Linfocitopenia T CD4+ associada à sideropenia: relato de caso

Importance of the issue: Idiopathic CD4 T lymphocytopenia is an unusual immune defect in which there is an unexplained deficit of CD4 T cells. This case presents a 39-year-old female patient, with CD4+ T lymphocytopenia, who was not infected with immunosuppressive viruses neither was she subjected to immunosuppressive therapies. Comments: While monitoring the patient, she was found to have very low serum ferritin and, after parenteral iron therapy, there were changes in CD4+ cell levels, indicating that, in this case, lymphocytopenia was secondary to sideropenia. The patient is being kept under strict control of serum iron and periodic immunological evaluation, and she has not showed any clinical and/or laboratory adverse events so far. It is known that iron deficiency is an important factor in the genesis of immunological changes that occur in patients with iron deficiency anemia. It is important to understand the effects of iron deficiency on the immune system due to its high prevalence worldwide. Moreover, it could also help to clarify several cases of idiopathic CD4 lymphocytopenia.Importância da questão: Linfocitopenia T CD4+ idiopática é um defeito imune incomum em que há um déficit inexplicável de células T CD4. Este caso clínico apresenta uma paciente do sexo feminino de 39 anos de idade, com linfocitopenia T CD4+, que não estava infectada por vírus imunossupressores nem foi submetida a terapias imunossupressoras. Comentários: Durante o acompanhamento da paciente, ela apresentou níveis muito baixos de ferritina sérica e, após a terapia parenteral de ferro, houve aumento da quantidade de células CD4+, indicando que, neste caso, linfocitopenia era secundária à sideropenia. A paciente estava sendo mantida sob rigoroso controle de ferro sérico e avaliação imunológica periódica, e não mostrou quaisquer eventos adversos clínicos e/ou laboratoriais até o momento. Com base em mudanças na reatividade imunológica dos pacientes observadas por outros pesquisadores após a suplementação com ferro, é evidente que a deficiência de ferro seria um fator importante na gênese das alterações imunológicas que ocorrem em pacientes com anemia ferropriva. É importante compreender os efeitos da deficiência de ferro no sistema imune, devido à sua elevada prevalência mundial. Essas informações também auxiliariam a esclarecer vários casos de linfocitopenia T CD4+ idiopática

Impaired NK cell cytotoxicity in multiple myeloma caused by the homozygous A91V polymorphism in the perforin gene: a case report: Citotoxicidade de células NK prejudicada no mieloma múltiplo causada pelo polimorfismo A91V em homozigose no gene da perforina: um relato de caso

We describe a 58-year-old man diagnosed with IgG/Kappa multiple myeloma (International Staging System III) treated for eight years with polychemotherapy (VAD schee) and autologous peripheral hematopoietic stem cell transplantation. The patient studied was homozygous C272T polymorphism (PRF1272T/T) by analysis of perforin gene by direct sequencing. This SNP is considered pathogenic and leads to the substitution of the amino acid alanine for valine in codon 91 of the perforin protein. The cytotoxic lymphocytes (CLs) of the patient and of the healthy wild homozygous individual were evaluated for their cytotoxic capacity. Our results show that PRF1272T/T effector cells had significantly reduced ability to induce specific lysis of K562 cells. The NK cells of the patient had three times less intracellular perforin than observed in the wild-type individual. The gene expression of PRF1 and FAS did not differ between the individuals, however the expression of GZMB was approximately 2.5 times higher in the patient. It was also observed that the T-BET expression was approximately 1.7-fold higher and IFN-γ expression was 4.5-fold higher in the PRF1272T/T patient. In conclusion, functional analysis of the CLs of the patient revealed a significant decrease in their cytolytic capacity as well as the amount of perforin present in NK cell granules

Impaired NK cell cytotoxicity in multiple myeloma caused by the homozygous A91V polymorphism in the perforin gene: a case report: Citotoxicidade de células NK prejudicada no mieloma múltiplo causada pelo polimorfismo A91V em homozigose no gene da perforina: um relato de caso

