Endogenous dimethylarginine as an inhibitor of nitric oxide synthesis. Nitric oxide (NO) is a widespread biological mediator with myriad functions. We have demonstrated that methylated arginines capable of inhibiting NO synthesis circulate in the plasma of healthy volunteers and are excreted unchanged in the urine. Up to 10 mg of asymmetric dimethylarginine is excreted in the urine every day, and this compound inhibits NO synthesis in vitro and in vivo, in animals and in humans. This finding raises the possibility that these compounds may act as endogenous regulators of the L-arginine:NO pathway in health and disease.


Ultraviolet-irradiated human endothelial cells elaborate nitric oxide that may evoke vasodilatory response. Human skin absorbs solar ultraviolet radiation which evokes vasodilation by a mechanism that is unknown. In this work, we show that ultraviolet (290-320 nm) irradiation of cultured human endothelial cells with doses (60 mJ/cm2) evoked nitric oxide (NO) release that was sustained for more than 60 min. In addition to augmenting NO, cyclic GMP production by human endothelial cells was also increased in a time-dependent manner. Ultraviolet stimulation of NO production was also observed with the enzyme NO synthase purified from the cytosol of human endothelial cells. These results indicate that ultraviolet radiation stimulates NO release, which stimulates cyclic GMP production by vascular smooth muscle, which results in a relaxation response.


New method of investigating functional roles of pressure-sensitive mechanoreceptor in human endothelial cells. The role of pressure-sensitive mechanoreceptors (PSMRs) in histamine-stimulated nitric oxide release from cultured human endothelial cells was investigated, using our new method (helium pack method). PSMRs were stimulated by various levels of pressure applied in addition to atmospheric pressure (40, 80, 120, and 160 mm Hg). Histamine-stimulated nitric oxide release was inhibited in a pressure-dependent manner. Our results suggest that PSMRs modulate nitric oxide release from endothelial cells.


Endogenous nitric oxide modulates sympathetic neuroeffector transmission in the isolated rabbit lateral saphenous vein. The rabbit isolated lateral saphenous vein (RLSV) has a heterogeneous population of alpha-adrenoceptors. Responses to electrical field stimulation, in the presence of cocaine, exhibit both alpha 1- and alpha 2-adrenoceptor-mediated components. The present study examined sympathetic neuroeffector transmission and the response to exogenous catecholamines after inhibition of nitric oxide (NO) synthesis with N omega-nitro-L-arginine methyl ester (L-NAME). A comparison of the response in the presence and absence of a functional endothelium was also carried out. L-NAME potentiated the first and second components of the response to nerve stimulation on the order of 300 and 500%, respectively. L-NAME also significantly potentiated responses to norepinephrine (NE), phenylephrine (PE), and UK 14304. Selective antagonism of the first phase was seen with prazosin (alpha 1-antagonist, 0.1 microM) and the second phase with rauwolscine (alpha 2-antagonist, 1 microM). In the presence of L-NAME, the remaining (uninhibited) components were potentiated. Removal of endothelial function induced by gentle rubbing of the intimal surface abolished potentiation to exogenous NE, PE, and UK14304 by L-NAME. However, a significant degree of potentiation of the neurogenic response was observed in the rubbed tissues in response to L-NAME. This suggests that there may be a nonendothelial source of NO that can modulate the neurogenic response to electrical field stimulation.


Effect of nitric oxide on membrane potential and contraction of rat aorta. Influence of nitric oxide (NO) on the membrane potential of rat aorta was assessed by blocking endothelial NO synthase with N omega-nitro-L-arginine (NOArg). Membrane potential was measured by two different methods: intracellular microelectrodes and [3H]tetraphenylphosphonium bromide ([3H]TPP+) uptake. Blocking of NO synthesis with NOArg (10(-4) M) depolarized the membrane by 4-6 mV. The NOArg-induced depolarization was suppressed by the NO donor SIN-1 (10(-5) M). Incubation with NOArg (10(-4) M) decreased the basal level of cGMP, and increased the basal 45Ca2+ influx as well as the sensitivity of contractile response to KCl. Results indicate that NO released by endothelial cells permanently hyperpolarizes the membrane of rat aorta smooth muscle cells and thereby may control the opening of voltage-dependent Ca2+ channels.


Histochemistry of guanylate cyclase, phosphodiesterase, and NADPH-diaphorase (nitric oxide synthase) in rat brain vasculature. The distribution of guanylate cyclase, phosphodiesterase, and NADPH-diaphorase [nitric oxide (NO) synthase] was studied in rat brain both at the light and electron microscopic level with special emphasis on the vascular system. We showed that the cGMP-generating enzyme is located in cells (glial cells and pericytes) surrounding cerebral vessels, but not in the endothelium. For NO synthase, a dual localization was observed. The enzyme is present in parts of the endothelium and in nerve endings apparently innervating larger brain vessels. We propose, therefore, that NO acts on guanylate cyclase both from a "synaptic" and endothelial source.


Structure of the bovine endothelin-B receptor gene and its tissue-specific expression revealed by northern analysis. We have isolated and characterized the gene encoding the bovine endothelin (ET) receptor ETB with seven putative membrane-spanning domains. The gene exists as a single copy of the bovine genome and extends over 36 kilobases. The coding region is divided into seven exons separated by six introns; the first exon encodes the first and second transmembrane domains and each of the following transmembrane domains is encoded by a separate exon. The 5' region of the transcript was examined by the ribonuclease protection analysis; multiple start sites were deduced with no apparent "TATA" box in the expected upstream region. Northern blot analysis demonstrated a widespread but tissue-specific distribution of the ETB receptor in bovine tissues.


Effects of storage solutions on contraction and relaxation of isolated saphenous veins. Saphenous veins are placed frequently in storage solutions before use as coronary bypass grafts. Controversy remains regarding which solution is optimal for the preservation of endothelium and vascular smooth muscle viability. Thus, this study measured the effect of four different storage solutions on saphenous vein smooth muscle and endothelial cell function. Saphenous vein segments from five baboons were stored for 3 h in one of four storage solutions consisting of physiological salt solution (PSS), University of Wisconsin solution (UWS), normal saline solution (NSS), or autologous whole blood (WB). Following storage, veins were suspended for isometric tension recording in tissue baths filled with PSS. Veins stored in PSS, UWS, and NSS showed similar contractile amplitude and agonist sensitivity to norepinephrine (10(-8) -10(-4) M) and KCl (20-100 mM). However, veins stored in WB contracted 31% less to KCl on average than those stored in the other three solutions (p < 0.05). The direct vascular muscle vasodilator, papaverine (10(-7) -10(-4) M), and the endothelium-dependent dilator, A23187 (10(-5) M), relaxed all vein preparations equally well. These results suggest that after storage in PSS, UWS, and NSS, contractile and relaxant vascular responses are similar in isolated saphenous veins. However, although veins stored in WB show comparable relaxation responses, they demonstrate an impaired vascular contraction immediately after storage. We conclude that storage of veins in blood may immediately modify vascular smooth muscle function.


Endothelium-dependent effects of platelet-activating factor in the coronary circulation. In experiments on anesthetized dogs, the effect of intracoronary administration of different doses of platelet-activating factor (PAF) has been investigated. One of the branches of the left coronary artery was catheterized via the main carotid artery. The coronary artery was autoperfused with blood from the subclavian artery. Intracoronary administration of small doses of PAF (100 and 200 ng/kg) caused a dose-dependent decrease in coronary resistance (CR) and an increase in coronary blood flow (CF). When 200 ng/kg of PAF was used, these changes were 30 and 34% vs. control, respectively. Dose increase to 300 ng/kg led to a biphasic reaction. Intracoronary administration of larger PAF doses produced opposite effects, i.e., increase in CR and decrease in CF. Chemical de-endothelialization by saponin (5 mg/5 ml, 1.5-2 min) transformed coronary dilation to small doses of PAF to constriction. The CR rose from 2.8 +/- 0.3 to 5.6 +/- 0.9 mm Hg/ml/min (p < 0.01) and CF dropped two-fold. The PAF receptor antagonists BN 52021 (6 mg/kg) and WEB 2086 (3 mg/kg) completely abolished PAF-induced coronary reactions. Hence, PAF-induced vasodilation of the canine coronary circulation is endothelium dependent.


Involvement of endothelium in vasodilating effects of vintoperol. Experiments were performed on vessels of the femoral vascular bed of anesthetized dogs and on isolated rings of the rat pulmonary artery, vena cava, and thoracic aorta. Vintoperol was administered intra-arterially (0.01, 0.1, and 0.3 mg/kg/min for 10 min) or added in vitro (10(-4) M). De-endothelialization by saponin (0.5 mg/ml for 5 min) of intact vascular beds or mechanical endothelium removal in rings decreased vasodilation or relaxation by 50-60% vs. control. In de-endothelialized vascular beds, vintoperol (0.3 mg/kg/min) increased blood flow by 18 +/- 5% but 47 +/- 4% under control conditions (p < 0.05). Methylene blue (10 mg/kg) reduced the increment of blood flow to vintoperol from 47 +/- 4 to 24 +/- 4% (p < 0.05). After de-endothelialization of the isolated pulmonary artery, relaxation of precontracted segments was reduced (21 +/- 4% vs. 56 +/- 5% under control conditions; p < 0.05). Vintoperol-induced relaxation of vascular rings was also inhibited by gossypol (2 x 10(-5) M) or methylene blue (5 x 10(-5) M); the level of inhibition (50-100%) or methylene blue (5 x 10(-5) M); the level of inhibition (50-100%) depended on the duration of exposure to the inhibitors. In conclusion, relaxation to vintoperol must be mediated by the release of endothelium-derived nitric oxide.


Function of vasoactive factors in the cerebral microcirculation. The properties of brain capillary endothelial cells (BCECs) have been analyzed. BCECs express two types of receptor sites for endothelins (ETs), and ETA-like receptor, and an ETB-like receptor that is not coupled to phospholipase C but whose occupancy activates Na+/H+ exchange activity. The ETA receptor is positively coupled to phospholipase C and negatively coupled to adenylate cyclase. BCECs, unlike aortic endothelial cells, express high-affinity receptor sites for C-type natriuretic peptide. They respond to exogenous nitric oxide (NO) and to NO donor molecules by large activations of soluble guanylate cyclase. They produce little cGMP in response to A23187 or to agonists of phospholipase C but do so after an exposure to interleukin-1. The physiological consequence of the high reactivity of BCECs to vasoactive factors is discussed.


