Croton cajucara crude extract and isolated terpenes: activity on Trypanosoma cruzi

Croton cajucara is a plant found in the Amazon region and is known for its medicinal properties. The effects of the methanolic extract of the stem bark of C. cajucara (MCC) and of the isolated terpenes, trans-dehydrocrotonin (t-DCTN) and acetyl aleuritolic acid (AAA), were investigated using four isolates of Trypanosoma cruzi. In assays with trypomastigotes, the extract was more active than the isolated compounds, presenting IC₅₀ in the range of 10 to 50 μg/mL. The trypanocidal effect of MCC, AAA and benznidazole was significantly higher in the GLT291 and C45 strains, which were recently isolated from wild animals. MCC and AAA caused a dose-dependent inhibition of epimastigote proliferation. In assays using intracellular amastigotes, AAA and MCC reduced the percent of infection and the endocytic index after 96 h of treatment, at concentrations that were non-toxic to the host cells. MCC inhibited the trypanothione reductase pathway in both epimastigotes and trypomastigotes of all the subpopulations. The absence of AAA activity on the trypanothione reductase pathway in epimastigotes of Dm28c suggests heterogeneity of the biochemical profile between this clone and the three strains. Epimastigotes and trypomastigotes (GLT291) were treated for 24 h with MCC or AAA, and both induced alterations of the plasma membrane, while AAA-treated epimastigotes also displayed mitochondrial damage.     

Immune cellular parameters of leprosy and human immunodeficiency virus-1 co-infected subjects

Leprosy and human immunodeficiency virus-1 (HIV-1) are examples of human infections where interactions between the pathogen and the host cellular immunity determine the clinical manifestations of disease. Hence, a significant immunopathological interaction between HIV-1 and leprosy might be expected. In the present study we explored several aspects of cellular immunity in patients co-infected with HIV-1 and Mycobacterium leprae. Twenty-eight individuals were studied, comprising four groups: healthy controls, HIV-1 and M. leprae co-infection, HIV-1 mono-infection, and M. leprae mono-infection. Subjects in the mono-infection and co-infection groups were matched as far as possible for bacillary load and HIV disease status, as appropriate. Peripheral blood mononuclear cells (PBMC) were analysed using six- and seven-colour flow cytometry to evaluate T-cell subpopulations and their activation status, dendritic cell (DC) distribution phenotypes and expression of IL-4 by T cells. The co-infected group exhibited lower CD4 : CD8 ratios, higher levels of CD8⁺ T-cell activation, increased Vδ1 : Vδ2 T cell ratios and decreased percentages of plasmacytoid DC, compared with HIV-1 mono-infected subjects. Across infected groups, IL-4 production by CD4⁺ T lymphocytes was positively correlated with the percentage of effector memory CD4⁺ T cells, suggesting antigenically driven differentiation of this population of T cells in both HIV-1 and M. leprae infections. Co-infection with M. leprae may exacerbate the immunopathology of HIV-1 disease. A T helper 2 (Th2) bias in the CD4⁺ T-cell response was evident in both HIV-1 infection and leprosy, but no additive effect was apparent in co-infected patients.     

Immunohistochemical profile and c-kit mutations in gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) are low-grade sarcomas arising from the interstitial cells of Cajal, harboring mutation of c-kit. We investigated the morphological, immunohistochemical, and molecular profile of 55 GISTs to establish the prevalence of mutations, their clinical significance, and diagnostic utility. c-kit mutations were investigated by evaluating the entire coding sequence of the gene with non-radioisotopic PCR-SSCP, and characterized with fluorescent cycle sequencing. Mutations were detected in 39 tumors (71%), the majority (67%) involving exon 11. Two tumors showed exon 9 mutations (one tumor located in the small intestine and one in the stomach), whereas two cases showed a polymorphism at the splicing site of exon/intron 1 present in healthy blood donors with a 3% frequency. CD117 was expressed in 53 tumors (96%); CD34 was positive in 42 cases (76%); 42 cases (76%) expressed both CD117 and CD34. c-kit mutations were similarly distributed in stromal tumors at low risk of aggressive behavior (78%), intermediate risk (66%), and high risk (71%). Fifteen tumors expressing CD117 showed wild-type kit gene, and on histological grounds, they were equally distributed among epithelioid and spindle cell morphology. One case neither expressed CD117 nor did it show c-kit mutation. Data suggest that both immunohistochemical and molecular evaluation may be useful in tumors likely to be classified as GISTs; molecular analysis appears valuable to support the diagnosis and to identify cases that can benefit from recent novel therapeutic tools.     

