Further observations on clinical, histopathological, and immunological features of borderline disseminated cutaneous leishmaniasis caused by Leishmania (Leishmania) amazonensis

Leishmania (Leishmania) amazonensis has for some time been considered as the causative agent of two distinct forms of American cutaneous leishmaniasis (ACL): localized cutaneous leishmaniasis (LCL), and anergic diffuse cutaneous leishmaniasis (ADCL). Recently, a new intermediate form of disease, borderline disseminated cutaneous leishmaniasis (BDCL), was introduced into the clinical spectrum of ACL caused by this parasite, and in this paper we record the clinical, histopathological, and immunological features of eight more BDCL patients from Brazilian Amazonia, who acquired the disease in the Pará state, North Brazil. Seven of them had infections of one to two years' evolution and presented with primary skin lesions and the occurrence of metastases at periods varying from six to 12 months following appearance of the first lesion. Primary skin lesions ranged from 1-3 in number, and all had the aspect of an erythematous, infiltrated plaque, variously located on the head, arms or legs. There was lymphatic dissemination of infection, with lymph node enlargement in seven of the cases, and the delayed hypersensitivity skin-test (DTH) was negative in all eight patients prior to their treatment. After that, there was a conversion of DTH to positive in five cases re-examined. The major histopathological feature was a dermal mononuclear infiltration, with a predominance of heavily parasitized and vacuolated macrophages, together with lymphocytes and plasma cells. In one case, with similar histopathology, the patient had acquired his infection seven years previously and he presented with the largest number of disseminated cutaneous lesions. BDCL shows clinical and histopathological features which are different from those of both LCL and ADCL, and there is a good prognosis of cure which is generally not so in the case of frank ADCL.

Effector function of human tumor-specific CD8 T cells in melanoma lesions: a state of local functional tolerance.

Although tumor-specific CD8 T-cell responses often develop in cancer patients, they rarely result in tumor eradication. We aimed at studying directly the functional efficacy of tumor-specific CD8 T cells at the site of immune attack. Tumor lesions in lymphoid and nonlymphoid tissues (metastatic lymph nodes and soft tissue/visceral metastases, respectively) were collected from stage III/IV melanoma patients and investigated for the presence and function of CD8 T cells specific for the tumor differentiation antigen Melan-A/MART-1. Comparative analysis was conducted with peripheral blood T cells. We provide evidence that in vivo-priming selects, within the available naive Melan-A/MART-1-specific CD8 T-cell repertoire, cells with high T-cell receptor avidity that can efficiently kill melanoma cells in vitro. In vivo, primed Melan-A/MART-1-specific CD8 T cells accumulate at high frequency in both lymphoid and nonlymphoid tumor lesions. Unexpectedly, however, whereas primed Melan-A/MART-1-specific CD8 T cells that circulate in the blood display robust inflammatory and cytotoxic functions, those that reside in tumor lesions (particularly in metastatic lymph nodes) are functionally tolerant. We show that both the lymph node and the tumor environments blunt T-cell effector functions and offer a rationale for the failure of tumor-specific responses to effectively counter tumor progression.

Treg-therapy allows mixed chimerism and transplantation tolerance without cytoreductive conditioning.

Establishment of mixed chimerism through transplantation of allogeneic donor bone marrow (BM) into sufficiently conditioned recipients is an effective experimental approach for the induction of transplantation tolerance. Clinical translation, however, is impeded by the lack of feasible protocols devoid of cytoreductive conditioning (i.e. irradiation and cytotoxic drugs/mAbs). The therapeutic application of regulatory T cells (Tregs) prolongs allograft survival in experimental models, but appears insufficient to induce robust tolerance on its own. We thus investigated whether mixed chimerism and tolerance could be realized without the need for cytoreductive treatment by combining Treg therapy with BM transplantation (BMT). Polyclonal recipient Tregs were cotransplanted with a moderate dose of fully mismatched allogeneic donor BM into recipients conditioned solely with short-course costimulation blockade and rapamycin. This combination treatment led to long-term multilineage chimerism and donor-specific skin graft tolerance. Chimeras also developed humoral and in vitro tolerance. Both deletional and nondeletional mechanisms contributed to maintenance of tolerance. All tested populations of polyclonal Tregs (FoxP3-transduced Tregs, natural Tregs and TGF-beta induced Tregs) were effective in this setting. Thus, Treg therapy achieves mixed chimerism and tolerance without cytoreductive recipient treatment, thereby eliminating a major toxic element impeding clinical translation of this approach.

