Mutations in the John Cunningham virus VP1 gene could predispose to the development of progressive multifocal leukoencephalopathy in multiple sclerosis patients undergoing treatment with natalizumab.

BACKGROUND: Patients with Multiple Sclerosis (MS) undergoing treatment with natalizumab (NTZ) are at risk of developing progressive multifocal leukoencephalopathy (PML) due to the reactivation of John Cunningham (JC) virus. A relevant characteristic among PML cases is the development of single nucleotide mutations in the VP1 gene of the causal JC virus. The identification of such mutations in timely manner can provide valuable information for MS management. OBJECTIVE: To identify mutations along the JC virus VP1 gene in MS patients undergoing treatment with NTZ, and correlate them with anti-JC virus antibody index. METHODS: Eighty-eight MS patients, one hundred twenty controls, and six patients with diagnosis of Human Immunodeficiency Virus (HIV) with and without secondary PML were included. JC virus was identified in peripheral blood mononuclear cells and cerebrospinal fluid by PCR. Amplification and sequencing of the entire length of the VP1 gene were performed in all positive clinical samples. RESULTS: In MS cases no mutations were observed in the JC virus VP1 gene, but it was positive in HIV controls with PML. Interestingly, the JC virus VP1 gene sequence derived from the HIV patients exhibited a non-silent substitution in position 186 (G â†’ C), leading to an amino acid change (Lys â†’ Asp). We did not find correlation between anti-JC virus antibody index and DNA viral detection. CONCLUSIONS: . The identification of single nucleotide mutants in the JC virus VP1 gene might be an early predictive marker to PML for efficient patient treatment and follow-up.

Surface properties and in vitro cytopathic effect of various strains of Trichomonas vaginalis.

The in vitro cytopathic effect of four strains of Trichomonas vaginalis on cultured epithelial monolayers was analyzed through electrophysiology and electron microscopy. Interaction of trichomonads of two virulent strains (GT-10 and GT-13) with cultured MDCK cell monolayers mounted in Ussing chambers produced a rapid decrease in transepithelial resistance to less than 30% of control values after only 15 min. By 30 min the electrical resistance was practically abolished by the virulent parasites. In contrast, of two attenuated strains of trichomonads (GT-3 and GT-7) analyzed under similar conditions, GT-3 trophozoites required 180 min to reduce transepithelial resistance to 9% of control values, while monolayers in contact with GT-7 parasites still showed 28% of control values at this time of incubation. Sequential scanning electron microscopy confirmed the much faster and widespread cytopathic effect of virulent parasites. In contrast, the slow lytic process produced by attenuated trophozoites was reduced to focal areas of direct contact with epithelial cells. Another difference was found by measurement of the surface charge of the four strains of T. vaginalis by means of cell microelectrophoresis. While the two virulent strains showed a negative surface charge, the two attenuated strains had no detectable surface charge at neutral pH. When parasites were incubated with cationized ferritin and studied with transmission electron microscopy the surface of virulent trichomonads appeared heavily labeled, whereas the surface of attenuated parasites had only sparse and irregular ferritin binding.

Ultrastructural study of Entamoeba invadens encystation and excystation.

In the life cycle of Entamoeba species, the cyst and all the processes associated to it have been poorly studied. Entamoeba invadens, a serpent's parasite, has been commonly accepted as a model for the study of encystation and excystation. Here we analyzed through scanning and transmission electron microscopy the in vitro morphological differentiation of both processes. During encystation, the formation of an irregular net of fibrillar material on the surface of precysts was observed. In thin sections of cryofixed and cryosubstituted specimens, abundant vacuoles containing a microfibrillar material of similar appearance to the structural components of the cyst wall were found in the cytoplasm. Assays with a calcofluor probe on cryosections of encysting trophozoites and precysts showed the presence of fluorescent circular cytoplasmic structures. In the cyst stage, the fluorescence was located on the surface. During excystation, the detachment of the metacyst from the cyst wall was observed through scanning electron microscopy. Metacysts endocyting amorphous material which may correspond to cyst wall residues were commonly found. By transmission electron microscopy the formation of a crescent-shaped space between the plasma membrane and the cyst wall was observed. Abundant small electrondense bodies were found in the cytoplasm. Many of them were in close apposition to the plasma membrane and frequently some of them were seen projecting towards this newly formed space. Our results suggest that the microfibrillar content of the vacuoles corresponds to the cyst wall material, that the electrondense bodies may be involved in the excystation process, and that part of the cyst wall residues may be endocyted by the parasite.

Giardia lamblia: effect of infection with symptomatic and asymptomatic isolates on the growth of gerbils (Meriones unguiculatus).

