Identification of Rare High-Avidity, Tumor-Reactive CD8+ T Cells by Monomeric TCR-Ligand Off-Rates Measurements on Living Cells.

The avidity of the T-cell receptor (TCR) for antigenic peptides presented by the peptide-MHC (pMHC) on cells is a key parameter for cell-mediated immunity. Yet a fundamental feature of most tumor antigen-specific CD8(+) T cells is that this avidity is low. In this study, we addressed the need to identify and select tumor-specific CD8(+) T cells of highest avidity, which are of the greatest interest for adoptive cell therapy in patients with cancer. To identify these rare cells, we developed a peptide-MHC multimer technology, which uses reversible Ni(2+)-nitrilotriacetic acid histidine tags (NTAmers). NTAmers are highly stable but upon imidazole addition, they decay rapidly to pMHC monomers, allowing flow-cytometric-based measurements of monomeric TCR-pMHC dissociation rates of living CD8(+) T cells on a wide avidity spectrum. We documented strong correlations between NTAmer kinetic results and those obtained by surface plasmon resonance. Using NTAmers that were deficient for CD8 binding to pMHC, we found that CD8 itself stabilized the TCR-pMHC complex, prolonging the dissociation half-life several fold. Notably, our NTAmer technology accurately predicted the function of large panels of tumor-specific T cells that were isolated prospectively from patients with cancer. Overall, our results demonstrated that NTAmers are effective tools to isolate rare high-avidity cytotoxic T cells from patients for use in adoptive therapies for cancer treatment.

Mycobacterium tuberculosis Infection in Young Children: Analyzing the Performance of the Diagnostic Tests

This study evaluated the performance of the Tuberculin Skin Test (TST) and Quantiferon-TB Gold in-Tube (QFT) and the possible association of factors which may modify their results in young children (0-6 years) with recent contact with an index tuberculosis case. Materials and Methods: A cross-sectional study including 135 children was conducted in Manaus, Amazonas-Brazil. The TST and QFT were performed and the tests results were analyzed in relation to the personal characteristics of the children studied and their relationship with the index case. Results: The rates of positivity were 34.8% (TST) and 26.7% (QFT), with 14.1% of indeterminations by the QFT. Concordance between tests was fair (Kappa = 0.35 P<0.001). Both the TST and QFT were associated with the intensity of exposure (Linear OR = 1.286, P = 0.005; Linear OR = 1.161, P = 0.035 respectively) with only the TST being associated with the time of exposure (Linear OR = 1.149, P = 0.009). The presence of intestinal helminths in the TST+ group was associated with negative QFT results (OR = 0.064, P = 0.049). In the TST- group lower levels of ferritin were associated with QFT+ results (Linear OR = 0.956, P = 0.036). Conclusions: Concordance between the TST and QFT was lower than expected. The factors associated with the discordant results were intestinal helminths, ferritin levels and exposure time to the index tuberculosis case. In TST+ group, helminths were associated with negative QFT results suggesting impaired cell-mediated immunity. The TST-&QFT+ group had a shorter exposure time and lower ferritin levels, suggesting that QFT is faster and ferritin may be a potential biomarker of early stages of tuberculosis infection.

Whole-body vibration decreases the proliferativeb response of TCD4+ cells in elderly individuals with knee osteoarthritis

The aim of this study was to investigate the effect of adding whole-body vibration (WBV; frequency = 35 to 40 Hz; amplitude = 4 mm) to squat training on the T-cell proliferative response of elderly patients with osteoarthritis (OA) of the knee. This study was a randomized controlled trial in which the selected variables were assessed before and after 12 weeks of training. Twenty-six subjects (72 ± 5 years of age) were divided into three groups: 1) squat training with WBV (WBV, N = 8); 2) squat training without WBV (N = 10), and 3) a control group (N = 8). Women who were ≥60 years of age and had been diagnosed with OA in at least one knee were eligible. The intervention consisted of 12 uninterrupted weeks of squatting exercise training performed 3 times/week. Peripheral blood mononuclear cells were obtained from peripheral blood collected before and after training. The proliferation of TCD4+ and TCD8+ cells was evaluated by flow cytometry measuring the carboxyfluorescein succinimidyl ester fluorescence decay before and after the intervention (∆). The proliferative response of TCD4+ cells (P = 0.02, effect size = 1.0) showed a significant decrease (23%) in the WBV group compared to the control group, while there was no difference between groups regarding the proliferative response of TCD8+ cells (P = 0.12, effect size = 2.23). The data suggest that the addition of WBV to squat exercise training might modulate T-cell-mediated immunity, minimizing or slowing disease progression in elderly patients with OA of the knee.

