Isolation, cultivation, and partial characterization of microvascular endothelium derived from human lung. Primary cultures of peripheral lung lobes were grown in a highly supplemented medium. Human lung endothelial cells (HLE) were isolated from the mixed population by FACS. The cells proliferated rapidly and were serially cultivated for at least 16 passages. Both early and late passage cells were positive for the standard endothelial markers. Factor VIII related-antigen (Factor VIII R-Ag), angiotensin-converting enzyme, acetylated low-density lipoprotein labeled with 1,1'-dioctadecyl-1,3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL) uptake, and bound the lectin Ulex europaeus agglutinin (UEA). Prostaglandin E2 was the major cyclooxygenase product of HLE, in contrast to human umbilical vein endothelial cells (HUVE), which synthesized PGI2 in excess of PGE2. Factor VIII R-Ag exhibited a diffuse cytoplasmic as well as an extracellular fibrillar distribution in HLE, in contrast to a vesicular (Weibel-Palade body) cytoplasmic distribution in HUVE. The HUVE did demonstrate some extracellular fibrillar Factor VIII R-Ag as well. Urokinase was the predominant plasminogen activator (PA) secreted by HLE, whereas tissue PA was predominant in HUVE cultures. HLE formed tube-like structures within 2 h of plating on a Matrigel matrix whereas HUVE formed larger tube-like structures only after 1 or more days. The properties described here indicate that human lung microvessel endothelium can be isolated and continuously grown from small tissue segments and express a number of properties that differ from those of HUVE. These studies provide further support for the concept that endothelial cells from different sources can exhibit considerable heterogeneity relating to their phenotypic and biochemical properties.


Identification of endogenous 5S,12S-dihydroxy-6,8,10,14-(E,Z,E,Z)-eicosatetraenoic acid (5S,12S-diHETE) in chick osteoblasts and epiphyseal cartilage. Cell homogenates were obtained from chick osteoblast-like cells and epiphyseal growth plates. The lipid fraction was extracted with ethyl acetate and separated by RP-phase HPLC. Reverse-phase HPLC analysis of this fraction showed a major peak displayed a conjugated tetraene structure and this endogenous synthetic compound could be partially blocked by heat treatment at 100 degrees C for 5 min. After methylation, this peak was further purified by SP-HPLC and a major peak was identified by an U.V. absorption at lambda max of 271 nm and shoulders at 261 and 281 nm. The compound was hypothetically proposed by gas chromatography and mass spectrometry (GC/MS) as the cyclohexadiene derivative of 5S,12S-dihydroxy-6,8,10,14-(E,Z,E,Z)-eicosatetraenoic acid (5S,12S-diHETE) with a ring closure at C6 and C11. These results demonstrated that the 5S,12S-diHETE is an endogenous eicosanoid in both intramembranous and endochondral skeletal tissues.


The effect of viral infection on eicosanoid formation and procoagulant activity of rat peritoneal macrophages. Role of the dietary fat type. The effect of dietary manipulation on eicosanoid formation in rat peritoneal macrophages was studied in relation to some of their effector functions: cellular procoagulant activity, production of reactive oxygen species (measured as chemiluminescence), and phagocytosis of antibody-coated erythrocytes. Rats were fed adequate diets for eight weeks containing mackerel oil (MO), sunflowerseed oil (SO) or hydrogenated coconut oil (HCO). The release of eicosanoids from resident macrophages stimulated by opsonized zymosan was significantly lower for the MO group as compared to the other dietary groups. Infection of the animals via intraperitoneal injection with rat cytomegalovirus resulted in a significant decrease in eicosanoid production in all groups, irrespective of dietary fat type. However, in the HCO group a partial restoration of TXB2 and HHT production could be observed at day 10 post infection. Resident macrophages obtained from the mackerel oil fed animals showed a significantly higher procoagulant activity than those from the other diet groups. Infection of the animals resulted in an increase in procoagulant activity in all groups. In contrast, no significant differences in chemiluminescence and in phagocytosis were detected between macrophages obtained from rats fed the different diet groups. It is concluded that peritoneal macrophages obtained from mackerel oil fed rats produce less eicosanoids and are more procoagulant than those from the other dietary groups, but a viral infection eradicates these differences. Therefore, a correlation between eicosanoid formation and effector functions studied could not be established.


Syntheses of leukotriene-derivatives.

Syntheses of leukotriene-derivatives. Some general synthetic routes for the synthesis of cysteinyl-leukotriene derivatives derived from stable building blocks are described. D6-LTE4, a metabolically stable isotopically labelled mass spectrometric internal standard, 20-hydroxy-LTE4, the unnatural 6-epi-LTE4; LTE3, a LT-derivative with 2-amino-thiophenol as a modified "amino-acid" and 14,15-dehydro-LTA4 were prepared. The compounds were tested in a LT-inhibition assay using a monoclonal antibody.


Characterization of an inositol 1,3,4,5-tetrakisphosphate 3-phosphatase from porcine brain. Some of the properties of a high affinity Ins(1,3,4,5)P4 3-phosphatase (Km approximately 400 nM) from the soluble fraction of pig brain are presented. Several inositol polyphosphates reduced the activity of the Ins-(1,3,4,5)P4 3-phosphatase. The most effective inhibitors were Ins(1,3,4, 5,6)P5 and InsP6 with Ki-values of about 60 nM and 3 nM, respectively. We could show that at least InsP6 is a likely substrate of the Ins(1,3,4,5)P4 3-phosphatase, which degraded InsP6 with a very low reaction velocity. This 3-phosphatase may be important for the metabolism of higher phos-phorylated inositol polyphosphates.


Inhibition of calcimycin-induced stimulation of cytosolic calcium in human PMN by a LTB4 receptor antagonist. This study investigates the significance of PMN-derived LTB4 for PMN activation by using a selective LTB4 antagonist (SC 41930). Human PMN were stimulated by platelet-activating-factor (PAF, 3 microM), a receptor dependent agonist, or by calcimycin (A 23187, 10 microM), a receptor independent agonist. PMN activation was determined by LTB4 release and changes in free cytosolic Ca++ levels. Pretreatment of the PMN with SC 41930 (0.1-10 microM) caused a concentration-dependent inhibition of agonist induced rise in cytosolic Ca++ with both PAF and calcimycin. Interestingly, at the same concentrations of SC 41.930, there was a concentration-dependent inhibition of LTB4 release. Control experiments with a cell-free 5-lipoxygenase preparation did not show any direct effect of SC 41930 on the enzyme under conditions when nordihydroguiaretic acid was active. The data demonstrate a nonselective inhibition of agonist induced PMN activation by the LTB4 receptor antagonist SC 41930 and suggest that formation of endogenous LTB4 is involved in a positive feed-back loop that is required for maximum stimulation of PMN function even by a calcium ionophore.


Functionally diverse purinergic P2Y-receptors mediate prostanoid synthesis in cultured rat astrocytes: the role of ATP-induced phosphatidyl-inositol breakdown. Cultured rat astrocytes possess purinergic P2Y-receptors. Stimulation of these receptors with ATP (10(-3) M) results in increased phosphatidylinositol biphosphate (PIP2)-breakdown and prostanoid formation. We have investigated the relevance of the PIP2-pathway in prostanoid synthesis. The intracellular Ca(2+)-mobilizing agent thapsigargin (TG) (10(-6) M) and the diacylglycerol (DAG)-mimetic tetradecaoylphorbol acetate (TPA) (10(-8)-10(-6) M) both stimulate prostaglandin D2 production. ATP-induced prostanoid formation can be mimicked by combined addition of TG and TPA, suggesting the importance of the second messengers IP3 and DAG, generated during P2Y-receptor mediated PIP2-breakdown. Inhibition of ATP-induced PIP2-hydrolysis by TPA (IC50 about 5 x 10(-8) M) or by 10(-4) M neomycine, however, does not affect astroglial prostanoid synthesis, showing that P2Y-receptor mediated prostanoid formation may occur also in the absence of PIP2-hydrolysis. These findings suggest that additional postreceptor mechanisms exist in the signal transduction chain of ATP-induced astroglial prostanoid synthesis. A possible involvement of phospholipase A2 and/or of Ca(2+)-channels, directly coupled to P2Y-receptors is proposed.


Regulation of leukotriene biosynthesis in human polymorphonuclear leukocytes. Since the formation of leukotrienes in human polymorphonuclear leukocytes is Ca-dependent we have determined the Ca-pools involved and the enzymes affected. With PAF, fMLP and LTB4 as agonists the Ca-transients measured with Fura-2 showed the same height of the intracellular Ca-signal but an external Ca-component in decreasing magnitude. In parallel the activity of 5-lipoxygenase was also decreasing. 5-lipoxygenase was linearly dependent on Ca with saturation of around 350 nMol/l already provided by the release of intracellular Ca. Phospholipase A2, however, started to activate at about this concentration. Hence arachidonate is a limiting factor in stimulated leukocytes due to a limited influx of extracellular Ca.


ACE-inhibition, kinins, and vascular PGI2 synthesis. In the present studies, ex vivo-, in vitro-, and in vivo-effects of three structurally different angiotensin I-converting enzyme (ACE) inhibitors on the kallikrein-kinin and eicosanoid systems are described. In the ex vivo- and in vitro-experiments using isolated rat aorta, vascular prostacyclin (PGI2) production is dose-dependently stimulated by the ACE inhibitors captopril, lisinopril, and ramipril. Furthermore, the ACE inhibitor-induced augmentation of vascular PGI2 synthesis observed in vitro was completely inhibited by the competitive bradykinin antagonist D-Arg[Hyp3,Thi5,8,D-Phe7]bradykinin suggesting that ACE inhibitors stimulate PGI2 generation by an enhancement of kinin activity. In the in vivo studies in healthy volunteers, we used platelet cyclic adenosine-5'-monophosphate (cAMP) and cyclic guanosine-5'-monophosphate (cGMP) as indirect parameters of the activity of prostacyclin and the endothelium-derived relaxing factor, respectively. Since platelet cAMP and cGMP were unaffected by an acute dose of 10 mg of lisinopril, our data do not support the concept that the interference of ACE inhibitors with the kallikrein-kinin-prostaglandin system observed ex vivo and in vitro participates in the haemodynamic effects of these agents in humans in vivo.


The role of the epidermal 12-hydroxyeicosatetraenoic acid receptor in the skin. The epidermal layer of the skin is the site of active arachidonic acid metabolism. The main product of epidermal keratinocytes is the 12-lipoxygenase derivative 12(S)-hydroxy-eicosatetraenoic acid (12(S)-HETE). Its biological effects in skin are mediated via specific, high affinity binding sites present on both keratinocytes and epidermal antigen presenting Langerhans cells. The main biological effect is chemotaxis of keratinocytes suggesting a physiological role of 12-HETE in cutaneous wound healing. Analysis of 12-HETE receptors in various cutaneous disease states revealed a dramatic defect in lesional and uninvolved psoriatic skin which may represent a central molecular defect in the pathophysiology of the disease.


