Amphotericin B induces apoptosis-like programmed cell death in Naegleria fowleri and Naegleria gruberi.

Naegleria fowleri and Naegleria gruberi belong to the free-living amoebae group. It is widely known that the non-pathogenic species N. gruberi is usually employed as a model to describe molecular pathways in this genus, mainly because its genome has been recently described. However, N. fowleri is an aetiological agent of primary amoebic meningoencephalitis, an acute and fatal disease. Currently, the most widely used drug for its treatment is amphotericin B (AmB). It was previously reported that AmB has an amoebicidal effect in both N. fowleri and N. gruberi trophozoites by inducing morphological changes that resemble programmed cell death (PCD). PCD is a mechanism that activates morphological, biochemical and genetic changes. However, PCD has not yet been characterized in the genus Naegleria. The aim of the present work was to evaluate the typical markers to describe PCD in both amoebae. These results showed that treated trophozoites displayed several parameters of apoptosis-like PCD in both species. We observed ultrastructural changes, an increase in reactive oxygen species, phosphatidylserine externalization and a decrease in intracellular potassium, while DNA degradation was evaluated using the TUNEL assay and agarose gels, and all of these parameters are related to PCD. Finally, we analysed the expression of apoptosis-related genes, such as sir2 and atg8, in N. gruberi. Taken together, our results showed that AmB induces the morphological, biochemical and genetic changes of apoptosis-like PCD in the genus Naegleria.

Prevalence of human papillomavirus in women from Mexico City.

INTRODUCTION: Cervical cancer is the most common cancer among Mexican women. The goal of the present study was to determine the prevalence and distribution of HPV types in women from Mexico City. METHODS: Our study was conducted in the Clinica de Especialidades de la Mujer de la Secretaría de la Defensa Nacional, Mexico. Random samples were taken from 929 healthy women requesting a cervical Papanicolaou examination. Detection and genotyping of HPV were performed by multiplex PCR, with the HPV4A ACE Screening kit (Seegene). RESULTS: 85 of nine hundred twenty-nine women (9.1%) were infected with HPV. Of HPV-positive women, 99% and 1% had high- and low-risk HPV genotypes, respectively. The prevalence of the 16 high-risk (HR) HPV types that were screened was 43% : 42% (18) were HPV positive and 14% (16) were HPV positive, which includes coinfection. Multiple infections with different viral genotypes were detected in 10% of the positive cases. Abnormal cervical cytological results were found in only 15.3% of HPV-positive women, while 84.7% had normal cytological results. CONCLUSIONS: We found a similar prevalence of HPV to previous studies in Mexico. The heterogeneity of the HPV genotype distribution in Mexico is evident in this study, which found a high frequency of HPV HR genotypes, the majority of which were HPV 18.

In vivo programmed cell death of Entamoeba histolytica trophozoites in a hamster model of amoebic liver abscess.

Entamoeba histolytica trophozoites can induce host cell apoptosis, which correlates with the virulence of the parasite. This phenomenon has been seen during the resolution of an inflammatory response and the survival of the parasites. Other studies have shown that E. histolytica trophozoites undergo programmed cell death (PCD) in vitro, but how this process occurs within the mammalian host cell remains unclear. Here, we studied the PCD of E. histolytica trophozoites as part of an in vivo event related to the inflammatory reaction and the host-parasite interaction. Morphological study of amoebic liver abscesses showed only a few E. histolytica trophozoites with peroxidase-positive nuclei identified by terminal deoxynucleotidyltransferase enzyme-mediated dUTP nick end labelling (TUNEL). To better understand PCD following the interaction between amoebae and inflammatory cells, we designed a novel in vivo model using a dialysis bag containing E. histolytica trophozoites, which was surgically placed inside the peritoneal cavity of a hamster and left to interact with the host's exudate components. Amoebae collected from bags were then examined by TUNEL assay, fluorescence-activated cell sorting (FACS) and transmission electron microscopy. Nuclear condensation and DNA fragmentation of E. histolytica trophozoites were observed after exposure to peritoneal exudates, which were mainly composed of neutrophils and macrophages. Our results suggest that production of nitric oxide by inflammatory cells could be involved in PCD of trophozoites. In this modified in vivo system, PCD appears to play a prominent role in the host-parasite interaction and parasite cell death.

