Jose Luis Ramirez

Predominance of lineage I among Trypanosoma cruzi isolates from Venezuelan patients with different clinical profiles of acute Chagas’ disease

Trypanosoma cruzi isolates from 23 acute chagasic patients from localities of Western Venezuela (state of Barinas) where Chagas’ disease is endemic were typed using ribosomal and mini‐exon gene markers. Results showed that isolates of the two major phylogenetic lineages, T. cruzi I and T. cruzi II, were isolated from these patients. Six isolates (26%) were typed as T. cruzi II and 17 (74%) as belonging to T. cruzi lineage I. Analysis of random amplified polymorphic DNA (RAPD) patterns confirmed these two groups of isolates, but did not disclose significant genetic intra‐lineage polymorphism. Patients infected by both T. cruzi I or T. cruzi II showed different clinical profiles presenting highly variable signs and symptoms of acute phase of Chagas’ disease ranging from totally asymptomatic to severe heart failure. The predominance of T. cruzi I human isolates in Venezuela allied to the higher prevalence of severe symptoms of Chagas’ disease (heart failure) in patients infected by this lineage do not corroborate an innocuousness of T. cruzi I infection to humans. To our knowledge, this is the first study describing predominance of T. cruzi lineage I in a large number of acute chagasic patients with distinct and well‐characterized clinical profiles.

Methylation patterns and K-ras mutations in tumor and paired serum of resected non-small-cell lung cancer patients

Gene methylation and K-ras mutations were examined in tumor and paired serum DNA of 50 resected non-small-cell lung cancer patients. RASSF1A, death associated protein kinase and target of methylation-induced silencing were methylated in 17/50 (34%), 23/50 (45%) and 18/50 (35%) tumors, respectively, and in 17/50 (34%), 20/50 (40%) and 17/50 (34%) sera, respectively. Methylation in tumor and serum were closely correlated (P=0.001), but no correlation was found with survival. Twelve K-ras mutations (cysteine) were found in serum and nine mutations were found in tumor (five cysteine, one alanine, one aspartic, one arginine, and one valine). K-ras mutations in serum correlated significantly with survival (P=0.01).

14-3-3σ Methylation in Pretreatment Serum Circulating DNA of Cisplatin-Plus-Gemcitabine-Treated Advanced Non–Small-Cell Lung Cancer Patients Predicts Survival: The Spanish Lung Cancer Group

PURPOSE Survival in patients with advanced non-small-cell lung cancer (NSCLC) who are treated with platinum-based chemotherapy is rather variable. Methylation-dependent transcriptional silencing of 14-3-3sigma, a major G2-M checkpoint control gene, could be a predictor of longer survival. PATIENTS AND METHODS A sensitive methylation-specific polymerase chain reaction assay was used to evaluate 14-3-3sigma methylation status in pretreatment serum DNA obtained from 115 cisplatin-plus-gemcitabine-treated advanced NSCLC patients. RESULTS 14-3-3sigma methylation was observed in all histologic types of 39 patients (34%). After a median follow-up of 9.8 months, median survival was significantly longer in the methylation-positive group (15.1 v 9.8 months; P = .004). Median time to progression was 8 months in the methylation-positive group and 6.3 months in the methylation-negative group (log-rank test, P = .027). A multivariate Cox regression model identified only 14-3-3sigma methylation status and Eastern Cooperative Oncology Group performance status as independent prognostic factors for survival. In an exploratory analysis, median survival for 22 methylation-positive responders has not been reached, whereas survival was 11.3 months for 29 methylation-negative responders (P = .001). CONCLUSION Methylation of 14-3-3sigma is a new independent prognostic factor for survival in NSCLC patients receiving platinum-based chemotherapy. It can be reliably and conveniently detected in the serum, thus obviating the need for tumor tissue analysis.

Detection of Trypanosoma cruzi and Trypanosoma rangeli Infection by Duplex PCR Assay Based on Telomeric Sequences

ABSTRACT We used the species specificity and repetitious nature of subtelomeric kinetoplastida sequences to generate a duplex PCR assay for the simultaneous detection of Trypanosoma cruzi and Trypanosoma rangeli in experimentally and naturally infected triatomine (Reduviid) bugs and in infected human subjects. The assay was species specific and was capable of detecting 1/20th of T. cruzi and 1/4th of T. rangeli cell equivalents without complementary hybridization. In addition, the PCR-based assay was robust enough for direct application to difficult biological samples such as Reduviid feces or guts and was capable of recognizing all T. cruzi and T. rangeli strains and lineages. Because the assay primers amplify entirely different target sequences, no reaction interference was observed, facilitating future adaptation of this assay to an automated format.

