Increased caspase-3 immunoexpression and morphology alterations in oenocytes and trophocytes of Apis mellifera larvae induced by toxic secretion of Epormenis cestri

Toxic honeydew produced by Flatidae Epormenis cestri in Uruguay has been shown to cause among honeybees (Apis mellifera) colonies a massive larva death called “River disease”, but the intrinsic mechanisms are still unknown. Because fat body cells, oenocytes and trophocytes, are known to regulated larvae metabolism, and to be affected by xenobiotics, we tested whether apoptosis of these cells can be an underlying cause of larvae death. Ten colonies were divided into two groups and fed with common honey or toxic honeydew obtained from colonies affected by “River disease”. Five-day-old larvae were collected and processed for histology and immunohistochemistry for caspase-3. The area, diameter, and immunostaining area in oenocytes and trophocytes were measured. The oenocyte and trophocyte cellular area decreased in the treated group (p=0.002; p<0.001 respectively) compared to the control group. The diameter of oenocytes (p=0.0002) and trophocytes (p<0.0001) decreased in the treated group. Caspase-3 was detected in cytoplasm in the control group but in the cytoplasm and nucleus in the treated group. The caspase-3 immunostaining area increased in oenocytes (p<0.002) and trophocytes (p<0.0001) of the treated group. The ingestion of toxic honeydew altered the morphology, localization and immunoexpression of caspase-3 in fat body cells, which suggests that the deregulation of the apoptotic mechanism affected the normal development in A. mellifera larvae

Impact of Aetiological Treatment on Conventional and Multiplex Serology in Chronic Chagas Disease

The main criterion for treatment effectiveness in Chagas Disease has been the seronegative conversion of previously reactive serology, generally achieved many years post-treatment. The lack of reliable tests to ensure parasite clearance and to examine the effect of treatment is the main difficulty in evaluating treatment for chronic Chagas disease. Decreases of conventional and non-conventional serological titers can be useful tools to monitor the early impact of treatment. We serially measured changes in antibody levels, including seronegative conversion as well as declines in titers in 53 benznidazole-treated and 89 untreated chronically T. cruzi-infected subjects. Seronegative conversion as well as decreases of titers was significantly higher in treated compared with untreated patients. A strong concordance was found between decreases of titers of conventional and non-conventional serologic tests post-treatment, reaffirming the findings. When seronegative conversion plus decreases of titers were considered altogether, the impact of treatment was higher, in a shorter follow-up period than previously considered. New tools for monitoring the effectiveness of treatment of chronic Chagas disease are necessary, and the results showed in this study is a contribution to researchers and physicians who assist patients suffering from this disease

Inhibitory Receptors Are Expressed by Trypanosoma cruzi-Specific Effector T Cells and in Hearts of Subjects with Chronic Chagas Disease

We had formerly demonstrated that subjects chronically infected with Trypanosoma cruzi show impaired T cell responses closely linked with a process of T cell exhaustion. Recently, the expression of several inhibitory receptors has been associated with T cell dysfunction and exhaustion. In this study, we have examined the expression of the cytotoxic T lymphocyte antigen 4 (CTLA-4) and the leukocyte immunoglobulin like receptor 1 (LIR-1) by peripheral T. cruzi antigen-responsive IFN-gamma (IFN-γ)-producing and total T cells from chronically T. cruzi-infected subjects with different clinical forms of the disease. CTAL-4 expression was also evaluated in heart tissue sections from subjects with severe myocarditis. The majority of IFN-γ-producing CD4+ T cells responsive to a parasite lysate preparation were found to express CTLA-4 but considerably lower frequencies express LIR-1, irrespective of the clinical status of the donor. Conversely, few IFN-γ-producing T cells responsive to tetanus and diphtheria toxoids expressed CTLA-4 and LIR-1. Polyclonal stimulation with anti-CD3 antibodies induced higher frequencies of CD4+CTAL-4+ T cells in patients with severe heart disease than in asymptomatic subjects. Ligation of CTLA-4 and LIR-1 with their agonistic antibodies, in vitro, reduces IFN-γ production. Conversely, CTLA-4 blockade did not improved IFN-γ production in response to T. cruzi antigens. Subjects with chronic T. cruzi infection had increased numbers of CD4+LIR-1+ among total peripheral blood mononuclear cells, relative to uninfected individuals and these numbers decreased after treatment with benznidazole. CTLA-4 was also expressed by CD3+ T lymphocytes infiltrating heart tissues from chronically infected subjects with severe myocarditis. These findings support the conclusion that persistent infection with T. cruzi leads to the upregulation of inhibitory receptors which could alter parasite specific T cell responses in the chronic phase of Chagas disease

Sequential combined treatment with allopurinol and benznidazole in the chronic phase of Trypanosoma cruzi infection: a pilot study

Even though the use of combined drugs has been proved to be effective in other chronic infections, assessment of combined treatment of antiparasitic drugs in human Chagas' disease has not been performed. Herein, a pilot study was conducted to evaluate the tolerance and side effects of a sequential combined treatment of two antiparasitic drugs, allopurinol and benznidazole, in the chronic phase of Trypanosoma cruzi infection

Sequential combined treatment with allopurinol and benznidazole in the chronic phase of Trypanosoma cruzi infection: a pilot study