Sustained hyperpolarization of cultured guinea pig coronary endothelial cells induced by adenosine. Electrophysiological experiments on cultured monolayers of guinea pig coronary endothelial cells were performed to substantiate the hypothesis that variation of the endothelial membrane potential may be a functionally important mechanism that contributes to the regulation of coronary blood flow. The endothelial cells were loaded with sodium by superfusion with K(+)-free solution at 37 degrees C for 5-30 min. Readmission of external K+ produced a transient hyperpolarization of up to 85 mV, which was due to stimulation of the electrogenic pump current. In most but not all of the monolayers, superfusion with 2 microM adenosine elicited a transient or a sustained hyperpolarization. The transient hyperpolarization had an amplitude of 15 +/- 6 mV. The sustained hyperpolarization had an amplitude of 11 +/- 3 mV. Our results are in line with the hypothesis that the hyperpolarization of the endothelium induced by release of adenosine into the perivascular space of the capillaries may be conducted electronically to the terminal arterioles and may cause vasodilation via current flow through myoendothelial gap junctions.


Purification of Aeromonas hydrophila major outer-membrane proteins: N-terminal sequence analysis and channel-forming properties. Four outer-membrane proteins of Aeromonas hydrophila were purified and their N-terminal sequences and channel-forming properties were determined. Three could be matched with proteins from other species. One was a maltoporin, as its level increased when cells were grown in maltose-containing media, and the channel it formed was blocked by maltose. Another was like OmpF and OmpC of Escherichia coli, except that its channel fluctuated much more rapidly. The third protein, which was produced in low-phosphate medium, exhibited several properties of the general anion porin PhoE. The fourth showed no similarity to any known proteins. It had a unique N-terminus and it formed small sharply-defined cation-selective channels. Two other proteins which corresponded to OmpW of Vibrio cholerae and E. coli OmpA were partly characterized.


The effect of ribosomal protein S1 from Escherichia coli and Micrococcus luteus on protein synthesis in vitro by E. coli and Bacillus subtilis. We have designed a set of nine plasmids containing the Bacillus pumilis cat gene with one of three Shine-Dalgarno (SD) sequences (weak, strong or stronger) and one of three initiation codons (AUG, GUG or UUG). These constructions have been used to determine the effect of ribosomal protein S1, SD and initiation codon sequences and Escherichia coli ribosomal protein S1 on translation in vitro by E. coli and B. subtilis ribosomes. Translation of these nine constructions was determined with three types of ribosomes: E. coli containing ribosomal protein S1, E. coli depleted of S1, and B. subtilis which is naturally free of S1. E. coli ribosomes were able to translate all nine transcripts with variable efficiencies. B. subtilis and S1-depleted E. coli ribosomes were similar to each other and differed from non-depleted E. coli ribosomes in that they required strong or stronger SD sequences and were unable to translate any of the weak transcripts. Addition of S1 from either E. coli or Micrococcus luteus, a Gram-positive bacterium, enabled S1-depleted E. coli ribosomes to translate mRNAs with weak SD sequences but had no effect on B. subtilis ribosomes. AUG was the preferred initiation codon for all ribosome types; however, B. subtilis ribosomes showed greater tolerance for the non-AUG codons than either type of E. coli ribosome. The presence of a strong or stronger SD sequence increased the efficiency by which E. coli ribosomes could utilize non-AUG codons.(ABSTRACT TRUNCATED AT 250 WORDS)


High copy number of the pUC plasmid results from a Rom/Rop-suppressible point mutation in RNA II. The plasmids pUC18 and pUC19 are pBR322 derivatives that replicate at a copy number several fold higher than the parent during growth of Escherichia coli at 37 degrees C. We show here that the high copy number of pUC plasmids results from a single point mutation in the replication primer, RNA II, and that the phenotypic effects of this mutation can be suppressed by the Rom (RNA one modulator)/Rop protein or by lowering the growth temperature to 30 degrees C. The mutation's effects are enhanced by cell growth at 42 degrees C, at which copy number is further increased. During normal cell growth, the pUC mutation does not affect the length or function of RNA I, the antisense repressor of plasmid DNA replication, but may, as computer analysis suggests, alter the secondary structure of pUC RNA II. We suggest that the pUC mutation impedes interactions between the repressor and the primer by producing a temperature-dependent alteration of the RNA II conformation. The Rom/Rop protein may either promote normal folding of the mutated RNA II or, alternatively, may enable the interaction of sub-optimally folded RNA II with the repressor.


An ELISA-like assay for hyaluronidase and hyaluronidase inhibitors. Hyaluronic acid (HA) is a prominent molecule in the extracellular matrix and is enriched whenever there is rapid tissue proliferation, regeneration and repair. HA is degraded in part by hyaluronidases (HA'ases) that are not well characterized. We have developed a novel ELISA-like rapid assay for HA'ases and their inhibitors. The assay is based on a high affinity biotinylated HA-binding peptide derived from tryptic digests of proteoglycan core protein of bovine nasal cartilage and the avidin-biotin reaction. HA-coated plates were incubated with serial dilutions of Streptomyces HA'ase, and the undegraded HA was measured. This established a standard curve for HA'ase activity against which all unknown enzyme samples were compared. The assay is easily modified to also serve a measure of HA'ase inhibitors. For detection of inhibitors, aliquots of sample were preincubated with a known activity of HA'ase and inhibition of HA degradation by the mixture was measured. We have used this assay to document the presence of potent HA'ase inhibitors in fetal calf sera. These techniques will aid in the purification and characterization of Ha'ases and their inhibitors.


Recognition of Entamoeba histolytica lipophosphoglycan by a strain-specific monoclonal antibody and human immune sera. Western blot analysis showed that the monoclonal antibody 2D7.10 recognized lipophosphoglycan (LPG) from Entamoeba histolytica HM-1:IMSS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern of [3H]galactose-labeled LPG and Western blot analysis of total lysate of E. histolytica with 2D7.10 revealed patterns similar to that of LPG with 2D7.10. This antibody could also immunoprecipitate purified LPG from the strain HM-1:IMSS after biosynthetically labeling with [3H]galactose and [32P]orthophosphate. However, no immunoprecipitation was observed when 2D7.10 was incubated with [32P]orthophosphate-labeled purified LPG from strain 200:NIH. Sera from patients suffering from invasive amoebiasis also immunoprecipitated 32P-labeled, purified LPG and could immunostain this molecule in Western blots. The human immune sera recognized carbohydrate epitopes but not the associated polypeptides of LPG, as evidenced by sensitivity to periodate digestion, mild acid hydrolysis but not to pronase treatment. It was earlier shown that 2D7.10 binds a carbohydrate epitope in a subset of axenized pathogenic strains of E. histolytica and that this epitope undergoes changes when cultured along with bacteria. These observations suggest that the E. histolytica LPG contains a strain-specific, variable epitope and that LPG is immunogenic in human.


Differential effect of right and left hemispheric lesions on two memory tasks: free recall of items and recall of spatial location. The effect of lateralized cerebral damage on free recall of items and recall of spatial location, under intentional and incidental learning conditions, was investigated. Eleven right brain-damaged (RBD) patients, 10 left brain-damaged (LBD) patients, 14 young and 11 elderly normal controls, participated in this study. The overall performance of the control groups was better than that of the patient groups. For all groups, free recall was better under intentional than under incidental learning condition. On recall of spatial location the learning condition had a differential effect on the groups. The RBD group performed better than the LBD group under intentional learning condition, while the reverse was found under incidental learning condition. The young-control group showed an advantage over the elderly-control group under intentional but not under incidental learning condition. The results are discussed in regard to different approaches to the distinction between automatic and effortful memory processes and their lateralization in the cerebral hemispheres.


[Subacute leukoencephalopathy after radio- and chemotherapy of lymphoblastic lymphoma]. In a patient aged 34 with lymphoblastic lymphoma lymphomatous infiltrations developed in the leptomeninges but were controlled with radiotherapy and chemotherapy. Recurrence of neurological signs after 7 months was recognized as relapse of lymphoma in the central nervous system, but in the brain extensive demyelination and necrotic changes were found. The authors relate the development of leucoencephalopathy to the hypersensitivity of the nervous tissue to chemotherapy after previous radiotherapy.


Distribution and origin of extrinsic nerve fibers containing calcitonin gene-related peptide, substance P and galanin in the rat upper rectum. The distributions of nerve fibers containing calcitonin gene-related peptide (CGRP), substance P (SP) and galanin (GAL) were examined in the rat rectum of mutants rats, aganglionic rats (AGRs), which completely lack the intramural nerve cells in the large intestine, and of their normal littermates. The origin of extrinsic peptide-containing nerve fibers was examined using retrograde tracing combined with immunohistochemistry in normal rats. In the rectum of normal rats, CGRP-, SP- and GAL-immunoreactive varicose fibers were observed throughout all layers of the rectal wall, and immunoreactive nerve cells were present in the enteric ganglia of colchicine-treated rats. In the aganglionic rectum of AGR, a rich supply of CGRP-immunoreactive fibers was observed in the mucosa, around the blood vessels, and in the submucous and intermuscular spaces. SP- and GAL-immunoreactive fibers in the aganglionic rectum showed a similar distribution to CGRP-immunoreactive fibers but were less dense. These results suggest that most of CGRP-positive fibers in the rectum are extrinsic whereas a large part of SP- or GAL-positive fibers are intrinsic. Fluoro-gold injected into the upper rectum of normal rat labelled nerve cells (less than 10% of total ganglion cells) in the lumbar (L1 and L2) and lumbosacral (L6 and S1) dorsal root ganglia. More than half of nerve cells in the dorsal root ganglia (L6 and S1) projecting to the rectum were immunoreactive for CGRP, and less than 10% were immunoreactive for SP or GAL. Comparison of serial sections of the dorsal root ganglion revealed that about half of the CGRP-immunoreactive cells were also positive for SP or GAL. These results indicate that SP- or GAL-positive neurons projecting to the rectum are scarce in the dorsal root ganglia. The present investigation suggests that CGRP-containing nerves are visceral afferents forming a major component of the sensory innervation of the rat rectum, and SP- and GAL-containing nerves which share their extrinsic origins appear to form a lesser proportion of the sensory innervation.


[A rare case of metastasis of Hodgkin's disease to the spinal cord]. A rare case of Hodgkin's lymphoma metastasis to the spinal cord is reported. The first signs of the metastasis appeared in the 6th year of cytostatic treatment. Diagnostic difficulties hampered causative treatment.


