Fábio Goulart de Andrade

An optimized one-tube, semi-nested PCR assay for Paracoccidioides brasiliensis detection

INTRODUCTION Herein, we report a one-tube, semi-nested-polymerase chain reaction (OTsn-PCR) assay for the detection of Paracoccidioides brasiliensis. METHODS We developed the OTsn-PCR assay for the detection of P. brasiliensis in clinical specimens and compared it with other PCR methods. RESULTS The OTsn-PCR assay was positive for all clinical samples, and the detection limit was better or equivalent to the other nested or semi-nested PCR methods for P. brasiliensis detection. CONCLUSIONS The OTsn-PCR assay described in this paper has a detection limit similar to other reactions for the molecular detection of P. brasiliensis, but this approach is faster and less prone to contamination than other conventional nested or semi-nested PCR assays.

Hemocyte quantitative changes in Anticarsia gemmatalis (Lepidoptera: Noctuidae) larvae infected by AgMNPV

The initial effects of the infection by AgMNPV in the total and differential counts of the hemocytes in Anticarsia gemmatalis (Lepidoptera: Noctuidae) larvae were studied. The total number of the hemocytes did not decrease in infected larvae, as it occurred in non infected larvae. In infected larvae, the hemocyte types showed the following frequencies: plasmatocytes - 47.8%, esferulocytes - 25.9%, granulocytes - 15.8%, oenocytoids - 7.2%, prohemocytes - 2.8%, vermicytes - 0,5%. Only the percentage of the granulocytes was different among infected and non infected larvae, indicating that these cells responded quickly to the initial viral infection. These results showed the effective role of the hemocytes in the response of the A. gemmatalis to the infection by AgMNPV. The comprehension of the immunological mechanisms of this insect is an important tool to understand its biological control.

Caracterização citológica dos hemócitos de Anticarsia gemmatalis (Lepidoptera, Noctuidae) em larvas resistentes ao vírus AgMNPV

The occurrence of Anticarsia gemmatalis (Hubner, 1932) larvae resistant to the virus AgMNPV in laboratory led to the study of the hemocytes of this insect, in order to evalue its participation in the mechanisms which enables the resistance to the virus. The resistant larvae with 6 to 11 developmental days old (3rd to 5th instars) were anesthetized by cold and after that were quickly cleaned in 70% alcohol. The hemolymph was collected through abdominal puncturing; the morphological analysis was made in phase contrast and colored smears with Seller solution. The total hemocytes counting (THC), was made in modified Neubauer chamber with not diluted hemolymph. For the differential hemocytes counting (DHC), was used diluted hemolymph in anticoagulant solution for insects. Six hemocytes types were identified: plasmatocytes (38.5%), granulocytes (22.6%), oenocytoids (20.4%), spherulocytes (14.5%), prohemocytes (2.3%) and vermiform (1.5%). The total number of hemocytes showed a significant increase during the studied larval period.

Direct renin inhibitor therapy and swimming training: hemodynamic and cardiac effects in hypertensive and normotensive rats.

Abstract Purpose: This study aimed to analyze the hemodynamic and cardiac effects of direct renin inhibitor (DRI) treatment and swimming training in hypertensive rats. Methods: Seventy-seven rats were divide into eight groups: sedentary normotensive (SN), trained normotensive (TN), sedentary normotensive treated with DRI (SN_DRI), trained normotensive treated with DRI (TN_DRI), sedentary hypertensive (SH), trained hypertensive (TH), sedentary hypertensive treated with DRI (SH_DRI), trained hypertensive treated with DRI (TH_DRI). Swimming training occurred for up to 60 min, five times a week for four weeks. The hypertensive animals were treated with 20 mg ċ kg−1 ċ day−1 L-NAME for four weeks. Groups treated with DRI received 10 mg ċ kg−1 ċ day−1 of aliskiren for four weeks. After the treatment period, all the animals underwent femoral artery catheterization surgery for direct measurement of cardiovascular variables. Results: The SH group presented hypertension (136.4 ± 5.0 mmHg) compared to the SN (107.1 ± 1.7 mmHg). The TH group showed lower mean arterial pressure (MAP) than the SH (121.1 ± 1.3 mmHg), but the treatment with DRI did not attenuate hypertension (128.2 ± 4.9 mmHg). The analysis of collagen areas demonstrated that treatment with DRI may attenuate cardiac remodeling in situations of hypertension, in the condition of treatment alone or combined with physical training. Conclusion: Both interventions in combination may be more effective at reducing cardiovascular risk in hypertensive subjects.

