Population genetic analysis of large sequence polymorphisms in Plasmodium falciparum blood-stage antigens

Plasmodium falciparum, the causative agent of human malaria, invades host erythrocytes using several proteins on the surface of the invasive merozoite, which have been proposed as potential vaccine candidates. Members of the multi-gene PfRh family are surface antigens that have been shown to play a central role in directing merozoites to alternative erythrocyte receptors for invasion. Recently, we identified a large structural polymorphism, a 0.58 Kb deletion, in the C-terminal region of the PfRh2b gene, present at a high frequency in parasite populations from Senegal. We hypothesize that this region is a target of humoral immunity. Here, by analyzing 371 P. falciparum isolates we show that this major allele is present at varying frequencies in different populations within Senegal, Africa, and throughout the world. For allelic dimorphisms in the asexual stage antigens, Msp-2 and EBA-175, we find minimal geographic differentiation among parasite populations from Senegal and other African localities, suggesting extensive gene flow among these populations and/or immune-mediated frequency-dependent balancing selection. In contrast, we observe a higher level of inter-population divergence (as measured by F(st)) for the PfRh2b deletion, similar to that observed for SNPs from the sexual stage Pfs45/48 loci, which is postulated to be under directional selection. We confirm that the region containing the PfRh2b polymorphism is a target of humoral immune responses by demonstrating antibody reactivity of endemic sera. Our analysis of inter-population divergence suggests that in contrast to the large allelic dimorphisms in EBA-175 and Msp-2, the presence or absence of the large PfRh2b deletion may not elicit frequency-dependent immune selection, but may be under positive immune selection, having important implications for the development of these proteins as vaccine candidates. (C) 2009 Elsevier B.V. All rights reserved.

CD8(+) T cell adjuvant effects of Salmonella FliCd flagellin in live vaccine vectors or as purified protein

Salmonella flagellin, the flagellum structural subunit, has received particular interest as a vaccine adjuvant conferring enhanced immunogenity to soluble proteins or peptides, both for activation of antibody and cellular immune responses. In the present study, we evaluated the Salmonella enterica FliCd flagellin as a T cell vaccine adjuvant using as model the 9-mer (SYVPSAEQI) synthetic H2(d)-restricted CD8(+) T cell-specific epitope (CS(280-288)) derived from the Plasmodium yoelii circumsporozoite (G) protein. The FliCd adjuvant effects were determined under two different conditions: (i) as recombinant flagella, expressed by orally delivered live S. Dublin vaccine strains expressing the target CS(280-288) peptide fused at the central hypervariable domain, and (ii) as purified protein in acellular vaccines in which flagellin was administered to mice either as a recombinant protein fused or admixed with the target CS(280-288) peptide. The results showed that CS(280-288)-specific cytotoxic CD8(+) T cells were primed when BALB/c mice were orally inoculated with the expressing the CS280-288 epitope S. Dublin vaccine strain. In contrast, mice immunized with purified FliCd admixed with the CS280-288 peptide and, to a lesser extent, fused with the target peptide developed specific cytotoxic CD8(+) T cell responses without the need of a heterologous booster immunization. The CD8(+) T cell adjuvant effects of flagellin, either fused or not with the target peptide, correlated with the in vivo activation of CD11c(+) dendritic cells. Taken together, the present results demonstrate that Salmonella flagellins are flexible adjuvant and induce adaptative immune responses when administered by different routes or vaccine formulations. (C) 2009 Elsevier Ltd. All rights reserved.