Description of a mechanistic approach to pain management in advanced cancer. Preliminary report. A mechanistic approach to advanced cancer pain management is proposed, based on the clinically perceived anatomical and pathophysiological mechanisms of pain generation. It is an extension of the World Health Organisation (WHO) analgesic ladder in which severity of pain is the principal determinant of analgesic choice. The mechanistic categories are: superficial somatic, deep somatic, visceral and neurogenic (mixed or pure, i.e., nociceptive component present or absent). Allocation of pain to the different categories is based on clinical history, physical findings and investigations to establish the site and extent of active primary or metastatic tumor deposits, and evidence of previous response to medication. Drug choice sequence is determined by the dominant pain mechanism judged to be present and not the severity of the pain. In order to describe this approach, mechanisms of pain, disease distribution and drug treatment have been analysed in the first 20 consecutive patients who consented to enter a longitudinal pain description and evaluation study on admission to an inpatient hospice unit. Despite a high exclusion rate from research standard monitoring due to severity of illness and related factors, the majority of eligible patients approached to enter the study did so, and the pain scoring was well tolerated. The implications of this for future research and clinical practice are discussed. In 6 patients only 1 pain mechanism was identified (visceral 4, deep somatic 2). Two mechanisms were present in 8 patients and 3 mechanisms in 6 patients. The deep somatic mechanism was identified in 15 patients, visceral mechanism in 13, neurogenic in 10 and superficial somatic in 2.(ABSTRACT TRUNCATED AT 250 WORDS)


Intrathecal co-administration of substance P and NMDA augments nociceptive responses in the formalin test. The effects of intrathecal administration of substance P and N-methyl-D-aspartate (NMDA) were studied in the formalin test in mice. Both substances were administered 5 min before injection of formalin into the hind paw. Co-administration of substance P and NMDA intensified the response in both the 1st (0-10 min) and the 2nd phase (20-30 min) of the formalin test, and increased the duration of the response. The increase in the response to formalin depended on the formalin concentration and was significant with 1% and 5% concentrations of formalin but not with a 0.05% concentration. No increase in the response was observed when NMDA or substance P was given alone. These findings indicate that concurrent activation of spinal NMDA and substance P receptors induces an enhancement of spinal transmission of nociception, and that this enhancement is dependent on the intensity or the quality of the peripheral stimulus.


[Adhesion and mobility of embryonic and tumoral cells]. Regulation of a number of adhesion molecules during neural crest cell migration was studied. The neural crest, a transient embryonic neural epithelium structure, undergoes mesenchymal transformation (epithelial-mesenchymal transition). The cells then migrate, giving rise to a variety of elements including the peripheral nervous system and melanocytes. During migration, neural crest cells do not express functional cell Adhesion Molecules but interact specifically with cell-binding domains in fibronectin molecules. A rat bladder carcinoma cell line was used as an in vitro model to study conversion of epithelial cells to a migratory fibroblast-like state. Conversion can be induced by culture on collagen or exposure to acidic Fibroblast Growth Factor (aFGF). Furthermore, constitutive fibroblast-like transformation can be induced by transfection with cDNA encoding aFGF. Growth factor-producing clones exhibit increased invasive and metastatic properties as compared with non-FGF-producing control cells. This model may provide increased understanding of the role of the different adhesion molecules in processes involving cell remodeling, such as tumor spread and development of metastases.


Exercise training: significance of regional alterations in serotonin metabolism of rat brain in relation to antidepressant effect of exercise. Alterations of 5HT and its chief metabolite 5HIAA were studied in four regions of the rat brain following acute 1-h swim exercise and 4 week chronic swim exercise (30 min/day, 6 days per week). Acute exercise significantly increased the synthesis and metabolism of 5HT in brain stem. Hypothalamus also showed increased levels of 5HT. However, no changes were observed in the cerebral cortex and hippocampus. Chronic exercise activated not only the synthesis but also the metabolism of 5HT in the cerebral cortex; this neuronal adaptation was sustained even 1 week after the termination of training. In brain stem, increased 5HT turnover was observed immediately after training. In hypothalamus, decrease in 5HT and 5HIAA levels occurred immediately after training, followed by a rebound increase in their levels after 1 week posttraining rest. In hippocampus, a delayed effect was observed, because 5HT level was unaltered immediately after the training, but its turnover decreased after 1 week rest. These findings have been discussed in an attempt to explain the antidepressant effect of exercise based on the 5HT deficiency theory of endogenous depression.


Positron emission tomography with 18F-dopa: interpretation and biological correlates in nonhuman primates. Positron emission tomography (PET) was carried out, with 18F-DOPA as a ligand, in normal control monkeys and "parkinsonian" monkeys who had been treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. The following approaches were used in data analysis: ratio of 18F accumulation in specific to nonspecific brain areas and 18F-DOPA influx constant obtained using either the actual plasma 18F-DOPA or the 18F activity in a nonspecific brain area as the input function. The results from these analyses were compared to one another and to biological parameters relevant to dopaminergic function. The striatum/cortex ratio and the rate constant calculated from plasma 18F-DOPA appeared to be the most sensitive analytic techniques.


Airway epithelial cells enhance eosinophil survival. Guinea pig and human eosinophils were co-cultured with or without epithelial cells (controls). Eosinophil survival was enhanced in the presence of the cultured epithelial cells in a number-dependent fashion. Further, supernatants from cultured epithelial cells also enhanced eosinophil survival. Both a monoclonal antibody to GM-CSF and indomethacin inhibited these epithelial-cell-mediated effects, and GM-CSF and PGE2 were shown to prolong eosinophil survival. These findings indicate that airway epithelial cells can enhance eosinophil survival. It is suggested that the mechanism may involve GM-CSF and PGE2 generation.


Scatter macular photocoagulation for subfoveal neovascular membranes in age-related macular degeneration. A pilot study. Forty eyes with age-related macular degeneration and a choroidal neovascular membrane involving the entire foveal avascular zone were enrolled in a pilot study to evaluate an unconventional photocoagulation technique that was postulated to stimulate inhibitors of neovascularization. Eighteen (45%) eyes were treated with extrafoveal scatter macular photocoagulation, and 22 (55%) eyes were treated with this type of photocoagulation and focal extrafoveal ablation of the choroidal neovascular membrane. The eyes were followed for at least 1 year and up to 4 years (average, 2.4 years). One to 7 months after treatment, approximately 45% of the membranes treated with each technique did not leak fluorescein during angiography. The absence of leakage was associated with better visual acuity, but this difference was not statistically significant. At the beginning of the study, the visual acuity was 20/200 or better in 32 eyes (80%) and 20/80 or better in 11 (28%). At the conclusion of the study, the visual acuity was 20/200 or better in 21 eyes (53%) and 20/80 or better in 7 (18%). After treatment, 24 eyes (60%) had the same or better (plus or minus two lines) visual acuities. Compared with eyes in natural history studies, those treated with scatter macular photocoagulation had less visual loss from baseline but did not recover acuity of 20/100 or better more frequently. There was no difference in results between these two methods of treatment.


Atypical parafoveal telangiectasis with subsequent anterior and posterior segment neovascularization. Bilateral parafoveal telangiectasis typically includes the presence of fluorescein staining of outer retinal capillaries, no macular ischemia, right-angled venules, and migration of pigment along these venules. In the bilateral form, atypical parafoveal telangiectasis with macular ischemia but without right-angled venules or migration of pigment along these venules has been reported in only five patients previously. An atypical case is presented of bilateral parafoveal telangiectasis complicated, not only by areas of macular nonperfusion, but also by peripheral ischemia with subsequent anterior and posterior segment neovascularization in the absence of any systemic abnormalities. As in previously reported cases, this patient had no evidence of right-angled venules or pigment migration. Pan-retinal photocoagulation resolved the rubeosis and caused regression of the retinal neovascularization.


Transurethral incision of the prostate: a viable alternative to transurethral resection. TUIP has been shown to be an effective method of relieving urinary outflow obstruction caused by BPH when prostatic size is 30 g or less. This is the situation for approximately 80% of patients undergoing TURP today. The long-term results of TUIP are quite favorable when compared with those of TURP. It is an easier technique to master than TURP. In addition, TUIP has a reduced operative time, little intraoperative and postoperative hemorrhage, reduced incidence of dilutional hyponatremia (TUR syndrome), and decreased length of hospitalization. TUIP also offers a lower risk of complications, especially with regard to impotence and retrograde ejaculation. This makes TUIP particularly well suited for patients in whom maintenance of sexual function is an important consideration. TUIP is also indicated in chronically ill, debilitated patients in whom the risk of anesthesia-related complications is high. Orandi and Graversen et al have shown that TUIP can be satisfactorily performed using only local anesthesia in high risk patients. However, TUIP is not without disadvantages. It is not effective for patients with a prominent median lobe or those with a markedly enlarged prostate gland (greater than 30 g). Another disadvantage is that no tissue is obtained for pathological analysis. Thus, all patients undergoing TUIP should be evaluated preoperatively with both a serum prostate-specific antigen determination and a meticulous digital rectal examination. As cost containment becomes a key factor for our health care system, it is quite likely that TUIP, as well as other less invasive procedures, will have expanded indications. Although the role of TURP in the management of very large prostates causing bladder outlet obstruction is not in question, smaller glands might be better managed with such a treatment as TUIP. As indicated earlier in this report, 80% of patients undergoing TURP could be effectively treated with TUIP--a less invasive, more cost effective treatment that has fewer associated side effects than TURP. Without question, TUIP is a grossly underutilized procedure in 1992. However, its future appears bright as more urologic surgeons are becoming aware of its many advantages and excellent long-term results.


[Suicide in the Ravensburg/Oberschwaben area. Results of a study of 508 suicides based on criminal police records]. On the basis of criminal police files we studied 508 suicides which happened between 1970 up to 1981 in the Ravensburg area in southern Germany. The police files also included medical records about in- or outpatient psychiatric treatment and also data about former violent behaviour. Mental disease as follows were most frequent: Depression 66% (diagnoses were made according to IDC-0 by two doctors under supervision of two senior psychiatrists; ICD-9: 300.4, 309.0 and 309.1 22%, ICD-9 296.1, 296.3 7.1% of the entire suicide group); neuroses and personality disorders 19%, addition, especially alcoholism, 28%. No psychiatric diagnosis could be made retrospectively in 10.6% (54 suicides). Sign in the presuicidal development like depressive symptoms, hopelessness and feelings of having no future, sleeping disturbances, feelings of guilt and anxiety, inner restlessness, but also changes in the direction of serenity and relaxation, treats of suicidal behaviour and reactions of the family and environment were reported showing a broad span of reactions from lack of perception to wrong interpretation. 15% of the suicides had also criminal activities in their former history. From a psychiatric point of view, improved diagnostic and therapeutic procedures in the treatment of the mentally ill, especially in the field of outpatient medical care of depressive and addictive patients, and better information of the relatives is to be demanded in order to prevent suicides.


