T follicular helper cells: Their development and importance in the context of helminthiasis.

The development of T follicular helper cells (Tfh) is a multifactorial process that occurs in multiple stages. After their activation the Tfh cells interact with the B cells to complete their differentiation. During this process, the Tfh cells begin to express canonical molecules such as the transcription factor B-cell lymphoma 6 protein, the CXC chemokine receptors type 5, and the inducible T-cell costimulator, as well as secreting other molecules such as IL-21. This whole process is regulated positively and negatively by several factors so that the best response is offered in the face of diseases of various origins, among them helminthiasis. In this context, the role of circulating Tfh, IL-4 and IgG subtypes is essential for an effective response against these pathogens. In this review, the migration process and the differentiation of Tfh, the regulation, their cell subtypes and the role of Tfh in the context of helminth infections will be addressed.

Differential Expression of IgM and IgD Discriminates Two Subpopulations of Human Circulating IgM+IgD+CD27+ B Cells That Differ Phenotypically, Functionally, and Genetically.

The origin and function of blood IgM+IgD+CD27+ B cells is controversial, and they are considered a heterogeneous population. Previous staining of circulating B cells of healthy donors with rotavirus fluorescent virus-like particles allowed us to differentiate two subsets of IgM+IgD+CD27+: IgMhi and IgMlo B cells. Here, we confirmed this finding and compared the phenotype, transcriptome, in vitro function, and Ig gene repertoire of these two subsets. Eleven markers phenotypically discriminated both subsets (CD1c, CD69, IL21R, CD27, MTG, CD45RB, CD5, CD184, CD23, BAFFR, and CD38) with the IgMhi phenotypically resembling previously reported marginal zone B cells and the IgMlo resembling both naïve and memory B cells. Transcriptomic analysis showed that both subpopulations clustered close to germinal center-experienced IgM only B cells with a Principal Component Analysis, but differed in expression of 78 genes. Moreover, IgMhi B cells expressed genes characteristic of previously reported marginal zone B cells. After stimulation with CpG and cytokines, significantly (p < 0.05) higher frequencies (62.5%) of IgMhi B cells proliferated, compared with IgMlo B cells (35.37%), and differentiated to antibody secreting cells (14.22% for IgMhi and 7.19% for IgMlo). IgMhi B cells had significantly (p < 0.0007) higher frequencies of mutations in IGHV and IGKV regions, IgMlo B cells had higher usage of IGHJ6 genes (p < 0.0001), and both subsets differed in their HCDR3 properties. IgMhi B cells shared most of their shared IGH clonotypes with IgM only memory B cells, and IgMlo B cells with IgMhi B cells. These results support the notion that differential expression of IgM and IgD discriminates two subpopulations of human circulating IgM+IgD+CD27+ B cells, with the IgMhi B cells having similarities with previously described marginal zone B cells that passed through germinal centers, and the IgMlo B cells being the least differentiated amongst the IgM+CD27+ subsets.

Oral infection of mice and host cell invasion by Trypanosoma cruzi strains from Mexico.

Oral infection by Trypanosoma cruzi has been responsible for frequent outbreaks of acute Chagas disease in the north of South America and in the Amazon region, where T. cruzi genetic group TcI predominates. TcI strains from different geographical regions have been used in oral infection in mice, but there is no information about strains from Mexico where TcI is prevalent. Here, we analyzed four Mexican strains as concerns the course of oral infection, the ability to invade host cells in vitro, and the profile of metacyclic trypomastigote surface molecules gp82 and gp90 that are implicated in parasite internalization. Oral infection of mice with metacyclic forms of all strains resulted in reduced blood and tissue parasitism, and mild to moderate inflammatory process in the heart/skeletal muscle. They expressed pepsin-resistant gp82 and gp90 molecules at high levels and invaded host cells poorly in full nutrient medium and efficiently under nutrient-deprived condition. The properties exhibited by Mexican strains were similar to those displayed by TcI strains from other geographical regions, reinforcing the notion that these features are common to the genetic group TcI as a whole.