We describe a 58-year-old man diagnosed with IgG/Kappa multiple myeloma (International Staging System III) treated for eight years with polychemotherapy (VAD schee) and autologous peripheral hematopoietic stem cell transplantation. The patient studied was homozygous C272T polymorphism (PRF1272T/T) by analysis of perforin gene by direct sequencing. This SNP is considered pathogenic and leads to the substitution of the amino acid alanine for valine in codon 91 of the perforin protein. The cytotoxic lymphocytes (CLs) of the patient and of the healthy wild homozygous individual were evaluated for their cytotoxic capacity. Our results show that PRF1272T/T effector cells had significantly reduced ability to induce specific lysis of K562 cells. The NK cells of the patient had three times less intracellular perforin than observed in the wild-type individual. The gene expression of PRF1 and FAS did not differ between the individuals, however the expression of GZMB was approximately 2.5 times higher in the patient. It was also observed that the T-BET expression was approximately 1.7-fold higher and IFN-γ expression was 4.5-fold higher in the PRF1272T/T patient. In conclusion, functional analysis of the CLs of the patient revealed a significant decrease in their cytolytic capacity as well as the amount of perforin present in NK cell granules

O papel do extrato EPs 7630 de Pelargonium sidoides na proliferação de linfócitos citotóxicos: The role of Pelargonium sidoides EPs 7630 extract in the proliferation of cytotoxic lymphocytes

Pelargonium sidoides é uma importante planta com propriedades medicinais nativa da África do Sul utilizada principalmente no tratamento de infecções respiratórias e comercializada sob a forma do extrato padronizado EPs 7630. Sua atividade farmacológica inclui efeitos antimicrobianos, antibacterianos, imunomoduladores e antivirais. Entretanto, falta compreender possíveis lacunas sobre as vias de atuação imunoduladoras, entre elas, se possui efeito na proliferação dos linfócitos citotóxicos. Portanto, o objetivo do trabalho foi analisar o efeito do extrato de P. sidoides na proliferação de linfócitos T CD8 e células natural killer (NK) e comparar estas taxas. Para isso, foram avaliadas células mononucleares de sangue periférico (PBMC) de 12 indivíduos saudáveis, sendo 6 do sexo feminino e 6 do masculino, entre 20 e 35 anos. A separação das PBMC foi realizada por centrifugação em gradiente de concentração com Ficoll-Paque™ PLUS e posteriormente incubadas com CFSE. Foram divididas nos seguintes grupos: células não tratadas, tratadas com 10μg/mL de extrato EPs 7630 de P. sidoides (TAKEDA) e controle veículo, contendo em cada um dos grupos células com e sem estímulo de CD3 e CD28. Por fim, as amostras foram adquiridas no citômetro de fluxo FACSCanto II (BD Biosciences). Quanto aos resultados obtidos, tanto nas células NK como nos linfócitos T CD8 o tratamento com EPs 7630 sem estímulo não proporcionou diferença nos parâmetros analisados. Entretanto, quando as células foram estimuladas com CD3 e CD28, obtivemos resultados mais distintos. As células NK apresentaram uma maior taxa de expansão quando tratadas com EPs 7630, assim como no percentual de células não divididas e de segunda geração em relação às células não tratadas. Mesmo com estímulo, os linfócitos T CD8 não apresentaram variações significativas nos parâmetros avaliados quanto ao tratamento com EPs 7630. Comparando as taxas de proliferação de células NK e linfócitos T CD8 tratadas com e sem estímulo, os linfócitos T CD8 tiveram médias mais altas no percentual de células não divididas e as células NK no percentual de células de segunda geração. Concluímos então que o extrato comercial à base de P. sidoides é capaz de modular o sistema imune inato por meio da proliferação de células NK. Logo, espera-se que este trabalho contribua para a divulgação e conhecimento desta alternativa terapêutica

O papel do extrato EPs 7630 de Pelargonium sidoides na proliferação de Linfócitos citotóxicos: The role of Pelargonium sidoides EPs 7630 extract in the proliferation of cytotoxic Lymphocytes

