In vitro susceptibility of Paracoccidioides brasiliensis yeast form to antifungal agents

A study was conducted to determine the susceptibility of P. brasiliensis yeast form to amphotericin B (A), ketoconazole (K), 5-fluorocytosine (5-FC) and rifampin (R). The three isolates tested produced minimal inhibitory concentrations (MICs) (mcg/ml) in the following range: A: 0.09-0.18; K: 0.001-0.007; 5-FC: 62.5-250 and R: 40-80. The minimal fungicidal concentrations (MFC) were several times higher than the corresponding MICs. Precise MFC for 5-FC were not obtained (> 500 mcg/ml). Combination of K plus A proved synergic, with the fractional inhibitory concentration (FIC) indices revealing synergy when the drugs were combined at the 1 to 1 and 1 to 5 MIC ratios. R (40 mcg/ml) appeared to antagonize K. These results indicate promise for the combined use of K plus A as a therapeutical regimen.

Viscerocutaneous form of loxoscelism and erythrocyte glucose-6-phosphate deficiency

In a period of time of five years, all patients who exhibited viscerocutaneous form of loxoscelism were investigated for erythrocyte glucose-6-phosphate deficiency, and in two patients out of seven it was found this deficiency. This finding suggests that this genetical enzyme deficiency could account for the hemolysis after Loxosceles bite, at least in some of the cases.

Biogeography, host specificity, and molecular phylogeny of the basidiomycetous yeast phaffia rhodozyma and its sexual form, xanthophyllomyces dendrorhous

Applied and Environmental Microbiology, Vol. 73, No.4

Hypoprothrombinemia in the compensated form of hepatosplenic schistosomiasis: further studies

Coagulation abnormality is frequently observed in schistosomiasis patients but its pathophysiology has not been established. We measured, by immunodiffusion. the prothrombin-antigen concentration in 56 individuals; of these 19 with demonstrated compensated form of hepatosplenic schistosomiasis, 17 with cirrhosis and 20 were control subjects. Transaminases, albumin, transthyretin, prothrombin time, antithrombin III, factor VII, and fibrinogen were also evaluated. All parameters were altered in the cirrhotic group but only albumin, prothrombin and antithrombin III levels were altered in the schistosomiasis group. Ninety percent of the patients with cirrhosis and sixty percent of the patients with schistosomiasis had abnormal plasma levels of albumin, transthyretin, prothrombin-antigen, and/or antithrombin III; an impaired hepatic synthesis was responsible for these results. Conversely forty percent of the schistosomiasis patients with normal plasma concentrations of both albumin and transthyretin had decreased mean plasma levels of both prothrombin and antithrombin III. These results suggest that either proth rombin and antithrombin III are more sensitive markers of impaired hepatic synthesis in schistosomiasis than are levels of albumin and transthyretin combined, or a low grade chronic consumption of clotting proteins also occurs. Considering the latter hypothesis it is possible that the thrombin formed would be inhibited by antithrombin III with the complexed thrombin-antithrombin III being cleared by the liver. Consequently the plasma levels of both prothrombin and antithrombin would be decreased, but the level of fibrinogen would be preserved.

Chlamydospore formation by Paracoccidioides brasiliensis mycelial form

To investigate the role of some adverse environmental conditions in chlamy-dospore formation by the mycelial form of P. brasiliensis, we cultured four P. brasiliensis isolates (18, Bt4, 1183, Pb9) at 25°C within solid agar medium either rich or poor in nutrients. Isolates 18 and 1183 were also cultured under anaerobiosis in a nitrogen atmosphere. Isolate 18 produced great number of terminal and intercalary chlamydospore after 7-10 days of culture in a medium poor in nutrients (2% agar with 0.1% dextrose and polypepton). The three other isolates also produced chlamydospores under the same conditions, but in lower numbers. Chlamydospore production by isolate 18 was abolished when the fungus was cultured in two agar media rich in nutrients (brain heart infusion and potato dextrose agar). Anaerobic incubation of isolate 18 under an atmosphere of N2 showed small mycelial outgrowth with numerous chlamydospores. At the electron microscopical level, the chlamydospores showed one or various nuclei and numerous mitochondria, indicating great potential for further development. Accordingly, chlamydospores produced multiple budding after only 24 h incubation at 35°C. The results demonstrate that under adverse environmental conditions P. brasiliensis mycelial form produces chlamydospores within a short period of time.

