Antigen-specific cytolysis of infected cells in murine candidiasis

Immune L3T4+ and Lyt-2+ lymphocytes play an important role in the acquired resistance of mice to challenge with virulent Candida albicans, and release macrophage-activating cytokines in response to yeast cells in vitro. To determine whether antigen (Ag)-specific cytotoxic T lymphocytes are generated during fungal infection, purified L3T4+ and Lyt-2+ lymphocytes from immunized mice were cultured in the presence of syngeneic accessory cells, Candida Ag, and IL-2. Yeast-infected bone marrow macrophages and peritoneal exudate neutrophils were used as target cells in a standard ⁵¹Cr release assay. Ag-specific, MHC-unrestricted lysis of infected macrophages was evident with immune Lyt-2+ cells after 5–10 days in culture. Under the same experimental conditions, the cytotoxic activity of L3T4+ cells was negligible, but its expression could be induced by the addition of anti-CD3 antibody.Culturing immune Lyt-2+ cells for shorter periods of time (1–2 days) resulted in preferential lysis of infected neutrophils. In addition, at limiting effector cell numbers, Ag-specific MHC-restricted lymphocytes with cytotoxic activity to infected macrophages could be identified. We suggest that C. albicans infection stimulates multiple cytotoxic T-cell precursors with varying recognition stringency, wich may have an important role in antifungal resistance in vivo.     

Ligand and cytokine dependence of the immunosuppressive pathway of tryptophan catabolism in plasmacytoid dendritic cells

Murine plasmacytoid dendritic cells (pDCs) have been credited with a unique ability to express indoleamine 2,3-dioxygenase (IDO) function and mediate immunosuppression in specific settings; yet, the conditions of spontaneous versus induced activity have remained unclear. We have used maneuvers known to up-regulate IDO in different cell types and have examined the relative efficacy and mechanisms of the induced activity in splenic pDCs, namely, after specific receptor engagement by CTLA-4-Ig, CD200-Ig or CD28-Ig, the latter in combination with silenced expression of the suppressor of cytokine signaling 3 (SOCS3) gene. We found that pDCs (CD11c+ mPDCA-1+ 120G8+) do not express IDO and are not tolerogenic under basal conditions. B7-1 engagement by CTLA-4-Ig, CD200R1 engagement by CD200-Ig and B7-1/B7-2 engagement by CD28-Ig in SOCS3-deficient pDCs were each capable of initiating IDO-dependent tolerance via different mechanisms. IFN-gamma was the major cytokine responsible for CTLA-4-Ig effects, and type I IFNs for those of CD200-Ig. Immunosuppression by CD28-Ig in the absence of SOCS3 required IFN-gamma induction and IFN-like actions of IL-6. Therefore, although pDCs do not mediate IDO-dependent tolerance constitutively, multiple ligands and cytokines will contribute to the expression of a tolerogenic phenotype by pDCs in the mouse.     

Different effect of statins on platelet oxidized-LDL receptor (CD36 and LOX-1) expression in hypercholesterolemic subjects.

Hydroxymethyl-glutaryl-CoA-reductase inhibitors (statins) reduce cardiovascular mortality by decreasing cholesterol as well as by non-lipid-related actions. Oxidized low-density lipoproteins (ox-LDL) are pro-atherogenic molecules and potent platelet agonists. CD36 and lectin-like ox-LDL receptor-1 (LOX-1) are specific ox-LDL receptors also expressed in platelets. This study was planned to address whether treatment with atorvastatin 10 mg/day, pravastatin 40 mg/day or simvastatin 20 mg/day could affect platelet CD36 and LOX-1 expression. Twenty-four patients for each treatment were evaluated after 3, 6, and 9 days and at 6 weeks for complete lipid profile (chromogenic), ox-LDL (ELISA), platelet P-selectin (P-sel), CD36, LOX-1 (FACS), and intracellular citrullin recovery (iCit) (HPLC). Data show hyperactivated platelets (P-sel absolute values, percent variation in activated cells, all p < 0.001), and CD36 and LOX-1 overexpression (all p < 0.001) in patients at baseline. P-sel, CD36, and LOX-1 were significantly decreased by atorvastatin and simvastatin (all p < 0.01) and related with iCit increase (r = 0.58, p < 0.001) and platelet-associated ox-LDL (r = 0.51, p < 0.01) at 9 days. Pravastatin reduced LOX-1 and P-sel (p < 0.05) at 6 weeks in relation with decreased LDL and ox-LDL (r = 0.39, p < 0.01 and r = 0.37, p < 0.01, respectively). These data suggest that atorvastatin and simvastatin reduce platelet activity by exposure of CD36 and LOX-1 before significant LDL reduction, whereas pravastatin action is detected later and in relation with LDL and ox-LDL lowering. Rapid and consistent reduction of CD36 and LOX-1 could be considered a direct anti-atherothrombotic mechanism related to the role of ox-LDL in platelet activation, platelet-endothelium interactions, and NO synthase activity.     

