay (ELISA) in an independent cohort. Optimal concentration thresholds of putative biomarkers were determined and the predictive efficacy for preterm birth was compared with that of fetal fibronectin. MAIN OUTCOME MEASURES: Prediction of spontaneous preterm labour within 7 days. RESULTS: Differentially expressed proteins were identified by proteomic analysis in women presenting with 'threatened' preterm labour without cervical change who subsequently delivered preterm (n = 12 women). ELISA validation using an independent cohort (n = 129 women) found albumin and vitamin D-binding protein (VDBP) to be significantly altered between women who subsequently experienced preterm birth and those who delivered at term. Prediction of preterm delivery within 7 days using a dual biomarker model (albumin/VDBP) provided 66.7% sensitivity, 100% specificity, 100% positive predictive value (PPV) and 96.7% negative predictive value (NPV), compared with fetal fibronectin yielding 66.7, 87.9, 36.4 and 96.2%, respectively (n = 64). Using the maximum number of screened samples, the predictive utility of albumin/VDBP yielded a sensitivity of 77.8%, specificity and PPV of 100% and NPV of 98.0% (n = 109). CONCLUSIONS: The dual biomarker model of albumin/VDBP is more efficacious than fetal fibronectin in predicting spontaneous preterm delivery in symptomatic women within 7 days. A clinical diagnostic trial is required to test this model on a larger population to confirm these findings and to further refine the predictive values.

Myostatin in the placentae of pregnancies complicated with gestational diabetes mellitus.

INTRODUCTION: Gestational diabetes mellitus (GDM) is characterised by maternal glucose intolerance and insulin resistance during pregnancy. Myostatin, initially identified as a negative regulator of muscle development may also function in the regulation of placental development and glucose uptake. Myostatin expression in placentae of GDM complicated pregnancies is unknown. However, higher myostatin levels occur in placentae of pregnancies complicated with preeclampsia. We hypothesise that myostatin will be differentially expressed in GDM complicated pregnancies. METHODS: Myostatin concentrations (ELISA) were evaluated in plasma of presymptomatic women who later developed GDM and compared to plasma of normal glucose tolerant (NGT) women. Furthermore, myostatin protein expression (Western blot) was studied in placentae of pregnant women with GDM (treated with diet or insulin) compared to placentae of NGT women. RESULTS: No significant difference in myostatin concentration was seen in plasma of pre-symptomatic GDM women compared to NGT women. In placenta significant differences in myostatin protein expressions (higher precursor; p < 0.05and lower dimer: p < 0.005) were observed in GDM complicated compared to NGT pregnancies. Furthermore, placentae of GDM women treated with insulin compared to diet have higher dimer (p < 0.005) and lower precursor (p < 0.05). Compared to lean women, placentae of obese NGT women were lower in myostatin dimer expression (p < 0.05). DISCUSSION: Myostatin expression in placental tissue is altered under stress conditions (e.g. obesity and abnormal glucose metabolism) found in pregnancies complicated with GDM. We hypothesise that myostatin is active in these placentae and could affect glucose homoeostasis and/or cytokine production thereby altering the function of the placenta.

Activin A as a marker of intrauterine infection in women with preterm prelabour rupture of membranes.

OBJECTIVE: To assess maternal serum activin A, an early phase response protein in systemic infection, as an early marker of intrauterine infection in women with preterm prelabour rupture of membranes (PPROM). STUDY DESIGN: A prospective cohort study of women with singleton pregnancies complicated by PPROM at 24 to 34 weeks' gestation. Serum was collected for activin A and cytokine measurements. Activin A was measured using commercial enzyme-linked immunosorbent assay. Cytokines were measured using commercial multiplex assay. Pregnancy outcomes including infection were determined by case-record review. RESULT: Eighteen women with PPROM were studied, with seven developing intrauterine infection. Serum activin A in women with and without infection did not differ. Peripheral white cell count, interleukin (IL)-6 and IL-10 were higher (P=0.03, 0.05 and 0.009, respectively) and IL-7 lower (P=0.04) 72 h before delivery in women with infection. CONCLUSION: Activin A is not a clinically useful marker of intrauterine infection in women with PPROM.