Chronic gastritis and Helicobacter pylori in digestive form of Chagas' disease

Patients with the digestive form of Chagas'disease frequently present chronic gastritis. As the microorganism Helicobacter pylori is now accepted as the most common cause of human chronic gastritis, the present work was undertaken to verify a possible relationship between the presence of this bacterium and inflammatory changes of antral mucosa in chagasic patients. Seventeen chagasics, with megaesophagus and or megacolon were studied. Fragments from two different regions of antral mucosa were obtained by endoscopy, fixed in 4% neutral formaldehyde and embedded in paraffin. The sections were stained by haematoxylin and eosin for histology analysis, and by carbolfuchsin for H. pylori identification. H. pylori was found in 16 (94.1%) chagasic patients, all of them presenting chronic gastritis. Superficial gastritis was seen in 9 (52.9%) while atrophic gastritis was present in 8 (47.1%) patients. H. pylori was present on gastric mucosa of 8 (100%) patients with atrophic gastritis and of 8 (88.8%) patients with superficial gastritis. We concluded that the microorganism H. pylori should be considered a possible factor connected with the etiopathogenesis of chronic superficial and atrophic gastritis frequently observed in patients with the digestive form of Chagas' disease.

Characterization of the non-apparent clinical form in the initial phase of schistosomiasis mansoni

In this paper the history of 115 recruits that had bathed simultaneously in streams contaminated with Schistosoma mansoni, during military maneuvers, is reported. Thirty four of the infected patients presented the initial phase of the infection diagnosed through epidemiologic, clinical and laboratorial parameters. Three out of the 34 patients did not reveal the clinical picture of the infection, thus being considered representatives of the non-apparent form of the disease. Differences between the intensity of blood eosinophilia, the area of immediate cutaneous reaction and the number of Schistosoma eggs eliminated in the stools proved not to be statistically significant (p>0.05) when the non-apparent and acute cases of schistosomiasis were compared. These cases actually may be considered evidences of the non-apparent form hitherto merely taken for granted in the literature.

Monitoring the ex-vivo expansion of human mesenchymal stem/stromal cells in xeno-free microcarrier-based reactor systems by MIR spectroscopy

Human mesenchymal stem/stromal cells (MSCs) have received considerable attention in the field of cell-based therapies due to their high differentiation potential and ability to modulate immune responses. However, since these cells can only be isolated in very low quantities, successful realization of these therapies requires MSCs ex-vivo expansion to achieve relevant cell doses. The metabolic activity is one of the parameters often monitored during MSCs cultivation by using expensive multi-analytical methods, some of them time-consuming. The present work evaluates the use of mid-infrared (MIR) spectroscopy, through rapid and economic high-throughput analyses associated to multivariate data analysis, to monitor three different MSCs cultivation runs conducted in spinner flasks, under xeno-free culture conditions, which differ in the type of microcarriers used and the culture feeding strategy applied. After evaluating diverse spectral preprocessing techniques, the optimized partial least square (PLS) regression models based on the MIR spectra to estimate the glucose, lactate and ammonia concentrations yielded high coefficients of determination (R2 ≥ 0.98, ≥0.98, and ≥0.94, respectively) and low prediction errors (RMSECV ≤ 4.7%, ≤4.4% and ≤5.7%, respectively). Besides PLS models valid for specific expansion protocols, a robust model simultaneously valid for the three processes was also built for predicting glucose, lactate and ammonia, yielding a R2 of 0.95, 0.97 and 0.86, and a RMSECV of 0.33, 0.57, and 0.09 mM, respectively. Therefore, MIR spectroscopy combined with multivariate data analysis represents a promising tool for both optimization and control of MSCs expansion processes.