Analysis of cysteinyl leukotrienes and leukotriene B4 by gas chromatography-(tandem) mass spectrometry. Gas chromatographic-(tandem) mass spectrometric (GC-MS(-MS)) methods for the analysis of cysteinyl leukotrienes (LTs) and leukotriene B4 (LTB4) in biological fluids were developed. The enzymatic synthesis of the stable-isotope labelled analogues [1,1-18O2] LTE4 and [14,15,17,17,18,18-2H6] LTB4 in high isotopic purity is described. Utilizing [1,1-18O2] LTE4 as an internal standard and GC-MS-MS enhanced cysteinyl LTs synthesis was found in patients with multiple trauma and psoriasis compared to healthy volunteers, respectively. The metabolism of LTB4 by human monocytes was investigated and two novel LTB4 metabolites were structurally identified by GC-MS as 10,11-dihydro-LTB4 and 10,11-dihydro-12-oxo-LTB4.


CT diagnosis of macrodystrophia lipomatosa. A case report. Radiographs and CT scans of a 45-year-old male with progressive enlargement of his right upper limb and shoulder are presented. Extensive soft-tissue hypertrophy with linear radiolucent bands (fat) limited to the lateral aspect of the limb were seen. Exostoses-like bony overgrowth were also seen along interphalangeal joints. At CT, hypertrophic adipose tissue intermingling with muscle fibers was demonstrated, a diagnostic finding distinguishing the lesion from plexiform neurofibrolipomatosis, Klippel-Trenaunay syndrome and other angiomatous lesions.


Plain radiography, renography, and 99mTc-DMSA renal scintigraphy before and after extracorporeal shock wave lithotripsy for urolithiasis. Eighteen patients were evaluated before and 5 weeks after the first treatment with extracorporeal shock wave lithotripsy (ESWL) using abdominal plain radiography, 131I-hippuran probe renography, and 99mTc-dimercaptosuccinic acid scintigraphy. In 6 patients no urolithiasis was present on the post ESWL plain radiograph, in 7 the size had decreased, and in 5 the stone mass was unchanged. The renograms were within normal range in the 6 patients who were cured by ESWL, whereas this was the case for only 4 of the 12 who still had renal calculi. In 2 patients pelvic stones had descended into the ureter after ESWL, and the renograms indicated obstruction. Another 3 patients had ureteral stones, whereas in the remaining 7 patients only pelvic stones were found on the plain radiographs. In no patient did the scintigrams reveal scars. It is concluded that abdominal plain radiography of the urinary tract and probe renography are complementary and sufficient in the monitoring of patients with urolithiasis post ESWL.


Optical isomers of a leukotriene B4 antagonist have differential effects on granulocyte diapedesis in the guinea pig dermis. Leukotriene B4 (LTB4) is a proinflammatory product of arachidonic acid metabolism that has been implicated in a number of inflammatory diseases. When injected intradermally into the guinea pig, LTB4 has been shown to elicit a dose-dependent infiltration of granulocytes as assessed by the level of the neutrophil marker enzyme myeloperoxidase. SC-41930 [7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)propoxy]-3,4-dihydro-8- propyl-2H-1-benzopyran-2-carboxylic acid] is a potent LTB4 receptor antagonist. When compounds were coadministered along with LTB4 (35 ng) into the dermal site, racemic SC-41930, (+)-SC-41930, and (-)-SC-41930 each inhibited granulocyte accumulation with ED50 values of 340 +/- 30, 98 +/- 5.7, and 1000 +/- 142 ng, respectively; when given intravenously inhibited with ED50 values of 0.5 +/- 0.06, 0.3 +/- 0.04, and 1.4 +/- 0.19 mg/kg, respectively; and when given intragastrically inhibited with ED50 values of 1.7 +/- 0.20, 1.4 +/- 0.23, and 3.0 +/- 0.41 mg/kg, respectively.


In vivo 1H, 31P-[1H] and 13C-[1H] magnetic resonance spectroscopy of malignant histiocytoma and skeletal muscle tissue in man. Human tumor and skeletal muscle tissue was studied by means of in vivo 1H, 31P, and 13C MRS. The examinations were performed with a 1.5 T whole-body MR scanner equipped with a second RF system. Localized 1H and broadband proton-decoupled 31P and 13C MR spectra were obtained in measurement times of 5, 10 and 33 min, respectively, from a malignant fibrous histiocytoma in a patient and from gastrocnemius muscle tissue in healthy volunteers. Proton decoupling enhanced the sensitivity (via the nuclear Overhauser effect) and the information available from in vivo 31P and 13C MR spectra significantly. The most information was obtained from 1H-decoupled 31P spectra. Observation of more than one spin species allows peak assignments to be verified mutually. The spectral data of the histiocytoma differ largely from that of muscle tissue and show a tumor with elevated pH value, normal level of nucleoside 5'-triphosphates, and high level of compounds involved in phospholipid turnover. The multinuclear in vivo MRS experiment may allow the non-invasive observation of almost the complete pathway of phospholipid synthesis and degradation in intact human tissue.


On the interpretation of proton NMR spectra from brain tumours in vivo and in vitro. Localized proton NMR spectroscopy in vivo allows focal studies of cerebral metabolites in both man and laboratory animals from image-defined regions as small as 1 mL or 64 microL, respectively. Although brain tumours lead to remarkable spectral alterations relative to normal brain, a number of problems may compromise the interpretation of the results. Potential complications arise from the chosen experimental conditions (method, TE, size and location of volume of interest), from regional metabolic heterogeneity in and around tumours, from differences between human tumours and animal models, and from discrepancies between in vivo and in vitro findings. Strategies and pitfalls are illustrated with use of selected examples from primary brain tumours, a rat tumour model and perchloric acid extracts of resected specimens.


Tumour suppressor genes.

Tumour suppressor genes. Genes responsible for the hereditary predisposition to a variety of human cancers have now been isolated. Their function seems to be part of complex signalling pathways involved in the control of cellular differentiation and the cell cycle. The presence of a single copy of these genes appears to be sufficient to ensure normal development, i.e. prevents tumorigenesis, and has earned them the name--tumour suppressor genes.


Oral ifosfamide-mesna: a clinical investigation in advanced non-small-cell lung cancer. The purpose of this study was to evaluate the toxicity and response efficacy of fixed-dose oral ifosfamide (OI)-mesna (M) in advanced, non-small-cell lung cancer (NSCLC). OI was given in four different fractionated-dose treatment schedules with a total dose per cycle of either 3.0 g/m2, 6.0 g/m2, 7.5 g/m2 or 10 g/m2 (equivalent to a daily dose of either 750 mg, 1000 mg or 1250 mg.) M was given p.o. by drinking ampules. In the 64 patients (pts) included, a total of 305 treatment cycles were administered with no evidence of severe neurotoxicity. Twenty-two pts (37%) developed mild to moderate CNS toxicity. Neither myelosuppression, alopecia, gastrointestinal toxicity nor urotoxicity were clinical problems. On schedule 2 (6 g/m2), 3 of 14 evaluable pts (21%) had partial remissions (PR), and on schedule 3 (7.5 g/m2) 4 pts (25%) showed PRs. The median duration of response was 9 months (mts) for pts on schedule 2, and 8 mts for pts on schedule 3. We conclude that OIM can easily be tolerated in the same dose usually given intravenously (7.5 g/m2/mts), when patients at high risk for developing CNS toxicity have been previously excluded from therapy. In order to reduce CNS toxicity, it is suggested that the total dose per cycle should not exceed 7.5 g/m2 (1000 mg daily) within a fractionated-dose, 14-day treatment schedule. We further conclude that the tumor response efficacy of OIM in NSCLC is comparable to the one achieved by intravenously-administered IM, whereby the total monthly OI dose should not be less than 6.0 g/m2 (750 mg daily).


1H NMR spectroscopy of subcutaneous tumors in mice: preliminary studies of effects of growth, chemotherapy and blood flow reduction. This study evaluates a number of methods for obtaining 1H NMR spectra with adequate suppression of lipid and water resonances in two subcutaneously implanted transplantable tumor models (RIF-1 and EMT6/SF). Spin-echo spectra with long TEs (270 ms; water suppressed by presaturation) eliminated lipid resonances from 1H spectra of RIF-1 and decreased lipid contamination in spectra of EMT6/SF; however, spectral sensitivity was substantially reduced. A shorter TE (135 ms) increased sensitivity but did not result in adequate suppression of the lipid peaks. In RIF-1, but not EMT6/SF, adequate lipid suppression was achieved by: (i) spatially selective presaturation of lipid, which in this tumor (but not in EMT6/SF) was localized in a thin region along the periphery of the tumor, followed by a 1-D spin-echo chemical shift imaging pulse sequence (TE = 135 ms); and (ii) 2-D spin-echo chemical shift imaging (TE = 270 ms; approximately 2 x 2 x 9 mm3 voxels). Preliminary 1H studies of the RIF-1 tumor indicate that: (i) there are no significant changes in metabolite levels relative to tumor water during 4 days of untreated tumor growth; (ii) tumor response to chemotherapy with 5-fluorouracil results in a decrease in intensity of all metabolite 1H resonances relative to tumor water, with total choline decreasing the most and lactate the least; and (iii) acute tumor blood flow reduction induced by administration of hydralazine results in doubling of the lactate intensity relative to water.(ABSTRACT TRUNCATED AT 250 WORDS)


Studies of human tumors by MRS: a review. The literature describing 31P, 1H, 13C, 23Na and 19F MRS in vivo in human cancers is reviewed. Cancers have typical metabolic characteristics in 31P and 1H MRS including high levels of phospholipid metabolites and a cellular pH more alkaline than normal. These alone are not specific for cancer but are diagnostic in appropriate clinical settings. Some metabolic characteristics appear to be prognostic indices and correlation with treatment response is emerging as an important potentially cost-effective use of MRS in oncology. 19F MRS examines pharmacokinetics of 5-fluorouracil and by demonstrating its retention predicts response of a cancer to treatment. Current needs include improvement of diagnostic specificity by use of techniques like multivoxel MRS, proton decoupling of 31P, short echo time and fat-suppressed 1H MRS, 13C MRS direct or via 1H-observe, and statistical analysis of multiple spectral features. Trials in large populations in well defined clinical settings are needed to determine if MRS can provide independent prognostic indices useful in cancer management.


