Induced regulatory T cells suppress Tc1 cells through TGF-β signaling to ameliorate STZ-induced type 1 diabetes mellitus

Type 1 diabetes mellitus (T1D) is a chronic autoimmune condition in which the immune system destroys insulin-producing pancreatic β cells. In addition to well-established pathogenic effector T cells, regulatory T cells (Tregs) have also been shown to be defective in T1D. Thus, an increasing number of therapeutic approaches are being developed to target Tregs. However, the role and mechanisms of TGF-β-induced Tregs (iTregs) in T1D remain poorly understood. Here, using a streptozotocin (STZ)-induced preclinical T1D mouse model, we found that iTregs could ameliorate the development of T1D and preserve β cell function. The preventive effect was associated with the inhibition of type 1 cytotoxic T (Tc1) cell function and rebalancing the Treg/Tc1 cell ratio in recipients. Furthermore, we showed that the underlying mechanisms were due to the TGF-β-mediated combinatorial actions of mTOR and TCF1. In addition to the preventive role, the therapeutic effects of iTregs on the established STZ-T1D and nonobese diabetic (NOD) mouse models were tested, which revealed improved β cell function. Our findings therefore provide key new insights into the basic mechanisms involved in the therapeutic role of iTregs in T1D.     

Histophysiological effects of fluid resuscitation on heart, lung and brain tissues in rats with hypovolemia

The efficacy of using colloids and crystalloids in the treatment of hypovolemia still remains controversial. An important aspect in treating hypovolemia is to re-establish normal tissue hemodynamics after fluid resuscitation. Production of nitric oxide (NO) or growth factors such as transforming growth factor beta (TGF-beta) has been identified as a key mechanism in physiological and pathological processes in the different systems. This study was designed to investigate the histophysiological effects of resuscitation with different plasma substitutes on the heart, lung and brain tissues following acute blood loss in male Sprague-Dawley rats weighing 250-280g (n=30). After anesthesia with sodium pentobarbital, the left femoral vein and artery were cannulated for the administration of volume expanders and for direct measurement of arterial pressure and heart rate. Twenty rats were bled (5ml/10min) and infused (5ml/10min) with one of four randomly selected solutions, (a) human albumin, (b) gelatin (Gelofusine), (c) dextran-70 (Macrodex); or (d) physiological saline (0.9% isotonic saline). Five control rats were bled without infusion. Tissue samples were taken and fixed in 10% formalin solution, then processed for embedding in paraffin wax. Sections were cut and stained with hematoxylin and eosin. Indirect immunohistochemical labelling was performed to reveal binding of primary antibodies against endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and TGF-beta. Mild immunoreactivity of eNOS was observed in endothelial cells of vessels in brain, heart and lung tissues. Increased immunoreactivities of eNOS, iNOS and TGF-beta were observed in the non-fluid resuscitated group in these organs; mild, moderate, moderate and strong immunoreactivities were seen in the albumin, gelatin, physiological saline and dextran-70 treated groups, respectively. Immunoreactivities of iNOS and TGF-beta in the non-fluid resuscitated group were increased significantly, in comparison to the other groups, apart from the dextran-70 treated group. The results of this study show that gelatin solution and physiological saline may be of use after acute blood loss, and dextran-70 is not the preferred resuscitation fluid in the early stages of acute blood loss. It was concluded that albumin solution is the preferred fluid for resuscitation.     

依那普利对自发性高血压大鼠心肌ACE和ACE2表达的影响

 目的 观察自发性高血压大鼠(spontaneously hypertensive rats, SHR)心肌的血管紧张素转换酶(angiotensin-converting enzyme, ACE)和ACE2的表达,以及依那普利干预的影响。方法 将15只SHR随机分为2组：SHR对照组(n=7)和依那普利组(n=8),分别给以安慰剂、依那普利15mg.kg-1.d-1灌胃干预4周。干预结束后处死大鼠,分离左心室,行RT-PCR、western blot蛋白质免疫印迹检测。同步取10只WKY大鼠作为正常血压对照组。结果SHR心肌的ACE的mRNA和蛋白质的表达都显著高于)WKY组(1.68±0.34 vs 0.33±0.12, P＜0.05;1.21±0.14 vs 0.71±0.11, P＜0.05),而ACE2 的mRNA和蛋白质表达皆明显低于WKY组(0.50±0.15 vs 1.16±0.24, P＜0.05; 0.71±0.24 vs 1.22±0.14, P＜0.05)。依那普利明显降低ACE的mRNA和蛋白质表达(0.44±0.19 vs 1.68±0.34, P＜0.01; 0.87±0.13 vs 1.21±0.14, P＜0.05),提升ACE2的mRNA表达(1.77±0.49 vs 0.50±0.15, P＜0.05),对ACE2的蛋白表达无明显影响(0.42±0.22 vs 0.71±0.24, P＞0.05)。结论 SHR心肌ACE明显升高,ACE2显著降低,有利于血压上调。依那普利能降低ACE,提升ACE2,可能是血管紧张素转换酶抑制剂(angiotensin-converting enzyme inhibitors, ACEI)的降压机制之一。     