Macrophage migration inhibitory factor is up-regulated in human first-trimester placenta stimulated by soluble antigen of Toxoplasma gondii, resulting in increased monocyte adhesion on villous explants

Considering the potential role of macrophage migration inhibitory factor (MIF) in the inflammation process in placenta when infected by pathogens, we investigated the production of this cytokine in chorionic villous explants obtained from human first-trimester placentas stimulated with soluble antigen from Toxoplasma gondii (STAg). Parallel cultures were performed with villous explants stimulated with STAB, interferon-gamma (IFN-gamma), or STAB plus IFN-gamma. To assess the role of placental MIF on monocyte adhesiveness to human trophoblast, explants were co-cultured with human myelomonocytic THP-1 cells in the presence or absence of supernatant from cultures treated with STAB (SPN), SPN plus anti-MIF antibodies, or recombinant MIF. A significantly higher concentration of MIF was produced and secreted by villous explants treated with STAB or STAB plus IFN-gamma after 24-hour culture. Addition of SPN or recombinant MIF was able to increase THP-1 adhesion, which was inhibited after treatment with anti-MIF antibodies. This phenomenon was associated with intercellular adhesion molecule expression by villous explants. Considering that the processes leading to vertical dissemination of T. gondii remain widely unknown, our results demonstrate that MIF production by human first-trimester placenta is up-regulated by parasite antigen and may play an essential role as an autocrine/paracrine mediator in placental infection by T. gondii.

Mannose-binding lectin complement pathway plays a key role in complement activation by Paracoccidioides brasiliensis

Paracoccidioides brasiliensis (Pb) is a dimorphic fungal pathogen that causes paracoccidioidomycosis the most severe deep mycosis from South America Although cell mediated immunity is considered the most efficient protective mechanism against Pb infection mechanisms of innate immunity are poorly defined Herein we investigated the interaction of the complement system with high and low virulence isolates of Pb We demonstrated that Pb18 a high virulence Pb Isolate when incubated with normal human serum (NHS) induces consumption of hemolytic complement and when immobilized promotes binding of C4b C3b and C5b-C9 Both low virulence (Pb265) and high virulence (Pb18) isolates consumed C4 C3 and mannose-binding learn (MBL) of MBL-sufficient but not of MBL-deficient serum as revealed by deposition of residual C4 C3 and MBL on immune complexes and mannan However higher complement components consumption was observed with Pb265 as compared with Pb18 The suggested relationship between low virulence and significant complement activation properties of Pb isolates was confirmed by the demonstration that virulence attenuation of Pb 18 results in acquisition of the ability to activate complement Conversely reactivation of attenuated Pb18 results in loss of the ability to activate complement Our results demonstrate for the first time that Pb yeasts activate the complement system by the lectin pathway and there is an Inverse correlation between complement activating ability and Pb virulence These differences could exert an influence on Innate immunity and severity of the disease developed by infected hosts (C) 2010 Elsevier Ltd All rights reserved

Fonsecaea pedrosoi infection induces differential modulation of costimulatory molecules and cytokines in monocytes from patients with severe and mild forms of chromoblastomycosis

