Physiologic expression of two superantigens in the BDF1 mouse.

The majority of endogenous superantigens in the mouse (including the Mls loci) is encoded by mouse mammary tumor proviruses (Mtv) carried in the germline. To understand the differences between the highly stimulatory viral superantigens such as Mls-1a (encoded by Mtv-7), which have biologic activity in vivo and in vitro, and the poorly stimulatory viral superantigens such as Etc-1 (encoded by Mtv-9), which are active only in vivo, the physiologic expression of each Ag was studied in the Mtv-7+ (Mls-1a+), Mtv-9+ (Etc-1+) C57BL/6 x DBA/2 F1 (BDF1) mouse. Using the T cell hybridomas, 1BVB11.40 (anti Etc-1) and 18bbm.19 (anti Mls-1a), we found that similar to Mls-1a, B cells from the spleen and from the thymus present the Etc-1 superantigen, whereas macrophages and dendritic cells do not. Small, resting B cells present the Mls-1a and Etc-1 superantigens poorly; however, the same cells treated with LPS or IL-4 are at least eightfold more efficient in the presentation of these gene products. Furthermore, the effects of LPS and IL-4 are synergistic, but this synergy is not fully explained by the enhancement of I-A and I-E expression. The depletion of IgM+ B cells from neonatal BDF1 mice prevents the clonal deletion of V beta 5+ and 11+ (Etc-1-reactive) cells but not the deletion of V beta 6+ and 8.1+ (Mls-1a reactive) T cells. Despite the persistence of Mls-1a-mediated clonal deletion in B cell-depleted BDF1 mice, these results taken together, argue that the highly stimulatory Mls-1a gene product and the weakly stimulatory Etc-1 gene product are expressed on similar cell types and that their presentation is regulated in a similar way by agents active with B lymphocytes. It is argued that the differences between the highly stimulatory and weakly stimulatory superantigens reflect differences in avidity between the relevant V beta domain and its class II MHC protein/superantigenic ligand.     

Toll receptors type-2 and CR3 expression of canine monocytes and its correlation with immunohistochemistry and xenodiagnosis in visceral leishmaniasis

The aim of the present study was to investigate TLR2 expression in peripheral blood monocytes from dogs naturally infected with Leishmania (Leishmania) infantum to determine whether it correlates with CD11b/CD18 (CR3) expression, and to evaluate the potential of dogs as sources of infection using phlebotomine xenodiagnosis. Forty eight dogs were serologically diagnosed with L. infantum infection by indirect immunofluorescence antibody test (IFAT) and enzyme linked immunosorbent assay (ELISA). Parasitological exams from bone-marrow aspirates were positive by PCR analysis. All dogs were clinical defined as symptomatic. Ear skin tissue samples were obtained for immunohistochemistry (IHQ) analysis. The potential of these dogs as a source of infection using phlebotomine xenodiagnosis (XENO) was evaluated. Flow cytometry was carried out on peripheral blood mononuclear cells using superficial receptors including CD14, CD11b, TLR2 and MHCII. IHQ ear skin tissue parasite load and XENO where done where we found a strict correlation (r = 0.5373). Dogs with higher expression of MFI of CD11b inside CD14 monocytes were represented by dogs without parasite ear tissue load that were unable to infect phlebotomines (IHQ⁻/XENO⁻). Dogs with lower expression of MFI of CD11b inside CD14 monocytes were represented by dogs with parasite ear tissue load and able to infect phlebotomines (IHQ⁺/XENO⁺) (p = 0,0032). Comparable results were obtained for MFI of MHCII (p = 0.0054). In addition, considering the population frequency of CD11b⁺TLR2⁺ and CD11b⁺MHCII⁺, higher values were obtained from dogs with IHQ⁻/XENO⁻ than dogs with IHQ⁺/XENO⁺ (p = 0.01; p = 0.0048, respectively). These data, together with the TLR2 and NO assays results (CD11b⁺TLR2⁺ and NO with higher values for dogs with IHQ⁻/XENO⁻ than dogs with IHQ⁺/XENO⁺), led to the conclusion that IHQ⁻/XENO⁻ dogs are more resistant or could modulate the cellular immune response essential for Leishmania tissue clearance.     