Study of lymphocyte subpopulations in bone marrow in a model of protein–energy malnutrition

ObjectiveProtein–energy malnutrition (PEM) is an important public health problem affecting millions of people worldwide. Hematopoietic tissue requires a high nutrient supply, and a reduction in leukocytes, especially lymphocytes, suggests that some nutritional deficiencies might be altering bone marrow function and decreasing its ability to produce lymphocytes. In this study, we evaluated the effect that PEM has on lymphocyte subtypes and the cell cycle of CD5⁺ cells.MethodsSwiss mice were subjected to PEM using a low-protein diet containing 4% protein. When the experimental group had lost about 20% of their original body weight, we collected blood and bone marrow cells and evaluated the hemogram, the myelogram, bone marrow lymphoid markers using flow cytometry, and the cell cycle in CD5⁺ bone marrow.ResultsMalnourished animals presented anemia, reticulocytopenia, and leukopenia with lymphopenia. The bone marrow was hypocellular, and flow cytometric analyses of bone marrow cells showed cells that were CD45⁺ (91.2%), CD2⁺ (84.9%), CD5⁺ (37.3%), CD3⁺ (23.5%), CD19⁺ (43.3%), CD22⁺ (34.7%), CD19⁺/CD2⁺ (51.2%), CD19⁺/CD3⁺ (24.0%), CD19⁺/CD5⁺ (13.2%), CD22⁺/CD2⁺ (40.1%), CD22⁺/CD3⁺ (30.3%), and CD22⁺/CD5⁺ (1.1%) in malnourished animals and CD45⁺ (97.5%), CD2⁺ (42.9%), CD5⁺ (91.5%), CD3⁺ (92.0%), CD19⁺ (52.0%), CD22⁺ (75.6%), CD19⁺/CD2⁺ (62.0%), CD19⁺/CD3⁺ (55.4%), CD19⁺/CD5⁺ (6.7%), CD22⁺/CD2⁺ (70.3%), CD22⁺/CD3⁺ (55.9%), and CD22⁺/CD5⁺ (8.4%) in control animals. Malnourished animals also presented more CD5⁺ cells in the G0 phase of cell cycle development.ConclusionMalnourished animals presented bone marrow hypoplasia, maturation interruption, prominent lymphopenia with depletion in the lymphoid lineage, and changes in cellular development. We suggest that these changes are some of the primary causes of lymphopenia in cases of PEM and partly explain the increase in susceptibility to infections found in malnourished individuals.     

Myelopoietic response in mice exposed to acute cold/restraint stress: modulation by Chlorella vulgaris prophylactic treatment

