In vitro activity of Acanthamoeba castellanii on human platelets and erythrocytes.

The effect of Acanthamoeba on human platelets and erythrocytes has not been fully elucidated. This paper reports that cell-free supernatants prepared from A. castellanii can activate human platelets, causing both a significant increase in the cytosolic free-calcium concentration and platelet aggregation. In addition, we demonstrated that platelet activation depends on the activity of ADP constitutively secreted into the medium by trophozoites. This study also showed that A. castellanii can affect human red blood cells, causing hemolysis, and provided evidence that hemolysis occurs in both contact-dependent and contact-independent ways; there are differences in kinetics, hemolytic activity, and calcium dependency between the contact-dependent and contact-independent mechanisms. Partial characterization of contact-independent hemolysis indicated that ADP does not affect the plasma membrane permeability of erythrocytes and that heat treatment of amoebic cell-free supernatant abolishes its hemolytic activity. These findings suggest that some heat-labile molecules released by A. castellanii trophozoites are involved in this phenomenon. Finally, our data suggest that human platelets and erythrocytes may be potential cell targets during Acanthamoeba infection.

Acanthamoeba castellanii promotion of in vitro survival and transmission of coxsackie b3 viruses.

This work was undertaken to determine whether Acanthamoeba could play a role in the survival and transmission of coxsackieviruses and focused on in vitro interactions between Acanthamoeba castellanii and coxsackie B3 viruses (CVB-3). Residual virus titer evaluations and immunofluorescence experiments revealed a remarkable CVB-3 adsorption on amoeba surfaces and accumulation inside cells. The survival of viruses was independent of the dynamics of amoeba replication and encystment. In addition, our results indicated that virus-infected amoebas can release infectious viruses during interaction with human macrophages. On the basis of these data, Acanthamoeba appears to be a potential promoter of the survival of coxsackieviruses and their transmission to human hosts.

In vitro evaluation of the effectiveness of the macrolide rokitamycin and chlorpromazine against Acanthamoeba castellanii.

The present study demonstrates the in vitro effectiveness of the macrolide rokitamycin and the phenothiazine compound chlorpromazine against Acanthamoeba castellanii. Growth curve evaluations revealed that both drugs inhibit trophozoite growth in dose- and time-dependent ways. The effects of both drugs when they were used at the MICs at which 100% of isolates are inhibited were amoebistatic, but at higher doses they were amoebicidal as well as cysticidal. Experiments showed that when rokitamycin was associated with chlorpromazine or amphotericin B, rokitamycin enhanced their activities. Furthermore, low doses of rokitamycin and chlorpromazine, alone or in combination, blocked the cytopathic effect of A. castellanii against WKD cells derived from the human cornea. These results may have important significance in the development of new anti-Acanthamoeba compounds.

ADP and other metabolites released from Acanthamoeba castellanii lead to human monocytic cell death through apoptosis and stimulate the secretion of proinflammatory cytokines.

Monocytes/macrophages are thought to be involved in Acanthamoeba infections. The aim of this work was to study whether soluble metabolites (ADP and other compounds) released by Acanthamoeba castellanii trophozoites could induce morphological and biochemical changes in human monocytic cells in vitro. We demonstrate here that ADP constitutively released in the medium by A. castellanii, interacting with specific P2y(2) purinoceptors expressed on the monocytic cell membrane, caused a biphasic rise in [Ca(2+)](i), morphological changes characteristics of cells undergoing apoptosis, caspase-3 activation, and secretion of tumor necrosis factor alpha (TNF-alpha). The same results were found in monocytes exposed to purified ADP. Cell damage and TNF-alpha release induced by amoebic ADP were blocked by the P2y(2) inhibitor suramin. Other metabolites contained in amoebic cell-free supernatants, with molecular masses of, respectively, >30 kDa and between 30 and 10 kDa, also caused morphological modifications and activation of intracellular caspase-3, characteristics of programmed cell death. Nevertheless, mechanisms by which these molecules trigger cell damage appeared to differ from that of ADP. In addition, other amoebic thermolable metabolites with molecular masses of <10 kDa caused the secretion of interleukin-1beta. These findings suggest that pathogenic free-living A. castellanii by release of ADP and other metabolites lead to human monocytic cell death through apoptosis and stimulate the secretion of proinflammatory cytokines.