HPV16 Tumor Associated Macrophages Suppress Antitumor T Cell Responses

Purpose: High-risk human papillomavirus (HPV) is the main etiologic factor for cervical cancer. The severity of HPV-associated cervical lesions has been correlated to the number of infiltrating macrophages. The objective of this work is to characterize the role of tumor-associated macrophages (TAM) on the immune cellular response against the tumor. Experimental Design: We used the HPV16 E6- and E7-expressing TC-1 mouse tumor model to study the effect of TAM on T-cell function in vitro, and depleted TAM, using clodronate-containing liposomes, to characterize its role in vivo. Results: TAM, characterized by the positive expression of CD45, F4/80, and CD11b, formed the major population of infiltrating tumor cells. TAM displayed high basal Arginase I activity, producing interleukin-10 (IL-10); they were resistant to iNOSll activity induction, therefore reversion to M1 phenotype, when stimulated in vitro with lipopolysaccharide/IFN gamma, indicating an M2 phentoype. In cultures of isolated TAM, TAM induced regulatory phenotype, characterized by IL-10 and Foxp3 expression, and inhibited proliferation of CD8 lymphocytes. In vivo, depletion of TAM inhibited tumor growth and stimulated the infiltration of tumors by HPV16 E7(49-57)-specific CD8 lymphocytes, whereas depletion of Gr1(+) tumor-associated cells had no effect. Conclusions: M2-like macrophages infiltrate HPV16-associated tumors causing suppression of antitumor T-cell response, thus facilitating tumor growth. Depletion or phenotype alteration of this population should be considered in immunotherapy strategies.

Estudo da distribuição e quantificação dos linfócitos CD8 e CD20 nas lesões inflamatórias periapicais crônicas

A lesão inflamatória periapical é uma patologia bastante frequente, sendo na maioria dos casos, consequência da cárie dental. O objetivo deste trabalho foi quantificar as populações linfocitárias CD8+ e CD20+ em lesões inflamatórias periapicais crônicas. Foram utilizadas 90 lesões inflamatórias periapicais. Através da técnica de imunohistoquímica pelo método da estreptoavidina-biotina, utilizou-se os marcadores CD8 e CD20 para identificação dos linfócitos T citotóxicos/supressores e linfócitos B, respectivamente. A contagem das células foi feita em 3 campos microscópicos da lâmina, mantendo-se um aumento de 400 vezes. A média da contagem das células CD8+ foi de 7,72 células para os cistos inflamatórios, enquanto que nos grupos cisto abscedado e abscesso crônico foi 11,25 e 11,62, respectivamente, com diferença estatisticamente significante. A média da contagem das células CD20+ foi 12,19; 11,06 e 12,91 células nos cistos abscedados, cistos inflamatórios e abscessos crônicos, respectivamente, sem diferença estatisticamente significante. As lesões inflamatórias periapicais supuradas apresentaram número maior de linfócitos CD8+ do que as lesões não supuradas e; a presença de supuração e proliferação epitelial nas lesões não interferiu na quantidade de linfócitos CD20+ presentes.

Estudo dos linfócitos CD20, CD8 e CD4 nas hiperplasias inflamatórias e sua relação com a infecção por candida sp

O objetivo do presente estudo é mapear e quantificar as populações de células CD 20, CD 8 e CD 4+ em Hiperplasias inflamatórias (HI) e estabelecer relação com a infecção por Candida sp. Foram utilizados 41 casos de HI do Laboratório de Patologia Bucal da UFRGS. Novos cortes de todos os casos foram submetidos à técnica de coloração do PAS, criando – se 2 grupos: com e sem infecção por Candida sp. Seguiu – se a marcação imunohistoquímica com os anticorpos monoclonais anti CD 20, anti CD 8 e anti CD 4, para se avaliar a localização, a distribuição e quantificação das células positivamente marcadas em 3 campos consecutivos (400x), escolhidos sobre a área de maior concentração do infiltrado inflamatório. Os resultados da recontagem dos campos mostraram que o examinador estava calibrado pelo teste “t”de Student. As células CD 8+ apresentaram localização próxima às hifas de Candida sp. e foram mais numerosas no grupo com infecção (diferença estatisticamente significante p= 732 x 10-20). As células CD 20 e CD 4 positivas não apresentaram relação com a infecção por Candida sp. Concluiu – se que as células CD 8+ apresentaram localização relacionada às hifas de Candida sp., além de uma razão células positivamente marcada/ linfócitos totais estatisticamente mais alta no grupo com infecção por Candida sp.