Changes in gene expressions elicited by physiological concentrations of genistein on human endometrial cancer cells.

The incidence of hormone-related diseases such as prostatic, breast, ovarian, and endometrial cancer is lower in Asian populations compared to Western countries. High consumption of soybean products that are rich in phytoestrogens, predominantly genistein, is postulated to be responsible for the lower incidence of hormone-related disease, although the mechanism through which this effect might be mediated is unclear. In this study, microarray analysis was used to identify the changes in gene expression elicited by treatment of the human endometrial cancer cell line, Ishikawa, with genistein at both physiologically achievable and supraphysiological concentrations. Genistein treatment at 5 microM concentration induced multiple changes in gene expression including some implicated in oncogenesis. In contrast, treatment with a supraphysiological concentration of genistein predominantly activated stress response genes and showed very limited overlap with the genes regulated at lower concentrations. Of the genes regulated by genistein, 9.3% were also regulated by 17beta-estradiol suggesting that genistein exerts its response via the estrogen pathway. These results indicate that at physiological concentrations, genistein is able to elicit pleiotropic effects on a variety of pathways believed to be involved in tumorigenesis. Supraphysiological concentrations of genistein, such as those used in many previous studies, elicit changes in gene expression that are unlikely to occur in vivo.

Translational proteomics: developing a predictive capacity -- a review.

Over the past decade, proteomics has undergone a rapid development and radiation, diversifying across the biochemical landscape. While no single technique yet delivers complete proteomic coverage, application-specific adaptations afford significant opportunity for discovery and the development of predictive capacity (e.g. surrogate biomarker and clinical diagnostics). Targeted proteomic approaches, protein profiling strategies using affinity capture mass spectrometry and solution array represent realistic opportunities to deliver predictive capacity. The aim of this review is to provide an overview of proteomic technologies and how the outcomes delivered by such platforms may be translated into applications of predictive utility in clinical and basic science. In particular, recent applications in protein/peptide profiling (solid-phase affinity capture mass spectrometry and the targeted approach of antibody arrays) and the opportunities they afford researchers within the discipline of reproductive biology to develop new diagnostic and prognostic tests and surrogate biomarkers to improve the delivery of women's health care are considered.

Factors affecting umbilical venous perfusion during experimental cord knotting.

The aim was to determine experimentally the factors that increase the risk of venous occlusion by applying a standardised tightening force to isolated perfused umbilical cords tied in a true knot in vitro. Umbilical cords were collected from patients undergoing Caesarean section. Cords were clamped, isolated and studied within 15 min. The umbilical vein was cannulated, the cord tied in a true knot and traction was applied using standard weights. The umbilical vein was perfused with modified Krebs solution at a constant pressure of 40 mmHg and the attached weight increased until perfusion ceased. The cord mass index (weight/length), hydration index/100-[(dry weight/wet weight)x100], and coiling index (coils/length) were determined. Cord morphometric analysis was performed on 193 cords. Intra uterine growth restriction was associated with decreased cord mass index (p=0.002) and increased coiling index (p=0.002). Venous perfusion experiments were performed on 75 cords. Using multivariate regression analysis, cord morphometric factors that increased the risk of cord occlusion were decreased cord mass index (p=0.008), decreased cord hydration index (p=0.004), and low venous flow capacity (p=0.001). During experimental cord knotting with applied traction, the susceptibility to venous occlusion was increased with low cord mass index, low cord hydration index and low venous flow capacity. These cord characteristics were associated with low fetal body weight and intrauterine growth restriction. An increased susceptibility to cord occlusion may contribute to the higher perinatal morbidity and mortality in growth restricted pregnancies.

Altered placental oxidative stress status in gestational diabetes mellitus.