Efeito da Migalina sobre atividade inflamatória com diferentes estímulos em linhagens de macrófagos J774 e derivados de medula óssea

Natural polyamines putrescine, spermine and spermidine are essential for cell growth and proliferation, protein and nucleic acid synthesis, cell matrix repair, adhesion and signaling processes. Mygalin was isolated from hemocytes of the spider Acanthoscurria gomesiana and identified as a bis-acylpolyamine analogue to spermidine, possessing microbicidal activity. Macrophages are innate immune response cells fundamental in the inflammatory process and are programmed to detect molecular patterns associated with pathogens. Immune cell surface molecules and cytokines are the key components for the achievement of the inflammatory response. Due to the limited knowledge of the role of Mygalin in the immune response, we investigated its immunomodulatory function in macrophages J774 and macrophages derived from C57BL / 6 mouse marrow MODM against several stimuli in different assays. The cells were either preincubated with Mygalin 40-160 μg/ml for 2 hours prior to stimulation or not with LPS 100 ng/ml, Toxoplasma gondii antigen 10-25-50 μg/ml, IFN-γ 1 ng/ml and Zimosan 10 μg/ml After 24 hours, hydrogen peroxide production was examined by the fluorimetric method with and without PMA (200 nM) and the alteration of the expression of the surface molecules by flow cytometry FACS. Supernatants from the J774 and MODM assays were harvested for quantify production of IL-12p40 ELISAand nitrite Griess. Treatment of cells with Mygalin and Toxoplasma gondii didn’t induce IL-12p40 and NO production. In J774 IFN-γ didn’t change the amount of NO in the control, but the LPS increased even in the presence of Mygalin and the same happened with LPS+IFN-γ. Zimosan increased NO production in J774, but IFN- γ reduced it. In MODM independent of the stimulus there was no alteration of the NO synthesis compared to control. Mygalin didn’t increase the production of IL-12p40 when the cells were stimulated by LPS and LPS+IFN-γ. Zimosan induced the synthesis of IL-12p40, but with IFN-γ the production reduced. We evaluated the effect of Mygalin on the production of hydrogen peroxide in MODM that Migalina reduced H2O2 production in the presence of PMA. Regarding the expression of CD11b: in J774 the LPS and LPS+IFN-γ decreased expression; F4 / 80: in MODM the LPS increased its synthesis; CD86: in J774 the LPS decreased expression even with IFN-γ, and in MODMall stimuli reduced synthesis even with Mygalin; CD284: no change in J774cells; CD206: LPS and LPS+IFN-γ there was a slight increase of the synthesis.The mechanisms involved are being analyzed for a better characterization ofthe role of macrolide in the macrophage.

Poliaminas naturais (putrescina, espermina e espermidina) são fundamentais para o crescimento e proliferação celular, na síntese de proteínas e de ácidos nucleicos, reparação da matriz celular, adesão e processos de sinalização. A Migalina foi isolada de hemócitos da aranha Acanthoscurria gomesiana e identificada como uma bis-acilpoliamina análoga a espermidina, possuindo atividade microbicida. Macrófagos são células da resposta imune inata fundamentais no processo inflamatório e são programadas para detectar padrões moleculares associados a patógenos. Moléculas de superfície de células imunes e as citocinas são componentes fundamentais para a realização da resposta inflamatória. Devido ao limitado conhecimento do papel da Migalina na resposta imune investigamos sua função imunomoduladora em macrófagos J774 e macrófagos derivados de medula de camundongo C57BL/6 (MODM) frente a vários estímulos em diferentes ensaios. As células foram pré- incubadas ou não com Migalina (40-160 μg/mL) por 2 horas antes da estimulação ou não com LPS (100 ng/mL), antígeno de Toxoplasma gondii (10- 25-50 μg/mL), IFN-γ (1 ng/mL) e Zimosan (10 μg/mL). Após 24 horas, a produção de peróxido de hidrogênio foi examinada pelo método fluorimétrico com e sem PMA (200 nM) e a alteração da expressão de moléculas de superfície por meio de citometria de fluxo (FACS). Os sobrenadantes dos ensaios de J774 e MODM foram colhidos para análises de IL-12p40 (ELISA) e nitrito (Griess). O tratamento das células com Migalina e Toxoplasma gondii não induziu a produção IL-12p40 e de NO. Em J774 o IFN-γ não alterou a quantidade de NO do controle, mas o LPS aumentou mesmo na presença de Migalina e o mesmo ocorreu com LPS+IFN-γ. O Zimosan aumentou a produção de NO em J774, mas o IFN-γ a reduziu. Em MODM independente do estímulo não houve alteração da síntese de NO comparado ao controle. A Migalina não aumentou a produção de IL-12p40 quando as células foram estimuladas com LPS e LPS+IFN-γ. Zimosan induziu a síntese de IL-12p40, mas na presença de IFN-γ a produção reduziu. Avaliamos o efeito da Migalina na produção de peróxido de hidrogênio em MODM no qual a Migalina reduziu a produção de H2O2 na presença de PMA. Com relação à expressão de CD11b: em J774 o LPS e LPS+IFN-γ diminuiu a expressão; F4/80: em MODM LPS aumentou sua síntese; CD86: em J774 LPS diminuiu a expressão mesmo com IFN-γ e em MODM todos os estímulos reduziu a síntese mesmo com Migalina; CD284: sem alteração nas células J774; CD206: LPS e LPS+IFN-γ houve um leve aumento da síntese. Os mecanismos envolvidos estão sendo analisados para melhor caracterização do papel da Migalina em macrófagos.