Short- and long-term beneficial effects of trimetazidine in patients with diabetes and ischemic cardiomyopathy

Background

Trimetazidine (TMZ) has been shown to partially inhibit free fatty acid oxidation by shifting substrate utilization from fatty acid to glucose. The aim of this study was to assess the effects of TMZ in patients with diabetes and ischemic cardiomyopathy.

Methods

Sixteen patients with diabetes and ischemic hypokinetic cardiomyopathy (all males) on conventional therapy were randomized to receive either placebo or TMZ (20 mg 3 times per day), each arm lasting 15 days, and then again to receive either placebo or TMZ for 2 additional 6-month periods, according to a double-blind, crossover design. At the end of each period, all patients underwent exercise testing, 2-dimensional echocardiography, and hyperinsulinemic/euglycemic clamp. Among the others, New York Heart Association class, ejection fraction, exercise time, fasting blood glucose, end-clamp M value (index of total body glucose disposal) and endothelin-1 levels were evaluated.

Results

Both in the short and long term (completed by 13 patients), on TMZ compared to placebo, ejection fraction (47 ± 7 vs 41 ± 9 and 45 ± 8 vs 36 ± 8%, P < .001 for both) and M value (4.0 ± 1.8 vs 3.3 ± 1.6, P = .003, and 3.5 ± 1.5 vs 2.7 ± 1.6 mg/kg body weight/min, P < .01) increased, while fasting blood glucose (121 ± 30 vs 136 ± 40, P = .02 and 125 ± 36 vs 140 ± 43, P = .19) and endothelin-1 (8.8 ± 3.8 vs 10.9 ± 3.8, P < .001 and 6.2 ± 2.4 vs 9.2 ± 4.3 pg/mL, P = .03) decreased. In the short term, 10 patients decreased 1 class on the NYHA scale during treatment with TMZ (P = .019 vs placebo). Eight patients decreased 1 NYHA class while on long-term TMZ treatment, while on placebo 1 patient increased 1 NYHA class and none improved (P = .018 vs placebo).

Conclusions

In a short series of patients with diabetes and ischemic cardiomyopathy, TMZ improved left ventricular function, symptoms, glucose metabolism, and endothelial function. Shifting energy substrate preference away from fatty acid metabolism and toward glucose metabolism by TMZ appears an effective adjunctive treatment in patients with diabetes with postischemic cardiomyopathy.     

A subset of anti-rotavirus antibodies directed against the viral protein VP7 predicts the onset of celiac disease and induces typical features of the disease in the intestinal epithelial cell line T84

Celiac disease (CD) is an autoimmune disorder of the small intestine triggered by environmental factors in genetically predisposed individuals. A strong association between type 1 diabetes (T1DM) and CD has been reported. We have previously shown that rotavirus infection may be involved in the pathogenesis of CD through a mechanism of molecular mimicry. Indeed, we identified a subset of anti-transglutaminase IgA antibodies that recognize the rotavirus viral protein VP7. In this study, we aimed at evaluating whether such antibodies may predict the onset of CD in children affected by T1DM. Moreover, to further analyze the link between rotavirus infection and pathogenesis of CD, we analyzed the effect of anti-rotavirus VP7 antibodies on T84 intestinal epithelial cells using the gene-array technique, complemented by the analysis of molecules secreted in the supernatant of stimulated cells. We found that anti-rotavirus VP7 antibodies are present in the vast majority (81 %) of T1DM-CD tested sera, but are detectable also in a fraction (27 %) of T1DM children without CD. Moreover, we found that anti-rotavirus VP7 antibodies are present before the CD onset, preceding the detection of anti-tTG and anti-endomysium antibodies. The gene-array analysis showed that purified anti-rotavirus VP7 antibodies modulate genes that are involved in apoptosis, inflammation, and alteration of the epithelial barrier integrity in intestinal epithelial cells, all typical features of CD. Taken together, these new data further support the involvement of rotavirus infection in the pathogenesis of CD and suggest a predictive role of anti-rotavirus VP7 antibodies.     