Danger signaling through the inflammasome acts as a master switch between tolerance and sensitization.

Efficient priming of adaptive immunity depends on danger signals provided by innate immune pathways. As an example, inflammasome-mediated activation of caspase-1 and IL-1beta is crucial for the development of reactive T cells targeting sensitizers like dinitrofluorobenzene (DNFB). Surprisingly, DNFB and dinitrothiocyanobenzene provide cross-reactive Ags yet drive opposing, sensitizing vs tolerizing, T cell responses. In this study, we show that, in mice, inflammasome-signaling levels can be modulated to turn dinitrothiocyanobenzene into a sensitizer and DNFB into a tolerizer, and that it correlates with the IL-6 and IL-12 secretion levels, affecting Th1, Th17, and regulatory T cell development. Hence, our data provide the first evidence that the inflammasome can define the type of adaptive immune response elicited by an Ag, and hint at new strategies to modulate T cell responses in vivo.

Characterization of the gene expression related to the process of DNA damage tolerance in Schistosoma mansoni

In the course of its complex life cycle, the parasite Schistosoma mansoni need to adapt to distinct environments, and consequently is exposed to various DNA damaging agents. The Schistosoma genome sequencing initiative has uncovered sequences from genes and transcripts related to the process of DNA damage tolerance as the enzymes UBC13, MMS2, and RAD6. In the present work, we evaluate the importance of this process in different stages of the life cycle of this parasite. The importance is evidenced by expression and phylogenetic profiles, which show the conservation of this pathway from protozoa to mammalians on evolution.

Immunological and microbiological activity of Davilla elliptica St. Hill. (Dilleniaceae) against Mycobacterium tuberculosis

Mycobacterium tuberculosis is responsible for over 8 million cases of tuberculosis (TB) annually. Natural products may play important roles in the chemotherapy of TB. The immunological activity of Davilla elliptica chloroform extract (DECE) was evaluated in vitro by the determination of hydrogen peroxide (H2O2), nitric oxide (NO), and tumor necrosis factor-alpha (TNF-alpha) release in peritoneal macrophages cultures. DECE was also tested for its antimycobacterial activity against M. tuberculosis using the microplate alamar blue assay. DECE (50, 150, 250 µg/ml) stimulated the production of H2O2 (from 1,79 ± 0,23 to 7,27 ± 2,54; 15,02 ± 2,86; 20,5 ± 2,1 nmols) (means ± SD), NO (from 2,64 ± 1,02 to 25,59 ± 2,29; 26,68 ± 2,41; 29,45 ± 5,87 µmols) (means ± SD) and TNF-alpha (from 2,44 ± 1,46 to 30,37 ± 8,13; 38,68 ± 1,59; 41,6 ± 0,90 units/ml) (means ± SD) in a dose-dependent manner and also showed a promising antimycobacterial activity with a minimum inhibitory concentration of 62,5 µg/ml. This plant may have therapeutic potential in the immunological and microbiological control of TB.

Trypanosoma (Duttonella) vivax: its biology, epidemiology, pathogenesis, and introduction in the New World - a review

The biology, epidemiology, pathogenesis, diagnostic techniques, and history of the introduction of Trypanosoma (Duttonella) vivax in the New World are reviewed. The two main immunological responses of trypanosome-infected animals - antibody production and immunodepression - are discussed in the context of how these responses play a role in disease tolerance or susceptibility. Isolation and purification of T. vivax are briefly discussed. The recent reports of bovine trypanosomiasis diagnosed in cattle on farms located in the Pantanal region of the states of Mato Grosso do Sul and Mato Grosso, Brazil, are also discussed.

