Protection by beta-carotene and related compounds against oxygen-mediated cytotoxicity and genotoxicity: implications for carcinogenesis and anticarcinogenesis.

beta-Carotene protects against photooxidative dermatitis in porphyric humans and mice by quenching of photoactivated species. Other actions of beta-carotene in vivo are explained on the basis of its ability to scavenge free radicals in vitro. For example, in guinea pigs treated with CCl4, beta-carotene decreases pentane and ethane production. Epidemiological studies link low serum beta-carotene levels to elevated risk of lung and other cancers, and in intervention trials, beta-carotene diminishes preneoplastic lesions. However, the dose/response relationships are not well established, and antineoplastic mechanisms await clarification. Given a radical quenching mechanism, beta-carotene should block tumor promotion, but more typically the site of action is progression and an even later role in invasion has not been ruled out. Some antineoplastic actions of carotenoids (such as increased rejection of fibrosarcomas in mice) are attributed to immunoenhancement; others may reflect conversion to retinoids and subsequent gene regulation. Carotenoids other than beta-carotene may act at an earlier stage of carcinogenesis or be more effective as anticarcinogens at certain target sites. As scavengers of hydroxyl radicals, canthaxanthin and astaxanthin are more effective than beta-carotene. Canthaxanthin is sometimes more effective than beta-carotene in chemoprevention, but it is sometimes completely ineffective. Lycopene quenches singlet oxygen more than twice as effectively as beta-carotene. However, the antineoplastic actions of lycopene or astaxanthin remain untested. Explorations of the interactions of carotenoids with other nutrients are just beginning. Dietary fat increases absorption of carotene but decreases antineoplastic effectiveness. Research is hampered by technical problems, including the unavailability of rigorous controls, the instability of carotenoids, and the heterogeneous phase structure induced by hydrophobic compounds in aqueous media. Areas of current controversy and promising approaches for future research are identified.


Antioxidant potential of ferulic acid.

Ferulic acid is a ubiquitous plant constituent that arises from the metabolism of phenylalanine and tyrosine. It occurs primarily in seeds and leaves both in its free form and covalently linked to lignin and other biopolymers. Due to its phenolic nucleus and an extended side chain conjugation, it readily forms a resonance stabilized phenoxy radical which accounts for its potent antioxidant potential. UV absorption by ferulic acid catalyzes stable phenoxy radical formation and thereby potentiates its ability to terminate free radical chain reactions. By virtue of effectively scavenging deleterious radicals and suppressing radiation-induced oxidative reactions, ferulic acid may serve an important antioxidant function in preserving physiological integrity of cells exposed to both air and impinging UV radiation. Similar photoprotection is afforded to skin by ferulic acid dissolved in cosmetic lotions. Its addition to foods inhibits lipid peroxidation and subsequent oxidative spoilage. By the same mechanism ferulic acid may protect against various inflammatory diseases. A number of other industrial applications are based on the antioxidant potential of ferulic acid.


Reduction of sperm whale ferrylmyoglobin by endogenous reducing agents: potential reducible loci of ferrylmyoglobin.

The reactivity of the endogenous antioxidants ascorbate, ergothioneine, and urate toward the high oxidation state of sperm whale myoglobin, ferrylmyoglobin-formed upon oxidation of metmyoglobin by H2O2--was evaluated by optical spectroscopy and SDS-PAGE analysis. Depending on whether these antioxidants were present in the reaction mixture before or after the addition of H2O2 to a metmyoglobin suspension, two different effects were observed: (a) In the former instances, ascorbate, ergothioneine, and urate reduced efficiently the oxoferryl moiety in ferrylmyoglobin to metmyoglobin and prevented dimer formation, a process which requires intermolecular cross-link involving specific tyrosyl residues. In addition, all the reducing compounds inhibited--albeit with different efficiencies--dityorosine-dependent fluorescence build up produced via dimerization of photogenerated tyrosyl radicals. (b) In the latter instances, the antioxidants reduced the preformed sperm whale ferrylmyoglobin to a modified metmyoglobin, the spectral profile of which was characterized by a blue shift of the typical 633 nm absorbance of native metmyoglobin. In addition, under these experimental conditions, the antioxidants did not affect dimer formation, thus indicating the irreversible character of the process. The dimeric form of sperm whale myoglobin--separated from the monomeric form by gel electrophoresis of a solution in which ergothioneine was added to preformed ferrylmyoglobin--revealed optical spectral properties in the visible region identical to that of the modified myoglobin. This suggests that the dimeric form of the hemoprotein is redox active, inasmuch as the oxoferryl complex can be reduced to its ferric form. These results are discussed in terms of the potential reactivity of these endogenous antioxidants toward the reducible loci of ferrylmyoglobin, the oxoferryl moiety, and the apoprotein radical.


Five year prospective study of the incidence, clinical features, and diagnosis of achalasia in Edinburgh.

With the increasing availability of manometry, patients with achalasia are often referred at an early stage when they lack the classic features of established disease. A prospective five year study of the presenting features of untreated achalasia referred to our department was undertaken. Twenty men and 18 women presented throughout adult life, with a mean age at the time of diagnosis of 44 years (range 17 to 76 years). The presenting symptoms were dysphagia: for solids (100%) and for liquids (97%), chest pain (74%), and weight loss (60%). Endoscopy was reported as normal in 15 patients and achalasia was suggested in only 21 of 33 barium examinations. Fourteen had been treated for gastrooesophageal reflux but none had been misdiagnosed as having cardiac or psychiatric disease. The annual incidence of achalasia in the Lothian region is 0.8/100,000 of population. Persistent dysphagia is the cardinal symptom of achalasia which presents throughout adult life. Nevertheless, recent onset achalasia is often misdiagnosed as gastrooesophageal reflux disease. Because endoscopy is frequently normal and the diagnosis is often not made by radiology, manometric investigation is necessary if the condition is to be recognised and treated at an early stage.


Helicobacter pylori secretes a chemotactic factor for monocytes and neutrophils.

Helicobacter pylori is associated with an inflammatory reaction in the stomach and duodenum, yet the mechanism of this inflammatory infiltrate is unknown. The ability of Helicobacter pylori to secrete a factor that attracts leucocytes is investigated. Helicobacter pylori conditioned supernatant attracted neutrophils and monocytes with 50-100% of the activity of control chemotactic factor, 10(-8) M formyl-methionine-leucine-phenylalanine. Strains derived from individuals with ulcer or non-ulcer associated H pylori infections displayed similar chemotactic activity. Preliminary characterisation shows that the factor has a molecular weight of less than 3000, is heat stable, is acid resistant, and can be diluted at least 10-fold. Checkerboard analysis confirmed that the activity was chemotactic rather than chemokinetic. This chemotactic activity could play a role in the pathogenesis of Helicobacter pylori gastritis.


Effect of Helicobacter pylori infection on 24 hour intragastric acidity in patients with gastritis and duodenal ulcer.

Helicobacter pylori status, gastric histology, and 24 hour acidity were studied in 35 gastritis patients, 21 duodenal ulcer patients, and 14 subjects with normal gastric mucosa. H pylori was identified in 21 of 35 patients with chronic active gastritis and in 19 of 21 duodenal ulcer patients, but in none of those with normal gastric mucosa. Mean scores of activity of gastritis were similar in H pylori positive gastritis and duodenal ulcer patients, but were significantly lower in H pylori negative gastritis patients (2.1 (0.8) and 2.3 (0.9) v 1.4 (0.7); p < 0.01, respectively). Median 24 hour hydrogen ion activity (interquartile range) was 21 (8.9-38.0) mmol/l in normal subjects and 23 (11.2-49.0) mmol/l, 19 (7.1-33.1) mmol/l, 44 (25.1-63.1) mmol/l, and 36 (31.6-39.8) mmol/l respectively in gastritis and duodenal ulcer patients with and without H pylori infection. During all predefined time periods, intragastric acidity was significantly higher in patients with H pylori positive duodenal ulcers compared with gastritis patients and normal subjects. However, there was no significant difference in intragastric acidity between the H pylori positive and negative gastritis patients. These results suggest that most of the subjects with chronic H pylori infection have normal gastric acidity.


Helicobacter pylori and gastric cancer: correlation with gastritis, intestinal metaplasia, and tumour histology.

This study aimed to examine the association between Helicobacter pylori, histological gastritis, and intestinal metaplasia in gastric cancers of different histological types. A total of 169 gastrectomy specimens received in one pathology department were studied. Altogether 156 were adenocarcinomas (intestinal type 87, diffuse type 50, mixed type 19). Gastritis occurred in 137 of 163 body specimens (84%) and in 126 of 131 antral specimens (96%). Its presence was unrelated to tumour histology. Atrophic gastritis was more common in both body and antral mucosa in intestinal type compared with diffuse type carcinoma. This was also true for intestinal metaplasia of the body, but not of the antral mucosa. H pylori was present in 101 of 163 (62%) body specimens and 56 of 131 (43%) antral specimens. In intestinal type carcinoma, H pylori was found in 52/84 (62%) body specimens and in 24/70 (34%) antral specimens, while the corresponding figures for diffuse type carcinoma were 29/48 (60%) and 17/38 (45%) respectively. Tumour histology therefore had no influence on the occurrence of H pylori. Tumour site had no effect on the presence or absence of gastritis, atrophic changes, intestinal metaplasia, or H pylori. While both H pylori and gastritis are associated with gastric cancer, the association is unrelated to tumour histology and may not be a causal one.


Oncogenes and onco-suppressor gene in adenocarcinoma of the oesophagus.

While the activation of the proto-oncogenes has been implicated in the development and progression of cancer of many tissues, the role of oncogenes in the development of oesophageal adenocarcinoma has not been defined. Fifteen patients who had undergone resection for oesophageal adenocarcinoma and 15 who had undergone oesophagectomy or biopsy for Barrett's oesophagus were studied. The latter patients also had adjacent normal gastric mucosa biopsied for comparison with the metaplastic oesophageal mucosa. The mucosal samples were snap frozen and subsequently stained with monoclonal antibodies to the following oncogene associated proteins; c-erbB2 (neu and CE-1) (external domain), c-erbB2 (NCL-CB11) (internal domain), c-src, c-ras, c-myc, c-fos, c-jun, and the onco-suppressor gene--p53. All tumours were well or moderately differentiated adenocarcinomas arising from the lower third of the oesophagus. Eleven specimens showed strong membraneous staining with both c-erbB2 (neu) and c-erbB2 (CBL-CB11). Seven specimens showed strong nuclear staining with p53 onco-suppressor gene. Three specimens were positive for c-ras and c-src, and two were positive for c-jun. In Barrett's epithelium, nine specimens were positive for c-erbB2 (neu and CB11), three were positive for c-src, two were positive for c-ras and c-jun, and one was positive for c-fos. Two of the gastric mucosal biopsy specimens expressed c-erbB2 weakly but no other oncogenes were found. The frequency of positive staining for c-erbB2 is very high, compared with the expression of these genes in other tumours. It is also concluded that errors in the onco-suppressor gene p53, and especially in the external and internal domains of c-erbB2, which is also often expressed in Barrett's mucosa, may be implicated in the development of adenocarcinoma of the oesophagus.