In this study, hematopoietic cells from mice pretreated with CVE and exposed to acute cold/restraint stress were stimulated in the presence of growth factors to form colonies, thus providing accurate information about the modulation of the green algae of the stress-induced changes in the hematopoietic response. Our results demonstrated that exposure to acute stress affected hematopoiesis. Mice exposed for a 2.5-hour time period of cold and restraint presented diminished clonal capacity for CFU-GM content per femur, which was decreased by as much as 50% compared with that in control mice, in spite of the significant increase in serum colony-stimulating activity (CSA). Treatment with 50 mg/kg CVE for 5 days, previously to the stress regimen, attenuates the effects of the stress, since comparable levels of myeloid progenitors were found in the bone marrow of both CVE/stress and control mice. Moreover, the sera from stressed mice pretreated with CVE further increased the CFU-GM formation. On the contrary, the spleen seemed to be less sensitive to acute stress in our experimental conditions. These findings are in line with our previous reports showing that the stress-induced reduction in bone marrow CFU-GM of rats exposed to electric shocks is mediated by activation of the HPA axis and by secretion of opioid agonists. No changes were observed in bone marrow, spleen and thymus total cell counts, and in relative organ weights. However, a 50% reduction in the body weight loss produced by the stress was observed in mice given the extract.     

Visual acuity and retinal function in patients with Bardet-Biedl syndrome

OBJECTIVE:

Bardet-Biedl syndrome is a genetic, multisystem disorder that causes severe visual impairment. This condition is characterized by retinal dystrophy, obesity, digit anomalies, renal disease, and hypogonadism. The purpose of this study was to analyze visual acuity and full-field electroretinogram findings in patients with the Bardet-Biedl syndrome phenotype.

METHODS:

The visual acuity of a group of 23 patients (15 males) with ages ranging from 6-36 years (mean = 15.8±6.4; median = 14.7) was assessed. Retinal function was evaluated by full-field electroretinography, and dark-adapted thresholds were assessed.

RESULTS:

Visual acuity in the better-seeing eye was 20/40 or better in 5 patients (21.7%), 20/50-20/150 in 13 (56.5%) patients, 20/200-20/400 in 2 (8.7%) patients and worse than 20/400 in one (4.3%) patient. The mean acuity in the better-seeing eye was 0.7±0.6 logMAR (20/100, Snellen equivalent). Scotopic rod and maximal responses were non-detectable in 21 (91.3%) patients, and cone responses were non-detectable in 15 (65.2%) patients. Elevated dark-adapted visual thresholds were observed in all 19 patients who were able to be assessed, with 10 (52.6%) patients having thresholds greater than 30 dB.

CONCLUSIONS:

In a relatively young cohort of patients with Bardet-Biedl syndrome, only 21% had 20/40 or better vision. ERG scotopic responses were absent in the majority of cases, with cone responses being observed in less than half of cases. These findings showed the early deleterious effects in retinal function and visual acuity caused by this condition.     

First report of typical Brazilian Toxoplasma gondii genotypes from isolates of free-range chickens (Gallus gallus domesticus) circulating in the state of Paraíba,Northeast Brazil

This study evaluated, for the first time, the genetic diversity of Toxoplasma gondii isolates from free-range chickens from the state of Paraíba, Northeast Brazil. Tissue samples from 33 chickens from properties in five municipalities of Paraíba (Esperança, Olho d’Água, Malta, Monteiro, and Patos) were bioassayed in mice. The brains of mice infected with T. gondii cysts were used for DNA extraction and genotyping. Genotyping was performed using 11 PCR-RFLP markers and 15 microsatellite (MS) markers. Complete genotyping results were obtained for 29 isolates, with nine genotypes detected by RFLP and 15 genotypes identified by MS. Three genotypes (#273, #274, and #277) have only been recently identified from pigs in the region. Brazilian clonal types BrII and BrIII were identified from one isolate each. Clonal types I, II, and III were not detected by RFLP. Genotype #13 (Caribbean 1), detected in 48.3% (14/29) of isolates from four of the five municipalities investigated, was the most prevalent genotype in the state of Paraíba. However, the MS analysis showed that of these 14 isolates, only four were unique genotypes, and considering the distance between the municipalities from where they were collected, it is possible that only seven are independent isolates while the others are clones. The other genotypes were restricted to different microregions. The results indicate that the Caribbean 1 lineage of T. gondii is circulating widely in Northeast Brazil. The genotypic diversity of T. gondii in the state of Paraíba is high, and microsatellite analysis revealed this diversity with higher resolution than PCR-RFLP.

