Expression of the human ADH2 gene: an unusual Sp1-binding site in the promoter of a gene expressed at high levels in liver. The sequence 5'-GTGGGTGTGGC (G3T) is important for the efficient initiation of transcription from the human ADH2 promoter. We show here that the purified transcription factor Sp1 binds with high affinity to the G3T site of ADH2 (encoding beta beta-alcohol dehydrogenase), even though the G3T sequence does not contain the canonical Sp1-binding site, GGGCGG. Proteins from mouse liver nuclei and purified Sp1 both footprint the same sequence of the ADH2 promoter with similar patterns. UV crosslinking demonstrates that the major G3T-binding protein in the liver extract is similar in size to Sp1. Mouse liver nuclear extract resembles purified Sp1 in its relative binding affinity to a series of oligodeoxyribonucleotides containing either the Sp1-binding site or variants of the G3T sequence. These data indicate that the G3T sequence can interact with Sp1 and that Sp1 may be important in the expression of ADH2. The G3T sequence from the closely related ADH3 gene (encoding gamma gamma-alcohol dehydrogenase) differs from that of ADH2 in the first two nucleotides; it binds both the liver protein and purified Sp1 with lower affinity. This might explain why ADH3 is expressed at lower levels than ADH2 in the liver.


Characterization of a gene that encodes a homologue of protein kinase in Arabidopsis thaliana. Cloning and analysis of a gene that encodes a homologue of protein kinase (PK) from Arabidopsis thaliana is reported. Oligodeoxyribonucleotides (oligos) corresponding to conserved regions in catalytic domains of various PKs were used for polymerase chain reaction (PCR) amplification with genomic DNA from A. thaliana as template, in an attempt to identify genes encoding PK in plants. We obtained several amplified DNA fragments that encoded part of a PK. We screened a genomic DNA library of A. thaliana with these oligos or PCR fragments as probes. Three genomic clones were obtained and one of them, named Atpk7, was sequenced and analyzed. Atpk7 was demonstrated by PCR to contain an intron. The mRNA transcribed from Atpk7 was detected predominantly in root tissue by Northern blot analysis. The transcription start point was determined by primer extension. The deduced amino acid (aa) sequence of the putative product of Atpk7 resembles those of S6 kinases, cyclic nucleotide-dependent PKs and calcium-dependent PKs. From this comparison of aa sequences, the ATPK7 protein is considered to be a member of a novel subfamily of Ser/Thr PKs in plants.


Cloning and characterization of cDNAs coding for heavy and light chains of a monoclonal antibody specific for pre-S2 antigen of hepatitis B virus. Binding specificity of a monoclonal antibody (mAb) (kappa, gamma 2b) H8 which can react with the pre-S2 peptide of hepatitis B virus (HBV) was determined by Western blot analyses. From the hybridoma cell line secreting mAb H8, poly(A)+ RNA was prepared and used as a template for cDNA synthesis and cloning. Full-length cDNAs coding for the heavy and kappa light chains of the mAb were cloned from the cDNA library and characterized by nucleotide (nt) sequence analyses and N-terminal amino acid sequencing. The sequence analyses revealed that both heavy and light chain-specific cDNAs are functional, and the variable regions of the heavy and light chains are members of mouse heavy chain subgroup III(c) and light chain group I, respectively. Comparison of the nt sequences with mouse immunoglobulin genes listed in the GenBank data base show that the cDNAs have not been previously reported. The cDNAs will be used for the construction of a therapeutic antibody for HBV infection.


Cloning and sequencing of the cDNA encoding tyrosinase of the Japanese pond frog, Rana nigromaculata. We cloned and sequenced the cDNA encoding tyrosinase (TYN) of the Japanese pond frog, Rana nigromaculata. The 3511-bp cDNA contained a 54-bp 5'-noncoding region, a 1596-bp open reading frame encoding TYN of 532 amino acids (aa), and a 1861-bp 3'-noncoding region. The aa sequence of frog TYN predicted from the cDNA sequence was homologous to that of mouse and human TYNs. The aa sequence including the copper-binding domain, which is likely the active center of TYN, was highly conserved among these three species and Neurospora crassa, Streptomyces antibioticus, and S. glaucescens. The frog TYN also contains possible glycosylation sites and conserved Cys at sites similar to those in the mouse and human TYNs. There are two hydrophobic regions at the N-terminus and near the C-terminus, which are likely the signal (leader) peptide and a transmembrane domain, respectively.


The use of a wild-type dihydrofolate reductase-encoding cDNA as a dominant selectable marker and induction of expression by methotrexate. The cDNA (DHFR) encoding the wild-type (wt) dihydrofolate reductase (DHFR) was used as a dominant selectable marker in the transfection of murine hybridoma Sp2/0-Ag14 cells by protoplast fusion. The initial clones contained 100-400 copies of integrated plasmid DNA, and the high level of wt DHFR protein produced enabled the cells to survive the drug selection at 100 nM methotrexate (MTX). The expression of the gene of interest was several fold higher than when the mutant DHFR with decreased MTX binding was used as the selection marker, presumably because the clones were more sensitive to the stress induced by MTX. When the clones were propagated at higher concentrations of MTX, expression of both DHFR and the gene of interest increased. This induction is freely reversible, and we have shown that it is controlled at the transcriptional level, by nuclear run-off transcription assays.


A rat brain-derived neurotrophic factor-encoding gene generates multiple transcripts through alternative use of 5' exons and polyadenylation sites. As a first step toward clarification of the transcriptional controls of the gene encoding brain-derived neurotrophic factor (BDNF), we cloned and sequenced a rat genomic DNA fragment carrying this gene. RNA blotting analysis using a probe derived from the 3'-flanking region of BDNF revealed that alternative use of 3'-polyadenylation sites generates at least two BDNF transcripts that differ in the size of the 3'-noncoding region. Furthermore, sequence analysis of the 5'-end of the BDNF cDNA revealed the presence of at least six different types of transcripts which were probably derived through alternative use of the multiple 5'-exons. Therefore, a single BDNF gene could produce multiple types of transcripts with different noncoding sequences through alternative use of both 5'-exons and 3'-transcription termination sites.


The human ribosomal protein S6 gene: isolation, primary structure and location in chromosome 9. Using PCR cloning we isolated the first intron of the human ribosomal protein S6 gene (hRPS6). By screening the human HeLa cell cDNA library in lambda ZAPII vector (Stratagene, La Jolla, CA), we identified and sequenced a partially spliced pre mRNA copy of hRPS6. The complete hRPS6 gene was isolated from a lambda DASH library with an intron-specific probe. The gene and flanking regions were sequenced, and the mRNA 5' end was mapped by primer extension experiments. The hRPS6 gene has 6 exons and 5 introns and is 3.6 kb long. Using intron-specific primers in PCR and a panel of human-hamster cell lines we localized the hRPS6 gene in human chromosome 9.


Cloning and sequencing of a human cDNA coding for dihydroorotate dehydrogenase by complementation of the corresponding yeast mutant. Dihydroorotate dehydrogenase (DHOdehase, EC 1.3.3.1) catalyses the fourth enzymatic step in de novo pyrimidine biosynthesis. A truncated human cDNA encoding this enzyme was isolated from a HeLa cell cDNA library by functional complementation of a corresponding deletion mutant from the yeast, Saccharomyces cerevisiae. The complementing clone contained a 1.5-kb poly(A)(+)-tailed insert with a 1191-bp open reading frame, hybridising with a unique human mRNA of 1.6 kb. The deduced amino acid sequence has 54%, 46% and 42% identity with Arabidopsis thaliana, Schizosaccharomyces pombe and Escherichia coli DHOdehases, respectively. In contrast, it has only 21% identity with the S. cerevisiae enzyme, which probably reflects the cytosolic location of the enzyme in the latter organism.


COPD: primary care management with drug and oxygen therapies. Chronic obstructive pulmonary disease related in most cases to extensive cigarette smoking is a leading cause of morbidity and mortality among patients age 55 and older. Smoking cessation, inhaled anticholinergic therapy, and oxygen supplementation (when indicated) are primary treatment modalities. Nicotine administration via a transdermal system or polacrilex is the pharmacologic method of choice for smoking cessation. Concomitant group psychotherapy increases the likelihood of success. Beta agonists, mucolytics, and antibiotics can be useful in selected subgroups. Steroids and theophylline are controversial in COPD and should be used with caution. Prognostic indicators, such as degree of reversible airway disease, aid in long-term care decisions.


Controversies in the management of cerebrovascular disease in older patients. Several ongoing studies are evaluating the optimal management of patients with cerebrovascular disease. The Carotid Artery Stenosis with Asymptomatic Narrowing: Operation Versus Aspirin (CASANOVA) study has shown that carotid endarterectomy is not recommended for asymptomatic patients with less than 90% carotid stenosis. The North American Symptomatic Carotid Endarterectomy Trial (NASCET) and the European Carotid Surgical Trial (ECST) have demonstrated that endarterectomy should be considered for patients who had recent carotid artery territory ischemic symptoms associated with angiographically defined stenosis of greater than 70%. These and other trials are expected to provide further data regarding management of cerebrovascular disease, including treatment of those patients with moderate (30 to 69%) carotid stenosis. Until that time, treatment decisions must be made on a case-to-case basis.


Healthier lifestyles: how to motivate older patients to change. A 1- to 3-minute clear discussion by the family physician of the risks of unhealthy lifestyles has been found to be quite successful in countering patients' denial of personal risk. Studies indicate that the physician's authority lends credibility to the need to make a change and is a strong motivator. An office-based physician counseling model has proven effective in motivating older patients to adopt healthier lifestyles. The model includes four basic steps: patient assessment, discussion of risk and delivery of a message to change, a prescription for change (planning and carrying out of a behavior change strategy), and prevention of relapse through a maintenance program.


