Expression and activity of caspases 1 and 3 in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are characterized by abnormal growth of committed progenitors in clonogenic assay, with reduced number of colonies and decreased colony/cluster ratio. It has been suggested that excessive apoptosis is the cause of marrow failure in MDS. We studied the expression of caspase-1 (interleukin-1beta-converting enzyme, ICE) and caspase-3 (CPP32/apopain) in marrow mononuclear cells, and the growth pattern of committed progenitors in a series of 83 MDS cases. The percentage of apoptotic cells as detected by TUNEL technique, and the percentage of caspase-3-positive cells were significantly higher in refractory anemia (RA) and RA with ringed sideroblasts (RAS) than in chronic myelomonocytic leukemia (CMML), refractory anemia with excess of blasts (RAEB) and RAEB in transformation (RAEB-T). Spontaneous growth of CFU-GM was associated with a higher percentage of blasts, and with a lower expression of caspase-3 and caspase-1. The yield of CFU-E, BFU-E, and CFU-GM (in the presence of growth factors) was decreased by comparison to normal marrow, but large individual differences were observed in all cytological categories. Inhibition of caspase-1 and caspase-3 activities by specific inhibitors resulted in a significant increase of the production of all types of colonies (up to 50-fold of control). In the presence of caspase-3 inhibitor, the number of BFU-E and CFU-E was in the range of normal values in most cases of RA and RAS. In addition, caspase-1 and -3 protease activities were detectable by fluorogenic assay in all cases studied. Western blot analysis confirmed the expression of caspase-3, including the cleaved (activated)-p17 form in most cases of RA/RAS analyzed. It is concluded that caspase-3 is implicated in the increased apoptosis observed in MDS and that inhibition of its activity can restore at least partially the growth of committed progenitors.

Selection of BCR/ABL-negative stem cells from marrow or blood of patients with chronic myeloid leukemia.

Philadelphia (Ph) or BCR/ABL-negative cells with immature phenotype (CD34-positive, DR-negative) can be recovered from patients with chronic myeloid leukemia (CML) in chronic phase. We used the technique described by Berardi et al (Science 1995; 267: 104-108) to select stem cells from marrow or blood of CML patients at diagnosis or during treatment with alpha-interferon. Mononuclear cells (MNC), and in some experiments CD34+ cells, were maintained for 7 days in the presence of 5-fluorouracil (5-FU), stem cell factor and interleukin-3. The number of viable cells recovered after culture was between 7.4 and 70.2 for 10(6) cells plated. These cells exhibited the following phenotype: CD34+, CD117+, CD38-, lineage-, and were able to generate cobblestone areas and secondary colonies in long-term culture (LTC), with a frequency similar to that of cells selected from normal marrow. Study by fluorescence in situ hybridization of LTC cells or secondary colonies showed no evidence of BCR/ABL rearrangement. Reverse transcriptase polymerase chain reaction studies on pooled LTC cells or secondary colonies were also negative. By contrast, LTC cells or secondary colonies obtained from CML CD34+ cells without culture in the presence of 5-FU were always positive for BCR/ABL rearrangement. Finally, 5-FU selected cells were able to engraft NOD/SCID mouse, as human cells were detected in blood and marrow 10 weeks post transplantation, which were BCR/ABL negative by RT-PCR. This method of culture makes it possible to select constantly BCR/ABL-negative cells with capacities of development in LTC assay and of NOD/SCID mouse engraftment.

Expression of apoptosis-controlling proteins in acute leukemia cells.

