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.

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.

T cell dysfunction in the diabetes-prone BB rat. A role for thymic migrants that are not T cell precursors.

Diabetes-prone BB (BB-DP) rats express several T cell dysfunctions which include poor proliferative and cytotoxic responses to alloantigen. The goal of this study was to determine the origin of these T cell dysfunctions. When BB-DP rats were thymectomized, T cell depleted, and transplanted with neonatal thymus tissue from diabetes-resistant and otherwise normal DA/BB F1 rats, the early restoration of T cell function proceeded normally on a cell-for-cell basis; i.e., peripheral T cells functioned like those from the thymus donor. Because the thymus in these experiments was subjected to gamma irradiation before transplantation and there was no evidence of F1 chimerism in the transplanted BB-DP rats, it appeared that the BB-DP T cell precursors could mature into normally functioning T cells if the maturation process occurred in a normal thymus. If the F1 thymus tissue was treated with dGua before transplantation, the T cells of these animals functioned poorly like those from untreated BB-DP rats. dGua poisons bone marrow-derived cells, including gamma radiation-resistant cells of the macrophage/dendritic cell lineages, while sparing the thymic epithelium. Therefore, the reversal of the T cell dysfunction depends on the presence in the F1 thymus of gamma radiation-resistant, dGua-sensitive F1 cells. Conversely, thymectomized and T cell-depleted F1 rats expressed T cell dysfunction when transplanted with gamma-irradiated BB thymus grafts. T cell responses were normal in animals transplanted with dGua-treated BB thymus grafts. With increasing time after thymus transplantation, T cells from all animals gradually expressed the functional phenotype of the bone marrow donor. Taken together these results suggest that BB-DP bone marrow-derived cells that are not T cell precursors influence the maturation environment in the thymus of otherwise normal BB-DP T cell precursors.

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.

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.

Induction of insulitis in athymic (nude) mice. The effect of NOD thymus and pancreas transplantation.

The NOD mouse is a model for human insulin-dependent diabetes mellitus. The disease is thought to have an autoimmune etiology because it is T-cell dependent and is characterized by mononuclear cell infiltration in and around the pancreatic islets of Langerhans. The mechanism by which autoreactive T-cells are generated is not fully understood, but it has been postulated that there is a breakdown in self-tolerance induction during intrathymic T-cell maturation. The aim of these studies was to determine whether transplantation of NOD thymus into diabetes-resistant mouse strains would generate islet-reactive T-cells. Neonatal thymus was pretreated either by irradiation or culture in 2-deoxyguanosine (dGua) and then transplanted into athymic BALB/c, CBA, and C57BL/6 nude mice. Generally, insulitis was not seen in the CBA or C57BL/6 recipients, but was found in 56% of BALB/c mice transplanted with an irradiated NOD thymus and in 46% BALB/c mice with a dGua-treated thymus. Similar experiments in which a NOD fetal pancreas was transplanted into nude BALB/c mice before NOD thymus transplantation showed a similar frequency and severity of insulitis in both the host pancreas and grafted NOD pancreas. This suggests that NOD islets are no more prone than the host islets to autoimmune attack and do not exacerbate insulitis. Overall, the data suggest that a defect of thymic origin (and correlating with the thymic epithelium) in the NOD mouse can lead to the development of autoreactive T-cells and specific islet cell damage. Autoreactivity appears to be restricted to the H-2Kd allele that is shared by NOD and BALB/c mice.

Insulin secretion by fetal human pancreatic islets of Langerhans in prolonged organ culture.

Fetal human pancreata, obtained from legally-induced abortions, were placed in organ culture to study their capacity to produce insulin over periods in vitro of between 18 and 40 days. Specimens obtained by hysterotomy usually showed increasing insulin secretion as measured by the insulin content of the media at each twice-weekly medium change. In most instances, relatively little insulin was produced during the first 7-10 days, but thereafter insulin secretion rapidly increased. In contrast, most specimens from prostaglandin-induced abortions showed high levels of insulin in the medium early in the culture period but little thereafter, indicating rapid release of insulin from damaged tissue. In some instances, after early insulin release by damaged tissue, some recovery of islet function occurred and insulin secretion again increased. The presence of differentiated endocrine cells was confirmed histologically. Tissue from each fetus was placed in a number of petri dishes, and for each individual fetus a qualitatively similar pattern of secretion was noted in each dish. These data suggest that representative and nondestructive monitoring of islet function is possible and may be important before such tissue is considered for use in islet transplantation in insulin-dependent diabetes.