Extracellular-superoxide dismutase type C (EC-SOD C) reduces myocardial damage in rats subjected to coronary occlusion and 24 hours of reperfusion. Extracellular-superoxide dismutase type C (EC-SOD C) is a secretory SOD isoenzyme which, in contrast to the intracellular CuZn SOD, has affinity to the endothelium and a long vascular half-life. In the present study, the effects of EC-SOD C and CuZn SOD on reperfusion-induced myocardial damage were determined in rats subjected to 10 min of left coronary artery ligation followed by 24 h of reperfusion. Recombinant human EC-SOD C (rh-EC-SOD C) or the corresponding volume of the vehicle was administered after completion of the coronary ligation. CuZn SOD was given in two equal doses, the first dose directly after ligation and the second one 6 h later. At the end of the reperfusion period the myocardial damage was quantified by measuring the creatine kinase concentration (CK) in the reperfused part of the left ventricular free wall (LVFW), and expressed as a percentage of the concentration in the non-ischemic septum. In the group given the vehicle, 47 +/- 10 (mean +/- SD) of the CK remained in the reperfused LVFW. In the rats receiving rh-EC-SOD C the corresponding values for each dose: 1.4, 4.2 and 12.6 mg/kg were 55 +/- 12 (ns), 55 +/- 12 (ns) and 65 +/- 12% (p less than 0.05, vs. vehicle, Dunnett's multiple comparison test), respectively. Administration of CuZn SOD (2 x 10 mg/kg) resulted in 58 +/- 16% (ns) CK remaining in the LVFW.(ABSTRACT TRUNCATED AT 250 WORDS)


Thioctic acid protects against ischemia-reperfusion injury in the isolated perfused Langendorff heart. Antioxidant properties of thioctic and dihydrolipoic acid have been demonstrated in membranes and low density lipoproteins (LDL) in vitro. In vivo studies with dietary supplementation of thioctic acid to rats showed that it can also protect tissues against oxidative damage. Presumably, this action is due to a thioctic acid dihydrolipoic acid (TA/DHLA) coupled antioxidant mechanism, which enhances the activity of other antioxidants (i.e. ascorbate, alpha-tocopherol) by regenerating them from their radical form. In the present study, thioctic acid proved to protect against ischemia/reperfusion injury to Langendorff perfused hearts. Hearts isolated from rats fed thioctic acid and subjected to ischemia exhibited better mechanical recovery (left ventricular developed pressure) after reperfusion and lower lactate dehydrogenase leakage. Thioctic acid supplementation also decreased the appearance of fluorescent lipid peroxidation products after ischemia/reperfusion, lowered the rate of 2,2'-azobis-(2,4-dimethylvaleronitrile) (AMVN) induced lipid peroxidation in heart homogenates, and prevented the loss of alpha-tocopherol. The total sulfhydryl group content in thioctic acid fed animals was higher and the decrease due to ischemia-reperfusion was not as marked in this group as observed in the control. These results show that dietary supplementation with thioctic acid in vivo provides protection against ischemia/reperfusion injury in the Langendorff heart model.


Trophic effect of gastrin on the enterochromaffin like cells of the rat stomach: establishment of a dose response relationship. Gastrin was given to rats by continuous subcutaneous infusion through implanted osmotic minipumps in doses covering a wide range of the dose response relationship for gastrin with regard to the trophic effect on the enterochromaffin like cells of the oxyntic mucosa. Thirty five rats were divided into five groups (each of seven rats), one group receiving a control solution of 1% albumin, the others receiving gastrin in 1% albumin at doses of 2.5, 5, 10, and 15 micrograms/kg/h, respectively. The plasma gastrin concentrations in the various groups increased in the same order of magnitude as expected from the gastrin doses given. Gastrin induced a dose dependent increase in enterochromaffin like cell density, oxyntic mucosal histamine concentration and histidine decarboxylase activity up to the dose of 5 micrograms/kg/h, where the increase levelled off. Hence, the dose response relationship for the trophic effect of gastrin on the enterochromaffin like cells seems to follow a polynomial rather than a linear function. These findings may also contribute to the understanding of the trophic effect of gastrin on enterochromaffin like cells in man with conditions associated with hypergastrinaemia.


Gastric B-cell mucosa associated lymphoid tissue lymphoma: a clinicopathological study in 56 patients. Clinico-pathological features of 56 patients with primary gastric lymphoma were evaluated retrospectively. All cases were regraded according to a classification of Isaacson et al into high grade and low grade B-cell mucosa associated lymphoid tissue lymphoma. A third group of mixed grade was recognised in 11 patients with low grade who also had occasional areas of high grade. Low grade and mixed grade patients had a 100% actuarial survival at 156 months, which was significantly better (p < 0.01) than that of 52% for patients with high grade disease. Different treatment methods--surgery, chemotherapy, or a combination of both--did not significantly affect survival. Low grade tumours occurred mainly in men with a history of several years, and who presented with non-specific gastric symptoms without remarkable exploratory or laboratory findings: most patients were in stage IE-IIE and achieved remission and cure. High grade can have a shorter history, systemic symptoms, abnormal exploratory and laboratory findings, gastric tumour masses, stage IV disease, and a worse outcome. The only significant prognostic factors for survival were the type of lymphoma and stage IV disease. These findings support the Isaacson classification system which separates two extreme groups of gastric lymphomas with different morphology, behaviour, and outcome. The presence of limited areas of high grade in a specimen showing low grade does not change the outcome but suggests that primary gastric lymphoma forms a continuum between these extreme types.


Changing pattern of gastric cancer in Oxfordshire. This study compares the incidence rates of histologically confirmed gastric carcinoma in Oxfordshire in two five year periods (1960-64, 1984-88). Data were available for 215 patients in the first period, and 200 in the second. The overall incidence fell from 18/100,000 to 15/100,000 but when analysed for site, the incidence of antral tumours fell from 10 to 4.5/100,000. In contrast, there was an increase from 2.8 to 5.2/100,000 of tumours of the cardia. These changes were more pronounced in men. There was a marked association between smoking and tumours of the cardia (relative risk 4.5). Helicobacter pylori was associated with 37.5% of tumours in the 1960s series compared with 25% in the later series. The changing patterns of incidence of gastric carcinoma may, in part, be related to changes in smoking habits and perhaps a change in incidence of H pylori infection.


Early gastric cancer: 46 cases treated in one surgical department. Forty six consecutive patients with early gastric cancer were treated between 1970 and 1990. The proportion of cases of early gastric cancer increased significantly (p < 0.01) from 1% of all cases in the first five year period to 15% in the last five year period, because of greater awareness of the condition and more widespread use of endoscopy. There were 33 men and 13 women, of median age 69 years (range 38-86). Most patients (91%) presented with symptoms indistinguishable from those of peptic ulceration. The median duration of symptoms was four months (range 0.1-36 months). All 46 patients were treated surgically. Three patients (6.5%) died after operation and a further 10 (22%) suffered postoperative complications. None of the surviving patients has been lost to follow up and 25 have been followed up for a minimum period of five years. Five year survival by life table analysis was 98%. These findings suggest that in Britain in the 1990s, as in Japan, it may be possible to diagnose an increasing proportion of patients with gastric cancer at a relatively early pathological stage, when most patients can be cured by radical surgical resection with lymphadenectomy.


Evidence of clonal variants of Helicobacter pylori in three generations of a duodenal ulcer disease family. Nine members of a family with a high incidence of duodenal ulcer disease were studied by interview, examination of hospital records, endoscopy, and antral biopsy. Helicobacter pylori was confirmed by CLO test, histology and culture. DNA extraction from pure isolates of H pylori was possible in six family members and strain typing was performed by restriction fragment length polymorphism. DNA restriction digestion was followed by vacublotting and then DNA hybridisation, using a cDNA probe complimentary to H pylori rRNA cistrons. Eight of the nine family members were H pylori positive by CLO test and histology. Five had duodenal ulcer disease. Three family members (one from each generation) harboured clonal variants of a single parent strain of H pylori but only two had duodenal disease. The other three members harboured different strains. Intrafamilial clustering of clonal variants of H pylori occurs in some duodenal ulcer disease families. Family members however, may develop duodenal disease irrespective of the colonising strain.


Eighteen month follow up of Helicobacter pylori positive children treated with amoxycillin and tinidazole. Sixty three children with dyspepsia (mean age 12 years, range one to 18, M/F 41/22) were Helicobacter pylori positive by histology of gastric antral biopsy specimens and were treated with a six week course of amoxycillin (50 mg/kg) and tinidazole (20 mg/kg). The endoscopic diagnoses were: normal (16), nodular gastritis (19), oesophagitis (four), duodenal ulcer (13), and gastric ulcer (11). H pylori was eradicated in 54 (87%) and histological gastritis resolved in 51 and was improved in the other three. Repeat investigation was offered at six monthly intervals. Reinfection was found in three of 34 (9%) at six months, in none of 22 at 12 months, and in two of 18 (11%) at 18 months, yielding an 18 month cumulative relapse rate of 20%. Children with persisting infection despite treatment remained positive during follow up. Serum H pylori IgG concentrations fell after treatment (p < 0.001), and for individual children during follow up there was a progressive decline, but an increased concentration indicated recurrence. After eradication of H pylori by combined amoxycillin and tinidazole treatment, only a minority of children relapse during the ensuing 18 months.


Fasting hypochlorhydria with gram positive gastric flora is highly prevalent in healthy old people. Fifteen healthy old people mean age 84 years (range 80-91 years), were examined to assess the effect of advanced age on the microecology of the upper gastrointestinal tract. Twelve of 15 (80%) were hypochlorhydric with pH 6.6 (0.3) (mean (SEM) and a mean bacterial count of 10(8) colony forming units (CFU) per ml (range 10(5)-10(10)) in fasting gastric aspirate. Normochlorhydric subjects had low counts (< or = 10(1) CFU/ml). The microbial flora was dominated by viridans streptococci, coagulase negative staphylococci, and Haemophilus sp. Only one subject harboured significant concentrations of Gram negative bacilli with Escherichia coli (10(4-5) CFU/ml) and Klebsiella (10(4-5)). Strict anaerobes were not found. The total concentration of short chain fatty acids in gastric aspirate was 10.6 (2.9) mmol/l (mean (SEM). Absence of significant, intraluminal fermentation of xylose to CO2 was shown by the 14C-d Xylose breath test, and ambulatory manometry showed preserved fasting motility pattern of the small intestine. Serum immunoglobulins were normal. Advanced age is accompanied by fasting hypochlorhydria and colonisation with mainly Gram positive flora in the upper gut. Other factors than old age and fasting hypochlorhydria are required for colonisation with Gram negative bacilli.


Concentrations of 5-ASA and Ac-5-ASA in human ileocolonic biopsy homogenates after oral 5-ASA preparations. Intramucosal 5-aminosalicylic acid (5-ASA) and acetylated 5-ASA (Ac-5-ASA) concentrations were determined in ileocolonic biopsy specimens from 61 patients with irritable bowel syndrome treated for one week with near equimolar doses of different slow release preparations of 5-ASA (Claversal, Asacol, or Pentasa) or azo-bound drugs (Salazopyrin, Dipentum). The transit time in these patients was accelerated by a laxative, metoclopramide, and colonic lavage. The presence of 5-ASA in the mucosa was confirmed by autofluorescence. The highest concentrations of 5-ASA were obtained after Asacol (mean (SEM), 298.5 (37.3) ng/mg wet wt), followed by Claversal 500 mg (108.8 (11.7) ng/mg wet wt) and Pentasa (25.7 (2.2) ng/mg wet wt). Very low concentrations only were observed after Claversal 250 mg (0.3 (0.03) ng/mg wet wt), Salazopyrine (1.2 (0.1) ng/mg wet wt), and Dipentum (11.0 (3.2) ng/mg wet wt). The results for Ac-5-ASA were similar but the concentrations were generally lower. Serum concentration-time curves over eight hours were obtained from 34 healthy volunteers after a single oral dose of 400 to 500 mg of the different drugs. For the slow release forms, an apparently inverse relationship was found between the area under the curve of the serum concentrations and the intramucosal concentrations, supporting the importance of the local availability of the drug. This inverse relationship was absent for the azo-bound drugs. Colonic washout induced mechanical removal of intraluminal 5-ASA with a secondary disturbance in absorption resulting in a rapid decline in the serum concentrations. However, only for Dipentum did this result in significantly lower 5-ASA mucosal concentrations. This is the first reported attempt to evaluate the mucosal availability of 5-ASA after different oral preparations. It shows that where transit time is accelerated higher mucosal concentrations occur after slow release preparations (except for Claversal 250 mg) than after azo-bound drugs. Additional studies are necessary to correlate these concentrations with clinical effects.


