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Biomedical subjects

V Tsutsumi

Publications and source records attributed to V Tsutsumi.

At least 37 records · Page 2Linked to original sources
PubMed1997

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.

Animals↗
PubMed1997

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.

Animals↗
PubMed1997

Human amebic liver abscess: expression of intercellular adhesion molecules 1 and 2 and of von Willebrand factor in endothelial cells.

Liver invasion by amebas with production of amebic liver abscess (ALA) is the most common extraintestinal lesion produced by the protozoan parasite Entamoeba histolytica. This hepatic damage is characterized by the presence of extensive tissue necrosis. However, little is known about the parasite and host factors involved in the process of tissue damage. During the early establishment of amebas in the liver parenchyma as well as during the extension of the tissue necrosis, parasites interact with sinusoidal endothelial cells. As a consequence of ameba-endothelial cell interactions, the latter can be activated and express proinflammatory factors that could be related to tissue destruction. We studied by immunohistochemistry the localization of antigenic molecules of E. histolytica trophozoites and of molecules such as intercellular adhesion molecule 1 (ICAM-1), ICAM-2, and von willebrand factor in activated endothelial cells of human ALA, which could be related to the pathophysiological mechanisms of tissue destruction in amebiasis.

Adolescent↗
PubMed1996

Hartmannella vermiformis isolated from the cerebrospinal fluid of a young male patient with meningoencephalitis and bronchopneumonia.

Meningoencephalitis and bronchopneumonia were documented in a patient from Peubla, Mexico. The patient began with symptoms and signs of a common flu and 12 days after the onset of his disease he was admitted to the hospital presenting symptoms and signs of meningoencephalitis. The clinical course evolved into an endocraneal hypertension syndrome with bronchopneumonia, coma and death. Wide-spectrum antibiotics, immunosuppressive and anti-tuberculosis therapy were unsuccessfully administered. Important antecedents were degree I malnutrition and repeated contact with polluted water. Post-mortem autopsy was not performed. Gram-positive cocci were isolated from the spinal fluid 2 days after admission, and then active amebae were isolated from three different samples of the spinal fluid at days 16, 18 and 19 after admission. Such samples were concentrated and inoculated onto specific culture media. Identification of amebae was based on their morphology and biochemistry. All amebae were Hartmannella vermiformis. Amebae were apparently not the cause of the disease and might be considered as an opportunistic colonizer which may have caused the evolution of the disease to become worse.

Acid Phosphatase↗
PubMed1995

Entamoeba histolytica: increase of enterotoxicity and of 53- and 75-kDa cysteine proteinases in a clone of higher virulence.

We compared the enterotoxicity and cysteine proteinases (CP) of the low-virulence Entamoeba histolytica HM1 strain with the highly virulent 1659 clone, derived from HM1 by hamster liver passages. Enterotoxicity of 50,000 freeze-thawed trophozoites was determined on 0.28-cm2 intestinal segments mounted in Ussing chambers; CP activity of Nonidet-P40 amebal lysates was assayed by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and carbobenzoxy-L-arginine-L-arginyl-p-nitroaniline, a CP-specific substrate. Treatment of gerbil cecum segments with amebal lysates caused an immediate fall of their electrophysiologic properties (potential difference, short-circuit current, and transmural resistance) whose decay rates were clearly faster with 1659 than with HM1 lysates. Nonimmune and immune antiamebic human sera and the CP-specific inhibitor E-64 (trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane) prevented the fall of the electrophysiologic properties. Gelatinases, less active in HM1 than in 1659 trophozoites, were better preserved in lysates containing 10 mM p-hydroxymercuribenzoate (pHMB) to prevent autoproteolysis: in lysates without pHMB nearly no gelatinase bands were observed in HM1 samples, whereas intense 30K, 35K, 44K, and 75K bands were seen in 1659 samples; in lysates with pHMB only 53K and 75K bands were found that were much more intense in 1659 samples, 75K being barely visible in HM1 samples. The overall CP activity was 17 times higher in 1659 than in HM1 lysates, was inhibited by E-64 (mean inhibitory dose, 20 microM), was stimulated by 2-mercaptoethanol (ME) 3.7 times in HM1 and 2.4 times in 1659 lysates, and was reactivated by ME in lysates containing pHMB. Most of the CP activity in HM1 lysates sedimented at 15,600g but predominated in 1659 supernatants. The increase of E. histolytica virulence thus correlates with a remarkable increase both of in vitro enterotoxicity and of two CPs (53K and 75K), suggesting that these proteinases are significant pathogenicity factors.

Animals↗
PubMed1994

Ultrastructural features of neurosecretory cells in the medulla externa of the crayfish eyestalk.

A conglomerate of 8-12 neurons in the medulla externa of the crayfish eyestalk was explored in their reaction to a polyclonal antibody against the tyrosinated octapeptide Red Pigment Concentrating Hormone (Tyr-RPCH). These are large neurons with diameters within a range of 33-43 microns and they were all positively stained with neutral red. By intracellular staining with lucifer yellow, the neurons were found to branch extensively within the medulla externa and the lamina ganglionaris of the eyestalk. Each neurite bifurcates at about 40 microns from the soma. Both branches run to the medial edge of the eyestalk; one proceeds distally to the lamina ganglionaris, while the other runs proximally to the medulla interna. Both end freely in multiple arborizations, covering from the medial to the lateral edges of the eyestalk. No branches were found to the sinus gland, the main neurohaemal organ of the eyestalk. A group of 4 neurons in the conglomerate consistently rendered positive reaction to the anti-Tyr-RPCH antibody (A-RPCH). They are superficially located in the cluster, and at the electron microscope, they showed the usual features of a secretory cell, i.e., clear and dense granules, an active and well-developed Golgi apparatus, and rough endoplasmic reticulum. The dense granules were larger (mean diameter: 101.5 nm) than the clear granules (mean diameter: 90.3 nm). The immunopositive reaction at the electron microscope was found to be largely confined to the dense-cored granules.

