Endogenously synthesized nitric oxide prevents endotoxin-induced glomerular thrombosis. Escherichia coli endotoxin (LPS) can induce the clinical syndrome of septic shock and renal cortical necrosis and can stimulate nitric oxide (NO) production from macrophages, vascular smooth muscle, and glomerular mesangial cells in vitro. NO is an endogenous vasodilator, which also inhibits platelet aggregation and adhesion. We therefore sought to determine whether LPS would stimulate NO production in vivo and, if so, whether this NO would modulate endotoxin-induced glomerular thrombosis. The stable NO end-products, NO2 and NO3, were measured in serum and urine collections from rats during baseline and after injection of LPS, with or without substances that modulate NO synthesis. The urinary excretion of NO2/NO3 was 1,964 +/- 311 nm/8 h during the baseline and increased to 6,833 +/- 776 nm/8 h after a single intraperitoneal injection of 0.1 mg/kg LPS (P < 0.05). The serum concentration of NO2/NO3 also significantly increased after LPS injection. Both the urine and serum stimulation was significantly prevented by the NO synthesis inhibitor, Nw-nitro-L-arginine methyl ester (L-NAME). L-Arginine, given with LPS+L-NAME significantly restored the NO2/NO3 levels in the urine. Ex vivo incubation of tissues from rats treated with LPS demonstrated NO production by the aorta, whole kidney, and glomeruli, but not cortical tubules. Histological examination of kidneys from rats given either LPS or L-NAME alone revealed that 2 and 4.5% of the glomeruli contained capillary thrombosis, respectively. In contrast, rats given LPS+L-NAME developed thrombosis in 55% of glomeruli (P < 0.001), which was significantly prevented when L-arginine was given concomitantly. We conclude that LPS stimulates endogenous production of NO in vivo and that this NO is critical in preventing LPS-induced renal thrombosis.


Cloning, characterization, and expression of a human calcitonin receptor from an ovarian carcinoma cell line. A human ovarian small cell carcinoma line (BIN-67) expresses abundant calcitonin (CT) receptors (CTR) (143,000 per cell) that are coupled, to adenylate cyclase. The dissociation constants (Kd) for the CTRs on these BIN-67 cells is approximately 0.42 nM for salmon CT and approximately 4.6 nM for human CT. To clone a human CTR (hCTR), a BIN-67 cDNA library was screened using a cDNA probe from a porcine renal CTR (pCTR) that we recently cloned. One positive clone of 3,588 bp was identified. Transfection of this cDNA into COS cells resulted in expression of receptors with high affinity for salmon CT (Kd = approximately 0.44 nM) and for human CT (Kd = approximately 5.4 nM). The expressed hCTR was coupled to adenylate cyclase. Northern analysis with the hCTR cDNA probe indicated a single transcript of approximately 4.2 kb. The cloned cDNA encodes a putative peptide of 490 amino acids with seven potential transmembrane domains. The amino acid sequence of the hCTR is 73% identical to the pCTR, although the hCTR contains an insert of 16 amino acids between transmembrane domain I and II. The structural differences may account for observed differences in binding affinity between the porcine renal and human ovarian CTRs. The CTRs are closely related to the receptors for parathyroid hormone-parathyroid hormone-related peptide and secretin; these receptors comprise a distinct family of G protein-coupled seven transmembrane domain receptors. Interestingly, the hCTR sequence is remotely related to the cAMP receptor of Dictyostelium discoideum (21% identical), but is not significantly related to other G protein-coupled receptor sequences now in the data bases.


Uptake of toxic silica particles by isolated rat liver macrophages (Kupffer cells) is receptor mediated and can be blocked by competition. Silica particles (quartz dust) are toxic to macrophages after their uptake into these cells. These experiments describe the opsonization mechanism(s) and macrophage receptor(s) involved in silica uptake. Freshly isolated rat liver macrophages (Kupffer cells) were incubated at 37 degrees C with silica particles in the presence or absence of autologous or heterologous plasma or purified plasma fibronectin and cell viability was assessed at various times. Within 60 min of coincubation, > 80% of macrophages were lysed in the presence of plasma or purified fibronectin but not in their absence (viability > 90%). Lysis was slower with defibronectinized plasma (28% in 60 min). Macrophages could be protected from lysis by addition of the monosaccharide N-acetyl-D-galactosamine but not by N-acetyl-D-glucosamine. Galactosylated serum albumin but not mannosylated albumin or native albumin exerted full protection from lysis. The pentapeptide GRGDS also prevented macrophage lysis in synergy with N-acetyl-galactosamine. Enzymatic deglycosylation of fibronectin reduced lysis significantly. These findings indicate an important opsonizing activity for fibronectin and dual recognition via the lectin-like galactose-specific binding activity of membrane-associated C-reactive protein and by integrin receptor(s). Binding experiments (at 4 degrees C) revealed initial binding as primarily galactose-inhibitable, suggesting integrin-mediated binding as a later event necessary for effective uptake.