Entamoeba histolytica: differential gene expression during programmed cell death and identification of early pro- and anti-apoptotic signals.

We have demonstrated that programmed cell death (PCD) in Entamoeba histolytica is induced in vitro by G418 aminoglycoside antibiotic. To ascertain if biochemical and morphological changes previously observed are paired to molecular changes that reflect a genetic program, we looked here for early differential gene expression during the induction of PCD. Using cDNA-amplified fragment length polymorphisms (AFLPs) and in silico derived analysis we showed in E. histolytica a differential gene expression during PCD induced by G418. The genes identified encoded for proteins homologous to Glutaminyl-tRNA synthase, Ribosomal Subunit Proteins 40S and 18S, Saposin-like, Silent Information Regulator-2 (Sir-2), and Grainins 1 and 2. Using real-time quantitative PCR (RT Q-PCR), we found that glutaminyl-tRNA synthetase, sir-2, grainins and saposin-like genes were strongly overexpressed after 30min of PCD induction, while its expression dramatically decreased up to 60min. On the other hand, overexpression of ribosomal genes increased only 7-fold of basal expression, showing a progressive down-regulation up to 90min. glutaminyl-tRNA synthetase, sir-2 and grainins could act as negative regulators of PCD, trying to control the biochemical changes related to PCD activation. Overexpression of saposin-like gene could act as up-regulator of some cell death pathways. Our results give evidence of the first genes identified during the early stage of PCD in E. histolytica that could be implicated in regulation of apoptotic pathways.

[Diagnosis of infection Chlamydia trachomatis by PCR among patients in the Specialty Clinic of Women of the National Defense Secretary]. / Diagnóstico de infección por Chlamydia trachomatis mediante PCR en pacientes que acuden a la Clínica de Especialidades de la Mujer de la Secretaría de la Defensa Nacional.

INTRODUCTION: Infection with Chlamydia trachomatis is a sexually transmitted bacterial disease most common in the world. MATERIAL AND METHODS: Determination through the PCR technique the presence of C. trachomatis in 98 cervical samples by amplifying a fragment of DNA plasmid cryptic. We investigated medical variables of interest and contraceptive methods in the population. Variables were compared between the positive and negative patients to infection with C. trachomatis using the test of Mann-Whitney U test and Chi square with the program SigmaStat version 2.0. We considered statistically significant as those values of p < 0.001. RESULTS: 20 samples were found positive for infection with C. trachomatis of the 98 patients studied, indicating a prevalence of 20.4% of the total group. The variables analyzed showed a statistically significant difference in the number of abortions, which were higher for patients positive for the infection. CONCLUSIONS: The prevalence found in this study suggests that infection with C. trachomatis is an issue of concern in public health, it requires the count on diagnostic techniques, such as PCR favoring an increase in the detection and early treatment in reducing the spread of the infection and long-term sequelae, so that justifies his provide detection in a broader way.

Las citocinas en la patogénesis por citomegalovirus / The role of the cytokines in the pathogenesis of cytomegalovirus-associated disease

La infección por Citomegalovirus Humano (CMVH) es una importante causa de morbilidad y mortalidad en pacientes inmunocomprometidos como son los pacientes con SIDA y receptores de órganos. La infección por CMVH comúnmente resulta de la reactivación del virus que se encuentra latente en el huésped. Esta reactivación de los virus genera síntomas y signos de diversas enfermedades inflamatorias tales como encefalitis, pneumonia intersticial, retinitis, hepatitis, gastritis y colitis. Los resultados de diversos experimentos en modelos murinos sugieren que las citocinas, tales como IFN-g, TNF-a, IL-6, IL-8, y el óxido nítrico participan en el desarrollo de estas enfermedades. Sin embargo, la razón de cómo y por qué la infección por CMVH aumenta la producción de estas citocinas es desconocido. La relación entre los niveles séricos elevados de citocinas y la detección de antígenos y ADN virales sugiere que estos factores solubles pueden contribuir a las complicaciones inmunopatológicas, por lo que la inhibición de la liberación o acción de estas citocinas puede ser una estrategia alternativa para prevenir la morbilidad asociada a la infección por CMVH en los pacientes inmunocomprometidos.