Gastric mucosal barrier: barrier to hydrogen ions imparted by gastric surfactant in vitro.

A simple experiment is described which shows how the highly surface active ingredient of gastric surfactant (DPPC) can be deposited on a filter paper to reduce the rate of transmission of hydrogen ions by one to two orders of magnitude. This finding is compatible with previous studies implying that the hydrophobic layer of surface active phospholipid provides the gastric mucosal barrier as a distinct physical entity.


Correlation between echographic gastric emptying and appetite: influence of psyllium.

The correlation between ultrasonographic gastric emptying and appetite was studied. Echographic evaluation of gastric emptying by measurement of the antral vertical diameter and assessment of sensations of hunger and satiety using analogue visual scales were performed simultaneously in 12 healthy volunteers. Measurements were carried out after the intake of 10.8 g psyllium or placebo in a randomised, crossover, double blind trial. The correlation between echographic gastric emptying and sensations of hunger and satiety was excellent (p < 0.001) after the intake of either psyllium or placebo. Psyllium significantly delayed gastric emptying from the third hour after a meal. It increased the sensation of satiety and decreased hunger at the sixth hour after the meal. The association between echographic measurement and visual scales is a simple method of evaluating the relationship between the stomach and appetite. The pharmacodynamic effect of psyllium should be confirmed by longterm therapeutic trials.


Cryptosporidium, chronic diarrhoea and the proximal small intestinal mucosa.

The association between Cryptosporidium, chronic diarrhoea and a proximal small intestinal mucosal enteropathy was reviewed over a six and a half year period. One hundred and twenty three children with cryptosporidiosis and no clinical evidence of immune deficiency were identified. 50% of children excreting only Cryptosporidium had chronic diarrhoea. Most cases (63%) of chronic diarrhoea occurred in the first two years of life. A mild to moderate enteropathy was present in all nine children undergoing a small intestinal biopsy and seven showed the presence of Cryptosporidium adhering to villous epithelium. All patients eventually recovered spontaneously. Cryptosporidium is a cause of chronic diarrhoea and a proximal small intestinal mucosal enteropathy in children without immune deficiency. Screening for the parasite should be part of the investigative procedures in children with chronic diarrhoea.


Antibodies to Saccharomyces cerevisiae in patients with Crohn's disease and their possible pathogenic importance.

Saccharomyces cerevisiae (baker's yeast) may play an important part in the pathogenesis of Crohn's disease. Because of this the levels of IgG and IgA antibodies against three S cerevisiae strains (NCYC 77, NCYC 79, and NCYC 1108) were assayed in 49 patients with Crohn's disease, 43 with ulcerative colitis, 14 with coeliac disease, and 21 healthy controls. Coded serum samples were tested by ELISA. Similar antibody patterns to all three strains were found. IgG and IgA antibody levels were significantly raised in patients with Crohn's disease compared with healthy controls (p < 0.001 and p < 0.0001 respectively) and with ulcerative colitis patients (p < 0.0001 and p < 0.0006 respectively). Raised IgA, but not IgG, yeast antibody levels were found in two patients with Crohn's disease who were intolerant to yeast, but these values were similar to those in other patients without yeast intolerance. In ulcerative colitis, both IgG and IgA levels were similar to normal controls. Patients with small bowel Crohn's disease had significantly higher IgG antibody levels than those with colonic disease (p < 0.01). High levels of IgG, but not IgA, antibody were present in patients with coeliac disease, the antibody responses being indistinguishable from those found in Crohn's disease. It is concluded that the presence of IgG antibody to S cerevisiae is characteristic but not specific to Crohn's disease. Although raised IgA antibody levels are more frequently found in Crohn's disease, their pathogenic importance remains to be established.


Epidemiological study of abdominal tuberculosis among Indian migrants and the indigenous population of Leicester, 1972-1989.

A retrospective, epidemiological study of abdominal tuberculosis in the city of Leicester from 1972 to 1989 is reported. Potential cases were identified from hospital medical records and endoscopy lists, in addition to the county notification register. The city population of 280,000 included over 75,000 South Asians. There were 146 cases among South Asians and six in Europeans, four of whom were British. The standardised incidence of abdominal tuberculosis in South Asians decreased significantly from 22.3 cases/10(5)/year during the 1970s to 9.2 cases/10(5)/year in the 1980s (chi 2 = 42, p < 0.001). The incidence during the 1980s was 10.7/10(5)/year in Hindus, 8.7/10(5)/year in Sikhs, and 4.6/10(5)/year in Muslims. The relative risk to Hindus was 2.3 fold greater, and for Sikhs 1.9 fold greater, than that for Muslims, a finding similar to that in pulmonary tuberculosis. The standardised incidence in Europeans was 0.2/10(5)/year and they had significantly less abdominal tuberculosis than South Asians (Z = 8.6, p < 0.001 and relative risk = 46). The standardised mortality ratio was significantly increased in Europeans (standardised mortality ratio = 755, 95% confidence interval 90-2730, chi 2 = 11.4, p < 0.001), but not in South Asians (standardised mortality ratio = 68, 95% confidence interval 20-160). Resection rates were similar between the two ethnic groups. Abdominal tuberculosis still occurs among migrants, and clinicians should remain alert to this in South Asians.


Epithelial cells bearing class II molecules stimulate allogeneic human colonic intraepithelial lymphocytes.

HLA-DR+ gut epithelial cells may present antigen to intraepithelial lymphocytes (IEL). This study aimed to isolate an IEL population from the human colon to activate CD3 + IEL by a human colonic epithelial cell line (HT-29), bearing different concentrations of class II antigen (HLA-DR). IEL were isolated by a mechanical method from six patients with ulcerative colitis (UC) and from 14 control patients. IEL were cocultured with HT-29 which had been induced to express class II molecules by gamma-interferon (IFN-gamma) in a dose dependent manner. The phenotype and the subsequent expression of activation markers by the IEL were determined to two colour flow cytometry. The IEL population had a CD4/CD8 ratio similar to that seen in tissue sections. In the mixed cell culture, the degree of IEL activation showed a positive correlation with the degree of HLA-DR expression by the HT29 cells and the IEL secreted a IFN-gamma like factor that in turn stimulated the HT-29. Thus, depending on their expression of HLA molecules, colonic epithelial cells are able to activate CD3+CD8+IEL.


Antiviral effect of prolonged intermittent lymphoblastoid alpha interferon treatment in chronic hepatitis B.

In a European multicentre study 40 patients with HBeAg positive chronic hepatitis B virus (HBV) infection were treated with 5 mega units of lymphoblastoid alpha-interferon daily according to the following regimen: a four week primer course, four weeks of rest and a second course lasting 16 to 30 weeks. After 52 weeks of follow up, a response (HBeAg seroconversion and HBV-DNA negativity) was observed in 22 patients (55%). HBsAg seroconversion occurred in five patients (12.5%). One patient exhibited a relapse for serum HBeAg and HBV-DNA after cessation of treatment. According to a response prediction model, the observed response rate was not related to the selection of patients likely to respond. The initial interferon course induced a reduction of the serum HBV-DNA and HBeAg levels of 87% and 18%, respectively, leading to a significantly lower level of viral replication activity at the start of the second longterm course compared with baseline. After 24 weeks of follow up (week 16 of the second course), 19 (48%) patients exhibited a response, 13 (32%) a partial response (HBeAg < 50% of initial level or HBV-DNA negative) and 8 (20%) no response. For eight of the 13 partial responders treatment was stopped at week 24 and viral replication rebounded to pretreatment values. In the last five partial responders prolongation of therapy up to week 38 led to a definite response and HBsAg seroconversion in three of the five patients. The results of this study suggest that a short primer course and prolongation of therapy may help to enhance the response rate of alpha-interferon therapy for chronic hepatitis type B.


Effects of ethanol, acetaldehyde and cholesteryl esters on pancreatic lysosomes.

Recent studies indicate that altered lysosomal function may be involved in the early stages of pancreatic injury. Chronic consumption of ethanol increases rat pancreatic lysosomal fragility. The aim of this study is to determine whether the lysosomal fragility observed after chronic ethanol consumption is mediated by ethanol per se, its oxidative metabolite acetaldehyde or cholesteryl esters (substances which accumulate in the pancreas after ethanol consumption). Pancreatic lysosomes from chow fed rats were incubated for 30 minutes at 37 degrees C with ethanol, acetaldehyde or phosphatidylcholine vesicles containing cholesteryl oleate. Lysosomal stability was then assessed by determination of: (a) Latency--that is, the per cent increase in lysosomal enzyme activity after addition of Triton X-100 and (b) Supernatant activity--that is, the proportion of lysosomal enzyme remaining in the supernatant after resedimentation of lysosomes. Acid phosphatase, N-acetyl glucosaminidase, beta-glucuronidase and cathepsin B were assayed as lysosomal marker enzymes. Lysosomes incubated with homogenising medium alone or equivalent volumes of phosphatidylcholine vesicles without cholesteryl oleate were used as controls. Cholesteryl oleate at concentrations of 15 and 20 mM increased pancreatic lysosomal fragility as shown by decreased latency and increased supernatant enzyme. In contrast, ethanol (150 mM) and acetaldehyde (5 mM) had no effect on lysosomal stability in vitro. These results suggest that increased pancreatic lysosomal fragility observed with ethanol may be mediated by cholesteryl ester accumulation rather than by ethanol or acetaldehyde.


Raised urea clearance in cirrhotic patients with high uric acid clearance is related to low salt excretion.

In cirrhotic patients without renal failure, salt retention could result from a decreased effective intravascular volume or could be a primary event leading to increased intravascular volume. Clearance of urea and uric acid depend on an effective intravascular volume. In the syndrome of inappropriate secretion of antidiuretic hormone (SIADH)--a state of increased intravascular volume--uric acid clearance is increased and that of urea is increased only when salt excretion is low. The intravascular volume of 60 consecutive cirrhotic patients without renal failure was estimated indirectly by studying the relationship between fractional excretion of filtered (FE) sodium, urea, and uric acid. Forty five per cent had a high FE uric acid (> 12%), which could mean a high intravascular volume, and presented with an FE urea that was inversely correlated with FE sodium (r = 0, 62; p < 0.001) as in SIADH, while in the controls the FE urea was positively correlated with FE sodium (r = +0, 46; p < 0.01). In patients who had a normal FE uric acid and low FE sodium (< 0.2%), the FE urea was significantly lower (40 (13)%, n = 20) than in subjects with high FE uric acid and a low FE sodium (61 (9)%, n = 16, p < 0.001); this last group also presented with lower mean blood urea concentrations (3.1 (1.2) mmol/l and 4.0 (1.8) mmol/l; p < 0.05) and a lower supine renin activity (p < 0.01). As observed in the SIADH, cirrhotic patient with high FE uric acid have raised FE urea only when salt excretion is low. It is believed that the low salt excretion is not caused by a decrease in effective intravascular volume and that this is increased in cirrhotic patients with raised FE uric acid.