High levels of circulating cytomegalovirus DNA reflect visceral organ disease in viremic immunosuppressed patients other than marrow recipients. Although viremia is a hallmark of disseminated cytomegalovirus (CMV) infection, not all viremic patients have visceral organ CMV disease. We used blot hybridization with a cloned subgenomic probe to quantitate viral DNA in blood leukocytes of 60 viremic patients (25 with solid organ transplants, 20 with AIDS, and 15 marrow recipients) who had different clinical manifestations of CMV infection. The results are expressed as pg of viral DNA/10 micrograms of leukocyte DNA. Patients with AIDS or with solid organ transplants who had CMV visceral organ disease had the largest amounts of viral DNA in their granulocytes (median 632 and 237 pg, respectively). These amounts were significantly greater than those in similar viremic patients without CMV visceral disease (17 and 21 pg; P < 0.005 and 0.002, respectively). All patients in the study with > 150 pg of CMV DNA in their granulocytes had visceral CMV disease. The amounts of viral DNA in granulocytes of AIDS and organ transplant patients with CMV retinitis were low (median 22 pg). Marrow transplant patients were unique in that the amounts of CMV DNA in granulocytes were low whether CMV visceral organ disease was present (17 pg) or absent (14 pg). We conclude that high levels of circulating CMV DNA in viremic AIDS and solid organ transplant patients reflect viral involvement of visceral organs but not the retina. In marrow recipients, the severity of CMV disease, even when fatal, is not reflected quantitatively in peripheral blood leukocytes.


Regulation of insulin receptor substrate-1 in liver and muscle of animal models of insulin resistance. Insulin rapidly stimulates tyrosine phosphorylation of a protein of approximately 185 kD in most cell types. This protein, termed insulin receptor substrate-1 (IRS-1), has been implicated in insulin signal transmission based on studies with insulin receptor mutants. In the present study we have examined the levels of IRS-1 and the phosphorylation state of insulin receptor and IRS-1 in liver and muscle after insulin stimulation in vivo in two rat models of insulin resistance, i.e., insulinopenic diabetes and fasting, and a mouse model of non-insulin-dependent diabetes mellitus (ob/ob) by immunoblotting with anti-peptide antibodies to IRS-1 and anti-phosphotyrosine antibodies. As previously described, there was an increase in insulin binding and a parallel increase in insulin-stimulated receptor phosphorylation in muscle of fasting and streptozotocin-induced (STZ) diabetic rats. There was also a modest increase in overall receptor phosphorylation in liver in these two models, but when normalized for the increase in binding, receptor phosphorylation was decreased, in liver and muscle of STZ diabetes and in liver of 72 h fasted rats. In the hyperinsulinemic ob/ob mouse there was a decrease in insulin binding and receptor phosphorylation in both liver and muscle. The tyrosyl phosphorylation of IRS-1 after insulin stimulation reflected an amplification of the receptor phosphorylation in liver and muscle of hypoinsulinemic animals (fasting and STZ diabetes) with a twofold increase, and showed a significant reduction (approximately 50%) in liver and muscle of ob/ob mouse. By contrast, the levels of IRS-1 protein showed a tissue specific regulation with a decreased level in muscle and an increased level in liver in hypoinsulinemic states of insulin resistance, and decreased levels in liver in the hyperinsulinemic ob/ob mouse. These data indicate that: (a) IRS-1 protein levels are differentially regulated in liver and muscle; (b) insulin levels may play a role in this differential regulation of IRS-1; (c) IRS-1 phosphorylation depends more on insulin receptor kinase activity than IRS-1 protein levels; and (d) reduced IRS-1 phosphorylation in liver and muscle may play a role in insulin-resistant states, especially of the ob/ob mice.


Interleukin-2 and interleukin-2 receptor expression in human corticotrophic adenoma and murine pituitary cell cultures. The production of IL-1 and IL-6 by pituitary cells has recently been demonstrated. In this study we investigated the expression of IL-2 and its receptor (IL-2R) by pituitary cells of different species. In Northern blots, a single hybridizing band of 1 kb, identical to that in normal stimulated lymphocytes, was obtained with specific IL-2 probes. In the mouse AT-20 pituitary tumor cell line, IL-2 mRNA expression was detected after stimulation with corticotropin-releasing hormone or phorbol myristate acetate. In human corticotrophic adenoma cells, basal IL-2 mRNA expression as well as IL-2 secretion were further stimulated by phorbol myristate acetate. Both adenoma and AtT-20 cells showed detectable amounts of IL-2R mRNA and by immunofluorescence, IL-2R membrane expression. In addition, dual immunofluorescence studies in rat anterior pituitary cells demonstrated colocalization of IL-2R with ACTH-positive cells and other cell types expressing the receptor. In addition to the action of lymphocyte-produced IL-2, this cytokine may have a paracrine or autocrine regulatory role within the pituitary. It remains to be established whether IL-2 production occurs in the normal pituitary or is intrinsic to the process of tumor development of these cells. IL-2 may be involved in the growth control of pituitary cells.