The expression of Bcl-2 family proteins (Bcl-2, Bcl-X, Bcl-XL, Bcl-Xs, BAX, BAD, MCL-1) and of Interleukin-1 converting enzyme (ICE)-related proteins (ICE, CPP32, ICH- 1) was analyzed in acute leukemia cells by flow cytometry. Most proteins studied were detectable in cell lines such as KG1a, HL60, K562 (myeloblastic), REH, RAJI and MOLT4 (lymphoblastic) and VAL (B-cell lymphoma). However, BCL-Xs and BAK were weakly expressed in K562, as were Bcl-X, BAD and BAK in the VAL line. In acute myeloid leukemia (66 cases studied), the proteins were expressed in most cases in a high percentage of cells, especially BAX and CPP32, without correlation with hematological characteristics. However, Bcl-2 was expressed in a higher percentage of cells in FAB M1 and M5 cases, and in CD34-positive cases, whereas Bcl-Xs was more frequently expressed in M3 cases. No differences were observed regarding fluorescence intensity. Higher percentages of Bcl-2-positive cells were associated with low remission rate, while expression of Bcl-Xs was predictive of high remission rate. In acute lymphoblastic leukemia (36 cases), all proteins studied were expressed in a majority of cases. Bcl-Xs was more frequently detected in T-cell type, and was also associated with a higher remission rate. These results suggest that apoptosis-controlling proteins may have a role in the pathogenesis and response to therapy of acute leukemia.

[Ovarian cysts. The respective role of clinical examination, echography, biological markers and cytology]. / Kyste de l'ovaire. Places respectives de l'examen clinique de l'échographie, des marqueurs biologiques et de la cytologie.

The authors assess the value of clinical examinations and para-clinical examinations carried out when an ovarian cyst is discovered. They try to find out whether a diagnosis of the aetiology is possible before the operation and particularly whether the cystic forms of cancer can be detected pre-operatively. The study is concerned with a retrospective assessment of 358 patients. The first line of investigation was seen to be ultrasound which is effective in 91% of cases. When the level of CA-125 is above 35 U/ml that suggests that there may be a malignant neoplasm present. The test is sensitive to about 78.6% and specific at 76%. 96 cysts were considered to be benign using clinical and para-clinical examinations and were punctured under ultrasound control or laparoscopically. The cytology could not be interpreted in 15%. Only one borderline tumour of the ovary was found. 25.3% of cysts that had been aspirated recurred and this is a high figure. Given these figures, the authors suggest a diagnostic and therapeutic program for ovarian cysts.

Propranolol for the prevention of recurrent variceal hemorrhage: a controlled trial.

We conducted a prospective, randomized single-blind trial of propranolol for the prevention of recurrent variceal bleeding. Seventy-nine patients shown to have variceal hemorrhage at endoscopy were included in the study within 72 hr following diagnosis. Fifty-seven patients had alcoholic cirrhosis, 10 cryptogenic cirrhosis, 6 posthepatitic cirrhosis, 4 biliary cirrhosis, 1 portal vein thrombosis without cirrhosis and 1 idiopathic portal hypertension. The severity of liver disease at inclusion was assessed according to the Pugh modification of the Child-Turcotte classification: 9 (11%) had Class A; 41 (52%) Class B, and 29 (37%) Class C disease. Patients were randomly assigned by sealed envelope to the propranolol group (42 patients) or the placebo group (37 patients). Propranolol dosage was titrated in order to produce plasma concentrations of propranolol of 50 to 150 ng per ml. beta-blockade was also confirmed by isoproterenol testing. The cumulative percentages of patients free of rebleeding 1 and 2 years after inclusion were 31 and 21% in the propranolol group, and 25 and 17% in the placebo group; both differences were not significant. Cumulative 1 and 2 years survival were also comparable: 64 and 54% in the propranolol group vs. 70 and 63% in the placebo group. There was no evidence for a therapeutic effect of propranolol after adjusting for potential confounding variables by multiple logistic regression. We conclude that propranolol is not effective for the prevention of variceal rebleeding, when administered early following the initial bleed, in cirrhotics unselected with respect to the severity of the liver disease.

Intrahepatic circulation in liver disease.