Perfil de memória e ativação de linfócitos T na leishmaniose visceral

Visceral leishmaniasis (VL) in Brazil is a disease caused by Leishmania infantum chagasi (L.i.chagasi). The clinical evolution post-infection depends on the vertebrate host immune response, which is genetically mediated. This study aimed to evaluate the immune response of individuals living in endemic area for VL in the state of the Rio Grande do Norte, considering individuals with VL under treatment (n = 9), recovered VL <1 year post treatment (n = 10), > 10 years posttreatment (n = 9), uninfected individuals living in endemic areas (n = 7), individuals that lost DTH response (n=6) and asymptomatic individuals for VL (n=9). Peripheral blood cells were evaluated in the presence and absence of soluble Leishmania antigens (SLA) and ex vivo, to determine activation, presence of regulatory cells and memory cells. The Leishmania parasitemia and anti-Leishmania antibodies were determined respectively by qPCR and ELISA. Cells from individuals with VL under treatment showed less cell activation after stimulation with SLA for the markers CD4/CD69, CD8/CD69 and CD8/CD25 compared with VL post treatment treatment (p <0.001). Apparently uninfected individuals have a higher cell activation than symptomatic VL (p <0.001), with the exception of CD8/CD25 marker (p = 0.6662). On the other hand, in the ex-vivo group, significant differences were observed for CD4/CD69, CD8/CD69 and CD8/CD25 between the 4 groups due to increased cell activation present in cells of individuals symptomatic LV (p <0.001). VL cells under treatment, ex vivo, have a lower percentage of memory cells (CD4/CD45RO and CD8/CD45RO) than individuals VL post-treatment or control group (p = <0.01). Likewise, individuals with symptomatic VL have fewer regulatory cells when stimulated by SLA [CD4/CD25 (p = 0.0022) and CD4/FOXP3 (p = 0.0016)] and in the ex-vivo group (p = 0.0017). Finally, DNA isolated from recovered VL contained Leishmania DNA, supporting the hypothesis of non-sterile clinical cure for Leishmania infection. Recovered VL, even 10 years after treatment have high levels of memory cells, which may be due to the presence of stimulation, either by reexposure to Leishmania or non-sterile cure

Leishmania (Viannia) shawi purified antigens confer protection against murine cutaneous leishmaniasis

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

Emprego da citometria de fluxo na avaliação do perfil imunofenotípico de pacientes com leucemia linfocítica crônica

Chronic lymphocytic leukemia (B-CLL) is a clonal proliferation of mature B lymphocytes characterized by indolent clinical course. Biologically this clonallity is characterized by low expression of surface immunoglobulin (sIg) with restriction to a single immunoglobulin light chain associated with high expression of CD5 antigen and positivity to B cell antigens lymphocytes such as CD19, CD20 and CD23 and negativity to FMC7. The immunological profile and morphological analysis of lymphoid cells are the main means for the differential diagnosis of B-CLL from other chronic lymphoproliferative diseases. The aim of this study was to evaluate the expression pattern of a variety of membrane antigens in leukemic cells originating from patients with B-CLL. In this study, peripheral blood samples from 80 patients with B-CLL were analyzed by multiparametric flow cytometry in addition to routine hematologic exams, using a panel of monoclonal antibodies (MoAb): CD45/CD14, CD3/CD19/CD45, CD4/CD8 / CD3, CD20/CD5/CD3, CD3/CD16-56/CD45, CD2/CD7, FMC7/CD23, CD103/CD22/CD20, HLADR/CD38, CD10/CD19, CD1a, CD11b and also IgM/gD, kappa and lambda immunoglobulin light chains for the detection of surface immunoglobulin and clonal restriction for immunoglobulin light chain. The Hematological data were obtained from the hematological analyzer and cytomorphological analysis in blood film stained by Leishmann. The study samples consisted of 45 men and 35 women, ages ranging from 55 to 84 years (mean 65 years). Complete white blood count showed count ranging from 10.0 to 42.0 x 109/l. (mean 50.0 x 109/l) and lymphocytes count greater than 5.0 x 109/l in all cases. The neoplastic cells displayed B-CLL phenotype (CD5+/CD19+/CD20+/HLADR+/CD23+) in the vast majority of the cases, associated to failed to stain for T cell markers (CD1a, CD2, CD4, CD3, CD7, CD8), CD103, CD14 and FMC7. Leukemic cells of most patients also expressed low intensity of IgM and IgD with restricted kappa light chain, in most cases (59,7%). This observation highlights the importance of immunophenotyping for correct diagnosis of chronic lymphoproliferative syndromes and the panel of MoAb used was sufficient for diagnostic confirmation of B-CLL