Human milk mucin inhibits rotavirus replication and prevents experimental gastroenteritis. Acute gastrointestinal infections due to rotaviruses and other enteric pathogens are major causes of morbidity and mortality in infants and young children throughout the world. Breast-feeding can reduce the rate of serious gastroenteritis in infants; however, the degrees of protection offered against rotavirus infection vary in different populations. The mechanisms associated with milk-mediated protection against viral gastroenteritis have not been fully elucidated. We have isolated a macromolecular component of human milk that inhibits the replication of rotaviruses in tissue culture and prevents the development of gastroenteritis in an animal model system. Purification of the component indicates that the antiviral activity is associated with an acidic fraction (pI = 4.0-4.6), which is free of detectable immunoglobulins. Furthermore, high levels of antiviral activity are associated with an affinity-purified complex of human milk mucin. Deglycosylation of the mucin complex results in the loss of antiviral activity. Further purification indicated that rotavirus specifically binds to the milk mucin complex as well as to the 46-kD glycoprotein component of the complex. Binding to the 46-kD component was substantially reduced after chemical hydrolysis of sialic acid. We have documented that human milk mucin can bind to rotavirus and inhibit viral replication in vitro and in vivo. Variations in milk mucin glycoproteins may be associated with different levels of protection against infection with gastrointestinal pathogens.


Verapamil ameliorates the clinical and pathological course of murine myocarditis. The effects of the calcium channel blocking agent, verapamil, were studied in a murine model of viral myocarditis. Three groups of 8-wk-old DBA/2 mice (n = 25 each) were inoculated with 10 plaque-forming units of encephalomyocarditis virus and randomized to three treatment regimens. Group 1 mice received verapamil intraperitoneally (5 mg/kg per d) for 7 d before infection, followed by verapamil orally (mean dose of 3.5 mg/mouse per d) in drinking water during infection. Group 2 mice received only verapamil orally starting on day 4 after infection, coincident with peak viremia. Group 3 (infected control) received no verapamil in regular drinking water after viral inoculation. Additional control animals were studied in group 4 (n = 21), consisting of uninfected control animals receiving intraperitoneal and oral verapamil at doses identical to group 1, and in group 5 (n = 21), consisting of uninfected and untreated controls. Animals were randomly killed from each group (n = 7) at 7, 14, and 28 d after infection. Routine histology was performed blindly on an apical slice of each heart and semi-quantitatively graded for inflammation, necrosis, calcification, and fibrosis on a scale of 0-4. Digital planimetry was performed to measure the absolute and relative areas of inflammation and necrosis. The pretreated animals in group 1 showed marked reduction in inflammation and necrosis (score of 3.7 +/- 1.4 vs. 8.7 +/- 2.0 in group 3 on day 14, P < 0.05) and were indistinguishable from the posttreated group 2 mice (score of 4.0 +/- 1.5 vs. 8.7 +/- 2.0 in group 3 on day 14, P < 0.05). All the uninfected control animals (groups 4 and 5) showed no myocardial lesions whether treated with verapamil or not. Quantitative planimetry confirmed decreased inflammation and necrosis (2.0 +/- 3.3% in group 1 and 3.5 +/- 3.1% in group 2 vs. 21.9 +/- 22.6% in group 3 on day 14). Untreated infected hearts injected with liquid silicone rubber exhibited extensive areas of focal microvascular constriction and microaneurysm formation; verapamil treatment in either group 1 or 2 completely abolished these abnormalities, resembling uninfected controls in groups 4 or 5. We conclude that verapamil, whether given before infection or after peak viremia in an encephalomyocarditis model of murine myocarditis, significantly reduces the microvascular changes and myocardial necrosis, fibrosis, and calcification leading to cardiomyopathy. This suggests the potentially important role of calcium and microvascular spasm in the pathogenesis of viral myocarditis leading to dilated cardiomyopathy, and may have future therapeutic implications.


Hepatic uptake of a modified low molecular weight heparin in rats. Fractionated and unfractionated heparins are widely used as antithrombotic agents. Because of their heterogeneous composition, it is difficult to study the pharmacokinetics of these drugs. We now report on a new method for labeling low molecular weight heparins with 131I by binding tyramine to the anhydromannose end of the molecules. We examined the pharmacokinetics of the compound by intravenous injection of 131I-tyramine-heparin into Wistar rats. About 18% of the activity was found in the liver, whereas 33% was detected in urine. Biological activity in terms of Factor Xa inhibition was measurable. Since evidence from cell culture experiments implies that reticuloendothelial cell system receptors might be involved in heparin metabolism, maleylated BSA, a substance known to block scavenger receptors, was injected before the radiolabeled heparin compound. The liver uptake was reduced from 17.4 to 4.8%. Injection of unfractionated heparin before tracer application caused a considerable increase in urine excretion of the tracer substance. To our knowledge, this is the first report that liver uptake of heparins is linked to scavenger receptor mediated mechanisms in vivo. This interaction of heparins with scavenger receptors might play an important role in the biology of the vessel wall.


Oxygen radical scavengers selectively inhibit interleukin 8 production in human whole blood. The hydroxyl radical (OH.) scavenger dimethyl sulfoxide (DMSO) was found to dose-dependently inhibit interleukin 8 (IL-8) production in LPS-stimulated human whole blood. At a concentration of 1% (vol/vol), DMSO blocked IL-8 release by approximately 90% in the presence of 1 microgram/ml LPS at a 24-h time point, but did not affect cell viability or reduce the production of tumor necrosis factor (TNF), interleukin 6, or interleukin-1 beta (IL-1 beta). DMSO was found to directly inhibit IL-8 expression at the level of transcription. Furthermore, this effect was not LPS-specific, in that IL-8 production was reduced by DMSO to a similar extent upon stimulation of blood with phytohemagglutinin, aggregated immune complexes, TNF, or IL-1 beta. Other oxygen radical scavengers that have been shown to inhibit OH.-dependent reactions (dimethyl thiourea, thiourea, mannitol, and ethanol) also inhibited IL-8 production. Conversely, addition of H2O2 caused a dose-dependent stimulation of IL-8 release. These results provide evidence that reactive oxygen metabolites play an important role in the regulation of IL-8 production and suggest that reduction of IL-8 release may contribute to the beneficial effects of antioxidants in experimental models of inflammation and ischemia/reperfusion injury.