Using the multiple indicator dilution approach, events occurring in the microvascular bed can be characterized in experimental animals with different types of cirrhosis and in man. Intrahepatic shunts can be found shunting blood away from sinusoids in both cirrhotic patients and cirrhotic animals. Such shunts were present in about one-third of cirrhotic patients with portal hypertension, and occurred mainly between the portal vein and hepatic veins. In cirrhotics, portohepatic anastomoses are usually large in diameter (more than 20 micron in diameter). Collagenization of the space of Disse and the progressive transformation of sinusoids into capillary-like channels decrease the extravascular space accessible to albumin and probably to other large molecules and protein-bound substances. However, unlike findings obtained in well-capillarized organs, these sinusoidal changes do not appear to limit the diffusion of sucrose, water, and lipophilic substances, such as lidocaine in the extravascular and intracellular spaces. The pattern observed for labeled sucrose curves following hepatic artery injection in cirrhotic patients could be secondary to the passage through the dense peribiliary capillary plexus originating from the enlarged arterial bed in cirrhosis. The difference in the perfusion of cirrhotic nodules with regard to the portal venous and hepatic artery routes introduces important new concepts in the overall mechanism of the elimination of endogenous and exogenous substances by the cirrhotic liver: blood entering the liver by the two afferent vessels will not flow through the same vascular bed before reaching the efferent hepatic veins.

Portal venous system measurements in portal hypertension.

The diameters of the portal, splenic, superior mesenteric, and coronary veins were measured during umbilicoportal venography in 64 cirrhotic patients with or without portal hypertension. The diameter of the portal vein did not increase along with the portohepatic gradient and even tended to decrease depending on the severity of hypertension and the opening of spontaneous portosystemic shunts. While there was no relationship between portohepatic gradient and splenic or superior mesenteric vein diameter, a coronary vein larger than 0.7 cm was associated with a portohepatic gradient greater than 10 mm Hg. This could be a useful sign of severe portal hypertension.

Hyperglobulinemia in alcoholic cirrhosis. Relationship with portal hypertension and intrahepatic portal-systemic shunting as assessed by Kupffer cell uptake.

The relative importance of portal hypertension and intrahepatic portal-systemic shunting in the pathogenesis of hyperglobulinemia is examined in 31 alcoholic cirrhotic patients and 6 patients with idiopathic portal hypertension. The degree of portal hypertension was evaluated by combined umbilicoportal and hepatic vein catheterization and the intrahepatic portal-systemic hunting was assessed by the Kupffer uptake of [125I]albumin microaggregates during a single passage through the liver. All patients had comparable severe portal hypertension and most had bled from ruptured varices; however, no demonstrable relationship could be found between portal hypertension and hyperglobulinemia. Elevated levels of serum gammaglobulins and immunoglobulins (mainly IgG) were observed only in cirrhotic patients, particularly those with markedly altered Kupffer cell uptake. It is concluded that intrahepatic portal systemic shunting as evaluated by the Kupffer cell uptake is more important than the collateral circulation secondary to portal hypertension in the pathogenesis of hyperglobulinemia in alcoholic cirrhotic patients.

Symposium on portal hypertension and its complications: current management. Preoperative assessment and predictors of encephalopathy.

None of the preoperative predictors of encephalopathy proposed so far to evaluate the risk of portacaval shunting in cirrhotic patients has been of value. The authors have found, in preliminary studies, that measurement of the hepatic extraction of indocyanine green (ICG), which correlates highly with the "functional" portal blood supply, could be of prognostic value: cirrhotic patients with a near-normal value for ICG extraction often have encephalopathy after portacaval shunting whereas those with a low ICG extraction value seldom have encephalopathy. These preliminary data suggest that cirrhotic patients with markedly decreased ICG extraction have a lesser risk of encephalopathy since their portal blood supply is already shunted away from hepatocytes before the operation because of anatomic changes in the liver microcirculation.

Treatment of resistant ascites by continuous ultrafiltration--reinfusion of ascitic fluid.