Oxidative stress has been clearly linked to type 2 diabetes mellitus, however, limited data are available on the involvement of oxidative stress in gestational diabetes mellitus (GDM), a disease of similar pathophysiology. The aim of this study was to investigate the status of placental oxidative stress in healthy pregnant women and women with GDM. The hypothesis to be tested was that tissue markers of oxidative stress are significantly increased in GDM compared to normal placental tissues. Markers of oxidative stress measured were the release of 8-isoprostane (8-epi-prostaglandin F(2alpha)) from human term placental explants (n=11), the activity of the antioxidant enzymes superoxide dismutase and glutathione peroxidase (n=10), and protein carbonyl content (n=12). Placental release of 8-isoprostane was 2-fold greater from women with GDM (P<0.001) compared to healthy pregnant women. Superoxide dismutase activity and protein carbonyl content were elevated in placentae obtained from women with GDM (P<0.04 and P<0.004 respectively), whilst there was no significant difference in the activity of glutathione peroxidase. These data demonstrate the presence of oxidative stress in the placenta from women with GDM, in addition to the induction of a key antioxidant, collectively indicating a state of existing oxidative stress in this condition.

Proteomic analysis of human plasma: failure of centrifugal ultrafiltration to remove albumin and other high molecular weight proteins.

Determination of specific low abundance proteins, usually by radiolabelled or enzyme-linked immunoassays in serum or plasma is widely used in diagnostic medicine. Substitution of these assays by a proteomic approach has been suggested, but this methodology has far from realised its potential as a diagnostic tool. The main protein fractions of plasma represent more than 80% of total protein, making the hundreds or even thousands of other proteins difficult to detect by two-dimensional electrophoresis (2-DE). Thus, loading sufficient sample to detect trace proteins invariably means excessive loading of albumin and other high abundance proteins. The aim of this study was to determine whether centrifugal ultrafiltration of whole plasma could be used to eliminate proteins exceeding a desired molecular weight cut-off. Cellulose filters with a 30 kDa molecular weight cut-off were used with whole plasma, and total protein was determined before and after ultrafiltration. Samples were processed by routine methods for 2-DE using 18 cm, pH 3-10 isoelectric focusing strips for the first dimension and 7-15% gradient gels for the second dimension followed by silver staining. Gel analysis of the retentate fraction (> 30 kDa) revealed a typical 2-DE plasma profile with most of the major landmark proteins in place and as expected, the gels lacked many of the smaller (< 30 kDa) proteins. Comparison with gels of the filtrate fraction (> 30 kDa) revealed very little difference. Not only were many of the higher molecular weight proteins still present, but some of the smaller < 30 kDa landmark proteins were absent. Overall, gels of both the retentate and filtrate fractions were less informative than gels of whole plasma.

Glucose-induced release of tumour necrosis factor-alpha from human placental and adipose tissues in gestational diabetes mellitus.