Mucus glycoprotein biosynthesis in the human gall bladder: inhibition by aspirin.

Aspirin, which inhibits mucin secretion in the gastrointestinal tract prevents gall stone formation in animals and may reduce gall stone recurrence in man. This study examines the effect of aspirin on mucin synthesis in human gall bladder explants. Two hundred explants were cultured with 3H-glucosamine (74 kBq/ml) for 24 hours at 37 degrees C. Mucin and other glycoproteins were isolated by papain digestion (72 hours) and exhaustive dialysis (144 hours) to remove non-incorporated radioactivity and digested protein. 3H-glucosamine was readily incorporated into glycoprotein. Pooled gall bladder explants were fractionated on a CsCl density gradient and by gel filtration on Sepharose 2B and 4B to confirm that >90% radioactivity was incorporated into mucin. Acetylsalicylic acid (230-666 micrograms/ml) significantly reduced total 3H-glucosamine incorporation (43-89%), p<0.01 (unpaired t test). Diclofenac (125-1250 micrograms/ml), similarly reduced incorporation by 45-97% p<0.001 (unpaired t test). Inhibition of mucin glycoprotein biosynthesis was irreversible with both drugs. Analysis of pooled samples on Sepharose 4B showed abolition of radioactive incorporation into mucin but no effect on incorporation into low molecular weight glycoprotein material (10% of total incorporation). This study provides a method for measuring human gall bladder mucin synthesis and shows its irreversible inhibition by acetylsalicylic acid and diclofenac at concentrations compatible with a therapeutic dose.


Inhibition of human gall bladder mucus synthesis in patients undergoing cholecystectomy.

Hypersection of gall bladder mucus is associated with gall stone formation in animal models. Aspirin inhibits both mucus synthesis and secretion, prevents gall stone formation in animals and reduces gall stone recurrence in man after dissolution therapy. Mucus biosynthesis in human gall bladder mucosal explants is inhibited by aspirin in vitro. We have studied the effects of aspirin in vivo. Fifty five patients with functioning gall bladder and stones have been randomised, 27 to group 1 (aspirin EC 300 mg once daily for seven days before cholecystectomy) and 28 to group 2 (controls). Gall bladder bile composition was analysed and mucus synthesis rates measured using 3H-glucosamine incorporation into mucosal explants cultured for 24 hours. Patient age, sex, and gall bladder histology were similar in both groups. There were no differences in stone composition, gall bladder bile calcium concentration, cholesterol saturation and cholesterol nucleation time. The mean 3H-glucosamine incorporation in aspirin treated patients was 1347 fmol/g wet weight as compared with 2008 fmol/g wet weight in controls (95% confidence interval 222-1100, p<0.005, unpaired t test). This reduction in biosynthesis was associated with gall bladder bile mucus concentrations of 7.6 mg/ml in patients and 7.1 mg/ml in controls (ns). Treatment with aspirin led to a significant reduction in mucus biosynthesis by the gall bladder mucosa. This action is consistent with a role for aspirin in the prevention of gall stones.


Postoperative bile leakage: endoscopic management.

Bile leakage is an infrequent but serious complication after biliary tract surgery. This non-randomised single centre study evaluated the endoscopic management of this problem in 55 consecutive cases. Treatment consisted of standard sphincterotomy and, if needed, subsequent stone extraction with or without endoprosthesis placement. The aim of all treatments was to facilitate bile flow into the duodenum. The biliary tract and the site of the leakage were visualised during endoscopic retrograde cholangiopancreatography (ERCP) in 98%. There was distal obstruction in 33--caused by retained gall stones in 15 patients and concomitant strictures in 18. Overall, 48 of 55 patients were treated endoscopically. An excellent outcome (clinical and radiological resolution of the bile leak) was achieved in 43 patients (90%). Five patients (10%) had continuing sepsis from which they died. Postoperative bile leakage can be diagnosed safely and effectively by ERCP and subsequent endoscopic management is successful in most cases.


Development of biliary sludge in patients on intensive care unit: results of a prospective ultrasonographic study.

Biliary sludge may be a precursor of gall stones in man. The aim of this study was to determine the incidence of biliary sludge in a prospective study of 36 patients admitted to the intensive care unit for longer than two days. The presence of biliary sludge was determined by ultrasonography. Biliary sludge developed in 17 patients (47%), after a mean of 5.5 days in the intensive care unit. Patients who developed biliary sludge spent longer in the intensive care unit (14.2 d (1.3)), compared with patients who did not (8.3 d (1.4)); (p = 0.003). Ten of the patients with biliary sludge had a recognised risk factor: total parenteral nutrition (five), abdominal surgery (two), or both (three). All neurosurgical patients (four) who required total parenteral nutrition developed biliary sludge. Seven patients with biliary sludge had no previously recognised risk factor, five of whom had severe head trauma or neurosurgery. In conclusion, biliary sludge develops frequently and rapidly in patients admitted to an intensive care unit. Neurosurgical procedures are associated with biliary sludge formation. (Sludge is commonly associated with the development of cholestatic liver biochemistry.)


Endotoxaemia and serum tumour necrosis factor as prognostic markers in severe acute pancreatitis.

Endotoxaemia and circulating tumour necrosis factor are important prognostic factors in severe sepsis and are implicated in the pathogenesis of septic shock. Because clinical and pathological features in acute pancreatitis are similar to septic shock this study sought to determine whether endotoxin and tumour necrosis factor were prognostic factors in 38 patients with prognostically severe acute pancreatitis. Endotoxaemia, present in 19/37 (51%) patients on day 1, was more common in nonsurvivors than survivors (10/11, 91% v 9/26, 35%, p = 0.003). Day 1 serum endotoxin concentrations were higher in patients with a severe outcome (median (interquartile range) 314 (173-563) pg/ml v 0 (0-185) pg/ml, p<0.01) and in non-survivors (266 (173-586) pg/ml v 0 (0-165) pg/ml, p<0.01). Serum tumour necrosis factor was detectable in 47 of 109 samples (43%) from 38 patients (median 35 pg/ml, range 5-943 pg/ml). Day 1 serum tumour necrosis factor correlated with a worse prognostic score and a severe outcome in all patients (n = 38, r = 0.36, p = 0.027; r = 0.33, p<0.05) and with mortality in patients with gall stones (n = 23, r = 0.50, p = 0.02). Our data suggest that endotoxin and tumour necrosis factor could be prognostic factors in severe acute pancreatitis.


Veralipride for hot flushes during gonadotropin-releasing hormone agonist treatment.

Hot flushes are the commonest symptom induced by gonadotropin-releasing hormone agonists (GnRHa). We performed an open observational trial to evaluate the efficacy of veralipride, an antidopaminergic drug, in reducing hot flushes in 25 premenopausal women treated with a GnRHa for endometriosis (8 subjects) or menorrhagia (17 subjects). The patients received goserelin depot for 6 months and veralipride was added for the third month. Hot flushes, severe in all women at 2 months, improved in both frequency and intensity in 92% of the subjects during veralipride administration. The benefit obtained persisted until the end of the GnRHa treatment.


Effect of testosterone on the development of bladder tumors and calculi in female rats.

In order to study the effect of testosterone on bladder calculi and tumor formation in female rats, Wistar rats were administered testosterone for 12, 18 or 24 weeks. Testosterone was found to increase the incidence of both bladder calculi and tumors in intact, but not in oophorectomized rats. It is suggested that testosterone in combination with estrogen may contribute to hyperplasia formation, in turn leading to an increased incidence of bladder calculi and tumors.


Lymphocyte subset and NK activity in cervical intra-epithelial neoplasia.

We have studied the systemic T-lymphocyte subset and, in particular, the natural-killer (NK) activity in 23 patients with cervical intra-epithelial neoplasia (CIN), and 11 controls (a history of normal cervical PAP smears and colposcopy patterns). The lymphocyte phenotypes were studied using a panel of monoclonal antibodies, and NK activity was evaluated as the percentage lysis of K 562 target cells. In patients with CIN we found a significant decrease in NK activity (p = 0.002) which was directly correlated with the grade of CIN, with no alterations in the absolute number of NK cells.


Prenatal cytogenetic diagnosis of 1,400 consecutive amniocenteses.

We report on the incidence of chromosomal abnormalities in 1,400 amniocenteses. Thirty-one cases (2.21%) were found to have various types of chromosomal abnormality. The majority of abnormal cases (54.84%) were trisomies. All individuals with trisomic fetuses chose to terminate the pregnancy with the exception of one who gave birth to a Down's syndrome child. However, fetuses with apparently balanced translocations were phenotypically normal at birth.


Successful delivery of twins in a woman with a unicornuate uterus.

The presence of a unicornuate uterus is a rare congenital condition which represents only 1-2% of uterovaginal anomalies. Previous reports have demonstrated an increase in the number of cases of primary infertility, pregnancy loss and preterm labor associated with the unicornuate uterus. Herein, we present a case in which a patient conceived following a thawed frozen embryo transfer. She had been given the option of selective reduction, because of the high risk associated with this pregnancy, but refused and her pregnancy successfully continued to 35 weeks and delivery of twins.


A lactating adenoma of the breast.

A lactating adenoma of the breast was first examined at the 24th week of gestation, and had doubled in size by the 37th week. After delivery, administration of bromocriptine (5 mg/day) reduced its size, and it was surgically removed on the 10th day. Histologically, the glandular epithelial cells of the adenoma showed the physiological changes normally seen during lactation, and retained much of their lactating activity (especially protein secretion). The myoepithelial cells contained an increased number of lysosomes and also some autophagosomes, implying that the tumor had partly undergone involution similarly to the normal breast tissue.


Tumor markers in gynecologic cancer.

The development of biochemical tumor markers has increased the use of antibody-dependent tumor marker assays in gynecologic oncology. Several monoclonal antibodies directed against novel epitopes on tumor-associated antigens have allowed the development of sensitive assays for serum markers. Assays for human chorionic gonadotrophin and TA-4 have been improved. CA 125 has provided a useful first-generation markers. Ovarian cystoadenocarcinoma-associated antigen and lipid-associated sialic acid have been developed for ovarian cancer, transforming growth factor for squamous cancer, and placenta protein 4 for endometrial and cervical cancer. The most widely applied procedures to identify these markers are immunofluorescent microscopy and immunocytochemical staining. Multiple markers and modalities may be required to increase the sensitivity of tumor detection. CA 15-3 and GCDFP-15 markers have been useful in detecting breast cancer. The application of radionuclide imaging will provide a new field for the diagnosis of gynecologic malignancies.