Mechanisms of Apoptosis of T-Cells in Human Tuberculosis

The role of TGF-β TNF-α FasL and Bcl-2 in apoptosis of CD4 T-cells during active TB was studied. Coculture of PBMC from TB patients with neutralizing antibodies to TGF-β or TNF-α decreased spontaneous (P ≤ 0.05) and MTB-induced (P≤ 0.02) T-cell apoptosis by 50–90%, but effects were not additive. Interestingly, only levels of TGF-β in supernatants correlated with rates of spontaneous and MTB-induced apoptosis. FasL surface and mRNA expression were higher in unstimulated and MTB-stimulated PBMC from patients than controls, and neutralization of FasL abrogated apoptosis of T-cells from patients only. Intracellular Bcl-2 protein was lower among unstimulated CD4 T-cells from patients than those from controls (P ≤ 0.02), and MTB stimulation reduced intracellular Bcl-2 content in CD4 T-cells from patients only (P ≤ 0.001). These findings may indicate that, during TB, predisposition of CD4 T-cells to apoptosis may involve both low expression of Bcl-2, and excessive expression of TGF-β TNF-α and FasL.     

Spontaneous Abortion in Immunodeficient SCID Mice

PROBLEM: Immunodeficient SCID mice on the CB-17 have been used to test the role of “rejection” in a xenogeneic blastocyst transfer model of recurrent miscarriage, but interpretation of the data requires knowing syngeneic within-species matings have a high success rate and do not require immunotrophic factors expected only in immunocompetent non-T-cell deficient mice. METHOD: Resorption rates were studied in a SCID CB-17 barrier facility that provided the mice used to test the role of immunology in the resorption model. RESULTS: Spontaneous resorption in syngeneically mated immunodeficient SCID mice on the CB-17 background occurred at an unexpectedly high rate and could not be prevented by treatment with anti-asialo GM1 antibody or GM-CSF, both of which are effective in ameliorating abortion in DBA/2J-mated CBA/J mice. Immunocompetent CB-17 +/+ mice showed an even higher rate of loss. The latter was also not affected by treatment with anti-asialo GM1 antibody or by GM-CSF and was not prevented by tetracycline (which is effective in the DBA/2-CBA/J system) or progesterone treatment. Mating experiments showed a scid/+ × scid//+ cross gave the highest rate of loss, and it appeared that the presence of +/+-type embryos in the uterus could be augmenting abortion with selective discrimination against scid/scid embryos. High abortion rates were associated both with appearance of a coagulase-negative Staphylococcus sp. in feces and with loss of one component of the SPF flora. Decidual tissue from mated CB-17 +/+ mice showed premature release of TNF-cc in absence of TGF-β2-related suppressor activity, and vascular lesions (fibrinoid necrosis), varying in extent, were associated with both scid/scid × scid/scid and +/+ × +/+ pregnancies. TNF-α also appeared prematurely in pregnant scid/scid mice, but the levels were lower (and areas of necrosis smaller than in +/+ × +/+ pregnancies). Outcrossing onto a C57B1/6 background dramatically reduced the abortion rate, indicating an important genetic effect on susceptibility with heterogeneity protecting against abortion. CONCLUSIONS: SCID mice on the CB-17 background do not have a high rate of successful syngeneic pregnancies, and a TNF-α induced vasculopathy may be responsible. Abortion was not caused by immunodeficiency leading to loss of immunotrophism because immunocompetent non-SCID CB-17 mice had a higher rate of loss. Factors augmenting the abortion rate included the presence of embryos of the +/+ genotype in the uterus and treatment with anti-asialo GM1 antibody. Abortion rates were not reduced by treatments effective in the DBA/2-mated CBA/J mouse model but were reduced by re-establishing a new colony with defined flora (a temporary effect) and by outcrossing mice with a different (C57B1/6) background. Together, the data suggest an infectious trigger (identity uncertain) of the vasculopathy and an important genetic influence on susceptibility with heterozygosity and a SCID mouse mutation providing against abortion a degree of protection.     