Eight patients with diuretic-resistant ascites due to cirrhosis were treated by reinfusion of concentrated ascitic fluid. In 11 procedures, with a mean duration of 21.9 hours, weight loss averaged 14.8 kg. Complications during reinfusion included septicemia in 1 procedure, left-sided heart failure in 5, pyrexia in 7 and coagulation abnormalities in 10. Ascites recurred within 2 months after reinfusion in all but one patient. Although this technique is an efficient and inexpensive method of treatment of ascites, it does not appear indicated in patients with cirrhosis and resistant ascites in view of the possibly serious complications associated with reinfusion and the poor long-term results.

Difference in the distribution of e antigen among different ethnic groups in a population of blood donors.

Sensitive techniques were used to detect e antigen and the corresponding antibody (anti-e) among 368 voluntary blood donors positive for hepatitis B surface antigen in the Montreal area and 310 people living in close contact with them. Neither e nor anti-e was found in the absence of markers of hepatitis B virus (HBV). Among the blood donors e antigen was detected in 23 and anti-e in 313, and 32 were negative for both markers. Of the 368 blood donors 330 were of French origin and 38 from other ethnic groups. The 23 e-positive subjects were unequally distributed among the ethnic groups: only 14 (4.2%) were recruited among the French group while 9 (23.7%) were recruited among other ethnic groups (P less than 0.001). This differences among ethnic groups might be related to the vertical or horizontal mode of dissemination of HBV infection.

Idiopathic portal hypertension.

Idiopathic portal hypertension is reported in five cases including one case of chronic arsenical intake and one case of chronic industrial vinyl chloride exposure. In all five cases the patients presented with gastrointestinal bleeding as the chief complaint. Physical examination was within normal limits except for splenomegaly in all. Results of liver function tests were normal, except for the relative clearance of sulfobromophtalein. A surgical liver biopsy specimen was obtained in all cases and showed moderate degrees of portal fibrosis, but no cirrhosis. Combined umbilicoportal, hepatic vein and superior mesenteric artery catheterization was performed in all cases. Hepatoportographies showed distortion of the intrahepatic portal venous system and cut-off of small portal venules. Porto-hepatic gradients ranged from 14.0 to 20.5 mm Hg. The portal hypertension was both sinusoidal and presinusoidal in nature but mainly presinusoidal. Hepatic extraction of indocyanine green and of albumin microaggregates was normal, thereby suggesting normal functional portal blood supply to the liver. The patients with arsenical or vinyl chloride exposure could not be differentiated from the other three patients with idiopathic portal hypertension. These results suggest that idiopathic portal hypertension may be related to domestic or industrial exposure to other hepatotoxins.

Detection of surface and core antigens of hepatitis B virus in the liver of 164 human subjects. A study by immunoperoxidase and orcein staining.

The surface (HBsAg) and core (HBcAg) antigens of hepatitis B virus (HBV) have been searched by optic microscopy in the liver specimens from patients hospitalized for various conditions and from 38 HGsAg chronic carriers. The study was done blindly using Shikata et al.'s orcein staining on fixed and frozen material and direct immunoperoxidase on frozen material with antisera specific for surface (anti-HBs) and core (anti-HBc) antigens of HBV. No liver staining could be found in the 98 HBsAg seronegative patients. Among the 28 HBsAg seropositive patients, only 3 showed positive staining: 1 patient with acute viral hepatitis showed nuclear staining with anti-HBc; 2 patients with postnecrotic cirrhosis showed cytoplasmic staining with anti-HBs and/or orcein, and one of them also showed nuclear staining with anti-HBc. In contrast, among the 38 chronic carriers, 25 showed positive cytoplasmic staining with anti-HBs and/or orcein, while one of them (with chronic aggressive hepatitis) also showed nuclear staining with anti-HBc. Anti-HBs and orcein staining are equally sensitive and specific for the detection of HBsAg in hepatocytes; discrepant results can be attributed to sampling error of distribution of HBsAg in small liver fragments.