Vulvar oedema among pregnant Mozambican women.

During a 3-year period, a total of 22 pregnant women with vulvar oedema were observed in a high-risk antenatal clinic (ANC) in Maputo. They were compared with 22 unselected normal ANC attenders, matched for age, parity, gestational length and area of living. Reported and observed genital ulcers were more prevalent in the oedema group than in the referent group. Reported and observed vaginal discharge was also significantly more common in the oedema group. Syphilis screening by VDRL was positive in 61.9% of oedema cases while positivity reached 5.0% in referents (p < 0.005). Cases found seropositive on screening were confirmed using Wassermann reaction (WR) in a reference laboratory, in which WR-positive cases underwent FTA-ABS analysis and IgM assay with solid-phase haemadsorption. IgM-positive individuals were significantly more prevalent among WR-positive oedema cases than among WR-positive referents (p < 0.05). It is concluded that among antenatal attenders in Maputo presenting with vulvar oedema, a significant proportion is associated with recent syphilis. Vulvar oedema should be considered as an important marker for seropositive syphilis during pregnancy.


Puerperal measurement of the symphysis-fundus distance.

Three groups of puerperal women were compared. The first group comprised 51 women with clinically evident endometritis-myometritis. This group was compared to 51 referents (group 2), matched for age, parity and days after delivery. Group 3 comprised 69 randomly selected, healthy puerperal women coming for postnatal check-up of their newborns. Groups 1 and 2 were compared regarding symphysis-fundus (SF) distance. It was found that puerperal women with clinical signs of endometritis-myometritis and healthy, matched referents (groups 1 and 2) did not differ in uterine size as measured by the SF distance. The uterine involution can be followed by simple anthropometry by assessment of the SF distance over the puerperal period. It is concluded that the allegedly swollen and enlarged uterus in puerpera with endometritis-myometritis post partum does not differ in fundal height from the uterus of healthy, normal puerpera.


Prepregnancy low body mass index is not a predictor of labor complications.

The course of delivery and fetal outcome in 149 women with forecoming normal pregnancy and a prepregnancy body mass index (BMI) less than 20.0 were compared to a similar group of women with BMI between 20.0 and 24.9 after matching for age and parity. None of the women was undernourished. Suspected intrauterine asphyxia was significantly more common in primipara with BMI below 18.0 than in their controls (6/12 vs. 1/12), and there was a trend towards fewer babies weighing at least 4,000 g in the women with low BMI. No other significant differences related to BMI were found. It is concluded, that constitutional low weight for height is not a predictor of complications during delivery, and no special observation of this group is recommended.


Reference values for resistance index and pulsatility index of uteroplacental Doppler flow velocity waveforms based on 612 uneventful pregnancies.

Using a 4-MHz continuous-wave Doppler device, standard rates were established for resistance index (RI) and pulsatility index (PI) of uterine and arcuate arteries of 612 patients with uneventful pregnancies and deliveries. From 18 to 41 weeks of gestation, neither RI nor PI of uterine or arcuate arteries proved to vary with gestational age, maternal heart rate, or maternal age. By contrast, a significant effect of placental location on the measurement results was found in both uterine and arcuate arteries. The differences between measurements on the placental or opposite site are more distinct in arcuate than in uterine arteries. Taking the 90th percentile as a localization gauge, cutoff levels of 0.52 (RI) and 0.98 (PI) were found in uterine arteries. In arcuate arteries, cutoff levels of 0.45 (RI) and 0.82 (PI) were found on the placental site or with a placenta without lateralization. On the nonplacental site of a lateralized placenta, the cutoff levels were 0.51 (RI) and 0.92 (PI).


Very high collagen increase in placentae of smoking mothers.

In 55 placentae, collected at term from clinically normal pregnancies, hydroxyproline was assayed. Thirty were from mothers who smoke and 25 from nonsmokers. In placentae of smokers, hydroxyproline concentration was 42.77 +/- 8.77 micrograms/mg of tissue and in these of nonsmokers 22.52 +/- 7.46 micrograms/mg. Such high a difference discloses a large increment in placental collagen production in smokers, which may account for some of the noxious effects of tobacco on pregnancy.


Labor characteristics of uncomplicated prolonged pregnancies after induction with intracervical prostaglandin E2 gel versus intravenous oxytocin.

Labor characteristics after intracervical application of 0.5 mg prostaglandin (PG) E2 gel (n = 83) versus intravenous administration of oxytocin (n = 82) for labor induction were investigated in uncomplicated prolonged pregnancies with unripe cervix. The induction to delivery time as well as the total oxytocin dose were significantly reduced in the PGE2 group (p < 0.001). Cesarean sections, instrumental deliveries and fetal distress had the same frequency, but the failures of trial were significantly higher in the oxytocin group than in the PGE2 group (20.7 vs. 6%, p < 0.01). Twenty-four percent of women needed a second PGE2 dose, and almost half of the women in the PGE2 group experienced 'spontaneous' labor. More neonates in the oxytocin group had 5-min Apgar scores < 7 (p < 0.05). Intracervical PGE2 gel application is superior to intravenous oxytocin in terms of shortening the induction-delivery interval and increasing the frequency of successful vaginal delivery. In addition, it is safe for mother and fetus.


Psychological reactions and sexual life after hysterectomy with and without oophorectomy.

A total of 678 women were interviewed about psychological reactions and sexual adjustment after abdominal hysterectomy with and without simultaneous oophorectomy. The response rate was high and complete questionnaires were returned by 86% of the women. A more positive attitude towards the operation was found in the group of women where the ovaries had been preserved. Women who had undergone oophorectomy experienced a deteriorated sexual life compared to women with preserved ovaries. This was observed regardless of age and in different parameters such as 'coital frequency' and 'experience of intercourse'. The negative effects of sexual life as a result of hysterectomy with simultaneous oophorectomy are important to bear in mind and should be discussed with the woman prior to surgery.


The soluble transferrin receptor: biological aspects and clinical usefulness as quantitative measure of erythropoiesis.

Iron transport in plasma is carried out by transferrin, which donates iron to cells through interaction with a specific membrane receptor. In this review, we summarize the current knowledge on the structure, intracellular cycle, distribution, and regulation of the tissue transferrin receptor. A soluble form of the transferrin receptor has been identified in animal and human serum. We review the molecular form and origin of the soluble receptor, its dosage in normal serum and its relationship with erythropoiesis, iron status, and cancer. Lastly, we discuss the clinical usefulness of the soluble transferrin receptor for the quantitative determination of marrow erythropoietic activity.


M-BCR breakpoint location does not predict survival in Philadelphia chromosome-positive chronic myeloid leukemia.

Some reports in the recent literature have shown that the site of molecular rearrangement within the M-BCR area may have a prognostic value in Ph1 + CML patients. A number of studies have, however, failed to demonstrate this finding. Here we report the molecular rearrangements of 107 patients and their clinical follow-up. Localization of the breakpoints was determined according to conventional criteria, after digestion with 2 to 4 restriction endonucleases and hybridization with 1 or 2 molecular probes. Sixty two patients were rearranged in the 5' area and 45 in the 3' area: there was no difference between the survival curves of the two groups, at least not after 3 years of follow-up. The site of the breakpoint within the M-BCR has no prognostic significance in our series.


Functional depletion of T cells by vincristine and methylprednisolone as an in vitro model for the prevention of graft versus host disease.

BACKGROUND AND METHODS. The outcome of mismatched bone marrow transplantation is still severely hampered by graft versus host disease (GVHD) and graft rejection. Procedures for the recognition and selective elimination of T cells are still unsatisfactory due to the increased incidence of graft failure and late rejection. Lymphocyte proliferation and generation of cytotoxic T cells (CTL) in response to allogeneic cells are considered good in vitro correlates of GVHD and have been used in the present study to asses the capacity of two drugs (vincristine, VCR, and methylprednisolone, MP) to affect the T cells involved in these reactions. RESULTS AND CONCLUSION. Treatment in vitro with VCR and MP has been shown to inhibit the functional capacity of peripheral blood lymphocytes to proliferate (mean reduction 95.8%) and to generate CTL in response to haploidentical stimulator cells. Responsiveness to antigens and mitogens was affected to a minor extent (mean reduction 40% and 65.5%, respectively), and the method allowed recovery of hemopoietic precursors. The results suggest that treatment of donor bone marrow with VCR and MP is worth studying as a new approach to the prevention of GVHD in haploidentical bone marrow transplantation.


Time-dependent sensitivity of rat CFU-GM to total body irradiation.

It has been shown that the light-darkness cycle affects the proliferative activity of the hemopoietic system, possibly acting on the distribution of the cells in the cell-cycle phases at different hours of the day. This could determine time-dependent modifications in ionizing radiation damage to hemopoietic progenitors. In this study the influence of the irradiation time on the radiosensitivity of rat CFU-GM was assessed by in vitro clonogenic assay. Rats were exposed to 3 Gy gamma rays at four time points (00:00, 06:00, 12:00, 18:00 hours). Bone marrow CFU-GM cultures were performed at various intervals ranging between 12 hours and 45 days after irradiation and compared with unirradiated controls. A marked decrease of femoral CFU-GM was observed in the five days following total body irradiation, regardless of the irradiation time point. From this interval on all the irradiated groups showed an increasing proliferation of CFU, which was particularly evident in the group irradiated during the day light period. At the latest intervals (45 days) the post-acute damage to hematopoietic progenitors lacked any evidence of time dependence. With the experimental model used, time scheduling does not seem to affect markedly either the acute depletion of CFU-GM in the 5 days following total body irradiation or the late consequence to the CFU-GM compartment.


Proliferation and maturation effects of in-vivo granulocyte-macrophage colony stimulating factor in acute non-lymphocytic leukaemia.

Granulocyte-macrophage colony stimulating factor (GM-CSF) can affect the treatment of acute non-lymphocytic leukaemia (ANLL) by supporting normal haemopoiesis and by enhancing the proliferation and the maturation of leukaemic cells. A human recombinant E. Coli synthesized GM-CSF was administered to 7 patients with ANLL at a dose of 5 or 10 micrograms/kg/day for 7 days, prior to antileukaemic treatment. Peripheral blood neutrophil and blast cell count was monitored daily. Changes in marrow blast cell morphologic features, mitotic index, and the reactivity to a panel of monoclonal antibodies were assessed after 3 and 7 days of treatment. Peripheral blood neutrophil and blast cell count increased in 6/7 cases. The mitotic index of marrow cells increased in 6/7 cases. A significant maturation occurred along the granulocytic line (2 cases) and the monocytic line (1 case). Mild to moderate eosinophilia developed in 3/7 cases. Early stem cell markers (CD 34 and HLA-DR) were not lost, or actually increased. Myelomonocytic markers (CD 33, CD 13, and CD 14) rose and fell. Expression of the multidrug resistance-associated 170 kd glycoprotein remained stable. These data showed that in vivo GM-CSF more consistently enhanced the proliferation than the maturation of leukaemic cells.