Cytomegalovirus immediate early genes prevent the inhibitory effect of cyclosporin A on interleukin 2 gene transcription. The use of cyclosporin A (CsA) as an immunosuppressive agent has markedly improved the clinical outcome in solid organ transplantation. However, posttransplantation infection remains a significant problem and may contribute to subsequent organ rejection. In this study the effect of cytomegalovirus (CMV) immediate early (IE) gene products on interleukin 2 (IL-2) gene transcription in the absence and presence of CsA was investigated using a transient transfection system. Jurkat T cells were transfected with plasmids expressing the CMV IE gene products or with a control plasmid. The presence of the CMV IE2 gene product abolished the inhibitory effect of CsA on IL-2 promoter activation and gene transcription. This effect was noted regardless of the time of CsA addition relative to the time of stimulation and was independent of CsA concentration. CsA had no effect on the CMV or the IL-2 receptor promoters. These studies suggest that the CMV IE gene products may play a role in graft rejection after solid organ transplantation.


Detection of intrauterine growth retardation in twins using individualized growth assessment: I. Evaluation of growth outcome at birth. The growth of 17 sets of twins was evaluated at 2 to 3 week intervals from 15 weeks to delivery by measurement of the head circumference, abdominal circumference, and thigh circumference, and estimation of weight. The birth characteristics of these twins were compared to those predicted by Rossavik growth models, derived from second-trimester ultrasound measurements, using the Growth Potential Realization Index (GPRI) and by comparison to population standards. Newborns were classified as normal or intrauterine growth retarded (IUGR) based on their Neonatal Growth Assessment Score, determined from GPRI values. All normal twins had birth weights that were appropriate-for-gestational-age and had few abnormal birth measurements. The birth weight differences between normal twin fetuses were all < 20%. IUGR twins were both small for gestational age (78%) and appropriate-for-gestational-age (22%), and all had 3 or 4 abnormal GPRI values. In only 40% of the cases was the birth-weight difference between a normal and an IUGR twin > 20%. No single anatomic parameter evaluated at birth adequately separated the normal twins from those with IUGR. These results indicate the need for multiple parameter Individualized Growth Assessment in the detection of IUGR in twins.


Detection of intrauterine growth retardation in twins using individualized growth assessment. II. Evaluation of third-trimester growth and prediction of growth outcome at birth. Second- and third-trimester growth in 34 twin fetuses was evaluated with ultrasonography by measurement of five anatomic parameters. Rossavik growth models, derived from second-trimester measurements, were used to specify expected third-trimester growth curves. Actual measurements were compared to predicted measurements by calculation of the percent deviations. Growth outcome at birth [normal, intrauterine growth retardation (IUGR)] was determined from Neonatal Growth Assessment Scores. Growth in the second trimester was similar in normal and IUGR twins. In the third trimester, abnormal negative deviations were larger and more numerous in IUGR twins. However, there was considerable individual variability and normal twins also had abnormal negative deviations. In IUGR twins, the first appearance of an abnormal negative deviation was quite variable (range: 28.6 weeks to 35.1 weeks), as was the parameter to show such a deviation. Prediction of neonatal outcome was poor using individual anatomic parameters but improved considerably with use of all five parameters. However, some fetuses were misclassified when only the number of abnormal negative deviations was used. The Prenatal Growth Assessment Score (PGAS), determined by both the number and magnitude of abnormal negative deviations, predicted neonatal outcomes with a sensitivity of 100% and specificity of 100%. On average, PGAS values were abnormal 5 weeks before delivery. These results indicate that normal and IUGR twins can be separated, using third-trimester growth patterns, if multiple parameter Individualized Fetal Growth Assessment is employed.


Sonographic findings in gallbladder ascariasis. Invasion of the adult roundworm, Ascaris lumbricoides, into the gallbladder is rare and was seen in 14(2.1%) of the 665 cases with hepatobiliary ascariasis. The diagnosis was suggested in all 13 cases in which sonography was performed and in 5 of the 11 cases at endoscopic retrograde cholangiography. Sonographic findings included a nonshadowing, long, echogenic structure in the form of a coil, an echogenic strip with central anechoic tube, an echogenic structure extending across the gallbladder giving it a septate appearance, and characteristic erratic, nondirectional, zig-zag movements of these echogenic structures in the gallbladder. Serial sonograms accurately predicted spontaneous exit of the worm. Pregnancy and anomalous origin of the cystic duct directly from the papilla of Vater facilitated worm invasion into the gallbladder. We conclude that real-time sonography offers a simple, rapid approach for the diagnosis and follow-up of patients, whereas endoscopic retrograde cholangiography has limited diagnostic value in this disorder.


Anomalous position of the gallbladder: ultrasonographic and scintigraphic demonstration in four cases. An anomalous position of the gallbladder (GB) is uncommon. Of 40,000 upper abdominal ultrasound examinations, 4 cases of ectopic gallbladder were noted. These were confirmed by hepatobiliary scintigraphy.


