Systemic IL-2/anti-IL-2Ab complex combined with sublingual immunotherapy suppresses experimental food allergy in mice through induction of mucosal regulatory T cells.

BACKGROUND: Therapeutic tolerance restoration has been proven to modify food allergy in patients and animal models and although sublingual immunotherapy (SLIT) has showed promise, combined therapy may be necessary to achieve a strong and long-term tolerance. AIMS: In this work, we combined SLIT with systemic administration of IL-2 associated with an anti-IL-2 monoclonal antibody (IL-2/anti-IL-2Ab complex or IL-2C) to reverse the IgE-mediated experimental allergy. MATERIALS AND METHODS: Balb/c mice were sensitized with cholera toxin and milk proteins and orally challenged with allergen to elicit hypersensitivity reactions. Then, allergic mice were treated with a sublingual administration of very low amounts of milk proteins combined with intraperitoneal injection of low doses of IL-2C. The animals were next re-exposed to allergens and mucosal as well as systemic immunological parameters were assessed in vivo and in vitro. RESULTS: The treatment reduced serum specific IgE, IL-5 secretion by spleen cells and increased IL-10 and TGF-ß in the lamina propria of buccal and duodenal mucosa. We found an augmented frequency of IL-10-secreting CD4+ CD25+ Foxp3+ regulatory T cells (Treg) in the submaxilar lymph nodes and buccal lamina propria. Tregs were sorted, characterized and adoptively transferred to naïve mice, which were subsequently sensitized. No allergy was experienced in these mice and we encouragingly discovered a faster and more efficient tolerance induction with the combined therapy compared with SLIT. CONCLUSION: The combination of two therapeutic strategies rendered Treg-mediated tolerance more efficient compared to individual treatments and reversed the established IgE-mediated food allergy. This approach highlights the ability of IL-2C to expand Tregs, and it may represent a promising disease-modifying therapy for managing food allergy.

C-terminal cleavage of human Foxp3 at a proprotein convertase motif abrogates its suppressive function.

Foxp3 plays a critical role in the development and function of regulatory T cells (Tregs). Differences in translational and post-translational processing of murine and human Foxp3 have been recently reported. Human Foxp3 exists as four isoforms generated by alternative splicing. Mouse Foxp3 only exists as one isoform, but can be proteolytically cleaved by N-terminal and/or C-terminal proprotein convertase subtilisin/kexins (PCSKs). Here, we show by transcriptome analysis that the proprotein convertases PCSK7, PCSK5 and Furin are present in human CD4(+) T cells with different expression patterns. Notably, after in vitro activation, only PCSK7 and Furin are expressed in Tregs and T effector cells (Teffs), with overexpression of PCSK7 in Tregs compared to Teffs. Human Foxp3 protein displays specific motifs that can be potentially cleaved by convertases. Consequently, we transduced human CD4(+) cells with Foxp3-expressing lentiviral vectors and assessed the generation of proteolytically cleaved Foxp3 forms by Western blot. Three different Foxp3 forms were detected, indicating that human Foxp3 can also be subjected to proteolytic cleavage at the N-terminal and C-terminal ends. These results prompted us to assess the suppressive activity associated with each forms. We observed that full length and N-cleaved Foxp3-transduced CD4(+) T cells similarly suppressed the in vitro proliferation of Teffs. However, the C-cleaved or N&C-cleaved Foxp3 forms afforded almost no suppressive function, indicating a crucial role of the human Foxp3 C-terminal region in Tregs suppressive activity, in marked contrast with the report of a superior suppressive activity for the C-cleaved murine Foxp3 compared to the full length.

Administration of low doses of IL-2 combined to rapamycin promotes allogeneic skin graft survival in mice.

Human CD4(+) CD25(+) FoxP3(+) regulatory T cells (Tregs) prevent allogeneic graft rejection by inhibiting T cell activation, as has been shown in mouse models. Recently, low-dose IL-2 administration was shown to specifically activate Tregs but not pathogenic conventional T cells, leading to resolution of type 1 diabetes in nonobese diabetic mice. We therefore tested the ability of low-dose IL-2 to prevent allogeneic skin graft rejection. We found that while IL-2 alone was inefficient in preventing rejection, combined with rapamycin, IL-2 treatment promoted skin graft survival both in minor disparate and semi-allogeneic skin graft combinations. Tregs are activated by this combined treatment while conventional CD4(+) cell expansion and activation are markedly inhibited. Co-administration of anti-CD25 antibodies dramatically reduces the effect of the IL-2/rapamycin treatment, strongly supporting a central role for Treg activation. Thus, we provide the first preclinical data showing that low-dose IL-2 combined with rapamycin can significantly delay transplant rejection in mice. These findings may form the rational for clinical evaluation of this novel approach for the prevention of transplant rejection.