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.

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.

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.

Persistent immunogenicity of rat thymic epithelium.

Culture of thymus tissue in 2-deoxyguanosine (2dGua) is thought to reduce tissue immunogenicity by selectively depleting highly immunogenic, thymic migrants of bone marrow origin. In the mouse 2dGua-treated thymus tissue survival is markedly enhanced compared with untreated tissue when transplanted under the kidney capsule of allogeneic recipients. These experiments were repeated in a rat model. As expected, DA strain neonatal thymus tissue was rejected when transplanted under the kidney capsule of normal allogeneic strain PVG rats. Surprisingly, acute rejection occurred when the tissue was cultured in 4 mM 2dGua, a dose capable of destroying rat thymocytes in vitro and 3 times the effective dose in mice. To test whether residual marrow-derived cells that escaped 2dGua treatment were responsible for inducing rejection, the treated DA tissue was "parked" in T cell-depleted PVG rats. Our working hypothesis was that the few remaining donor-derived cells of marrow origin would be overgrown by host-type cells. When 2dGua-treated DA thymus tissue was transplanted into T cell-depleted PVG recipients rejection did not occur. However this DA tissue, parked for as long as 200 days in T cell-depleted rats, was acutely rejected when retransplanted into normal PVG recipients. These results suggest that rat thymic epithelium devoid of marrow-derived cells in innately immunogenic.

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.

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.

Secretion of CTLA4Ig by an SV40 T antigen-transformed islet cell line inhibits graft rejection against the neoantigen.

In a model of transplantation rejection, we tested whether a graft manipulated to secrete an immunomodulator could protect itself from immune destruction, thus waiving the need for administration of exogenous immunosuppressants to the recipient. An insulinoma cell line, NIT, having the nonobese diabetic (NOD) genotype but also expressing the SV40 large T antigen, was transfected with CTLA4Ig in an attempt to block the CD28/B7 costimulatory pathway between antigen-presenting calls and T lymphocytes near the site of the graft. The SV40 T antigen is potent at inducing graft rejection. NIT.CTLA4Ig and control transfectants were transplanted subcutaneously into young NOD mice to determine whether CTLA4Ig secretion would abet the survival of the insulinoma graft. CTLA4Ig protein was secreted abundantly in vitro (3-5 microg/ml) and this phenotype was maintained in vivo. Tumor growth was monitored visibly, by palpation, by measuring blood glucose levels, and by death of the host from hypoglycemia caused by unregulated insulin production of the growing insulinoma. Cell growth was similar for NIT.CTLA4Ig7 and control transfectants in immunodeficient mice (nude, irradiated, or SCID mice), indicating that there was no intrinsic growth advantage of the NIT.CTLA4Ig cells. In immunocompetent NOD mice however, the survival/growth of the NIT.CTLA4Ig graft was significantly better than that of the controls. Histopathology was consistent with this finding. Donor-specific second-set grafts were acutely rejected, indicating that tolerance was not induced. CTLs were generated even when the graft secreted CTLA4Ig; there was no clear difference in in vitro immune responses generated by NIT.CTLA4Ig and control cells. We conclude that blockade of the B7 costimulation pathway by graft manipulation can contribute to transplantation success.

Induction of limited growth and differentiation of early thymic precursor cells by thymic epithelial cell lines.

The early thymic precursor population of adult mice (low CD4 precursor) has the potential to produce T cells, B cells and dendritic cells if transferred into the appropriate inductive environment of an irradiated recipient. To assess its developmental potential in vitro, this population was isolated and cultured, alone and with various stromal cell lines. Cultured alone, these precursor cells all died rapidly. Co-culture with 3T3 fibroblasts gave good survival but no growth. Co-culture with thymic cortical epithelial cell lines induced significant proliferation after an initial 50% cell loss. However, the supernatant of these cortical epithelial cell lines caused only limited proliferation after very extensive cell death. Examination of the surface phenotype of the cultures on the cortical epithelial layer showed some changes which were compatible with very early steps of thymocyte development, but none of the features of more developed T cells were seen. A proportion of the proliferating cells developed some of the surface markers and morphology of dendritic cells. Immature myeloid cells also grew in these cultures; these appeared to derive from a small number of myeloid progenitors, possibly contaminants within the preparation, and their outgrowth required only soluble factors released by the cortical epithelial cells.