The host defense mechanism in chromoblastomycosis has not been thoroughly investigated. It has been suggested that cell- mediated immunity in patients with long- standing chromoblastomycosis is somehow impaired. As a result, these individuals became unable to develop an efficient immune reaction. Many studies have shown that monocyte- derived macrophages exhibit critical activities in immunity to microorganisms. Moreover, the ability of cells from the monocytic lineage to process and present antigens, to produce cytokines, and to provide costimulatory signals confirms their pivotal role in the initiation of specific immune responses. In the present study, it was observed that monocytes from patients with a severe form of disease had a higher production of IL- 10 and a lower expression of HLA- DR and costimulatory molecules when stimulated with specific antigen or LPS. Immune modulation with recombinant IL- 12 or anti- IL- 10 can restore the antigen- specific Th1- type immune response in chromoblastomycosis patients by up- regulating HLA- DR and costimulatory molecules in monocytes. Therefore, our data show that monocytes from patients with different clinical forms of chromoblastomycosis present distinct phenotypic and functional profiles. This observation suggests possible mechanisms that control the T cell response and influence their role in the development of pathology.

Influence of Th1/Th2 cytokines and nitric oxide in murine systemic infection induced by Sporothrix schenckii

In an attempt to elucidate the effects of Sporothrix schenckii infection on the immune response, our laboratory has developed a murine model of disseminated sporotrichosis. Helper T cells can be further subdivided into Th1 and Th2 phenotypes. The differentiation of two subsets of T lymphocytes is driven by IL-12 and IL-4 cytokines, respectively. Th1 cells produce IFN-gamma that activate macrophages and promote cell-mediated immunity. In addition, we found low levels of iNOS and NO production in the initial (1st and 2nd weeks) and final (9th and 10th weeks) periods of the infection, in contrast with the period of week 4 to 7 of elevated values. The determination of IFN-gamma and IL-12 are in agreement with NO/iNOS detection, showing the presence of cellular immune response throughout the infectious process. However, the production of IL-4 shows an increase in levels after the 5th and 6th weeks suggesting a participation of Th2 response in this period as well. Regarding these results, the study demonstrated that in experimental sporotrichosis infection the cellular immune response participated throughout the period analyzed as a nitric oxide dependent mechanism. In contrast, the presence of Th2 response began in the 5th week, suggesting the participation of humoral immune response in advanced stages of sporotrichosis.

Lymphocyte transformation assay for C neoformans antigen is not reliable for detecting cellular impairment in patients with Neurocryptococcosis

Background: Cryptococcus neoformans causes meningitis and disseminated infection in healthy individuals, but more commonly in hosts with defective immune responses. Cell-mediated immunity is an important component of the immune response to a great variety of infections, including yeast infections. We aimed to evaluate a specific lymphocyte transformation assay to Cryptococcus neoformans in order to identify immunodeficiency associated to neurocryptococcosis (NCC) as primary cause of the mycosis.Methods: Healthy volunteers, poultry growers, and HIV-seronegative patients with neurocryptococcosis were tested for cellular immune response. Cryptococcal meningitis was diagnosed by India ink staining of cerebrospinal fluid and cryptococcal antigen test (Immunomycol-Inc, SP, Brazil). Isolated peripheral blood mononuclear cells were stimulated with C. neoformans antigen, C. albicans antigen, and pokeweed mitogen. The amount of H-3-thymidine incorporated was assessed, and the results were expressed as stimulation index (SI) and log SI, sensitivity, specificity, and cut-off value (receiver operating characteristics curve). We applied unpaired Student t tests to compare data and considered significant differences for p<0.05.Results: The lymphotoxin alpha showed a low capacity with all the stimuli for classifying patients as responders and non-responders. Lymphotoxin alpha stimulated by heated-killed antigen from patients with neurocryptococcosis was not affected by TCD4+ cell count, and the intensity of response did not correlate with the clinical evolution of neurocryptococcosis.Conclusion: Response to lymphocyte transformation assay should be analyzed based on a normal range and using more than one stimulator. The use of a cut-off value to classify patients with neurocryptococcosis is inadequate. Statistical analysis should be based on the log transformation of SI. A more purified antigen for evaluating specific response to C. neoformans is needed.