Avaliação dos marcadores celulares por citometria de fluxo nos portadores de leucemia mieloide aguda atendidos no Hemocentro do Rio Grande do Norte-Hemonorte

The acute myeloid leukemia (AML) is a disease in which malignant myeloblasts expand, build up and suppress normal hematopoietic activity would represent a major diagnostic challenge. With the advent of immunophenotyping by flow cytometry, the diagnosis of these tumors have become more faithful, facilitating the treatment and monitoring of patients. The objectives of this study: diagnosis and classification of AML based on immunophenotyping by flow cytometry with a panel of AcMo specific for acute leukemias, set the frequency of AML in samples from patients with acute leukemias sent to the Department of Hematology Blood Center of Rio Grande do Norte - HEMONORTE, establish standards of antigen expression for different subtypes of acute leukemia and its correlation with the newly diagnosed cases refractory to treatment and recurrence of the disease, standardization of methods for detection and labeling of surface antigens by flow cytometry and intracytoplasmic flow, and observe the frequency of acute leukemia with aberrant phenotypes rare. During the study, 351 were diagnosed acute leukemia, and 179 (51%) classified as AML and 172 (49%) and ALL, which were excluded from the present work. Of the 179 AML, 92 (51.4%) were female and 87 (48.6%) were male, with ages ranging from 3 to 95 years of ag, with higher incidence in individuals in the age group of 41 to 65. Splenomegaly was the clinical finding more present, a total of 147 cases (82.1%), followed by hepatomegaly present in 132 cases (73.7%). The hemorrhagic events were observed in 55 cases (30.7%). Lymphadenopathy in turn was detected in 20 of 179 cases (11.2%). In order to classify subtypes of AML, we used a large panel of monoclonal antibodies, obtaining the following results: AML M0, 02 (1.1%) AML M1, 40 (22.3) AML M2, 60 (33.5) AML M3, 22 (12.3%) AML M4, 10 (5.6) AML M5, 13 (7.3%) AML M6 06 (3.4%) and AML M7 01 (0.6%). We observed some cases with aberrant expression of some antigens such as CD7, CD4, CD19, CD3, CD5 and TdT, CD 7 was present in 30 (16.8%), CD4 in 5 (2.8%), the CD 3 in 5 (2.8%), the CD19 in 3 (1.7%), the CD5 in 3 (1.7%) and TDT was in 7 (3.9%) cases of AML .the CD8 and CD79a was present in only a 1 case.

Humoral immune response of water buffalo monitored with three different antigens of Toxocara vitulorum