[Results of percutaneous chemonucleolysis in lumbar disk hernia]. The percutaneous interventions have gained widespread acceptance for operative treatment of certain lumbar disc herniations. During recent years, percutaneous nucleotomy and laser discolysis replaced more and more the older technique of chemonucleolysis. Reports of anaphylactic shocks and severe neurological complications as well as unsatisfaction with the results of chemonucleolysis were responsible for this change. The basic mechanical concept of reduction of the intradiscal pressure remains the same with the newer methods. At the Inselspital percutaneous nucleotomy replaced chemonucleolysis in 1990. The results of chemonucleolysis were analysed with the aim of quantifying the efficiency of the percutaneous approach among our patient population. A total of 111 patients underwent chemonucleolysis between 1984 and 1989. The indication for the intervention was based on a several month history of lumbosciatica and a correlating radiological finding of disc protrusion or limited herniation. One case of anaphylaxis was the only serious perioperative complication. Upon follow-up examination approximately 2 months later, 54% of the patients were satisfied with the result, 27% reported an unchanged condition, and 19% had more pain than prior to the intervention. These results appear insufficient for an intervention associated with a non-negligible risk. Reduction of the nucleus pulposus appears often not to be an efficient mechanical concept for the treatment of disc protrusions and herniations.


[Psychosocial factors and follow-up of depressive disease]. In a one-year follow-up study 187 inpatients were studied using structured and semistructured interviews (Diagnostic Interview Schedule, Münchner Ereignisliste, Social Interview Schedule). In order to predict the course of depression we focused on psychosocial factors and their influence on the course of depression. The severity of depression at discharge and negative life events during the follow-up period could be identified as the main influence factors. Results suggest reciprocal influence between illness and social conditions, especially illness and marital relations. Marital dysfunctioning at follow-up was reported significantly more often by patients with unfavourable outcome, whereas patients who recovered did not differ from the control group in their marital conditions. The results suppose the value of social variables in the prediction of the course of illness. A few implications on these findings for therapeutic process are discussed.


[Are sex differences in schizophrenia stable on a transnational basis?]. The ABC-Schizophrenia-Study (Häfner et al. 1991), investigating a large, representative sample of 267 first admissions, did not find any gender differences in the lifetime morbidity risk for this disorder or in the frequency of nuclear symptoms in male and female schizophrenic patients. Marked gender differences, however, could be detected regarding the age at onset and its distribution and regarding illness behavior and certain secondary symptoms. The transnational stability of these findings was tested with data from the WHO studies "On Assessment and Reduction of Psychiatric Disability" (including 510 patients from 7 centres) and on "Determinants of Outcome of Severe Mental Disorders" (with 1,292 patients from 11 centres). Studying pooled data sets from the studies minimized centre-specific selection effects on sampling. Major results of the ABC-Schizophrenia-Study were validated: the lack of a gender difference in the morbidity risk (cumulative lifetime incidence rates) for schizophrenia; lower mean age at first admission in males; an early, marked peak in the distribution of the age at onset in males; a later and broader peak in this distribution in females with a second peak after age 40-45; equal frequency of nuclear symptoms in both sexes; gender differences in some specific contents of delusions, secondary affective symptoms, social adjustment and illness behavior.


[Personality of schizophrenic and schizoaffective patients]. At time of discharge 33 patients with ICD-9 schizophrenia and 16 patients with ICD-9 schizoaffective psychosis were assessed by the Freiburg Personality Inventory (FPI). Psychological profiles were analyzed individually and compared to controls. In addition, a hierarchical cluster analysis was carried out. Psychopathological findings at time of discharge were documented by the AMDP system. Impact of psychopathology on response behavior in the FPI was not evident. There were but few schizophrenic or schizoaffective patients without any abnormal FPI score. For the schizophrenics abnormal (high and low) scores could especially be found on the scales "Nervousness", "Depressiveness" and "Masculinity", for the schizoaffective patients on the scales "Nervousness", "Depressiveness", "Excitability", "Extraversion" and "Neuroticism". With regard to personality both groups turned out to be heterogenous. The schizophrenics differed significantly from the controls by higher means on the scales "Nervousness" and "Depressiveness" and a lower mean on the scale "Masculinity". The schizoaffective patients differed significantly from the controls by higher means on the scales "Depressiveness" and "Inhibition". Hierarchical cluster analysis resulted in 3 clusters for the schizophrenics: 1. introverted-schizoid type (42%), 2. extraverted-aggressive type (36%), 3. normal type (22%), and 2 clusters for the schizoaffective patients: 1. emotional labile type (56%), 2 normal type (44%). Results are compared to literature.


Myopathy associated with monoclonal gammopathy and leukoencephalopathy. We report here two patients presenting a myopathy associated with a monoclonal gammopathy (of IgG lambda or IgA kappa type) and, in one case, also with a leukoencephalopathy. Possible etiopathological mechanisms between these neurological diseases and an immune disregulation are discussed.


A study of quality of life in cancer patients receiving palliative chemotherapy. Although primary treatment for cancer has been associated with psychosocial distress, less research has focused on patients with advanced disease. Traditionally, the outcomes of treatment have been assessed using biomedical criteria, including tumour regression, progression and survival. It is argued that these data are inadequate to understand the impact of cancer upon the patient. Instead, quality of life considerations are crucial when treatments are aversive, especially when the aims are palliative rather than curative. Fifty-three patients with advanced breast cancer or ovarian cancer were studied prospectively for 6 months to assess whether the site and method of chemotherapy administration influenced their quality of life. Patients received palliative chemotherapy either at home or in hospital. Quality of life was operationalized as measurement of anxiety, depression, self-esteem, health locus of control, physical performance and symptoms. In addition, semi-structured interviews explored social roles, relationships, and perceptions of treatment. Hospital administered chemotherapy was perceived to be most distressing. Regression analysis indicated that anxiety and depression accounted for most of the variance in quality of life. Patients who died during the study 13 (24%) experienced considerable psychological and physical morbidity. Women over 60 years, experienced less psychological and physical distress. Quality of life broadens the criteria by which cancer treatments are evaluated, to include the experience of the patient.


[Use and abuse of visual aids--presentation technique]. In today's health service, doctors cannot avoid teaching or lectures. Visual aids are an important part of any successful presentation. Adequate overheads and slides can be easily made using ordinary office equipment and simple cameras. Modern computers and laser printers are also an exciting medium for producing visual aids. Nevertheless, if the talk is to achieve its purpose, good pictures must be presented in the correct way, along with an absorbing commentary. Both these aspects of presentation technique require careful preparation, but are seldom taught to medical students or junior staff as a necessary tool for their future work as doctors.


Neovascularization effect with He-Ne laser in the rat trachea. The tracheal tissue of a rat was heterotopically transplanted beneath the abdominal skin of a syngenetic rat, and the transplanted graft was irradiated with a He-Ne laser (2.41 J/cm2). Four or seven days following transplantation, Chinese ink staining was used to ascertain the number of newly generated vessels. Rats sacrificed on day 4 were divided into 3 groups, according to the number of laser irradiation treatments: A0, A2, and A4. Similarly, rats sacrificed on day 7 were divided into groups B0, B2, and B4. Among the A groups, vascular counts were 49.5 +/- 31.1 in group A0 (n = 6), 108.9 +/- 34.7 in group A2 (n = 7), and 90.9 +/- 24.6 in group A4 (n = 4). Neovascularization was significantly more pronounced in the irradiated groups that in the non-irradiated group. In the B groups, vascular counts were 148 +/- 58 in group B0 (n = 6), 146 +/- 56 in group B2 (n = 5), and 147 +/- 49 in group B4 (n = 5). There was no significant difference between the irradiated and non-irradiated B groups. Laser irradiation was determined to stimulate neovascularization, and the possibility of a clinical application for this procedure was suggested.


Comparison of staining and concentration techniques for detection of Cryptosporidium oocysts in cat faecal specimens. Modified Ziehl-Neelsen (MZN), auramine-phenol (A-P) and fluorescein isothiocyanate-labelled (FITC-labelled) monoclonal antibody (MAb) techniques were compared for detection of Cryptosporidium parvum oocysts in cat faecal specimens inoculated with known numbers of C. parvum oocysts. Of the three techniques, the FITC-labelled MAb technique detected more oocysts than the MZN and A-P techniques (P < 0.05), but A-P was more efficient than MZN (P < 0.05). Comparison of sucrose flotation, zinc sulphate (ZnSO4) flotation and formol-ether (F-E) sedimentation techniques revealed that F-E was the most efficient of the three (P < 0.05) for concentration of C. parvum oocysts from cat faecal specimens. On average, the F-E technique recovered 37% of oocysts from the original sample, whereas the sucrose and ZnSO4 flotation techniques recovered 33% and 11%, respectively. The findings of this study suggest that MZN and A-P staining are both useful for screening C. parvum oocysts in cat faecal materials containing 10(6) oocysts or more, but FITC-labelled MAb should be used when the number of oocysts is low. Also, the F-E sedimentation technique is recommended for concentrating oocysts in cat faecal specimens.


D2 dopamine receptor genotype and cerebrospinal fluid homovanillic acid, 5-hydroxyindoleacetic acid and 3-methoxy-4-hydroxyphenylglycol in alcoholics in Finland and the United States. If a genetic association between the D2 dopamine receptor genotype and alcoholism is mediated by altered dopamine function, then a stronger association might be found in alcoholics who are deviant in indices of dopamine function and by comparing alcoholics to nonalcoholics matched for ethnic origin. Therefore, we evaluated the D2/TaqI polymorphism in 29 impulsive violent alcoholic Finns, 17 nonimpulsive violent alcoholic Finns and 36 Finnish controls free of mental disorders, alcoholism and substance abuse. In 37 of the alcoholics, we measured cerebrospinal fluid (CSF) homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA) and 3-methoxy-4-hydroxyphenylglycol. There was no relationship between D2/Taq 1 genotype and concentrations of these monoamine metabolites in this group, which exhibits lower CSF HVA and 5-HIAA as compared to controls. There was also no genotypic difference between Finnish alcoholics and nonalcoholic controls. The lack of relationship between D2/Taq1 genotype and HVA concentration was replicated in 24 Caucasian alcoholics in the United States.


Voltage, calcium, and stretch activated ionic channels and intracellular calcium in bone cells. Embryonic chick bone cells express various types of ionic channels in their plasma membranes for as yet unresolved functions. Chick osteoclasts (OCL) have the richest spectrum of channel types. Specific for OCL is a K+ channel, which activates (opens) when the inside negative membrane potential (Vm) becomes more negative (hyperpolarization). This is consistent with findings of others on rat OCL. The membrane conductance constituted by these channels is called the inward rectifying K+ conductance (GKi), or inward rectifier, because the hyperpolarization-activated channels cause cell-inward K+ current to pass more easily through the membrane than outward K+ current. Besides GKi channels, OCL may express two other types of voltage-activated K+ channels. One constitutes the transient outward rectifying K+ conductance (GKto), which is activated upon making the membrane potential less negative (depolarization) but has a transient nature. This conductance favors transient K+ conduction in the cell-outward direction. The GKto also occurs in a small percentage of cells in osteoblast (OBL) and periosteal fibroblast (PFB) cultures. The other OCL K+ conductance, the GKCa, is activated by both membrane depolarization and a rise in [Ca2+]i. GKCa channels are also present in the other chick bone cell types, that is, OBL, osteocytes (OCY), and PFB. Furthermore, in excised patches of all bone cell types, channels have been found that conduct anions, including Cl- and phosphate ions. These channels are only active around Vm = 0 mV. While searching for a membrane mechanism for adaptation of bone to mechanical loading, we found stretch-activated channels in chick osteoclasts; other investigators have found stretch-activated cation channels (K+ or aselective) in rat and human osteogenic cell lines. In contrast to other studies on cell lines or OBL from other species, we have not found any of the classic macroscopic voltage-activated calcium conductances (GCa) in any of the chick bone cells under our experimental conditions. However, our fluorescence measurements of [Ca2+]i in single cells indicate the presence of Ca2+ conductive pathways through the plasma membrane of osteoblastic cells and osteoclasts, consistent with other studies. We discuss possible roles for GKi, GKCa, and anion channels in acid secretion by OCL and for stretch-activated channels in OCL locomotion.