Catabolism of exogenous deoxyinosine in cultured epithelial amniotic cells.

Uptake and catabolism of purine nucleosides have been commonly considered as means to salvage the purine ring for nucleic acid synthesis, usually neglecting the destiny of the pentose moiety. With the aim to ascertain if deoxyribose derived from exogenous DNA can be utilised as a carbon and energy source, we studied the catabolism of exogenous deoxyinosine in a cell line derived from human amnion epithelium (WISH). Intact WISH cells catabolise deoxyinosine by conversion into hypoxanthine. The nucleoside enters the cell through a nitrobenzylthioinosine-insensitive equilibrative transport. Deoxyinosine undergoes a phosphorolytic cleavage inside the cell. The purine base diffuses back to the external medium, while the phosphorylated pentose moiety can be further catabolised to glycolysis and citric acid cycle intermediates. Our results indicate that the catabolism of the deoxynucleoside can be considered mainly as a means to meet the carbon and energy requirements of growing cells.

By releasing ADP, Acanthamoeba castellanii causes an increase in the cytosolic free calcium concentration and apoptosis in wish cells.

The role played by soluble molecules that may participate in acanthamoebal cytopathogenicity has yet to be fully characterized. We demonstrate here that Acanthamoeba castellanii trophozoites constitutively release ADP in the medium. Cell-free supernatants prepared from A. castellanii, by interaction with specific P(2y2) purinoceptors expressed on the Wish cell membrane, caused a biphasic rise in [Ca(2+)](i), extensive cell membrane blebbing, cytoskeletal disorganization, and the breakdown of nuclei. Cell damage induced by amoebic supernatants was blocked by the P(2y2) inhibitor Suramin. The same results were found in Wish cells exposed to purified ADP. These findings suggest that pathogenic free-living A. castellanii may have a cytopathic effect on human epithelial cells through ADP release, by a process that begins with a rise of cytosolic free-calcium concentration, and culminates in apoptosis.

Deoxyadenosine metabolism in a human colon-carcinoma cell line (LoVo) in relation to its cytotoxic effect in combination with deoxycoformycin.

We have assessed the intracellular metabolism of 2'-deoxyadenosine in a human colon-carcinoma cell line (LoVo), both in the absence and in the presence of deoxycoformycin, the powerful inhibitor of adenosine deaminase. The combination of 2'-deoxyadenosine and deoxycoformycin has been reported to inhibit the growth of LoVo cells in culture. In this paper we demonstrate that the observed toxic effect is strictly dependent on cell density. In the absence of deoxycoformycin, 2'-deoxyadenosine is primarily deaminated to 2'-deoxyinosine and then converted into hypoxanthine. In the presence of the inhibitor, the deoxynucleoside, in addition to a phosphorylation process, undergoes phosphorolytic cleavage giving rise to adenine. The conversion of 2'-deoxyadenosine to adenine might represent a protective device, emerging when the activity of adenosine deaminase is reduced or inhibited. There is much evidence to indicate that the enzyme catalyzing this process may be distinct from methylthioadenosine phosphorylase and S-adenosyl homocysteine hydrolase, which are the enzymes reported to be responsible for the formation of adenine from 2'-deoxyadenosine in mammals.

Acanthamoeba castellanii metabolites increase the intracellular calcium level and cause cytotoxicity in wish cells.