Humoral immune response of water buffalo naturally infected with Toxocara vitulorum was monitored using three different antigens of this parasite in serum and colostrum of buffalo cows and calves. Soluble extract (Ex) and excretory/secretory (ES) larval antigens and perienteric fluid antigen (Pe) of adult T vitulorum were used to measure the antibody levels by an indirect ELISA. Serum of 7-12 buffalo cows for the first 365 days and colostrum of the same number of buffalo cows for the first 60 days of parturition, and serum of 8-10 buffalo calves for the first 365 days afterbirth were assayed. The ELISA detected antibodies against all three T vitulorum antigens in the colostrum and serum of 100% of buffalo cows and calves examined. The highest antibody levels against Ex, ES and Pe antigens were detected in the buffalo cow sera during the perinatal period and were maintained at high levels through 300 days after parturition. on the other hand, colostrum antibody concentrations of all three antigens were highest on the first day post-parturition, but decreased sharply during the first 15 days. Concomitantly to the monitoring of immune response, the parasitic status of the calves was also evaluated. In calves, antibodies passively acquired were at the highest concentrations 24 h after birth and remained at high levels until 45 days coincidentally with the peak of T vitulorum infection. The rejection of the worms by the calves occurred simultaneously with the decline of antibody levels, which reached their lowest levels between 76 and 150 days. Thereafter, probably because of the presence of adults/larvae stimulation, the calves acquired active immunity and the antibodies started to increase slightly in the serum and plateaued between the days 211 and 365. All three antigens were detected by the serum antibodies of buffalo calves; however, the concentration of anti-Pe antibody was higher than anti-EX and anti-ES, particularly after 90 days of age. By conclusion, the buffalo cows develop immunity and keep high levels of antibodies against T vitulorum-Ex, ES and Pe antigens and these antibodies are transferred to their calves through the colostrum. This passively acquired immunity does not protect the calves against the acquisition of the infection, but these antibodies, passively or actively acquired, may have an important role during worm rejection by the calves and prevention of intestinal reinfection. (C) 2004 Elsevier B.V. All rights reserved.

Dendritic cell are able to differentially recognize Sporothrix schenckii antigens and promote Th1/Th17 response in vitro

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

Production of Trichophyton mentagrophytes antigens and their characterization in mice

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

Taenia saginata: Production and characterization of monoclonal antibodies against Taenia saginata metacestode antigens

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

Expressão imuno-histoquímica do cd8, foxp3, Tgf β, tnf α e nf-кb em displasias epiteliais e Carcinomas epidermóides orais

The Oral Epithelial Dysplasia (OED) is the lesion that precedes or co-exists with the Oral Squamous Cell Carcinoma (OSCC), presenting molecular and/or histological similar alterations. The divergences about the malignization potential of OEDs and the role of inflammation in this process make hard the early diagnosis and evaluation of OSCCs aggressiveness. Thus, it became the goal of this study to evaluate the role of inflammation in oral carcinogenesis and tumoral aggressiveness. For this purpose a morphological study was performed in 20 OED cases and 40 OSCC cases to detect the malignization potential of OEDs and the histologic malignancy grading (HMG) of OSCCs, analyzing superficial masses for dismorphism evaluation and the invasive front for evaluation of tumoral growing; and immunohistochemical, using anti-CD8, anti-FOXP3, anti-TGFβ, anti-TNFα and anti-NF-кB antibodies, comparing their with the types lesion, histological degree and intensity of the inflammatory infiltrate. The results were statistically significant for the parameters: cell maturity (p=0,0001), masses presence (p=0,038) and dismorphism (p=0,037), when associated to HMG. To compare the expression of the markers with the types lesion, a significantly higher expression of CD8 (p=0,001) and NF-кB (p=0,002) in the OED, and also a smaller expression of the epithelial TGFβ in the severe OEDs (p=0,011), without significant expression between OSCC degrees. By relating the expression of the studied markers with the inflammatory infiltrate intensity, a positive relation was observed with: inflammatory TNFα(p=0,003), epithelial TNFα and NF-кB (p=0,051 and p=0,004), in OEDs; and with CD8 (p=0,021) and TNFα (p=0,015) in conjunctive OSCCs; and a negative relation with epithelial TNFα (p=0,034) in OSCCs. No significant relation was found between FOXP3 with any of the studied variables. These findings lead to the conclusion that, the study of the invasive front is as important as the study of superficial masses for the evaluation of tumoral aggressiveness; the intensity of the inflammatory infiltrate has no use as a parameter for prognostic evaluation of OSCC in routine exams, but, the molecular events detected in this study may be necessary to give basis for determining the malignant potential in OEDs and aggressiveness in OSCCs