A novel microcarrier bead model to investigate bone cell responses to mechanical compression in vitro. Periosteal cells grown in macroporous microcarrier bead columns were cyclically loaded with compressive force using a newly developed model. In response to 1/2 h of cyclic compression, RNA synthesis increased significantly by twofold, from 113.4 +/- 19.6 to 260.7 +/- 363 (p < 0.01) after 16 h, whereas DNA synthesis did not increase significantly after 24 h. The microcarrier bead model was calibrated using a linear volume displacement transducer with a range of strain magnitudes applied across the column of 1000-6000 microstrain.


Regulatory cells and cytokines involved in primary B lymphocyte production. Based upon the above data, it is now possible to formulate a working model that defines the stages of B cell development on which stromal cells and their products act. During the initial stages of this process, pro-B cells which do not express Ig heavy or light chain protein or other non-Ig B lineage associated molecules develop into B220 and c mu expressing pre-B cells in response to a low (< 10 kD) molecular weight stromal cell derived factor. No defined interleukin or colony stimulating factor, including molecules such as KL and IL-7, can replace stromal cell conditioned medium in mediating this developmental step. There appears to be little cell proliferation associated with the differentiation of pro-B cells into pre-B cells. However, our data indicate that as precursors develop into B220 expressing B cell progenitors, they become sensitive to the proliferation stimulating effects of IL-7 and KL. These results are in accord with findings that progenitor cells that have undergone DJH rearrangements are particularly sensitive to KL and IL-7(18,19). The analysis of pre-B cells present in individual lymphoid colonies indicates that once cells have rearranged and expressed their Ig heavy chain genes, they are no longer sensitive to KL and IL-7. These observations are based on the fact that receptors for these cytokines are not expressed in stromal cell dependent pre-B cells and are consistent with kinetic studies showing that the maturation of pre-B cells into surface Ig expressing B lymphocytes is not dependent upon cell proliferation21.(ABSTRACT TRUNCATED AT 250 WORDS)


[The onset mechanism of experimental autoimmune uveoretinitis induced by interphotoreceptor retinoid-binding protein]. In order to analyze the onset mechanism of experimental autoimmune uveoretinitis (EAU), two experimental models were used; one was EAU induced by one injection of purified bovine interphotoreceptor retinoid-binding protein (IRBP) with complete Freund's adjuvant in Lewis rat, and the other was an IRBP-induced autoimmune uveoretinitis that occurred spontaneously in nude (nu/nu) mice at 4 weeks of age reconstituted by the grafting of rat embryonic thymus (TG nude mouse). EAU develops when the IRBP-reactive lymphocytes in the regional lymph-nodes are activated. Activation begins when the T lymphocyte recognizes the peptide for the epitope bound to a major histocompatibility complex (MHC) molecule in the antigen-presenting cell by way of the T-cell receptor (TCR). In EAU, ten peptide residues p1182-1191 of the IRBP amino acid sequence, were revealed to be sufficiently capable of lymphocyte activation for EAU, and it was also shown that amino acid positions 1182W (tryptophane), 1185G (glycine), 1186V (valine) and 1188P (proline) of IRBP play important roles as the epitopes or agretopes in developing EAU. On the other hand, two amino acids of IRBP, amino acid positions 1182W (tryptophane) and 1194P (proline) were shown to be the agretopes inducing autoimmune uveoretinitis in the TG nude mouse. A study of the variable region of the TCR with a residual p1182-1194 specific T-cell line from the TG nude mouse revealed that as many as 96% utilized the T-cell receptor V beta 6 gene and that the peptide-MHC molecule complex was recognized by restricted receptors. Adhesion molecules such as ICAM-1 and LFA-1 were also found to play an important role as cofactors in activation of lymphocytes in the antigen-recognition process of EAU. Uveoretinitis seemed to result from an immune reaction in the eye occurring when the T lymphocyte arrives there, activating the immunological process. ICAM-1 and LFA-1 were also found to be involved in the infiltration process of inflammatory cells: our immunohistological examination revealed that ICAM-1 was present in the retinal pigment epithelium and epithelium of the ciliary body composing the blood-ocular barrier. In contrast, LFA-1 was expressed in the infiltrating cells. Finally, the tolerance of IRBP was discussed and it was experimentally demonstrated that the absence of IRBP-induced uveoretinitis in human beings and certain experimental animals resulted from endogenous IRBP serving as a tolerogen; we assumed that the breakdown of this self-tolerance would induce EAU due to thymic dysfunction or IRBP antigen injection.


[Immunotherapy in ocular diseases].

[Immunotherapy in ocular diseases]. Basic and clinical studies on immunotherapy in immune-mediated ocular disorders, i.e. uveitis, allograft rejection in corneal transplantation and allergic conjunctivitis, were carried out using a variety of immunosuppressants, including immunophilin ligands (FK506 and cyclosporine). 1. In an animal model for uveitis, experimental autoimmune uveitis (EAU), immunophilin ligands were demonstrated in the rat and monkey to have unique immunological activities: (1) intense and prolonged suppression of EAU development, (2) therapeutic effects by treating animals only after disease onset, (3) selective suppression on cellular immune response to S-antigen, (4) induction of immunological tolerance and activation of antigen specific suppressor cells. Combination therapy with low doses of immunophilin ligand and other immunosuppressant was tested to achieve better effects with less side effects. A low dose of cyclosporine (2 mg/kg/day) with bucillamine (20 mg/kg/day) which suppresses antigen-presenting activity by macrophages caused much stronger suppression of EAU than the therapy with either cyclosporine or bucillamine alone. Similarly, a low dose of FK506 (0.1 mg/kg/day) with dexamethasone (0.01 mg/kg/day) caused stronger suppression of EAU. A multi-center clinical open trial of FK506 in refractory uveitis was carried out in Japan. A total of 40 cases of active uveitis in the posterior segment of the eye were treated with FK506 (0.05, 0.1 or 0.2 mg/kg/day) and the mean observation period was 26.2 +/- 12.4 weeks. FK506 therapy improved uveitis in 60% of all cases including 47% of patients resistant to previous therapy with cyclosporine. FK506 significantly suppressed the number of uveitis attacks in patients with Behçet's disease. As for the side effects, 22.5% of patients showed abnormal values of renal function on FK506. The trough level of FK506 in whole blood correlated with adverse side effects as well as with therapeutic effect on uveitis, and it should be maintained between 15 and 25 ng/ml. 2. Effects of immunophilin ligands on the allograft rejection in corneal transplantation was examined in the rat. Fisher rat were used as donors and Lewis rats as recipients. This combination caused 100% rejection by 2-3 weeks after surgery as indicated by (1) edema, opacity and neovascularization in the graft, (2) infiltration of a variety of immune cells demonstrated by immunohistological examination and (3) high mixed leukocyte reaction (MLR). Systemic administration of cyclosporine (10 mg/kg/day) or FK506 (0.3, 1, 3 mg/kg/day) suppressed the allograft rejection. In addition, topical instillation of FK506 (0.3%) was effective to prolong the graft survival as long as the topical therapy continued.(ABSTRACT TRUNCATED AT 400 WORDS)


[Prostatic specific antigen (PA) in mass screening for prostate cancer]. The usefulness of prostatic specific antigen (PA) was compared with that of prostatic acid phosphatase (PAP). PA was determined in the serum of 2,183 patient examined by the mass screening for prostate cancer from 1987 to 1990. The serum samples of these patients were obtained from our serum bank. PA was measured by the E test "TOSOH" II (PA). The relationship of PA and PAP to prostate size estimated by digital rectal examination (DRE) and ultrasound tomography (US), and age was investigated. PA and PAP correlated with aging and prostate size estimated by DRE. However PA was more apparently related with these things. The correlation between PA and prostatic size estimated by US was relatively high (r = 0.53), but the correlation between PAP and prostate size estimated by US was low (r = 0.20). When the upper limit of normal range was set at 6.0 ng/ml, the sensitivity, specificity and efficiency was 64%, 97% and 62%, respectively. PA was more sensitive than PAP and could be more useful since none of the patients with prostate cancer was PAP positive and PA negative. We conclude that PA should be a reliable tumor marker in our mass screening system.


Papillary cystic tumor of the pancreas. A clinicopathologic study of 20 cases with cytologic, immunohistochemical, ultrastructural, and flow cytometric observations, and a review of the literature. Twenty cases of papillary cystic tumor of the pancreas were studied (19 female patients, one male patient; median age, 19.5 years). Most tumors developed in the head or body of the pancreas as well-circumscribed, large masses. Gross examination showed that they were solid, cystic, and hemorrhagic. Preoperative fine-needle aspiration biopsy anticipated the diagnosis in four cases. Histologic examination showed that uniform cells formed solid sheets, and loss of cohesion produced pseudopapillae. Hemorrhage, foam cells, cholesterol granulomas, and entrapped nests of pancreatic parenchyma were often found. Fifteen cases studied immunohistochemically were reactive for vimentin and alpha-1-antitrypsin, 13 expressed neuron-specific enolase, 2 expressed cytokeratin, and 1 expressed S-100 protein. None were reactive for pancreatic hormones, opioid peptides, hormonal receptors, or neuroendocrine markers. Electron microscopic examination in five cases showed oval nuclei, moderate amounts of rough endoplasmic reticulum, and many mitochondria; it also showed that annulate lamellae were common. No diagnostic secretory granules were found. DNA study in nine cases revealed a diploid GO/1 peak in eight and hyperdiploid (diploid index = 1.1) DNA content in one case. Fourteen patients with follow-up were free of disease (mean, 2.6 years). Papillary cystic tumor of the pancreas possibly originates from primordial pancreatic cells and lacks definite evidence of endocrine or exocrine differentiation.