Potential antidiabetic activity of benzimidazole derivative albendazole and lansoprazole drugs in different doses in experimental type 2 diabetic rats

Background/aim The aim of this study is to determine the effects of different concentrations of albendazole and lansoprazole, which were benzimidazole derivatives, on endocrinologic and biochemical parameters in experimental type 2 diabetic (T2D) rats. Materials and methods In this study, 46 male Wistar Albino rats were used. Animals were divided as healthy control (0.1 mL/rat/day saline, s.c, n = 6), diabetes control (0.1 mL/rat/day saline, s.c, n = 8), diabetes+low-dose albendazole (5 mg/kg, oral, n = 8), diabetes+high-dose albendazole (10 mg/kg, oral n = 8), diabetes+low-dose lansoprazole (15 mg/kg, subcutaneous, n = 8), and diabetes+high-dose lansoprazole (30 mg/kg, subcutaneous, n = 8). All groups were treated for 8 weeks. The blood samples were analyzed by autoanalyzer and ELISA kits for biochemical and endocrinological parameters, respectively. Results Glucose, HbA1c, triglyceride, low density cholesterol (LDL), leptin, and Homeostatic Model Assessment for insulin resistance (HOMA-IR) levels increased and insulin and HOMA-β levels decreased in the diabetic rats compared to the healthy control group. The glucose, HbA1c, and triglyceride levels were partially decreased; however, insulin and HOMA-β levels were increased by low-dose albendazole therapy. The high dose of lansoprazole treatment increased insulin level. Conclusion The lansoprazole and albendazole treatments can be a potential drug or combined with antidiabetic drugs in T2D treatment by Adenosine 5 ′ -monophosphate activated protein kinase (AMPK), peroxisome proliferator-activated receptor (PPAR), incretin-like effect and other antidiabetic mechanisms. It may be beneficial to create an effective treatment strategy by developing more specific substances with benzimidazole scaffold.     

Delayed effects of enalapril on structural characteristics of the glomerular apparatus in the kidneys of NISAG rats

Structural characteristics of renal glomeruli were studied in adult hypertensive NISAG rats (hereditary stress-induced hypertension) receiving antihypertensive drug enalapril, an inhibitor of angiotensin-converting enzyme, on days 28–58 of life. Treatment with enalapril (25 mg/kg perorally) in the early period of postnatal ontogeny produced delayed hypotensive and nephroprotective effects. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 141, No. 2, pp. 124–127, February, 2006     

Fibrosis of the lung and other tissues: new concepts in pathogenesis and treatment

Tissue fibrosis can lead to significant organ dysfunction and resulting patient morbidity and mortality. Unfortunately, the therapeutic repertoire is currently limited, nonspecific, and largely ineffective. While the pathogenesis is incompletely understood, evidence is accumulating that immune and cytokine mediated mechanisms are critical. In this review, data will be provided to support the role of Type 2 cytokines in the pathogenesis of fibrosis. The importance of the role of the pro-fibrogenic cytokine TGF-beta and CD40-CD40 ligand mediated fibroblast activation will also be evaluated. Finally, novel therapeutic options based on inhibiting these pathways will be described.     