     

Imaging Trypanosoma cruzi within tissues from chagasic patients using confocal microscopy with monoclonal antibodies

Confocal fluorescence microscopy combined with differential interference contrast imaging of tissues from chagasic patients enabled the unequivocal identification of the parasite Trypanosoma cruzi. Using different monoclonal antibodies that indicate the parasite form and replication stage in conjunction with DNA labelling, specimens derived from distinct clinical forms of the disease were examined. Intracellular amastigote forms of the parasite were clearly detected in heart, brain, skin, lung, and kidney. Dividing amastigotes as well as trypomastigote forms were recognized in samples obtained from patients undergoing either acute-phase or some form of reactivation caused by immunosuppression. Received: 22 December 1998 / Accepted: 11 March 1999     

Evaluation of autonomic function underlying slow postexercise heart rate recovery

The reduction in heart rate (HR) during the first minute of recovery immediately after a graded maximal exercise stress test (GXT) has recently been found to be a powerful and independent predictor of cardiovascular and all-cause mortality. Reduced vagal activity has been postulated as the cause, but this has not been proven in a population with slow HR recovery (HRR). PURPOSE: To investigate autonomic contributions to HRR using time-frequency analysis in a group of individuals demonstrating slow HRR. METHODS: HRR was defined as the difference in HR between peak exercise and 1 min later; a value < or = 18 bpm was set as threshold and considered abnormal. A modified continuous wavelet transform (CWT) was used to perform time-dependent spectral analysis during the baseline steady state and the following non-steady-state conditions created by GXT. This method provides dynamic measures of low-frequency (LF) and high-frequency (HF) peaks associated with autonomic activity. Individuals (N = 20) with a previous slow HRR underwent a second GXT within 3 months after their initial test. An additional eight subjects whose first GXT disclosed normal HRR were taken as a control group. RESULTS: Seven of 20 subjects demonstrated slow HRR (14 +/- 5 bpm) on the repeat test, and 13 subjects displayed normal HRR (29 +/- 5 bpm). Subjects with slow HRR in both GXT displayed significantly (P < 0.05) lower HF and LF fluctuations during recovery than those with normal HRR. CONCLUSIONS: Attenuated HRR after GXT, assessed by CWT, is indeed associated with abnormal vagal reactivation and prolonged sympathetic stimulation after termination of maximal exercise.     

Effects of splenic artery occlusion on portal pressure in patients with cirrhosis and portal hypertension.

The specific contribution of splenic blood inflow to portal hypertension in patients with cirrhosis is still unclear. In this study, we investigated this contribution by assessing the hemodynamic effects of transient splenic artery occlusion. In 15 cirrhotic patients, portal pressure gradient (PPG) was measured just before inserting a transjugular intrahepatic portosystemic shunt (TIPS), in baseline conditions, for 15 minutes after splenic artery occlusion and 5 minutes after recovery. Splenic artery occlusion caused a significant decrease in PPG (range, -4% to -38%, median -20%, P < 0.001) which promptly returned to baseline values after recovery of the splenic inflow. The decrease in PPG showed a significant correlation with spleen volume (r = 0.70, P < 0.005), liver volume (r = -0.63; P < 0.01), and spleen/liver volume ratio (r = 0.82, P < 0.001). Seven out of eight patients with a spleen/liver volume ratio greater than 0.5 had a marked decrease in PPG (>20%), whereas none of patients with a ratio lesser than 0.5 had a marked PPG response. In conclusion, in cirrhotic patients with portal hypertension, splenic artery occlusion causes a significant reduction in portal pressure (PPG). The drop in PPG is directly related to spleen volume and indirectly related to liver volume. The spleen/liver volume ratio accurately predicts the drop in PPG and may be used to identify patients who could obtain a significant advantage from surgical and nonsurgical procedures decreasing splenic inflow.