Biomedical subjects
Publications and source records attributed to S Bruno.
To determine the risk of cohabitant HCV infection, we investigated the sera of 101 family members of 53 anti-HCV antibody positive chronic liver disease patients. Altogether 14.8% of the cohabitants were also anti-HCV antibody positive, compared to a prevalence of 1.4% in the general population. These results suggest that hepatitis-C-virus may spread by person-to-person infection.
We retrospectively investigated anti-HCV prevalence in a series of 160 consecutive patients with primary biliary cirrhosis who presented between 1980 and 1989. Of these, 19 (12%) were positive for anti-HCV by C-100 ELISA. Serum IgG levels were significantly higher in anti-HCV-positive patients and correlated to optical density values. A serum sample was again collected from all the patients from the same series who were seen in 1990 for follow-up, after a median period of 32 months. Anti-HCV positivity was found to be substantially unchanged in this subgroup of patients when the freshly drawn blood samples were retested with C-100 ELISA, while it increased from 10% to 17% when second generation ELISA was used. Three of the C-100 ELISA positive samples were C-100 RIBA reactive, and six of the second generation ELISA positive samples were 4-RIBA reactive. The HCV genome was not detected in any of the seven anti-HCV C-100 ELISA and second generation ELISA positive sera which were studied by polymerase chain reaction, including four cases confirmed by 4-RIBA. Life expectancy, as determined by survival analysis, did not differ significantly between anti-HCV-positive and -negative patients. These findings suggest that anti-HCV positivity does not influence the clinical presentation and course of primary biliary cirrhosis.
Erythrocyte antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) and reduced glutathione, serum ceruloplasmin, and serum trace elements (copper, zinc, iron, and selenium) related to antioxidant enzymes were assayed in subjects with alcoholic liver disease of different degrees of severity. The erythrocytes of subjects with moderate and severe alcoholic liver cirrhosis had an unbalanced antioxidant system (normal superoxide dismutase, low catalase and glutathione peroxidase activities, and low glutathione content). Serum ceruloplasmin levels were in the normal range. Levels of the serum trace elements zinc and selenium were significantly low in subjects with moderate and severe cirrhosis, whose red cell half-life was also significantly short, as measured by radioactive chromium. These data suggest that the erythrocytes of subjects with moderate and severe alcoholic liver cirrhosis are less protected against oxidant stress. The particular erythrocyte antioxidant system and serum trace element pattern may play a role in the genesis of hemolytic disorders and of alcoholic hepatic damage.
The expression and stability of the proliferation-associated nuclear antigen detected by Ki-67 antibody have been investigated in human promyelocytic leukaemic HL-60 cells in relation to their progression through the cell cycle. Expression of this antigen was minimal in late G1 and early S phase cells. The antigen accumulated in the cells predominantly during S phase, and its rate of increase per cell accelerated during the second half of this phase. The accumulation of Ki-67 antigen during S exceeded the increase in DNA content, and thus the Ki-67/DNA ratio rose 80% from late G1 to G2 + M. This antigen rapidly disappeared from post-mitotic cells. The half-life of this protein estimated in post-mitotic cells during stathmokinesis induced by vinblastine appeared to be shorter than 1 h. This rapid turnover should be compared with the relatively long (6-8 h) duration of G1 of the studied cells. In cells in which de novo protein synthesis was inhibited by 0.1 microgram/ml cycloheximide, the half-life of the Ki-67 antigen was also found to be about 1 h regardless of the cell position in the cell cycle. Thus, the data suggest that variations in the level of this protein during the cell cycle are a consequence of its different synthesis rate rather than phase-specific changes in the rate of its degradation. Because the late G1 and very early S phase cells express the antigen at levels only slightly above background, it is possible that, when using Ki-67 antibody as a marker of the cell growth fraction, some late G1 cells can be erroneously classified as non-cycling cells.
