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TNF blockade constitutes an effective therapy for inflammatory bowel disease, yet increases the risk for infection, including active tuberculosis. The DECTIN2 family C-type lectin receptors MINCLE, MCL, and DECTIN2 sense mycobacterial ligands and activate myeloid cells. In mice, upregulation of DECTIN2 family C-type lectin receptor after stimulation with Mycobacterium bovis Bacille Calmette-Guérin requires TNF. Here, we investigated whether TNF controls inducible C-type lectin receptor expression in human myeloid cells. Monocyte-derived macrophages were stimulated with Bacille Calmette-Guérin and the TLR4 ligand lipopolysaccharide, and expression of C-type lectin receptor was analyzed. Bacille Calmette-Guérin and lipopolysaccharide strongly upregulated messenger RNA expression of DECTIN2 family C-type lectin receptor but not of DECTIN1. Bacille Calmette-Guérin and lipopolysaccharide also induced robust production of TNF. Recombinant TNF was sufficient to upregulate expression of DECTIN2 family C-type lectin receptor. Blocking TNF with the TNFR2-Fc fusion protein etanercept abrogated, as expected, the effect of recombinant TNF and impaired induction of DECTIN2 family C-type lectin receptor by Bacille Calmette-Guérin and lipopolysaccharide. Flow cytometry confirmed upregulation of MCL at the protein level by recombinant TNF and showed inhibition of Bacille Calmette-Guérin–induced MCL by etanercept. To investigate the impact of TNF on C-type lectin receptor expression in vivo, we analyzed peripheral blood mononuclear cells of patients with inflammatory bowel disease and observed downregulation of MINCLE and MCL expression after therapeutic TNF blockade. Together, TNF is sufficient to upregulate DECTIN2 family C-type lectin receptor in human myeloid cells and contributes to this process after encounter with Bacille Calmette-Guérin or lipopolysaccharide. Impaired C-type lectin receptor expression in patients receiving TNF blockade may dampen the sensing of microbes and defense against infection.

Objectives The physiological number and distribution of mast cells (MCs) in the pediatric gastrointestinal (GI) tract is not well defined and reference values of normality are missing. To define a physiological and disease defining cut-off, a systematic histological exploration of MC distribution from the esophagus to the rectum in healthy as well as in patients with gastrointestinal food allergies (GFA) was performed. Methods Nine pediatric subjects that exhibited unremarkable histopathological evaluations or underwent endoscopy for surveillance reasons after a previous polypectomy of single colonic juvenile polyps served as reference cohort. In all of these subjects a chronic inflammatory disease (e.g. inflammatory bowel disease, celiac disease) or allergy was excluded. In addition, a group of 15 patients with gastrointestinal complaints suspected to be caused by a GFA were investigated. Immunohistochemistry was performed from all biopsies using CD117 (c-Kit) as a reliable marker to identify MCs in the lamina propria. Results There were distinct differences of MC counts in all parts of the pediatric GI tract. The highest counts of MCs in both symptomatic patients and control cohort, were found in the duodenum, terminal ileum, cecum and ascending colon. The lowest counts were found in the esophagus. Significant disparities between GFA and healthy subjects were found in the gastric corpus (22.1 ± 4.0 /HPF vs. 32.0 ± 10.1 /HPF; p = 0.034) and ascending colon (44.8 ± 10.4 /HPF vs. 60.4 ± 24.3 /HPF; p = 0.047). Conclusions Mucosal MC counts in the pediatric GI tract are higher than previously reported, with a considerable overlap between healthy and GFA patients. These results provide detailed information on distribution and numbers of MCs in pediatric allergic patients while allowing estimates of physiological values in childhood for the first time. With regard to diagnostic procedures in GFA further laboratory parameters have to be integrated.