Search of intravascular hemolysis in patients with the cutaneous form of loxoscelism

Haptoglobin assay, a highly sensitive method to detect intravascular hemolysis was carried out in the sera of 19 patients referred to Hospital Vital Brazil with the cutaneous form of loxoscelism in order to investigate the occurrence of mild intravascular hemolysis. Data from this series did not show decreased levels haptoglobin, ruling out intravascular hemolysis in these patients with cutaneous form of loxoscelism.

Immune reconstitution after autologous hematopoietic stem cell transplantation

Abstract The investigation of the web of relationships between the different elements of the immune system has proven instrumental to better understand this complex biological system. This is particularly true in the case of the interactions between B and T lymphocytes, both during cellular development and at the stage of cellular effectors functions. The understanding of the B–T cells interdependency and the possibility to manipulate this relationship may be directly applicable to situations where immunity is deficient, as is the case of cancer or immune suppression after radio and chemotherapy. The work presented here started with the development of a novel and accurate tool to directly assess the diversity of the cellular repertoire (Chapter III). Contractions of T cell receptor diversity have been related with a deficient immune status. This method uses gene chips platforms where nucleic acids coding for lymphocyte receptors are hybridized and is based on the fact that the frequency of hybridization of nucleic acids to the oligonucleotides on a gene chip varies in direct proportion to diversity. Subsequently, and using this new method and other techniques of cell quantification I examined, in an animal model, the role that polyclonal B cells and immunoglobulin exert upon T cell development in the thymus, specifically on the acquisition of a broader repertoire diversity by the T cell receptors (Chapter IV and V). The hypothesis tested was if the presence of more diverse peptides in the thymus, namely polyclonal immunoglobulin, would induce the generation of more diverse T cells precursors. The results obtained demonstrated that the diversity of the T cell compartment is increased by the presence of polyclonal immunoglobulin. Polyclonal immunoglobulin, and particularly the Fab fragments of the molecule, represent the most diverse self-molecules in the body and its peptides are presented by antigen presenting cells to precursor T cells in the thymus during its development. This probably contributes significantly to the generation of receptor diversity. Furthermore, we also demonstrated that a more diverse repertoire of T lymphocytes is associated with a more effective and robust T cell immune function in vivo, as mice with a more diverse T cell receptors reject minor histocompatiblility discordant skin grafts faster than mice with a shrunken T cell receptor repertoire (Chapter V). We believe that a broader T cell receptor diversity allows a more efficient recognition and rejection of a higher range of external and internal aggressions. In this work it is demonstrated that a reduction of TCR diversity by thymectomy in wild type mice significantly increased survival of H-Y incompatible skin grafts, indicating decrease on T cell function. In addiction reconstitution of T-cell diversity in mice with a decreased T cell repertoire diversity with immunoglobulin Fab fragments, lead to a increase on TCR diversity and to a significantly decreased survival of the skin grafts (Chapter V). These results strongly suggest that increases on T cell repertoire diversity contribute to improvement of T cell function. Our results may have important implications on therapy and immune reconstitution in the context of AIDS, cancer, autoimmunity and post myeloablative treatments. Based on the previous results, we tested the clinical hypothesis that patients with haematological malignancies subjected to stem cell transplantation who recovered a robust immune system would have a better survival compared to patients who did not recover such a robust immune system. This study was undertaken by the examination of the progression and overall survival of 42 patients with mantle cell non-Hodgkin lymphoma receiving autologous hematopoietic stem cell transplantation (Chapter VI). The results obtained show that patients who recovered higher numbers of lymphocytes soon after autologous transplantation had a statistically significantly longer progression free and overall survivals. These results demonstrate the positive impact that a more robust immune system reconstitution after stem cell transplantation may have upon