Gerbils (Meriones unguiculatus) were intragastrically inoculated with axenic Giardia lamblia cultures from symptomatic and asymptomatic children. All isolates were able to colonize the duodenum. However, the colonization capacity of the symptomatic isolates was significantly higher compared to that of the asymptomatic ones. Despite the different colonization capacity of the isolates, the growth curves of infected animals were significantly lower than those of controls. The study demonstrates that acute giardia infections are capable of altering the corporal development of the host. These results may suggest that not only symptomatic, but also asymptomatic giardiasis in children, often unnoticed by parents and clinicians, could be causing a silent detriment in their nutritional status.

Pathogenesis of intestinal amebiasis: from molecules to disease.

In spite of a wealth of knowledge on the biochemistry and cellular and molecular biology of Entamoeba histolytica, little has been done to apply these advances to our understanding of the lesions observed in patients with intestinal amebiasis. In this review, the pathological and histological findings in acute amebic colitis are related to the molecular mechanisms of E. histolytica pathogenicity described to date. Infection of the human colon by E. histolytica produces focal ulceration of the intestinal mucosa, resulting in dysentery (diarrhea with blood and mucus). Although a complete picture has not yet been achieved, the basic mechanisms involved in the production of focal lytic lesions include complex multifactorial processes in which lectins facilitate adhesion, proteases degrade extracellular matrix components, porins help nourish the parasite and may also kill incoming polymorphonuclear leukocytes and macrophages, and motility is used by the parasite to invade deeper layers of the colon. In addition, E. histolytica has developed mechanisms to modulate the immune response during acute infection. Nevertheless, much still needs to be unraveled to understand how this microscopic parasite has earned its well-deserved histolytic name.

Morphological and cytochemical characterization of female Anopheles albimanus (Diptera: Culicidae) hemocytes.

Hemocytes of 2- to 3-d-old female Anopheles albimanus Wiedemann are described by morphology, cytochemistry, and functional criteria. Supplemented Grace's insect medium in a modified Foley's perfusion method was used to obtain hemolymph from An. albimanus. Morphological analysis indicated 3 types of hemocytes were present, prohemocytes, plasmatocytes, and granular cells. Prohemocytes were small round cells with a high nuclear/cytoplasmic ratio. Plasmatocytes were the most abundant cell types in the hemolymph, and appeared as small to large and spindle-shaped cells with round or elongate nucleus, variable number of vacuoles, small granules, and pseudopodia. Granular cells were small to large and round with a large number of cytoplasmic granules, vacuoles, and numerous filopodia. Ultrastructurally, prohemocytes were undifferentiated with abundant free ribosomes and with few small electron-dense granules. Plasmatocytes were rich in mitochondria, rough endoplasmic reticulum, free ribosomes, small electron-dense granules, numerous peripheral vacuoles and with an important organelle polarization. Granular cells contained numerous large electron-dense granular inclusions and vacuoles. Cytochemical studies showed that plasmatocytes and granular cells have cationic bactericidal proteins. Only granular cells showed phenoloxidase and probably lysosomal activities. In vitro functional studies demonstrated that both plasmatocytes and granular cells were able to attach to glass slides, and only plasmatocyte had phagocytic activity and motility. These results characterize the hemocytes of An. albimanus and suggest that plasmatocytes and granular cells may have a role in defense responses to foreign organisms.

Entamoeba dispar: ultrastructure, surface properties and cytopathic effect.

The cytological features of Entamoeba dispar, recently recognized by biochemical and molecular biology criteria as a distinct species, were compared to those of Entamoeba histolytica When cultured under axenic conditions, living trophozoites of E. dispar strain SAW 76ORR clone A were more elongated in form, had a single frontal pseudopodium, and showed a noticeable uroid. In sections of E. dispar trophozoites stained with Toluidine blue, characteristic areas of cytoplasmic metachromasia were seen due to the presence of large deposits of glycogen, seldom found in E. histolytica strain HM1:IMSS. Under the light microscope the periphery of the nucleus in E. dispar was, lined by finer, more regularly distributed dense granules. With transmission electron microscopy the surface coat of E. dispar was noticeable thinner. In addition. E. dispar had a lower sensitivity to agglutinate with concanavalin A and a higher negative surface charge, measured by cellular microelectrophoresis. The cytopathic effect of E. dispar was much slower, analyzed by the gradual loss of transmural electrical resistance of MDCK epithelial cell monolayers mounted in Ussing chambers. Whereas in E. histolytica phagocytosis of epithelial cells plays an important role in its cytopathic effect. E. dispar trophozoites placed in contact with MDCK cells showed only rare evidence of phagocytosis. The results demonstrate that the morphology of E. dispar is different to that of E. histolytica, both at the light microscopical and the ultrastructural levels. In addition they show that E. dispar in axenic culture has a moderate cytopathic effect on epithelia] cell monoLayers. However, when compared to E. histolytica, the in vitro lytic capacity of E. dispar is much slower and less intense.

Trichomonas vaginalis: in vitro phagocytosis of lactobacilli, vaginal epithelial cells, leukocytes, and erythrocytes.