Kidney transplantation in mice is a complicated and challenging surgery procedure. There are very few publications demonstrating the key steps of this operation. Therefore, this article introduces the technique and points out the surgical caveats associated with this operation. In addition, important modifications in comparison to the conventional procedure are demonstrated. Firstly, a patch of the abdominal aorta is cut and prepared so that the proximal bifurcations of the renal artery, including the ureteral artery are transected together with the donor kidney en bloc. This reduces the risk of a ureter necrosis and avoids the development of a urinary tract occlusion. Secondly, a new method of the vascular anastomosis is demonstrated that allows the operator to flexibly increase or decrease the size of the anastomosis after renal transplant reperfusion has already been initiated. This avoids the development of vessel strictures and intraabdominal bleeding. Thirdly, a technique that enables the anastomosis of the delicate donor ureter and the recipient bladder that does not cause a trauma is shown. Adopting this protocol can shorten the operation time and reduces the damage to the recipient's bladder, thereby significantly increasing the operation success rate for the recipient mice.

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the established technique for a modified blood circuit reconstruction in a heterotopic abdominal heart transplantation model is presented. This method uses the intrathoracic inferior vena cava (IIVC) instead of the pulmonary artery of the donor heart for the anastomosis to the inferior vena cava of the recipient. It is facilitating and improving success rates for abdominal heart transplantation in mice.

Allograft-specific regulatory T cells (Trₑg cells) are crucial for long-term graft acceptance after transplantation. Although adoptive Trₑg cell transfer has been proposed, major challenges include graft-specificity and stability. Thus, there is an unmet need for the direct induction of graft-specific Trₑg cells. We hypothesized a synergism of the immunotolerogenic effects of rapamycin (mTOR inhibition) and plerixafor (CXCR4 antagonist) for Trₑg cell induction. Thus, we performed fully-mismatched heart transplantations and found combination treatment to result in prolonged allograft survival. Moreover, fibrosis and myocyte lesions were reduced. Although less CD3⁺ T cell infiltrated, higher Trₑg cell numbers were observed. Noteworthy, this was accompanied by a plerixafor-dependent plasmacytoid dendritic cells-(pDCs)-mobilization. Furthermore, in vivo pDC-depletion abrogated the plerixafor-mediated Trₑg cell number increase and reduced allograft survival. Our pharmacological approach allowed to increase Trₑg cell numbers due to pDC-mediated immune regulation. Therefore pDCs can be an attractive immunotherapeutic target in addition to plerixafor treatment. CA extern

BackgroundThe role of B cells in inflammatory bowel disease (IBD) is ambiguous, as B cells may have both pathogenic and protective functions in IBD. We studied B cell subsets before and after initiation of an anti-tumor necrosis factor alpha (anti-TNFα) therapy in pediatric IBD. The aim of the study was to examine the behavior of B cells in pediatric IBD patients undergoing an anti-TNFα therapy and, more specifically, to clarify their association with a successful or an unsuccessful infliximab (IFX) treatment.MethodsA total of N = 42 pediatric IBD patients (Crohn disease, n = 30; ulcerative colitis, n = 12) for whom an anti-TNFα therapy with and without a concomitant azathioprine (AZA) medication was administered were recruited. Fourteen healthy age-matched children served as control patients. Blood samples were collected before initiation of the anti-TNFα therapy, before the fourth infusion at the end of the induction phase, and after 6 and 12 months under therapy maintenance. Flow cytometry (CD20, CD27, CD38, CD138) and intracellular staining (interleukin 10 [IL10], TNFα, granzyme B) were performed. Responders to successful IFX therapy were classified exhibiting a fecal calprotectin level of below 100 µg/g or achieving levels of ResultsBefore initiation of anti-TNFα therapy, flow cytometry revealed increased percentages of naïve B cells whereas transitional B cells were reduced compared with those in the healthy control patients. The IL10-producing B cells of both ulcerative colitis and Crohn disease patients were reduced at the initiation of IFX therapy, whereas TNFα-producing transitional CD24hiCD38hi B cells in ulcerative colitis patients were increased compared with those in healthy control patients. After 12 months of therapy, we detected a significant increase of IL10-producing transitional B cells in responding patients.The IFX trough levels in the responding patients showed a significant increase until 6 months after IFX initiation, attaining mean values of 9.9 µg/mL, whereas the IFX dosage was significantly lower than that in the nonresponding patients. The IFX trough levels in AZA-treated patients reached earlier therapeutic levels than in patients without AZA comedication, whereas during the course of the IFX therapy, comedication with AZA had no significant effect on the outcome.ConclusionsAttaining a normalization of IL10 production among CD24hiCD38hi B cells after 12 months of therapy may represent additional information about the reconstitution of a patient’s immune system in responding patients. The achievement of an IFX trough level of ~10 µg/mL at 6 months of treatment is associated with a successful anti-TNFα therapy. In addition, AZA comedication supports an earlier achievement of therapeutic IFX trough levels.