Clinical presentation of tuberculoid leprosy in an epidermodysplasia verruciformis patient

Epidermodysplasia verruciformis (EV) is triggered by a variety of mechanisms that at least partly include genetic background. We present a Brazilian man with a 30-year history of flat, wart-like lesions with clinical, histopathological, and evolutive aspects consistent with papillomavirus (HPV)-associated EV. Histological analysis of the wart lesions showed epidermis with hyperkeratosis, regular acanthosis, hypergranulosis, and cells with abundant basophilic cytoplasm. Moreover, a perivascular lymphocytic infiltrate was found in the superficial dermis, consistent with a viral wart. Type-2-HPV DNA was detected in various fragments of skin-wart lesions using the polymerase chain reaction (PCR). Two years after the EV diagnosis, the patient presented with an anesthetic well-demarcated, erythematous and mildly scaly plaque on his right forearm. A histopathological analysis of this lesion demonstrated the presence of a compact tuberculoid granuloma. Ziehl-Neelsen staining demonstrated the presence of rare acid-fast bacilli and confirmed the tuberculoid leprosy diagnosis. The patient's Mitsuda Intradermal Reaction was positive. To elucidate the possible mechanism involved in this case of EV, we genotyped the HLA genes of this patient. DQB genotyping showed the polymorphic HLA alleles DQB1*0301 and 0501. The patient was treated with a paucibacillary multidrug therapy scheme, and the disease was cured in six months. This report describes an EV patient with an M. leprae infection, confirming that tuberculoid leprosy patients possess a relatively specific and efficient cell-mediated immunity against the bacillus and, therefore, localized forms of the disease. Moreover, we show the possible involvement of the polymorphic HLA alleles DQB1*0301 and 0501 in EV induction mechanisms.

Effects of propolis from Brazil and Bulgaria on fungicidal activity of macrophages against Paracoccidioides brasiliensis

Paracoccidioidomycosis is the most important systemic mycosis in Latin America. Its etiological agent, Paracoccidoides brasiliensis, affects individuals living in endemic areas through inhalation of airborne conidia or mycelial fragments. The disease may affect different organs and systems, with multiple clinical features, with cell-mediated immunity playing a significant role in host defence. Peritoneal macrophages from BALB/c mice were stimulated with Brazilian or Bulgarian propolis and subsequently challenged with P, brasiliensis. Data suggest an increase in the fungicidal activity of macrophages by propolis stimulation, independently from its geographic origin, (C) 2002 Elsevier B.V. Ireland Ltd. All rights reserved.

The Effect of Propolis on Th1/Th2 Cytokine Expression and Production by Melanoma-bearing Mice Submitted to Stress

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Paracoccidioidomycosis associated with acquired immunodificiency syndrome: report of seven cases

São apresentadas as características clínicas e evolutivas de sete pacientes (cinco masculinos e dois do sexo feminino), a maioria dos quais usuários de drogas ilícitas endovenosa, com paracoccidioidomicose associada à Síndrome da Imunodeficiência Adquirida (SIDA/AIDS). em quatro pacientes a paracoccidioidomicose comprometia os pulmões isoladamente, nos demais a doença era generalizada com envolvimento cutâneo. Apenas dois pacientes eram procedentes recentes da zona rural. O que nos faz presumir que nos demais a paracoccidioidomicose doença resultou da reativação de focos latentes da infecção. Dado o papel da imunidade medida por células na defesa do hospedeiro contra o Paracoccidioides brasiliensis, é de se prever crescente ocorrência da associação paracoccidioidomicose -SIDA/AIDS nas áreas endêmicas para ambas as enfermidades.