Effect of anagrelide on platelet count and function in patients with thrombocytosis and myeloproliferative disorders.

Anagrelide is a quinazolin compound developed initially as an inhibitor of platelet aggregation. Since "in vivo" studies demonstrated that it was responsible for thrombocytopenia in humans, anagrelide has been used recently in a small number of patients with thrombocytosis and myeloproliferative disorders. Platelet count was well controlled in the large majority of patients, and only minimal side effects were observed. Eight patients (5 with essential thrombocythemia, 2 with chronic granulocytic leukemia, and 1 with idiopathic myelofibrosis) received anagrelide (induction dose 4 mg/die; mean maintenance dose 2 mg/die; mean observation time 26 weeks). Complete blood counts were determined 4 times during the first month, and subsequently every month. "In vivo" and "ex vivo" platelet function was studied before anagrelide and after 4 and 10 days of therapy. Platelet count was reduced and maintained below 500 x 10(9)/L in 5 of 8 patients. Headache, palpitation/tachycardia, gastrointestinal symptoms and a decrease in hemoglobin were the side effects. Anagrelide did not modify the leukocyte count or "in vivo"/"ex vivo" platelet function. Anagrelide may control thrombocytosis in patients with myeloproliferative disorders, even when traditional drugs have failed. When required, anti-aggregating drugs may be associated with anagrelide, since it has no effect on platelet function.


Prophylaxis of venous thrombosis after gynaecological surgery: a controlled pilot study of defibrotide.

Defibrotide (Def), a new antithrombotic drug, has been proposed as a prophylactic agent in postoperative DVT. Most of the studies to date, however, have either not been controlled or have used unverifiable systems for asymptomatic DVT diagnosis. This randomized pilot study compared Def versus standard low-dose calcium heparin (CH) prophylaxis after gynaecological surgery, using objective criteria for DVT diagnosis. Forty-one pts received 400 mg Def intramuscularly twice a day starting the day before surgery; 40 pts received 5000 IU CH s.c. twice daily beginning 2h before surgery. The two groups were well matched for all relevant risk factors. DVT was diagnosed by means of the 125I fibrinogen uptake test (FUT) and venography. Blood coagulation and fibrinolysis tests were also carried out perioperatively. Isotopic DVT (FUT-positive for two consecutive days) was recorded in 6 (14.6%) of the Def and 5 (12.5%) of the CH groups. In cases where FUT was positive for at least three consecutive days (4 in Def and 1 in CH), venography confirmed DVT in 3 cases (all in the Def group). No side-effects were recorded in either group and the amounts of transfused blood were not different. No significant differences in blood coagulation or fibrinolysis tests were recorded, except for higher fibrinogen levels on the 8th post-operative day in the Def group. These results do not indicate any trend suggesting that Def, as a prophylactic agent in gynaecological surgery, offers any clinical or practical advantages over standard low-dose heparin prophylaxis.


Retrospective analysis of platelet serology investigations performed in 1990 by twenty laboratories belonging to the "Cooperative Group for the Study of Platelet Immunology".

Retrospective co-operative analysis of platelet serology investigations was performed during 1990 by twenty laboratories from eighteen institutions. Data regarding the serological investigations, the methods used and the clinical diagnosis of the patients investigated during 1990 were collected and analyzed. A total of 2591 patients was investigated, including 505 potentially alloimmunized subjects and 2086 subjects with autoimmune disease-related conditions. Alloantibodies were detected in 46% of the patients, while autoantibodies were detectable in 30% of the autoimmune conditions. The antibodies were mostly of the IgG class; however, IgM were detected in 19% of alloimmunized patients and in 28% of those with detectable autoantibodies. Each laboratory used one or more methods. In all nine methods were used. Collection of the multi-center retrospective results gave an approximate picture of the diagnostic potential of the Group, which is ready for the next phase of the cooperative activities that will consist of serological workshops for technical standardization and antibody characterization.


High-dose methotrexate administration and acute liver damage in children treated for acute lymphoblastic leukemia. A prospective study.

Methotrexate-induced hepatotoxicity following chronic low-dose administration has been extensively reported. Current protocols now include high-dose methotrexate (HDMTX), but there are few studies providing data on its acute hepatotoxicity in childhood leukemia. To evaluate the prevalence of HDMTX-induced acute hepatotoxicity, sixty-eight consecutive children with ALL were prospectively studied from diagnosis to the end of HDMTX courses with biochemical and clinical evaluation performed at regular intervals. Prevalence of HDMTX-induced acute hepatotoxicity was 1.47% (1/68 patients). ALT values did not change in 22% (15/68) and decreased in 76.4% (52/68) after HDMTX infusion. Mean ALT levels calculated in all the patients decreased significantly during HDMTX administration when compared to the values reached during induction (p less than 0.0001). Direct hyperbilirubinemia was present only in the child with HDMTX-related hepatotoxicity. The use of HDMTX in the treatment of childhood ALL is not associated with major evidence of direct acute hepatotoxic effects, while it may modify the pattern of preexisting liver diseases.


Successful use of high-dose cyclophosphamide in a child with severe autoimmune hemolytic anemia.

We report the case of a five-month-old boy with a life-threatening autoimmune hemolytic anemia that was unresponsive to conventional therapy with steroids, high-dose immunoglobulin, azathioprine and splenectomy. Despite these therapies, the patient's condition worsened, requiring 2-3 blood transfusions/day, since the hemoglobin level was constantly below 4 g/dl. We eventually increased immunosuppression by giving high-dose cyclophosphamide and the child showed a striking, sudden improvement, followed by complete recovery. No major long-term complications were observed.


Post-transfusional human retrovirus infection in 41 Italian beta-thalassemic patients.

Previous studies have demonstrated that HTLV-I is present in Italy both in endemic form in Southern Apulia and in epidemic form among the population of intravenous drug addicts. In the present paper we intend to evaluate the risk for transfusional HTLV-I transmission in our country, as well as the already known risk for HIV1. A population of 41 polytransfused Italian beta-thalassemic patients was examined by serological methods and PCR (polymerase chain reaction) for human retrovirus infection. Genomic DNA from PBMCs was analyzed by PCR with primer pairs specific for the HTLV-I gag, pol and env regions, and the HTLV-II env region. Two patients were found to be weakly seroreactive to p19 and p24 HTLV-I/HTLV-II proteins by Western blot. The analysis of genomic DNA from PBMCs by PCR revealed sequence homology to HTLV-I only in these two patients. On the contrary, PCR with primer pairs specific for HTLV-II showed no beta-thalassemic patient was infected by this retrovirus. Surprisingly, Western blot analysis for detecting anti-HIV1 antibodies in these polytransfused subjects showed a seropositivity in two patients (not the same found to be infected with HTLV-I) in spite of a screening for HIV1 antibodies in the blood bank. These findings suggest that in Italy polytransfused people should still be considered at risk for HIV1 as well as HTLV-I infection, even if the incidence cannot be evaluated from such a small sample. The authors stress the importance of a through medical history of potential blood donors to eliminate possibly infected subjects.


Delta + 27 homozygosis in a Sicilian family.

During a screening program to identify at risk couples for beta-thalassemia first-trimester prenatal diagnosis, we were able to detect, by polymerase chain reaction (PCR) and direct genomic sequencing of the PCR product, a homozygosis for the G-T substitution at the first nucleotide of codon 27 of the delta-globin gene in a pregnant Sicilian woman. The possibility of showing an interaction between delta and beta thalassemia is relevant for a thalassemia prevention program because it may hide a beta-thal carrier state.


Prognostic relevance of ploidy and proliferative activity evaluation by flow cytometry in poor prognosis non-Hodgkin's lymphomas.

The DNA content and S-phase fraction (SPF) of pathological paraffin-embedded lymph node tissue from 47 patients with poor prognosis non-Hodgkin's lymphoma (NHL) were analyzed by flow cytometry (FCM). Fifteen of 42 evaluable cases proved to be aneuploid. The DNA aneuploidy was not correlated with age, histology, stage, presence of systemic symptoms, clinical response or overall survival (OS) of patients. Mean SPF evaluated in 35 cases was 22.9%. After 78 months of follow-up, nine patients with diploid low SPF tumors had a significantly longer disease-free survival (DSF) with respect to the group of patients (33 cases) with aneuploid or diploid high SPF tumors (100% vs 59%; log rank test, p = 0.04).


Age as a prognostic factor in primary non Hodgkin lymphoma of the stomach.

We present 20 cases of primary non-Hodgkin lymphoma of the stomach. Histological classification, staging, age of patients and therapeutical approach were evaluated. Median overall survival was 30 months, and 5-year survival was 44%. In our small series, the age of the patients plays an important role: median overall survival is 18 months in patients greater than 60 y.o. (compared to 90 months in patients less than 60 y.o.) and 5-year survival 12.5% (compared to 62%).


Long-term treatment of refractory HIV-related immune thrombocytopenia in a patient with haemophilia A.

Haemophilia A patient developed symptomatic immune thrombocytopenia 5 years after HIV seroconversion without any progression of the viral disease. He displayed major bleeding with less than 30 x 10(9) platelets/l. No increase in platelet count was obtained using steroids, azidothymidine and alpha-interferon, while the patient was responsive only to high-dose intravenous immunoglobulins (IVGG). The patient remained responsive to IVGG for 1 year, and the repeated infusions of immunoglobulins were effective in safely maintaining the platelet count, with peak counts above 100 x 10(9)/l. On the contrary, after a single course of six plasma exchanges the patient became symptomatic and completely refractory to IVGG during the next month. In conclusion, IVGG could be effectively used in a long-term regimen in haemophiliacs with refractory HIV-ITP to avoid the risk of haemorrhages and to delay splenectomy.


Ascorbic acid for the treatment of chronic refractory idiopathic thrombocytopenic purpura (ITP).

We describe our experience with ascorbic acid in the treatment of chronic refractory ITP. Nine patients, 5 males and 4 females aged 27-74 years, 4 of whom were previously splenectomized, received the drug at a daily dose of 2 grams. After 2-12 months (median 4) of treatment, a partial response was observed in only one patient, while no response was registered in the other eight. Our data, analyzed together with those of the literature, allow us to conclude that ascorbic acid may not be considered a drug of interest in the treatment of chronic refractory ITP.


Colloidal gold immunostaining for the detection of platelet reactive allo- and autoantibodies. Use on fresh and frozen cells.

