Apoptosis of Purified CD4+ T Cell Subsets Is Dominated by Cytokine Deprivation and Absence of Other Cells in New Onset Diabetic NOD Mice

BACKGROUND: Regulatory T cells (Treg) play a significant role in immune homeostasis and self-tolerance. Excessive sensitivity of isolated Treg to apoptosis has been demonstrated in NOD mice and humans suffering of type 1 diabetes, suggesting a possible role in the immune dysfunction that underlies autoimmune insulitis. In this study the sensitivity to apoptosis was measured in T cells from new onset diabetic NOD females, comparing purified subsets to mixed cultures. PRINCIPAL FINDINGS: Apoptotic cells are short lived in vivo and death occurs primarily during isolation, manipulation and culture. Excessive susceptibility of CD25(+) T cells to spontaneous apoptosis is characteristic of isolated subsets, however disappears when death is measured in mixed splenocyte cultures. In variance, CD25(-) T cells display balanced sensitivity to apoptosis under both conditions. The isolation procedure removes soluble factors, IL-2 playing a significant role in sustaining Treg viability. In addition, pro- and anti-apoptotic signals are transduced by cell-to-cell interactions: CD3 and CD28 protect CD25(+) T cells from apoptosis, and in parallel sensitize naïve effector cells to apoptosis. Treg viability is modulated both by other T cells and other subsets within mixed splenocyte cultures. Variations in sensitivity to apoptosis are often hindered by fast proliferation of viable cells, therefore cycling rates are mandatory to adequate interpretation of cell death assays. CONCLUSIONS: The sensitivity of purified Treg to apoptosis is dominated by cytokine deprivation and absence of cell-to-cell interactions, and deviate significantly from measurements in mixed populations. Balanced sensitivity of naïve/effector and regulatory T cells to apoptosis in NOD mice argues against the concept that differential susceptibility affects disease evolution and progression

Effector and Naturally Occurring Regulatory T Cells Display No Abnormalities in Activation Induced Cell Death in NOD Mice

BACKGROUND: Disturbed peripheral negative regulation might contribute to evolution of autoimmune insulitis in type 1 diabetes. This study evaluates the sensitivity of naïve/effector (Teff) and regulatory T cells (Treg) to activation-induced cell death mediated by Fas cross-linking in NOD and wild-type mice. PRINCIPAL FINDINGS: Both effector (CD25(-), FoxP3(-)) and suppressor (CD25(+), FoxP3(+)) CD4(+) T cells are negatively regulated by Fas cross-linking in mixed splenocyte populations of NOD, wild type mice and FoxP3-GFP trangeneess. Proliferation rates and sensitivity to Fas cross-linking are dissociated in Treg cells: fast cycling induced by IL-2 and CD3/CD28 stimulation improve Treg resistance to Fas-ligand (FasL) in both strains. The effector and suppressor CD4(+) subsets display balanced sensitivity to negative regulation under baseline conditions, IL-2 and CD3/CD28 stimulation, indicating that stimulation does not perturb immune homeostasis in NOD mice. Effective autocrine apoptosis of diabetogenic cells was evident from delayed onset and reduced incidence of adoptive disease transfer into NOD.SCID by CD4(+)CD25(-) T cells decorated with FasL protein. Treg resistant to Fas-mediated apoptosis retain suppressive activity in vitro. The only detectable differential response was reduced Teff proliferation and upregulation of CD25 following CD3-activation in NOD mice. CONCLUSION: These data document negative regulation of effector and suppressor cells by Fas cross-linking and dissociation between sensitivity to apoptosis and proliferation in stimulated Treg. There is no evidence that perturbed AICD in NOD mice initiates or promotes autoimmune insulitis

