Evaluation of herpesvirus members on hospital admission in patients with systemic lupus erythematous shows higher frequency of Epstein-Barr virus and its associated renal dysfunction / Avaliação de membros da família Herpesviridae na admissão hospitalar de pacientes com lúpus eritematoso sistêmico mostra maior frequência do vírus Epstein-Barr e sua disfunção renal associada

Abstract Introduction: Members of the Herpesviridae family have been described in patients with systemic lupus erythematous (SLE), but the clinical impact on renal function is not well known. Methods: HSV1, HSV2, VZV, EBV, CMV, HHV-6, HHV-7, and HHV-8 were evaluated by molecular biology on admission in blood samples from 40 consecutive SLE patients hospitalized for lupus activity. Results: Patients were 90.0% female, 77.5% non-white, with average age of 32.7 ± 13.6 years. We found positivity for EBV (65.0%), CMV (30.0%), HSV-1 (30.0%), HHV-6 (12.5%), and HHV-7 (7.5%). For all viruses, age, SLEDAI, hematological tests, ferritin, LDH, C-reactive protein, and erythrocyte sedimentation rate (ESR) were not significant. However, EBV positivity was a significant factor for higher serum creatinine (3.0 ± 2.8 vs. 0.9 ± 0.8; P = 0.001) and urea (86 ± 51 vs. 50 ± 46; P = 0.03). Moreover, positive cases for EBV only or with combined co-infections (66.7%-CMV; 58.3%-HSV-1) or negative for EBV only were evaluated by Kruskal-Wallis test again showed statistical significance for serum creatinine and urea (both P ≤ 0.01), with posttest also showing statistical differences for renal dysfunction and EBV presence (alone or in combined co-infections). The presence of EBV viral load was also significant for nephrotic-range proteinuria, renal flare, and the need for hemodialysis. Conclusion: Members of the Herpeviridae family (mainly EBV, HSV-1 and CMV) are common on hospital admission of SLE patients, reaching 65% for EBV, which seems to be associated with renal dysfunction and could reflect a previous association or overlapping disease, which is not well understood.

Resumo Introdução: Membros da família Herpesviridae tem sido descritos em pacientes com lúpus eritematoso sistêmico (LES), mas o impacto clínico na função renal não é bem conhecido. Métodos: Avaliou-se HSV1, HSV2, VZV, EBV, CMV, HHV-6, HHV-7, HHV-8 por biologia molecular na admissão em amostras sanguíneas de 40 pacientes com LES consecutivos hospitalizados por atividade lúpica. Resultados: Pacientes 90,0% mulheres, 77,5% não brancos, idade média 32,7 ± 13,6 anos. Encontramos positividade para EBV (65,0%), CMV (30,0%), HSV-1 (30,0%), HHV-6 (12,5%), HHV-7 (7,5%). Para todos os vírus, idade, SLEDAI, exames hematológicos, ferritina, LDH, proteína C reativa, velocidade de hemossedimentação não foram significativos. Entretanto, positividade para EBV foi estatisticamente significativo para creatinina (3,0 ± 2,8 vs. 0,9 ± 0,8; P = 0,001) e ureia (86 ± 51 vs. 50 ± 46; P = 0,03) séricas mais elevadas. Ademais, casos positivos para EBV isolado ou com coinfecções combinadas (66,7%-CMV; 58,3%-HSV-1) ou negativos apenas para EBV foram avaliados pelo teste Kruskal-Wallis e novamente mostraram significância estatística para creatinina e ureia séricas (ambas P ≤ 0,01), com pós-teste mostrando também diferenças estatísticas para disfunção renal e presença de EBV (sozinho ou em coinfecções combinadas). A presença de carga viral do EBV também foi significativa para proteinúria de faixa nefrótica, inflamação aguda, necessidade de hemodiálise. Conclusão: Membros da família Herpeviridae (principalmente EBV, HSV-1, CMV) são comuns na admissão de pacientes com LES, chegando a 65% para EBV, que parece associar-se à disfunção renal podendo refletir associação prévia ou doença sobreposta, o que não é bem compreendido.