Chronic carriers of hepatitis B antigen (HBsAg). Histological, biochemical, and immunological findings in 31 voluntary blood donors.

Among 289 HBsAg carriers detected by the Montreal Red Cross Blood Transfusion Service and seen by our group, 31 submitted voluntarily to liver biopsy. These 31 carriers have now been followed for 10-33 months (mean: 23) and all remained positive for HBsAg. 15 of these 31 subjects had lived in institutions during infancy or childhood and none were drug users. Histological examinations revealed 24 cases of chronic persistent hepatitis (CPH), 2 cases of chronic aggressive hepatitis, 2 with steatosis, and 3 with normal liver. On repeated determination, 16 of the 31 subjects had at least one elevated transaminase level. Transaminases levels could not be correlated with the histological diagnosis. 4 cases had positive antinuclear antibodies, all in the CPH group, a finding that could not be correlated with any clinical, biological, or histological findings. The search for other autoantibodies and the immunoglobulin determinations were totally unrewarding. Thus, it appears that chronic HBsAg carriers in Montreal voluntary blood donors often have chronic hepatitis, usually persistent, occasionally aggressive; liver biopsy still remains the most useful approach in the evaluation of these HBsAg carriers. The HBsAg-carrier state seems to be well tolerated, but further long-term studies are needed to understand the natural history of this condition.

Extraction of 125I-albumin microaggregates from portal blood. An index of functional portal blood supply in cirrhotics.

Portal and hepatic indicator dilution curves (IDC) were obtained after injection of a mixture of 51Cr-labeled red blood cells ([51Cr]RBC) and 125I-albumin microaggregates (125I-AMA) into the cranial mesenteric artery in dogs. The extraction (E) of 125I-AMA from portal blood was measured during one passage through the hepatic reticuloendothelial system. Using [51Cr]RBC as a vascular reference substance, E-125I-AMA was calculated by comparing simultaneous [51Cr]RBC and 125I-AMA portal and hepatic IDC, and was expressed as percentage of 125I-AMA flowing through the portal vein. In 44 experiments (15 dogs), the colloid was almost completely extracted (E-125I-AMA = 92.3 +/- 1.0% (mean +/- SE)). This approach was applied in 15 patients with severe portal hypertension undergoing combined umbilicoportal, hepatic vein, and superior mesenteric artery catheterization. Eleven patients had alcoholic cirrhosis (AC) and 4 patients had idiopathic noncirrhotic portal hypertension (IPH). Using [51Cr]RBC-IDC, the portal fraction of hepatic blood flow varied between 34.1 and 100% (mean 62.6%) in AC patients and between 56.5 and 91.2% (mean 74.2%) in IPH patients. E-125I-AMA varied from 5.2 to 100% (mean 45.1%) in AC patients, although normal values were obtained in IPH patients (mean 93.2%). In all patients the extraction of Indocyanine green (E-ICG) was calculated using a continuous infusion for the estimation of hepatic blood flow. E-ICG was decreased in AC patients (mean 22.1%), although normal values were obtained in IPH patients (mean 49.5%). A highly significant correlation was found between E-125I-AMA and E-IGC (r = 0.977, P less than 0.001). Also, a significant correlation was found in all patients between E-125I-AMA and the relative clearance of ICG (r = 0.906, P less than 0.001). The correlations between the extraction or clearance of substances removed by two different cell population suggest that their decreases are mainly due to changes in liver microcirculation. In cirrhotics, the decreased E-125I-AMA can be related to part of portal blood bypassing Kupffer cells (intrahepatic portohepatic shunts) and/or to sinusoidal changes responsible for ineffective phagocytosis. Thus, E-125I-AMA can be used as an estimation of the functional portal blood supply to the liver in cirrhotics. Using portal and hepatic IDC after injection of [51Cr]RBC and 125I-AMA into the superior mesenteric artery, the portal fraction of hepatic blood flow and the functional portal blood supply can be estimated simultaneously in patients with portal hypertension before portacaval shunts.