Comparison of 5-aminosalicylic acid and N-acetylaminosalicylic acid uptake by the isolated human colonic epithelial cell. Isolated human colonic epithelial cell suspensions were incubated with either 0.1 mM 5-aminosalicylic acid (5-ASA) or 0.1 mM acetylaminosalicylic acid (Ac-ASA) for up to two hours. Intra- and extracellular 5-ASA and Ac-ASA were measured by high performance liquid chromatography. Mean 5-ASA uptake in one hour was 160.5 nmol/g dry weight, compared with an Ac-ASA uptake of only 5.75 nmol/g dry weight. No unchanged 5-ASA was detected inside the cell. Repeated washing had no effect on the intracellular Ac-ASA concentration. This discrepancy in drug uptake may explain why Ac-ASA seems to be ineffective when given to patients with ulcerative colitis.


Tests of renal function in patients with quiescent colitis: effects of drug treatment. Mesalazine has structural similarities to aspirin and phenacetin and is nephrotoxic when given intravenously in high doses to rats. A number of cases of nephrotoxicity has been reported recently in patients taking oral mesalazine. Sensitive indicators of renal function in a group of patients maintained on long term, delayed release mesalazine and a comparable group on sulphasalazine have been studied. Sixty two patients (32 men, aged 28-82 years) with quiescent colitis were studied. Thirty four had been maintained on delayed release mesalazine 1.6 (0.8-2.4) g/day for 2.9 (0.5-6.9) years and 28 on sulphasalazine 2 (2-3) g/day. Groups were comparable for age, sex, disease duration, and disease extent. Renal function was assessed by: urine microscopy; creatinine clearance; the urinary excretion of two markers of glomerular toxicity, albumin and transferrin; and the urinary excretion for two markers of tubular toxicity, N-acetyl-beta-D-glucosaminidase (NAG) and alpha 1-microglobulin. There were no significant differences in renal function between the two treatment groups. Furthermore, no correlations were found between measures of renal function and either cumulative mesalazine dose or mesalazine treatment duration. In this study, long term maintenance treatment with delayed release mesalazine was no more nephrotoxic than continued treatment with sulphasalazine.


Inhibition of binding of interferon-gamma to its receptor by salicylates used in inflammatory bowel disease. 5-Aminosalicylic acid (5ASA), 4ASA, their N-acetylated metabolites N-acetyl-5ASA and N-acetyl-4ASA, olsalazine, and colchicine impair interferon-gamma (IFN gamma) induced HLA-DR expression on a colonic cell line, HT-29. The mechanism of this effect is now reported. HT-29 cells were cultured with 50 U/ml IFN gamma with or without drug, and northern blot analysis was performed using a probe for the beta chain of the DR molecule. IFN gamma led to a noticeable increase in HLA-DR mRNA which was attenuated by the drugs. Analysis of the specific binding of increasing concentrations of 125I-IFN gamma by non-linear regression showed a Kd of 1.35 x 10(-10) M and 2.3 x 10(5) binding sites per HT-29 cell. Binding of 125I-IFN gamma was reduced by incubation with increasing concentrations of unlabelled IFN gamma but not with IFN alpha. Incubation with therapeutic concentrations of drugs led to the following reductions in binding: 10 mM 5ASA, 20% (p < 0.001); 10 mM N-acetyl-5ASA, 24% (p < 0.01); 10 mM 4ASA, 21% (p < 0.005); 10 mM N-acetyl-4ASA, 29% (p < 0.001); and 1 mM olsalazine, 29% (p < 0.001). Colchicine (10(-7) M) and 10(-5) M prednisolone had no effect. Incubation with higher concentrations of the drugs revealed a dose-response effect on binding with complete inhibition by 100 mM 4ASA and 10 mM olsalazine, and lesser degrees of inhibition by 100 mM 5ASA, N-acetyl-5ASA, and N-acetyl-4ASA. At concentrations found in the rectal lumen, the salicylates used in inflammatory bowel disease impair the binding of IFN gamma to its receptor on colonic epithelial cells.


IgG subclass distribution in serum and rectal mucosa of monozygotic twins with or without inflammatory bowel disease. Serum samples from 26 monozygotic twin pairs concordant or discordant with regard to inflammatory bowel disease, and rectal biopsies from 42 twins of the same subject group, were examined for IgG subclasses. They were all compared with normal controls. Almost all affected twins were in clinical remission. Paired immunofluorescence staining of the rectal mucosa showed that those with ulcerative colitis had a significantly higher (p < 0.01) proportion of IgG1 producing mucosal immunocytes than normal controls (78.1% v 55.9%). Conversely, the IgG2 cell fraction was significantly reduced (15.9% v 34.6%). Healthy twins from ulcerative colitis pairs tended to show a raised proportion of IgG1 cells and the IgG2 cell fraction was significantly reduced (p < 0.05). In discordant ulcerative colitis twin pairs, no difference appeared in the cellular IgG subclass pattern between healthy and affected twins. Furthermore, the proportion of IgG1 in these healthy and diseased twins showed good correlation (T = 0.867). The results in rectal mucosa of twins with Crohn's disease were widely scattered and affected twins did not differ significantly from normal controls. Healthy twins, however, showed a marginally raised IgG1 cell proportion, but no correlation was seen between the IgG subclass fractions in discordant Crohn's disease twin pairs. The serum concentrations of IgG1 and IgG2 did not differ from normal controls in twins of either category. These results suggested that in ulcerative colitis, the aberrant mucosal production of IgG1 and IgG2 does not depend on active disease, but is apparently at least partially explained by a genetic impact. Conversely, the mucosal IgG subclass pattern in Crohn's disease appears to be determined mainly by exogenous variables.


Plasma polyunsaturated fatty acid pattern in active inflammatory bowel disease. Plasma fatty acid patterns were assessed by gas liquid chromatography in 73 patients with active inflammatory bowel disease and 107 healthy controls. The influence of the disease activity on fatty acid profile was also investigated. Plasma fatty acid patterns in patients with ulcerative colitis and Crohn's disease were similar. Plasma C18:3n3 and C22:6n3 were significantly higher in active ulcerative colitis (p = 0.0143 and p < 0.00001 respectively) and in Crohn's disease (p < 0.00001 for both) than in controls, whereas C20:3n6 was significantly lower in patients than in controls, both in ulcerative colitis (p = 0.0001) and in Crohn's disease (p = 0.0041). In more severe disease, plasma polyunsaturated fatty acid concentrations fell with a significant stepwise decrease in the desaturation index (p = 0.0031 in ulcerative colitis and p = 0.0355 in Crohn's disease). Even in patients with severe disease, however, plasma n3 fatty acids (C18:3n3 and C22:6n3) never fell below those of healthy controls. These findings suggest that in active inflammatory bowel disease, an increased biosynthesis might coexist with an increased consumption of polyunsaturated fatty acids. These observations may be of relevance in the pathogenesis of the disease as polyunsaturated fatty acids are involved in tissue eicosanoid synthesis and cellular membrane function, including that of immunocompetent cells. These results also question the rationale of using n3 polyunsaturated fatty acids in the treatment of inflammatory bowel disease.


Prevalence of anti-neutrophil antibody in primary sclerosing cholangitis and ulcerative colitis using an alkaline phosphatase technique. The detection of a nuclear anti-neutrophil antibody in patients with primary sclerosing cholangitis (PSC), using an immunoperoxidase technique, was recently reported by us. Subsequently, detection of a cytoplasmic anti-neutrophil antibody was reported by others, using a two stage procedure of enzyme linked immunosorbent assay followed by an immunofluorescent method. Detection of cytoplasmic anti-neutrophil antibody in PSC, which, in contrast to that two stage procedure, uses a simple one step immuno-alkaline phosphatase method is now reported. Normal human neutrophils were cytocentrifuged, ethanol fixed, and then incubated with coded patients' sera. Rabbit anti-human immunoglobulin conjugated with alkaline phosphatase was used to detect the bound antibody. Fast red was used to visualise the reaction. Twenty three of 30 (77%) PSC patients showed positive granular cytoplasmic staining (with some perinuclear accentuation) with a network of cytoplasmic filaments. Fifteen of 45 (33%) ulcerative colitis patients and 1 of 3 chronic active hepatitis patients showed similar staining. Thirty five of 152 patients with ulcerative colitis, chronic active hepatitis, and a variety of other liver diseases showed a different pattern of cytoplasmic labelling, with no surrounding filaments. Seventy nine patients, including seven PSC patients and 33 normal subjects, were negative. In comparison, 86% of PSC patients, 57% of patients with primary biliary cirrhosis, 50% of normal subjects, and well over 60% of patients with ulcerative colitis, Crohn's disease, alcoholic liver disease, and chronic active hepatitis were positive using the one step immunofluorescent method. This method is more specific for PSC than those described in recent reports and may be of diagnostic importance.


Sclerosing cholangitis and biliary tract calculi--primary or secondary? The clinical features of 61 patients with sclerosing cholangitis were reviewed. This group included 23 patients with biliary tract calculi, commonly considered as excluding the diagnosis of primary sclerosing cholangitis. The aim of this study was to compare these 23 patients (group A) with 38 patients with sclerosing cholangitis free of calculi (group B). Both groups had the following features in common: (i) age at presentation, (ii) incidence of inflammatory bowel disease, (iii) extent of radiological disease, (iv) prevalence of HLA-B8 and DR3 haplotype, (v) incidence of cholangiocarcinoma, and (vi) progression to hepatic transplantation (mean follow up 49.9 months). All patients in group A were symptomatic at diagnosis compared with 23 of the 38 patients (61%) in group B. Recurrent ascending cholangitis occurred in 12 patients in group A (52%) and two patients (5%) in group B. The similarity between the two groups was maintained when the nine patients in group A who developed calculi after sclerosing cholangitis was diagnosed were excluded. It is concluded that choledocholithiasis is part of the spectrum of primary sclerosing cholangitis and that it is not necessary to invoke choledocholithiasis as the initial lesion of the bile ducts in such patients.