Histology and ultrastructure of the fat body of Anticarsia gemmatalis (HÜBNER, 1818) (LEPIDOPTERA: NOCTUIDAE)

The aim of this study was to analyze the morphologically the fatty body of fourth-instar Anticarsia gemmatalis larvae under light, transmission and scanning electron microscopy. Two distinct portions of the fat body were detected: the parietal (PA) and perivisceral (PV). The PA, the parietal portion, presented a long-stripped shape located below the tegument and lateral to the digestive tube. The PV, rarely observed, was in dorsal region, adhered to digestive wall. Both the portions were constituted of only one cellular type, the trophocytes. These cells in the PA were organized in one layer of thickness showing cylindrical contiguously morphology, whereas the PV was comprised by a mass of small cells, superposed as clusters. Both the portions were covered by a layer of connective tissue, grouping the trophocytes and keeping them separated from the hemolymph. The cytoplasm of the trophocytes from the PA presented acidophilic stain, while the basophilic cytoplasmic of the trophocytes from the PV was due to the large amount of rough endoplasmic reticulum. From the results, it could be concluded that the fat body presented morphological and ultrastructural differences according to the portion and that these features could characterize distinct functions.

Study of differential expression of miRNAs in lung tissue of mice submitted to experimental infection by Paracoccidioides brasiliensis

Abstract MicroRNAs (miRNAs) are small single stranded RNA sequences involved in post‐transcriptional regulation of different biological and physiological processes. Paracoccidioidomycosis (PCM) is an infection caused by Paracoccidioides brasiliensis, and it is a major cause of mortality due to systemic mycoses in Brazil. To date, there have been few reports on the role of miRNAs in the immune response against fungi, especially PCM. The objective of this study was to evaluate the differential expression of miRNAs related to the inflammatory response associated with pulmonary infection by P. brasiliensis. For this purpose, lungs from BALB/c mice, intravenously infected with P. brasiliensis (2.7×107 yeast cells/ml, n = 12) and noninfected BALB/c mice (n = 8), were collected at the 28 and 56 day after infection. The lung parenchyma presented a great number of yeast cells, granulomas, and edema at 28 days and a framework of resolution of the inflammatory process after 56 days. The mRNAs gata‐3, ror‐&ggr;t, foxp3, and IL‐6 were positively regulated at the moment at the 56 day, while the TGF‐&bgr;1 mRNA was positively regulated at both moments. The miRNAs 126a‐5p, 340‐5p, 30b‐5p, 19b‐3p, 221‐3p, 20a‐5p, 130a‐3p, and 301a‐3p, 466k presented the greatest increase in expression levels 28 days after infection, and the miRNAs let‐7f‐5p, let‐7a‐5p, 5p‐26b, let‐7e‐5p and 369‐3p, 466k presented a greater increase in levels of expression 56 days after infection. This study shows a set of differentially expressed miRNAs possibly involved in the immune response in mice during pulmonary infection by P. brasiliensis.

Efeito do treinamento físico no pulmão de ratos submetidos à ingestão alcoólica

Chronic alcohol consumption causes alterations in the lung tissues characterized by edema and formation of large inflammatory infiltrate. The purpose of this work was to evaluate the effect of physical exercise on lung injuries caused by chronic alcohol intake in Wistar rats. MATERIAL AND METHODS: Thirty-two male Wistar rats (261.1 ± 1.3 g) received sugarcane distilled alcoholic beverage diluted (30%, v/v, alcohol group) or tap water (control group) for 120 days. After this period, five animals of each group were sacrificed. The remaining animals received water and were sorted in four groups: alcoholic and sedentary (AS), control and sedentary (CS), alcoholic and trained (AT) and control and trained (CT). The AT and CT groups were submitted to a swimming exercise protocol with progressive daily increase in the training time until 20 minutes per day, five times per week, for five weeks. For the same period, AS and CS groups were maintained at sedentary state. RESULTS: after the alcoholic intake period, the alcohol group presented decreased (P<0.05) body weight and increased relative lung weight (P<0.05). Lungs of alcoholic group showed characteristics of edema and inflammatory infiltrate. The CS and CT groups did not present morphological changes. AT animals showed increased inflammation and number of hyper pigmented macrophages in relation to CT group. CONCLUSION: exercise can increase lung inflammation when applied in animals with inflammatory injury induced by chronic alcohol consumption.