Immunological imbalance between IFN-³ and IL-10 levels in the sera of patients with the cardiac form of Chagas disease

The immune response is crucial for protection against disease; however, immunological imbalances can lead to heart and digestive tract lesions in chagasic patients. Several studies have evaluated the cellular and humoral immune responses in chagasic patients in an attempt to correlate immunological findings with clinical forms of Chagas disease. Moreover, immunoglobulins and cytokines are important for parasitic control and are involved in lesion genesis. Here, cytokine and IgG isotype production were studied, using total epimastigote antigen on sera of chagasic patients with indeterminate (IND, n = 27) and cardiac (CARD, n = 16) forms of the disease. Samples from normal, uninfected individuals (NI, n = 30) were use as controls. The results showed that sera from both IND and CARD patients contained higher levels of Trypanosoma cruzi-specific IgG1 (IgG1) antibodies than sera from NI. No difference in IgG2 production levels was observed between NI, IND and CARD patients, nor was a difference in IL-10 and IFN-³ production detected in the sera of IND, CARD and NI patients. However, IND patients displayed a positive correlation between IL-10 and IFN-³ levels in serum, while CARD patients showed no such correlation, indicating an uncontrolled inflammatory response in CARD patients. These findings support the hypothesis that a lack of efficient regulation between IFN-³ and IL-10 productions in CARD patients may lead to cardiac immunopathology.

Clinical, bacteriological and immunological follow-up of household contacts of leprosy patients from a post-elimination area - Antioquia, Colombia

Follow-up of the household contacts (HHC) of leprosy patients is still the best strategy for early detection of leprosy. HHC from a post-elimination region of Colombia studied in 2001-2002 were re-contacted in 2007. They were tested at both times by clinical examination, bacillary index (BI), PCR from a slit skin smear (SSS) and anti PGL-1 IgM titres. Thirty-two of 61 HHC (52%) were re-contacted. Nine HHC (28%) showed sero-conversion and one had a skin lesion (BI negative, nested PCR positive). Periodic evaluation of HHC can contribute to the detection of infected HHC as well as new and early leprosy cases.

Early and late skin reactions to radiotherapy for breast cancer and their correlation with radiation-induced DNA damage in lymphocytes

INTRODUCTION Radiotherapy outcomes might be further improved by a greater understanding of the individual variations in normal tissue reactions that determine tolerance. Most published studies on radiation toxicity have been performed retrospectively. Our prospective study was launched in 1996 to measure the in vitro radiosensitivity of peripheral blood lymphocytes before treatment with radical radiotherapy in patients with breast cancer, and to assess the early and the late radiation skin side effects in the same group of patients. We prospectively recruited consecutive breast cancer patients receiving radiation therapy after breast surgery. To evaluate whether early and late side effects of radiotherapy can be predicted by the assay, a study was conducted of the association between the results of in vitro radiosensitivity tests and acute and late adverse radiation effects. METHODS Intrinsic molecular radiosensitivity was measured by using an initial radiation-induced DNA damage assay on lymphocytes obtained from breast cancer patients before radiotherapy. Acute reactions were assessed in 108 of these patients on the last treatment day. Late morbidity was assessed after 7 years of follow-up in some of these patients. The Radiation Therapy Oncology Group (RTOG) morbidity score system was used for both assessments. RESULTS Radiosensitivity values obtained using the in vitro test showed no relation with the acute or late adverse skin reactions observed. There was no evidence of a relation between acute and late normal tissue reactions assessed in the same patients. A positive relation was found between the treatment volume and both early and late side effects. CONCLUSION After radiation treatment, a number of cells containing major changes can have a long survival and disappear very slowly, becoming a chronic focus of immunological system stimulation. This stimulation can produce, in a stochastic manner, late radiation-related adverse effects of varying severity. Further research is warranted to identify the major determinants of normal tissue radiation response to make it possible to individualize treatments and improve the outcome of radiotherapy in cancer patients.

Immunological and non-immunological effects of cytokines and chemokines in the pathogenesis of chronic Chagas disease cardiomyopathy

The pathogenesis of Chagas disease cardiomyopathy (CCC) is not well understood. Since studies show that myocarditis is more frequent during the advanced stages of the disease, and the prognosis of CCC is worse than that of other dilated cardiomyopathies of non-inflammatory aetiology, which suggest that the inflammatory infiltrate plays a major role in myocardial damage. In the last decade, increasing evidence has shown that inflammatory cytokines and chemokines play a role in the generation of the inflammatory infiltrate and tissue damage. CCC patients have an increased peripheral production of the inflammatory Th1 cytokines IFN-³ and TNF-± when compared to patients with the asymptomatic/indeterminate form. Moreover, Th1-T cells are the main producers of IFN-³ and TNF-± and are frequently found in CCC myocardial inflammatory infiltrate. Over the past several years, our group has collected evidence that shows several cytokines and chemokines produced in the CCC myocardium may also have a non-immunological pathogenic effect via modulation of gene and protein expression in cardiomyocytes and other myocardial cell types. Furthermore, genetic polymorphisms of cytokine, chemokine and innate immune response genes have been associated with disease progression. We will review the molecular and immunological mechanisms of myocardial damage in human CCC in light of recent findings.