Intrauterine growth restriction (IUGR) is associated with an increased risk of renal diseases in adulthood. However, while low-birth-weight-infants often undergo accelerated postnatal growth, the impact of postnatal environmental factors such as nutrition and early postnatal stressors on renal development and function remains unclear. In this context, Neuropeptide Y (NPY) may act as a critical factor. NPY is a sympathetic coneurotransmitter involved in blood pressure regulation and tubular function. Yet, little is known about the expression and function of endogenous NPY in the kidney and the functional relevance for the transmission of persistent postnatal-induced effects.(1) IUGR was induced in Wistar rats by isocaloric protein restriction in pregnant dams. (2) Litter size was reduced to 6 (LSR6) or 10 (LSR10) male neonates. To differentiate the effect of postnatal nutrition and stressors, we additionally included home-cage-control animals without any postnatal manipulation. Animals were sacrificed at Day 70.Litter size reduction (LSR) to 6 but not IUGR increased messenger RNA expression of endogenous NPY and down-regulated the NPY-receptors Y1 and Y2. Furthermore, dipeptidylpeptidase IV (DPPIV)--an enzyme that cleaves NPY--was decreased after LSR. Expression and the phosphorylation of mitogen-activated protein kinase 42/44 (intracellular signalling pathway of the receptor Y1) were altered. An impaired renal function with pronounced kaliuresis and natriuresis was observed at Day 70 after LSR.Postnatal nutrition and stressors such as LSR lead to dysregulated signalling of NPY. These data demonstrate that factors in the early postnatal environment exert important changes in the tubular function, which may predispose to corresponding pathology.

Objective Leptin and its receptor (Ob-R) are co-expressed in human placenta suggesting auto- and paracrine mechanisms of the hormone. So far it is unclear, how changes in the placental environment affect Ob-R expression. Hence, the main purpose of the study was to investigate leptin receptor expression and regulation under hypoxic conditions. The influences of hypoxia and leptin on signal transduction and cell proliferation in chorioncarcinoma cell lines as well as primary villous trophoblasts were determined. Results We found a time-dependent induction of leptin receptor mRNA and protein in placental cells under hypoxic conditions. In contrast, soluble leptin receptor expression did not change under oxygen deprivation. Leptin treatment neither activated the p42/p44 nor the STAT3 pathway in placental cells, being independent of hypoxic or normoxic conditions. Furthermore, leptin added to the culture medium in high concentrations was unable to interfere with the rate of proliferation. Conclusion Our data demonstrate that hypoxia leads to an increase of Ob-R expression in placental cells. Interestingly, leptin-dependent signal transduction and proliferation remained unaffected. A possible role of the soluble leptin receptor in modulating free leptin levels will be discussed.

Leptin is a circulating hormone that is secreted primarily by adipose tissue. However, recent studies have demonstrated leptin production by other tissues, including placenta, stomach, kidney, liver, and lung, a process not only activated by stimuli such as insulin or corticosteroids, but also by hypoxia, which is mediated by the hypoxia inducible factor-1. In contrast to this fact, smokers have lower plasma leptin levels. The purpose of this study was to determine whether tissue hypoxygenation [induced by lack of oxygen] or inhalation of carbon monoxide (CO) are sufficient to up-regulate leptin in fat cells as well as in peripheral organs such as lung, liver, and kidney of rats. In hypoxic rats, leptin expression was unchanged or even reduced in adipose tissue. In contrast, in liver, kidney, and lung we observed an increase in leptin expression compared with normoxic controls, whereas plasma levels were unchanged. When animals were exposed to CO, generating a functional anemia known to activate the HIF-1-dependent transcription, a significant decrease in leptin gene expression in adipose tissue and in all organs tested was observed. Plasma leptin concentrations after CO exposure were significantly diminished compared with those in control animals. These findings suggest that tissue hypoxygenation up-regulates leptin expression in nonadipose tissue. However, this is not sufficient to raise plasma leptin levels in rats. Inhalation of CO leads to a significant decrease in leptin mRNA and protein concentration in the plasma of the animals, suggesting a negative effect of CO on leptin transcription.