Intravenous 6-thioguanine or cisplatin, fluorouracil and leucovorin for advanced non-small cell lung cancer: a randomized phase II study of the cancer and leukemia group B. This randomized phase II study was designed to evaluate the activity of intravenous 6-thioguanine (6-TG) as a single agent and the combination of cisplatin and 5-fluorouracil (5-Fu) modulated by oral leucovorin (PFL) in patients with advanced non-small cell lung cancer (NSCLC). Eligible patients had measurable or evaluable stage III B or IV NSCLC, had no received prior chemotherapy and had a performance status of 0-2. Patients were randomized to treatment with intravenous 6-TG at 55 mg/m2 administered over 30 minutes for 5 consecutive days and repeated every 35 days, or PFL chemotherapy with cisplatin 100 mg/m2 on day 1, 5-FU 800 mg/m2/day as a continuous intravenous infusion over 5 days and oral leucovorin administered at 100 mg every 4 hours during the entire duration of the cisplatin and 5-FU infusions. PFL was repeated every three weeks. Ninety-five eligible patients were randomized, 46 to 6-TG and 49 to PFL. Response rates were 4% for 6-TG (95% confidence interval 0.5%-14.8%, 1 partial, and 1 complete response) and 29% (16.6%-43.3%) for PFL (all partial). The median time to treatment failure was 2 and 4 months, respectively, and the median survival times were 6 and 10 months, respectively. Toxicities with 6-TG were, generally, mild to moderate but severe or life-threatening granulocytopenia was observed in 21% of patients. With PFL, mucositis was dose-limiting, and 78% of patients had severe or life-threatening mucositis. This led to dose reduction of 5-FU and leucovorin during subsequent cycles or treatment termination in 82% of patients.(ABSTRACT TRUNCATED AT 250 WORDS)


Hodgkin's disease and Epstein-Barr virus. Seroepidemiological and molecular biological studies have established an association of Hodgkin's disease with Epstein-Barr virus (EBV). Recently, EBV genomes and gene products, most notably the latent membrane protein (LMP), have been detected in approximately 50% of the cases. The findings suggest that EBV may contribute to the pathoetiology of a sizable proportion of Hodgkin's disease cases.


Soluble CD8, CD25 and CD30 antigens as prognostic markers in patients with untreated Hodgkin's lymphoma. In a search for serum markers with prognostic value and specificity for disease activity in Hodgkin's lymphoma, we evaluated the clinical significance of soluble suppressor/cytotoxic T-cell antigen (sCD8), soluble interleukin-2 receptor (sCD25) and soluble Hodgkin's associated antigen (sCD30) levels in the serum of 90 patients with untreated Hodgkin's lymphoma (HD). HD patients in advanced stages had significantly higher sCD8 and sCD25 levels than normal controls. The rate of detectable sCD30, which was absent in healthy controls, depended on stage and histological subtype, and was 22% for the entire group. Low sCD25 levels (< 1000 U/ml) predicted an excellent prognosis (100% event-free survival), while patients with high sCD8 levels (> 750 U/ml) or the demonstration of sCD30 had a poor outcome. Therefore, all three markers have prognostic significance. However, only sCD30 correlated strictly with disease activity and seems to be of value for the follow-up of patients in remission.


Biology of Hodgkin's disease.

Biology of Hodgkin's disease. The biology of Hodgkin's disease is one of the most intriguing subjects in lymphoma research. The presence of only a small proportion of neoplastic cells and a vast majority of reactive cells reflects the presence of complex interactions between these cell types. In this paper we discuss findings indicating that Reed-Sternberg cells may be virally transformed cells that have evaded a cytotoxic immune response and induce an ineffective delayed type hypersensitivity reaction.


Immunoscintigraphy in Hodgkin's disease and anaplastic large cell lymphomas: results in 18 patients using the iodine radiolabeled monoclonal antibody HRS-3. Immunoscintigraphy (IS) using the HRS-3 Hodgkin associated monoclonal antibody (MoAb) was performed in 18 patients with Hodgkin's Disease (HD) at staging or restaging. Either F(ab')2 fragments (14 patients) or whole HRS-3 (4 patients) labeled with 77-260 Mbq 131-I were used. Whole body images, including anterior and posterior views, were taken from a digital gamma camera, within 4 to 8 hours after injection and then daily for 5 days. In one patient IS was discontinued due to iodine intolerance. Seventeen patients were evaluable: 14 showed a true positive result including 2 cases which were reviewed as anaplastic large cell lymphoma (ALCL). Nodal, splenic, bone marrow and muscle involvements were imaged, many of these sites were previously unsuspected. In 7 patients with true positive findings the Pressman Specificity Index, as measured from biopsied material, ranged from 1.5-3 in 4 patients and from 5 to greater than 100 in 3 patients. Imaging was equivocal or failed in 1 patient (lymph nodes). In 2 patients, IS imaging was truly negative due to the absence of active HD, and a false negative result occurred once (inguinal node). In none of the patients a false positive image was observed. In order to rule out non-specific iodine uptake a control, anti-ACE MoAb, labeled with 125-Iodine was injected simultaneously in 10 patients. The evaluation of the study gave a sensitivity of 87% and a good specificity. IS using radioiodine labeled MoAbs is feasible and represents a reliable non-invasive diagnostic method for the staging and follow-up of HD and ALCL.


Oncogenes in Hodgkin's disease.

Oncogenes in Hodgkin's disease. The following manuscript reviews data presented at the Cologne meeting relating to oncogene expression in Hodgkin's disease. Presented data ranged from investigations of oncogene expression in cell lines, where transcripts of unique size were identified and lineage related expressions of transcription factors described to detailed cytogenetic investigations of fresh Hodgkin's biopsy tissue. Particular attention was centred on discrepancies in the described expression of t(14; 18) and the molecular demonstration of translocated bcl-2 breakpoints in Hodgkin's disease. A large volume of data was presented relating to the relative expression of bcl-2 breakpoints by either genomic hybridization or hybridization following DNA amplification, the expression of the bcl-2 protein or the defined cytogenetic presence of the translocation. Certain other cytogenetic abnormalities of interest in Hodgkin's disease were discussed.


Experimental therapy in Hodgkin's disease. Hodgkin/Reed-Sternberg (H-RS) cells express lymphoid activation markers like CD25 and CD30 which are present only on a small minority of normal cells. Currently, most experimental approaches in Hodgkin's lymphoma are aimed at targeting H-RS cells via monoclonal antibodies against CD25 and CD30: immunotoxins constructed by linking the antibody moiety chemically to deglycosylated ricin A-chain destroy up to 60% of small H-RS tumors in mice. The most potent immunotoxin is currently being scaled up for clinical trials. Other experimental strategies use bispecific constructs that, after binding to the cell surface of H-RS cells, convert prodrugs into their toxic counterparts, or employ monoclonal antibodies for active immunotherapy.


Subclassification of class I antiarrhythmic drugs: enhanced relevance after CAST. Class I antiarrhythmic drugs are traditionally divided into three subclasses--Ia, Ib, and Ic--on the grounds of differences in kinetics of interaction with the sodium channel and different effects on the duration of the action potential. The CAST study has highlighted our growing awareness of the proarrhythmic potential of this group of agents, particularly the Class Ic subgroup. Class I drugs can cause arrhythmias either by slowing conduction to critical levels, thus enhancing the possibility of reentrant arrhythmias, or in some cases by prolonging action potential duration, leading to early afterdepolarizations, which probably underlie triggered automaticity. Evidence is presented that the Class Ic compounds may be inherently more proarrhythmic than the Ib compounds, because of their lesser ability to depress ischemic myocardium selectively. Arguments are advanced for the continued use of a slightly modified subclassification of Class I antiarrhythmic drugs.


Acute and chronic effects of lisinopril on renal and systemic hemodynamics in hypertension. Acute and chronic effects of the converting enzyme inhibitor lisinopril on renal and systemic hemodynamics were studied in 12 patients with mild to moderate essential hypertension. After a washout period, cardiac output (measured by Doppler echography), renal plasma flow, and glomerular filtration rate (measured by isotopic techniques) were determined before and after oral administration of 20 mg lisinopril (visit 1). The same protocol was repeated after 3 months of lisinopril therapy 20 mg once daily (visit 2). Acute administration of lisinopril, both in untreated hypertensive patients (visit 1) and during long-term treatment (visit 2), decreased blood pressure (p < 0.05) and increased renal plasma flow (p < 0.05), while cardiac output and glomerular filtration rate were unchanged. Comparison of baseline parameters between visits 1 and 2 showed that chronic treatment with lisinopril decreased blood pressure and renal vascular resistance and that these effects were still significant 24-hours postdosage. We conclude that lisinopril is an effective antihypertensive agent with favorable renal hemodynamic effects.


Neuronal organization and plasticity in adult monkey visual cortex: immunoreactivity for microtubule-associated protein 2. Immunocytochemical staining for microtubule-associated protein 2 (MAP 2) was used to examine the morphology of neurons, the organization of neuronal groups, and the neurochemical plasticity of cells in adult monkey area 17. MAP 2-immunostained neurons are present through the depth of area 17 but are most intensely immunoreactive in layers IVB and VI. From layer IVB, separate groups of MAP 2-positive cells invade layers IVA and IVC alpha. Clusters of cells protrude upward from superficial layer IVB and occupy the central core regions of the cytochrome oxidase (CO)-stained honeycomb in layer IVA, while large neurons typical of layer IVB are distributed in irregular clusters in the subjacent layer IVC alpha. The somata in the layer IVA honeycomb cores give off immunostained dendrites which remain largely within the core regions. Patches of MAP 2-positive neurons are also present in layers II and III, where they coincide with the CO-stained puffs. Intravitreal injections of tetrodotoxin (TTX) into one eye of adult monkeys produce stripes of alternating light and dark MAP 2 immunostaining in layer IVC. Stripes of light immunostaining coincide with stripes of light CO staining, and correspond to reduced MAP 2 immunoreactivity within cortical neurons dominated by the TTX-injected eye. In layers II and III, the MAP 2 immunostaining of patches overlying the injected-eye columns is similarly reduced. No change occurs in the MAP 2 immunostaining of layer IVA. These data suggest that the anatomical and physiological heterogeneity of layers IVA and IVC alpha arises from the periodic invasion of neurons characteristic of layer IVB, that the neurons in layer IVA have dendrites confined to thalamocortical-recipient or nonrecipient zones and that the expression of MAP 2 changes in adult cortical neurons following the loss of retinal input.


Glomus tumour of the palate: case report and review of the literature. Glomus tumours are benign vascular tumours, usually located in the skin. Intra-oral glomus tumours are rare. A glomus tumour of the palate is reported, only the fifth such case out of a total of 14 intra-oral glomus tumours in the world medical literature.


Oromandibular-limb hypogenesis syndrome: type II A, hypoglossia-hypodactylia--report of a case. A case of oromandibular-limb hypogenesis syndrome, Type II A, hypoglossia-hypodactylia is reported. The features essential for the diagnosis of this congenital defect include a reduction in tongue size, hypoplastic mandible and variable limb anomalies.


The mobile receptor hypothesis revisited: a mechanistic role for hormone receptor lateral mobility in signal transduction. Recent application of the technique of fluorescence photobleaching recovery to direct measurement of the lateral mobility of plasma membrane-localized hormone receptors has shed new light on the role of receptor lateral mobility in signal transduction. Receptors for insulin and EGF have been known for some time to be largely immobile at physiological temperatures. This presumably relates to their signal transduction mechanism, which appears to require intermolecular autophosphorylation (receptor aggregation) for activation. In contrast, G-protein coupled receptors must interact with other membrane components to bring about signal transduction, and it is interesting in this regard that the adenylate cyclase (AC) activating vasopressin V2-receptor is highly laterally mobile at 37 degrees C. It has recently been possible to reversibly modulate the V2-receptor mobile fraction (f) to largely varying extents, and to demonstrate thereby a direct effect on the maximal rate of in vivo cAMP production at 37 degrees C in response to vasopressin. A direct correlation between f and maximal cAMP production indicates that f may be a key parameter in hormone signal transduction in vivo, especially at sub-KD (physiological) hormone concentrations, with mobile receptors being required to effect G-protein activation.