This paper explores the interaction of two strains of Trichomonas vaginalis, of high and low virulence, with the cell types present in the microenvironment of the parasite during human infections. With the use of transmission and scanning electron microscopy the sequence of internalization by T. vaginalis of Döderlein's lactobacilli, and of vaginal epithelial cells, leukocytes, and erythrocytes was documented. Furthermore, the degradation of ingested material by colocalization of acid phosphatase activity in phagocytic vacuoles was demonstrated. Phagocytosis of all cell types analyzed was found in both strains studied, although the highly virulent strain internalized target cells more rapidly than the less virulent one. Ultrastructural evidence indicated that phagocytosis takes place through two distinct mechanisms, only one involving the formation of a phagocytic stoma, characteristic of professional phagocytes. T. vaginalis phagocytosis may be both an efficient means of obtaining nutrients for the parasite and a significant factor in the pathogenesis of trichomonal infections of the human genitourinary tract.

Entamoeba histolytica: liver invasion and abscess production by intraperitoneal inoculation of trophozoites in hamsters, Mesocricetus auratus.

Intraperitoneal inoculation of axenically cultured Entamoeba histolytica trophozoites constitutes an easy to perform, highly reproducible procedure for inducing amebic liver abscesses in hamsters. Efficiency in abscess production (95% of infected animals after 1 week) was similar to data reported using direct intrahepatic or intraportal inoculation. The morphological sequence of infection shows that amebas in the peritoneal cavity initially produce a large exudate constituted mainly of acute inflammatory cells. These cells form a rim of polymorphonuclear leukocytes surrounding the amebas, which adhere to the trophozoite and can sometimes be observed polarized to one end of the parasite, suggesting capping of surface receptors. Early stages are also characterized by the production of distant inflammatory reactions in the hepatic portal spaces. At 6 h postintraperitoneal inoculation, larger foci of inflammatory reactions surrounding amebas are developed in the peritoneum, extending to and damaging the liver surface membranes as well as the serosa of other internal organs. Thereafter, tissue damage progresses deeper into the liver parenchyma, and a few days later, coalescing granulomas and large necrotic areas are observed in the liver tissue. Based on the present morphological time-sequence study, we suggest that inflammatory cells associated with E. histolytica trophozoites play an important role in commencing the damage of liver sheaths and producing the subsequent parenchymal lesions. The simplicity and reliability of this model are important factors to consider when large numbers of experimentally induced amebic liver abscesses are needed.

In vivo and in vitro experimental intestinal amebiasis in Mongolian gerbils (Meriones unguiculatus).

One of the main drawbacks of experimental amebiasis is the lack of an adequate animal model for invasive intestinal lesions. Mongolian gerbils are useful because both intestinal and hepatic amebiasis can be produced experimentally with Entamoeba histolytica trophozoites. In this paper we show results obtained with in vivo and in vitro models of intestinal amebiasis in gerbils. We inoculated gerbils intracecally with monoxenic cultures of a highly virulent E. histolytica HM1:IMSS substrain. In the in vivo model an increase in mucus production was observed during the first 6 h of interaction. Microulcerative mucosal lesions appeared at 24-72 h postinoculation. Inflammatory infiltrate and edema of the lamina propria were associated with superficial foci of necrosis. At 96 h the cecal mucosa had an almost normal appearance and live amebas were no longer detected. In the in vitro model, early damage was detected in cecal strips mounted in Ussing chambers as a rapid fall in potential difference, short-circuit current, and transepithelial resistance that correlated with the extent of the microscopic lesions produced. The latter consisted of cellular edema and the appearance of small, translucent vacuoles at the base of epithelial cells. Further damage led to loss of intercellular junctions, destruction of interglandular epithelial cells, and edema of the lamina propria. The present results demonstrate that the gerbil is useful as an experimental model for the analysis of early stages of invasive intestinal amebiasis both in vivo and in vitro.

In vitro Entamoeba histolytica adhesion to human endothelium: a comparison using two strains of different virulence.

Extraintestinal dissemination of Entamoeba histolytica is frequently manifested by the life-threatening amebic liver abscess (ALA). The hepatic establishment of amebas implies invasion of blood vessels and contact with the endothelium. By means of a fluorescence-based quantitative adhesion assay, we assessed the binding to human endothelial cells of two E. histolytica strains of different virulence. The highly virulent strain (L-A) adhered substantially more strongly to unstimulated endothelium than the non-virulent one (BG3). Attachment of L-A was increased by treatment of endothelial cells with interleukin-1 beta (IL1 beta). Other proinflammatory cytokines such as interferon-gamma (IFN gamma) and tumor necrosis factor-alpha (TNF alpha) did not modify the spontaneous adhesion capacity of amebas. For purposes of comparison we also performed adhesion of the parasites to skin fibroblasts. Adhesion to this cell type was quite low (< 10%). Parasite virulence, differential adhesive capacity to endothelial cells, and modulation of the latter phenomenon by proinflammatory factors (IL1 beta) may influence the evolution and outcome of extraintestinal amebiasis, especially hepatic abscesses.