Immunological and parasitological parameters after treatment with dexamethasone in murine Schistosoma mansoni

This work aimed to evaluate the effect of diphenyl dimethyl bicarboxylate (DDB) and dexamethasone alone and in combination with praziquantel on various parasitological, immunological and pathological parameters reflecting disease severity and morbidity in murine schistosomiasis. DDB and dexamethasone had no effect on worm burden but altered tissue egg distribution. This indicates that, under the schedule used, neither drug interfered with the development of adult worms or oviposition, but both can modulate liver pathology. Dexamethasone resulted in a greater reduction in granuloma size than did DDB. Dexamethasone-treated mice also showed lower levels of serum gamma interferon (IFN-γ), interleukin-12 (IL-12) and IL-4, together with higher IL-10 levels, than infected untreated control animals. These data suggest that dexamethasone is a convenient and promising coadjuvant agent that results in decreased morbidity in murine schistosomiasis.

Molecular and immunological characterization of gluten proteins isolated from oat cultivars that differ in toxicity for celiac disease.

A strict gluten-free diet (GFD) is the only currently available therapeutic treatment for patients with celiac disease (CD). Traditionally, treatment with a GFD has excluded wheat, barley and rye, while the presence of oats is a subject of debate. The most-recent research indicates that some cultivars of oats can be a safe part of a GFD. In order to elucidate the toxicity of the prolamins from oat varieties with low, medium, and high CD toxicity, the avenin genes of these varieties were cloned and sequenced, and their expression quantified throughout the grain development. At the protein level, we have accomplished an exhaustive characterization and quantification of avenins by RP-HPLC and an analysis of immunogenicity of peptides present in prolamins of different oat cultivars. Avenin sequences were classified into three different groups, which have homology with S-rich prolamins of Triticeae. Avenin proteins presented a lower proline content than that of wheat gliadin; this may contribute to the low toxicity shown by oat avenins. The expression of avenin genes throughout the development stages has shown a pattern similar to that of prolamins of wheat and barley. RP-HPLC chromatograms showed protein peaks in the alcohol-soluble and reduced-soluble fractions. Therefore, oat grains had both monomeric and polymeric avenins, termed in this paper gliadin- and glutenin-like avenins. We found a direct correlation between the immunogenicity of the different oat varieties and the presence of the specific peptides with a higher/lower potential immunotoxicity. The specific peptides from the oat variety with the highest toxicity have shown a higher potential immunotoxicity. These results suggest that there is wide range of variation of potential immunotoxicity of oat cultivars that could be due to differences in the degree of immunogenicity in their sequences.

Characterization of the immunological properties of " Plasmodium falciparum " and " Plasmodium berghei " circumsporozoite proteins : antibody responses and recognition by specific CD8+ T cells