Comparison of the molecular species patterns of phosphatidic acid, CDP-diacylglycerols and phosphatidylinositol in potato tuber, pea leaf and soya-bean microsomes: consequences for the selectivity of the enzymes catalyzing phosphatidylinositol biosynthesis. Microsomes prepared from pea leaf, potato tuber or germinated soya-beans, were incubated for 30 min with [14C]glycerol 3-phosphate. In the three tissues, phosphatidic acid (PA), CDP-diacylglycerols (CMP-PA) and phosphatidylinositol (PI) were labelled and could be separated by TLC. After methylation of phosphatidic acid, or treatment of CMP-PA by a nucleotidase, the molecular species composition of the three lipid classes could be determined by radio-HPLC. The similarity observed between the distributions of radioactivity among CMP-PA and PA molecular species, in the three tissues, indicates that the enzyme CTP:PA cytidylyltransferase did not present any selectivity towards any molecular species of PA. In contrast, only two molecular species containing palmitic acid (16:0/18:2 and 16:0/18:3) were labelled in PI whereas labelled PA and CMP-PA contained molecular species possessing stearic acid (18:0/18:2, 18:0/18:3 and 18:0/18:1). This indicates that the enzyme PI-synthase utilizes preferentially those molecular species of CMP-PA containing palmitic acid as substrates. However, mass analyses of PI prepared from the microsomes of the three tissues used in this study, indicated the presence of molecular species containing stearic acid (18:0/18:2 and 18:2/18:2). Except in soya-bean microsomes (where 18:0/18:2-PI represented 16% of total PI), those last molecular species were always present in small amounts.


Reversibility of the reactions catalyzed by cholinephosphotransferase and ethanolaminephosphotransferase solubilized from rat-brain microsomes. The incorporation of CMP into CDP-ethanolamine and CDP-choline, catalyzed by ethanolaminephosphotransferase (EC 2.7.8.1) and cholinephosphotransferase (EC 2.7.8.2), respectively, has been studied in solubilized preparations of rat-brain microsomes. Mn2+ ions were required for the maximal activity of both enzymes. The CMP concentration needed to reach the half-maximal reaction rate was 1.6 microM for both activities. The rate of incorporation of CMP into CDP-choline and CDP-ethanolamine was increased by increasing the concentration of phosphatidylcholine and phosphatidylethanolamine, respectively, in detergent-phospholipid micellar systems. The rate of the reaction at pH 6.5 was comparable with that measured at pH 8.5, whereas the rate of synthesis of phosphatidylcholine and phosphatidylethanolamine, catalyzed by the same enzymes, increased with pH. Ethanolaminephosphotransferase, which catalyzes the synthesis of phosphatidylethanolamine from CDP-ethanolamine and diacylglycerol, was co-eluted with the enzyme activity catalyzing the reverse reaction, when solubilized microsomes were submitted to anion exchange chromatography on DEAE Bio-Gel A. Cholinephosphotransferase was inactivated during the chromatographic procedure.


Signal-transducing G proteins and antidepressant drugs: evidence for modulation of alpha subunit gene expression in rat brain. Signal-transducing G proteins, heterotrimers formed of alpha, beta, and gamma subunits, are central to the coordination of receptor-effector communication. They are derived from a large gene family, and recent cloning and sequencing of cDNAs encoding the alpha subunits, which confer receptor and effector specificity on the heterotrimer, have defined four major classes, Gs, Gi, Gq, and G12, with at least 16 isotypes. The G proteins that coordinate receptor-effector activity are especially important in the central nervous system (CNS), where they serve widespread, critical roles in the regulation of neuronal function, maintain the functional balance between neurotransmitter systems, and, as such, represent attractive potential targets for antidepressant drugs. We describe an integrated series of animal and cell culture studies aimed at testing the hypothesis that alterations in G protein function may contribute the complex neuroadaptive mechanisms involved in the clinical actions of antidepressants, and demonstrate that long-term administration of a wide spectrum of antidepressant drugs regulate G alpha s, G alpha i1, G alpha i2, G alpha o, G alpha q, and G alpha 12 mRNA and protein expression in various areas of the rat brain. Additionally, we present the polymerase chain reaction-(PCR) mediated cross-species partial cDNA cloning and sequencing of rat and human G alpha o and rat G alpha 12, illustrate the regional distribution of G alpha mRNA and protein in rat brain, and provide evidence that different classes of antidepressants alter expression and/or stability of the recently identified G alpha 12 mRNA. We conclude that long-term treatment with antidepressant drugs exerts differential effects on G alpha mRNA and protein expression in rat brain, thus modifying signal transduction as an integral part of complex neuroadaptive mechanisms that may underlie their therapeutic efficacy. The development of novel drugs with G proteins as primary targets remains an attractive prospect for the future.


Endogenous digitalis-like factors (EDLF) in obese individuals: preliminary results. We measured by RIA the serum and urinary digoxin-like immunoreactivity (EDLF) in 8 subjects with severe obesity and in 10 healthy, non-obese individuals (as a control group), to evidentiate whether circulating and urinary levels of EDLF are increased in obesity. For each individual, we measured the mean EDLF on two sera collected consecutively on two successive mornings, between 8-9 a.m. and the daily urinary EDLF excretion. Every subject collected his/her 24 hour urine in 5 different timed fractions. For each urine fraction, we measured the excretion of EDLF, electrolytes (Na and K), and creatinine. In obese people, the mean serum digoxin-like immunoreactivity (no. 8, 27.3 +/- 8.7 ng/L de) was significantly higher (unpaired t test, p = 0.0002) than in the controls (no. 10, 12.0 +/- 7.3 ng/L de), whereas control and obese subjects had superimposable 24 hour EDLF urinary excretion. Urinary excretion of EDLF significantly changed throughout the day in normals, but not in obese people. In a multiple stepwise regression analysis, urinary K+ and Na+ significantly (p less than 0.01) contributed to the regression with urinary EDLF (EDLF = 76.9 + 0.67 K+ - 0.24 Na+; R = 0.601, no. 40) in obese individuals. The in vivo kinetic and metabolic pathways of EDLF are conceivably different in obese and normal subjects. A difference in the production rate, binding to plasma proteins and/or removal mechanisms could explain the findings of higher circulating levels with normal EDLF urinary excretion in obese persons.(ABSTRACT TRUNCATED AT 250 WORDS)


Plasminogen: a structural review.

Plasminogen: a structural review. Plasminogen is the zymogen form of plasmin, a broad specificity serine protease whose activity contributes to a variety of normal and pathological conditions, including intravascular thrombolysis and extracellular proteolysis. Plasminogen contains seven structural units or 'domains', each of which confer specific properties on the molecule. The kringle domains possess fibrin-binding functions and, together with the N-terminal peptide, regulate the ability of plasminogen to adopt at least three dissimilar conformations. These conformational forms influence the rate of formation, following activation by plasminogen activators, of the plasmin active site within its C-terminal serine protease domain. Structural and functional analogies are postulated between these plasminogen structures and the conformations of other proteins related by sequence homology.


[Cytomegaloviruses in the bronchoalveolar lavage fluids of immunocompromised patients: microbiological results and clinical significance]. We have evaluated the microbiologic output and clinical significance of the detection of cytomegalovirus in 111 bronchoalveolar lavage specimens from immunosuppressed patients with pneumonitis. The samples were simultaneously processed by conventional tube culture and the rapid shell-vial centrifugation culture assay. Cytomegalovirus was recovered from 30 specimens (27%). The rapid shell-vial procedure was more sensitive than the tube culture, but in two cases cytomegalovirus was isolated only in tube cultures. Cytomegalovirus was considered clinically significant in only 3 from 13 HIV positive patients. All culture positive, HIV negative patients received treatment with ganciclovir. However, ganciclovir was never used on culture negative, HIV negative patients and cytomegalovirus related morbi-mortality was not found in these patients. A prospective study is needed to conclude if a cytomegalovirus negative culture also has a treatment exclusion value in HIV positive patients.


Clinical aspects of platelet transfusion. Several clinical complications of platelet transfusions relate to contaminating donor leucocytes, and a number of strategies have been devised to leucodeplete platelet products before transfusion. Both alloimmunization to class I human leucocyte antigens (HLA), which causes febrile transfusion reactions and refractoriness to transfused platelets, and transmission of cytomegalovirus have been shown to be reduced by 3-log10 leucodepletion by filtration. Lesser degree of leucodepletion, e.g. by platelet preparation from buffy coats, will control febrile transfusion reactions, but will not reliably prevent other complications. The clinical implications and cost-effectiveness of different strategies of platelet production remain a matter of debate.


Vulvar and vaginal disorders.

Vulvar and vaginal disorders. Vulvovaginitis is the most common reason for a woman to see her gynecologist. The most common causes are bacterial vaginosis, Candida species, and Trichomonas vaginals, respectively. Human papillomavirus has become a major diagnostic and therapeutic problem. Human papillomavirus DNA testing suggests that exposure to human papillomavirus occurs frequently at birth and intermittently throughout a woman's life after the onset of sexual activity. Interferon has been shown to be effective in treating some forms of human papillomavirus.


Functional integrity of proximal tubule cells. Effects of hypoxia and ischemia. Effects of warm hypoxia and ischemia on electrophysiologic properties of isolated perfused mouse proximal straight tubules were studied. Oxyrase (5 to 10 microliters/mL) was added to the hypoxic and ischemic solutions to lower the oxygen tension to 5 mm Hg. The ischemic solution also simulated acidosis, K+ and lactate accumulation, and substrate deprivation. Twenty-minute tubular perfusion with the hypoxic and ischemic solutions (lumen and bath) at 37 degrees C did not significantly alter basolateral membrane potential, basolateral K+ transference number, or intracellular Na+ activity from control values of -69 +/- 1 mV (N = 91), 0.71 +/- 0.01 (N = 15), and 15.2 +/- 0.8 mM (N = 12), respectively. However, the hypoxic and ischemic perfusions decreased transepithelial potential by 40% (hypoxia: -1.7 +/- 0.1 to -1.1 +/- 0.1 mV [N = 30; P < 0.001]; ischemia: -1.4 +/- 0.1 to -0.82 +/- 0.05 mV [N = 17; P < 0.001]). A similar extent of reduction in transepithelial resistance was observed (hypoxia: 14.3 +/- 1.0 to 9.2 +/- 1.1 omega.cm2 [N = 7; P < 0.005]; ischemia: 12.6 +/- 1.2 to 8.1 +/- 1.0 omega.cm2 [N = 6; P < 0.03]). In addition, neither apical (R(ap)) nor basolateral (Rbl) cell membrane resistances were significantly altered after the ischemic perfusion (control: R(ap) = 369 +/- 48 omega.cm2; Rbl = 92 +/- 11 omega.cm2 [N = 63]; reperfusion: R(ap) = 454 +/- 88 omega.cm2; Rbl = 101 +/- 16 omega.cm2 [N = 21]). It was concluded that tubular cells are able to maintain their electrogenic ionic transport after short-term exposure to hypoxic or ischemic conditions. However, cell-to-cell junctions are damaged by these insults, which could possibly increase leakage and decrease the efficiency of the active transport.