Previous studies have shown that trophozoites of the pathogenic free-living amoeba Acanthamoeba castellanii rapidly lyse a variety of cells in vitro. However, the role played by cytolitic molecules that may participate in Acanthamoebal cytopathogenicity has yet to be completely elucidated. The aim of this work was to study whether soluble molecules released by A. castellanii trophozoites could induce cytopathic effect in human epithelial cells in vitro. The results obtained indicate that A. castellanii trophozoites constitutively elaborate and release soluble factors that immediately elicit a cytosolic free-calcium increase in target cells. This phenomenon is induced by low molecular weight amoebic metabolites and depends on a transmembrane influx of extracellular calcium. Morphological changes, cytoskeletal damage, cell death and cytolysis followed the elevation of cytosolic free-calcium levels. Calcium ions are very important for cell homeostasis, in fact, they control the functions of a variety of cellular responses, including secretion, cell proliferation and apoptosis. Our results suggest that the substained elevation of the cytosolic free-calcium in response to A. castellanii metabolites might play a fundamental role in target cell damage during Acanthamoeba infections.

[Human fibrin glue in the treatment of residual parenchymal surface after total pericystectomy for hepatic echinococcus]. / La colla di fibrina umana nel trattamento della superficie parenchimale residua a pericistectomia totale per echinococco epatico.

The aim of this study was to evaluate the presumed efficacy of fibrin sealant in limiting bleeding and biliary leakage from liver residual surface after total pericystectomy for hydatid disease. Forty-five patients (group A) who underwent total pericystectomy in our Institution from 1986 to 1995 and liver residual surface treated with conventional techniques and fibrin sealant for control of haemorrhage and bile leakage were selected. A control group (B) was carefully selected, matching the main characteristics of patients in group A: it consisted of 44 patients, who underwent total pericystectomy from 1981 to 1993 and in which fibrin sealant was not used. Postoperative hospital stay, morbidity, mortality, abdominal drainage discharge, perioperative variations of hemoglobin and hematocrit readings and the need for postoperative blood transfusion were evaluated in the two groups. A statistical analysis was performed. We found no statistical significance for the considered parameters in the two groups. Markedly no significative difference was found in morbidity, abdominal drainage discharge and need for postoperative blood transfusion. Our results do not allow a definite assessment of the actual role of fibrin sealant in rising efficacy on control of bleeding and biliary leakage from residual liver surface to total pericystectomy obtained with conventional haemostatic techniques. We believe that a previously planned controlled prospective trial could give the needed further elements to precisely evaluate the role of fibrin sealant in the surgical treatment of hydatid disease of the liver.

[Stomach cancer in the elderly patient]. / Il cancro dello stomaco nell'anziano.

Gastric cancer is a typical disease of old age, in fact about one half of the patients affect by it are aged over 65. Elderly patients imply a problematical choice of surgical treatment due to the general and specific risk and to life expectancy. In order to evaluate the specific features of gastric cancer in aged people and to share their experience in choosing the surgical treatment, a series of 50 patients with gastric cancer selected on the age > or = 75 years and observed from 1970 to 1993, was reviewed by the authors. Some features have been settled such as sex ratio approaching the unity, a prevalence of tumors located in the distal third of the stomach, the higher incidence of intestinal type and a wide incidence of intestinal type and a wide incidence of patients in III or IV stage. No surgical procedure was undertaken in eleven patients. The remaining 39 patients (78%) underwent a surgical procedure. In 22 patients (56.4) a resection was performed: 17 (77.3%) underwent a subtotal gastric resection, 11 curative and 6 palliative, and 5 (22.7%) a total gastrectomy out of necessity for tumor localization. In 17 patients (43.6%) a bypass procedure was carried out, while in 7 (17.9%) the surgical procedure was a simple laparatomy. Postoperative morbidity incidence was 17.9%, mortality rate 10.2%. Actuarial 5 years survival rate for curative resection was 41.5%. Median survival time was 13 months for patients who underwent a palliative resection and 6 months after bypass procedures. The data suggest that subtotal gastrectomy, also as palliative procedure, fits better to geriatric patients' requirements and is able to offer a satisfactory quality of life, to prevent cancer complications and to determine a longer survival.

Kinetic changes of alpha B crystallin expression in neoplastic cells and syngeneic rat fibroblasts at various subculture stages.