the survival of patients with haematological malignancies. In a similar clinical research framework, this dissertation also includes the study of the impact of recovering normal serum levels of polyclonal immunoglobulin on the survival of patients with another B cell haematological malignancy, multiple myeloma, after autologous stem cell transplantation (Chapter VII). The relapse free survival of the 110 patients with multiple myeloma analysed was associated with their ability to recover normal serum levels of the polyclonal compartment of immunoglobulin. These results suggest again the important effect of polyclonal immunoglobulin for the (re)generation of the immune competence. We also studied the impact of a robust immunity for the response to treatment with the antibody anti CD20, rituximab, in patients with non- Hodgkin’s lymphoma (NHL) (Chapter VIII). Patients with higher absolute counts of CD4+ T lymphocytes respond better (in terms of longer progression free survival) to rituximab compared to patients with lower number of CD4+ T lymphocytes. These observations highlight again the fact that a competent immune system is required for the clinical benefit of rituximab therapy in NHL patients. In conclusion, the work presented in this dissertation demonstrates, for the first time, that diverse B cells and polyclonal immunoglobulin promote T cell diversification in the thymus and improve T lymphocyte function. Also, it shows that in the setting of immune reconstitution, as after autologous stem cell transplantation for mantle cell lymphoma and in the setting of immune therapy for NHL, the absolute lymphocyte counts are an independent factor predicting progression free and overall survival. These results can have an important application in the clinical practice since the majority of the current treatments for cancer are immunosuppressive and implicate a subsequent immune recovery. Also, the effects of a number of antineoplastic treatments, including biological agents, depend on the immune system activity. In this way, studies similar to the ones presented here, where methods to improve the immune reconstitution are examined, may prove to be instrumental for a better understanding of the immune system and to guide more efficient treatment options and the design of future clinical trials. Resumo O estudo da rede de inter-relações entre os diversos elementos do sistema immune tem-se mostrado um instrumento essencial para uma melhor compreensão deste complexo sistema biológico. Tal é particularmente verdade no caso das interacções entre os linfócitos B e T, quer durante o desenvolvimento celular, quer ao nível das funções celulares efectoras. A compreensão da interdependência entre linfócitos B e T e a possibilidade de manipular esta relação pode ser directamente aplicável a situações em que a imunidade está deficiente, como é o caso das doenças neoplásicas ou da imunossupressão após radio ou quimioterapia. O trabalho apresentado nesta dissertação iniciou-se com o desenvolvimento de um novo método laboratorial para medir directamente a diversidade do reportório celular (Capítulo III). Reduções da diversidade do reportório dos receptores de células T têm sido relacionadas com um estado de imunodeficiência. O método desenvolvido utiliza “gene chips”, aos quais hibridizam os ácidos nucleicos codificantes das cadeias proteicas dos receptores linfocitários. A diversidade é calculada com base na frequência de hibridização do ácido nucleico da amostra aos oligonucleótidos presentes no “gene chip”. De seguida, e utilizando este novo método e outras técnicas de quantificação celular examinei, num modelo animal, o papel que as células policlonais B e a imunoglobulina exercem sobre o desenvolvimento linfocitário T no timo, especificamente na aquisição de um reportório diverso de receptores T (Capítulos IV e V). Testei, então, a hipótese de que a presença no timo de péptidos mais diversos, como a imunoglobulna policlonal, induzisse a génese de precursores T mais diversos. Demonstrámos que a diversidade do compartimento T é aumentado pela presença de imunoglobulina policlonal. A imunoglobulina policlonal, e particularmente os fragmentos Fab desta molécula, representam as moléculas autólogas mais diversas presentes nos organismos vertebrados. Estes péptidos são apresentados por células apresentadoras de antigénio às células precursoras T no timo, durante o desenvolvimento celular T. Tal, provavelmente, contribui para a génese da diversidade dos receptores. Também demonstrámos que a presença de um reportório mais diverso de linfócitos T se associa a um incremento da função imunológica T in vivo. Uma diversidade de receptores T mais extensa parece permitir um reconhecimento e