Study of the major phenotype of large granular T-cell lymphoproliferative disorder. Six cases of large granular T-cell lymphoproliferative disorder with a selected immunophenotype (CD3+, CD4-, CD8+, CD16+) were studied to characterize a homogeneous group of patients. It was found that most of these patients did not exhibit the clinical features frequently described in large granular T-cell lymphoproliferative disorder--recurrent infection, rheumatoid arthritis, and splenomegaly. The laboratory tests usually positive in large granular T-cell lymphoproliferative disorder, including rheumatoid factor and anti-nuclear antibodies, also were frequently negative. The pathognomonic features were found to be neutropenia and large granular lymphocytosis with positive killer cell markers. All six cases showed T-cell receptor gene rearrangement that indicated a monoclonal proliferation of lymphoid cells, which were natural killer-like T cells by immunophenotyping. B cells were essentially absent in all cases. It should be emphasized that bone marrow aspirates are as informative as peripheral blood samples for the diagnosis of large granular T-cell lymphoproliferative disorder; indeed, phenotypes of blood and marrow in one case were identical in terms of percentages of markers. In this selected group of patients, the clinical courses were indolent with uncomplicated outcomes. In three patients, chemotherapy did not induce an obvious clinical response, but all patients' conditions remained stable with only supportive care.


Treatment of perennial allergic rhinitis with cetirizine. A study was made of 30 patients with perennial extrinsic rhinitis sensitized only to dust mites who were treated with cetirizine for 15 days, at a dose of 10 mg/24 hours. The following parameters were evaluated in each patient on day -1 before initiating treatment and on day 15: histamine 1/100 prick-test, Dermatohophagoides pteronyssinus prick-test, physical examination, assessment of nasal edema and rhinorrhea by anterior rhinoscopy, and of the degree of nasal obstruction by active anterior rhinomanometry. Each patient was given a form for daily clinical self-evaluation of the following subjective symptoms: sneezing, rhinorrhea, nasal obstruction, and nasal itchiness. The occurrence of side effects was evaluated. Cetirizine reduced significantly the area of the histamine and D. pteronyssinus papules elicited by prick-test. Clinical symptoms decreased significantly, with sneezing, nasal itchiness and rhinorrhea being greatly relieved, and nasal obstruction, evaluated by means of active anterior rhinomanometry performed before and after 15 days of treatment, being less alleviated. There was scant incidence of side effects in patients treated with cetirizine.


Hand mirror variant of adult acute lymphoblastic leukemia. Evidence for a mixed leukemia. Blast cells from a female patient with acute lymphoblastic leukemia-hand mirror variant were examined using various techniques, including light and ultrastructural morphologic examination, cytochemical analysis, surface antigen characterization, cytogenetic analysis, and gene rearrangement studies. The blast cells were found to be pre-B cells (CD19+ and Tdt+) that also expressed the myeloid antigens CD13 and CD33 and demonstrated a heavy chain immunoglobulin gene rearrangement. Cytogenetic studies revealed a t(11;19) translocation previously described in biphenotypic leukemias. A subset of acute lymphoblastic leukemia-hand mirror cells has been previously defined and includes predominately female patients with an indolent course. The authors' findings place this case, a mixed leukemia, within that subgroup. The possibility of mixed lineage should be considered in future cases of hand mirror variants of adult acute lymphoblastic leukemia. Furthermore, hand mirror morphologic features in any case of acute leukemia should alert the hematopathologist/hematologist to the possibility of mixed lineage.


Assessment of histamine release from basophils in whole blood by benzylpenicilloyl poly-L-lysine in penicillin-sensitized patients. Histamine release from basophil granulocytes in whole blood by benzylpenicilloyl poly-L-lysine (PPL) was investigated in 7 patients with penicillin allergy. All patients presented with systemic immediate hypersensitivity reactions after i.v. administration of penicillin G. Total histamine (of 7 patients) ranged from 27.5 ng/ml to 62.1 ng/ml (mean 43.2 ng/ml). The spontaneous histamine release ranged from 0.15% to 5.1% (mean 1.8%) of the total content. Addition of PPL in various concentrations resulted in values between 0.8 and 9.6%. Although PPL is a reliable allergen for prick- and intradermal testing in the diagnosis of penicillin allergy--demonstrating a histamine liberation in the skin--the in vitro experiment using the same allergen showed no histamine release above 10%. Using a threshold of 5% out of 7 patients, 4 (57%) would show a positive histamine release. Therefore it might indicate that in penicillin allergy a threshold of 5% must be used. In addition, basophils in whole blood and skin mast cells may be activated differently.


Clinical evaluation of in vitro leukocyte histamine release in allergy to muscle relaxant drugs. We have evaluated the in vitro leukocyte histamine release tests for the diagnosis of allergy to muscle relaxant drugs in 40 patients (Group A) and a control group of 44 subjects with negative leukocyte histamine release (Group B). Non-IgE dependent histamine release, expressed as a percentage of the total blood histamine, was 3.94% +/- 0.49 in Group B. The upper limit of positivity was estimated to be 5% (mean + 2 SD). Leukocyte histamine release tests were positive in 65% of the patients from Group A. The concordance between LHR and QAS-RIA was 64%. The maximal histamine release was observed at dilutions of 10(-2)-10(-4) in 20 of the 26 positive cases. The maximal histamine release was 43.8% +/- 23.3. The spontaneous histamine release was as low as 1.7% +/- 1.1. Cross-reactivity among the 5 different muscle relaxant drugs has been investigated and compared by intradermal testing. The muscle relaxant drugs which gave the lower skin reaction (M2) and the drug responsible for shock (M1) were selected for the study of in vitro leukocyte histamine release. Of 20 M2. All of the 10 cases had negative ID tests with M2. Three of these patients subsequently underwent general anesthesia with the muscle relaxant chosen as harmless (M2) without any clinical reaction.


Egg and milk allergy in adults. Diagnosis and characterization. Nineteen adult patients representing a total of 24 medical histories of immediate adverse reactions to egg or cows' milk underwent 1) standardized questioning about signs/symptoms occurring less than 2 h after ingestion of egg or milk, 2) skin prick test, RAST and histamine release test, and 3) titrated, oral, double-blind, placebo-controlled challenge (DBPCFC) with fresh egg or milk. Eleven medical histories (46%) were confirmed by DBPCFC in 10 patients (53%). All DBPCFC-positive patients experienced gastrointestinal symptoms, and in 80% of the patients, gastrointestinal symptoms were accompanied by respiratory or skin symptoms. Threshold doses varied between 50 mg and 250 g, with 4 patients presenting objective signs following 5 g or less. DBPCFC-positive patients reported significantly more symptoms and had a significantly higher number of positive tests than had DBPCFC-negative patients. None of the tests were in significant concordance with DBPCFC, although RAST showed a sensitivity of 100%. Thus, DBPCFC cannot be substituted in the diagnosis of milk and egg allergy in adults. The use of titrated, fresh foods in DBPCFC proved to be a safe and well-controlled procedure.


Analysis of antigenic determinants shared by two different allergens recognized by human T cells: house dust mite (Dermatophagoides pteronyssinus) and chironomid midge (Chironomus yoshimatsui). To analyse the cross-reactivity of T-cell-mediated immunity between Dermatophagoides pteronyssinus (Dp) and Chironomus yoshimatsui (Cy), the most common allergens in Japan, we established antigen-specific human T-cell lines and clones. Some but not all of the Cy-induced T-cell lines showed a significant proliferative response not only to Cy, but also to Dp. No T-cell line responded to other unrelated antigens. When we stimulated the Dp-induced T-cell clones with Cy, 3 of the 40 clones (7.5%) showed a significant proliferation, and 2 of the 3 clones produced interleukin-4 and interferon-gamma, indicating their helper function. Cross-reactivity was diminished significantly after the absorption of Dp antigen in an anti-Cy affinity column. The cross-reactive epitopes were thought to be expressed on the Dp molecule of 45-53 kD. The presence of helper T cells reactive to both allergens suggests a possibility that this cross-reactivity might be involved in part in the high incidence of allergy to the 2 major allergens.


Transcriptional inhibition by carcinogenic chromate: relationship to DNA damage. Hexavalent chromium compounds are carcinogenic to humans, are potent inducers of tumors in experimental animals, and can neoplastically transform cells in culture. In this study, the effects of sodium chromate on the expression of the 78-kDa glucose-regulated protein (GRP78) gene and on general transcription were investigated with respect to the DNA damage induced by this agent. DNA single-strand breaks, DNA-protein cross-links, and chromium-DNA adducts were present in CHO cells immediately after 2 h of treatment with sodium chromate. Subsequently, these types of damage were repaired at different rates. Single-strand breaks were essentially repaired after 8 h. By 24 h posttreatment, no cross-links remained in cells exposed to 30 or 150 microM chromate, although cells treated with the 300-microM concentration still contained cross-links at that time. DNA-chromium adducts remained unrepaired for at least 32 h. The moderate constitutive level of GRP78 mRNA was not affected by chromate. Chromate did, however, suppress induction of this gene by tunicamycin in a concentration-and time-dependent manner. Thirty micromolar sodium chromate (96% survival), which caused the least DNA damage, had no effect on GRP78 induction, general RNA synthesis, or mRNA synthesis. Induction of GRP78 was suppressed immediately and 12 h after treatment with 150 microM chromate (54% survival), although there was a partial recovery of induction at 24 h after treatment, which correlated with the repair of DNA-protein cross-links. In contrast, both total cytoplasmic RNA and mRNA synthesis were suppressed by approximately 60-75% for at least 32 h by 150 microM chromate. At the 300-microM concentration (8% survival), where DNA-protein cross-links persisted beyond 24 h, GRP78 induction was totally suppressed for at least 24 h, while total RNA and mRNA synthesis were suppressed by 80-90% for at least 32 h. Overall, the effects of chromate on GRP78 induction correlated most closely with the presence of DNA-protein cross-links, but suppression of total RNA and mRNA synthesis correlated with the presence of DNA-chromium adducts. These results indicate that chromate exerts differential effects on the induction of the GRP78 gene and on general transcription.


Regulatory mutations and human genetic disease. Mutations in gene promoter/regulatory regions represent an important class of lesion causing human genetic disease. Such mutations are associated with either increases or decreases in transcriptional activity mediated by the altered binding behaviour of trans-acting protein factors to specific DNA sequences in the promoter region. Although most promoter mutations are individually very infrequent, some occur at polymorphic frequencies. Both categories of lesion are likely to be important in clinical medicine and their study has already led to new insights into the mechanisms underlying the regulation of human genes.