Factors related to early mortality in cirrhotic patients bleeding from varices and treated by urgent sclerotherapy. Variceal haemorrhage in cirrhotic patients carries a high early mortality even when balloon tamponade or emergency sclerotherapy are applied. The aim of this study to identify patients dying within six weeks of their first variceal haemorrhage. One hundred and twenty one patients with parenchymal cirrhosis presenting with the first variceal bleeding episode between June 1983 and December 1988 were studied. Nineteen patients were excluded for various reasons. Emergency sclerotherapy was carried out in cases of active bleeding or where there were endoscopic signs of recent bleeding, and then regularly repeated afterwards. Of the 24 variables studied and included in a multivariate analysis using a logistic regression model, three had an independent prognostic value: encephalopathy, prothrombin time, and the number of blood units transfused within the 72 hours of time zero. The subsequent regression equation was able to predict 89% of the patients who will die and 97% of the patients who will still be alive six weeks after their first variceal haemorrhage treated by sclerotherapy. Pugh score was less discriminatory than these last three variables in terms of accuracy of adjustment, goodness of fit to the model, receiver operating characteristic curves, and percentage correct prediction. To measure the accuracy of the prediction rule, our model was applied to another series of 28 cirrhotic patients admitted with their first variceal bleeding during the next period (January 1989 to May 1990). Death and survival were correctly predicted in respectively 82% and 94% of the cases. The use of this score is recommended for the selection of patients with high early mortality after variceal bleeding despite sclerotherapy, and for the design of new therapeutic trials.


Splenic function in alcoholic liver disease. Splenic function was assessed in 42 patients with alcoholic liver disease by counting the percentage of erythrocytes with indentations or pits, seen by differential interference contrast microscopy. These pits represent cellular debris normally removed by the spleen. The findings were compared with 42 age and sex matched controls. Mean (SEM) pitted red cell counts in the patients was 2.7 (0.4)% and in the controls 0.7 (0.07)% (p < 0.001). In all of the eight reformed drinkers (five with biopsy proven cirrhosis), cell counts were normal. Six patients with alcoholic liver disease had had serious infections within the past year. Of these, one had had a recent pneumococcal pneumonia and another of the patients died from overwhelming pneumococcal septicaemia. Both of these patients had evidence of functional hyposplenism as judged by high pitted erythrocyte counts. A total of 18 patients were considered to have pitted red cell counts above the normal, and 11 of these had proven cirrhosis and/or gross ascites. This study is the first to show the presence of functional hyposplenism in alcoholic liver disease and provides further evidence of the predisposition that these patients have to infection. At present, it is unclear whether the hyposplenism is a direct toxic effect of alcohol or the result of cirrhosis; further studies are warranted.


Recurrence of hepatitis B and delta hepatitis after orthotopic liver transplantation. The clinical course of 10 liver transplant recipients who had hepatitis B virus (HBV) and five recipients with HBV and D (delta) infection before transplantation is described. Six patients who underwent eight transplants died. The estimated one and two year survival rates in patients with HBV only before transplantation were 74% and 67% respectively. The estimated one and two year survival in patients with HBV and HDV infection beforehand was 100%. Graft infection by HBV occurred in 8 of 10 patients infected with HBV only; and in 4 of 5 patients with previous HBV and HDV infection. There was a widely variable time from transplantation to the appearance of HBV markers in liver or serum, ranging from 6-331 days. Hepatitis D antigen (HDAg) appeared in three grafts very rapidly after transplantation at 4, 8, and 37 days respectively. Graft infection by HBV was accompanied by significant liver injury in six allografts in five recipients. In particular, there was a striking morphological appearance in five infected livers in which the hepatocytes became progressively enlarged and distorted as they accumulated huge amounts of hepatitis B surface and core antigens (HBsAg, HBcAg). These features were accompanied by pericellular fibrosis and cholestasis but little associated inflammation. This syndrome carried a poor prognosis. A gradual progression to cirrhosis occurred in one additional liver. Finally, recurrent HBV infection was a principal or a contributing factor in all deaths. The presence of HBcAg and inflammation in he native liver increased the risk of HBV induced tissue damaged in the graft whereas HDV infection in the host liver seemed to reduce the risk of significant HBV induced tissue damage in the allograft. These data suggest that post transplant HBV infection is accompanied by a variety of changes in the liver allograft, some of which are unique to the transplanted liver and may result in impaired allograft function.


Liver disease in a district hospital remote from a transplant centre: a study of admissions and deaths. The profile of liver disease admissions and associated deaths in a district general hospital was studied to determine whether patients with end stage liver disease are appropriately referred for consideration of liver transplantation. Admission details were provided by the Office of Population Censuses and Surveys (OPCS) and their accuracy was assessed by case note analysis. According to OPCS, 77 patients with liver disease were admitted on 113 occasions between 1 January 1987 and 31 December 1989. The case notes of 74 (96%) were retrieved and examined. Only 64 (86%) had primary liver disease. Twenty four (31%) died of liver failure. Alcohol was the aetiological agent in two thirds. According to accepted criteria, 11 patients were suitable for liver transplant assessment but only three had been referred to a transplant centre. Of the remaining eight, five died during the study period. Two of the three patients referred died without transplantation; one underwent transplant and survived. There is discrepancy between OPCS data and true disease aetiologies, with approximately 40% under reporting of alcoholic liver disease. If this population is representative of the situation nationally, substantial numbers of patients with end stage liver disease might benefit from liver transplantation, but are not referred to a centre.


Effect of lipid infusion on bile composition and lithogenicity in patients without cholesterol gall stones. A prospective study was performed to investigate the effect of short term lipid infusion on bile composition and its lithogenicity in humans. Thirty five patients shown to be free of cholesterol gall stones participated in the study. Starting 48 hours before surgery they were infused randomly with a lipid emulsion of either long chain triglycerides (LCT) or a mixture of medium and long chain triglycerides (MCT/LCT) (50%/50%) for six hours each 24 hours. A group of patients infused with a solution of 5% glucose in NaCl 0.9% served as a control. Bile samples were obtained by puncture of the gall bladder during operation. Both lipids caused an increase in biliary cholesterol and phospholipids but this effect was more pronounced and significant (p < 0.001) only with the MCT/LCT emulsion. The fatty acid composition of biliary phospholipids was not affected by either lipid infusion. The cholesterol saturation index increased significantly (p < 0.005) with the MCT/LCT emulsion and there was shortening in the nucleation time but this was not significant. There was no effect on the distribution of cholesterol between micelles and vesicles. This study shows that infusion of MCT/LCT lipid emulsion can cause lithogenic changes in bile composition in humans and may thus contribute to sludge formation and cholelithiasis during long term parenteral nutrition.


Increased activity of ionised calcium in gall bladder bile in gall stone disease. The actual activity of ionised calcium (Ca2+) in gall bladder bile determined with an ion-selective electrode was significantly higher in patients with gall stone disease (n = 15) than in patients without gall stones (n = 10) (0.43 mmol/kg v 0.31 mmol/kg; p < 0.05). No change in the Ca2+ activity in any of the gall bladder bile samples was observed during equilibration with CO2. During titration with HCl/NaOH, however, the Ca2+ activity fell with increasing pH in a biphasic manner, with the breaking point occurring at a significantly lower median pH in patients with gall stones than in patients without (pH 7.1 v 8.2; p < 0.0001). The combination of a higher activity of calcium in bile and precipitation of bile salts taking place at a lower pH in patients with gall stone disease than in patients without gall stones suggests a major role for calcium and pH in the pathogenesis of gall stones. Strict anaerobic sampling is not necessary for the measurements of Ca2+ in gall bladder bile, because the Ca2+ was not significantly affected by the changes in pCO2. The metabolic studies suggest, however, that simultaneous measurements of the activity of Ca2+ and pH is important in order to interpret data for the calcium activity in gall bladder bile.


Value of exfoliative cytology for investigating bile duct strictures. The cause of a biliary tract stricture may be difficult to determine radiologically. Exfoliative biliary cytology was evaluated in 62 patients (median age 65 years, range 30-94) with biliary tract strictures presenting to the Hepatobiliary Unit between January 1984 and December 1989. Bile samples were taken during endoscopic retrograde cholangiopancreatography (ERCP) in 42 patients, percutaneous cholangiography in 14, and both in six. The site of stricturing was upper third of the bile duct in 43% (n = 27), middle third in 10% (n = six), and lower third in 47% (n = 29). Of the 47 patients with radiological appearances of a malignant stricture, 22 (47%) had histological confirmation by biopsy either under computed tomography guidance, at endoscopy, at operation, or at necropsy. Fourteen of the 47 patients had positive cytology (30%). In seven patients cytology alone established the presence of malignancy (15%) and in the other seven positive cytology was confirmed by histology. The addition of cytology to tissue biopsy therefore allowed malignancy to be confirmed in 29 of the 47 patients (62%). None of the 15 patients subsequently shown to have benign disease had positive cytology. Sensitivity of the technique was 30% and specificity 100%. Samples for exfoliative cytology are simple to obtain, the results are highly specific and should be a routine part of the investigation of biliary strictures.


Management of common bile duct stones with a biliary endoprosthesis. Report on 40 cases. Endoscopic placement of a biliary endoprosthesis has been proposed for the management of choledocholithiasis when stone extraction is difficult or considered hazardous. Over a two year period this approach was used in 40 such patients. There were 24 women and 16 men with a median age of 76 years. In seven patients with severe cholangitis no attempt was made to extract the stones. Twenty three (57.5%) patients underwent a sphincterotomy and four (10%) needle knife papillotomy. The endoprosthesis insertion was considered a temporary measure in 13 (32.5%) patients and definitive treatment in 27 (67.5%). Bile duct drainage was established in all patients. Early complications occurred in six patients (15%), but were without sequelae. Late complications developed in eight (20%) of the patients and included biliary colic (four), cholangitis (three), and cholecystitis (one). Two patients (one cholangitis and one cholecystitis) died as a consequence of the complication. Only patients without a sphincterotomy developed cholangitis. A total of eight patients (20%) underwent surgery (one as an emergency) and nine a repeat endoscopic retrograde cholangio pancreatography (two as an emergency) to clear the duct. The remaining 23 patients are asymptomatic at a median of 13 months (range five to 24 months). Biliary endoprosthesis insertion for choledocholithiasis is an important alternative means of establishing drainage in selected cases, and is probably the optimum method of management for the elderly and or debilitated patients with previous cholecystectomy. Caution must be exercised, however, in patients with an in situ gall bladder.


Extracorporeal shock wave lithotripsy of bile duct stones: a single institution experience. Extracorporeal shock wave lithotripsy treatment with Dornier HM3 or MPL 9000 machines was applied in 37 patients with problematic bile duct stones. General anaesthesia was not required. After one extracorporeal shock wave lithotripsy session 14/37 patients (38%) were spontaneously stone free, and additional endoscopic extraction (eight of 37) and retreatments with extracorporeal shock wave lithotripsy (seven cases) increased the stone free rate to 29/37 (78%). In three patients with intrahepatic stones, the bile ducts could not be evaluated decisively at cholangiography and ultrasonography, but they were all symptom free at 15 to 38 months follow up. If these three patients are added to the radiologically stone free patients, the overall clinical success rate was 32/37 (86%). There were no serious complications, hospital admissions, or 30 day mortality as a result of extracorporeal shock wave lithotripsy or endoscopic procedures. It is concluded that extracorporeal shock wave lithotripsy is a valuable adjunct to the non-surgical treatment of bile duct stones.