Blood viscosity changes in experimentally Trypanosoma cruzi-infected rats.

Microcirculatory alterations would explain focal lesions found in Chagas' cardiomyopathy. Trypanosoma cruzi (T. cruzi) infection induces host blood properties modifications and defensive responses capable of producing blood hyperviscosity, an ischemic risk factor able to affect microvascular blood flow. We studied whole blood viscosity (eta(b)) and plasmatic and cellular factors influencing it in rats, 7 and 14 days after experimental infection with T. cruzi. Increased plasma viscosity (eta(p)) was found in infected versus control rats and it was correlated with high blood parasite levels at 7 days and enhanced gamma-globulin fraction concentration at 14 days. The hematocrit, mean corpuscular volume (MCV) and eta(b) were higher in 14 days infected rats vs. 7 days and control animals. Also, electron microscopy observation showed morphological changes in red blood cells (RBC) at 7 and 14 days post-infection, with increased proportion of echinocyte and stomatocyte shapes transformation. In our rat model of Chagas' disease, BPL, increased plasmatic protein concentration, enhanced MCV and RBC shapes transformation would determine blood hyperviscosity, cause of microvascular blood flow abnormalities.

Hepatitis B vaccination and central nervous system demyelination: an immunological approach.

Demyelination events or multiple sclerosis following hepatitis B virus (HBV) vaccination have been reported. We therefore compared the T-cell response to HBsAg in patients with CNS demyelination following HBV vaccination and in HBV-vaccinated healthy individuals. Our data showed no differences in terms of T-cell proliferation or cytokine production between these groups and may help to allay concerns that HBV vaccination might trigger a deleterious immune response.

Effect of the dithiocarbamate pesticide zineb and its commercial formulation azzurro. I. Genotoxic evaluation on cultured human lymphocytes exposed in vitro.

The in vitro cytogenetic effects exerted by the dithiocarbamate fungicide zineb and one of its commercial formulations currently used in Argentina, azzurro, were studied in whole blood human lymphocyte cultures. The genotoxicity of the fungicides was measured by analysis of the frequency of chromosomal aberrations and sister chromatid exchanges (SCEs) and cell cycle progression assays. Both zineb and azzurro activities were tested within the range 0.1-100.0 microg/ml immediately after in vitro lymphocyte stimulation. Only concentrations of 50.0 and 100.0 microg/ml zineb and azzurro induced a significant increase in SCE frequency over control values. Furthermore, this genotoxicity appears to be correlated with its cytotoxicity, measured as cell cycle kinetics, since both a significant delay in cell cycle progression and a significant reduction in proliferative rate index were only observed in those cultures treated with these fungicide concentrations. For both chemicals, a progressive dose-related inhibition of the mitotic activity of cultures was observed when increasing the fungicide concentration. Moreover, only the mitotic activity statistically differed from control values when doses of zineb or azzurro <10 microg/ml were employed. For both fungicides the mitotic index reached the minimal value at doses of 100 microg/ml. Both products induced a significant dose-dependent increase in the number of abnormal cells, chromatid-type and chromosome-type aberrations as well as in the total number of aberrations in the 0.1-100.0 microg/ml dose range. Based on these results, the evaluation of zineb as a controversial genotoxic/non-genotoxic compound for human health should be reconsidered. Instead, we demonstrate that the fungicide induces large DNA alterations and should be considered as a clastogenic mutagen.

Trypanocidal drug benznidazole impairs lipopolysaccharide induction of macrophage nitric oxide synthase gene transcription through inhibition of NF-kappaB activation.

In murine macrophages, inducible NO synthase II (NOSII) gene expression is promoted at a transcriptional level by LPS and/or IFN-gamma with benznidazole (BZL), a trypanocidal drug, acting to down-regulate NOSII gene induction and hence inhibiting NO production. By performing transient transfection experiments, we now report that BZL also inhibited the expression of NOSII gene promoter or multimerized NF-kappaB binding site controlled reporter genes. By contrast, no effect was observed on the expression of a reporter gene under the control of the NOSII promoter-derived IFN regulatory factor element. EMSAs demonstrated that BZL inhibited the nuclear availability of NF-kappaB in stimulated macrophages. NF-kappaB is activated in macrophages by phosphorylation, ubiquitination, and subsequent proteolysis of IkappaB. Within this setting, Western blot was also performed to show that BZL blocked IkappaBalpha degradation. Collectively, these results demonstrate that BZL is able to specifically inhibit macrophage NF-kappaB activation after LPS plus IFN-gamma stimulation.