Pelargonium sidoides é uma importante planta com propriedades medicinais nativa da África do Sul utilizada principalmente no tratamento de infecções respiratórias e comercializada sob a forma do extrato padronizado EPs 7630. Sua atividade farmacológica inclui efeitos antimicrobianos, antibacterianos, imunomoduladores e antivirais. Entretanto, falta compreender possíveis lacunas sobre as vias de atuação imunoduladoras, entre elas, se possui efeito na proliferação dos linfócitos citotóxicos. Portanto, o objetivo do trabalho foi analisar o efeito do extrato de P. sidoides na proliferação de linfócitos T CD8 e células natural killer (NK) e comparar estas taxas. Para isso, foram avaliadas células mononucleares de sangue periférico (PBMC) de 12 indivíduos saudáveis, sendo 6 do sexo feminino e 6 do masculino, entre 20 e 35 anos. A separação das PBMC foi realizada por centrifugação em gradiente de concentração com Ficoll-Paque™ PLUS e posteriormente incubadas com CFSE. Foram divididas nos seguintes grupos: células não tratadas, tratadas com 10μg/mL de extrato EPs 7630 de P. sidoides (TAKEDA) e controle veículo, contendo em cada um dos grupos células com e sem estímulo de CD3 e CD28. Por fim, as amostras foram adquiridas no citômetro de fluxo FACSCanto II (BD Biosciences). Quanto aos resultados obtidos, tanto nas células NK como nos linfócitos T CD8 o tratamento com EPs 7630 sem estímulo não proporcionou diferença nos parâmetros analisados. Entretanto, quando as células foram estimuladas com CD3 e CD28, obtivemos resultados mais distintos. As células NK apresentaram uma maior taxa de expansão quando tratadas com EPs 7630, assim como no percentual de células não divididas e de segunda geração em relação às células não tratadas. Mesmo com estímulo, os linfócitos T CD8 não apresentaram variações significativas nos parâmetros avaliados quanto ao tratamento com EPs 7630. Comparando as taxas de proliferação de células NK e linfócitos T CD8 tratadas com e sem estímulo, os linfócitos T CD8 tiveram médias mais altas no percentual de células não divididas e as células NK no percentual de células de segunda geração. Concluímos então que o extrato comercial à base de P. sidoides é capaz de modular o sistema imune inato por meio da proliferação de células NK. Logo, espera-se que este trabalho contribua para a divulgação e conhecimento desta alternativa terapêutica

Cholelithiasis and its complications in sickle cell disease in a university hospital

Abstract Introduction: The clinical manifestations of sickle cell disease are related to the polymerization of hemoglobin S. The chronic hemolysis caused by this condition often causes the formation of gallstones that can migrate and block the common bile duct leading to acute abdomen. Objective: This study aimed to evaluate the profile of patients with sickle cell disease and cholelithiasis. Methods: Patients with sickle cell disease were separated into groups according to the presence or absence of cholelithiasis. Socioepidemiological and clinical characteristics, such as gender, age, use of hydroxyurea and the presence of other hemoglobinopathies were researched in the medical records of patients. Results: A hundred and seven patients with sickle cell anemia were treated at the institution. Of these, 27 (25.2%) had cholelithiasis. The presence of cholelithiasis was higher in the 11–29 age group than in younger than 11 years and over 29 years. No association was found for the presence of cholelithiasis with gender, use of hydroxyurea or type of hemoglobinopathy (hemoglobin SS, hemoglobin SC or sickle beta-thalassemia). Sixteen of the patients had to be submitted to cholecystectomy with 14 of the surgeries being performed by laparoscopy. Complications were observed in three patients and one patient died for reasons unrelated to the surgery. Conclusion: A quarter of patients with sickle cell disease had gallstones, more commonly in the 11- to 29-year age range. Patients should be monitored from childhood to prevent cholelithiasis with preoperative, intra-operative and postoperative care being crucial to reduce the risk of complications in these patients

Specific human ATR and ATM inhibitors modulate single strand DNA formation in Leishmania major exposed to oxidative agent

Leishmania parasites are the causative agents of a group of neglected tropical diseases known as leishmaniasis. The molecular mechanisms employed by these parasites to adapt to the adverse conditions found in their hosts are not yet completely understood. DNA repair pathways can be used by Leishmania to enable survival in the interior of macrophages, where the parasite is constantly exposed to oxygen reactive species. In higher eukaryotes, DNA repair pathways are coordinated by the central protein kinases ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR). The enzyme Exonuclease-1 (EXO1) plays important roles in DNA replication, repair, and recombination, and it can be regulated by ATM- and ATR-mediated signaling pathways. In this study, the DNA damage response pathways in promastigote forms of L. major were investigated using bioinformatics tools, exposure of lineages to oxidizing agents and radiation damage, treatment of cells with ATM and ATR inhibitors, and flow cytometry analysis. We demonstrated high structural and important residue conservation for the catalytic activity of the putative LmjEXO1. The overexpression of putative LmjEXO1 made L. major cells more susceptible to genotoxic damage, most likely due to the nuclease activity of this enzyme and the occurrence of hyper-resection of DNA strands. These cells could be rescued by the addition of caffeine or a selective ATM inhibitor. In contrast, ATR-specific inhibition made the control cells more susceptible to oxidative damage in an LmjEXO1 overexpression-like manner. We demonstrated that ATR-specific inhibition results in the formation of extended single-stranded DNA, most likely due to EXO1 nucleasic activity. Antagonistically, ATM inhibition prevented single-strand DNA formation, which could explain the survival phenotype of lineages overexpressing LmjEXO1. These results suggest that an ATM homolog in Leishmania could act to promote end resection by putative LmjEXO1, and an ATR homologue could prevent hyper-resection, ensuring adequate repair of the parasite DNA