Cyst fluid from human autosomal dominant polycystic kidneys promotes cyst formation and expansion by renal epithelial cells in vitro. Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive renal enlargement, culminating in renal insufficiency in over one half of affected individuals. The highly variable onset and clinical course of ADPKD may be due to factors extrinsic to the genetically defined renal cysts. In this study, cyst fluid samples from 12 nonazotemic and 18 azotemic ADPKD subjects were examined for in vitro biologic activity that promotes cellular proliferation and the secretion of fluid by renal epithelial monolayers, two pathogenetic mechanisms that have critical roles in the formation and the rate of expansion of renal cysts. Cyst fluid added to culture medium (final concentrations, 1 to 20%) caused Madin-Darby canine kidney cells and human kidney cortex (HKC) cells derived from primary cultures to form cysts in Type I collagen matrix. Cyst fluid stimulated the net transepithelial secretion of fluid by polarized monolayers composed of these same cells. Absolute levels of fluid secretory activity determined by MDCK bioassay were correlated directly with the rate of fluid secretion by HKC cell monolayers and with the extent of cyst formation by MDCK and HKC cells embedded in collagen matrix. The secretory activity of urine was negligible; secretory activity was detectable in the serum of normal and ADPKD subjects, but the levels were much lower than in cyst fluid. cAMP agonists prostaglandins E1 and E2, arginine vasopressin, and 8-Br-cAMP stimulated fluid secretion by MDCK and HKC monolayers, but these substances did not cause HKC cells to form cysts in collagen matrix, whereas cyst fluid did. Among other naturally occurring growth factors and autacoids, only epidermal growth factor and transforming growth factor alpha stimulated cyst formation by HKC cells; however, the capacity of cyst fluid to stimulate fluid secretion was not affected by treatment with antiserum to epidermal growth factor. It was concluded that potent, and possibly unique, substances in the cyst fluids of individuals with ADPKD support and augment biologic processes in renal epithelial cells that may be important in the promotion of progressive cyst expansion.


Herbicide-resistant Indica rice plants from IRRI breeding line IR72 after PEG-mediated transformation of protoplasts. The commercially important Indica rice cultivar Oryza sativa cv. IR72 has been transformed using direct gene transfer to protoplasts. PEG-mediated transformation was done with two plasmid constructs containing either a CaMV 35S promoter/HPH chimaeric gene conferring resistance to hygromycin (Hg) or a CaMV 35S promoter/BAR chimaeric gene conferring resistance to a commercial herbicide (Basta) containing phosphinothricin (PPT). We have obtained so far 92 Hgr and 170 PPTr IR72 plants from protoplasts through selection. 31 Hgr and 70 PPTr plants are being grown in the greenhouse to maturity. Data from Southern analysis and enzyme assays proved that the transgene was stably integrated into the host genome and expressed. Transgenic plants showed complete resistance to high doses of the commercial formulations of PPT.


Studies on the clinical significance of the clonal origins of recurrent hepatocellular carcinoma. In order to understand the clonal origins of recurrent hepatocellular carcinoma (HCC), as well as their clinical significance, the DNA contents of 44 resected recurrent HCCs from 21 patients were quantitatively measured using image analysis techniques. In 8 patients (38.1%), the recurrent HCCs had DNA contents which were obviously different from those in their primary tumors, suggesting new clonal origins; 13 (61.9%) had similar DNA contents, suggesting the same clonal origins. In these 2 groups, the mean intervals between 2 or 3 operations were 25 +/- 9.9 months and 7.9 +/- 3.1 months (P < 0.01), respectively, and the mean postoperative survival times were 51 +/- 20.8 months and 27.7 +/- 14.2 months (P < 0.05), respectively. The results indicate that the majority of recurrent HCCs originate from unresected fragments of their primary tumors, while some of them clearly originate from new tumor clones. Finally, reoperative resection is still considered an effective treatment for recurrent HCC, especially for those which originate from new tumor clones.


cDNA cloning of a tetraubiquitin gene, and expression of ubiquitin-containing transcripts, in aleurone layers of Avena fatua. A lambda gt 11 cDNA library, constructed from poly(A)+ mRNA isolated from Avena fatua aleurone layers incubated with 1 microM gibberellin A1 (GA1) for 4 days, was screened with an anti-idiotypic antiserum raised against the GA-specific monoclonal antibody MAC 182. One positive clone was isolated, sequenced and shown to encode a tetraubiquitin based on the deduced amino acid sequence. This polyubiquitin cDNA exhibited a high degree of homology to a cloned wheat hexaubiquitin in its 3'-non-coding region. Analysis of total RNA isolated from A. fatua aleurone layers, treated without or with a range of concentrations of GA1 from 10(-11) to 10(-6) M, by northern blotting using the cDNA probe revealed 8 different ubiquitin-containing transcript classes all of which are constitutively expressed in aleurone and are regulated by GA1.


Construction of an Rb gene expression plasmid. We obtained an 844 bp Bg1II fragment from an Rb cDNA clone and inserted it into the expression vector pWR-13 to construct an Rb gene expression plasmid. When the Rb Bg1II fragment was fused in-frame into pWR-13, it was operated by a Lac Z promoter and produced a fusion protein which consisted of expressed Rb protein and a small peptide from Lac Z. The recombinants were transformed into E. coli with the CaCl2 method, screened by in situ hybridization, and restriction mapped. Total cellular protein of transformed clones was analyzed by SDS-PAGE and Commassie blue staining. The sense clones showed a unique band at 28,000. On Western blot, this band specifically reacted with 125I-labelled antibody against synthetic Rb peptide. This protein comprised more than 5% of total bacterial protein.


Endocytosis and lysosomal delivery of tissue plasminogen activator-inhibitor 1 complexes in Hep G2 cells. Receptor-mediated endocytosis of tissue-type plasminogen activator (t-PA)-plasminogen activator inhibitor type 1 (PAI-1) complexes results in their clearance by Hep G2 cells. After complexes are internalized, the t-PA component is degraded. However, neither the locus of intracellular catabolism nor the fate of PAI-1 has been elucidated. To characterize these aspects of t-PA-PAI-1 catabolism, the subcellular distribution of a prebound cohort of ligand molecules was delineated after internalization at 37 degrees C. 125I-t-PA.PAI-1 and t-PA.125I-PAI-1 were compared in separate experiments. After ligand uptake, intracellular vesicles were separated on density gradients. Internalized 125I-t-PA.PAI-1 concentrated initially in endosomes. After 20 minutes of uptake, the complex began to appear in lysosomes. Subsequently, low molecular weight labeled ligand fragments were detected in culture media. A panel of lysosomotropic agents, including primaquine, chloroquine, ammonium chloride, and a combination of leupeptin and pepstatin A, inhibited degradation. When t-PA.125I-PAI-1 rather than 125I-t-PA.PAI-1 was internalized, strikingly different results were observed. Although the kinetics of internalization and the intracellular itinerary were indistinguishable for the differently labeled complexes, the 125I-PAI-1 component of t-PA.125I-PAI-1 resisted rapid degradation. After a rapid loss of t-PA, the 125I-PAI-1 moiety persisted in lysosomes for up to 180 minutes. Thus, internalized t-PA.PAI-1 is targeted to lysosomes in which PAI-1 is relatively more stable than t-PA.


Tumor necrosis factor and interleukin-1 induce expression of the verocytotoxin receptor globotriaosylceramide on human endothelial cells: implications for the pathogenesis of the hemolytic uremic syndrome. The epidemic form of the hemolytic uremic syndrome (HUS), beginning with an acute gastroenteritis, has been associated with a verocytotoxin-producing Escherichia coli infection. The endothelial cell is believed to play an important role in the pathogenesis of HUS. Endothelial cell damage by verocytotoxin-1 (VT-1) in vitro is potentiated by the additional exposure of inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha). Preincubation of human umbilical vein endothelial cells (HUVEC) with TNF-alpha resulted in a 10- to 100-fold increase of specific binding sites for 125I-VT-1. Furthermore, interleukin-1 (IL-1), lymphotoxin (TNF-beta), and lipopolysaccharide (LPS) also markedly increase VT-1 binding. Several hours' exposure to TNF-alpha was enough to enhance the number of VT-1 receptors on the endothelial cells for 2 days. The TNF-alpha-induced increase in VT-1 binding could be inhibited by simultaneous addition of the protein synthesis inhibitor cycloheximide. Glycolipid extracts of TNF-alpha-treated cells tested on thin-layer chromatography demonstrated an increase of globotriaosylceramide (GbOse3cer), a functional receptor for VT-1, which suggests that preincubation of human endothelial cells with TNF-alpha leads to an increase in GbOse3cer synthesis in these cells. We conclude from this study that TNF-alpha and IL-1 induce one (or more) enzyme(s) that is (are) rate-limiting in the synthesis of the glycolipid VT-1 receptor, GbOse3cer. These in vitro studies suggest that, in addition to VT-1, inflammatory mediators play an important role in the pathogenesis of HUS.


15-Hydroxyeicosatetraenoic acid-mediated potentiation of thrombin-induced platelet functions occurs via enhanced production of phosphoinositide-derived second messengers--sn-1,2-diacylglycerol and inositol-1,4,5-trisphosphate. We investigated whether biologically relevant concentrations of the mono-hydroxyeicosatetraenoic acids (mono-HETEs) modulate platelet functions. We report that 15-HETE, an eicosanoid produced by endothelial cells, granulocytes, and lymphocytes, potentiated platelet aggregation, nucleotide release, and elevation in intracellular calcium levels induced by a threshold concentration of thrombin (0.025 U/mL). Significant potentiation effects on these responses were observed at concentrations between 1 and 100 nmol/L. 15-HETE at these concentrations enhanced thrombin-induced platelet aggregation by 32% to 57%, nucleotide release by 40% to 65%, and elevation of intracellular calcium by 31% to 52% (P < .05 to .01). Both 12-HETE and 5-HETE, the structural isomers of 15-HETE, also potentiated thrombin-induced platelet aggregation and nucleotide release. While 12-HETE showed a small but significant effect at 100 pmol/L, 5-HETE had effects similar to those of 15-HETE at micromolar concentrations. To understand the mechanism of the HETE modulation of platelet functions, we studied the effect of 10 and 100 nmol/L 15-HETE on the production of sn-1,2-diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (1,4,5-IP3). 15-HETE enhanced thrombin-induced production of DAG and 1,4,5-IP3 in a time- and concentration-dependent manner. 15-HETE also potentiated agonist-induced phosphorylation of the 47-Kd platelet protein. These studies demonstrate an important modulatory role for 15-HETE on platelet functions. Since this eicosanoid is elevated in pathologic states associated with platelet hyperfunction, including diabetes mellitus and atherosclerosis, an elucidation of its mechanism(s) of action appears relevant to our understanding of the genesis of atherothrombotic vascular disease.