Fresh washed platelets are commonly used as target cells for the detection of platelet reactive antibodies. The use of stored platelets would allow a faster execution of the tests and a rapid selection of phenotyped cells. Glass-adherent platelet monolayers were freeze stored. The antigen-antibody reaction between platelet antigens and platelet reactive allo- or autoantibodies was revealed by a modified antiglobulin test, using colloidal gold-labeled anti-human IgG. Immunogold staining (IGS) was compared with the platelet suspension immunofluorescence test (PSIFT) and with the solid-phase red cell adherence assay (SPRCA). The reactivity of fresh and freeze-stored platelets was compared. IGS proved to be slightly less sensitive (10%) than PSIFT and SPRCA: The results obtained with fresh and freeze-stored cells were scored identically. The immunogold staining assay on freeze-stored platelets seems to be a convenient technical approach for platelet serology.


A new mutation in the beta-globin gene (IVS II-850 G-C) found in a Yugoslavian beta-thalassemia heterozygote.

The recent development of laboratory techniques that can rapidly characterize the molecular defects of beta-thalassemia has resulted in the discovery of more than 100 different point mutations in the beta-globin gene. These mutations are population specific. About 20 of them account for over 90% of beta-thal genes in the world. The other mutations are usually found in single families. In this paper we describe a case with a novel mutation at position IVS II-850 (G-C) as a cause of beta-thalassemia. Direct sequencing of PCR amplified DNA was used for the detection of the mutation. ASO probes were synthesized for dot-blot hybridization. Expression of the mutated allele was evaluated through Northern blot and RNA-PCR analyses. This mutation was found in four members of a family, who exhibited severe microcytosis and hypochromic anemia, with an average alpha/beta ratio of 2.0. The sequencing of PCR amplified DNA showed a G-C mutation at position IVS II-850 of the beta-globin gene. Dot blot analyses confirmed the presence of this substitution in all four carriers. Northern blot and RNA-PCR analyses did not reveal any abnormally spliced mRNA species. The G-C substitution at position IVS II-850 is the third mutation in the invariant AG dinucleotide of the acceptor splice site of the second intron of the beta-globin gene. It abolishes normal splicing, which leads to abnormally processed mRNA. It is a relatively rare mutation since it was not detected among the uncharacterized beta-thal chromosomes from Yugoslavia.


Evidence for the co-existence of immunocomplexes and platelet specific antibodies in HIV infection-related thrombocytopenia in narcotic drug addicts. Experimental results and immunopathogenetic discussion.

The development of symptomatic or asymptomatic thrombocytopenia is not uncommon in HIV seropositive drug addicts. Platelet binding immunoglobulins, as immune complexes or as platelet specific antibodies, were studied in eleven patients. Whole sera anche serum factions obtained by PEG sedimentation and gel filtration were investigated using methods able to characterize the IgG and IgM immunoglobulin classes. Immune complexes-containing fractions were used in competition assays with some monoclonal antibodies with receptor specificities. Immune complexes able to bind both autologous and allogenic platelets were detected in all patients, while platelet specific IgG or IgM autoantibodies were detected in most, but not in all, patients. The results obtained with the various individual tests were sometimes discrepant, owing to the low avidity binding of platelet-reacting immunoglobulins. Anti-low-affinity FcRII monoclonal antibody moderately reduced the platelet binding of IgG containing immune-complex fractions. In thrombocytopenic, HIV seropositive drug addicts immunecomplexes and platelet specific antibodies usually coexist and possibly have different pathogenetic potential in the development of thrombocytopenia.


Cytotoxic effects of dacarbazine in patients with acute myelogenous leukemia: a pilot study.

Preclinical studies performed in our laboratory showed that mouse leukemias become highly immunogenic following in vivo treatment with Decarbazine. This observation led to a successful immunochemotherapy protocol in mice using Dacarbazine plus cytoreductive chemotherapy. Therefore an in vivo and in vitro pilot study was conducted in patients (pts) with resistant or relapsed acute myelogenous leukemia (AML). The DNA-repair enzyme O6-alkylguanine-DNA-alkyl-transferase (OGAT) and the in vitro chemosensitivity to Temozolomide, a Dacarbazine derivative, were evaluated in leukemic blasts. Nine pts received Dacarbazine (0.4-0.8 g/sqm/day) on days 0, 1 and 2. On day 7, noticeable blast reduction occurred in 4 pts: pt 1 was in partial remission on day 21, pt 6 still showed bone marrow leukemia, pt 8 died of sepsis on day 8, pt 9 is still in aplasia on day 25. Low OGAT levels and consistent sensitivity to Temozolomide in vitro were found in the blasts of pts responsive to Dacarbazine. Subsequently, pts 1-7 underwent Ara-C (1g/sqm/day) plus Mitoxantrone (6mg/sqm/day) treatment for 6 days. Four pts entered complete remission after 27-45 days of aplasia. Failures were due to hypoplastic death, absolute drug resistance, or hypoplasia followed by blast cell regrowth. These data point out that Dacarbazine can induce a marked reduction of blast cells as well as severe myelotoxicity in leukemic patients.


Hb-M "Hyde Park": a de novo mutation, identified by mass spectrometry and DNA analysis.

Structural hemoglobinopathies usually are inherited as autosomic dominant traits; de novo mutations are uncommon. Analytical and preparative procedures for the characterization of an abnormal hemoglobin are complex and time-consuming. Mass spectrometer analysis allows a rapid identification of the amino acid substitution. A cyanotic 7-year-old girl was found to have 16% methemoglobin. Laboratory data showed the presence of an abnormal hemoglobin, which was isolated by collecting the abnormal peak from DEAE and globin chains from CM52. The amino acid substitution was rapidly identified by FAB mass spectroscopic analysis, leading to the recognition of HbM Hyde Park. These data were confirmed by molecular analysis (Southern blot and DNA sequencing). Neither the parents nor a sister showed any abnormality; non-paternity was excluded by blood group serology and HLA typing. This is a case of HbM Hyde-Park arising as a de novo mutation. FAB mass spectroscopic analysis is a rapid and useful analytical method for identifying aminoacid substitution.


Loss of surface HLA class I molecules in leukemic myeloblasts is correlated with an increased leukocyte concentration at onset.

Reduced expression of HLA class I molecules has been demonstrated for many human neoplasias and is correlated with increased malignancy. We investigated the correlation between HLA expression on blasts and the number of peripheral blood leukocytes (WBC) at onset in 34 patients with acute non lymphoblastic (ANLL) leukemia. Leukemic blasts at onset were serologically typed for HLA-A,B specificities, and the patients were classified in three groups according to the number of detectable surface HLA antigens. The amount of WBC at onset in patients lacking HLA antigens was significantly higher than in patients with no antigenic loss. A decrease in HLA class I molecules on the cell surface is correlated with a greater expansion of the leukemic clone.


Primary gastrointestinal lymphoma: a clinicopathological study of 58 cases.

Primary non Hodgkin's lymphoma (NHL) of the gastrointestinal tract (GI) is the most frequent extranodal lymphoma accounting for approximately 40% of all extranodal primary NHL. The role of surgery and other treatment modalities in the management of these patients is still controversial. We reviewed the records of 68 patients with primary GI-NHL. Ten patients had incomplete records and were excluded from further evaluation. The records of 58 patients were considered, and all were available for analysis and follow-up. The most frequent site of involvement was the stomach (47 patients), followed by ileum (7 patients), large bowel (3 patients) and duodenum (1 patient). Malignant lymphomas of follicular center cell origin represented the most prevalent histologic types, accounting for 58% (34 of 58) of all cases. Stage, evaluated according to the criteria of Musshoff, was Ie in 15 cases, IIe in 16, IIIe in 7, and IV in the remaining 20 cases. The median survival for the entire group of 58 patients was 54 months, with 46% of patients surviving at 5 years. The median survival was 71 months for patients in stage I-II, 60 for patients in stage III, and 25 for patients in stage IV (p = 0.016). Moreover, we found significantly improved survival in patients undergoing surgical tumor resection (p = 0.003). Even if at the present time the optimal management of primary GI-NHL is difficult to assess, our data suggest that it is prudent to advise resection followed by adjuvant CT in most patients, whereas CT alone should be considered only when surgery cannot be performed.


[Cochlear implantation].

Since the Israeli cochlear implant program was started in June 1989, 74 candidates have been evaluated for implantation. The main causes for hearing loss were meningitis (16 patients), aminoglycoside toxicity (9), congenital (6), viral (e.g. mumps) (6), and middle ear infection. Candidates were rejected mainly due to psychosocial factors (40%) and audiological parameters (30%). 16 postlingual deaf were implanted. In 7 of them (43.7%) the results were excellent, in 4 (25.0%) good, in 3 (18.7%) fair; 1 case is currently in a training program and 1 is a failure. There were 2 major complications: facial nerve palsy which necessitated surgical decompression, and insertion of the electrodes into the hypotympanic air cells, corrected by reoperation.


An unusually prolonged case of heparin-induced thrombocytopenia and disseminated intravascular coagulation.

An unusually prolonged case of heparin-induced severe thrombocytopenia and decompensated disseminated intravascular coagulation (DIC) is described. The patient, treated with heparin at a dosage of 25,000 units/day for 3 days and 12,500 units/day for an additional 4 days because of a clinically suspected deep venous thrombosis, developed (4 days after the discontinuation of heparin) a clinical and laboratory picture of severe DIC, manifested by subcutaneous hematomas and ecchymoses. Platelet count was 24 x 10(9)/l, fibrinogen level 89 mg/dl and fibrin-degradation products between 3,200 to 6,400 ng/ml. A thorough laboratory and instrumental evaluation failed to demonstrate any underlying disorder. No heparin-dependent aggregating factor was detected in the serum of the patient. The patient recovered spontaneously. Whereas fibrinogen and fibrin-degradation products reverted to normality within four weeks, platelet count normalization was delayed until the sixth week after heparin discontinuation.


B-cell acute lymphoblastic leukemia (B-ALL): a report of 17 pediatric cases.

B-cell acute lymphoblastic leukemia (B-ALL) presents FAB L3 morphology and surface Ig, CD19, CD20, CD24. This pattern may show some morphological and immunological heterogeneity. Seventeen pediatric cases of B-ALL at onset, treated in twelve AIEOP Centers (Italian Association for Pediatric Hematology and Oncology), are presented. Clinical and hematological features were characterized by low WBC counts at presentation, high M/F ratio, older age and association with extramedullary involvement. The overall survival curve at 78 months is 40%. All patients showed blasts positive for surface Ig (sIg), DR, CD19, and CD24. Ten/17 cases presented the classical features of B-ALL: FAB L3 morphology, sIg+ restricted to light chains, CD20+, cytoplasm mu (c mu)-, CD10-, TdT-. The remainder showed some differences in this pattern, such as non-L3 morphology (3 cases), absence of CD20 (3 cases), CD10+ (4 cases), TdT+ (3 cases), c mu+ (1 case), lack of surface light chains (1 case). This rare ALL subset seems to be characterized by a high phenotype heterogeneity, indicating various degrees of differentiation.