AIMS: The cytokine tumour necrosis factor-alpha (TNF-alpha) has been implicated in the pathogenesis of insulin resistance in Type 2 diabetes mellitus, but limited data are available in relation to gestational diabetes mellitus (GDM), a disease in which similar biochemical abnormalities exist. We investigated the effect of exogenous glucose on the release of TNF-alpha from placental and adipose (omental and subcutaneous) tissue obtained from normal pregnant women, and women with GDM. METHODS: Human tissue explants were incubated for up to 24 h and TNF-alpha concentration in the incubation medium quantified by ELISA. The effect of normal (5 mmol/l) and high (15 and 25 mmol/l) glucose concentrations on the release of TNF-alpha was assessed. RESULTS: In placental and subcutaneous adipose tissues obtained from women with GDM (n = 6), TNF-alpha release was significantly greater under conditions of high glucose compared with normal glucose (placenta, 25 mmol/l 5915.7 +/- 2579.6 and 15 mmol/l 4547.1 +/- 2039.1 vs. 5 mmol/l 1897.1 +/- 545.5; subcutaneous adipose tissue, 25 mmol/l 423.5 +/- 207.0 and 15 mmol/l 278.5 +/- 138.7 vs. 5 mmol/l 65.3 +/- 28.5 pg/mg protein; P < 0.05). In contrast, there was no stimulatory effect of high glucose on TNF-alpha release by tissues obtained from normal pregnant women (n = 6) (placenta, 25 mmol/l 1542.1 +/- 486.2 and 15 mmol/l 4263.3 +/- 2737.7 vs. 5 mmol/l 5422.4 +/- 1599.0; subcutaneous adipose tissue, 25 mmol/l 189.8 +/- 120.4 and 15 mmol/l 124.5 +/- 32.3 vs. 5 mmol/l 217.9 +/- 103.5 pg/mg protein). CONCLUSIONS: These observations suggest that tissues from patients with GDM release greater amounts of TNF-alpha in response to high glucose. As TNF-alpha has been previously implicated in the regulation of glucose and lipid metabolism, and of insulin resistance, these data are consistent with the hypothesis that TNF-alpha may be involved in the pathogenesis and/or progression of GDM.

Protection of xenografts by a combination of immunoisolation and a single dose of anti-CD4 antibody.

Immunoisolation is the separation of transplanted cells from cells of the immune system using a semipermeable membrane. Using one such immunoisolation capsule-the TheraCyte device-we have assessed the survival of encapsulated xenogeneic tissue in vivo as well as the contribution of CD4+ve T cells to encapsulated xenograft rejection. The foreign body reaction to the TheraCyte capsule in vivo was assessed by transplanting empty capsules into normal mice. These capsules elicit a foreign body response by the host animal. Encapsulated CHO, NIT-1, and PK-15 cells were placed in culture and in immunodeficient mice to investigate their growth characteristics in the TheraCyte device. These cell lines survive both in culture and in immunodeficient SCID mice. Xenogeneic PK cells were also transplanted into normal C57BL/6 mice. These cells do not survive in normal mice despite the absence of direct contact between infiltrating and encapsulated cells. In addition, the survival of encapsulated cells in mice treated with a single dose of anti-CD4 antibody was examined. This was assessed using two systems: 1) histological analysis of capsule sections; 2) a quantitative luciferase reporter system using PK cells transfected to express luciferase. In both cases, anti-CD4 antibody contributed to prolonged encapsulated xenogeneic cell survival. Encapsulated xenogeneic cells survive in immunodeficient mice but not normal mice. Treatment of normal mice with anti-CD4 antibody results in prolonged survival of xenogeneic cells that can be measured using a luciferase reporter system. These results highlight the contribution of CD4+ve T cells to encapsulated xenograft rejection.

The effect of vascular coiling on venous perfusion during experimental umbilical cord encirclement.

OBJECTIVE: The aim of these studies was to compare venous perfusion in umbilical cords subjected to a standardized tight encirclement force. Comparisons were made between cords from normal pregnancies and those complicated by gestational diabetes mellitus and intrauterine growth restriction. STUDY DESIGN: The cannulated cord segment was wrapped around a plastic container, which in turn was attached with nylon string to a hanging graduated measuring cylinder in which known volumes of water could be applied for weight. The cord was perfused with Krebs solution to a constant venous perfusion pressure of 40 mm Hg. Weights of 100-g increments were applied until total cessation of venous perfusion was observed. The weight, length, number of vascular coils, and degree of hydration were recorded for each cord. The coiling index was defined as the number of vascular coils per 10 cm of cord. RESULTS: Regression analysis of 34 cords (normal, n = 16; gestational diabetes mellitus, n = 12; intrauterine growth restriction, n = 6) identified a significant inverse correlation (P =.0003, Spearman rank correlation) between coiling index and the minimum weight required to occlude venous perfusion. Cords from pregnancies complicated by intrauterine growth restriction displayed a higher frequency of vascular coiling and were more easily occluded (median weight, 350 g) than were cords from pregnancies complicated by gestational diabetes mellitus, which were less coiled and tended to resist occlusion (median weight, 1100 g). CONCLUSION: During experimental cord encirclement there was a significant inverse relationship between vascular coiling and susceptibility to cord venous occlusion when traction was applied to the encirclement.