Effects of vitamin D on renal fibrosis in diabetic nephropathy model rats

This study is to investigate the effects of vitamin D on renal fibrosis in rat diabetic nephropathy models, as well as the changes and interactions in the expressions of renal fibrogenesis- and inflammation-related genes. Rat diabetic nephropathy models were established by high-fat diets, which were subjected to TGF-β1 manipulation, as well as vitamin D treatment. H&E staining, Masson staining, and TEM detection were performed to assess the effects of vitamin D treatment and/or TGF-β1 manipulation on pathological changes in the renal tissues in these rat diabetic nephropathy models. Immunohistology and real-time PCR were used to evaluate the expressions of TGF-β1, MCP-1, CTGF, and VDR. Histological staining and TEM detection showed that, in both TGF-β1 over-expressed and interfered groups, vitamin D administration alleviated the renal fibrosis, compared with the vehicle treatment. Similar results were observed with the immunohistological staining. Real-time PCR analysis indicated that, when TGF-β1 was over-expressed in diabetic nephropathy, the expressions of MCP-1 and CTGF were also up-regulated, which would be decreased by the treatment of vitamin D. On the other hand, when TGF-β1 was interfered in DN, the expressions of MCP-1 and CTGF were relatively down-regulated, which would be further lowered by vitamin D administration. The mRNA expression of VDR was elevated by vitamin D treatment in these diabetic nephropathy models. Active vitamin D₃ and lentivirus-mediated TGF-β1 interference could effectively reduce the renal fibrosis and protect the renal function in diabetic nephropathy rat models, which makes a promising therapeutic strategy for the disease.     

Estrogen's effects on central and circulating immune cells vary with reproductive age

Previous work from this lab has shown that estrogen attenuates inflammatory cytokine production following brain lesions in young adult female rats, but not in older, reproductive senescent females. The present study was designed to elucidate whether these effects result from estrogen's actions on brain-resident immune cells (microglia) or on circulating immune cells recruited to the brain from blood. Microglia, harvested from the olfactory bulbs of ovariectomized young adult and reproductive senescent animals, were pretreated with 17beta-estradiol and subsequently with the bacterial endotoxin LPS. LPS treatment significantly increased the pro-inflammatory cytokine IL-1beta in microglial cultures harvested from young and senescent females, but estrogen treatment had no effect on cytokine expression in either group. In young adult-derived microglia, LPS treatment also increased nitric oxide (NO), which was attenuated by estrogen, and MMP-9, which was not affected by estrogen. Reproductive senescent-derived microglia cultures had higher basal expression of NO and MMP-9 activity as compared to those from young adult microglial cultures, although LPS did not further stimulate these inflammatory markers. In blood cultures, LPS stimulated a dose-dependent increase in the inflammatory cytokine TNF-alpha expression in both young adult and reproductive senescent animals. Estrogen replacement significantly attenuated TNF-alpha induction by LPS in blood cultures derived from young adult females. Paradoxically, estrogen replacement increased LPS-induced TNF-alpha expression in blood cultures derived from reproductive senescent animals as compared to age-matched controls. The age and estrogen dependent effects on circulating immune cells found in whole blood cultures closely mimic the effects of estrogen on cytokine expression in the young and senescent animals that we reported in vivo, supporting the hypothesis that the immunosuppressive actions of estrogen replacement on neural injury may result from hormone-action on circulating immune cells.     

Human intestinal epithelial cells down-regulate IL-8 expression in human intestinal microvascular endothelial cells; role of transforming growth factor-beta 1 (TGF-β1)

Cytokines produced from intestinal epithelial cells may function as signals to neighbouring immune cells. In the present study we analysed the effects of colonic epithelial cell lines (HT-29, Caco-2, HCT-116, Colo-320) and freshly isolated intestinal epithelial cells on IL-8 expression in the SV-40T transfected human microvascular endothelial cell line (HMEC-1). Epithelial cell-conditioned media and transwells preventing physical contact between epithelial and endothelial cells were used. TGF-β1 and IL-8 levels were determined by ELISA and Northern blot analysis. Increasing concentrations of IL-1β led to increasing production of IL-8. The addition of epithelial cell-conditioned medium or epithelial cells to HMEC-1 cells in a two-compartment co-culture system resulted in a strong decrease in IL-8 at the protein and mRNA level. Decrease of IL-8 was markedly stronger when epithelial cells were co-cultured in contact with HMEC-1 cells, indicating that not only soluble factor(s) play a role in the induction of IL-8 suppression in HMEC-1 cells. MoAbs against TGF-β1 partially inhibited down-regulation of endothelial IL-8 expression. In further studies, IL-8 expression in freshly isolated human intestinal microvascular endothelial cells (HIMEC) was also down-regulated by intestinal epithelial cells. Our results demonstrate that intestinal epithelial cells down-regulate IL-8 expression in HMEC-1 cells. TGF-β1 is a candidate factor of epithelial–endothelial communication in the colonic mucosa.     