Increased TNF-alpha production by peripheral blood mononuclear cells in patients with Krabbe's disease: effect of psychosine

BACKGROUND: Inflammatory and/or immune activation occurs both in animal models (twitcher mice) and in the brain of patients with Globoid cell leukodystrophy (GLD) or Krabbe's disease (KD). In this study we evaluated in vitro the cytokine profile of KD patients and the effect of psychosine, the toxic metabolite which plays a role in the demyelination process in these patients. MATERIALS AND METHODS: We studied cytokine production by peripheral blood mononuclear cells (PBMCs) isolated from four KD patients, diagnosed on the basis of clinical criteria. Cells were cultured and stimulated with appropriate agents and the supernatants collected before and after the addition of psychosine. Tumour necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8) and monocyte chemoattractant factor (MCP)-1) production was evaluated (ELISA method) and compared with a group of 10 normal subjects. RESULTS: We found a significant increase of TNF-alpha release by PBMCs of KD patients compared with healthy subjects; TNF-alpha production was significantly increased after LPS addition. Psychosine was able to induce a further significant increase (P < 0.05) only in cells obtained from KD patients and not from control subjects. No changes were found in IL-8 and MCP-1 production. CONCLUSIONS: The increased TNF-alpha production permits us to confirm the presence of an inflammatory-immune stimulus in KD patients, which may be induced and potentiated by the pathogenetic metabolite psychosine.     

Cell-mediated immunity to chemically xenogenized tumors--IV. Production of lymphokine activity by, and in response to, highly immunogenic cells

To determine whether a novel pattern of lymphokine production might be involved in the superior immunogenicity of chemically xenogenized tumors over that of parental cells, we tested a panel of murine tumors xenogenized by DTIC for production of soluble factors with lymphokine-like activity and induction of lymphokine release from na?ve or specifically sensitized lymphocytes. In the L5178Y tumor system, a majority of xenogenized but not parental clones produced an IL-1-like factor, and this was associated, as a rule, with class II antigen expression and antigen-presenting ability. However, no such properties were exhibited by the xenogenized variants of P815 and L1210Ha cells, which nevertheless occasionally expressed other lymphokine (GM-CSF, IL-3) activities. On examining the ability of xenogenized and parental tumors to cause release of IL-1, IL-2, IL-3, IFN-gamma, TNF/LT and GM-CSF from T-cells, we found, as a rule, an increased lymphokine production when lymphocytes primed in vivo to a xenogenized tumor were restimulated in vitro with the same or parental cells.     

Gamma interferon modifies CD4+ subset expression in murine candidiasis.

A single injection of monoclonal antibody to gamma interferon administered in conjunction with a live Candida albicans yeast cell vaccine resulted in the detection of nonprotective Th2 rather than protective Th1 responses and altered the early expression of interleukin 4 and gamma interferon mRNA in CD4+ cells.     

Tumor-specific L3T4+ and Lyt-2+ lymphocytes in mice primed to mutagenized cell variants.

We have investigated the tumor-specific reactivity of different T-cell subsets from mice primed with clonal variants of L5178Y and P815 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). In both tumor systems, anti-parental tumor immunity and protection against non-immunogenic clones were only induced by vaccinating the hosts with highly immunogenic cell variants, and the effect correlated with the detection of TATA-specific delayed-type hypersensitivity (DTH) reactions. The footpad reaction was transferable with spleen cell populations from immunized mice, and enrichment of splenic lymphocytes in L3T4+ but not Lyt-2+ lymphocytes increased the footpad swelling. Unfractionated spleen cell populations from immunized mice released high amounts of IL-2 and IFN-gamma in vitro in response to parental antigens. Purified L3T4+ and Lyt-2+ lymphocytes also produced IFN-gamma when incubated in vitro with the parental tumors and accessory cells. It is suggested that the mechanisms of anti-parental tumor immunity induced by MNNG-treated variants may be similar to those described previously for triazene-xenogenized L5178Y/DTIC cells, and may involve induction of a tumor-specific DTH reaction and IFN-gamma-mediated stimulation of non-specific tumoricidal effects.     

DNase I mediates internucleosomal DNA degradation in human cells undergoing drug-induced apoptosis

Internucleosomal DNA fragmentation following the activation of endonucleases is the common end point of apoptosis. DNase I, a Ca(2+) / Mg(2+)-dependent endonuclease ubiquitously expressed in mammalian tissues, is believed to play a role in this process. To analyze the in vivo function of this enzyme in human cells, we have generated a cell line with targeted disruption of the DNase I gene, as well as several stable cell lines which overexpress the DNase I gene. Inactivation of the human DNase I gene was obtained in the Jurkat T cell clone JA3, characterized by high susceptibility to apoptotic cell death induced by pharmacological stimuli. JA3 cells, after disruption of the DNase I gene, became resistant to apoptotic stimuli. DNase I was overexpressed in the human cell lines JA3, K562 (erythroleukemia), M 14 (melanoma) and CEM (T cell lymphoma). Remarkably, stable overexpression of DNase I gene resulted in accelerated apoptosis in JA3 cells and induced apoptosis in K562, CEM and M14 cell lines, which are otherwise resistant to internucleosomal DNA degradation following pharmacological stimuli. Our study provides the first in vivo evidence that DNase I mediates internucleosomal DNA degradation in human cells undergoing drug-induced apoptosis.