rejeição mais eficientes de um maior número de agressores internos e externos. Demonstrámos que ratinhos com receptores de células T (RCT) com maior diversidade rejeitam transplantes cutâneos discordantes para antigénios minor de histocompatibilidade mais rapidamente do que ratinhos com um menor reportório T (Capítulo V). Por outro lado, uma redução da diversidade do RCT, causada por timectomia de ratinhos de estirpes selvagens, mostrou aumentar significativamente a sobrevivência de transplantes cutâneos incompatíveis para o antigénio H-Y (antigénio minor de histocompatibilidade), indicando uma diminuição da função linfocitária T. Além disso, a reconstituição da diversidade dos linfócitos T em ratinhos com uma diversidade de reportório T diminuída, induzida pela administração de fragmentos Fab de imunoglobulina, conduz a um aumento da diversidade dos RCT e a uma diminuição significativa da sobrevivência dos enxertos cutâneos (Capítulo V). Estes resultados sugerem que o aumento do reportório de células T contribui para uma melhoria das funções celulares T e poderão ter implicações importantes na terapêutica e reconstitutição imunológica em contexto de SIDA, neoplasias, autoimunidade e após tratamentos mieloablativos. Baseado nos resultados anteriores, decidimos testar a hipótese clínica de que doentes com neoplasias hematológicas sujeitos a transplantação de precursores hematopoiéticos e com recuperação imunológica precoce após transplante teriam uma sobrevivência mais longa do que doentes que não recuperassem tão bem a sua imunidade. Analisámos a sobrevivência global e sobrevivência sem doença de 42 doentes com linfoma não Hodgkin de células do manto sujeitos a transplante autólogo de precursores hematopoiéticos (Capítulo VI). Os resultados obtidos mostraram que os doentes que recuperaram contagens mais elevadas de linfócitos imediatamente após o transplante autólogo, apresentaram uma sobrevivência global e sem progressão mais longa do que doentes que não recuperaram contagens linfocitárias tão precocemente. Estes resultados demonstram o efeito positivo de uma reconstitutição imunológica robusta após transplante de presursores hematopoiéticos, sobre a sobrevivência de doentes com neoplasias hematológicas. Do mesmo modo, estudámos o efeito que a recuperação de níveis séricos normais de imunoglobulina policlonal tem na sobrevivência de doentes com outras neoplasias hematológicas de linfócitos B, como o mieloma múltiplo,após transplante autólogo de precursos hematopoiéticos (Capítulo VII). A sobrevivência livre de doença dos 110 doentes com mieloma múltiplo analizados está associada com a sua capacidade de recuperar níveis séricos normais do compartmento policlonal de imunoglobulina. Estes resultados pioneiros indicam a importância da imunoglobulina policlonal para a génese de competência imunológica. Também estudámos o impacto de um sistema imunitário eficiente sobre a resposta ao tratamento com o anticorpo anti CD20, ituximab, em doentes com linfoma não Hodgkin (LNH) (Capítulo VIII). Os resultados mostram que doentes com valores mais elevados de linfócitos T CD4+ respondem melhor (em termos de maior sobrevida livre de doença) ao rituximab, do que doentes com valores mais baixos. Estas observações ilustram a necessidade de um sistema imunitário competente para o benefício clínico da terapêutica com rituximab em doentes com LNH. Em conclusão, o trabalho apresentado nesta dissertação demonstra que as células B e a imunoglobulina policlonal promovem a diversidade das células T no timo e melhoram a função linfocitária T periférica. Concomitantemente, também demonstrámos que, no contexto de reconstituição imune, por exemplo, após transplante autólogo de precursores hematopoiéticos em doentes com linfomas de células do manto, o número absoluto de linfócitos é uma factor independente da sobrevivência. Os resultados demonstram, também, a importância dos valores de linfocitos T na resposta ao tratamento com rituximab no caso de doentes com LNH. O mesmo princípio se prova pelo facto de que doentes com mieloma múltiplo sujeitos a transplante autólogo de precursores hematopoiéticos que recuperam valores normais séricos de imunoglobulinas policlonais, terem melhores taxas de resposta em comparação com doentes que não recuperam valores normais de imunoglobulinas policlonais. Estes resultados podem ter importantes aplicações na prática clínica dado que a maioria dos tratamentos de doenças neoplásicas implica imunossupressão e, subsequente, recuperação imunológica. Estes estudos podem ser um instrumento fundamental para uma melhor compreensão do sistema imune e guiar uma escolha mais eficiente de opções terapêuticas bem como contribuir para a concepção de futuros estudos clínicos.