Identification of a new congenital defect of platelet function characterized by severe impairment of platelet responses to adenosine diphosphate. This study characterizes a congenital hemorrhagic disorder caused by a platelet function defect with the following features: (1) severely impaired platelet aggregation and fibrinogen or von Willebrand factor (vWF) binding induced by adenosine diphosphate (ADP); (2) defective aggregation, release reaction, and fibrinogen or vWF binding induced by other agonists; (3) normal aggregation and release reaction induced by high concentrations of thrombin or collagen; (4) no further inhibition by ADP scavengers of aggregation, release reaction, and fibrinogen or vWF binding, comparable with those observed for normal platelets in the presence of ADP scavengers; (5) normal membrane glycoprotein (GP) composition and normal binding of the anti-GP IIb/IIIa monoclonal antibody 10E5; (6) no acceleration by ADP of binding of the anti-GP IIb/IIIa monoclonal antibody 7E3; (7) normal platelet-fibrin clot retraction if induced by thrombin or reptilase plus epinephrine, absent if induced by reptilase plus ADP; (8) no inhibition by ADP of the prostaglandin E1-induced increase in platelet cyclic adenosine monophosphate, but normal inhibition by epinephrine; (9) defective mobilization of cytoplasmic Ca2+ by ADP; (10) normal binding of 14C-ADP to fresh platelets, but defective binding of [2-3H]-ADP to formalin-fixed platelets. This congenital platelet function defect is characterized by selective impairment of platelet responses to ADP, caused by either decreased number of platelet ADP receptors or abnormalities of the signal-transduction pathway of platelet activation by ADP.


Determination of the DNA content of the Reed-Sternberg cell of Hodgkin's disease by image analysis. The nature of the Reed Sternberg (RS) cell, the malignant cell of Hodgkin's disease (HD), remains unknown. Cytogenetic studies have yielded ambiguous results regarding the chromosomal profile of this cell. In an attempt to further clarify the ploidy status of the RS cell, we analyzed the DNA content of CD30-positive RS cells and RS cell variants in HD lesions from 32 patients using an image analysis system. A diploid and/or near-diploid (DNA index [DI], 1.0 +/- 0.2) and a tetraploid (2.0 +/- 0.2) RS cell population were identified in 9 and in 11 of the 32 cases examined, respectively. An aneuploid RS cell population was identified in 8 of the 32 cases examined. The remaining four cases contained two RS cell subpopulations with different DNA content, each one representing more than 15% of the total RS cell population. There was no significant correlation between the DNA content of the RS cells and the category of HD. Furthermore, analysis of multiple biopsies of an individual patient taken from different lymphoid organs at the same or different time periods showed a constant DNA profile. Our data indicate that RS cells can express variable DNA content and suggests that multiple subpopulations of RS cells with different DNA content may simultaneously coexist within the same HD lesion in some patients. In addition, the RS cell population within each patient appears to express a specific DNA content profile, possibly representing unique clones. These highly individualized profiles potentially may be useful as markers to follow the clinical course of patients with HD.


Protein phosphatase inhibitors okadaic acid and calyculin A alter cell shape and F-actin distribution and inhibit stimulus-dependent increases in cytoskeletal actin of human neutrophils. The phosphatase inhibitors okadaic acid and calyculin A were found to elicit or to modify several neutrophil responses, suggesting that dephosphorylation plays a regulatory role. The concentrations of okadaic acid (> or = 1 mumol/L) that were effective on neutrophil functions (shape changes and marginal stimulation of pinocytosis) were shown to stimulate the incorporation of 32PO4 into many neutrophil proteins several-fold. Calyculin A was effective at 50-fold lower concentrations. In the presence of the inhibitors, the cells exhibited a nonpolar shape and the polarization response induced by chemotactic peptide was inhibited. Both phosphatase inhibitors also induced the association of F-actin with the cell membrane. A steady-state phosphatase activity is thus involved in maintaining shape and F-actin localization of resting cells. Inhibitors alone had no significant effect on the amount of cytoskeleton-associated actin. The increase in cytoskeletal actin observed at 30 minutes of stimulation with phorbol ester or 5 to 30 minutes of stimulation with chemotactic peptide, however, was abolished by okadaic acid or calyculin A, suggesting an important role of a phosphatase. In contrast, the early increase in cytoskeleton-associated actin observed at 1 minute of stimulation with peptide was not affected. This finding indicates that the increased association of actin with the cytoskeleton in the early and the later stages of neutrophil activation may be mediated by different signalling pathways.


The effect of methotrexate (MTX) on the small intestine of the mouse. IV. The Golgi apparatus, phosphatases and esterases. The Golgi apparatus and alkaline and acid phosphatase and nonspecific esterase activities were studied in the jejunal epithelium of adult male albino mice (Mus musculus) under normal conditions and after MTX treatment. In the control, the Golgi apparatus took the form of rods, spheres and crescents occupying the supranuclear region. After MTX, the Golgi apparatus, in most of the cells, was hypertrophied. In the control cells, alkaline and acid phosphatase and nonspecific esterase activities were moderate and localized supranuclearly, but were intense in the brush border and basement membrane. After MTX, all three enzyme activities increased, with a marked reaction in the brush border and basement membrane. The increase in alkaline phosphatase may mean that more phosphate transport is needed in the active phosphorylation process or in the transfer of MTX macromolecules across the cell membrane, or it may be due to MTX-induced disorganization of metabolism. The increase in acid phosphatase activity denotes an increase in catabolic processes resulting from imbalance of lysosomal function, while the rise in nonspecific esterase activity could be related to fatty acid metabolism, or it might be due to the detoxicant function of esterases. In all control and MTX-treated specimens, the supranuclear concentration of these enzymes coincided with the localization of the Golgi apparatus.


The role of free radicals and neutrophil elastase in development of pulmonary emphysema. Extracellular proteolysis is hypothesized to be the major cause of pulmonary emphysema and oxygen-derived free radicals and neutrophil elastase are thought to play an important role in its pathogenesis. In this study, peripheral polymorphonuclear leukocytes (PMNs) obtained from 16 patients with emphysema generated a significantly larger amount of superoxide and elastase activity than those obtained from normal controls. A significant correlation was observed between elastase activity and superoxide release. In addition, the superoxide release showed a negative correlation with the disease duration. The superoxide release appeared to correlated with a decline of FEV1.0 over the course of several years in 8 patients. It seems likely that activated PMNs play an important role in the development of pulmonary emphysema.


Late-onset homozygous protein C deficiency manifesting cerebral infarction as the first symptom at age 27. We report a 31-year-old female who had repeated thrombosis and was diagnosed as having congenital homozygous protein C deficiency based on decreased protein C antigen and activity, and the findings of family history. This patient had shown no symptom of thrombosis until the age of 27 years, when she had cerebral infarction as the first symptom. Low molecular weight heparin was useful for disseminated intravascular coagulation (DIC) that complicated protein C deficiency in this patient.


The rate of DNA damage and aging.

The rate of DNA damage and aging. A new theory of aging based on the rate of DNA damage is presented, and the relationship between the rate of oxidative DNA damage and maximum life span (MLS) of mammalian species is explored. In humans the level of oxidative DNA damage, as measured by urinary biomarkers, can be modulated by caloric restriction and dietary composition. Consequently, longevity may depend not only on the basal metabolic rate but also on dietary caloric intake and the type of diet. The theory may provide the basis for a practical approach for reduction of degenerative diseases in general, extension of life expectancy, and optimization of individual lifestyles.


Variable alpha-tocopherol stimulation and protection of glutathione peroxidase activity in non-transformed and transformed fibroblasts. Studies on glutathione metabolism in an established baby hamster kidney cell line (BHK-21/C13) and in its polyoma virus-transformed counterpart (BHK-21/PyY), have revealed a significant stimulation of intracellular glutathione peroxidase activity (Se-independent plus Se-dependent) by alpha-tocopherol supplementation (14 microM). This stimulation was found to be much greater in the transformed cells. Other GSH-requiring enzyme activities (namely glutathione reductase and glutathione transferase) were unaltered by alpha-tocopherol treatment, suggesting a degree of specificity in its action on GSHpx. In unsupplemented growth media, the GSHpx activity in both cell lines was significantly decreased by an oxidative stress. However, the same stress applied to the alpha-tocopherol-supplemented cells had no effect on the stimulated GSHpx activity, suggesting a protection afforded by the alpha-tocopherol.


Active oxygen species, articular inflammation and cartilage damage. Rheumatoid arthritis and osteoarthritis are age-related diseases, in which degenerative changes (arthrosis) and superimposed inflammatory reactions (arthritis) lead to progressive destruction of the joints. Active oxygen species derived from various sources play a role in this process, which may be influenced by appropriate treatment with antioxidants and free radical scavengers.


Oxidative damage to collagen.

Oxidative damage to collagen. Extracellular matrix molecules, such as collagens, are good targets for oxygen free radicals. Collagen is the only protein susceptible to fragmentation by superoxide anion as demonstrated by the liberation of small 4-hydroxyproline-containing-peptides. It seems likely that hydroxyl radicals in the presence of oxygen cleave collagen into small peptides, and the cleavage seems to be specific to proline or 4-hydroxyproline residues. Hydroxyl radicals in the absence of oxygen or hypochlorous acid do not induce fragmentation of collagen molecules, but they trigger a polymerization of collagen through the formation of new cross-links such as dityrosine or disulfure bridges. Moreover, these cross-links can not explain the totality of high molecular weight components generated under these experimental conditions, and the nature of new cross-links induced by hydroxyl radicals or hypochlorous acid remains unclear.


Mitochondrial production of oxygen free radicals in the heart muscle during the life span of the rat: peak at middle age. Mitochondria extracted from Wistar rat hearts at 3, 14-18 and 24 months of age showed no change in state 3-mitochondrial respiration measured in the presence of glutamate or succinate. Again no changes were found in the SMP-O2- production at the level of the rotenone-inhibited region, whilst at the level of the antimycin-inhibited region there was a marked increase in O2- production in the group of 14-18-month-old rats. In the same age period, the production of mitochondrial H2O2 supported by glutamate or succinate and the level of GSSG increased in comparison to the young group, accompanied by a decrease in the GSH level. Mitochondrial TBARS levels did not change during a life span, while a progressive accumulation in the mitochondrial lipofuscin content with age was measured.


Pulmonary blastoma--a rare tumour.

Pulmonary blastoma--a rare tumour. A 57-year-old man was found to have a tumour in the right lower lobe of the lungs, which was not classifiable by biopsy. The tumour could only be partially removed by surgical resection. The diagnosis of a pulmonary blastoma was made from the resected tissue. Clinically, rapid progress occurred with invasion in the mediastinal space and the epigastrium. In spite of radiation therapy, the patient died about 2 1/2 months after surgery of respiratory insufficiency. Autopsy confirmed a pulmonary blastoma with extensive infiltration of the mediastinal space and upper abdomen as well as metastases in the regional lymph nodes, pleura, peritoneum, thyroid gland, heart and central nervous system. The present report of a pulmonary blastoma should draw attention to this extremely rare tumour. It should be included in the differential diagnosis, because the survival time can be increased if the correct diagnosis is made very early.