Differential effects of low and high-dose estradiol on airway reactivity in ovariectomized rats

The aim of this animal study was to test the hypothesis that low and high doses of 17beta-estradiol (E2) may differentially influence airway responsiveness. Ovariectomized female rats received either placebo or E2 (10 or 100 microg/kg per day) for 21 days. The concentration of inhaled acetylcholine (ACh) required to double pulmonary resistance (EC200 RL) was calculated as the in vivo index of airway responsiveness. Ex vivo airway responsiveness was evaluated by the cumulative concentration-response curve (CCRC) of isolated tracheal segments. Rats treated with low-dose E2 were less responsive to ACh than rats given either placebo or high-dose E2 (P=0.003). Ex vivo, low-dose E2 treatment decreased (P=0.01) and high-dose E2 increased the potency of ACh (P<0.001) compared to placebo. E2 treatment did not alter smooth muscle cross-sectional area or epithelium thickness. Accumulation of liquid within the tracheal mucosa was moderately enhanced by high-dose E2 treatment compared with animals given either placebo or low-dose E2 (P=0.03). We conclude that E2 treatment has differential, dose-dependent effects on airway responsiveness to acetylcholine.     

Effect of peptide Vilon on the content of transforming growth factor-β and permeability of microvessels during experimental chronic renal failure

We studied the effect of Vilon in rats 2, 4, and 6 months after the onset of chronic renal failure. Subcutaneous injection of Vilon significantly decreased serum concentration of transforming growth factor-β1 and permeability of mesenteric microvessels in rats 2 months after the onset of chronic renal failure. Our results indicate that the preparation produces a potent homeostatic effect in the early period of chronic renal failure.__________This revised version was published online in August 2005 with the addition of the issue titleTranslated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 139, No. 1, pp. 28–31, January, 2005     

Transforming growth factor-beta-mediated regulation of BK virus gene expression

The increasing prevalence of BK virus (BKV)-associated diseases in immunosuppressed patients has prompted an investigation of the immune response to BKV, especially the role of cytokines in regulating viral replication. We examined the effect of TGF-beta, a cytokine that is stimulated by certain immunosuppressive therapies, on BKV gene expression during lytic infection of renal proximal tubule epithelial cells. Viral gene expression, and specifically the activity of the BKV early promoter, is regulated by TGF-beta in a strain-dependent manner. Promoter activity is upregulated in the presence of TGF-beta for the TU strain of BKV, and not for the Dik, Dunlop, or Proto-2 strains. Using site-directed mutagenesis, we have identified a small segment of the TU promoter that is required for stimulation in response to TGF-beta. These results demonstrate that BKV strains can respond differently to cytokine treatment and suggest that TGF-beta may play a role in the reactivation of BKV.     

Connective tissue growth factor expression is regulated by histamine in lung fibroblasts: potential role of histamine in airway remodeling

BACKGROUND: In the inflamed lung of allergic asthma, an aberrant injury-repair response is accompanied by structural changes in the airway, known as airway remodeling. TGF-beta and its downstream mediator connective tissue growth factor (CTGF) are playing a key role in these processes, resulting in irreversible airway remodelling. OBJECTIVE: As histamine is a key mediator of allergic reactions, we investigated whether histamine is involved in airway remodeling. METHODS: The effect of histamine and TGF-beta1 on proliferation of lung fibroblast cells IMR-90 was studied by [(3)H]-thymidine proliferation assay. The regulation of CTGF by histamine and TGF-beta1 in lung fibroblasts was analyzed by RT-PCR, real-time PCR, Western blot analysis, and promoter analysis and characterized by specific histamine-receptor antagonists. RESULTS: Histamine and TGF-beta1 enhanced proliferation of lung fibroblast cells IMR-90. Both induced CTGF mRNA and protein expression with different time kinetics. Whereas TGF-beta1 induced maximal CTGF expression after 12 hours (347% +/- 23%), histamine-induced maximal CTGF expression was lower and delayed (maximum expression of 204% +/- 11% after 48 hours). Histamine and TGF-beta1 stimulated the CTGF promoter and the TGF-beta-response element in the CTGF promoter. The histamine-induced CTGF expression was mediated through the histamine receptor (HR1) and could be completely abolished by TNF-alpha. CONCLUSIONS: These findings demonstrate that histamine plays a potential role in the induction of airway remodeling mediated by the induction of lung fibroblasts proliferation and CTGF expression. CLINICAL IMPLICATIONS: This mechanism could be important for prophylactic strategies aiming at airway remodeling and could be a new indication for antihistamine treatment.