Modulation of the Response to Mycobacterium leprae and Pathogenesis of Leprosy.

The initial infection by the obligate intracellular bacillus Mycobacterium leprae evolves to leprosy in a small subset of the infected individuals. Transmission is believed to occur mainly by exposure to bacilli present in aerosols expelled by infected individuals with high bacillary load. Mycobacterium leprae-specific DNA has been detected in the blood of asymptomatic household contacts of leprosy patients years before active disease onset, suggesting that, following infection, the bacterium reaches the lymphatic drainage and the blood of at least some individuals. The lower temperature and availability of protected microenvironments may provide the initial conditions for the survival of the bacillus in the airways and skin. A subset of skin-resident macrophages and the Schwann cells of peripheral nerves, two M. leprae permissive cells, may protect M. leprae from effector cells in the initial phase of the infection. The interaction of M. leprae with these cells induces metabolic changes, including the formation of lipid droplets, that are associated with macrophage M2 phenotype and the production of mediators that facilitate the differentiation of specific T cells for M. leprae-expressed antigens to a memory regulatory phenotype. Here, we discuss the possible initials steps of M. leprae infection that may lead to active disease onset, mainly focusing on events prior to the manifestation of the established clinical forms of leprosy. We hypothesize that the progressive differentiation of T cells to the Tregs phenotype inhibits effector function against the bacillus, allowing an increase in the bacillary load and evolution of the infection to active disease. Epigenetic and metabolic mechanisms described in other chronic inflammatory diseases are evaluated for potential application to the understanding of leprosy pathogenesis. A potential role for post-exposure prophylaxis of leprosy in reducing M. leprae-induced anti-inflammatory mediators and, in consequence, Treg/T effector ratios is proposed.

Evaluation of herpesvirus members on hospital admission in patients with systemic lupus erythematous shows higher frequency of Epstein-Barr virus and its associated renal dysfunction.

INTRODUCTION: Members of the Herpesviridae family have been described in patients with systemic lupus erythematous (SLE), but the clinical impact on renal function is not well known. METHODS: HSV1, HSV2, VZV, EBV, CMV, HHV-6, HHV-7, and HHV-8 were evaluated by molecular biology on admission in blood samples from 40 consecutive SLE patients hospitalized for lupus activity. RESULTS: Patients were 90.0% female, 77.5% non-white, with average age of 32.7 ± 13.6 years. We found positivity for EBV (65.0%), CMV (30.0%), HSV-1 (30.0%), HHV-6 (12.5%), and HHV-7 (7.5%). For all viruses, age, SLEDAI, hematological tests, ferritin, LDH, C-reactive protein, and erythrocyte sedimentation rate (ESR) were not significant. However, EBV positivity was a significant factor for higher serum creatinine (3.0 ± 2.8 vs. 0.9 ± 0.8; P = 0.001) and urea (86 ± 51 vs. 50 ± 46; P = 0.03). Moreover, positive cases for EBV only or with combined co-infections (66.7%-CMV; 58.3%-HSV-1) or negative for EBV only were evaluated by Kruskal-Wallis test again showed statistical significance for serum creatinine and urea (both P ≤ 0.01), with posttest also showing statistical differences for renal dysfunction and EBV presence (alone or in combined co-infections). The presence of EBV viral load was also significant for nephrotic-range proteinuria, renal flare, and the need for hemodialysis. CONCLUSION: Members of the Herpeviridae family (mainly EBV, HSV-1 and CMV) are common on hospital admission of SLE patients, reaching 65% for EBV, which seems to be associated with renal dysfunction and could reflect a previous association or overlapping disease, which is not well understood.

Genetic and Clinical Predictors of Age of ESKD in Individuals With Autosomal Dominant Tubulointerstitial Kidney Disease Due to UMOD Mutations.