Treatment with benznidazole and its immunomodulating effects on Trypanosoma cruzi-infected rats.

Because benznidazole (BZL) was found to downregulate nitric oxide (NO) and cytokine synthesis by murine macrophages, we analyzed the potential immunological repercussions of BZL treatment in Trypanosoma cruzi-infected rats. To evaluate whether the effects of BZL were also observed in the presence of an immunostimulating cytokine, four groups of acutely infected rats were subjected to one of the following 20-day therapeutic schedules: (1) a curative BZL oral regimen, (2) recombinant interferon (IFN-gamma) injections, (3) a suboptimal BZL regimen (25% of curative dose), (4) the latter plus IFN-gamma. All BZL doses markedly reduced NO-derived metabolites either in the circulation or in cultured macrophage supernatants. This was observed in rats simultaneously treated with IFN-gamma, which contrasted with the augmented NO production seen in animals given this cytokine alone. The untreated rats, and groups receiving monotherapy with IFN-gamma or 25% BZL, had increased circulating interleukin (IL)-1beta and IL-2 levels, which were reduced in those given BZL plus IFN-gamma. Although combined treatment failed to cause the virtually undetectable blood parasite levels induced by optimal BZL doses, chronic myocardial lesions were reduced to the same extent as in those receiving the curative schedule. The beneficial effects of BZL in this trypanosomiasis may also depend on some immunomodulating influences.

Effects of halothane reexposure in female mice and their offspring.

Female CBi mice subjected to multiple exposures to halothane inhalation anesthesia before mating were investigated for the potential effects of such intervention on a specific antibody response mounted by them and their offspring. An assessment of the toxicologic and reproductive performance of female mice undergoing anesthesia was also performed. Adult female mice received three episodes of halothane anesthesia at weekly intervals. Seventy-two hours after the last dose, mice were subjected to the following procedures: 1) study of the specific humoral immune response to sheep red blood cells (SRBC); 2) hematologic, hepatologic, and histopathologic studies; and 3) mating with syngeneic sires. Halothane-treated females had increased amounts of specific antibody secreting B cells, with liver studies showing evidence of microscopic fatty changes and decreased lipid peroxidation. Anesthesia did not alter reproductive performance but lowered offspring survival. Offspring displayed depressed antibody response after challenge with SRBC at weaning and at 60 d of age. The anti-SRBC antibody response that was found to be enhanced in halothane anesthetized females, seemed to be conversely impaired when studied in the offspring.

Benznidazole, a drug employed in the treatment of Chagas' disease, down-regulates the synthesis of nitrite and cytokines by murine stimulated macrophages.

Benznidazole (BZL) is a nitroheterocyclic drug employed in the chemotherapy of Chagas' disease, a protozoan disease caused by Trypanosoma cruzi. Because this parasite mostly replicates in macrophages, we investigated whether BZL was likely to modify the synthesis of macrophage mediators such as nitrite, tumour necrosis factor-alpha (TNF-alpha), IL-1beta, IL-6 and IL-10. Control and stimulated murine macrophages (lipopolysaccharide (LPS) and/or interferon-gamma (IFN-gamma)) were treated with BZL and measurements were carried out in culture supernatants collected 24 h later. Synthesis of nitrite, IL-6 and IL-10 was maximal upon combined stimulation with LPS + IFN-gamma, whereas lower amounts of the three mediators were detected when both stimuli were given alone. BZL treatment significantly reduced nitrite, IL-6 and IL-10 production, to undetectable levels in some cases, particularly IL-6 and IL-10. LPS was the most potent stimulus of IL-1beta and TNF-alpha production, followed by LPS + IFN-gamma and IFN-gamma in decreasing order. BZL partly inhibited TNF-alpha synthesis, but this effect was smaller than that observed for nitrite, IL-6 and IL-10. LPS-induced production of IL-1beta was also affected by BZL. Semiquantification of gene expression for inducible nitric oxide synthase (iNOS) showed that BZL completely inhibited iNOS gene induction by IFN-gamma, and resulted in respective inhibitions of 30% and 50% with LPS- and LPS + IFN-gamma-stimulated cells. BZL was not cytotoxic on macrophage cultures, as shown by the lactate dehydrogenase activity. Besides its trypanocidal activity, BZL may also alter the balance between pro- and anti-inflammatory mediators with important consequences for the course of T. cruzi infection.