[Treatment of chronic non-A, non-B-C hepatitis]. Preventive measures, and particularly screening for anti-HCV donors, have diminished the incidence of HCV infections, however, a vaccine against the disease is necessary for an effective prevention. The most active treatment at this time, seems to be alpha-interferon, at the dosage of 3 MU during 6 months. Possibly larger doses during longer periods of time might improve response and recurrence indexes, but not sufficiently as to give indiscriminate treatment to all patients at great cost and with many adverse reactions. While waiting for better predictive factors, the most convenient therapy remains the indicated dosage during 6 months, followed by periodic assessment during 2-3 months of transaminase levels: if they remain normal, the patient should be reassessed later on, if there is a recurrence, another course of treatment should be tried; non-responders should not be treated again with single interferon; other combinations or different treatments should be tried.


Simulation of animal cell metabolism.

Simulation of animal cell metabolism. A simulation of hybridoma growth and antibody production has been developed. It is capable of simulating all major variables of interest (e.g., specific growth rate, cell yield, sugars and amino acids profile, and antibody yield). This simulation is the most complete reported to date including such factors as cell composition, media composition, substrate and product effects, osmolarity etc. The stimulation of a large range of experimental data for hybridomas illustrates that this simulation is a powerful tool in the rational assessment of factors influencing the growth and metabolism of hybridoma cells.


Biological characteristics of CD7 positive acute myelogenous leukaemia. We studied the biological characteristics of CD7+ acute myelogenous leukaemia (AML). We diagnosed nine out of 88 consecutive AML cases as CD7+ AML based on myeloperoxidase positivity and surface antigen expression. In eight of these nine cases more than 20% of leukaemic blasts were found to coexpress both CD7 and a myeloid-associated antigen, CD33, by a two-colour flow-cytometric assay, while in the remaining case more than 90% of blasts were positive for CD7 and myeloperoxidase. CD7+ AML was most frequently observed in M1 among AML subtypes according to the FAB classification. An early stage-specific antigen, CD34 was also expressed on leukaemic blasts from eight of these nine cases. Neither the T-cell receptor (TcR)-beta nor the TcR-gamma gene was clonally rearranged in any of the cases. We then studied the proliferative responses to stimulation by various growth factors. Among interleukin-3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), and granulocyte-CSF (G-CSF), IL-3 showed the strongest stimulatory effect on DNA synthesis and leukaemic blast colony formation in 8/9 and 6/8 CD7+ AML cases examined, respectively. On the other hand, the strongest stimulatory effect exerted by IL-3 on blast colony formation was observed in only six out of the 33 CD7- AML cases examined. Furthermore, CD7+ AML blasts could proliferate in response to stem cell factor (SCF); SCF alone showed stimulatory effects on blast colony formation (7/8 cases), and in 5/7 SCF-responding cases, stimulatory effects of SCF were more potent than those of IL-3. In addition, SCF enhanced blast colony formation synergistically with IL-3 in four of these seven cases. These data suggest that progenitor cells of CD7+ AML may possess the biological properties characteristic of immature haematopoietic stem cells.


Massive abdominal lymphadenopathy in hairy cell leukaemia: a report of 12 cases. Lymphadenopathy is an uncommon finding in hairy cell leukaemia (HCL). We report 12 HCL patients in whom relapse was associated with massive abdominal lymphadenopathy. All but one had long-standing HCL (range 3-25 years; median 10 years); in one it was discovered at presentation. Nine patients had been splenectomized and seven had previously been treated with 2'deoxycoformycin (DCF) and/or alpha-interferon (alpha IFN): three had achieved complete remission and four a partial response. The computerized tomography (CT) scan appearances were similar in all cases with a primary lymph node mass centred around the coeliac axis and involving upper para-aortic and retropancreatic regions. Histology and/or cytology confirmed nodal involvement by HCL in six patients. Large immature hairy cells were seen in both lymph nodes and bone marrow, suggesting a degree of transformation. Nine patients were treated with DCF: one had complete resolution, six responded with 50-90% reduction of the lymphadenopathy, one did not respond and one is still on treatment; alpha-IFN was used concomitantly or sequentially in two of the responders. One responding patient died of sepsis after four injections of DCF. Three patients received either alpha- or beta-IFN alone with no response. One elderly patient was not treated. Abdominal lymphadenopathy could be part of the natural history of HCL and/or may represent a transformation analogous to that seen in other low-grade lymphoproliferative disorders. Routine abdominal CT scanning should be part of the work up of all patients with HCL.


Mouse erythrocyte rosette formation with malignant human B-lymphocytes re-evaluated: still a useful marker for differentiating mature B-cell malignancies. We have re-evaluated mouse rosette formation (MRF) as a marker for B-CLL by estimating the fraction of mouse rosette forming B-lymphocytes (identified by CD20 monoclonal antibodies) in normal donors and malignant CD20+ cell proliferations (ALL, AML, B-NHL, B-HCL and B-CLL). Whereas this ratio was increased in B-CLL, all other CD20 positive malignancies showed mean ratios of less than 0.1. As part of a Danish multi-centre study, we furthermore prospectively analysed 86 patients and found that the mouse/CD20 ratio divided the 78 patients with monoclonal B-cell populations suspected of B-CLL in two distinct groups. In the low ratio group, three patients were categorized as leukaemized B-NHL and one as PLL. The remaining three patients with low ratio were clinically and immunologically (by SmIg density and CD5 expression) B-CLL patients suggesting a frequency of MR-negative B-CLLs of approximately 5%. In the high ratio group two of 70 patients were diagnosed as B-NHLs. Thirdly, MRF was a valuable parameter in patients, where transformation of disease is suspected, since it preceded clinical changes by several months. Thus, MRF is still a useful marker in the age of monoclonal antibodies.


Studies of oral neutrophil levels in patients receiving G-CSF after autologous marrow transplantation. Patients are at risk of mucositis and infections in the oral cavity during the neutropenic period after chemotherapy, which are significant causes of morbidity. In phase I/II studies with the haemopoietic growth factor granulocyte colony stimulating factor (G-CSF), a reduction in post-chemotherapy mucositis has been observed in addition to haematologic effects. To understand this phenomenon better in patients receiving G-CSF following high-dose chemotherapy with autologous bone marrow transplantation (ABMT), we studied the effects of G-CSF on levels of neutrophils recoverable from the oral cavity using a quantitative mouthrinse assay. In normal subjects, mouthrinses contained 472 +/- 329 x 10(3) neutrophils/mouthrinse. After chemotherapy followed by ABMT, mouthrinse neutrophil levels decreased to undetectable levels during the neutropenic period, but recovered 1-2 and 3-9 d before circulating neutrophil levels reached 0.1 and 1 x 10(9)/l respectively, whether or not patients received G-CSF. In patients who received G-CSF, the mean cumulative mucositis score was reduced from 35 +/- 9 to 21 +/- 12 (P < 0.05), and the maximum mean daily mucositis score was reduced from 2.8 +/- 0.5 to 1.7 +/- 0.9 (P < 0.01), compared to patients who did not receive G-CSF after ABMT. These studies provide in vivo evidence that neutrophils produced during G-CSF therapy are available to leave the circulation and enter tissues where their function is required for host defence. Since the usual temporal relationship between oral and peripheral blood neutrophil recovery was preserved during G-CSF administration after ABMT, these data support the hypothesis that the reduction in post-ABMT mucositis observed with G-CSF therapy may reflect a beneficial effect of G-CSF on the kinetics of oral mucosal neutrophil recovery in addition to the effect of G-CSF to accelerate peripheral blood neutrophil recovery.


Inhibition by heparin and derivatized dextrans of Staphylococcus aureus adhesion to fibronectin-coated biomaterials. Recent data on cardiovascular device-centered infections suggest that some plasma and extracellular matrix proteins contribute to bacterial adhesion and colonization on biomaterials. We previously developed an in vitro assay to study the Staphylococcus aureus adhesion-promoting effect of surface-adsorbed fibronectin on flat PMMA coverslips coated with a monolayer amount of fibronectin. We screened the potential anti-adhesive properties of a group of substituted dextrans, previously shown to exhibit potent anticoagulant and anticomplementary activities. In comparison to unsubstituted dextran which showed no significant (< 20%) adhesion inhibition at 1 mg/ml, dextrans increasingly substituted with carboxylic and benzylamide groups (CMBD) exhibited increasing anti-adhesive activities. Three CMBD derivatives showing an increasing proportion (5-14%) of benzylamide groups showed inhibition of bacterial adhesion increasing from 33 to 51% at 1 mg/ml. Another category of substituted dextrans having a variable proportion (2-26%) of sulfonated benzylamide groups (CMBDS) produced active inhibition of S. aureus adhesion. In comparison to these heparin-like dextran derivatives, native heparin produced inhibition values of S. aureus adhesion which were intermediate between those of CMBD and CMBDS compounds. Furthermore, the anti-adhesive activity was still expressed when substituted dextrans were preincubated with fibronectin-coated PMMA but washed away at the time when radiolabeled bacteria were added to the adhesion assay. This indicates that the anti-adhesive effects of CMBDS could be exerted at the level of the S. aureus binding site of fibronectin. In conclusion, S. aureus adhesion on fibronectin-coated biomaterials can be efficiently blocked in vitro by soluble compounds such as dextran derivatives.


Structural abnormalities of the cricopharyngeus muscle in patients with pharyngeal (Zenker's) diverticulum. Recent manometric and radiological studies suggest that the upper oesophageal sphincter has poor compliance in patients with a pharyngeal (Zenker's) diverticulum. To test the hypothesis that this phenomenon is related to structural changes within the cricopharyngeus muscle we examined, histologically, muscle strips from 14 patients with a Zenker's diverticulum and compared them with control tissue obtained at autopsy from 10 non-dysphagic individuals. The cricopharyngeus muscle from patients and controls differed from inferior constrictor muscle by virtue of type 1 fibre predominance and greater fibre size variability. Ragged red fibres and nemaline bodies are a normal finding in the cricopharyngeus. Marked differences were observed in the cricopharyngeus muscle of Zenker's patients which demonstrated fibro-adipose tissue replacement and fibre degeneration. It is concluded that these structural changes may account for the observed diminished upper oesophageal sphincter opening and dysphagia in patients with Zenker's diverticulum.


Colony-stimulating factors in the clinic. Recombinant purified human haemopoietic growth factors are available for clinical trials and some have been licensed for therapeutic use. Some haemopoietic lineages (erythroid, neutrophilic, monocyte-macrophagic) can be selectively stimulated in order to ameliorate the cytopenias that follow cytotoxic treatment, or that characterize some haematological syndromes, and to stimulate mature cell function. Advances in the knowledge of receptor-ligand interactions and of transduction mechanisms, plus the production of synthetic or mutant molecules that may mimic, potentiate or antagonize the effects of the natural growth factors, should make novel therapeutic approaches possible.