INTRODUCTION: Autosomal dominant tubulo-interstitial kidney disease due to UMOD mutations (ADTKD-UMOD) is a rare condition associated with high variability in the age of end-stage kidney disease (ESKD). The minor allele of rs4293393, located in the promoter of the UMOD gene, is present in 19% of the population and downregulates uromodulin production by approximately 50% and might affect the age of ESKD. The goal of this study was to better understand the genetic and clinical characteristics of ADTKD-UMOD and to perform a Mendelian randomization study to determine if the minor allele of rs4293393 was associated with better kidney survival. METHODS: An international group of collaborators collected clinical and genetic data on 722 affected individuals from 249 families with 125 mutations, including 28 new mutations. The median age of ESKD was 47 years. Men were at a much higher risk of progression to ESKD (hazard ratio 1.78, P < 0.001). RESULTS: The allele frequency of the minor rs4293393 allele was only 11.6% versus the 19% expected (P < 0.01), resulting in Hardy-Weinberg disequilibrium and precluding a Mendelian randomization experiment. An in vitro score reflecting the severity of the trafficking defect of uromodulin mutants was found to be a promising predictor of the age of ESKD. CONCLUSION: We report the clinical characteristics associated with 125 UMOD mutations. Male gender and a new in vitro score predict age of ESKD.

Simultaneous circulation of arboviruses and other congenital infections in pregnant women in Rio de Janeiro, Brazil.

BACKGROUND: Arboviruses (Zika, dengue and chikungunya) represent a major risk for pregnant women, especially because their vertical transmission can lead to neurological damage in newborns. Early diagnosis can be difficult due to similar clinical presentation with other congenital infections that are associated with congenital abnormalities. OBJECTIVES: To investigate the circulation of arboviruses and other pathogens responsible for congenital infections, reporting clinical aspects and geographic distribution of maternal rash in a metropolitan region of Rio de Janeiro (Brazil). METHODS: Cross-sectional study with pregnant women presenting rash attended at the Exanthematic Diseases Unit (Niterói, Rio de Janeiro) from 2015 to 2018. Diagnosis of arboviruses was performed by real-time PCR (RT-qPCR) and laboratorial screening for syphilis, toxoplasmosis, rubella, cytomegalovirus and HIV was assessed. Demographic data was used for georeferencing analysis. FINDINGS: We included 121 pregnant women, of whom Zika virus was detected in 45 cases (37.2%), chikungunya in 33 (27.3%) and dengue in one (0.8%). Five patients presented syphilis, and we observed one case each of listeria, cytomegalovirus, and a syphilis-toxoplasmosis case. Similarity of clinical symptoms was observed in all groups; however, 84.8% of patients with chikungunya presented arthralgia. Following the decline of Zika cases, chikungunya infection was mostly observed during 2017-2018. Considering pregnant women infected with arboviruses and other infections, 41% resided in urban slums, mostly in Niterói. MAIN CONCLUSIONS: Simultaneous circulation of arboviruses and other agents responsible for congenital infections were observed; however, we did not identify co-infections between arboviruses. In this scenario, we emphasize the importance of adequate prenatal care to provide an accurate diagnosis of maternal rash.

Pp65 antigenemia and cytomegalovirus diagnosis in patients with lupus nephritis: report of a series.

INTRODUCTION: In contrast to organ transplantation, few studies correlate the monitoring of pp65 antigenemia with a diagnosis of cytomegalovirus (CMV) in patients with systemic lupus erythematosus (SLE). OBJECTIVE: To highlight the importance of CMV outside transplantation, we monitored pp65 antigenemia in a series of SLE patients. METHODS: From March 2015 to March 2016, SLE patients presenting kidney involvement, fever, and an unclear infection at hospital admission were monitored through pp65 antigenemia. The pp65 antigenemia assay, revealed by immunofluorescence, was correlated with clinical and laboratory findings. RESULTS: We included 19 patients with a suspected unclear infection. A positivity for pp65 antigenemia was found in seven patients (36.8%). The mean age was 33.5 ± 11.2 years, 16 (84%) were females, and 16 (84%) were black. Lymphopenia, anemia, and higher scores of SLEDAI were significantly more common in pp65-positive patients. Five patients received antiviral therapy with ganciclovir. Although receiving specific CMV treatment, one patient died because of suspected CMV disease. CONCLUSIONS: Pp65 antigenemia might be relevant in SLE patients, and studies with a greater number of patients are needed in order to establish sensitivity and specificity of pp65 antigenemia in different clinical contexts of SLE patients.