Familial coexistence of achalasia and non-achalasic oesophageal dysmotility: evidence for a common pathogenesis. In five of seven siblings of healthy parents, dysphagia developed during adolescence or early adult life. A barium swallow was normal in one patient but showed appearances considered to be consistent with achalasia in all others. Oesophageal manometry was successfully performed in four of the five patients, including the patient with symptoms but normal radiological appearance. One patient had achalasia, two had oesophageal body motor dysfunction associated with a hypertensive, but normally relaxing lower oesophageal sphincter, and one had diffuse oesophageal spasm alone. The occurrence of three different oesophageal dysmotility disorders within members of a single sibship suggests that these conditions are intimately related and probably genetically determined as an autosomal recessive trait.


Non-steroidal anti-inflammatory drug-induced jejunal and colonic diaphragm disease: a report of two cases. Two patients with intestinal submucosal diaphragm disease associated with non-steroidal anti-inflammatory drugs are presented. The jejunum was affected in one, and in the other the ascending colon. Most previously reported cases have shown ileal disease and no case to date has shown involvement of the colon. The reasons for the localisation of these diaphragms remain unclear.


Ulcerative colitis complicated by Wenckebach atrioventricular block. Extracolonic manifestations of inflammatory bowel disease are common and diverse. Cardiac complications, however, are rare and of these pericarditis is the most frequently described association. A 57 year old man with a 20 year history of ulcerative colitis presented with a four day history of retrosternal chest pain and exertional dyspnoea. Electrocardiogram showed Wenckebach atrioventricular block. Three days later he developed bloody diarrhoea and sigmoidoscopy showed active proctocolitis. He was treated with oral prednisolone after which the chest pain and diarrhoea settled within 48 hours. At outpatient review two weeks later he was completely well and the electrocardiogram had returned to normal.


Sclerosing cholangitis associated with multifocal fibrosis: a case report. The association of sclerosing cholangitis, retroperitoneal fibrosis, and Riedel's thyroiditis has been reported twice before, and on both occasions the authors successfully used steroids to control the fibrotic process. A further case of fibrosis with this triad of organ involvement in which a combination of surgery and steroid treatment has arrested disease progression is described. This suggests an inappropriate immune response in this type of fibrotic overlap syndrome.


Piroxicam induced submassive necrosis of the liver. Several widely used non-steroidal anti-inflammatory drugs have been reported as causing severe hepatitis. Three cases of severe acute hepatitis have been reported in association with piroxicam. A fatal submassive necrosis that occurred in a 68 year old lady who had received piroxicam for 15 months is described. A 48 year old man who developed submassive hepatic necrosis six weeks after beginning piroxicam but was successfully treated with orthotopic liver transplantation is also reported. Piroxicam may induce submassive necrosis of the liver, probably as an idiosyncratic reaction.


[Effects of ambroxol HCl on the guinea pig tracheal mucous secretion and the rat pulmonary surfactant secretion]. The effects of orally administered ambroxol HCl (ambroxol) on guinea pig tracheal mucous secretion and rat pulmonary surfactant secretion were investigated histologically and biochemically. Ambroxol significantly increased the number of active goblet cells in guinea pig tracheal epithelium and total mucopolysaccharide level. Moreover, ambroxol significantly increased the neutral mucopolysaccharide level and PAS-positive substance in the guinea pig tracheal submucosal glands. Ambroxol did not show a significant effect on the content of the total phosphatidylcholine in rat lung lavage fluid, while ambroxol significantly increased the ratio of disaturated phosphatidylcholine to total phosphatidylcholine. From these results, it is suggested that ambroxol increases both the tracheal mucous secretion, especially the neutral mucopolysaccharide, and pulmonary surfactant secretion and these effects reflect part of the expectorant mechanism of the drug.


[Effects of drugs on the convulsions induced by the combination of a new quinolone antimicrobial, enoxacin, and a nonsteroidal anti-inflammatory drug, fenbufen, in mice]. The effects of drugs on the convulsions induced by the combination of a new quinolone antimicrobial, enoxacin, and a nonsteroidal anti-inflammatory drug, fenbufen, were studied in mice. The combination of enoxacin at 30 or 100 mg/kg, p.o. and fenbufen at 100 mg/kg, p.o. induced convulsions; and the mice died as a result of the convulsions. Pretreatment with either phenobarbital, phenytoin, valproic acid intraperitoneally, or morphine intravenously did not influence the convulsions. A high dose of diazepam or clonazepam prolonged the survival time, but could not prevent the mice from dying. After the occurrence of convulsions induced by enoxacin with fenbufen, administration of the excitatory amino acid antagonist MK-801 at 1 mg/kg, i.v. extended the survival time, even though all the mice died as a result of the convulsions. Simultaneous intravenous injections of MK-801 and diazepam suppressed the convulsions. This suppression was stronger than that produced by MK-801 or diazepam, injected separately. However, no mouse survived at the end. From these results, participation of both GABA-ergic and excitatory amino acidergic systems in the convulsions induced by enoxacin and fenbufen was discussed.


[Analgesic effects of Tsumura-shuuji-bushi-matsu and mesaconitine]. "Tsumura-shuuji-bushi-matsu" (TJ-3021) is a herbal medicine produced from Aconiti tuber through autoclaving to decrease its toxicity. In this study, the analgesic effects of TJ-3021 and mesaconitine (MA) was examined in rats and mice. TJ-3021 (300 mg/kg, p.o.) or MA (0.5 mg/kg, p.o.) depressed the acetic acid-induced writhing significantly. In Randall-Selitto's method, TJ-3021 (1000 mg/kg) increased the pain threshold ratio in the inflamed foot significantly but not in the normal foot. In the hot plate method or on adjuvant-induced arthritic pain, TJ-3021 (1000 mg/kg) significantly increased the pain threshold, and its effect was less than that of "nama-bushi-matsu" (TNB), which was produced from the same Aconiti tuber without autoclaving. Repeated cold stress (RCS) for 65.5 hr decreased the pain threshold ratio on the paw pressure in rats by 60%. TJ-3021 (300 mg/kg) significantly increased the pain threshold ratio in RCS rats, and its activity was almost the same degree as that of TNB. MA (0.5 mg/kg) significantly increased the pain threshold ratio in RCS rats or on the adjuvant-induced arthritic pain; and in RCS rats, it was more potent than morphine (1 mg/kg, p.o.).


[The vasospasmolytic effects of nicorandil, cromakalim and pinacidil on 3,4-diaminopyridine-induced phasic contractions in canine coronary arteries as an experimental vasospasm model]. The spasmolytic mechanisms of nicorandil, a novel antianginal drug, were investigated using 3,4-diaminopyridine (3,4-DAP)-induced phasic contractions of isolated canine coronary arteries in comparison with those of cromakalim and pinacidil. Nicorandil (10(-4) M), cromakalim (10(-6) M) and pinacidil (10(-5) M) suppressed the phasic contractions. Pretreatment with glibenclamide (10(-6) M), a specific blocking agent of ATP-sensitive K+ channel, eliminated the suppression of phasic contractions by these drugs; glibenclamide completely eliminated the suppression by cromakalim, while the eliminations against nicorandil and pinacidil were incomplete. The recoveries of peak tensions were only 56.8% and 76.1% for nicorandil and pinacidil, respectively. Nicorandil and pinacidil may suppress the phasic contractions via K+ channel opening and additional mechanisms. Methylene blue (10(-7)-10(-5) M) alone, a guanylate cyclase inhibitor, had no effect on the suppression of phasic contractions by nicorandil. In the presence of glibenclamide (10(-6) M), however, the pretreatment with methylene blue significantly augmented the recovery of peak tension for nicorandil. These results indicate that K+ channel openers may suppress the phasic contractions induced by 3,4-DAP via ATP-sensitive K+ channels, and that additionally, nicorandil may suppress the phasic contractility through guanylate cyclase stimulation, as a nitrate.


[Effects of tiropramide hydrochloride on the isolated detrusor and intravesical pressure of the bladder in situ in rats]. The effects of tiropramide on the isolated detrusor and intravesical pressure of the bladder in situ in rats were compared with those of flavoxate, oxybutynin and terodiline. The IC50 values (x 10(-5) M) of tiropramide for carbachol (CCh)-, K+ (60 mM)-, Ba2+ (10 mM)-, and electrical stimulation-induced contractions were 3.6, 4.2, 5.8, and 2.9, respectively. The four antispasmodics used (2 and 4 mg/kg, i.v., each) abolished the rhythmic bladder contractions in situ in anesthesized rats. Of the four compounds, oxybutynin was most potent and no significant differences were observed between the inhibitory effects of tiropramide, flavoxate and terodiline. The administration of flavoxate (30 and 60 mg/kg) into the duodenum little influenced the rhythmic bladder contractions. Tiropramide, flavoxate, oxybutynin and terodiline (8 and 12 mg/kg, i.v., each) dose-dependently prolonged the time to the volume-evoked micturition reflex, and the activity of tiropramide was not statistically different from those of the other three antispasmodics. Under unilateral pelvic and bilateral hypogastric nerve transection, both of the contractions induced by electrical stimulation of the peripheral and central cut ends of the pelvic nerve were dose-dependently inhibited to the same extent by tiropramide and terodiline. These results suggest that the effects of tiropramide on the function of urinary bladder in rats may be mainly due to direct actions on the smooth muscle, and that tiropramide is more potent than flavoxate and less potent than oxybutynin and terodiline.


[Inhibitory effects of Siamese Tinospora crispa extracts on the carrageenin-induced foot pad edema in rats (the 1st report)]. The naturally occurring Tinospora crispa (T.c.) at Chiang Mai in Thailand has been found to inhibit carrageenin-induced foot pad edema. Compared with the control group, oral administration of the 50% methanol extract (10 mg/kg) from its stems inhibited the edema by 38% in volume, which was induced 4 hr after stimulation by carrageenin in rats. This inhibitory effect on edema formation has been most significant in the n-butanol soluble fraction compared with the ethyl ether- or water-soluble ones, and the action of the n-butanol fraction has been observed in a dose-dependent manner in the range of 1 to 30 mg/kg, p.o. Administration of this fraction by the s.c., or i.p. route also inhibited the carrageenin-induced edema formation to the same degree as that by the oral route. The 3 mg/kg, s.c. dosage of the fraction corresponds roughly to 250 mg/kg sulpyrine and 10 mg/kg diphenhydramine, s.c. Moreover, the fraction administered i.v. also reduced LPS-induced fever in rabbits, and had an antagonistic effect equivalent to 100 mg/kg sulpyrine and 1 mg/kg morphine hydrochloride (in i.p.-administration). The comprehensive anti-inflammatory substance(s) contained in T.c. stems are moderately non-polar compounds soluble in n-butanol and absorbable from both walls of the gut and vessels. The usefulness of a modified dosage of this plant extract for clinical treatment of various types of inflammation is highly suggested.