Type II phospholipase A2 in preterm human gestational tissues.

Maternal genital tract infections and the associated inflammatory response may contribute to the onset of many cases of preterm labour. Type II phospholipase A(2)(PLA2) hydrolyses glycerophospholipids, releasing free fatty acid for conversion into potent biological mediators, such as prostaglandins, which play a significant role in both the onset and progression of human labour and the activation of inflammatory reactions. The aim of this study was to quantify immunoreactive (ir) Type II PLA2 in placenta, amnion and choriodecidua collected from women delivering prematurely or due to histological chorioamnionitis, and to compare levels to those at term. Tissues were assayed for ir Type II PLA2 by ELISA and expressed as ng/mg tissue protein. Ir Type II PLA2 tissue content was significantly higher in preterm (n=26) amnion and choriodecidua, but not in the placenta when compared to tissues obtained at term (n=42). When the data were stratified with respect to labour status, ir Type II PLA2 content was significantly higher in the preterm not-in-labour group (NIL, n=17) than the preterm in labour group (IL, n=9) in the amnion. When the NIL group was analysed with respect to membrane rupture, women who had spontaneously ruptured membranes (n=6) expressed significantly greater ir Type II PLA2 than those that had intact membranes (n=11) in both the amnion and choriodecidua but not in the placenta. No significant difference was observed between the preterm IL group (n=9) and the group with histological chorioamnionitis (n=14). The data obtained in this study support a role for Type II PLA2 in association with spontaneous rupture of membranes.

Protective effect of CTLA4Ig secreted by transgenic fetal pancreas allografts.

BACKGROUND: Pancreas allotransplantation offers a cure for insulin-dependent diabetes mellitus. Systemic immunosuppression used to prevent immune destruction of the graft has side-effects, including increased susceptibility to infection and neoplasia. These unwanted effects may be limited by engineering the graft to secrete immunomodulatory molecules, to achieve local immunosuppression. Several studies have shown that transient local CTLA4Ig results in partial protection of allogeneic grafts. Our intent has been to determine whether sustained secretion of transgenic CTLA4Ig from pancreatic islets is able to protect against allograft rejection. METHODS AND RESULTS: Mouse CTLA4 (test=CTLA4Ig) or CD5 leader sequence (control=CD5LIg) was fused to the Fc of mouse IgG2c, and expressed transgenically under the control of the rat insulin promoter in C57BL/6 mice carrying the bml mutation of H-2K(b) (B6.C-H-2(bm1)). This resulted in expression in pancreatic islets. We used ELISA quantification of transgene products secreted into the supernatants of cultured fetal pancreata to select high (CTLA4Ig(hi)) and low (CTLA4Ig(lo)) expresser transgenic mice. Cultured fetal pancreata were transplanted under the kidney capsule of wholly allogeneic CBA recipient mice. CTLA4Ig(hi) but not CTLA4Ig(lo) expresser grafts showed enhanced survival compared with control CD5LIg grafts at 6 weeks posttransplant, provided the recipient mice were transiently depleted of CD4 T cells (by a single low-dose injection of GK1.5) before transplantation. CONCLUSIONS: Sustained local secretion of CTLA4Ig from transgenic grafts in combination with transient systemic CD4 T-cell depletion can enhance allograft acceptance.

Local secretion of a chimeric anti-CD4 antibody protects against graft rejection in the NOD mouse.