A prospective comparative study of transvaginal ultrasonography and digital examination for cervical assessment in the third trimester of pregnancy. Digital examination of the cervix has traditionally been the "gold standard" for cervical assessment. However, it can be subject to inter- as well as intra-observer variation. The usefulness of transvaginal ultrasonography for cervical assessment is examined in this study. Although differences between measurements of transvaginal ultrasonography and digital examinations were observed, transvaginal ultrasonography appears to be a potentially useful and reproducible technique for assessing cervical changes in pregnancy and for prediction of onset of labor.


Comparative analysis of human papillomavirus detection by dot blot hybridisation and non-isotopic in situ hybridisation. To determine the relative diagnostic performance of non-isotopic in situ hybridisation (NISH) and a dot-blot assay for detecting human papillomavirus (HPV) on exfoliated cervical cells; and to correlate the results with cytopathological assessment. Cervical smears and cytological samples were obtained from 122 patients during the same clinical examination and the presence of HPV sequences determined by NISH and dot-blot analysis, respectively. Dot-blot analysis gave an autoradiographic signal in 15 of 121 (12.4%) cases, while NISH detected viral genomes in 38 of 114 (33.3%) cases. Even in the presence of koilocytosis, where vegetative replication of the virus occurs, NISH was positive in over twice as many cases as dot-blot analysis (NISH 90%, dot-blot 40%), while in smears within normal cytological limits, where the viral copy number is likely to be considerably lower, the differences were more striking (NISH 31%, dot-blot 5%). These data show that NISH on cytological smears is more sensitive than a standardised dot-blot hybridisation assay for detecting HPV infection in cytological material and is therefore a more appropriate screening tool.


Invasive breast carcinoma with granulomatous response and deposition of unusual amyloid. To report an unusual case of invasive breast ductal carcinoma associated with non-caseating epithelioid granuloma and unusual deposition of amyloid. Formalin fixed, paraffin wax embedded tissue from breast and lymph nodes were stained with a variety of methods. Representative tissue fragments were sampled and fixed in 2.5% buffered glutaraldehyde, postfixed in 1% osmium tetroxide, dehydrated and embedded in Araldite. Thin sections were viewed under a Phillips 400T transmission electron microscope. Multinucleated giant Langhans' cells were found in the granulomatas tissue in both breast carcinoma and metastatic axillary lymph node carcinoma. Electron microscopic examination showed "tubular" amyloid deposition intermingled with invasive carcinoma and granuloma. "Tubular amyloid" was characterised by a mesh of non-branching curving fibrils with hollow profiles. These tended to be located in the cell membranes. The presence of an epithelioid granulomatous reaction and deposition of "tubular" amyloid in an invasive breast carcinoma could be related to an abnormal immunological response.


Confirmation of second generation anti-hepatitis C virus enzyme immunoassays by antigenic cross-validation. To determine if a scheme for validating enzyme immunoassay (EIA) results could be devised that did not require costly and methodically elaborate supplemental assays. Samples (n = 525) from patients with haemophilia A, leukaemia, and chronic liver disease and at increased risk of hepatitis C virus infection were tested by EIA-1 (Ortho Diagnostics), an assay which uses recombinant HCV fusion proteins as antigens, and by EIA-2 (United Biomedical), an assay based on synthetic HCV oligopeptide antigens. Samples (n = 193) were repeatedly reactive in both EIAs. Of these, 190 (98%) yielded reactivities in both of two supplemental assays used, one an immunoblot assay (RIBA) using recombinant HCV polypeptides similar to EIA-1 antigens, and the other a neutralisation EIA (EIA-2N) based on antigenic competition with HCV peptides similar to EIA-2 antigens. The three samples not reactive in supplemental tests exhibited low EIA optical density (OD) values (signal/cutoff ratios of less than 3). Hence, all specimens reactive and yielding high OD values in both EIAs were also reactive in supplemental assays. Twenty four samples were reactive in EIA-1 only and nine (38%) of these were reactive in RIBA. Fourteen of the 15 (93%) specimens reactive in EIA-1 but not RIBA were derived from patients with chronic liver dysfunction. Two samples were reactive in EIA-2 only, of which one was reactive in EIA-2N and none in RIBA. Compared with EIA-2, EIA-1 yielded more validated reactive samples and resulted in more non-validated reactivities. It is therefore suggested that for clinical diagnosis: (i) EIA-1 be used for anti-HCV testing and EIA-2 for validation of EIA-1 reactivities; (ii) samples concordantly reactive in EIA-1 and EIA-2 and displaying high OD readings be considered HCV antibody positive without supplemental testing; (iii) supplemental testing by RIBA be limited to samples reactive in EIA-1 but equivocal or unreactive in EIA-2 and those concordantly reactive but exhibiting low absorbance readings.


Immunoreactivity of alpha smooth muscle actin in salivary gland tumours: a comparison with S100 protein. Antibodies against alpha smooth muscle actin (ASMA) and S100 protein were applied to paraffin wax embedded sections from 40 salivary gland tumours and seven normal salivary glands. The results indicate that ASMA is useful in the diagnosis of epithelial-myoepithelial carcinoma, but is otherwise only of limited use in diagnostic practice. An unexpected finding was the failure of ASMA to react with myoepitheliomas.