Clinical evaluation of a newly established anti-HCV assay for the diagnosis of hepatitis C in Japan. To develop a more dependable method of diagnosing hepatitis C, serum anti-hepatitis C virus (HCV) was examined by using a new assay (anti-HCV second generation). The results were compared with those of either the conventional assay (anti-HCV first generation) or HCV-RNA analysis. With the first generation assay, anti-HCV was detected in 69% of post-transfusion acute hepatitis (AH), 44% of sporadic AH, 50% of needlestick exposed AH, 72% of chronic hepatitis (CH), 77% of liver cirrhosis (LC) and 86% of hepatocellular carcinoma (HCC). These results were remarkably increased by using the second generation assay (92% in post-transfusion AH, 72% in sporadic AH, 100% in needlestick exposed AH, 96% in CH, 96% in LC and 97% in HCC). Furthermore, in the early stages of AH (from 1-5 weeks after onset), anti-HCV was not detected in all 18 patients by the first generation assay, but was found in 10 of them by using the second generation assay. The failure to detect anti-HCV with the first generation assay was mainly due to a lack of the core region coding peptide (C22-3) in this assay. In the AH-resolving group, anti-HCV second generation did not disappear, but the titre tended to be lower than that in the CH-developing group. Thus, the second generation assay for anti-HCV was considered to be a more useful tool for not only the diagnosis of hepatitis C but also for determining prognosis.


[Role of neoadjuvant chemotherapy in the treatment of advanced head and neck tumors. Results of two randomized therapeutical trials conducted at the Institut Curie on 208 patients]. From March 1983 to December 1989, 208 patients with locally advanced squamous cell carcinoma of the head and neck were successively included into two trials randomizing induction chemotherapy versus no pre-irradiation treatment. The chemotherapy regimen of the first trial, which included 100 patients, consisted of two cycles of a combination of cisplatin, bleomycin, vindesine, mitomycin C; while that of the second trial, which included 108 patients, consisted of three cycles of a combination cisplatin, 5-fluorouracil (continuous infusion) and vindesine. Local treatment was the same in two trials: 'primary' radiotherapy in all patients. The response was then evaluated at 55 Gy; in the case of poor response, surgery was performed, otherwise radiotherapy was continued to full doses (possibly followed by salvage surgery). The tumor and lymph node responses to chemotherapy (complete and partial response) were higher in the second trial and in the first one: 70% versus 50% for primary lesions, 47% versus 25% for lymph nodes. The toxicity of the two chemotherapy regimens was minimal. In the two trials, an initial major response to chemotherapy predicted subsequent efficacy of irradiation in 80% of the patients. Complete response rate at the end of irradiation correlates with the previous response to the chemotherapy. With a median follow-up of 60 months with the first chemotherapy regimen and 30 months with the second, overall survival and disease-free interval did not significantly differ in the two groups of patients, with or without chemotherapy. The incidence of distant metastasis was significantly reduced (P < 0.03) in the chemotherapy arms. This negative trial encourages the design of new chemotherapy protocols according to new schemes of treatment. For advanced stages of head and neck cancers (T3, T4, N2, N3), we recently launched a pilot study combining platinum and irradiation, but according to a concomitant schedule.


Alpha-fetoprotein in patients with hepatocellular carcinoma after transcatheter arterial embolization. To assess the usefulness of alpha-fetoprotein (AFP) in monitoring treatment effects of transcatheter arterial embolization (TAE) in hepatocellular carcinoma (HCC) patients, a total of 31 sets of AFP levels after TAE in 21 HCC patients were analysed by linear regression between logarithmic AFP levels and days. Eleven sets of AFP data with poor linear declination were accompanied with poor TAE results except in one patient who had chronic hepatitis with acute exacerbations. Twenty sets of data with good linear declination in the first month after TAE indicated good TAE results. Seven of them showed no evidence of tumour recurrence nor elevated AFP levels within a follow-up of 6 months. The mean, standard deviation and range of half-lives of AFP in the non-recurrent group were 5.0, 1.6 and 2.9-7.2 days, respectively. The others experienced late tumour recurrence that was detected by rebound of AFP levels except one who had another non-AFP-secreting HCC. Thus, the results might be used as a reference in monitoring the treatment effects of TAE and the timing selection of repeated TAE.


Immunoblot analysis of the R-form lipopolysaccharide from Salmonella S forms. Lipopolysaccharide (LPS) preparations from Salmonella S-form bacteria contain, in addition to S-form LPS, variable amounts of R-form material, lacking the O-specific polysaccharide. In the present study, we investigated the R-form LPS present in 22 LPS preparations derived from different Salmonella S-form strains belonging to 8 serological groups. The R-form part of the LPSs was separated by SDS-polyacrylamide gel electrophoresis and its antigen specificity analyzed by subsequent immunoblotting using Salmonella R-antisera (anti-Ra, -Rb1, -Rb2, -Rc). The results show that different R-determinants were present in one and the same LPS preparation. The pattern of reaction varied considerably among individual strains. This variation was also present among strains of the same serogroup. Approximately one half of the R-form LPSs exhibited a weak or no reaction with Ra antiserum. In contrast, almost all R-form LPSs showed a significant reaction with Rb1 antiserum. The electrophoretic mobility of the R-form material was very similar, exhibiting only minor differences among the different LPS preparations. In most cases, migration was similar to that of authentic Salmonella Ra-LPS; in some cases, the migration was somewhat faster resembling that of Rb1-LPS.


Species specific monoclonal antibodies to Bacteroides fragilis lipopolysaccharide protect mice from severe infection. Four monoclonal antibodies which reacted in a species specific manner with Bacteroides fragilis were isolated. They recognised at least two different epitopes of B. fragilis lipopolysaccharide. The monoclonal antibodies protected non-immune mice from longlasting bacteraemia and abscess formation induced by the intraperitoneal administration of B. fragilis in combination with an infection-potentiating agent. The monoclonal antibodies were as efficient as an anti-B. fragilis hyperimmune serum. Only antibodies administered intraperitoneally or intramuscularly were protective.


Binding of Chlamydia trachomatis serovar L2 to collagen types I and IV, fibronectin, heparan sulphate, laminin and vitronectin. Chlamydia trachomatis serovar L2 elementary bodies were tested for binding to 125I-labelled components of the extracellular matrix. C. trachomatis bound high levels of collagen type I and heparan sulphate, whereas the binding to collagen type IV, fibronectin, laminin and vitronectin was substantially less. Preliminary characterization showed that chlamydial surface proteins are essential for binding to occur. Our results suggest that a selective affinity for C. trachomatis to a limited number of components of the extracellular matrix may have important implications mediating attachment to host cell surfaces and localization of chlamydiae in other tissues, such as joints.


Monoclonal antibodies directed to the O antigen of Salmonella serogroup E cross-react with lipopolysaccharides of Salmonella serogroups C, F and S. Four murine hybridoma clones secreting monoclonal antibodies (mAb) directed against lipopolysaccharide (LPS) antigen of S. newington, serogroup E1, were obtained after a fusion of spleen cells of mice immunized with formaldehyde-killed bacteria and mouse myeloma cells of the X63-Ag8.653 line. Antigen binding properties and specificity of the mAb were studied in bacterial agglutination tests, passive hemolysis and its inhibition, passive hemagglutination tests, passive hemolysis and its inhibition, passive hemagglutination and immunoenzyme tests (ELISA and immunoblotting). Three of the mAb (24E6, 29E1 and 45F6) were agglutinating and were active in all tests used, while mAb 31H12 did not agglutinate bacteria but revealed a high reactivity in the immunoenzyme reactions. It was found that the mAb reacted with LPS and Salmonella strains from serogroup E (E1, E2, E3 and E4) as well as from serogroups C (C1 and C4), F and S thus showing that the O3 antigen possesses more than one epitope, one of which is represented on the LPS antigens of the serovars from the cross-reacting groups mentioned. According to the known chemical the most probable recognized epitope consists of mannose with beta-linkage to the next monosaccharide residue in the LPS chain.


Expression and purification of a soluble tissue factor fusion protein with an epitope for an unusual calcium-dependent antibody. The use of bacterial signal peptides to target recombinant mammalian proteins to the periplasmic space of Escherichia coli (to promote proper disulfide bond formation) has met with variable success. We report the design and use of a bacterial expression vector to direct recombinant fusion proteins to the periplasmic space of E. coli: it contains the signal peptide from the pelB gene of Erwinia carotovora linked to a small peptide epitope for an unusual calcium-dependent antibody (HPC4). HPC4 binds to the epitope in a Ca(2+)-dependent manner, but the epitope itself does not bind Ca2+. We have used this system to express a biologically active, soluble form of tissue factor, the protein responsible for triggering the blood clotting cascade. Soluble tissue factor was secreted into the culture medium at 1-2 mg/liter, from which it could be readily purified using immobilized HPC4 antibody. The HPC4 epitope could be removed by digestion with thrombin or factor Xa, although a free amino terminus was not required for function since soluble tissue factor was equally active with the epitope still in place. This vector/epitope system permits large-scale expression and purification of recombinant soluble tissue factor and should be generally applicable to the isolation of other recombinant proteins. Furthermore, the epitope confers Ca(2+)-dependent binding of the fusion protein to HPC4 antibody while avoiding the creation of a new metal binding site on the fusion protein itself. Tb3+ can bind in this Ca2+ site near Trp, allowing this site to serve as a means of attaching a fluorescent probe to tissue factor.


Phosphorylatable and epitope-tagged human erythropoietins: utility and purification of native baculovirus-derived forms. The hematopoietic glycopeptide erythropoietin (EPO) is a prime regulator of red cell production in mammals, yet the precise nature of its interaction with specific cell surface receptors is poorly understood. Towards defining domains of EPO that are involved in receptor activation, we have developed (i) conditions for the expression of recombinant human EPO (rhEPO) at high levels in SF9 cells using modified 2- and 5-liter stirred reactors, (ii) a two-step procedure for the purification of this EPO without denaturation, and (iii) forms of EPO tagged with either a hemagglutinin influenza virus epitope or a consensus sequence for in vitro phosphorylation. Compared to EPO expressed in mammalian cells, rhEPO from SF9 cells in N-glycosylated with simple, neutral oligosaccharides of limited size, yet as purified presently using nondenaturing procedures, possesses exceptionally high in vitro activity (> or = 500,000 U/mg). Thus, this form of EPO should prove advantageous for direct physicochemical analyses. Regarding epitope-tagged and phosphorylatable EPOs, forms modified at the amino terminus (Ala1) fully retained receptor binding and in vitro biological activities. In contrast, forms modified at the carboxy terminus (Cys161) were inactive and did not compete for receptor binding, indicating that integrity of this domain is essential for receptor recognition. For active amino-terminal-modified forms, the specific binding of MAb 12CA5 to native HAI-EPO and the utility of 32P-labeled PHOS-EPO in receptor binding and internalization studies also were demonstrated. The development of these unique, highly active forms of human EPO should advance studies of essential interactions between this cytokine and its cell surface receptor.