Determination of normal expression patterns of CD86, CD210a, CD261, CD262, CD264, CD358, and CD361 in peripheral blood and bone marrow cells by flow cytometry.

In 2010, new monoclonal antibodies were submitted to the 9th International Workshop on Human Leukocyte Differentiation Antigens, and there are few studies demonstrating normal expression patterns of these markers. Thus, the objective of this study was to determine the normal patterns of cell expression of CD86, CD210a, CD261, CD262, CD264, CD358, and CD361 in peripheral blood (PB) and bone marrow (BM) samples by flow cytometry. In the present study, CD86 was expressed only in monocytes and B lymphocytes in PB and in monocytes and plasma cells in BM. Regarding CD210a expression, in PB samples, monocytes and NK cells showed weak expression, while neutrophils, B and T lymphocytes, and basophils showed weak and partial expression. In BM samples, expression of CD210a was observed in eosinophils, monocytes, and B and T/NK lymphocytes. Weak expression of CD210a was also observed in neutrophilic cells and plasma cells. All B cell maturation stages had weak expression of CD210a except for immature B cells, which did not express this marker. In the present study, no cell type in PB samples showed positivity for CD261 and, in BM samples, there was very weak expression in neutrophilic series, monocytes, and B lymphocytes. Conversely, plasma cells showed positivity for CD261 with a homogeneous expression. For CD262, there was weak expression in monocytes, neutrophils, and B lymphocytes in PB samples and weak expression in monocytes, B lymphocytes, and plasma cells in BM samples. The evaluation of CD264 showed very weak expression in B cells in PB samples and no expression in BM cells. Very weak expression of CD358 was observed in neutrophils, monocytes, and B lymphocytes in PB and BM samples. In addition, in BM samples, plasma cells and T lymphocytes showed weak expression of CD358. In relation to the maturation stages of B cells, there was weak expression in pro-B cel, pre-B cell, and mature B cell. In the present study, it was possible to observe expression of CD361 in all cell types analyzed in PB and BM samples. The analyzed markers presented varied profiles of expression and, in some cases, these profiles were different from those observed in other studies. Further studies are needed to evaluate these molecules, mainly in relation to a possible application in the diagnosis of hematological malignancies or as new therapeutic targets for the treatment of hematological neoplasms or autoimmune diseases.