A model for studying the effects of mouthrinses on de novo plaque formation. The aim of the present study was to describe a 4-day no oral hygiene model to assess the pattern of de novo plaque formation and to use this model to appraise the potential of some mouthwash preparations to retard or inhibit plaque formation in the human dentition. 10 subjects were recruited for the trial. During a preparatory period, the participants were exposed to repeated professional plaque control and given oral hygiene instruction to eliminate signs of gingivitis. At the end of the preparatory period, each participant received a final professional tooth cleaning and was subsequently told to abstain from mechanical plaque control efforts for the next 4 days. They were asked to rinse twice daily for 60 s with 10 ml varying test solutions. On Day 4, the volunteers were exposed to a new clinical examination and the presence and amount of plaque were examined by the use of the plaque index system (P1I). The participants were subsequently given a professional tooth cleaning and asked to exercise proper self performed plaque control during the next 10 days. A new test period was then initiated. 6 different mouthwash preparations were tested in each subject namely, (1) placebo (a negative control rinse), (2) Veadent mouthrinse, (3) Listerine mouthrinse, (4) 0.06% triclosan + polyvinyl phosphonic acid (PVPA), (5) 0.06% triclosan + phenolic flavor and (6) 0.12% chlorhexidine digluconate (a positive control rinse). The results from the study revealed that the mean P1I values for individuals, groups of teeth and tooth surfaces provide an adequate but gross overall estimation of the potential of a given mouthrinse to retard/inhibit plaque build up. More detailed information on the effects of the test rinses could be obtained by data describing the % distribution of different P1I score categories; a high frequency of score 0 describes the potential of a mouthrinse to maintain tooth surfaces free from plaque while a low frequency of score 2/3 describes the ability of a treatment to retard/prevent gross plaque formation. The plaque pattern displays finally allowed assessment of the magnitude of plaque prevention, in comparison to the positive and negative controls, that could be achieved by a given compound in various parts and surfaces of the dentition. In this model, all test rinses (i) were significantly more effective than the placebo rinse in retarding de novo plaque build up and (ii) had a minor effects on plaque build up in the maxillary molars and at the approximal surfaces.(ABSTRACT TRUNCATED AT 400 WORDS)


IgG subclass antibodies to Porphyromonas gingivalis in patients with destructive periodontal disease. A case: control study. We have estimated by ELISA the levels of IgG subclass antibodies against P. gingivalis strain W83 whole cells in 35 cases of adult periodontitis and 35 age, sex, ethnic origin and plaque index-matched healthy controls. The mean levels of IgG1 (p < 0.0003) and IgG2 (p < 0.0416) were significantly elevated in the cases. Many sera had no detectable IgG4 antibodies but by categorising IgG4 responses into high, low and absent, this subclass was more often present (p < 0.002) in the cases. Analysis of the paired differences between cases and controls showed that only IgG1 (p < 0.0005) and IgG4 (p < 0.003) levels were significantly greater in the cases. The data support the role of P. gingivalis as a key pathogen in adult periodontitis and high levels of IgG4 antibodies might possibly provide a marker of patients with active disease.


Occurrence of periopathogens in smoker and non-smoker patients. The aim of the present study was to elucidate the interrelationship between cigarette smoking and the occurrence of periopathogens, i.e., Actinobacillus actinomycetemcomitans, Bacteroides gingivalis and Bacteroides intermedius. The study was based on 145 patients with clinically severe periodontitis, 52 men (age range 32-73 years) and 93 women (age range 32-74 years). 83 patients were smokers (> or = 15 cigarettes/day) and 62 non-smokers. Bacterial samples were collected from one site with a probing depth > or = 6 mm for each individual. There were no statistically significant differences in bacterial counts between smokers and non-smokers (p > 0.05). The relative frequencies of smokers and non-smokers positive for A. actinomycetemcomitans were 31% and 31%, for B. gingivalis 42% and 44%, and for B. intermedius 65% and 53%, respectively. The differences between smoking groups were not statistically significant (p > 0.05). The occurrence in different combinations of these bacteria was also determined. There were no statistically significant differences between smoking and combinations of periopathogens (p > 0.05). The results suggest that smoker and non-smoker patients do not differ with regard to occurrence, relative frequency or different combinations of A. actinomycetemcomitans, B. gingivalis and B. intermedius.


Plasma bactericidal activity of a new C-5 methyl fluoroquinolone after oral doses of 400 and 800 mg. The plasma bactericidal activity of a new C-5 methyl fluoroquinolone, OPC-17116, was determined after once-daily oral ingestion of 400 mg and 800 mg in normal, healthy volunteers. OPC-17116 at a 400-mg dose produced plasma bactericidal titers greater than or equal to 1:16 at 12 hours against Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Haemophilus influenzae, and Moraxella catarrhalis. OPC-17116 bactericidal titers against Pseudomonas aeruginosa were 1:2 or 1:4 at 6 and 12 hours. The plasma bactericidal titers against Streptococcus pyogenes and Streptococcus pneumoniae were 1:4 or greater, but bactericidal titers against Staphylococcus aureus were 1:2 at 12 hours and less than 1:2 at 24 hours. The 800-mg dose of OPC-17116 produced bactericidal titers of at least 1:32 at 12 hours for the Enterobacteriaceae, Haemophilus, and Moraxella, and 1:4 for S. pyogenes and S. pneumoniae, but bactericidal titers against S. aureus were 1:2. These data would suggest that an 800-mg dose of OPC-17116 taken orally once daily would provide adequate concentrations to treat infections due to the pathogens examined in this study.