Pp65 antigenemia and cytomegalovirus diagnosis in patients with lupus nephritis: report of a series / Antigenemia pp65 e diagnóstico de citomegalovírus em pacientes com nefrite lúpica: série de casos

ABSTRACT Introduction: In contrast to organ transplantation, few studies correlate the monitoring of pp65 antigenemia with a diagnosis of cytomegalovirus (CMV) in patients with systemic lupus erythematosus (SLE). Objective: To highlight the importance of CMV outside transplantation, we monitored pp65 antigenemia in a series of SLE patients. Methods: From March 2015 to March 2016, SLE patients presenting kidney involvement, fever, and an unclear infection at hospital admission were monitored through pp65 antigenemia. The pp65 antigenemia assay, revealed by immunofluorescence, was correlated with clinical and laboratory findings. Results: We included 19 patients with a suspected unclear infection. A positivity for pp65 antigenemia was found in seven patients (36.8%). The mean age was 33.5 ± 11.2 years, 16 (84%) were females, and 16 (84%) were black. Lymphopenia, anemia, and higher scores of SLEDAI were significantly more common in pp65-positive patients. Five patients received antiviral therapy with ganciclovir. Although receiving specific CMV treatment, one patient died because of suspected CMV disease. Conclusions: Pp65 antigenemia might be relevant in SLE patients, and studies with a greater number of patients are needed in order to establish sensitivity and specificity of pp65 antigenemia in different clinical contexts of SLE patients.

RESUMO Introdução: Diferentemente do transplante de órgãos, poucos estudos correlacionam o monitoramento da antigenemia pp65 com o diagnóstico de citomegalovírus (CMV) em pacientes com lúpus eritematoso sistêmico (LES). Objetivo: De modo a destacar a importância do CMV para além do transplante, monitorizamos a antigenemia pp65 em uma série de pacientes com LES. Métodos: De março de 2015 a março de 2016, pacientes com LES que apresentaram acometimento renal, febre e infecção indeterminada na internação foram monitorados através da antigenemia pp65. O ensaio de antigenemia, revelada por imunofluorescência, foi correlacionado com achado clínicos e laboratoriais. Resultados: Foram incluídos 19 pacientes com suspeita de infecção indeterminada. Positividade para antigenemia pp65 foi encontrada em sete pacientes (36,8%). A idade média foi de 33,5 ± 11,2 anos; 16 (84%) eram do sexo feminino e 16 (84%) eram negros. Linfopenia, anemia e escore de SLEDAI mais elevado foram significativamente mais comuns em pacientes pp65 positivos. Cinco pacientes receberam terapia antiviral com ganciclovir. Apesar de receber tratamento específico para CMV, um paciente com suspeita de doença por CMV veio a óbito. Conclusões: Antigenemia pp65 pode ser relevante em pacientes com LES, e estudos com maior número de pacientes são necessários para estabelecer a sensibilidade e a especificidade da antigenemia pp65 em diferentes contextos clínicos envolvendo pacientes com LES.

Clinical correlates of pp65 antigenemia monitoring in the first months of post kidney transplant in patients undergoing universal prophylaxis or preemptive therapy

Abstract Introduction: Human cytomegalovirus is a major cause of morbidity in kidney transplant patients. Objectives: We aimed to study viral replication and serological response in the first months post kidney transplant in patients undergoing universal prophylaxis or preemptive therapy and correlate the findings with the clinical course of Human cytomegalovirus infection. Patients and methods: Independent from the clinical strategy adopted for managing Human cytomegalovirus infection, prophylaxis versus preemptive therapy, the pp65 antigenemia assay and serological response were assessed on the day of transplantation, and then weekly during the first three months of post-transplant. Results: From the 32 transplant recipients, 16 were positive for pp65 antigenemia, with a similar incidence rate in each group. There were no positive results in the first three weeks of monitoring; the positivity rate peaked at week eight. There was a trend for a higher and earlier frequency of positivity in the universal prophylaxis group in which the course of the Human cytomegalovirus infection was also more severe. Despite the differences in clinical picture and in the initial immunosuppressant schedule, the serological response was similar in both groups. Conclusion: Routine monitoring during the first three post-transplant months has a positive impact on the early detection of Human cytomegalovirus viral replication allowing for timely treatment in order to reduce morbidity of the disease. The strategy of universal therapy employing intravenous ganciclovir was associated to a worse clinical course of the Human cytomegalovirus infection suggesting that the use of >10 cells/2 × 105 leukocytes as a cut-off in this setting may be inappropriate.