We have previously observed that the DNA topoisomerase I inhibitor camptothecin (CAM), or DNA topoisomerase II inhibitors teniposide (TEN) and amsacrine (m-AMSA) trigger endonucleolytic activity in myelogenous (HL-60 or KG1), but not lymphocytic (MOLT-4) leukaemic cell lines. DNA degradation and other signs of apoptotic death were seen as early as 2-4 h after cell exposure to these inhibitors. Cells replicating DNA (S phase) were selectively sensitive whereas cells in G1 were resistant; the sensitivity of G2 or M cells could not be assessed in these studies. The present studies were aimed at revealing whether DNA repair replication induced by ionizing radiation can sensitize the cells, and to probe the sensitivity of cells arrested in G2 or M, to these inhibitors. The data show that gamma-irradiation (0.5-15 Gy) of HL-60 cells does not alter their pattern of sensitivity, i.e. G1 cells, although engaged in DNA repair replication, still remain resistant to CAM compared with the S phase cells. Likewise, irradiation of MOLT-4 cells also does not render them sensitive to either CAM or TEN, regardless of their position in the cell cycle. Irradiation, however, by slowing the rate of cell progression through S, increased the proportion of S phase cells, and thus made the whole cell population more sensitive to CAM. HL-60 cells arrested in G2 either by irradiation or treatments with Hoechst 33342 or doxorubicin appear to be more resistant to CAM relative to S phase cells. Also resistant are cells arrested in M by vinblastine. The data suggest that some factor(s) exist exclusively in S phase cells, which precondition them to respond to the inhibitors of DNA topoisomerases by rapid activation of endogenous nuclease(s) and subsequent death by apoptosis. HL-60 cells in G1, G2 or M, or MOLT-4 cells, regardless of the phase of the cycle, appear to be protected from such a mechanism, and even induction of DNA repair replication cannot initiate DNA degradation in response to DNA topoisomerase inhibitors. These data, together with the evidence in the literature that topoisomerase I may be involved in DNA repair, suggest that a combination of these inhibitors with treatments that synchronize cells in the S phase and/or recruit quiescent cells to proliferation, including radiation, may be of value in the clinic.
To determine whether the overload associated with myocardial infarction and ventricular failure in rats is coupled with activation of DNA synthesis in the remaining left and right ventricular myocytes, flow cytometric analysis was performed on myocyte nuclei prepared from both ventricles 7 and 30 days after coronary occlusion. In addition, oral captopril was administered in separate groups of control and experimental rats to establish whether a relation existed between attenuation of ventricular loading and magnitude of DNA synthesis in myocytes. Results demonstrated that left ventricular failure and right ventricular dysfunction at 7 days after infarction were biventricularly associated with marked increases in the number of myocyte nuclei in the G2M phase of the cell cycle. In contrast, the fraction of nuclei in the G0+G1 phase decreased. In comparison with the earlier time point, the 30-day interval was characterized by a significant magnitude of cardiac hypertrophy, a moderate amelioration of ventricular pump function, and a decrease in the percentage of myocyte nuclei in the G2M phase in both ventricles. However, 30 days after infarction, the number of right ventricular myocyte nuclei in the S and G2M phases remained elevated with respect to control animals. Captopril therapy reduced the extent of ventricular loading and the population of myocyte nuclei in the cell cycle at 7 days. In conclusion, DNA synthesis in myocyte nuclei may represent an important adaptive component of the myocardial response to infarction.
Exposure of human promyelocytic leukemic HL-60 cells to the topoisomerase I inhibitor camptothecin (CAM) triggers endonucleolytic activity and apoptotic death of these cells. The nucleolytic effect is seen 2-4 h after drug addition and is highly selective to cells progressing through S phase. Concomitant with degradation of DNA, which is preferential to the nucleosomal DNA linker sections, extensive proteolysis takes place in these cells. Cellular RNA, however, is initially degraded to a much lesser degree than DNA or protein. Both endonucleolysis and proteolysis triggered by CAM in S-phase HL-60 cells can be prevented by the protease inhibitors N-tosyl-L-phenylalanylchloromethyl ketone (TPCK), N-tosyl-L-lysylchloromethyl ketone (TLCK) or partly by N-tosyl-L-arginine methyl ester (TAME), added simultaneously with CAM, or up to 30 min after exposure to CAM, at their respective concentrations known to inhibit proteases. The protective effect of these protease inhibitors on DNA degradation cannot be due to the suppression of cell progression through S phase because cells still replicate DNA in their presence, albeit at a reduced rate. Furthermore, TPCK and TLCK protect rat thymocytes against endonucleolysis induced by prednisolone. In the latter cell system, (considered a classic model of apoptosis), endonucleolysis, which primarily affects G0/G1 cells, is unrelated to cell progression through S phase. The present data suggest that the endonucleolysis and proteolysis which accompany apoptotic cell death are coupled, and the proteolytic step is needed for DNA degradation to occur.