EXPERIMENTAL PARACOCCIDIOIDOMYCOSIS OF HAMSTER INOCULATED IN THE CHEEK POUCH

We compared the granuloma morphology and immune response of hamsters inoculated with Paracoccidioides brasiliensis (Pb) into the cheek pouch, which lacks lymphatic drainage, and into the footpad, which is rich in lymphatics. Our objective was to better understand the modulation of Pb granuloma in an immunocompetent animal inoculated in an immunologically privileged site. The humoral immune response (ELISA) and cell mediated immunity (footpad test) became positive on days 7 and 14, respectively in animals inoculated into footpad and on days 35 and 60 in animals inoculated into the pouch. Typical epithelioid granulomas were observed at both sites on day 14. The number of fungi gradually decreased from the beginning of the experiment in footpad lesions, but only after day 35 in pouch granulomas, when cell mediated immunity was detectable. The results indicate that typical epithelioid paracoccidioidomycotic granulomas may develop in the absence of a detectable immune response; however, they are incapable of controlling fungal reproduction. Lack of lymphatic drainage delays the appearance of a detectable immune response, but with time fungi escape from the pouch, elicit an immune response and reach other organs. Our results further indicate the importance of the lymphatics in the pathogenesis of paracoccidioidomycosis.

Correlation among immune response, morphogenesis of the granulomatous reaction and spleen lymphoid structure in murine experimental paracoccidioidomycosis

We studied the correlation among cellular immune response, the pattern of lung granulomatous lesions and alterations in spleen lymphoid structure in Swiss mice inoculated intravenously with Paracoccidioides brasiliensis strain 18. The animals were evaluated at 24, 48 and 96 h after infection and further studied weekly for 18 weeks by: (i) the macrophage migration inhibition test with phytohemagglutinin (PHA) and P. brasiliensis antigen (PbAg); and (ii) histopathology of the lung and spleen lesions. One group of animals was gamma -irradiated (8 Gy), infected under the same conditions and evaluated for the pattern of lung granulomatous lesions and spleen lymphoid structure at 24, 48 and 96 h after infection. During the first week of infection, the non-irradiated animals presented a positive response to PHA and PbAg, compact granulomas in the lungs and a typical hyperplasia of the spleen white pulp. However, from weeks 2 to 5, a depression of the cell-mediated immunity (CMI) response to PHA and PbAg was observed in association with granulomas presenting only large mononuclear cells and lacking both giant cells and a peripheral halo of small mononuclear cells. This pattern of granuloma formation was similar to that seen in gamma -irradiated animals, whose cells involved in CMI were absent. After week 7, the non-irradiated animals showed granulomas characterized by the presence of giant cells and a peripheral halo of small mononuclear cells. This type of granuloma was formed concomitantly with recovery of the CMI and of the lymphoid structure of the spleen. The results showed a correlation among granulomas composed of large mononuclear cells, hypoplasia of the splenic tissue and impaired CMI. This correlation indicated that although granuloma morphogenesis per se does not depend on the activation of CMI, this response is important at later stages during modulation of the cellular composition of the granulomas.

Experimental pulmonary paracoccidioidomycosis in the Syrian hamster: morphology and correlation of lesions with the immune response.

A model for pulmonary paracoccidioidomycosis in the hamster is described. The disease was induced by intratracheal inoculation of 1.7 x 10(5) viable yeast forms of P. brasiliensis. Lung histopathology, dissemination lesions and humoral and cellular immune responses were investigated at intervals up to 24 weeks after infection. Humoral immunity was studied by immunodiffusion and complement fixation tests. Cell-mediated immunity was evaluated in vitro by the macrophage migration inhibition test in the presence of phytohaemagglutinin and P. brasiliensis soluble antigen, and in vivo by the paracoccidioidin test. Thirty out of 35 infected animals (85.7%) developed pulmonary paracoccidioidomycosis. Dissemination lesions were observed in regional lymph nodes (82.8%), liver (8.5%) and spleen (5.7%). Lung involvement was mainly around bronchi and vessels. Regional lymph nodes were severely involved from the fourth week on, acquiring a pseudotumoral aspect at later stages. Specific antibodies were detected from the fourth week on, with titres increasing progressively. The cellular immune response to phytohaemagglutinin was intact throughout the experiment and the response to P. brasiliensis antigen was already detectable by the second week and remained positive to the end of the experiment. The skin test became positive from the fourth week on. Inoculation by the intratracheal route represents a highly effective way of infecting hamsters with P. brasiliensis, with the induction of localized disease, good antibody production and intact cell immunity.