Clinical correlates of pp65 antigenemia monitoring in the first months of post kidney transplant in patients undergoing universal prophylaxis or preemptive therapy.

INTRODUCTION: Human cytomegalovirus is a major cause of morbidity in kidney transplant patients. OBJECTIVES: We aimed to study viral replication and serological response in the first months post kidney transplant in patients undergoing universal prophylaxis or preemptive therapy and correlate the findings with the clinical course of Human cytomegalovirus infection. PATIENTS AND METHODS: Independent from the clinical strategy adopted for managing Human cytomegalovirus infection, prophylaxis versus preemptive therapy, the pp65 antigenemia assay and serological response were assessed on the day of transplantation, and then weekly during the first three months of post-transplant. RESULTS: From the 32 transplant recipients, 16 were positive for pp65 antigenemia, with a similar incidence rate in each group. There were no positive results in the first three weeks of monitoring; the positivity rate peaked at week eight. There was a trend for a higher and earlier frequency of positivity in the universal prophylaxis group in which the course of the Human cytomegalovirus infection was also more severe. Despite the differences in clinical picture and in the initial immunosuppressant schedule, the serological response was similar in both groups. CONCLUSION: Routine monitoring during the first three post-transplant months has a positive impact on the early detection of Human cytomegalovirus viral replication allowing for timely treatment in order to reduce morbidity of the disease. The strategy of universal therapy employing intravenous ganciclovir was associated to a worse clinical course of the Human cytomegalovirus infection suggesting that the use of >10 cells/2×105 leukocytes as a cut-off in this setting may be inappropriate.

The Role of Heme and Reactive Oxygen Species in Proliferation and Survival of Trypanosoma cruzi.

Trypanosoma cruzi, the protozoan responsible for Chagas disease, has a complex life cycle comprehending two distinct hosts and a series of morphological and functional transformations. Hemoglobin degradation inside the insect vector releases high amounts of heme, and this molecule is known to exert a number of physiological functions. Moreover, the absence of its complete biosynthetic pathway in T. cruzi indicates heme as an essential molecule for this trypanosomatid survival. Within the hosts, T. cruzi has to cope with sudden environmental changes especially in the redox status and heme is able to increase the basal production of reactive oxygen species (ROS) which can be also produced as byproducts of the parasite aerobic metabolism. In this regard, ROS sensing is likely to be an important mechanism for the adaptation and interaction of these organisms with their hosts. In this paper we discuss the main features of heme and ROS susceptibility in T. cruzi biology.

Heme-induced ROS in Trypanosoma cruzi activates CaMKII-like that triggers epimastigote proliferation. One helpful effect of ROS.

Heme is a ubiquitous molecule that has a number of physiological roles. The toxic effects of this molecule have been demonstrated in various models, based on both its pro-oxidant nature and through a detergent mechanism. It is estimated that about 10 mM of heme is released during blood digestion in the blood-sucking bug's midgut. The parasite Trypanosoma cruzi, the agent of Chagas' disease, proliferates in the midgut of the insect vector; however, heme metabolism in trypanosomatids remains to be elucidated. Here we provide a mechanistic explanation for the proliferative effects of heme on trypanosomatids. Heme, but not other porphyrins, induced T. cruzi proliferation, and this phenomenon was accompanied by a marked increase in reactive oxygen species (ROS) formation in epimastigotes when monitored by ROS-sensitive fluorescent probes. Heme-induced ROS production was time- and concentration-dependent. In addition, lipid peroxidation and the formation of 4-hydroxy-2-nonenal (4-HNE) adducts with parasite proteins were increased in epimastigotes in the presence of heme. Conversely, the antioxidants urate and GSH reversed the heme-induced ROS. Urate also decreased parasite proliferation. Among several protein kinase inhibitors tested only specific inhibitors of CaMKII, KN93 and Myr-AIP, were able to abolish heme-induced ROS formation in epimastigotes leading to parasite growth impairment. Taken together, these data provide new insight into T. cruzi- insect vector interactions: heme, a molecule from the blood digestion, triggers epimastigote proliferation through a redox-sensitive signalling mechanism.