Animals↗
PubMed1994

Entamoeba histolytica: electron-dense granule secretion, collagenase activity and virulence are altered in the cytoskeleton mutant BG-3.

HM-1:IMSS, a pathogenic strain of Entamoeba histolytica, and its mutant BG-3, identified by resistance to cytochalasin D, were tested for their capacity to: (i) secrete electron-dense granules; (ii) adhere and digest native type I collagen gels; and (iii) produce liver abscesses in new-born hamsters. The results demonstrate that the mutant has low adherence to collagen, low electron-dense granule secretion and collagenolytic activity, and low capacity to produce liver lesions in vivo, compared with the parental strain HM1:IMSS.

Animals↗
PubMed1994

Electrophysiology and immunity of the mucosa in intestinal infections.

We present here some of the major concepts and approaches to study the electrophysiology of the intestinal mucosa, and review the pathophysiology of intestinal infections caused by enteropathogenic bacteria, protozoa--especially our own work on experimental amebiasis using intestinal preparations mounted in the Ussing chamber--and nematodes, and finally discuss briefly the immunophysiology of the intestinal mucosa.

Amebiasis↗
PubMed1993

A fluorescence-based quantitative adhesion assay to study interactions between Entamoeba histolytica and human enterocytes. Effect of proinflammatory cytokines.

In order to study the initial events during infection of target cells by the enteric pathogen Entamoeba histolytica, we developed a quantitative adhesion assay based on the use of a human colonic cell line (CaCo-2) and biotinylated amoebae tagged with fluorescein. To prevent the strong and rapid lytic activity of Entamoeba histolytica on colonic cells, which would otherwise impede the study of the primary adhesion steps, parasites were mildly fixed, biotinylated and labelled with streptavidin-FITC. After labelled parasites have bound to enterocytes, nonadhered amoebae are removed by washing and attached parasites quantified by means of an automated fluorescence plate reader. The bioassay is simple, nonhazardous and can be completed in 1.5 h. We were able to detect ranges from 200 to 20,000 fluorescent parasites per microwell in a 96-well plate, containing approximately 10(5) colonic cells. Fluorescence intensity (arbitrary units) increased in direct relationship to the number of parasites added per well, and was not limited by the size of the culture plate (96, 24 or six wells). As an example of the value of this assay, two proinflammatory cytokines (interleukin-1, (IL-1 beta) and interferon-gamma (IFN-gamma) known to influence the adhesion properties of endothelial and epithelial cells, were used to assess their effects upon enterocyte-entamoeba binding. The increase in amoebae binding revealed by cytokine treatment to enterocytes suggests that the parasite may take advantage of inflammatory stimuli in order to increase its binding to colonic epithelium. We believe this rapid, sensitive and simple method offers the potential for large scale screening assays to study the immunobiology of this protozoal infection by analysing the mechanisms involved in the primary interactions between Entamoeba histolytica and enterocytes.

Animals↗
PubMed1993

Entamoeba histolytica: electrophysiologic and morphologic effects of trophozoite lysates on rabbit colon.

Although in acute intestinal amoebiasis the events occurring during the initial interaction of Entamoeba histolytica trophozoites with the mucosa of the large intestine probably determine the invasion by the parasites, an appropriate experimental model to study them does not exist. To develop such a model we used rabbit colonic segments (0.28 cm2) mounted in Ussing-type chambers. These preparations had electrophysiological properties (potential difference, short-circuit current, and electrical resistance) similar in magnitude and duration to those reported for stripped colonic mucosa. Lysates from 50,000 Entamoeba invadens PZ strain trophozoites (virulent to reptiles) added to the mucosal side of preparations had no effect, whereas lysates from the same number of E. histolytica HM1 strain trophozoites (virulent to humans) caused an immediate decay of electrophysiological properties. E. histolytica lysates induced the formation of translucent vacuoles at the bases of epithelial cells and separation between the epithelium and the lamina propria. The specificity and speed of the electrophysiologic effects of E. histolytica lysates and their correlation with the microscopic lesions suggest that this model may be useful to analyze the initial pathogenic events of intestinal amoebiasis.

Animals↗
PubMed1992

Active immunization in hamsters with live Entamoeba histolytica trophozoites of low virulence.

Live trophozoites of Entamoeba histolytica of low virulence (LV) were inoculated intraportally in hamsters. One week later they were challenged intrahepatically with highly virulent (HV) trophozoites of the same strain and sacrificed 1 week later. Significant protection manifested by the production of smaller lesions was observed in immunized animals. Antiamebic IgG levels were significantly higher in the immunized group. Histologically, livers of immunized animals presented small lesions containing foamy macrophages, epithelioid cells, fibroblasts and devoid of amebas. Lymphocyte and plasma cell infiltrates were seen periportally.

Animals↗