Immunohistochemical distribution and biological activity of pituitary adenylate cyclase-activating polypeptide (PACAP) in the central nervous system of the frog Rana ridibunda. The primary structure of frog pituitary adenylate cyclase-activating polypeptide (PACAP) has recently been determined and the results show that the sequence of PACAP has been highly conserved during evolution. In particular, the structure of the 1-27 fragment of PACAP is identical in frog and mammals. Using an antiserum raised against PACAP27, we have investigated the distribution of PACAP-containing neurons in the central nervous system of the frog Rana ridibunda by the immunofluorescence technique. The main populations of immunoreactive perikarya were located in the medial and ventral diencephalon, i.e., the preoptic nucleus, the ventral and dorsal infundibular nuclei, the nucleus posterocentralis thalami, and the ventral and ventrolateral areas of the thalamus. In the telencephalon, sparse cell bodies were found in the nucleus accumbens septi, the amygdala, the pallial commissure, and the bed nucleus of the pallial commissure. In the hindbrain, the torus semicircularis, the nucleus profundus and the nucleus anteroventralis tegmenti of the mesencephalon also contained populations of PACAP-immunoreactive perikarya. Beaded nerve fibers were observed throughout the brain. Occasionally they formed bundles, e.g., from the ventral infundibulum to the external vascular layer of the median eminence, from the central thalamus to the optic tectum, and rostrocaudally, from the nucleus accumbens septi to the nucleus entopeduncularis. Other areas, such as the interpeduncular nucleus, the nucleus isthmi and the roots of cranial nerves V and VIII in the medulla oblongata, were also densely innervated. The adenylate cyclase-stimulating activity of PACAP was tested by using a static incubation technique for hypothalamic slices.(ABSTRACT TRUNCATED AT 250 WORDS)


Modulation of the calcium pump of the kidney and testes of rats infected with Trypanosoma congolense. The activity of the CaMgATPase (Ca-pump) of the kidney and testes of Wistar rats infected with Trypanosoma congolense was studied during the course of infection. The activity of the enzyme in both organs was found to decrease with increase in parasitaemia. The transition temperature (Tc) decreased and activation energy (Ea) of the enzyme increased with increase in parasitaemia. The relevance of the Ca-pump in the pathogenesis of trypanosomiasis is discussed.


Primary hepatic tumours in cattle. A classification of 66 cases. A study of 66 primary hepatic tumours was carried out on cattle. These consisted almost entirely of adult females. Fifty hepatocellular tumours (10 adenomas and 40 carcinomas), 10 cholangiocellular tumours (three adenomas and seven carcinomas), two cavernous haemangiomas, two haemangioendothelial sarcomas, one fibroma and one Schwannoma were diagnosed. The 50 hepatocellular tumours were classified into adenomas (20 per cent) and carcinomas (80 per cent), both prevalent in the solitary macronodular form. The hepatocellular carcinomas were divided into six groups based on their distinctive histological arrangement and the morphology of the neoplastic cells: trabecular (55 per cent), pseudoglandular (10 per cent), solid (12 per cent), scirrhous (12 per cent), pleomorphic (7 per cent) and fibrolamellar (2 per cent). Twenty-seven of 50 hepatocellular tumours (54 per cent) were associated with features of blood-filled lakes resembling telangiectasis within the neoplastic tissue. Metastasis was uncommon (10 per cent of hepatocellular carcinomas) and grading according to Edmondson's system (1958) showed a low level of anaplasia in most carcinomas (55 per cent). An association with cirrhosis was never found.


Morphological characteristics of a transplantable nephroblastoma (NB-Y) in F344 rats and the relation of tumour growth to hyper-reninaemia in NB-Y-bearing rats. A transplantable tumour, designated NB-Y, was established from a spontaneous nephroblastoma in an F344 rat. NB-Y was serially passaged in syngeneic rats by subcutaneous implantation up to the 49th generation. The transplants grew into nodules with an average diameter of 5 cm and average weight of 92.9 g 4 weeks after implantation. The primary tumour and NB-Y consisted mainly of sheets or clusters of undifferentiated blastemal cells, which reacted immunohistochemically for vimentin but not for keratin. Renin-containing cells were observed in the small blood vessel walls within the primary tumour, but neoplastic cells of both primary tumour and NB-Y failed to stain for renin. Plasma renin activity was significantly higher (40.7 ng per ml per h) in transplanted rats 4 weeks after implantation compared with non-transplanted controls (28.0 ng per ml per h). Hyperplastic juxtaglomerular cells were often observed in rats bearing NB-Y. Sinusoidal dilatation was present in the liver, adrenal glands, pituitary gland and bone marrow of recipients, suggesting abnormal blood flow provoked via the renin-angiotensin system. The present study revealed the development of hyper-reninaemia in NB-Y-bearing rats, but its pathogenesis remains unknown.