[Psychological aspects of cochlear implantation].

Psychological aspects of cochlear implantation were studied in 20 adults and children. Patients were interviewed before admission and were given tests and questionnaires to answer with regard to overall wellbeing, intellectual functioning, hopes and beliefs, expectations with regard to the implant and the support they were getting (last 2 also for a family member). Those who passed the screening were referred for surgery, and those who didn't were referred for therapy. 6 months after implantation the patient and the family member were again interviewed and answered questionnaires with regard to adjustment to the implant and the patient's satisfaction with it. In those who benefitted from the implant there were no psychopathological features, there was good cognitive functioning, a moderate level of concentration and attention span and a great degree of flexibility in personality traits. Crucial to better adjustment was quality of family support. Following cochlear implantation there was considerable improvement in life style.


Chronic lymphocytic leukemia coexisting with chronic myelomonocytic leukemia.

A case with coexisting chronic lymphocytic leukemia (CLL) and chronic myelomonocytic leukemia (CMML) is described. A 74-year-old man with a typical B-CLL also showed sustained peripheral blood and bone marrow monocytosis. Typical myelodysplastic changes and monosomy 7 were also found. Cytographic and immunophenotypic analysis confirmed the presence of two distinct cell populations, i.e., lymphoid and monocytoid. Both malignancies presented an extraordinarily benign prognosis. It remains uncertain whether monocytosis was either the expression of a distinct myelomonocytic clone or the progeny of a B/monocytic bipotential precursor able to feed both leukemic phenotypes.


[Psychosocial characteristics of phenylketonuric women: birth defect prevention].

Women with phenylketonuria (PKU) are at high risk for having offspring with mental retardation, microcephaly, heart defects and low birth weight. These adverse outcomes can be prevented by a low-phenylalanine diet started before conception and continued throughout pregnancy. In view of the frequency of poor dietary compliance in women with PKU, a psychosocial model was developed that delineates developmental stages with specific behavioral goals for them to follow. In the present study 15 women with PKU over the age of 16 were followed for 3 years and compared to groups of their healthy acquaintances and of diabetic women. Structured interviews and standard questionnaires were used to study factors hypothesized as being related to the subjects' adjustment and to achievement of their PKU-related behavioral goals. After 1 year most of the PKU subjects were not planning a pregnancy, making their main behavioral goal the prevention of an unplanned pregnancy. Their knowledge of the risks of maternal PKU and family planning was unsatisfactory. PKU subjects had more conservative attitudes about sex and contraception than the controls. The psychosocial profile of PKU subjects pinpointed their special needs and indicated the kinds of specific intervention that might help them adhere to the recommended treatment and prevent birth defects in their offspring.


Prolymphocytic leukemia: a very satisfactory response to treatment with recombinant interferon alpha.

We report the case of a patient with prolymphocytic leukemia in whom a lengthy survival of 5 years was observed after treatment with splenic irradiation and chemotherapy. The patient obtained a surprising improvement with a significant reduction in the absolute count of the prolymphocytes and considerable reduction in splenomegaly after 3 months' therapy with interferon alpha.


Antithrombin III biological activity and emotional stress in patients with coronary artery disease.

We studied the effect of emotional stress (mental arithmetic for 10 minutes) in ten postinfarction patients and in ten age-matched, apparently healthy subjects as controls. Blood samples for the determination of epinephrine and AT III levels were taken in basal conditions, at the end of mental stress and after 30 minutes of recovery. Mental stress induced a significant increase in epinephrine levels and a significant decrease in AT III levels in control subjects. Both parameters returned to baseline values after 30 minutes of recovery. On the contrary, in postinfarction patients AT III levels of recovery were still significantly lower than those of baseline, suggesting a reduced ability to restore the original concentration of this physiologic inhibitor. Our data can contribute to a better understanding of the complex relationships among phychosocial factors, the haemostatic system and vascular disease.


[Physical training in coronary patients with poor left ventricular function].

Physical training is beneficial in patients with coronary artery disease, and in those after myocardial infarction in whom left ventricular function and ejection fraction (LVEF) are relatively well preserved. However, little is known about the effect of physical training in coronary patients with impaired left ventricular function. 12 patients after myocardial infarction (range 42-60 years) with LVEF of 30% or less at rest were evaluated after 11 months of isotonic physical training. Mean LVEF increased from 28.0% to 33.0% (p less than 0.002) at rest and from 29.4% to 33.2% (p less than 0.002) on effort. Exercise tolerance increased 228 seconds by the end of the study (p less than 0.001); the double product at symptom limit effort increased (p less than 0.001) and the double product at the same work load decreased (p less than 0.002). During training there was no cardiovascular deterioration and all patients returned to work within a mean of 3.7 months. The results show that patients with severe left ventricular impairment should not necessarily be excluded from cardiac rehabilitation programs involving physical training.


[Calcific periarthritis in patients with endstage renal disease on chronic dialysis].

2 patients with end-stage renal disease undergoing dialysis developed calcific periarthritis. A 25-year-old man on hemodialysis developed arthritis of 2 right metacarpophalangeal joints and a 65-year-old man on chronic ambulatory peritoneal dialysis suffered from pain and tenderness in the left buttock. Treatment with nonsteroidal antiinflammatory drugs in the first case and periarticular injection of methylprednisolone (Depomedrol) in the second were successful.


[Complications of laparoscopic cholecystectomy].

92% of our first 60 laparoscopic cholecystectomies were successful. Postoperative complications included fever in 10 cases (17%), urinary retention in 5 (8%), intraabdominal abscess in 2 (3%), biliary leakage in 1 (2%) and unexplained abdominal pain in 10 (17%). Although laparoscopic cholecystectomy has obvious advantages, it also has major as well as minor complications.


[Echinococcus cyst of the breast imitating carcinoma].

A 3 x 4 cm breast mass in a 38-year-old woman was surgically removed and was found to be an echinococcal cyst; thorough investigation failed to reveal any other hydatid cysts. Echinococcal cyst should be considered in the differential diagnosis of the much more common malignant breast mass.


[Acute dopamine deficiency and malignant neuroleptic-like syndrome].

A 60-year-old woman, under treatment for parkinsonism with dopicar, was hospitalized in a stuporous catatonic state. After several days, while dopicar was being tapered off, she developed a clinical picture similar to malignant neuroleptic-like syndrome, including continuous high fever, elevated blood pressure and increased perspiration, and the parkinsonian symptoms became more severe. Neuroleptic medication was not used, but renewal of dopicar treatment relieved this typical syndrome. It is suggested that a relative deficiency of dopamine is involved in the pathogenesis of malignant neuroleptic-like syndromes.


[Bloody nipple discharge in an infant].

A milky discharge from the nipple and breast hypertrophy are often seen in mature infants, but bloody nipple discharge is very rare in infancy and childhood. In adults, a bloody discharge may be associated with breast carcinoma, but in infants it is a benign, self-limited condition that should be managed conservatively. Surgical procedures should be avoided, because injury to the breast bud may cause permanent damage. We report a 3-month-old girl who presented with a bloody discharge from the left nipple. The discharge diminished gradually in the course of time and ceased completely at the age of 9 months.


[Occupational scleroderma due to organic solvent exposure].

Progressive systemic sclerosis (PSS; scleroderma) is a multisystem disease characterized by inflammation, fibrosis and degeneration of the integument, with similar changes and vascular lesions in the heart, lungs, kidneys, gastrointestinal tract and synovia. Its etiology is not clear. Several occupational exposures have been implicated as potential causes of PSS and scleroderma-like diseases. Among them are vinyl chloride monomer, silica dust, epoxy resin, and benzene and other solvents, aromatic and aliphatic, specifically chlorinated (trichloroethylene, perchloroethylene and trichloromethane). We present a patient whose illness was diagnosed as occupationally induced PSS. During 13 years of work renovating carburetors he was heavily exposed to trichloromethane. To the best of our knowledge this is the first reported case of PSS due to exposure to organic solvents in Israel; very few cases have been reported from abroad.


[Peripheral serotonin receptors: respiratory and circulatory modulation].

The wide distribution of serotonin (5-HT) receptors and 5-HT-mediated effects suggest that 5-HT has important physiological and/or pathophysiological roles in the peripheral nervous system. Recent studies about the localization and release of 5-HT in the peripheral nervous system suggest that 5-HT likely plays a functional role in regulating discharge of autonomic neurons and modulating sensory afferent neurons. The electrophysiological approach has the merit of measuring a direct effect of 5-HT on its receptor. In this review, much of our described work focuses on effects mediated via 5-HT3-receptors on respiration and circulation using electrophysiological techniques, and we consider whether these actions of 5-HT have physiological and/or pathophysiological significance.


[Molecular pharmacology of endothelin in the cardiovascular system].

Endothelin-1 (ET-1), ET-2 and ET-3 are 21-amino acid peptides that act to stimulate contraction of many smooth muscle tissues including blood vessels, uterus, bladder, and intestine. In the case of each ET, the 21-amino acid bioactive form is produced from each precursor (referred to as big ET) via specific conversion between Trp-Val (ET-1, ET-2) or Trp-Ile (ET-3). Northern blot analysis of the mRNA for the ET isoform as well as determination of the peptide itself revealed that they are independently expressed in various tissues, indicating that each isoform may play separate physiological or pathophysiological roles. However, the vascular endothelial cells appear to produce only ET-1. Since ET-1 was first discovered, the majority of investigations so far reported have been concerned with this particular peptide. This review will focus on ET-1 with respect to the regulation of its biosynthesis in endothelial cells and its precise pharmacological actions on the cardiovascular system including the cerebral, coronary and renal circulations. Sub-classification of ET receptors was made from molecular biological, biochemical as well as pharmacological points of view. The signal transduction for mechanisms of action was also explained in detail.


[The recent development and the present status of K+ channel opener].

The development of vasodilator drugs that open the K+ channels in blood vessels has been of great academic and practical interest. The discoveries of the ATP-sensitive K+ channel and the glibenclamide-sensitive K+ channel have promoted these interests. In relation to this channel, the cardioprotective effectiveness of a K+ channel opener (Aprikalim) in doses that did not change haemodynamics or collateral blood flow were demonstrated in infarct dog heart. The effects were antagonized by glibenclamide. Thus, ATP-sensitive K+ channels seem to play an important role in this effect. Clinical evaluations of the K+ channel openers are reviewed. The hypotensive effects of the drugs are well-recognized. At present, however, the clinical usefulness of K+ channel openers has not been accepted widely, because of their side-effects including reflex tachycardia, edema, flushing and headache. An approach to reduce these side-effects is critical if these K+ channel openers are to be used as good hypotensive drugs. The K+ channel opener nicorandil has been evaluated as a highly effective antianginal drug. It seems likely that the clinical benefits of nicorandil result from both its K+ channel opening properties and its ability to stimulate smooth muscle guanylate cyclase. Clinical data on the pure-selective K+ channel opener cromakalim (lemakalim) as an antianginal drug is limited. However, on the basis of the vasodilator profile of this drug, it is expected to be useful for this purpose. The application of K+ channel openers to treat other disorders such as bladder instability is limited because of its hypotensive action.