The heme uptake process in Trypanosoma cruzi epimastigotes is inhibited by heme analogues and by inhibitors of ABC transporters.

Heme (iron protoporphyrin IX) is an important molecule involved in many biological reactions, including oxygen transport, respiration, photosynthesis and drug detoxification. Trypanosoma cruzi parasites, the etiological agent of Chagas' disease, take up heme from the environment to supply their nutritional needs because they do not synthesize this cofactor. However, the mechanisms involved in heme transport across biological membranes are poorly understood. Indeed, in T. cruzi, no heme transporter has yet been characterized. In the present work, we evaluate the heme uptake processes by T. cruzi epimastigotes using fluorescent heme-analogues. Heme uptake decreased significantly when cells were pretreated with different concentrations of SnPPIX, PdMPIX or ZnMPIX, this observed competition suggests that they are taken up by the same transport system. We studied the growth behavior of epimastigotes using the same heme-analogues and the treatments with SnPPIX or PdMPIX impaired cell growth but when heme was added to the culture medium the observed inhibition was partially reversed. In addition, we tested how the heme uptake processes are affected by the presence of different transporter inhibitors. When the cells were treated with inhibitors and then incubated with heme, heme uptake decreased significantly for all treatments. These results constitute a strong indication for the existence of a protein associated with porphyrin transport in T. cruzi, possibly ATP-binding cassette transporters (ABC-transporter).

Análise de uma heme oxigenase funcional em Trypanosoma cruzi / Analysis of a heme oxygenase functional in Trypanosoma cruzi

O Trypanosoma cruzi é o agente etiológico da doença de Chagas, transmitida através de insetos vetores triatomíneos durante a alimentação do hospedeiro vertebrado. Os triatomíneos ingerem numa única alimentação cerca de 10 mM de heme ligado à hemoglogina. O heme é uma importante molécula no metabolismo dos organismos. Um mecanismo intracelular importante no controle de sua homeostase é a degradação enzimática pela Heme Oxigenase (HO) formando biliverdina (Bv), monóxido de carbono e ferro. Como esta enzima não está presente no genoma de T. cruzi, esse trabalho tem por objetivo identificar uma atividade funcional de HO neste parasito, uma vez que dados do nosso laboratório mostram a presença de biliverdina nas incubações dessas células com heme. No presente trabalho testamos o efeito do SnPPIX (inibidor da HO-1), CoPPIX (indutor da HO-1) e Bv sobre a proliferação da forma epimastigota do parasito. A adição de SnPPIX diminuiu a proliferação do parasito tanto na ausência quanto na presença de heme. Quando a Bv foi adicionada à cultura esse efeito foi revertido; a Bv aumenta a proliferação celular na presença de heme. Por outro lado, a adição de CoPPIX não interferiu na proliferação. Posteriormente, mostramos através da técnica de immunoblotting, utilizando anticorpo monoclonal contra a HO-1, um aumento da expressão de uma proteína em resposta ao heme. Diferentemente das HO-1 já descritas que possuem massa molecular de 32 kDa, a única banda reconhecida pelo anticorpo apresenta 45 kDa. Analisamos também a expressão da HO-1 na presença de CoPPIX, SnPPIX e biliverdina, e somente o CoPPIX foi capaz de modular os níveis de expressão da HO-1. A análise estrutural através da técnica de imunocitoquímica mostrou uma maior expressão da enzima na presença de heme, e que a HO-1 de T. cruzi pode ter mais de uma localização, apresentando marcação citoplasmática e glicossomal. A fim de investigar a sequência da HO-1 de T. cruzi, o DNA genômico foi extraído para amplificação ...