Calcification of the aortic wall in hypercalcemic rabbits. The mineralization process was investigated in the aortic wall of hypercalcemic rabbits. The elevated calcium level in serum was induced by intramuscular injection of vitamin D3. The animals were killed at different times of the experiment (max. 246 d). The freeze-dried tissue homogenates were used for elemental composition studies by means of proton induced X-ray emission (PIXE) and atomic absorption spectroscopy. The structural information was obtained from infrared (IR) and X-ray diffraction (XRD) spectra. Moreover, the ascending part of the aortic arch was separated and used for micro-PIXE (PIXE in combination with proton microprobe) and histochemical examinations. It was found that hypercalcemia (blood serum Ca content elevated by about 20%) induced calcification of the aortic wall. The mineral phase within the aortic wall consisted of Ca-P salts. The Ca/P ratio continuously increased during the experiment and approached 2 after 246 d of the vitamin D3 treatment. The IR and XRD studies made possible the identification of the complex phase composition of the samples. The hydroxyapatite crystals were detected after 196 days, however, in earlier phases of the experiment, amorphous calcium phosphate, dicalcium phosphate dihydrate and octacalcium phosphate were also observed. On the basis of the data obtained, the mechanism of the precipitation and growth of inorganic deposits in the tunica media of the aortic wall was discussed.


The MRS1 gene of S. douglasii: co-evolution of mitochondrial introns and specific splicing proteins encoded by nuclear genes. We have developed a rapid and simple methodology to locate yeast genes within cloned inserts, obtain partial sequence information, and construct chromosomal disruptions of these genes. This methodology has been used to study a nuclear gene from the yeast S. douglasii (a close relative of S. cerevisiae), which is essential for the excision of the mitochondrial intron aI1 of S. douglasii (the first intron in the gene encoding subunit I of cytochrome oxidase), an intron which is not present in the mitochondrial genome of S. cerevisiae. We have shown that this gene is the homologue of the S. cerevisiae MRS1 gene, which is essential for the excision of the mitochondrial introns bI3 and aI5 beta of S. cerevisiae, but is unable to assure the excision of the intron aI1 from the coxI gene of S. douglasii. The two genes are very similar, with only 13% nucleotide substitutions in the coding region, transitions being 2.5 times more frequent than transvertions. At the protein level there are 86% identical residues and 7% conservative substitutions. The divergence of the MRS1 genes of S. cerevisiae and S. douglasii, and the concomitant changes in the structure of their mitochondrial genomes is an interesting example of the co-evolution of nuclear and mitochondrial genomes.


Signal transduction in murine B16 melanoma cells. The relationship between lung colonization and signal transduction was investigated for six B16 melanoma variants. A range of experimental metastatic potential (as determined by lung colonization), forskolin-stimulated cyclic AMP accumulation and FCS-stimulated protein kinase C activity was found. The major findings were that: (1) cells with the highest agonist-stimulated cyclic AMP production were those with the highest level of membrane-associated protein kinase C activity; (2) clones which differed in protein kinase C levels and distribution did so in the presence but not in the absence of foetal calf serum; and (3) no simple relationship was seen between either signal transduction system and lung colonization for all six variants. Altered ras expression was also excluded as an explanation for the differences in signal transduction and lung colonization potential which were observed. We conclude that differences in signal transduction in vitro between these cells do not relate simply to lung colonization potential in vivo.


Tissue-specific expression of the skeletal alpha-actin gene involves sequences that can function independently of MyoD and Id. The skeletal alpha-actin gene is a member of the sarcomeric contractile protein gene family and is specifically expressed in differentiated muscle. The skeletal alpha-actin gene is regulated efficiently by enhancer and regulatory sequences between nucleotide positions -1282 and -87. In the present study we have shown that the sequences 3' of nucleotide position -87 can functionally interact with the SV40 enhancer in a tissue-specific manner and can restrict the ubiquitous function of the SV40 enhancer to myogenic cells. Site-specific cassette mutagenesis was used to delimit the sequences upstream of the TATA motif (-32), between nucleotide positions -64 and -37, that mediate efficient expression in myogenic cells in the presence of the SV40 enhancer. The skeletal alpha-actin promoter was trans-activated by the helix-loop-helix (HLH) transcription factors MyoD, MRF-4, and Myogenin, in pluripotential 10T1/2 fibroblasts and trans-repressed by the HLH protein Id (inhibitor of differentiation) in myogenic C2C12 cells. This trans-regulation required sequences upstream of -87 and occurred independently of the two consensus E boxes (CANNTG) at positions +18 and +71. The -64/-37 region interacted with purified Sp1 and an unidentified protein(s), proximal regulatory factor(s) I (PRF-I). We conclude that the muscle-specific expression of the skeletal alpha-actin promoter is not simply determined by MyoD elements and enhancer and regulatory sequences, but that the minimal promoter contains important determinants of cell-specific transcription that can function independently of the helix-loop-helix transcription factors.


Deletion and insertion mutants of HBsAg particles. We have found previously that hybrid 22-nm HBsAg particles can be created by insertion of short antigenic sequences into the HBV major envelope protein. We have now performed a detailed deletion mutagenesis of the S gene of HBV encoding HBsAg. Deletion of the 51 C-terminal amino acids including most of the third and all of the fourth hydrophobic domain of the S protein did not affect particle assembly and secretion. However, secretion of 22-nm particles was abolished by minor deletions in the N-terminal region. Insertion and deletion/substitution mutants carrying a poliovirus epitope at the N-terminus and the preS1 region at the C-terminus have been characterized.


Recurrent herpetic keratitis during topical acyclovir application. Dendritic herpetic keratitis developed in a 49-year-old patient during topical acyclovir treatment. A positive herpes simplex culture was obtained. After acyclovir was replaced by trifluorothymidine and interferon, the dendritic lesion disappeared and herpes simplex culture became negative. Six months later a carcinoma of the larynx was diagnosed. The acyclovir-resistant herpetic keratitis may be associated with the carcinoma because resistant herpes simplex virus strains are predominantly described in patients suffering from immune deficiency.


Comparative molecular biology of flaviviruses and hepatitis C virus. Currently available sequence information suggests that the genome organization of hepatitis C virus is similar to that of flaviviruses. A positive-stranded genomic RNA contains a single long open reading frame (ORF) which is flanked by 5' and 3' noncoding sequences. This RNA codes for structural proteins at the 5' end (starting with the capsid protein) and a set of nonstructural proteins in the remainder of the genome. The latter provide essential virus-specific functions for the viral life cycle, such as protease, helicase, and RNA replicase activities. The sequence motifs characteristic of the corresponding functional protein domains are separated by similar spacings in the nonstructural regions of hepatitis C virus and flaviviruses. The structural region of the hepatitis C virus appears to consist of a capsid protein which is larger than that of flaviviruses and two putative envelope proteins which are presumably different in molecular weight and much more heavily glycosylated than their counterparts in flaviviruses. A study group of the International Committee on the Taxonomy of viruses proposes to include hepatitis C virus as a genus into the family 'flaviviridae'.


Detection and characterization of hepatitis C virus sequence in the serum of a patient with chronic HCV infection. A cDNA fragment corresponding to the nonstructural gene region of Hepatitis C virus was cloned and sequenced. cDNA was obtained by reverse transcription of viral RNA extracted from serum of a German patient with chronic post transfusion hepatitis. "Nested" PCR resulted in a cDNA fragment of 345 nt. The sequence showed a homology of 96% to the American prototype HCV.


Hepatitis C virus (HCV) and autoimmune liver diseases. Anti-HCV tests were positive in 18-45% of sera from patients with autoimmune chronic active hepatitis. High gammaglobulin levels may result in false positive results, however, some sera show true positivity. PCR testing of such sera is necessary in order to determine whether HCV is directly involved in specific forms of the disease.


Antibodies to hepatitis C virus in primary biliary cirrhosis. We investigated the prevalence of anti-HCV in 160 consecutive patients with primary biliary cirrhosis. By ELISA, 19 (12%) were positive, as compared to a 68% prevalence in 135 patients with chronic non-A, non-B hepatitis. Serum IgG levels were significantly higher in the anti-HCV positive group. By RIBA, seropositivity was confirmed for 4 patients, whereas 7 were indeterminate. A slight, non-significant reduction of life expectancy was found in anti-HCV positive patients. Until reliable and independent confirmatory tests become available, definitive conclusions on the importance of anti-HCV positivity in primary biliary cirrhosis are improper.


Prevalence of antibodies against hepatitis C virus in primary biliary cirrhosis and autoimmune chronic hepatitis. We have determined the prevalence of antibodies against hepatitis C virus (anti-HCV) in 45 patients with primary biliary cirrhosis (PBC) and 6 with autoimmune chronic active hepatitis (AI-CAH). Anti-HCV was positive in two cases of PBC, both with a history of previous blood transfusion, and in one patient with AI-CAH, only during an active phase of the disease.


Confirmation of anti-HCV EIA reactivities by RIBA and neutralization assay among blood donors and patients with chronic liver disease and hepatocellular carcinoma. The aim of our study was to confirm by Recombinant Immunoblot Assay (RIBA) and by neutralization assay the repeat positive reactions found by two commercially available EIAs (Ortho and Abbott) when testing samples from volunteer blood donors, patients with chronic liver disease and with hepatocellular carcinoma. Our data show a high confirmatory rate among patients with chronic viral NANBH and HCC, while among donors and patients with CLD other than NANBH the percentage of presumptive EIA positive reactions confirmed by RIBA and/or neutralization assay is much lower. In our experience, the neutralization assay appears to be somewhat more sensitive than RIBA, especially when samples show low EIA optical densities.


Hepatitis in pre-school children: prevalent role of cytomegalovirus. Virological and serological investigations were performed on 8 children with clinical and/or laboratory signs of hepatitis. Cytomegalovirus (CMV) appeared as the most frequently involved etiologic agent, since it was associated with 5 severe or chronically-evolving cases. Out of the other 3 patients with non-CMV associated hepatitis, all completely recovering, two had clinically typical Epstein-Barr virus infections, while the remaining patient had an asymptomatic HBV infection.


Prevalence of antibody to hepatitis C virus in acute non-A, non-B hepatitis in patients from different epidemiological categories. The prevalence of antibodies to hepatitis C virus (HCV) was determined in 65 patients with acute non-A, non-B hepatitis (NANBH). The results suggest that HCV is the most common causative agent in posttransfusion NANBH and in drug-related hepatitis. Detection of HCV antibodies does not appear to be a particularly useful diagnostic criterion due to the kinetics of the immune response in the course of the disease.