[Effects of Gomishi and Shosaiko-to on lipid peroxidation of rat brain]. Gomishi and Shosaiko-to were administered to the rats at a dose of 10-100 mg/kg daily for 2 weeks, and their effects on lipid peroxidation of rat brain were compared with that of alpha-tocopherol. Administration of Gomishi and Shosaiko-to showed almost the same suppressive action on the lipid peroxidation. Gomishi and Shosaiko-to exhibited a radical-trapping action on a stable free radical, 1, 1-diphenyl-2-picrylhydrazyl (DPPH), which was estimated photometrically. The effects of Gomishi or Shosaiko-to at concentrations of 10(-3) to 10(-5)g/ml on lipid peroxidation of rat brain homogenates were investigated. The lipid peroxidation was inhibited by the addition of these drugs, and the suppressive effect was also dependent on the concentration. These suppressive effects with Shosaiko-to were stronger than those of Gomishi. These results suggest that the radical trapping action of Gomishi or Shosaiko-to is the likely mechanism suppressing brain lipid peroxidation; Particularly, the suppressive effect on the brain's lipid peroxidation by Shosaiko-to is at least in part due to its radical trapping action and inhibition of O2-. production.


[Effects of traditional Chinese medicines (dai-saiko-to, sho-saiko-to and hachimi-zio-gan) on spontaneously diabetic rat (WBN/Kob) with experimentally induced lipid and mineral disorders]. To clarify the therapeutic effects of several traditional Chinese medicines to improve disorders of carbohydrate, lipid and mineral metabolism, spontaneously diabetic rats (WBN/Kob) were treated with Vit. D2, 1 x 10(5) I.U./kg b.w./day, for 4 days, and then fed a hyperlipidemic diet containing traditional Chinese medicines for 6 weeks. The following results were obtained: 1) In the diabetic rats, the 3 traditional Chinese medicines further decreased the blood glucose level at 120 min after glucose loading in the glucose tolerance test. 2) The drugs increased the inorganic phosphate in the liver and normalized mineral metabolic disorder. 3) Hachimi-zio-gan decreased the cholesterol content in the kidney, and Sho-saiko-to decreased the cholesterol content in the elastin fraction (elastin-cholesterol) of the kidney. Such experimental results suggest that traditional Chinese medicines may be effective against the pathological conditions of diabetes mellitus that involve disorders of lipid and mineral metabolism.


[Effects of sodium hyaluronate on the nociceptive response of rats with experimentally induced arthritis]. Antinociceptive effects of sodium hyaluronate (Na-HA) were studied on the basis of improvement in the graded abnormal gait elicited by arthritis induced by intra-articular administration of monosodium urate crystal (MSU) to rats. One hour before MSU injection, intra-articular administration of a 1.0% solution of Na-HA with different molecular weights, ranging from 4.70 x 10(5) to 2.02 x 10(6) (HA-200), improved the score of abnormal gait in a molecular weight-dependent manner in the experimental arthritis model. Similarly, administrations of HA-200 at concentrations ranging from 0.1 to 1.0% prior to MSU treatment resulted in improvement of the score in abnormal gait in a dose-dependent manner. To elucidate the antinociceptive mechanisms of Na-HA, effects of pretreatment with Na-HA (1.0%) of different molecular weights on prostaglandin E2 (PGE2) and bradykinin (BK) releases in synovial fluid 3 hr after MSU injection were studied. Increases in PGE2 and BK concentration in the synovial fluid were depressed in a molecular weight-dependent manner by Na-HA (1.0%) pretreatment. These results indicate that Na-HA attenuates the nociceptive responses inflicted by the MSU-induced arthritis. Such an antinociceptive effect may be due to the inhibition of PGE2 and BK synthesis in the synovial joint of rats.


A calcium-binding protein in bile and gallstones. Calcium salts are often present in the center of all types of gallstones. Matrix proteins are known to be essential for biomineralization and may therefore also be important in the formation and growth of gallstones. Other researchers have described an anionic peptide fraction of a biliary lipoprotein complex in bile and a low-molecular weight acidic glycoprotein present in gallstones. Our goal was to determine whether such a protein was present in bile and whether this protein has any calcium-binding properties. We identified a pigment-associated, highly acidic protein that precipitates from bile on addition of CaCl2 0.5 mol/L. In addition, the protein is selectively concentrated in cholesterol and pigment stones. We have, therefore, confirmed the findings of these other researchers, and we have extended the study of this protein's interactions with calcium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates a single band (molecular weight < or = 14 kD) that reacts positively with cationic stains. The protein was shown to inhibit the precipitation of CaCO3 from a supersaturated solution. The capacity to bind calcium was further confirmed by autoradiography with 45Ca++ and by a membrane adsorption-binding assay. Calcium-induced aggregation was demonstrated by equilibrium dialysis and by quasielastic light scattering studies. Protein measured by Lowry's assay method and amino acid analysis constitutes only 2% to 4% of the harvested material. We speculate that a substantial lipid component may also be present.(ABSTRACT TRUNCATED AT 250 WORDS)


Hepatitis B virus precore mutants are identical in carriers from various ethnic origins and are associated with a range of liver disease severity. Hepatitis B virus carriers in Israel are mostly HBeAg negative, of whom 5% to 10% have circulating hepatitis B virus. Recently, a hepatitis B virus variant with a stop codon in the precore region was identified, and it was suggested that specific mutations are associated with fulminant or severe chronic active hepatitis. We have analyzed serum samples from HBeAg-positive and HBeAg-negative patients by polymerase chain reaction, using primers spanning the precore/core region. Nucleotide sequence analysis (by direct sequencing) from amplified hepatitis B virus DNA demonstrated that viral genomes from all HBeAg-negative patients contain G to A mutation (nucleotide 1896), leading to the formation of a stop codon. An additional G to A mutation was identified three nucleotides downstream (nucleotide 1899). These patients are of various ethnic origins, with no unique clinical characteristics and with normal liver histology, chronic hepatitis or cirrhosis. No mutation at the precore/core region was observed in the HBeAg-positive patients. In conclusion, the precore mutations identified in hepatitis B virus carriers in Israel are identical regardless of the carrier's ethnic origin and are associated with mild-to-severe liver disease.


Prevalence, classification and natural history of gastric varices: a long-term follow-up study in 568 portal hypertension patients. To determine the prevalence and natural history of gastric varices, we prospectively studied 568 patients (393 bleeders and 175 nonbleeders) with portal hypertension (cirrhosis in 301 patients, noncirrhotic portal fibrosis in 115 patients, extrahepatic portal vein obstruction in 117 patients and hepatic venous outflow obstruction in 35 patients). Primary (present at initial examination) gastric varices were seen in 114 (20%) patients; more were present in bleeders than in non-bleeders (27% vs. 4%, respectively; p < 0.001). Secondary (occurring after obliteration of esophageal varices) gastric varices developed in 33 (9%) patients during follow-up of 24.6 +/- 5.3 mo. Gastric varices (compared with esophageal varices) bled in significantly fewer patients (25% vs. 64%, respectively). Gastric varices had a lower bleeding risk factor than did esophageal varices (2.0 +/- 0.5 vs. 4.3 +/- 0.4, respectively) but bled more severely (4.8 +/- 0.6 vs. 2.9 +/- 0.3 transfusion units per patient, respectively). Once a varix bled, mortality was more likely (45%) in gastric varix patients. Gastric varices were classified as gastroesophageal or isolated gastric varices. Type 1 gastroesophageal varices (lesser curve varices) were the most common (75%). After obliteration of esophageal varices, type 1 gastroesophageal varices disappeared in 59% of patients and persisted in the remainder; bleeding from persistent gastroesophageal varices was more common than it was from gastroesophageal varices that were obliterated (28% vs. 2%, respectively; p < 0.001). Type 2 gastroesophageal varices, which extend to greater curvature, bled often (55%) and were associated with high mortality. Type 1 isolated gastric varices patients had only fundal varices, with a high (78%) incidence of bleeding.(ABSTRACT TRUNCATED AT 250 WORDS)


The effect of oral-administered lactulose on colonic nitrogen metabolism and excretion. The influence of lactulose on organic acid fermentation, nitrogen metabolism and excretion in the colon associated with its mechanism of action on hepatic encephalopathy was investigated. Orally administered lactulose in increasing amounts (0 to 20 to 40 to 80 to 160 gm/day) to 12 healthy volunteers decreased ammonia production in 16.6% fecal homogenates incubated 6 hr and 24 hr at 37 degrees C (mean +/- S.E.M.: from 7 +/- 1 to 0 +/- 0 and from 13 +/- 2 to 0 +/- 0 mmol/L, respectively). Every dose of lactulose was given for 3 days with intervals of 1 to 2 wk, and 24-hr stools were collected on day 3. Fecal concentrations of ammonia decreased (from 50 +/- 9 to 11 +/- 3 mmol/L), but ammonia excretions increased (from 6 +/- 2 to 17 +/- 4 mmol/24 hr). Total fecal concentrations of nitrogen decreased (from 1,043 +/- 78 to 300 +/- 136 mmol/L), but excretions of nitrogen increased fourfold (from 111 +/- 21 to 457 +/- 113 mmoL/24 hr) because of the increase in stool mass. Fecal pH declined (from 6.9 +/- 0.1 to 4.9 +/- 0.1), but total organic acids (short-chain fatty acids and DL-lactate; range = 105 to 148 mmol/L) and osmolality in feces (417 to 450 mOsm/L) did not change, although the colonic fermentation of lactulose had a major impact on the proportions between the nontoxic acetate (increased from 65% +/- 2% to 89% +/- 3%) and the potentially neurotoxic 3-6-carbon fatty acids (decreased from 35% +/- 2% to 11% +/- 2%).(ABSTRACT TRUNCATED AT 250 WORDS)


Pulmonary hemorrhage and glomerulonephritis in primary biliary cirrhosis. We observed life-threatening intrapulmonary hemorrhages and focal proliferative glomerulonephritis in a 41-yr-old woman with primary biliary cirrhosis. The severity of the symptoms necessitated blood transfusions and mechanical ventilation; the patient improved with the help of corticosteroid therapy. No formal evidence of either Goodpasture's syndrome or any other well-defined systemic vasculitis could be found. Neutrophil cytoplasmic antibodies were initially positive and became undetectable after 3 mo of immunosuppressive treatment without relapse. This association has not been described previously and may be added to the list of extrahepatic immune-mediated conditions associated with primary biliary cirrhosis.


Pallidal hyperintensity on magnetic resonance imaging in cirrhotic patients: clinical correlations. Patients with cirrhosis show increased signal intensity in the globus pallidus on T1-weighted magnetic resonance imaging of the brain. This abnormal appearance of the basal ganglia has been related to the severity of liver failure and to the presence of portal-systemic shunting, although its cause and clinical significance remain unknown. We prospectively assessed the metabolic, neurological and neuropsychological statuses of 30 stable cirrhotic patients and correlated these clinical variables with computed measurements of globus pallidus signals. Some metabolic variables denoting disease severity appeared to be significantly related to image changes, although the strongest association was found with plasma ammonia levels. After adjustment for ammonia level, on multiple regression analysis, the other variables were not significant. Furthermore, pallidal changes were associated with specific neurological symptoms and neurological functions, symptoms and functions that also had a significant correlation with ammonia levels. Our findings suggest that globus pallidus signal abnormality could arise as a marker of brain impairment related to hyperammonemia.