SUMMARY Interest in developing intervention strategies against malaria by targeting the liver stage of the Plasmodium life cycle has been fueled by studies which show that sterile protective immunity can be achieved by immunization with radiation-attenuated sporozoites. Anti-malarial drugs and insecticides have been widely used to control the disease, but in the hope of developing a more cost-effective intervention strategy, vaccine development has taken centre stage in malaria research. There is currently no vaccine against malaria. Attenuated sporozoite-induced immunity is achieved by antibodies and T cells against malaria liver stage antigens, the most abundant being the circumsporozoite protein (CSP), and many vaccine formulations aim at mimicking this immunity. However, the mechanisms by which the antibody and T cell immune responses are generated after infection by sporozoites, or after immunization with different vaccine formulations are still not well understood. The first part of this work aimed at determining the ability of primary hepatocytes from BALB/c mice to process and present CSP-derived peptides after infection with P. berghei sporozoites. Both infected hepatocytes and those traversed by sporozoites during migration were found to be capable of processing and presenting the CSP to specific CD8+ T cells in vitro. The pathway of processing and presentation involved the proteasome, aspartic proteases and transport through a post-Endoplasmic Reticulum (ER) compartment. These results suggest that in vivo, infected hepatocytes contribute to the elicitation and expansion of a T cell response. In the second part, the antibody responses of CB6F1 mice to synthetic peptides corresponding to the N- and C-terminal domains of P. berghei and P. falciparum CS proteins were characterized. Mice were immunized with single peptides or a combination of N- and C-terminal peptides. The peptides were immunogenic in mice and the antisera generated could recognize the native CSP on the sporozoite surface. Antisera generated against the N-terminal peptides or against the combinations inhibited sporozoite invasion of hepatocytes in vitro. In vivo, more mice immunized with single P. berghei peptides were protected from infection upon a challenge with P. berghei sporozoites, than mice immunized with a combination of N- and C-terminal peptides. Furthermore, P. falciparum N-terminal peptides were recognized by serum samples from people living in malaria-endemic areas. Importantly, recognition of a peptide from the N-terminal fragment of the P. falciparum CSP by sera from children living in a malaria-endemic region was associated with protection from disease. These results underline the potential of using such peptides as malaria vaccine candidates. RESUME L'intérêt de développer des stratégies d'intervention contre la malaria ciblant le stade pré-erythrocytaire a été alimenté par des études qui montrent qu'il est possible d'obtenir une immunité par l'injection de sporozoites irradiés. Les médicaments et les insecticides anti-paludiques ont été largement utilisés pour contrôler la maladie, mais dans l'espoir de développer une stratégie d'intervention plus rentable, le développement de vaccins a été placé au centre des recherches actuelles contre la malaria. A l'heure actuelle, il n'existe aucun vaccin contre la malaria. L'immunité induite par les sporozoites irradiés est due à l'effet combiné d'anticorps et de cellules T qui agissent contre les antigènes du stade hépatique dont le plus abondant est la protéine circumsporozoite (CSP). Beaucoup de formulations de vaccin visent à imiter l'immunité induite par les sporozoites irradiés. Cependant, les mécanismes par lesquels les anticorps et les cellules T sont génerés après infection par les sporozoites ou après immunisation avec des formulations de vaccin ne sont pas bien compris. La première partie de ce travail a visé à déterminer la capacité de hépatocytes primaires provenant de souris BALB/c à "processer" et à présenter des peptides dérivés de la CSP, après infection par des sporozoites de Plasmodium berghei. Nous avons montré que in vitro, les hépatocytes infectés et ceux traversés par les sporozoites pendant leur migration étaient capables de "processer" et de présenter la CSP aux cellules T CD8+ spécifiques. La voie de présentation implique le protéasome, les protéases de type aspartique et le transport à travers un compartiment post-reticulum endoplasmique. Ces résultats suggèrent que in vivo, les hépatocytes infectés contribuent à l'induction et à l'expansion d'une réponse immunitaire spécifique aux cellules T. Dans la deuxième partie, nous avons caractérisé les réponses anticorps chez les souris de la souche CB6F1 face aux peptides N- et C-terminaux des protéines circumsporozoites de Plasmodium berghei et Plasmodium falciparum. Les souris ont été immunisées avec les peptides individuellement ou en combinaison. Les peptides utilisés étaient immunogéniques chez les souris, et les anticorps produits pouvaient reconnaître la protéine CSP native à la surface des sporozoites. In vitro, les sera contre les peptides N-teminaux et les combinaisons étaient capables d'inhiber l'invasion de hépatocytes par les sporozoites. In vivo, plus de souris immunisées avec les peptides individuels de la CSP de P. berghei étaient protégées contre la malaria que les souris immunisées avec une combinaison de peptides N- et C-terminaux. De plus, les peptides N-terminaux de la CSP de P. falciparum ont été reconnus par les sera de personnes vivant dans des régions endémiques pour la malaria. Il est intéressant de voir que la reconnaissance d'un peptide N-terminal de P. falciparum par des sera d'enfants habitant dans des régions endémiques était associé à la protection contre la maladie. Ces résultats soulignent le potentiel de ces peptides comme candidats-vaccin contre la malaria.