Objectives: Even though the use of combined drugs has been proved to be effective in other chronic infections, assessment of combined treatment of antiparasitic drugs in human Chagas' disease has not been performed. Herein, a pilot study was conducted to evaluate the tolerance and side effects of a sequential combined treatment of two antiparasitic drugs, allopurinol and benznidazole, in the chronic phase of Trypanosoma cruzi infection. Patients and methods: Changes in total and T. cruzi-specific T and B cells were monitored during a median follow-up of 36 months. Allopurinol was administered for 3 months (600 mg/day) followed by 30 days of benznidazole (5 mg/kg/day) in 11 T. cruzi-infected subjects. Results: The combined sequential treatment of allopurinol and benznidazole was well tolerated. The levels of T. cruzi-specific antibodies significantly decreased after sequential combined treatment, as determined by conventional serology and by a multiplex assay using recombinant proteins. The frequency of T. cruzi-specific interferon-γ-producing T cells significantly increased after allopurinol treatment and decreased to background levels following benznidazole administration in a substantial proportion of subjects evaluated. The levels of total naive (CD45RA + CCR7 + CD62L+) CD4 + and CD8 + T cells were restored after allopurinol administration and maintained after completion of the combined drug protocol, along with a decrease in T cell activation in total peripheral CD4 + and CD8 + T cells. Conclusions: This pilot study shows that the combination of allopurinol and benznidazole induces significant modifications in T and B cell responses indicative of a reduction in parasite burden, and sustains the feasibility of administration of two antiparasitic drugs in the chronic phase of Chagas' disease.Fil: Pérez Mazliah, Damián Eduardo. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Álvarez, M. G.. Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal de Agudos "Eva Perón"; ArgentinaFil: Cooley, G.. University of Georgia; Estados UnidosFil: Lococo, B. E.. Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal de Agudos "Eva Perón"; ArgentinaFil: Bertocchi, G.. Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal de Agudos "Eva Perón"; ArgentinaFil: Petti, M.. Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal de Agudos "Eva Perón"; ArgentinaFil: Albareda, María Cecilia. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Armenti, A. H.. Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal de Agudos "Eva Perón"; ArgentinaFil: Tarleton, R. L.. University of Georgia; Estados UnidosFil: Laucella, Susana Adriana. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Viotti, Rodolfo Jorge. Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal de Agudos "Eva Perón"; Argentin

Evolutive behavior towards cardiomyopathy of treated (nifurtimox or benznidazole) and untreated chronic chagasic patients Evolução à miocardiopatia dos infectados chagásicos crônicos tratados (nifurtimox ou benzonidazol) e não tratados

The aim of this work was to compare the evolution of chronic chagasic untreated patients (UTPs) with that of benznidazole or nifurtimox-treated patients (TPs). A longitudinal study from a low endemic area (Santa Fe city, Argentina) was performed during an average period of 14 years. Serological and parasitological analyses with clinical exams, ECG and X-chest ray were carried out. At the onset, 19/198 infected patients showed chagasic cardiomyopathy (CrChM) while 179 were asymptomatic. In this latter group the frequency of CrChM during the follow-up was lower in TPs compared with UTPs (3.2% vs 7%). Within the CrChM group, 2/5 TPs showed aggravated myopathy whereas this happened in 9/14 UTPs. Comparing the clinical evolution of all patients, 5.9% of TPs and 13% of UTPs had unfavourable evolution, but the difference is not statistically relevant. Serological titers were assessed by IIF. Titers equal to or lower than 1/64 were obtained in 86% of the TPs, but only in 38% of UTPs. The differences were statistically significant (geometric mean: 49.36 vs. 98.2). Antiparasitic assessment of the drugs (xenodiagnosis) proved to be effective. The low sensitivity in chronic chagasic patients must be born in mind. Despite treated patients showed a better clinical evolution and lower antibody levels than untreated ones, it is necessary to carry on doing research in order to improve therapeutic guidelines, according to the risk/benefit equation and based on scientific and ethical principles.<br>Para comparar a evolução dos infectados chagásicos crônicos não tratados (UTPs) e tratados (TPs) com nifurtimox ou benznidazol, fez-se um estudo longitudinal numa área de baixa endemicidade (cidade de Santa Fe, Argentina), com média de seguimento de 14 anos. Em cada controle foram feitas análises sorológicas e parasitológicas, exames clínicos, eletrocardiográficos e radiológicos. No inicio, 19/198 infectados apresentaram miocardiopatia chagásica crônica (MChCr), enquanto 179 eram assintomáticos. A frequência de MChCr no seguimento destes últimos foi 3,2% para os tratados e 7% para os não tratados. Dos pacientes com MChCr no início, 2/5 dos tratados e 9/14 dos não tratados agravaram sua miocardiopatia. Comparando a evolução clínica dos infectados, 5,9% dos tratados e 13% dos não tratados tiveram evolução desfavorável, mas esta diferença não é estatisticamente significativa. Na avaliação sorológica por IFI, 86% dos TPs e só 38% dos UTPs apresentaram títulos menores ou iguais a 1/64. As diferenças são estatisticamente significativas (média geométrica: 49,36 vs 98,2). Os xenodiagnósticos seriados realizados, demonstraram efetividade das drogas (considerar a baixa sensibilidade deste método na etapa crônica). Os pacientes tratados mostraram melhor evolução clínica e mais baixos níveis de anticorpos do que os não tratados. É preciso continuar as investigações para estabelecer pautas terapêuticas mais claras sobre a relação risco-benefício, sustentadas nos princípios científicos e éticos