Alpha B crystallin, a structural at variable levels, in many extraocular tissues where it plays a protective role in stress conditions. In fact, heat or toxic shocks, as well as pathological states, increase alpha B crystallin levels in many cell types. Here we show that alpha B crystallin expression is also modulated in subcultures of rat fibroblasts and Galliera sarcoma cells. Western blots analysis with anti alpha B crystallin antibodies reveals the presence of the protein in both cell populations, although the kinetic pattern of expression is different. Galliera fibroblasts constitutively express the protein up to the 70th subculture and afterwards the synthesis ceases. On the other hand, Galliera sarcoma cells do not contain alpha B crystallin in the early stages of the culture, but there is a progressive increases between the 20th and 40th cell subculture. Differences also exist concerning the intracellular distribution: alpha B crystallin is diffusely localized in the cytoplasm of fibroblasts while in sarcoma cells it localizes mainly to the perinuclear region. Alpha B crystallin is totally recovered as soluble protein in the supernatants obtained after low speed centrifugation of fibroblast homogenates, while in sarcoma cells a portion of the protein is also recovered in the insoluble pellet. Intracellular pH measurements show an alkaline cytosol in sarcoma cells compared to fibroblasts. Heat shock treatment of fibroblast subcultures constitutively expressing alpha B crystallin induces an over-expression of the protein, while in fibroblasts whose biosynthetic capacity is lost, heat shock is unable to activate the crystallin gene. Correlation between alpha B crystallin expression and proliferative rate shows that highly proliferating fibroblasts do not express alpha B crystallin, while neoplastic cells do.

Plasma and platelet taurine are reduced in subjects with insulin-dependent diabetes mellitus: effects of taurine supplementation.

Plasma and platelet taurine concentrations were assayed in 39 patients with insulin-dependent diabetes mellitus (IDDM) and in 34 control subjects matched for age, sex, and both total and protein-derived daily energy intake. Platelet aggregation induced by arachidonic acid in vitro at baseline and after oral taurine supplementation (1.5 g/d) for 90 d was also studied. Plasma and platelet taurine concentrations (mean +/- SEM) were lower in diabetic patients (65.6 +/- 3.1 mumol/L, or 0.66 +/- 0.07 mol/g protein) than in control subjects (93.3 +/- 6.3 mumol/L, or 0.99 +/- 0.16 mol/g protein, P < 0.01). After oral supplementation, both plasma and platelet taurine concentrations increased significantly in the diabetic patients, reaching the mean values of healthy control subjects. The effective dose (mean +/- SEM) of arachidonic acid required for platelets to aggregate was significantly lower in diabetic patients than in control subjects (0.44 +/- 0.07 mmol compared with 0.77 +/- 0.02 mmol, P < 0.001, whereas after taurine supplementation it equaled the mean value for healthy control subjects (0.72 +/- 0.04 mmol). In in vitro experiments, taurine reduced platelet aggregation in diabetic patients in a dose-dependent manner, whereas 10 mmol taurine/L did not modify aggregation in healthy subjects.

Alpha B crystallin is constitutively expressed in cultures of bovine articular chondrocytes.

alpha B crystallin is a small heat shock protein constitutively expressed in the mammalian lens and in a variety of extraocular tissues. We report here the presence of alpha B crystallin also in bovine articular chondrocytes by means of an immunoblot and immunofluorescence analysis carried out with anti-alpha B crystallin polyclonal antibodies. The expression level of alpha B crystallin can be further induced by a short heat shock treatment of chondrocytes as well as cell treatment with cadmium bromide or calcium ionophore A 23187. The level of alpha B crystallin expression is not modified by treating chondrocytes with interleukin-1 and phorbol 12-myristate 13-acetate. In some preparations the antibodies recognise two bands of alpha B crystallin, probably corresponding to different degrees of protein phosphorylation, but in cells treated with phorbol ester a single band is constantly observed, indicating a complete phosphorylation of alpha B crystallin.

Cytotoxicity of lazaroid U-75412E in human epithelial cell line (Wish).