Variability of atrial natriuretic peptide plasma levels in ascitic cirrhotics: pathophysiological and clinical implications. Ascitic cirrhotic patients are a heterogenous population with respect to factors that may affect plasma human atrial natriuretic peptide levels (such as degree of plasma volume and plasma levels of angiotensin II, vasopressin and norepinephrine). Thus the proven variability of plasma human atrial natriuretic peptide values in ascitic cirrhotic patients may be due also to the selection of patients, not only to the study conditions. The response to standardized stepped-care medical treatment of ascites makes it possible to characterize ascitic cirrhotic patients with different patterns of renal sodium excretion, intrarenal sodium handling, plasma renin activity, plasma aldosterone and thus, probably, effective circulating volume. Consequently, we evaluated human atrial natriuretic peptide plasma levels in controls (n = 23), in ascitic cirrhotic patients who underwent spontaneous diuresis (group A, n = 7) and in cirrhotic patients who required diuretic treatment (group B, n = 44). The last group was then divided into two subgroups. Subgroup B-R (n = 25) included patients who responded to spironolactone alone, whereas subgroup B-NR (n = 19) included patients who did not respond to 500 mg/day spironolactone. All patients were maintained on identical normocaloric restricted sodium intake (80 mEq/day) throughout the study. Ascitic cirrhotic patients, as a whole, had higher values of human atrial natriuretic peptide than did controls (70.8 +/- 46.6 pg/ml vs. 41.7 +/- 16.3 pg/ml, p < 0.025). No difference was found in human atrial natriuretic peptide/plasma renin activity between the two groups (87 +/- 160 pg/ng/hr vs. 44 +/- 73 pg/ng/hr, p = NS).(ABSTRACT TRUNCATED AT 250 WORDS)


Hepatic mitochondrial glutathione depletion and progression of experimental alcoholic liver disease in rats. Long-term ethanol feeding has been shown to selectively reduce hepatic mitochondrial glutathione content by impairing mitochondrial uptake of this thiol. In this study, we assessed the role of this defect in evolution of alcoholic liver disease by examining the mitochondrial glutathione pool and lipid peroxidation during progression of experimental alcoholic liver disease to centrilobular liver necrosis and fibrosis. Male Wistar rats were intragastrically infused with a high-fat diet plus ethanol for 3, 6 or 16 wk (the duration that resulted in induction of liver steatosis, necrosis and fibrosis, respectively). During this feeding period, the cytosolic pool of glutathione remained unchanged in the ethanol-fed animals compared with that in pair-fed controls. In contrast, the mitochondrial pool of glutathione selectively and progressively decreased in rats infused with ethanol for 3, 6 or 16 wk, by 39%, 61% and 85%, respectively. Renal mitochondrial glutathione level remained unaffected throughout the experiment. Serum ALT levels increased significantly in the ethanol-fed rats at 6 wk and remained elevated at 16 wk. In the mitochondria with severely depleted glutathione levels at 16 wk, enhanced lipid peroxidation was evidenced by increased malondialdehyde levels. Thus a progressive and selective depletion of mitochondrial glutathione is demonstrated in the liver in this experimental model of alcoholic liver disease and associated with mitochondrial lipid peroxidation and progression of liver damage.


Preformed lymphocytotoxic antibodies: the effects of class, titer and specificity on liver vs. heart allografts. The effect on liver and heart allograft survival (ACI rats to Lewis rats) was studied after three methods of recipient presensitization and after different intervals between sensitization and transplantation. With comparable lymphocytotoxic antibody titers, liver allografts always survived longer than heart grafts. The titer, class and specificity of the antibodies varied with the method of sensitization. Four skin grafts produced IgG and IgM lymphocytotoxic antibody titers of 1:2,000 to 4,000. The IgG fraction was shown to have hepatic vascular endothelial specificity by indirect immunofluorescence. These primed recipients hyperacutely rejected both heart and liver allografts, which showed vascular deposition of IgG antibodies. Survival of either kind of graft was inversely proportional to the lymphocytotoxic antibody titer and length of time after placement of the last skin graft. Presensitization with a single heterotopic heart graft produced an even higher mixed IgG and IgM lymphocytotoxic antibody titer of 1:8,000 but with less IgG vascular endothelial specificity. These animals also hyperacutely rejected heart or liver grafts with tissue deposition of IgG but less consistently and with a weaker correlation with lymphocytotoxic antibody titers and time after sensitization. Sensitization with two pretransplant blood transfusions produced the lowest titer (1:500 to 1,000) and the least IgG vascular endothelial specificity. Liver allograft survival was routinely enhanced in these animals, and little effect was seen on heart grafts. Collectively, the experiments showed that the liver is not only resistant to antibody-mediated rejection relative to the heart but is more easily enhanced.(ABSTRACT TRUNCATED AT 250 WORDS)


Liver regeneration and the effect of exogenous putrescine on regenerative activity after partial hepatectomy in cirrhotic rats. There are conflicting data regarding the ability of the liver to regenerate after partial hepatectomy in animals and humans with cirrhosis. The purpose of this study was to document liver regeneration after partial hepatectomy in a carbon tetrachloride rat model of cirrhosis and to determine whether exogenous putrescine, a polyamine that has been reported to stimulate liver regeneration in animal models of acute liver failure, enhances regenerative activity in cirrhosis. Liver fibrosis and cirrhosis were produced by weekly intragastric gavage with carbon tetrachloride in 130 adult male rats. Vehicle-gavaged rats (n = 12) served as healthy controls. At surgery and at 4 and 8 hr after 70% hepatectomy, rats received normal saline solution or 1 or 10 mg/kg putrescine by intraperitoneal injection. Another group (n = 32) of carbon tetrachloride-treated rats was given putrescine (100 mg/kg) or normal saline solution twice daily for 10 days before partial hepatectomy and at 0, 4 and 8 hr after partial hepatectomy. Liver regeneration was documented 24 and 48 hr after partial hepatectomy on the basis of restitution of liver mass, ornithine decarboxylase activity and [3H]thymidine incorporation into liver DNA. Automated image analysis of the resected liver specimens separated carbon tetrachloride-treated rats into two subgroups: those with bridging fibrosis (fibrotic group) and those with micronodular cirrhosis (cirrhotic group). Restitution of liver mass and ornithine decarboxylase activity at 24 and 48 hr after partial hepatectomy were similar to carbon tetrachloride-treated rats (both fibrotic and cirrhotic) and vehicle-treated healthy controls.(ABSTRACT TRUNCATED AT 250 WORDS)


Hemodynamic effects of calibrated stenosis of the superior mesenteric artery in conscious rats with portal vein stenosis. Because superior mesenteric arterial blood flow is increased in portal hypertension and plays a role in elevated portal pressure, mechanical reduction of artery diameter should induce decreases in portal pressure and superior mesenteric arterial blood flow. In this study, calibrated superior mesenteric artery stenosis (induced with a 22-gauge needle) was performed in rats simultaneously with portal vein stenosis or 2 wk after creation of portal vein stenosis. Hemodynamic studies were performed 3 wk after induction of portal vein stenosis in conscious, unrestrained rats. At that time, neither weight loss nor digestive tract alterations were observed in rats with arterial stenosis. In neither group of rats with arterial stenosis was portal tributary blood flow significantly different from that of normal rats; nor was it significantly lower than in rats with portal vein stenosis without arterial stenosis. In both groups of rats with arterial stenosis, portal pressure was significantly lower (12.1 +/- 1.6 mm Hg and 12.5 +/- 1.8 mm Hg, respectively) than in rats subjected to portal vein stenosis (15.4 +/- 1.5 mm Hg) but significantly higher than in controls (7.2 +/- 1.0 mm Hg). In rats with arterial stenosis, cardiac index was also significantly lower than that in rats with portal vein stenosis but higher than that in controls. In conclusion, this study shows that both early and late superior mesenteric artery stenosis significantly reduce the degree of portal hypertension and the hyperkinetic state of rats with extrahepatic portal hypertension. Thus we can speculate that superior mesenteric artery stenosis might provide a new therapeutic approach for portal hypertension.


Role of mesenchymal cell populations in porcine serum-induced rat liver fibrosis. The role of liver mesenchymal cell populations in porcine serum-induced rat liver fibrosis were studied morphologically and immunohistochemically. Five-week-old rats were intraperitoneally injected with porcine serum twice a week and examined at various intervals between 3 and 24 wk after the initial injection. At an early phase, numbers of fibroblasts and extracellular matrix increased in the walls of central veins and in portal and capsular connective tissues. In the walls of central veins, the number of "second-layer cells" (i.e., the fibroblasts located at the second layer of the wall) increased. Connective tissue septa, accompanying some fibroblasts, extended from these interstitial tissues into the hepatic parenchyma, and their foremost edges came into direct contact with the perisinusoidal stellate cells. The sinusoids adjacent to the newly formed septa collapsed and later disappeared; this process resulted in the formation of hepatic limiting plates along the septa. At a more advanced stage, the interstitial fibroblasts and septal cells-which were derived from interstitial fibroblasts and the stellate cells-increased and became multilayered, constructing three-dimensional cell networks. These networks, together with increased collagen fibrils and elastic fibers, constitute the fibrotic dense connective tissue. In the control rat, smooth muscle cells were positive on vimentin, desmin and smooth muscle-alpha-actin staining. The stellate cells, second-layer cells, capsular and portal fibroblasts were shown to be vimentin and desmin positive and smooth muscle-alpha-actin negative. In the fibrotic liver, septal(fibroblastic) cells were vimentin and desmin positive and smooth muscle-alpha-actin negative. We conclude that not only the perisinusoidal stellate cells but also the interstitial fibroblasts, including the second-layer cells, play substantial role in the development of porcine serum-induced septal fibrosis in rat liver.


Resident mast cells are the main initiators of anaphylactic leukotriene production in the liver. During anaphylaxis the sensitized liver can have substantial capacity for leukotriene production. However, the intrahepatic cellular source for these potent eicosanoid mediators has been unclear so far. We therefore analyzed the appropriate role of resident liver cells in organ-specific generation of leukotrienes by immunohistochemical localization of 5-lipoxygenase, by measurement of cysteinyl leukotriene production in animals or isolated livers and by histochemical monitoring of mast cells in rat, guinea pig and mouse livers, respectively. During anaphylaxis in vivo, these species all generated large amounts of leukotrienes. Immunohistochemistry with rat liver demonstrated resident mast cells as the predominant cell type in liver containing 5-lipoxygenase. Rat and guinea pig livers contained numerous mast cells and produced substantial amounts of leukotrienes on antigen challenge; in contrast, mouse livers neither showed detectable mast cells nor generated leukotrienes when stimulated analogously. Infusion of histamine or serotonin (1 mmol/L each) or of the degranulating substance P (8 mumol/L) did not elicit leukotriene generation in rat livers. Furthermore, substantial degranulation of liver mast cells by compound 48/80 (0.5 mg/kg body mass) was paralleled by only modest leukotriene formation (63 +/- 10 pmol in bile/kg body mass/30 min). These results indicate that during anaphylaxis mast cells are the main intrahepatic cells initiating leukotriene production and that such leukotriene generation is likely to be independent of mast cell degranulation or the release of histamine or serotonin.