Avaliação da interação in vitro do fungo oportunista, emergente, paecilomyces lilacinus, agente causal da hialohifomicose, com células apresentadoras de antígenos humanas (macrófagos e células dentríticas) / Assessment of in vitro interaction of opportunistic emerging fungus, Paecilomyces lilacinus, causal agent of hyalohyphomycosis with human antigen-presenting cells (macrophages and dendritic cells)

Paecilomyces lilacinus, um dos agentes causais da hialohifomicose, é um fungo filamentoso, assexuado. Está amplamente distribuído pelo mundo e frequentemente é encontrado como contaminante, proveniente do ar, em espécimes clínicos e em soluções consideradas estéreis. Acomete principalmente pacientes comprometidos imunologicamente, porém pacientes imunocompetentes também podem apresentar manifestações clínicas da doença, sendo esse fungo, atualmente, considerado como um importante patógeno oportunista humano. O presente trabalho teve por objetivo a investigação da interação de conídios de P. lilacinus, proveniente de caso clínico de hialohifomicose humana, com células apresentadoras de antígenos (APCs - macrófagos e células dendríticas). Conídios interagiram com macrófagos e células dendritícas de doadores humanos saudáveis, em diferentes concentrações (conídio/APCs) e tempos de incubação para avaliação qualitativa e quantitativa da interação por microscopia óptica e para estudo do percentual de células expressando moléculas de superfície celular que atuam na resposta imune do hospedeiro. Sobrenadantes provenientes da interação das APCs com os conídios foram coletados para o estudo das citocinas IL1-beta e TNF-alfa...

Os resultados demonstraram que o fungo foi capaz de infectar de modo semelhante ambas as APCs e que a concentração de 1:1 (conídio/APCs) foi a melhor proporção entre as células para avaliar o processo de fagocitose e as etapas de desenvolvimento do fungo assim como o tempo de 6 horas de interação foi o melhor tempo para a certificação da internalização dos conídios pelas APCs. Após o processo de internalização dos conídios pelas APCs os mesmos começaram a se dilatar, formar tubos germinativos e hifas septadas que se ramificaram formando um micélio que destruiu as células humanas no período de 24 horas de observação. A avaliação quantitativa da fagocitose dos conídios pelas APCs demonstrou que não houve diferenças significativas entre o percentual fagocítico dos macrófagos e células dendríticas de cada doador como também entre os doadores. A interação do fungo com as APCs na presença de L-Name não modificou o processo de desenvolvimento do conídio no interior das células humanas, quando comparado a interação das células sem L-Name. Em relação aos marcadores de superfície celular a presença dos conídios não aumentou o percentual de macrófagos expressando CD14, porém aumentou as células expressando B7.1 e DC-SIGN. As células dendríticas apresentaram um percentual semelhante de células positivas para B7.1,na ausência e presença do fungo, e um aumento significativo do percentual de células DC-SIGN positivas na presença dos conídios. Os conídios de P. lilacinus foram capazes de estimular a secreção da citocina IL1-beta pelos macrófagos e células dendríticas e é possível que eles tenham inibido a produção de TNF-alfa pelos macrófagos. Então, os dados aqui apresentados demonstram a capacidade invasiva para APCs e de estimulação de moléculas de superfície importantes na resposta imune do hospedeiro por P. lilacinus, até o momento desconhecida...

Molecular mechanisms of Trypanosoma cruzi infection by oral route

Frequent reports on outbreaks of acute Chagas' disease by ingestion of food contaminated with parasites from triatomine insects illustrate the importance of this mode of transmission. Studies on oral Trypanosoma cruzi infection in mice have indicated that metacyclic trypomastigotes invade the gastric mucosal epithelium. A key molecule in this process is gp82, a stage-specific surface glycoprotein that binds to both gastric mucin and to target epithelial cells. By triggering Ca2+ signalling, gp82 promotes parasite internalisation. Gp82 is relatively resistant to peptic digestion at acidic pH, thus preserving the properties critical for oral infection. The infection process is also influenced by gp90, a metacyclic stage-specific molecule that negatively regulates the invasion process. T. cruzi strains expressing high gp90 levels invade cells poorly in vitro. However, their infectivity by oral route varies considerably due to varying susceptibilities of different gp90 isoforms to peptic digestion. Parasites expressing pepsin-susceptible gp90 become highly invasive against target cells upon contact with gastric juice. Such is the case of a T. cruzi isolate from an acute case of orally acquired Chagas' disease; the gp90 from this strain is extensively degraded upon short period of parasite permanence in the gastric milieu. If such an exacerbation of infectivity occurs in humans, it may be responsible for the severity of Chagas' disease reported in outbreaks of oral infection.