Liver-cell dysplasia is a well known histological entity with preneoplastic significance in experimental hepatic carcinogenesis. However, while the association of liver-cell dysplasia with hepatitis B virus can be considered as established, it is still controversial whether this lesion represents a premalignant condition in cirrhotic patients. Efforts have been made to render its morphological assessment more reliable, but no firm conclusions can be drawn from the available clinical studies, which are mainly retrospective or based on autopsy series. Preliminary results from a prospective study argue that liver-cell dysplasia is associated with an increased risk to hepatocellular carcinoma. The emergence of liver-cell dysplasia as a preneoplastic lesion in cirrhotic patients will have some impact in the future on their management, including selection for closer monitoring in early detection of hepatocellular carcinoma and for liver transplantation.
Rat thymocytes were treated in culture with prednisolone or the DNA topoisomerase I or II inhibitors, camptothecin (CAM) or teniposide (TN), and proportions of cells in different phases of the cell cycle were estimated by flow cytometry using a staining methodology which makes it possible to discriminate between G0 and G1 cells, as well as to recognize the cells which undergo apoptosis. The appearance of apoptotic cells in cultures treated with pharmacological concentrations of these drugs, observed as early as 3-6 hr after treatment, coincided with the selective loss of G0 cells in these cultures, while no significant changes in the proportion of S or G2+M cells were apparent. Agarose gel electrophoresis of DNA isolated from the treated cells indicated degradation of the internucleosomal spacer sections, typical of the endonucleolytic activity which accompanies apoptotic cell death. The data indicate that G0 thymocytes were particularly sensitive to agents that induce apoptosis while cells progressing through the cell cycle were resistant. This suggests that under in vivo conditions (immunological response), the selective death of G0 cells may promote the clonal expansion of stimulated thymocytes which enter the cell cycle. Together with our earlier studies on the effects of CAM and TN on MOLT-4 and HL-60 leukemic cell lines, these data indicate that both, phenotypic- and and cell cycle phase specific- factors modify the ability of cells to respond to toxic agents, including chemotherapeutics by apoptosis.(ABSTRACT TRUNCATED AT 250 WORDS)
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The Authors report a case of voluminous hemorrhagic pseudocyst of the left suprarenal gland. The anatomo-pathological classification of adrenal cysts is described, in particular with reference to the etiopathogenetic hypotheses regarding the formation of hemorrhagic pseudocysts. Lastly, the importance of using instrumental tests for a correct diagnosis is underlined and the surgical indications of accidentally discovered asymptomatic cysts are discussed.
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Lovastatin (LOV), the drug recently introduced to treat hypercholesteremia, inhibits the synthesis of mevalonic acid. The effects of LOV on the cell cycle progression of the human bladder carcinoma T24 cell line expressing activated p21ras were investigated. At a concentration of 2-10 microM, LOV arrested cells in G1 and also prolonged--or arrested a minor fraction of cells in--the G2 phase of the cell cycle; at a concentration of 50 microM, LOV was cytotoxic. The cytostatic effects were reversed by addition of exogenous mevalonate. Cells arrested in the cycle by LOV were viable for up to 72 hr and did not show any changes in RNA or protein content or chromatin condensation, which would be typical of either unbalanced growth or deep quiescence. The expression of the proliferation-associated nuclear proteins Ki-67 and p105 in these cells was reduced by up to 72% and 74%, respectively, compared with exponentially growing control cells. After removal of LOV, the cells resumed progression through the cycle; they entered S phase asynchronously after a lag of approximately 6 hr. Because mevalonate is essential for the posttranslational modification (isoprenylation) of p21ras, which in turn allows this protein to become attached to the cell membrane, the data suggest that the LOV-induced G1 arrest may be a consequence of the loss of the signal transduction capacity of p21ras. Indeed, while exposure of cells to LOV had no effect on the cellular content of p21ras (detected immunocytochemically), it altered the intracellular location of this protein, causing its dissociation from the cell membrane and translocation toward the cytoplasm and nucleus. However, it is also possible that inhibition of isoprenylation of proteins other than p21ras (e.g., nuclear lamins) by LOV may be responsible for the observed suppression of growth of T24 cells.
The Authors describe a rare case of obstructive jaundice secondary to solitary nonparasitic cyst of the liver. The anatomopathology, embriological and clinical aspects are analyzed with emphasis on the importance of the instrumental investigation in differential diagnosis of malignancy. Finally they describe the indications to various surgical and non surgical treatment.