Objective Hypoxia and insulin are known key players in the activation leptin transcription and translation in vivo and in vitro . These insulin- and hypoxia-dependent effects are leptin transcription are mediated via independent elements on the leptin-promotor, even more coincubation of the two stimuli in vitro results in a supraadditive effect on leptin transcription. The aim of this study was to examine whether hyperinsulinemia is able to interfere with the hypoxia-driven expression of leptin in adipose and extra-adipose tissue in vivo . Research methods and procedures We used the KK/HlJ mouse strain as a model for hyperinsulinemia and C57BL/6J mice as control. These two groups were exposed to hypoxia for 12 h. Serum levels of insulin and leptin were analyzed by ELISA, mRNA expression of leptin was measured via real-time PCR. Results In the hyperinsulinemic KK/HlJ mice, hypoxia was not able to further increase the amount of leptin in serum. Instead, a significant decrease of insulin levels was detected, while serum leptin and insulin levels increased in C57BL/6J. Analysis of leptin mRNA expression in subcutaneous fat, mesenteric fat and kidney revealed that hypoxia induces leptin transcription in kidneys of C57/BL6 but not in hyperinsulinemic animals. In contrast, leptin expression in adipose tissue was not increased during hypoxia. Discussion We conclude that leptin regulation during hypoxia in vivo depends at least in part on the modulating role of insulin. The hypoxia driven induction of insulin expression in C57/BL6 animals may be responsible for the stimulation of leptin transcription. In contrast, already hyperinsulinemic animals showed no induction — neither of insulin nor leptin after short-term hypoxia.

Leptin is a regulator on placenta and conceptus during pregnancy. Hyperinsulinism and hypoxia induce partially overlapping pathophysiological disturbances during pregnancy. As insulin and hypoxia are known inducers of leptin secretion, we asked whether these two stimuli have synergistic effects. By analyzing mRNA levels of leptin after stimulation of BeWo cells with insulin in the presence or absence of oxygen, we found a supraadditive effect when incubating hypoxic cells with insulin. As shown by Western-blot of hypoxia-inducible-factor-1 alpha (HIF-1 alpha), the additive effects of these stimuli were not mediated by an increased stabilization of the HIF-complex. We therefore asked what elements of the leptin promoter are responsible for these effects. When deleting a 0.6 kb fragment of the cloned leptin promoter, a so far unknown loss of insulin-dependent activation of transcription, as well as a loss of the supraadditive effect of insulin and hypoxia could be observed. These results provide strong evidence that insulin and hypoxia act as agonists on the human leptin transcription but on two different regulatory elements.

The hypoxia-inducible factor 1α (HIF-1α), a member of the PAS superfamily, is a global regulator of cellular and systemic O2 homeostasis as well as embryonic development. As the activity of HIF-1α is increased by a lowered oxygen tension in vivo and in vitro, we established a cell line producing high amounts of HIF-1α under normoxic conditions. As this overexpression was inducible by doxycycline, we can now provide a system to study HIF-1α-dependent gene regulation under normoxic as well as hypoxic conditions. We were able to show that the doxycycline-induced induction of the target gene HIF-1α – followed by the message of its target genes erythropoietin and vascular endothelial growth factor – is a dose- and time-dependent process. As the inducible overexpression of HIF-1α did not increase the rate of apoptosis, it provides a helpful new tool in drug discovery and tumor research to differentiate between hypoxia-dependent and hypoxia-independent pathways during HIF-1α-dependent gene regulation and HIF-1α-dependent effects on apoptosis.