Trypanosoma cruzi, the ethiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate host. These hematophagous insects ingest blood about 6 to 12 times its original weight, reaching in a single meal about 10mM heme bound to hemoglobin. Heme (iron protoporphyrin IX) is an important molecule in metabolism of all living organisms. One important intracellular mechanism to control heme homeostasis is its enzymatic degradation by heme oxygenase (HO). HO catalyzes the degradation of heme to biliverdin (Bv), carbon monoxide and iron. HO is absent in T. cruzi genome, thus we have been investigating the presence of a functional HO in this parasite, since our previous results showed a presence of biliverdin in heme-treated epimastigotes. In the present work, we evaluated the effect of SnPPIX, a HO-1 inhibitor, CoPPIX, a HO inducer, and Bv upon T. cruzi epimastigotes proliferation. The addition of SnPPIX decreased the parasite proliferation in the absence or in the presence of heme. When Bv was added to the culture this effect was reversed; Bv increases the parasite proliferation in the presence of heme. On the other hand, CoPPIX did not interfered on proliferation. Furthermore, we showed through immunoblotting, using an anti-HO-1 monoclonal antibody, an increase in the protein expression in heme-treated epimastigotes. Differently of described HO-1 that has a mass molecular of a 32 kDa, we showed a 45 kDa protein, the only band recognize by the HO-1 antibody. HO-1 expression analysis in the presence of CoPPIX, SnPPIX and biliverdin, showed that only CoPPIX was able to modulate its expression level. Ultrastructural immunocytochemistry analysis suggests a higher expression of the enzyme in heme-treated epimastigotes, and that T. cruzi HO-1 might have a dual distribution, since the anti-HO-1 antibody labeled both cytosol and glycosomes. In order to investigate the T. cruzi HO-1 gene sequence, we isolated genomic DNA ...

Envolvimento da CaMKII no efeito proliferativo do heme(Fe-Protoporfirina IX)em Trypanosoma cruzi / Envolvement of the CaMKII in the proliferativo effect of the heme(Fe-Protoporfirina IX)em Trypanosoma cruzi

O Trypanossoma cruzi é o agente etiológico da Doença de Chagas, que é transmitida através de insetos vetores triatomíneos durante a alimentação no hospedeiro vertebrado. A doença de Chagas é um grave problema na América Latina, e apesar disso a quimioterapia existente para a doença ainda não é satisfatória, sendo pouco eficaz em seus estágios avançados, além de ser extremamente tóxica para o hospedeiro. Os triatomíneos chegam a ingerir em sangue, de 6 a 12 vezes o seu peso original, ingerindo numa única alimentação cerca de 10 mM de heme ligado à hemoglobina. Como a forma epimastigota de T. cruzi vive em constante presença de heme, começamos a investigar sua participação na proliferação do parasito como molécula sinalizadora. No presente trabalho testamos diversos inibidores de proteínas quinases in vitro. Somente o KN93 (2M), inibidor de quinases dependentes de Ca2+/CaM (multifuncionais), teve um efeito inibitório da ação do heme, enquanto os demais inibidores não alteraram a proliferação destas células. Quando utilizado o KN92, um análogo sem atividade inibitória, este não apresentou efeito, mostrando então a especificidade do KN93. A fim de identificar qual CaMK está envolvida na proliferação mediada por heme, testamos o peptídeo Myr-AIP, um inibidor altamente específico e potente, derivado do substrato da CaMKII, e observamos que a adição do inibidor na cultura bloqueou a proliferação dos parasitos na presença de heme, confirmando o efeito específico sobre a proliferação mediada por heme, e o envolvimento da via da CaMKII. Através da técnica de western blotting mostramos o aumento da fosforilação da CaMKII na presença de heme, confirmando o envolvimento da CaMKII nesse processo. Posteriormente, ensaiamos a atividade da CaMKII utilizando uma CaMKII recombinante comercial. Vimos que a adição de heme 300 ?M no meio de reação aumentou a atividade enzimática cerca de 10 vezes, corroborando o resultado obtido no western blotting, e sugerindo a existência de dif...