DNA content analysis allows discrimination between Trypanosoma cruzi and Trypanosoma rangeli.

Trypanosoma cruzi, a human protozoan parasite, is the causative agent of Chagas disease. Currently the species is divided into six taxonomic groups. The genome of the CL Brener clone has been estimated to be 106.4-110.7 Mb, and DNA content analyses revealed that it is a diploid hybrid clone. Trypanosoma rangeli is a hemoflagellate that has the same reservoirs and vectors as T. cruzi; however, it is non-pathogenic to vertebrate hosts. The haploid genome of T. rangeli was previously estimated to be 24 Mb. The parasitic strains of T. rangeli are divided into KP1(+) and KP1(-). Thus, the objective of this study was to investigate the DNA content in different strains of T. cruzi and T. rangeli by flow cytometry. All T. cruzi and T. rangeli strains yielded cell cycle profiles with clearly identifiable G1-0 (2n) and G2-M (4n) peaks. T. cruzi and T. rangeli genome sizes were estimated using the clone CL Brener and the Leishmania major CC1 as reference cell lines because their genome sequences have been previously determined. The DNA content of T. cruzi strains ranged from 87,41 to 108,16 Mb, and the DNA content of T. rangeli strains ranged from 63,25 Mb to 68,66 Mb. No differences in DNA content were observed between KP1(+) and KP1(-) T. rangeli strains. Cultures containing mixtures of the epimastigote forms of T. cruzi and T. rangeli strains resulted in cell cycle profiles with distinct G1 peaks for strains of each species. These results demonstrate that DNA content analysis by flow cytometry is a reliable technique for discrimination between T. cruzi and T. rangeli isolated from different hosts

Genetic variation of FcγRIIa induces higher uptake of Leishmania infantum and modulates cytokine production by adherent mononuclear cells in vitro

IntroductionSingle nucleotide variations (SNVs) are specific genetic variations that commonly occur in a population and often do not manifest phenotypically. However, depending on their location and the type of nucleotide exchanged, an SNV can alter or inhibit the function of the gene in which it occurs. Immunoglobulin G (IgG) receptor genes have exhibited several polymorphisms, including rs1801274, which is found in the FcgRIIa gene. The replacement of A with T results in a Histidine (H) to Arginine (R) substitution, altering the affinity of the IgG receptor for IgG subtypes and C-reactive protein (CRP). In this study, we analyzed rs1801274 and its functional implications concerning L. Infantum uptake and cytokine production.MethodsWe genotyped 201 individuals from an endemic area for visceral leishmaniasis to assess the presence of rs1801274 using Taqman probes for a candidate gene study. Additionally, we included seventy individuals from a non-endemic area for a functional study. Subsequently, we isolated and cultivated one-week adherent mononuclear cells (AMCs) derived from the peripheral blood of participants residing in the non-endemic region in the presence of L. infantum promastigotes, with and without antigen-specific IgG and/or CRP. We analyzed the rate of phagocytosis and the production of nitric oxide (NO), tumor necrosis factor (TNF)-a, interleukin (IL)-10, IL-12 p70, IL-1b, IL- 6, and IL-8 in the culture supernatants.Results and discussionIn participants from the endemic region, the A/G (H/R isoform) heterozygous genotype was significantly associated with susceptibility to the disease. Furthermore, SNVs induced a change in the phagocytosis rate in an opsonin-dependent manner. Opsonization with IgG increased the production of IL-10, TNF-a, and IL-6 in AMCs with the H/R isoform, followed by a decrease in NO production. The results presented here suggest that the rs1801274 polymorphism is linked to a higher susceptibility to visceral leishmaniasis