Prediction of seizure control in non-ketotic hyperglycemic induced seizures

<p>Abstract</p> <p>Background</p> <p>To study the factors predictive for seizure control in non-ketotic hyperglycemic induced seizures (NKHS).</p> <p>Methods</p> <p>We studied 21 patients who were clinically diagnosed as NKHS at Khon Kaen University hospital, Thailand. Multiple linear regression analysis was used to identify the factors predictive for seizure control.</p> <p>Results</p> <p>Most patients had no previous history of diabetes and presented with repetitive partial seizures. The mean number of seizure attacks was 45 times prior to admission. The average duration to terminate seizure was 36 hours and significantly predicted by frequency of seizures (estimate 0.9, p value 0.013).</p> <p>Conclusion</p> <p>Frequency of seizures is the only predictive factor for the success of seizure control in NKHS.</p

Effect of hyperbaric oxygen on mesenchymal stem cells for lumbar fusion in vivo

<p>Abstract</p> <p>Background</p> <p>Hyperbaric oxygen (HBO) therapy has been proved in improving bone healing, but its effects on mesenchymal stem cells (MSCs) <it>in vivo </it>is not clear. The aims of this study are to clarify whether the HBO therapy has the same enhancing effect on MSCs with regard to bone formation and maturation and to ascertain whether the transplanted MSCs survive in the grafted area and contribute to new bone formation.</p> <p>Methods</p> <p>Twenty-three adult rabbits underwent posterolateral fusion at L4-L5 level. The animals were divided into three groups according to the material implanted and subsequent treatment: (1) Alginate carrier (n = 6); (2) Alginate-MSCs composite (n = 11); and (3) Alginate-MSCs composite with HBO therapy (n = 6). After 12 weeks, spine fusion was examined using radiographic examination, manual testing, and histological examination. Using a PKH fluorescence labeling system, whether the transplanted MSCs survived and contributed to new bone formation in the grafted area after HBO therapy was also examined.</p> <p>Results</p> <p>The bilateral fusion areas in each animal were evaluated independently. By radiographic examination and manual palpation, union for the Alginate, Alginate-MSCs, and Alginate-MSCs-HBO groups was 0 of 12, 10 of 22, and 6 of 12 respectively. The difference between the Alginate-MSCs and Alginate-MSCs-HBO groups was not significant (P = 0.7997). The fluorescence microscopy histological analysis indicated that the transplanted PKH67-labeled MSCs survived and partly contributed to new bone formation in the grafted area.</p> <p>Conclusions</p> <p>This study demonstrated that the preconditioned MSCs could survive and yield bone formation in the grafted area. HBO therapy did not enhance the osteogenic ability of MSCs and improve the success of spine fusion in the rabbit model. Although there was no significant effect of HBO therapy on MSCs for spine fusion, the study encourages us to research a more basic approach for determining the optimal oxygen tension and pressure that are required to maintain and enhance the osteogenic ability of preconditioned MSCs. Further controlled <it>in vivo </it>and <it>in vitro </it>studies are required for achieving a better understanding of the effect of HBO treatment on MSCs.</p

CD95 co-stimulation blocks activation of naive T cells by inhibiting T cell receptor signaling

CD95 is a multifunctional receptor that induces cell death or proliferation depending on the signal, cell type, and cellular context. Here, we describe a thus far unknown function of CD95 as a silencer of T cell activation. Naive human T cells triggered by antigen-presenting cells expressing a membrane-bound form of CD95 ligand (CD95L) or stimulated by anti-CD3 and -CD28 antibodies in the presence of recombinant CD95L had reduced activation and proliferation, whereas preactivated, CD95-sensitive T cells underwent apoptosis. Triggering of CD95 during T cell priming interfered with proximal T cell receptor signaling by inhibiting the recruitment of ζ-chain–associated protein of 70 kD, phospholipase-γ, and protein kinase C-θ into lipid rafts, thereby preventing their mutual tyrosine protein phosphorylation. Subsequently, Ca2+ mobilization and nuclear translocation of transcription factors NFAT, AP1, and NF-κB were strongly reduced, leading to impaired cytokine secretion. CD95-mediated inhibition of proliferation in naive T cells could not be reverted by the addition of exogenous interleukin-2 and T cells primed by CD95 co-stimulation remained partially unresponsive upon secondary T cell stimulation. HIV infection induced CD95L expression in primary human antigeen-presenting cells, and thereby suppressed T cell activation, suggesting that CD95/CD95L-mediated silencing of T cell activation represents a novel mechanism of immune evasion