Microencapsulation technology: a powerful tool for human embryonic stem cells expansion and cryopreservation

Dissertation presented to obtain a Master degree in Biotechnology at the Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Peptic disease and Helicobacter pylori are highly prevalent in patients with the indeterminate form of chagas’ disease: report of 21 cases

Given that chagasic patients in the indeterminate form of this disease, can have abnormal motility of the digestive tract and immunologic abnormalities, we decided to assess the frequency of peptic disease and Helicobacter pylori (Hp) infection in these individuals. Twenty-one individuals, 13 males and 8 females, mean age 37.6 ± 11.1 years, were examined. Biopsies of the duodenum, antrum, lesser and greater gastric curvature and esophagus were performed. The endoscopic findings were of chronic gastritis in 20 (95.2%) patients, duodenal ulcer in 3 (14.3%), gastric and duodenal ulcer in 3 (14.3%), gastric ulcer alone in 1 (4.8%), esophagitis in 5 (23.8%), and duodenitis in 5 (23.8%). The diagnosis of infection by the Hp was done by the urease test and histologic examination. Hp infection was found in 20 (95.2%) individuals: in 20 out of them in the antrum, in 17 in the lesser curvature, and in 17 in the greater curvature. Hp was not found in the esophagus and duodenum. The only individual with no evidence of infection by Hp was also the only one with normal endoscopic and histologic examinations. The histologic examinations confirmed the diagnoses of gastric ulcer as peptic, chronic gastritis in 20 patients, duodenitis in 14, and esophagitis in 9. In this series the patients had a high frequency of peptic disease, which was closely associated with Hp infection

Periodontal aspects of the juvenile form of paracoccidioidomycosis

Three cases of the juvenile form of paracoccidioidomycosis are reported. Emphasis has been given to the oral manifestations, particularly the periodontal involvement. The main periodontal findings were: generalized and progressive alveolar bone destruction leading to gingival recession with exposure of the tooth roots, and spontaneous tooth losses. The gingival mucosa was predominantly smooth, erithematous and slightly swollen. These aspects, although rare, may be the earliest signs of the disease and sometimes its only manifestation.

Psychometric Properties of the Portuguese Version of the Posttraumatic Growth Inventory Short Form Among Divorced Adults

The aim of this study was to develop and validate a Portuguese version of the Short Form of the Posttraumatic Growth Inventory (PTGI-SF). Using an online convenience sample of Portuguese divorced adults (N = 482), we confirmed the oblique five-factor structure of the PTGI-SF by confirmatory factor analysis. The results demonstrated the measurement invariance across divorce initiator status groups. Total score and factors of PTGI-SF showed good internal consistency, with the exception of the New Possibilities factor, which revealed an acceptable reliability. The Portuguese PTGI-SF showed a satisfactory convergent validity. In terms of discriminant validity, posttraumatic growth assessed by the Portuguese PTGI-SF was a distinct factor from posttraumatic psychological adjustment. These preliminary findings suggest the cultural adaptation and also psychometric properties of the present Portuguese PTGI-SF to measure posttraumatic growth after personal crisis.

Comparative morphometry and morphology of Anopheles aconitus Form B and C eggs under scanning electron microscope

Comparative morphometric and morphological studies of eggs under scanning electron microscope (SEM) were undertaken in the three strains of two karyotypic forms of Anopheles aconitus, i.e., Form B (Chiang Mai and Phet Buri strains) and Form C (Chiang Mai and Mae Hong Son strains). Morphometric examination revealed the intraspecific variation with respect to the float width [36.77 ± 2.30 µm (Form C: Chiang Mai strain) = 38.49 ± 2.78 µm (Form B: Chiang Mai strain) = 39.06 ± 2.37 µm (Form B: Phet Buri strain) > 32.40 ± 3.52 µm (Form C: Mae Hong Son strain)] and number of posterior tubercles on deck [2.40 ± 0.52 (Form B: Phet Buri strain) = 2.70 ± 0.82 (Form B: Chiang Mai strain) < 3.10 ± 0.32 (Form C: Chiang Mai strain) = 3.20 ± 0.42 (Form C: Mae Hong Son strain)], whereas the surface topography of eggs among the three strains of two karyotypic forms were morphologically similar.