The role of vitamin A on the inhibitors of nonheme iron absorption: Preliminary results

Abstract The interaction of vitamin A and inhibitors of iron absorption from a basal breakfast containing bread from either 100 g of precooked maize flour or 100 g of wheat flour + 50 g of cheese + 10 g of margarine was studied. These breads were labeled with either 55 Fe or 59 Fe. This basal breakfast was given alone on the first day of the study, and a beverage containing coffee or tea at different concentrations was administered with this breakfast on the following days. In the first three experiments performed, the bread was made from commercially available flours, fortified with iron as ferrous fumarate and vitamins. It can be noticed that whereas the iron absorption from the breakfast containing wheat bread was significantly reduced when given with different concentrations of coffee beverages, the bioavailability of iron from the breakfast containing precooked maize bread remained the same in spite of being administered with increasing concentrations of coffee beverages. The only ingredient present in precooked maize bread and not in wheat bread was vitamin A. In the other experiments, iron and vitamin A were added to the non-fortified precooked maize flour in our laboratory. In presence of vitamin A, nonheme iron absorption from the basal breakfast containing either coffee or tea was not statistically different from the breakfast without coffee, meaning that vitamin A can overcome the inhibition of coffee and tea on iron absorption and also prevents the inhibitory effect of phytates. The high performance liquid chromatography and spectrophotometric studies seem to indicate that during the digestive process, iron and vitamin A form a new prouct or complex, that keeps iron soluble even at pH6. All these data suggest that vitamin A binds iron liberated during digestive process and acts as a quelating agent, keeping iron soluble in the intestinal lumen and preventing the inhibition of polyphenols and phytates on nonheme iron absorption.

Expression of Fluorescent Genes in Trypanosoma cruzi and Trypanosoma rangeli (Kinetoplastida: Trypanosomatidae): Its Application to Parasite-Vector Biology

Abstract Two Trypanosoma cruzi-derived cloning vectors, pTREX-n and pBs:CalB1/CUB01, were used to drive the expression of green fluorescent protein (GFP) and DsRed in Trypanosoma rangeli Tejera, 1920, and Trypanosoma cruzi Chagas, 1909, isolates, respectively. Regardless of the species, group, or strain, parasites harboring the transfected constructs as either episomes or stable chromosomal integrations showed high-level expression of fluorescent proteins. Tagged flagellates of both species were used to experimentally infect Rhodnius prolixus Stal, 1953. In infected bugs, single or mixed infections of T. cruzi and T. rangeli displayed the typical cycle of each species, with no apparent interspecies interactions. In addition, infection of kidney monkey cells (LLC-MK2) with GFP-T. cruzi showed that the parasite retained its fluorescent tag while carrying out its life cycle within cultured cells. The use of GFP-tagged parasites as a tool for biological studies in experimental hosts is discussed, as is the application of this method for copopulation studies of same-host parasites.

Genetic profiling of a central Venezuelan population using 15 STR markers that may be of forensic importance

The AmpFlSTR Identifiler kit has recently been accepted for use in DNA databasing of forensic samples in the FBIs National DNA Index System. In the present study, we used this kit to analyze the allele distribution of 15 short tandem repeat markers (STR) in individuals living in Caracas city, Venezuela. The allele frequencies of two of these STR, D2S1338 and D19S433, have not previously been reported for this or any other Latin American population. The results indicate that for the population here studied, the 15 STR tested are useful markers for paternity testing and forensic casework.

Persistent infections by Leishmania (Viannia) braziliensis.

Here we review the phenomenon of persistency in Leishmania (Viannia) braziliensis infections. In other Leishmania species where appropriate animal models exist, considerable advances in the understanding of basic immunologic mechanisms of persistency have been made; for a review see Aebisher (1994). On the contrary, the evidences of persistence in infections with L. braziliensis rest on studies of human clinical cases many of which we summarized and discussed in this work.

Heat treatment on heme iron and iron-containing proteins in meat: Iron absorption in humans from diets containing cooked meat fractions

Abstract The present study was undertaken to characterize the effect of heat on iron compounds and iron-containing proteins of rabbit meat. We also studied human iron absorption from beef meat precipitates and investigated changes in the cysteine content in beef and rabbit meat caused by cooking processes. Supernatant and precipitate fractions were obtained by an extraction procedure that included homogenization and repeated centrifugations. A 50% decrease of soluble iron was produced by cooking the meat. Cooking also reduced the heme iron content of the meat by 62%. Chromatographic separation of soluble meat extracts showed changes in ferritin, hemoglobin, and myoglobin elution profiles in cooked meat compared with raw meat. Determinations of the cysteine content in raw or cooked meat samples showed a statistically significant reduction ( P

Vitamin A Reduces the Inhibition of Iron Absorption by Phytates and Polyphenols

In searching for an explanation for the rapid response to iron-fortification programmes, we focused on the interaction of vitamin A and inhibitors of iron absorption from a basal breakfast containing bread from either pre-cooked maize flour or wheat flour plus cheese and margarine. These breads were labeled with either 59Fe or 55Fe. These experiments demonstrated that vitamin A prevented the inhibiting effect of polyphenols and phytates on iron absorption. It was also demonstrated that vitamin A had the same effect on iron absorption as phytase.