Analysis of a monoclonal rat antibody directed to the alpha-chain variable region (V alpha 3) of the mouse T cell antigen receptor

Three rat mAb, RR3-15, RR3-16, and RR3-18, were established by fusing spleen cells from a rat immunized with the male Ag-specific cytolytic T cell clone, OH6, to mouse myeloma cells. The mAb was identified by their capacity to focus the cytolytic activity of the OH6 CTL clone on nonspecific target cells via FcR-FcR interaction. That all three mAb recognized the OH6 TCR was confirmed by immunoprecipitation studies in which each antibody precipitated a 90 kDa disulfide-linked heterodimer characteristic of the TCR. Surface immunofluorescence staining of a panel of T cell lines and splenic T cell populations showed that RR3-16 reacted not only to the OH6 T cell clone but also to a minor fraction of normal T cells. This reactivity was found to be due to the expression of a gene in the V alpha 3 family. However, RR3-16 did not react with all T cell lines and clones known to express genes from the V alpha 3 family. cDNA sequences of three independent RR3-16+ T cell hybridomas analyzed by polymerase chain reaction were identical to the previously published V alpha 3 sequence of the CTL clone C9. Thus, the mAb RR3-16 is specific for a single member of the TCR V alpha 3 gene family. Analysis of the expression of RR3-16+ TCR in CD4+ and CD8+ subsets of peripheral T cells demonstrated preferential expression on CD8+ T cells, suggesting regulated expression of this particular TCR V alpha gene.     

Characterization of a novel mutation in the cardiac ryanodine receptor that results in catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmogenic disease that manifests as syncope or sudden death during high adrenergic tone in the absence of structural heart defects. It is primarily caused by mutations in the cardiac ryanodine receptor (RyR2). The mechanism by which these mutations cause arrhythmia remains controversial, with discrepant findings related to the role of the RyR2 binding protein FKBP12.6. The purpose of this study was to characterize a novel RyR2 mutation identified in a kindred with clinically diagnosed CPVT.Single-strand conformational polymorphism analysis and direct DNA sequencing were used to screen the RyR2 gene for mutations. Site-directed mutagenesis was employed to introduce the mutation into the mouse RyR2 cDNA. The impact of the mutation on the interaction between RyR2 and a 12.6 kDa FK506 binding protein (FKBP12.6) was determined by immunoprecipitation and immunoblotting and its effect on RyR2 function was characterized by single cell Ca²⁺ imaging and [³H]ryanodine binding.A novel CPVT mutation, E189D, was identified. The E189D mutation does not alter the affinity of the channel for FKBP12.6, but it increases the propensity for store-overload-induced Ca²⁺ release (SOICR). Furthermore, the E189D mutation enhances the basal channel activity of RyR2 and its sensitivity to activation by caffeine.The E189D RyR2 mutation is causative for CPVT and functionally increases the propensity for SOICR without altering the affinity for FKBP12.6. These observations strengthen the notion that enhanced SOICR, but not altered FKBP12.6 binding, is a common mechanism by which RyR2 mutations cause arrhythmias.Key words: arrhythmia, calcium, death sudden, genetics, ion channels     

Immunochemotherapy in American cutaneous leishmaniasis: immunological aspects before and after treatment

In this study, we evaluated the immune response of patients suffering from cutaneous leishmaniasis treated with two distinct protocols. One group was treated with conventional chemotherapy using pentavalent antimonium salts and the other with immunochemotherapy where a vaccine against cutaneous leishmaniasis was combined with the antimonium salt. Our results show that, although no differences were observed in the necessary time for complete healing of the lesions between the two treatments, peripheral blood mononuclear cells from patients treated by chemotherapy showed smaller lymphoproliferative responses at the end of the treatment than those from patients in the immunochemotherapy group. Furthermore, IFN-gamma production was also different between the two groups. While cells from patients in the chemotherapy group produced more IFN-gamma at the end of treatment, a significant decrease in this cytokine production was associated with healing in the immunochemotherapy group. In addition, IL-10 production was also less intense in this latter group. Finally, an increase in CD8+ -IFN-gamma producing cells was detected in the chemotherapy group. Together these results point to an alternative treatment protocol where healing can be induced with a decreased production of a potentially toxic cytokine.     