Elevated creatine kinase activity in primary hepatocellular carcinoma

BACKGROUND: Inconsistent findings have been reported on the occurrence and relevance of creatine kinase (CK) isoenzymes in mammalian liver cells. Part of this confusion might be due to induction of CK expression during metabolic and energetic stress. METHODS: The specific activities and isoenzyme patterns of CK and adenylate kinase (AdK) were analysed in pathological liver tissue of patients undergoing orthotopic liver transplantation. RESULTS: The brain-type, cytosolic BB-CK isoenzyme was detected in all liver specimens analysed. Conversely, CK activity was strongly increased and a mitochondrial CK (Mi-CK) isoenzyme was detected only in tissue samples of two primary hepatocellular carcinomas (HCCs). CONCLUSION: The findings do not support significant expression of CK in normal liver and most liver pathologies. Instead, many of the previous misconceptions in this field can be explained by interference from AdK isoenzymes. Moreover, the data suggest a possible interplay between p53 mutations, HCC, CK expression, and the growth-inhibitory effects of cyclocreatine in HCC. These results, if confirmed, could provide important hints at improved therapies and cures for HCC

Erythropoietin Ameliorates Rat Experimental Autoimmune Neuritis by Inducing Transforming Growth Factor-Beta in Macrophages

Erythropoietin (EPO) is a pleiotropic cytokine originally identified for its role in erythropoiesis. In addition, in various preclinical models EPO exhibited protective activity against tissue injury. There is an urgent need for potent treatments of autoimmune driven disorders of the peripheral nervous system (PNS), such as the Guillain-Barré syndrome (GBS), a disabling autoimmune disease associated with relevant morbidity and mortality. To test the therapeutic potential of EPO in experimental autoimmune neuritis (EAN) - an animal model of human GBS – immunological and clinical effects were investigated in a preventive and a therapeutic paradigm. Treatment with EPO reduced clinical disease severity and if given therapeutically also shortened the recovery phase of EAN. Clinical findings were mirrored by decreased inflammation within the peripheral nerve, and myelin was well maintained in treated animals. In contrast, EPO increased the number of macrophages especially in later stages of the experimental disease phase. Furthermore, the anti-inflammatory cytokine transforming growth factor (TGF)-beta was upregulated in the treated cohorts. In vitro experiments revealed less proliferation of T cells in the presence of EPO and TGF-beta was moderately induced, while the secretion of other cytokines was almost not altered by EPO. Our data suggest that EPO revealed its beneficial properties by the induction of beneficial macrophages and the modulation of the immune system towards anti-inflammatory responses in the PNS. Further studies are warranted to elaborate the clinical usefulness of EPO for treating immune-mediated neuropathies in affected patients

TNF signalling drives expansion of bone marrow CD4+ T cells responsible for HSC exhaustion in experimental visceral leishmaniasis

Visceral leishmaniasis is associated with significant changes in hematological function but the mechanisms underlying these changes are largely unknown. In contrast to naïve mice, where most long-term hematopoietic stem cells (LT-HSCs; LSK CD150+ CD34- CD48- cells) in bone marrow (BM) are quiescent, we found that during Leishmania donovani infection most LT-HSCs had entered cell cycle. Loss of quiescence correlated with a reduced self-renewal capacity and functional exhaustion, as measured by serial transfer. Quiescent LT-HSCs were maintained in infected RAG2 KO mice, but lost following adoptive transfer of IFNγ-sufficient but not IFNγ-deficient CD4+ T cells. Using mixed BM chimeras, we established that IFNγ and TNF signalling pathways converge at the level of CD4+ T cells. Critically, intrinsic TNF signalling is required for the expansion and/or differentiation of pathogenic IFNγ+CD4+ T cells that promote the irreversible loss of BM function. These finding provide new insights into the pathogenic potential of CD4+ T cells that target hematopoietic function in leishmaniasis and perhaps other infectious diseases where TNF expression and BM dysfunction also occur simultaneously