The 21-aminosteroids (or lazaroids) are a recently synthesized class of compounds demonstrated to protect tissue against damage induced by trauma and/or ischemia. Currently, very little is known about the biological effects of lazaroids. In this work the action of lazaroid U-75412E on a human epithelial cell line (Wish) was evaluated. The data obtained showed an inhibition of cell growth and a dose- and time-dependent decrease of cell viability. Furthermore, a dose- and time-dependent increase of cells in the G2/M phase with the appearance of apoptotic cells was observed by flow cytometric analysis. Nuclear fragmentation was also evident. Lactate dehydrogenase release and scanning electron microscopy experiments suggested that plasma membrane integrity was altered by this compound. The immunofluorescence technique and transmission electron microscopy images also showed intracellular damage, such as alteration of microtubular arrangement, mitochondrial swelling and the presence of vacuoles. This study demonstrated that 1 microM U-75412E was unable to modify these parameters, while higher concentrations (6-75 microM) had a cytotoxic effect on Wish cells.

Enhancement of the antitrichomonal activity of 5-nitroimidazole derivatives by hydrogen peroxide.

The in vitro sensitivity of nine Trichomonas vaginalis isolates to commonly employed 5-nitroimidazoles (metronidazole, nimorazole, ornidazole and tinidazole) was evaluated in absence and in presence of sub-inhibitory concentrations of hydrogen peroxide (H2O2). Co-incubation with H2O2 and 5-nitroimidazole compounds decreased the MIC values of the strains exhibiting cross-resistance to these drugs. It was suggested that H2O2 produced in the inflammatory process during trichomonal infection could enhance the therapeutic effect of 5-nitroimidazole drugs.

Phenotypic variation of surface antigenic determinants in Trichomonas vaginalis detected by monoclonal antibodies.

We produced a large panel of murine monoclonal antibodies against surface determinants of Trichomonas vaginalis using the hybridoma technique. An immunoenzymatic technique (E.L.I.S.A.) was used to screen positive hybrid cells producing specific antibodies against the protozoan surface. Eleven monoclonal antibodies (Mabs) out of seventy-seven positives were further characterized. We tested antibody reactivity in order to investigate the antigenic variance among 13 different strains of Trichomonas vaginalis of different geographic origin. To elucidate the complexity of antigenic expression in Trichomonas vaginalis, further characterization of the antigenic pattern in our 13 clinical isolates was carried out by immunoblotting techniques. We demonstrate that some monoclonal antibodies react with antigens varying in molecular weight in the different strains tested. We also demonstrate the pivotal role of protozoan proteases in antigenic rearrangement.

Hydrogen peroxide-induced cytotoxicity in cultured epithelial cells (WISH): A functional and morphological study.

The effect of hydrogen peroxide on cultured epithelial cells (WISH) was investigated with particular emphasis on cell functions: cell morphology and cytoskeletal components were also studied. The presence of low concentrations of H(2)O(2) (0.1-0.4 mM) in the culture medium markedly inhibited cell growth, although WISH contained catalase and glutathione-peroxidase activities. After 1 hr of incubation with H(2)O(2) up to 5 mM, the majority of the cells were still alive, but reincubation in normal medium for 24 hr clearly reduced cell viability. Cell adhesion was dose-dependently reduced by H(2)O(2) treatment (0.1-0.5 mM) for 4 hr. Incubation with 1.5 mM-H(2)O(2) gave rise to a bleb appearance on the cell surface and to mitochondrial swelling, as shown, respectively, by scanning and transmission electron microscopy. Immunofluorescence studies revealed changes in microtubules and microfilaments, which are two of the main cytoskeletal components. The modification of microtubules was also confirmed by Western blot analysis of WISH protein homogenates submitted to SDS-PAGE. WISH treated with 1.5 mM-H(2)O(2) showed decreased levels of GSH compared with control cells: glutathione transferase activity was reduced, whereas other enzymes of the glutathione cycle were unchanged.