BACKGROUND: Engineering a graft to secrete its own immunosuppressive antibodies may minimize the risks associated with current high dose systemic immunosuppression. METHODS AND RESULTS: A beta cell insulinoma cell line (NIT-1) was transfected with genes encoding a chimeric anti-CD4 antibody. The NIT-1 cells secreted functional chimeric anti-CD4 antibody that bound to the CD4 molecule on mouse thymocytes and inhibited in vitro proliferation of CD4+ve T cells. Both test and control transfected cell lines grew at a similar rate in immunodeficient mice. In immunocompetent NOD mice, NIT-1 cells are normally rejected by a cellular immune response against the SV40 T antigen. Although control transfected NIT-1 cells were rapidly rejected by NOD mice, anti-CD4 secreting NIT-1 cells grew significantly better and were able to form tumors at the site of injection. CONCLUSIONS: The local secretion of chimeric anti-CD4 antibody from transfected cells can contribute to graft survival in our transplantation model.

Inhibition of mitochondrial oxidative phosphorylation induces hyper-expression of glutamic acid decarboxylase in pancreatic islet cells.

It has been hypothesised that mitochondrial dysfunction in pancreatic beta cells could produce hyper-expression of glutamic acid decarboxylase (GAD), a major autoantigen in insulin-dependent diabetes mellitus (IDDM) (Degli Esposti, M. and Mackay, I.R. Diabetologia 40: 352-356, 1997). Here we report that specific inhibition of mitochondrial respiration enhances the expression of GAD in both foetal mouse pancreatic tissue and hamster HIT-T15 cells. Inhibitors of NADH-ubiquinone oxidoreductase (complex I) seem to be particularly effective in increasing the expression of GAD in both foetal mouse pancreas and HIT-T15 hamster beta cells, especially in the presence of nutrients such as arginine and glucose. These results represent the first evidence that GAD expression is enhanced under conditions that are toxic to pancreatic beta cells, and establish a link between mitochondrial dysfunction and expression of IDDM autoantigens.

Transgenic expression of CD95 ligand on islet beta cells induces a granulocytic infiltration but does not confer immune privilege upon islet allografts.

Binding of CD95 (Fas/APO-1) by its ligand (CD95L) commonly induces apoptosis. Apoptosis of activated T cells, induced by CD95L expressed in the rodent testis, has been proposed to be the mechanism of immune privilege [Bellgrau, D., Gold, D., Selawry, H., Moore, J., Franzusoff, A. & Duke, R. C. (1995) Nature (London) 377, 630-632]. To test whether CD95L could protect pancreatic islet grafts from rejection, we made transgenic mice expressing murine CD95L on their islet beta cells and transplanted fetal pancreata under the kidney capsules of allogeneic animals. Expression of CD95L failed to protect the grafts from rejection. However, transgenic mice developed a granulocytic infiltration in their pancreata. These results demonstrate a pro-inflammatory function of CD95L and suggest that expression of CD95L may not be sufficient to protect organ allografts.

Transgenic expression of mouse proinsulin II prevents diabetes in nonobese diabetic mice.

IDDM in humans and in nonobese diabetic (NOD) mice is a T-cell-dependent autoimmune disease in which the beta-cells of the pancreatic islets are destroyed. Several putative beta-cell autoantigens have been identified, but insulin and its precursor, proinsulin, are the only ones that are beta-cell specific. (Pro)insulin may be a key autoantigen in IDDM. To address the role of proinsulin in the development of IDDM, we generated NOD mice transgenic for the mouse proinsulin II gene driven off a major histocompatibility complex (MHC) class II promoter to direct expression of the transgene to MHC class II bearing cells, including those in the thymus, with the aim of deleting proinsulin-reactive T-cells. The mononuclear cell infiltration of the islets (insulitis) is almost completely absent, and diabetes is prevented in these transgenic NOD mice. The mononuclear cell infiltration of the salivary glands (sialitis) and immune responses to ovalbumin (OVA) are not altered, indicating that the protective effect of the transgene is specific for islet pathology and not due to general immunosuppression. We conclude that autoimmunity to proinsulin plays a pivotal role in the development of IDDM.