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Using multiparameter flow cytometry we have measured the nuclear DNA content of exponentially growing HL-60 cells in conjunction with protein content, nuclear forward light scatter, DNA in situ sensitivity to denaturation, DNA accessibility to 7-aminoactinomycin D (7-AMD), and content of the proliferation-associated proteins: cyclin (PCNA), p105, p34, and Ki-67. Multivariate analysis of the data made it possible to correlate changes in each parameter with the degree of cell advancement through S phase (amount of replicated DNA). A decrease of the protein/DNA ratio, lowered DNA accessibility to 7-AMD, increased sensitivity of DNA to denaturation, and increased ability of isolated nuclei to scatter light all paralleled cell progression through S phase. These changes indicate that during S phase chromatin progressively condenses and suggest that the condensation is associated with the efflux of nonhistone proteins from the nucleus. The increase in the content of the antigen detected by the Ki-67 antibody was observed to exceed the increase in DNA content during S phase and the rate of the Ki-67/DNA increase was higher during the second half of S phase. Thus, this protein appears to be primarily synthesized during S, especially late in S phase, and is degraded in G1. The ratio of cyclin (PCNA)/DNA remained rather constant whereas the contents of p105 and p34 proteins, when expressed per unit of DNA, both decreased during S phase. The data indicate that significant changes in structure and composition of chromatin take place during S phase and suggest that the composition of chromatin associated with the nonreplicated DNA is different compared to chromatin associated with the newly replicated DNA.
In 1984 we started a prospective controlled trial comparing endoscopic sclerotherapy (ES) with the distal splenorenal shunt (DSRS) in the elective treatment of variceal hemorrhage in cirrhotic patients. The study population included 40 patients with cirrhosis and portal hypertension referred to our department from October 1984 to March 1988. These patients were drawn from a pool of 173 patients who underwent either elective surgery or endoscopic sclerotherapy during this time. Patients were assigned to one of the two groups according to a random-number table: 20 to DSRS and 20 to ES. During the postoperative period, no DSRS patient died, while one ES patient died of uncontrolled hemorrhage. One DSRS patient had mild recurrent variceal hemorrhage despite an angiographically patent DSRS. Four ES patients suffered at least one episode of gastrointestinal bleeding: two from varices and two from esophageal ulcerations. Five ES patients developed transitory dysphagia. Long-term follow-up was complete in all patients. Two-year survival rates for shunt (95%) and ES (90%) groups were similar. One DSRS patient rebled from duodenal ulcer, while three ES patients had recurrent bleeding from esophagogastric sources (two from varices and one from hypertensive gastropathy). One DSRS and two ES patients have evolved a mild chronic encephalopathy; four DSRS and two ES patients suffered at least one episode of acute encephalopathy. Two ES patients had esophageal stenoses, which were successfully dilated. Preliminary data from this trial seem to indicate that DSRS, in a subgroup of patients with good liver function and a correct portal-azygos disconnection, more effectively prevents variceal rebleeding than ES. However no significant difference in the survival of the two treatment groups was noted.
Progressive in vitro culturing of interleukin-3 (IL-3) dependent normal murine mastocytes (PB-3) resulted in a variant cell line (PB-1) able to grow without exogenous IL-3 and which was tumorogenic in syngenic mice. Bivariate flow cytometry was used to evaluate the c-myc protein and DNA content of PB-3 and PB-1 cells. The c-myc protein was detected by specific monoclonal antibodies. Kinetic characteristics of PB-3 and PB-1 cell lines, namely, the duration of the G1, S and G2 + M cell cycle phases were also evaluated using the bromodeoxyuridine (BrdU) pulse-chase method and BrdU/DNA flow cytometry. Levels of c-myc protein in PB-1 cells were about two-fold higher than those of PB-3 cells in all cell cycle phases. Mean duration of the cell cycle (Tc) was 15.3 h for PB-3 cells and 12.4 h for PB-1 cells. Shortening in Tc for the transformed cells was due to a decrease of nearly 30% in mean duration of the G1 phase (from 8 h to 5.7 h). No significant differences were found in the duration of the S and G2 + M phases. These results indicate that acquired IL-3 independency in vitro and tumorogenicity of PB-1 cells were accompanied by a doubling of c-myc protein level and by a parallel shortening, or bypass, of the regulatory events within the G1 phase of the cell cycle.