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.

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.

Prevention of autoimmunity in nonobese diabetic (NOD) mice by neonatal transfer of allogeneic thymic macrophages.

Nonobese diabetic (NOD) mice spontaneously develop insulin dependent diabetes mellitus. The disease results from an autoimmune process which involves mononuclear cells surrounding and eventually infiltrating the pancreatic islets of Langerhans. Macrophages are thought to be the first cells to infiltrate the islets and are actively involved in the disease process because diabetes is prevented if host macrophages are depleted or inactivated. Several lines of evidence also suggest that NOD macrophages are phenotypically and functionally abnormal. In this study, allogeneic (CBA) macrophages derived from the thymus were inoculated into newborn NOD mice and these were followed for more than 250 days. Spontaneous diabetes was significantly reduced in female NOD mice (6% diabetic versus 45% of controls). Insulitis was also significantly reduced in both male and female mice compared to their control counterparts, and in most cases there were virtually no inflammatory cells in the pancreas. Allogeneic skin grafting and mixed leukocyte cultures indicated that the recipients were not tolerant of donor antigens, and donor-derived cells were not detected in the lymphoid tissues by either flow cytometry or immunohistochemistry. The results show that macrophages from diabetes-resistant donors will prevent insulitis and diabetes in most recipients, however, the mechanism for the protection is unclear, but does not appear to be due to long-term tolerance induction.

The effect of cyclophosphamide treatment on lymphocyte subsets in the nonobese diabetic mouse: a comparison of various lymphoid organs.

The nonobese diabetic (NOD) mouse is a model for human Type 1 diabetes mellitus. Pancreatic beta-cell destruction in NOD mice is mediated by an autoimmune process which can be accelerated by cyclophosphamide (CP). We studied the phenotype of lymphocytes from central, peripheral and regional lymphoid tissues in prediabetic NOD and C3H mice before and after a single large dose of CP. All lymphoid organs showed a greatly diminished cell number and most alterations appeared early after CP and were transient, but an aggressive insulitis was not seen in NOD mice until 14 d after injection. The pancreatic islets in C3H mice remained intact and were not infiltrated. NOD female mice, which are most prone to spontaneous and CP-induced diabetes, exhibited the most unusual lymphoid kinetics after treatment with CP. Their thymus and spleen showed the least relative drop in total cell number and the most rapid rate of recovery. The thymus of these mice was also found to have an increased proportion of CD3+ thymocytes while CD4/CD8 double positive thymocytes decreased 7 d after CP. At 14 d after CP the number of IL-2R+ thymocytes had surpassed that of normal levels. The most dramatic observation was the rapid recovery and overshoot in the number of pancreatic lymph node cells of female NOD mice which coincided with aggressive insulitis.

Lymphocyte subsets in thymus and peripheral lymphoid tissues of aging and diabetic NOD mice.

The nonobese diabetic (NOD) mouse spontaneously develops insulin dependent diabetes mellitus. The disease is associated with a leucocytic infiltration of the pancreatic islets of Langerhans and it is believed that during the development of autoimmune diabetes, the insulin-secreting islet beta-cells are destroyed by autoreactive T lymphocytes. We investigated the alteration of lymphocyte subsets in central and peripheral lymphoid organs of NOD female mice with increasing age beginning before the onset of insulitis and ending well after the onset of diabetes. The spleen, inguinal and pancreatic lymph nodes all increased in cell number, especially after the onset of insulitis (8 weeks), and all decreased after the onset of diabetes. Flow cytometric studies showed a widening of the visible side scatter profile of female NOD lymph node cells which coincided with the initiation of insulitis. Anti-CD4 and anti-CD8 double staining of thymocytes revealed a large increase in the double negative population and a corresponding decrease in the double positive population, but this occurred long after the onset of diabetes. Generally, there was an increase in the CD4:CD8 ratio in the peripheral lymphoid organs during the onset of insulitis which was largely due to an increase in the CD4 T cell population while the ratio decreased after the onset of diabetes. In the spleen this was mostly due to an increase in CD8 T cells. The pancreatic lymph nodes, which theoretically might reflect what is happening in the pancreas, showed an unexpected decrease in overall cell number and a decrease in T-cells (especially CD4 T cells), while B cells were increased.(ABSTRACT TRUNCATED AT 250 WORDS)