Prevalence of anti-HCV antibodies in patients with acute nonA-nonB viral hepatitis. In a group of 55 patients with NANBH, 81% were found to be reactive for HCV antibodies. In addition, many patients who had not been subject to parenteral risk of infection were also found to be reactive. Statistically, HCV positive patients have an increased tendency to develop chronic hepatitis.


Prevalence of antibody to hepatitis C virus in chronic HBsAg carriers. The presence of the anti-HCV antibody was investigated in sera from 102 chronic HBsAg carriers. The subjects varied as to the characteristics of the clinical states. It was found that HCV coinfection was more common in HBsAg positive intravenous drug addicts than in other parenteral risk groups. It also appears that HCV may be the causative agent of chronic liver disease in HBsAg carriers with undetectable HBV (and possibly HDV) replication.


Prevalence of hepatitis C virus (HCV) antibodies in haemodialysis patients. The prevalence of antibodies to HCV and the course of hepatitis have been determined in 357 haemodialysed patients treated at a single institution. The prevalence of HCV infection increases with the duration of haemodialysis and with the use of blood transfusions, yet there is high frequency of HCV seropositivity even without blood transfusions. Evolution of HCV hepatitis to chronicity is frequent and biological signs of chronic hepatopathy can coexist with absence of alanine aminotransferase (ALT) abnormalities.


Prevalence of anti-HCV in two Tanzanian villages. The presence of anti-HCV antibodies was investigated in sera from a total of 123 inhabitants of two Tanzanian villages. In one of the villages, 72.2% of the sera and in the other village, 82.6% of the sera were found to be anti-HCV positive. These values are dramatically higher than other reported prevalences, whereby cross-reactivity between HCV and Flaviviruses as well as possible transmission by arthropod vectors cannot be ruled out.


Hepatitis C virus and hepatocellular carcinoma. Epidemiological, clinical and laboratory data point to a role of hepatitis C virus infection in hepatocellular carcinoma. The connection appears to be indirect and to be mediated by cirrhosis. Thus, geographical differences can be observed, based on the locally prevalent etiological factors for cirrhosis. In the end, prospective studies of hepatitis C virus infected persons will be needed to elucidate the role of this agent in liver cancer.


Clinical significance of the polymerase chain reaction (PCR) assay in chronic HBV carriers. PCR was evaluated as a clinical tool for use in accurate identification of the specific etiologic agent in chronic HBV carriers. The method was found to be valuable in diagnosis and for monitoring therapy, as well as for elucidation of genotypic variants of HBV in chronic HBV cases. By this means an HBV defective variant with alterations in the preS1/preS2 sequence was detected and is consequently described here.


Results of the surveillance policy of stage I non-seminomatous germ cell testicular tumours. A series of 115 patients with clinical Stage I non-seminomatous germ cell testicular tumours were managed with orchiectomy and close surveillance (median follow-up 36 months, range 3-119); 34 (29.5%) relapsed, 21 within 6 months, 29 within a year and the latest at 28 months. At relapse all patients were treated with platinum or analogue-based drug combinations, supplemented in 7 by retroperitoneal node dissection; 30 patients achieved durable remissions and 2 have had further relapses successfully treated. Two died; both had malignant teratoma intermediate with primary stage T1 and vascular and/or lymphatic invasion of primary tumour. At a median follow-up time of 36 months, the survivors (98.3%) demonstrate no evidence of disease, these results matching the outcome of other methods of management. Vascular and/or lymphatic invasion was associated with an enhanced relapse rate but specific histology, T stage of the primary and pre-orchiectomy serum alpha-fetoprotein status did not appear to favour relapse. The first sign of relapse was tumour marker alone in 10 patients, radiological features alone in 12, or both in 10 patients. However, in 2 cases the relapse was first detected clinically. Furthermore, pre-orchiectomy and relapse marker status did not correlate well. These points emphasise the importance of all aspects of follow-up, none of which can be safely omitted.


Peptide hormones and sport: misuse and detection. In 1989 the Medical Commission of the International Olympic Committee (IOC) introduced the new doping class of 'peptide hormones and analogues,' which include human chorionic gonadotrophin (hCG) and related compounds, adrenocorticotrophic hormone (ACTH), human growth hormone (hGH), all the releasing factors of these listed hormones, and erythropoietin (Epo). Currently there are no IOC approved definitive tests for these hormones but highly specific immunoassays combined with suitable purification techniques may be sufficient to warrant IOC approval. The importance of measuring hCG and luteinizing hormone (LH) in the control of testosterone misuse is discussed and strategies for the detection of hGH, ACTH and Epo administration are suggested.


Differential expression of growth-associated protein (GAP-43) mRNA in rat primary sensory neurons after peripheral nerve lesion: a non-radioactive in situ hybridisation study. An alkaline phosphatase-labelled anti-sense oligodeoxynucleotide probe specific for growth-associated protein messenger RNA (GAP-43 mRNA) was used for non-radioactive in situ hybridisation histochemistry to follow relative changes in GAP-43 mRNA content in lumbar primary sensory neurons (L4-6) after unilateral ligation of the sciatic nerve. In normal dorsal root ganglia (DRG) 16% of neurons expressed GAP-43 mRNA, and these cells belonged to a sub-group of intermediate-sized (32-50 microns diameter) and large (> 50 microns) neurons. The hybridisation signal detected in these cells was weak to moderate. One day after nerve ligature a significant increase in the number of GAP-43 mRNA expressing neurons in the ipsilateral DRG was detected involving particularly the very small (12-20 microns) cells, and small cell population (20-32 microns), though the hybridisation signal was less pronounced in this latter cell group. A significant increase in the cellular content of GAP-43 mRNA was detected in both cell groups when compared to the normal DRG by 2 days after the lesion. At later times (4, 7, and 10 days postinjury) the intermediate-sized and large cell subpopulations also showed an increase in the number of GAP-43 mRNA positive neurons, followed by a significant rise in their content of GAP-43 mRNA. However, they did not reach the same intensity of hybridisation signal as seen in the small and very small neurons. All DRG neurons showed a maximum of GAP-43 mRNA expression by 10 days postsurgery. At longer times there was a slight decrease in the content of GAP-43 mRNA towards 14 days postinjury, but mRNA levels remained elevated up to 28 days after nerve ligature, the longest time point examined in this study. The different onset and levels of GAP-43 gene expression in the rat primary sensory neurons after lesion of their peripheral branch axons further characterize the different subclasses of these cells and may reflect their different involvement in the plastic changes following peripheral nerve injury.


Tyrosine kinase regulation of a molluscan circadian clock. On a formal level the clocks regulating circadian and cell division cycles are related in that both have been modeled as limit cycle oscillations (Science, 211 (1981) 1002-1013; Brain Res., 504 (1989) 211-215; Proc. Natl. Acad. Sci. USA, 88 (1991) 7328-7332). Furthermore, in several organisms each clock system is able to modulate the other (Science, 211 (1981) 1002-1013). However, in spite of the similarities at the formal level, and the connections at the physiological level, no common cellular elements have been identified linking the two processes. In the current series of experiments we show that one key element of cell cycle regulation, tyrosine phosphorylation/dephosphorylation is intimately associated with circadian rhythm generation in the eye of the marine snail, Bulla gouldiana. The importance of tyrosine kinase activity in the generation of circadian rhythms provides a possible point of similarity between the fundamental biochemical mechanisms underlying both circadian and cell cycle clocks.


Effect of nitric oxide on mitogenesis and proliferation of cerebellar glial cells. In the brain, nitric oxide (NO) has been identified as a messenger molecule and a mediator of excitatory amino acid-induced neurotoxicity. In this study, the effects of NO on serum-induced mitogenesis and cell proliferation of the cerebellar glial cells were assessed. NO-generating agent, S-nitroso-N-acetylpenicillamine (SNAP) increased intracellular cyclic guanosine monophosphate (cGMP) levels. Furthermore, 2 chemically dissimilar NO-generating agents, SNAP and sodium nitroprusside (SNP) inhibited serum-induced thymidine incorporation and cell proliferation. The antimitogenic effect of NO was mimicked by 8-bromo-cGMP and blocked by hemoglobin, a known inhibitor of NO. The effect of NO was not cytotoxic, since the cells were not stained with Trypan blue and did not show increased release of lactate dehydrogenase in the culture supernatants. However, NO-treated cells showed decreased conversion of tetrazolium to blue formazan suggesting that NO inhibited mitochondrial activity in the glial cells. These results demonstrate that NO inhibits serum-induced mitogenesis and cell proliferation of cultured rat cerebellar glial cells.


Selective forebrain fiber tract lesions implicate ventral hippocampal structures in tonic regulation of paraventricular nucleus corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) mRNA expression. The hippocampus appears to be involved in tonic regulation of the hypothalamo-pituitary-adrenocortical axis via interactions with corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP)-containing neurons of the hypothalamic paraventricular nucleus (PVN). To further investigate the anatomical basis of such interactions, lesions were made to forebrain fiber tracts in position to communicate inhibitory information from the hippocampus to the PVN. Total fimbria-fornix transections (TFF) and lateral fimbria-fornix lesions (LFF) both significantly increased CRH mRNA levels in the medial parvocellular PVN, as assayed by semi-quantitative in situ hybridization histochemistry. Medial fimbria-fornix lesions or section of the medial corticohypothalamic tracts (MCHT) did not influence CRH mRNA levels. The LFF group showed increases in both AVP mRNA and ACTH secretion, whereas no other lesion was effective in this regard. The results suggest: (1) hippocampal efferents conferring tonic inhibition of the HPA axis probably originate in regions contributing to the lateral extent of the fornix, representing structures in the ventral subiculum and ventral extent of CA1; (2) projections from the hippocampus to the medial basal hypothalamus (travelling in the MCHT) are unlikely to affect HPA function; (3) hippocampus may influence the PVN CRH/AVP neuron at multiple levels, in that LFF and TFF lesions have differential effects on PVN AVP mRNA levels and ACTH secretion.


Tonic stimulation of GABAB receptors in the nucleus tractus solitarius modulates the baroreceptor reflex. Previous studies have indicated that tonic stimulation of GABAB receptors in the nucleus tractus solitarius (NTS) contributes to the regulation of arterial blood pressure (AP). The present studies examined the hypotheses that (1) tonic stimulation of GABAB receptors in the NTS provides a tonic attenuation of the baroreceptor reflex and (2) enhanced stimulation of these GABAB receptors markedly attenuates the baroreceptor reflex resulting in an increase in AP. In chloralose-anesthetized rats electrical stimulation of the aortic depressor nerve elicited frequency-dependent decreases in AP and heart rate (HR). These responses were markedly attenuated, but not eliminated, by injection of the GABAB receptor agonist baclofen into the ipsilateral NTS. In contrast, the GABAA receptor agonist muscimol completely inhibited aortic depressor nerve-evoked responses. Blockade of GABAB receptors in the NTS by local injection of CGP-35348 elicited a dose-dependent decrease in AP, and a dose-dependent blockade of the pressor response elicited by injection of baclofen into the NTS. These results support the hypothesis that GABA acts tonically on GABAB receptors in the NTS to attenuate the baroreceptor reflex, thereby contributing to the regulation of AP.