[Molecular pharmacology of intracellular Ca2+ messenger system in cardiovascular function: development and application of selective inhibitors].

The importance of Ca2+ for the transmission of information inside living cells is well-recognized. The mobilized Ca2+ binds to the intracellular Ca2+ binding proteins to transmit the Ca2+ signal. Calmodulin is the most important Ca2+ binding protein to exert pleiotropic effects on various cellular functions by activating multiple enzymes. To determine the physiological significance of the Ca2+ messenger system, specific inhibitors are quite essential. We have been originally developing novel and selective inhibitors, and analyzing the several processes involved in information flow in the Ca(2+)-dependent regulatory system of cell functions. The aim of this review is to introduce our research strategy for the production of selective inhibitors towards calmodulin-dependent enzymes and to discuss recent progress in the understanding of the multiple calmodulin-dependent pathways that mediate the action of exogenous physiological stimuli. We summarize much of the pharmacological evidence that has led to our current knowledge of calmodulin-regulated cell functions, with emphasis on aspects that may be relevant to drug design. These H-series inhibitors are one of the most powerful tools as molecular probes for pharmacological approaches, and will shed light on the physiological significance and molecular mechanisms of the calmodulin-dependent pathways in various cell functions.


[Central effect of Ca2+ channel blockers: multiple sites of action].

To develop a new concept of central acting drugs, the modulation of brain Ca2+ flux must be considered as one of the important factors. This is because excessive Ca2+ influx to neuronal cells damages or kills these cells, and also because abnormal intracellular Ca2+ concentrations induce several types of mental disorders. Recently, both pre-clinical and clinical studies indicated that some Ca2+ channel blockers (Ca antagonists) will be useful for the treatment of grand mal, manic depressive insanity, panic disorder and anxiety. Furthermore, it has been estimated by animal studies and clinical pharmacology that ischemia-induced neuronal death can be prevented by the treatment with a Ca antagonist. However, the latter data, especially, has been mainly explained by pharmacological effects on the cerebrovascular system, not because of possible direct central actions. To invoke the notion of direct central action, it must be assumed that Ca antagonists might pass the blood-brain barrier (BBB). This potentiality that some Ca antagonists (i.e., flunarizine, nicardipine, nimodipine, etc.) can pass the BBB has been initially explored. If substantiated, such direct central effects of Ca antagonists may explain both the psychotropic effects and neuronal protection by these agents. To investigate the actual therapeutic effects of Ca2+ antagonists on psychotropic disorders and neuronal death, a suitable animal model and reasonable methods and criteria must be established. Then, both preclinical and clinical studies can be expected to relate to atypical central acting drugs modulating the brain Ca2+ channels, and also to the development of new pharmacological properties of Ca2+ antagonists.


[Novel types of cardiotonic drugs].

Since the use of cardiac glycosides for heart failure therapy is limited by their narrow margin of safety, numerous efforts have been made to find and develop novel cardiotonic agents that are superior to the cardiotonic glycosides. Positive inotropic drugs acting on beta-adrenoceptors and inhibitors of cAMP phosphodiesterase have been extensively studied for the treatment of patients with heart failure. The main mechanism of these agents is elevation of cAMP tissue levels. Furthermore, Ca sensitizers such as sulmazol, pimobendan, MCI-154, END 53998 and DPI 201-106 are of interest, since such a mechanism of action may be beneficial for the failing heart. Recently, cardiotonic substances with a novel mechanism of action such as gingerol and xestoquinone have been isolated from natural sources. Natural products, purealin, goniodomin and okadaic acid, have proven to be valuable pharmacological tools for studies on cardiac muscle contraction.


[Thrombus models and mechanisms of action of anti-platelet drugs].

Thrombus formation is initiated by platelet aggregation, followed by activation of the blood coagulation system, so that anti-thrombotic drugs can be classified into anti-platelet drugs, anti-coagulation drugs and fibrinolytic drugs. A variety of thrombus models in animals have been reported. Microthrombus formation in arterioles in the microcirculation is useful for analyzing the nature of the thrombus and the process of thrombus formation. Platelet aggregation can be divided into two types: (a) reversible aggregation (Rev-Aggr), in which platelets do not release their granular contents and are able to return to the resting discoid shape and (b) irreversible aggregation (Irrev-Aggr), in which platelets release their granular contents. Irrev-Aggr is inhibited by aspirin and not by PGI2 at the maximum aggregation, whereas Rev-Aggr is not inhibited by aspirin and inhibited by PGI2 even at the maximum aggregation. Thrombi corresponding to either type of platelet aggregation can be produced in arterioles in the microcirculation. An ADP-induced thrombus, which is composed of Rev-Aggr of platelets and disaggregated automatically, is not inhibited by indomethacin, but inhibited by PGI2. In contrast, a stable thrombus, which is composed of Irrev-Aggr of platelets and keeps its initial shape for a longer period, is sensitive to indomethacin, but resistant to PGI2. Thus, anti-thrombotic drugs should be developed and used for targeting the individual processes of thrombus formation.


[Studies on anti-nephritic effects of plant components (5). Effects of pherodendrin on original and crescentic-type anti-GBM nephritis in rats].

Effects of pherodendrin (OB-5) on anti-GBM nephritis were investigated. OB-5 (50 mg/kg/day, i.p.) prevented the urinary protein excretion in original and crescentic-type anti-GBM nephritis in rats. In addition, OB-5 also inhibited the elevation of serum creatinine, urea nitrogen and cholesterol contents in both models of nephritis. Histopathological observations indicated that OB-5 prevented the hypercellularity, crescent formation, adhesion and fibrinoid necrosis in the glomeruli of nephritic rats. OB-5 and cyclosporine A, a positive control drug, prevented the increase in the number of OX-1, CD8 and ED-1 positive cells in the glomeruli. These results indicated that OB-5 may be effective in human glomerulonephritis, and anti-nephritic mechanisms of OB-5 may be due to its inhibition of the activation of macrophages or cytotoxic T cells.


[Effects of the new cardiotonic agent loprinone hydrochloride (E-1020) on left ventricular diameter in normal and experimental heart failure dogs and its action potential characteristics in isolated guinea pig cardiac muscles and sinus nodes].

In anesthetized dogs, loprinone hydrochloride (LP, 10 approximately 100 micrograms/kg, i.v.) dose-relatedly increased cardiac contractility (LV dP/dt max), enhanced cardiac index and decreased systemic vascular resistance with relatively small reduction in mean aortic pressure (MAP) and a mild increase in heart rate (HR). LP also decreased left ventricular internal diameter (ID), increased fractional shortening (LVFS) and caused a leftward shift and an increase in the slope of the LV end-systolic pressure-ID relation. In the heart failure models induced by propranolol, LP rapidly reversed the cardiac depression and the enlargement in cardiac size. The extent of changes in MAP and HR by LP was smaller than those observed with several other cardiotonic agents. In guinea pig cardiac muscles, LP (10(-6)-10(-4) M) produced a concentration-related increase in contractile force and the maximum rate of rise of Ca(2+)-action potential. In the sinus nodes, LP induced a concentration-related decrease in action potential duration and increase in the slope of slow diastolic depolarization, resulting in an increase in beating rate. These influences of LP on sinus nodes were smaller than those of milrinone. These results indicate that LP increases LVFS, reduces cardiac size and improves the heart failure due to its cardiotonic and vasodilating properties. Moreover, electrophysiological studies suggest that at least a part of the lower chronotropic effects of LP may be due to the mildness of its direct stimulating effects on sinus nodes.


[Effects of KW-6055, a novel benzylpyridine derivative, on central cholinergic systems].

We examined the effect of KW-6055 [alpha-(p-butyrylamino-o-nitrobenzyl) pyridine], which has anti-amnesic activity, on the central cholinergic systems of rat frontal cortex using in vivo brain microdialysis. 1) KW-6055 (40, 160 mg/kg, p.o.) increased the extracellular level of ACh in normal rats (257 +/- 23, 202 +/- 24%). The stimulating effect of KW-6055 on ACh release was abolished by tetrodotoxin treatment, supporting that the released ACh was due to neuronal firing. Reserpine pretreatment decreased the effect of KW-6055, indicating that KW-6055 acted on cholinergic neurons through catecholaminergic neurons. 2) In basal forebrain-lesioned rats, KW-6055 (40 mg/kg, p.o.) significantly increased the extracellular level of ACh (251 +/- 22%) for more than 2 hr, which was longer than in normal rats. In conclusion, these results suggest that the stimulating activity on ACh release may be involved in the mechanism of the anti-amnesic effects of KW-6055.


[Study on the kinetics of bradykinin level in the wound produced by thermal injury in the ear burn model in mice].

The kinetics of bradykinin derived from localized thermal injury on vascular-permeability were investigated in mice with experimental ear burn. The leakage of plasma into the ear tissue by accelerated vascular-permeability reached the maximum at 3 hr and decreased gradually until 6 hr after the thermal injury. The contents of bradykinin increased by the thermal injury and the increment continued up to 24 hr. Although the bradykinin contents in the burned ear decreased by using a protease inhibitor, the hyperpermeability of injured ear vessels was unchanged by protease inhibitors. These results suggest that bradykinin is concerned with the pain reaction rather than vascular-permeability.


Chromosomal rearrangements and speciation of sportive lemurs (Lepilemur species).

Theoretical configurations of meiotic chromosomes of potential hybrids between the different Lepilemur species were examined, and the classification of this genus was reviewed in the light of this information. Among the chromosomal rearrangements that occurred during the chromosomal evolution of the sportive lemurs, only those which would generate a pronounced reproductive barrier were considered in relation to the geographic distribution of this genus. The analysis showed that the pattern of geographic distribution is compatible with the inferred chronological occurrence of these chromosomal rearrangements in the phylogenetic tree of the genus Lepilemur.


The size of the neocortex in relation to ecology and social structure in monkeys and apes.

In an attempt to reveal factors associated with neocortical development in monkeys and apes (anthropoids), relationships between the relative size of the neocortex and differences in ecology and social structure were examined for 24 genera of 11 subfamilies. Relative sizes of the neocortex (RSNs) in a given group were assessed as the difference between actual neocortical volume and the volume expected from an allometric relationship between neocortical volume and the volume of the rest of the brain. We found that RSNs are related to diet and social structure: frugivorous anthropoids had higher values of RSNs than folivorous anthropoids, and polygynous anthropoids had significantly higher values of RSNs than monogynous anthropoids. Furthermore, RSNs were positively correlated with the size of the troop. These results suggest that development of the neocortex is associated with both diet and social structure in anthropoids.