Loss of STI1‐mediated neuronal survival and differentiation in disease‐associated mutations of prion protein

Cellular prion protein (PrP^C ) is widely expressed and displays a variety of well‐described functions in the central nervous system (CNS ). Mutations of the PRNP gene are known to promote genetic human spongiform encephalopathies, but the components of gain‐ or loss‐of‐function mutations to PrP^C remain a matter for debate. Among the proteins described to interact with PrP^C is Stress‐inducible protein 1 (STI 1), a co‐chaperonin that is secreted from astrocytes and triggers neuroprotection and neuritogenesis through its interaction with PrP^C . In this work, we evaluated the impact of different PrP^C pathogenic point mutations on signaling pathways induced by the STI 1‐PrP^C interaction. We found that some of the pathogenic mutations evaluated herein induce partial or total disruption of neuritogenesis and neuroprotection mediated by mitogen‐activated protein kinase (MAPK )/extracellular signal‐regulated kinases 1 and 2 (ERK 1/2) and protein kinase A (PKA ) signaling triggered by STI 1‐PrP^C engagement. A pathogenic mutant PrP^C that lacked both neuroprotection and neuritogenesis activities fail to promote negative dominance upon wild‐type PrP^C . Also, a STI 1‐α7‐nicotinic acetylcholine receptor‐dependent cellular signaling was present in a PrP^C mutant that maintained both neuroprotection and neuritogenesis activities similar to what has been previously observed by wild‐type PrP^C . These results point to a loss‐of‐function mechanism underlying the pathogenicity of PrP^C mutations.

     

Type I IFNs stimulate nitric oxide production and resistance to Trypanosoma cruzi infection

The participation of type I IFNs (IFN-I) in NO production and resistance to Trypanosoma cruzi infection was investigated. Adherent cells obtained from the peritoneal cavity of mice infected by the i.p. route produced NO and IFN-I. Synthesis of NO by these cells was partially inhibited by treatment with anti-IFN-alphabeta or anti-TNF-alpha Abs. Compared with susceptible BALB/c mice, peritoneal cells from parasite-infected resistant C57BL/6 mice produced more NO (2-fold), IFN-I (10-fold), and TNF-alpha (3.5-fold). Later in the infection, IFN-I levels measured in spleen cell (SC) cultures from 8-day infected mice were greater in C57BL/6 than in infected BALB/c mice, and treatment of the cultures with anti-IFN-alphabeta Ab reduced NO production. IFN-gamma or IL-10 production by SCs was not different between the two mouse strains; IL-4 was not detectable. Treatment of C57BL/6 mice with IFN-I reduced parasitemia levels in the acute phase of infection. Mice deprived of the IFN-alphabetaR gene developed 3-fold higher parasitemia levels in the acute phase in comparison with control 129Sv mice. Production of NO by peritoneal macrophages and SCs was reduced in mice that lacked signaling by IFN-alphabeta, whereas parasitism of macrophages was heavier than in control wild-type mice. We conclude that IFN-I costimulate NO synthesis early in T. cruzi infection, which contributes to a better control of the parasitemia in resistant mice.     

Acute Chagas Disease: New Global Challenges for an Old Neglected Disease

Chagas disease is caused by infection with the protozoan Trypanosoma cruzi, and although over 100 years have passed since the discovery of Chagas disease, it still presents an increasing problem for global public health. A plethora of information concerning the chronic phase of human Chagas disease, particularly the severe cardiac form, is available in the literature. However, information concerning events during the acute phase of the disease is scarce. In this review, we will discuss (1) the current status of acute Chagas disease cases globally, (2) the immunological findings related to the acute phase and their possible influence in disease outcome, and (3) reactivation of Chagas disease in immunocompromised individuals, a key point for transplantation and HIV infection management.     

IL-12 induces monocyte IL-18 binding protein expression via IFN-gamma

IL-18 is a Th1 cytokine that synergizes with IL-12 and IL-2 in the stimulation of lymphocyte IFN-gamma production. IL-18 binding protein (IL-18BP) is a recently discovered inhibitor of IL-18 that is distinct from the IL-1 and IL-18 receptor families. In this report we show that IL-18BPa, the IL-18BP isoform with the highest affinity for IL-18, was strongly induced by IL-12 in human PBMC. Other Th1 cytokines, including IFN-gamma, IL-2, IL-15, and IL-18, were also capable of augmenting IL-18BPa expression. In contrast, IL-1alpha, IL-1beta, TNF-alpha, IFN-gamma-inducible protein-10, and Th2 cytokines such as IL-4 and IL-10 did not induce IL-18BPa. Although monocytes were found to be the primary source of IL-18BPa, the induction of IL-18BPa by IL-12 was mediated through IFN-gamma derived predominantly from NK cells. IL-18BPa production was observed in cancer patients receiving recombinant human IL-12 and correlated with the magnitude of IFN-gamma production. The IFN-gamma/IL-18BPa negative feedback loop identified in this study may be capable of broadly controlling immune activation by cytokines that synergize with IL-18 to induce IFN-gamma and probably plays a key role in the modulation of both innate and adaptive immunity.