United States' tort costs continue to escalate. Tort costs in the United States totaled $132 billion in 1991, or 2.3% of the nation's total economic output; and medical malpractice accounted for $9.1 billion of that total, according to new data from Tillinghast, a division of Towers Perrin, New York City.


Cancer at the crossroads.

Cancer at the crossroads. A number of elements are transforming cancer care in hospitals. First is ongoing, rapid technological advance: Experts contend that biotechnology therapies will shift the site of cancer treatment from external, systemic treatments like radiation and chemotherapy, to targeted, tumor-specific drugs that boost the immune system. That change alone holds many implications for oncology management. In addition, an aging population, longer cancer survival rates, reimbursement concerns, and the increasing involvement of patients and their families in treatment decision making, will all have an impact on the way cancer care is designed and delivered. The time to plan, say experts, is now. Meanwhile, innovative treatment plans, patient care approaches, and collaborative efforts among institutions are moving hospital oncology into the 1990s in a dramatic way.


Are trustees prepared to lead through health reform maze? Not all hospital trustees realize the vital leadership role they must play as their hospitals move toward the future, say CEOs. As a result, executives understand that they have a major educative role to play in bringing their boards up to speed on health care reform issues--quickly.


New AHA guidebook provides framework for implementing clinical practice guidelines. A new guidebook from the AHA promises to help hospitals move from the abstract to day-to-day clinical practice when it comes to adopting clinical practice guidelines. At the same time, new federal guidelines offer another source from which hospitals can directly receive new information.


Healthy investor interest in non-rated hospital bonds. Despite persistent rumors of a "credit crunch," tax-exempt financing remains a viable source of capital for many hospitals that can't obtain investment-grade ratings. In fact, say experts, there's still strong investor interest in debt issued by lower-credit quality hospitals.


CEOs link IS visions to hospital strategic plans. More and more hospital CEOs are recognizing the importance of aligning the goals for their information systems with those of their strategic plans. In addition, information management experts offer advice on how CEOs can participate in IS selections without getting bogged down in details.


Prolactin as an immunoregulatory hormone in mammals and birds. The immunoregulatory function of prolactin (PRL) and the mechanism of its action in mammals seem to be well documented. Reciprocal interdependence between PRL secretion and immune system function is essential for normal ontogeny, development and aging. PRL receptors in lymphocytes participate in the transduction of its regulatory signal into the intracellular enzymatic machinery including that of the nucleus, leading to the expression of some genes and to the synthesis of new proteins. Activation of phosphoinositide turnover and subsequent increase in protein kinase-C activity seems to be a possible mechanism acting in the regulatory influence of PRL on mammalian immune cells. These cells in turn, under mitogen or antigen stimulation, secrete a substance with PRL-like activity. The regulatory function of PRL within the avian immune system is less well known, but it seems to have some features in common with those in mammals. Direct mitogenic action on thymocytes and splenocytes in the chicken might indicate the existence of PRL receptors in these cells and could explain the immunostimulatory effect of PRL observed in vivo, which is dependent on the time of hormone administration. As the avian PRL stimulates mitogenesis of rat Nb2 lymphoma cells, the mechanism of direct PRL action on immune cells in mammals and birds seems to be similar. PRL in chickens also modifies the level and the diurnal rhythm of corticosterone which, in turn, influences the immunoregulatory effect exerted by PRL. Thus, PRL seems to be an important factor, influencing directly or indirectly the avian immune system.


Melatonin induced increase in gamma-interferon production by murine splenocytes. Previously, we demonstrated that production of gamma-interferon (gamma-IFN) by the mouse splenocytes isolated at night was higher than from those isolated in the morning. In this paper we show that melatonin increased gamma-IFN production by murine splenocytes. Moreover, this stimulating effect was significantly higher (10 times) in the cells isolated at night than in those isolated in the morning (2 times).


Chemo-immunotherapy in patients with metastatic melanoma using sequential treatment with dacarbazine and recombinant human interleukin-2: evaluation of hematologic and immunologic parameters and correlation with clinical response. We have treated 18 patients with metastatic malignant melanoma (MM) with high-dose IL-2 administered by continuous iv infusion in combination with dacarbazine (DTIC), and correlated the clinical response with various hematologic and immunologic parameters. Two regimens differing in the sequence of treatment were employed, and 1-6 treatment cycles were given, depending on patient response. Two patients had a complete response (CR, 46+m, 14m), two patients a partial response (PR, 16m,6m), one a minimal response and four had a stable disease lasting 2-7 months, thus the response rate (CR+PR) was 22%. None of the following parameters, tested prior to initiation of the therapy and 1-2 days after termination of each course of IL-2, correlated with the clinical response: WBC counts (total and differential), levels of blood CD4 and CD8 T cells, NK cells, monocytes and B cells, production of IL-1 and IL-1 inhibitor by monocytes, responsiveness to 3 mitogens, NK/LAK cell activity, and serum levels of IL-1 alpha, IL-2, soluble IL-2 receptor, and TNF alpha. The only prognostic parameter was the greater increase in the level of IL-2 receptor (Tac)-bearing lymphocytes in the responding patients after 1-3 cycles of IL-2. The data suggests that non-specific immune parameters have no prognostic value for patients undergoing IL-2-based immunotherapy.


Global degranulation of rat mast cells stimulated with DNP-polystyrene. Mediator release was studied in rat peritoneal mast cells sensitized with a mouse monoclonal anti-DNP IgE antibody, and stimulated with DNP-ornithine covalently attached to radio-derivatized polystyrene petri dishes. Cells releasing serotonin at maximal rates were investigated by transmission electron microscopy. Generalized exocytosis of granules could be observed, suggesting non-directional release of mediators, and non-compartmentalized action of second messengers in mast cells stimulated with polystyrene-bound DNP. Stimulation of sensitized mast cells by DNP covalently bound to the rigid polystyrene surface is consistent with extrinsic mechanisms proposed for Fc(epsilon)RI receptor action, and suggests that internalization of Fc(epsilon)RI is not needed for triggering cell degranulation.


Phagocytosis and microbicidal capacity of human monocytes in the course of HIV infection. Phagocytosis and bactericidal activity of human monocytes were studied in asymptomatic HIV carriers and in patients with clinically apparent HIV infection, diagnosed as persistent generalised lymphadenopathy (PGL) or AIDS-related complex (ARC). Monocytes of asymptomatic HIV carriers manifested no significant changes while a decreased phagocytosis was shown by monocytes in the majority of patients with PGL or ARC. The latter patients also exhibited a decreased bactericidal activity of the cells. Tuftsin and even serum from healthy donors were found to normalise the disturbed monocyte function, the effect of tuftsin being more pronounced. Nevertheless, the examined sera contained neither factors which could stimulate nor factors which could inhibit phagocytosis. The obtained results indicated that the productive phase of HIV infection is associated with disturbed phagocytosis and disturbed microbicidal activity of monocytes, reflecting deficiency of serum factor(s) needed for the normal function of the cells.


Degenerative changes in the A375 melanoma line induced by transforming growth factor beta 1. A375 human melanoma cell cultures grown in the presence of TGF beta contained greatly reduced cell numbers and exhibited drastic alterations in cell morphology compared to the control cultures. Preincubation of the cells with the cytokine for only 18 h was sufficient to induce these changes irreversibly. Examination of TGF beta-treated cells in the electron microscope revealed large numbers of lipid-filled vacuoles in the cytoplasm, greatly contracted nuclei and some loss of the otherwise abundant microvilli. Thus TGF beta may have a direct toxic effect on the A375 melanoma cells.


Fast filtration enzyme immunoassay for haptens. An immunometric method for determination of hapten concentration in fluids has been developed. High-affinity hapten-specific enzyme-labeled monoclonal antibodies are mixed with a sample containing hapten, then the mixture is filtered through a membrane with immobilized hapten. The level of enzyme activity retained by the membrane is inversely proportional to the concentration of hapten in a sample. The assay has been developed for theophylline, digoxin and phenobarbital. The coefficient of variation is less than 5% and the test takes about 2 min.


Specific anti-A responses of SCID mice populated with human lymphoid cells from peripheral blood, umbilical cord, bone marrow and spleen after immunization with group A erythrocytes [corrected]. Mice with severe combined immunodeficiency (SCID) were injected i.p. with 10 x 10(6) or 50 x 10(6) human leukocytes obtained from adult peripheral blood, umbilical cord blood, bone marrow and spleen from six group O individuals, together with allogeneic group A erythrocytes. Mice were bled every two weeks and levels of human IgG, IgM and anti-A were determined in the murine plasma. Spleen cells elicited the highest Ig levels (up to 5.2 mg/ml IgG) and umbilical cord the least (0-0.16 mg/ml IgG); maximum IgG levels were obtained at about 6-8 weeks after injection. Anti-A was detected in mice receiving adult peripheral blood or spleen leukocytes and immunizing erythrocytes 4-6 weeks after injection. Mice injected with the higher dose of leukocytes gave the best anti-A responses, but were more likely to develop tumours after 8 weeks.


Tetrapeptide QDPR is a minimal immunodominant epitope within the preS2 domain of hepatitis B virus. A hepatitis B virus preS2 deletion library with the preS2 sequence fused to the coat protein of the RNA phage fr (fr CP) as a carrier has been constructed and used for the approximate localization of epitope recognized by a panel of murine monoclonal anti-preS2 antibodies. DNA copies of putative preS2 epitopes were synthesized and cloned within the fr CP gene. Tetrapeptide Gln-Asp-Pro-Arg (QDPR) corresponding to the preS (132-135) sequence was found to be the minimal sufficient recognition site for one of the monoclonal antibodies, S26. The closely related tetrapeptide EDPR did not mimic the epitope activity of QDPR.


Characterization of IgM molecules in light-chain deficient variants of a B-cell tumor. Characterization of a membrane-IgM-negative variant cell line derived from the murine B-cell line 38C-13 revealed the absence of light chains and the presence of polypeptides with an apparent molecular size of 18 kDa and 14 kDa, previously denoted omega and iota and characteristic of pre-B cells. These polypeptides assemble with the mu chains into complexes with apparent molecular sizes of about 100 kDa and 200 kDa. It has been previously shown that light-chain-deficient variants of the 38C cell line undergo 'secondary' light chain rearrangements. It is suggested, therefore, that complexes of mu and the 'surrogate' light chains omega and iota play a role in this process. As these complexes do not reach the cell surface we would like to propose that the mechanism of secondary rearrangement is intracellularly controlled.