Atypical fibrous histiocytoma of the skin and subcutis in childhood and adolescence. The authors have observed 15 examples of a distinctive fibrohistiocytic lesion in children and adolescents which they chose to designate as "atypical fibrous histiocytoma" (AFH). Patient ages ranged from 1 to 19 years (mean 9.3 yr.). Only two cases were encountered in the first year of life, but 7 were seen in children under the age of 10 yr. The anatomic distribution of AFH showed a tendency for a truncal origin (66%), and none was located in the skin of the face, neck, or scalp. Tumor sizes ranged from 1 to 3 cm, and one-third were 2 cm or greater in maximum dimension. Histologically, AFH was characterized by a multinodular, dermal or dermal-subcuticular proliferation of spindle cells, with tapered, cytologically bland nuclei. However, nucleocytoplasmic ratios were increased when compared with those of normal fibroblasts. Nuclear chromatin was dispersed or vesicular; nucleoli were seen in a minority of cases, but mitotic activity was regularly present. Admixed giant cells were present but infrequent, cellularity was dense, and a storiform growth pattern was consistently seen. Mean followup in this group of cases averaged 75 mo. Seven patients (47%) had tumor recurrences after initial excision; in two of these, tissue margins had been free of involvement. The authors conclude that AFH of childhood is analogous to a lesion that has previously been reported as "benign fibrous histiocytoma" in adults. Complete excision and regular postoperative surveillance are recommended for these tumors.


Myoepithelial differentiation in benign sweat gland tumors. Demonstrated by a monoclonal antibody to alpha-smooth muscle actin. One hundred and two cases of benign sweat gland tumors of the skin were studied for the presence of myoepithelial cells specifically identified by a monoclonal antibody to alpha-smooth muscle actin on paraffin-embedded tissues. The monoclonal antibody gave a positive result in 12 of 12 cases of cylindroma, 14 of 16 cases of spiradenoma, 2 of 2 cases of apocrine tubular adenoma (papillary eccrine adenoma), 5 of 5 cases of apocrine hidrocystoma, 5 of 5 cases of hidradenoma papilliferum, and in 10 of 10 cases of syringocystadenoma papilliferum. Rare myoepithelial cells were detected in only 1 of 10 cases of mixed tumor, apocrine type. There was no immunoreactivity for alpha-smooth muscle actin in eccrine hidrocystoma (2 cases), mixed tumor of eccrine type (2 cases), syringoma (7 cases), hidroacanthoma simplex (1 case), eccrine poroma (14 cases), clear cell hidradenoma (15 cases), and in 1 case of eccrine syringofibroadenoma. Our data support the concept that myoepithelial cells are seen in most sweat gland tumors considered to differentiate toward the secretory coil of the normal sweat gland. In contrast, myoepithelial cells are absent in tumors showing differentiation toward the excretory (ductal) component of the gland.


Sweat gland carcinoma with mucinous and infiltrating duct-like patterns. We report a rare case of mucinous carcinoma of the skin with mammary infiltrating carcinoma-like patterns. An 82-year-old Japanese male had a gourd-shaped tumor on his scalp. Histopathologically, the posterior portion of the tumor showed small lobules of cuboidal tumor cells with no atypia floating in mucinous lakes. In the anterior portion, there were solid lobules, cords, and strands of anaplastic tumor cells infiltrating into the surrounding stroma. Enzyme- and immunohistochemistry and electron microscopy confirmed the eccrine origin of this tumor. It is suggested that mucinous carcinoma of the skin can occur in association with diverse histological patterns, analogous to mucinous carcinoma of the breast.


Effect of fat from whole soybeans on performance of dairy cows fed rations differing in fiber level and particle size. Two trials were conducted with lactating dairy cows to determine effects on intake, performance, and chewing activity of supplemental fat in early lactation diets that differed in fiber level and particle size. In trial 1, whole raw soybeans were added at 11.6% of ration DM to alfalfa silage-based TMR containing either finely chopped silage or the same silage with 8.1% coarsely chopped alfalfa hay. Each combination of soybeans (0 or 11.6%) and silage (fine or hay added) was fed as an isocaloric, isonitrogenous TMR to eight Holstein cows in early lactation in a replicated Latin square design with 4-wk periods. Addition of soybeans decreased DMI for fine silage. With silage plus hay, soybean addition decreased milk yield and protein content but increased fat test so that FCM was unchanged. Silage plus hay promoted chewing activity with no interaction of forage particle size with fat addition on chewing activity. In trial 2, TMR based on alfalfa and corn silage contained either 25 or 29% NDF and 0 or 11.6% soybeans. Otherwise, trials 1 and 2 were similar. Soybean addition decreased DMI with low NDF diets. Addition of fat from soybeans had no effect on milk yield or composition, but low fiber decreased fat test and chewing activity. When .4 to .5 kg/d of supplemental fat from whole raw soybeans was fed, higher dietary NDF and larger particle size promoted greater intake with no effect on FCM yield.


Replacement of forage or concentrate with combinations of soyhulls, sodium bicarbonate, or fat for lactating dairy cows. A lactation study was performed from wk 4 to 19 of lactation to evaluate the ability of soyhulls with or without 1% sodium bicarbonate to replace corn silage and the ability of soyhulls, roasted soybeans, and rumen-inert fat to replace concentrate. All diets contained similar concentrations of NE(L) (tabular value), CP, and degradable protein. When forage NDF was reduced to 62.5% of total dietary NDF (32 to 36% NDF, depending on analytical method) with soyhulls, milk production and total tract nutrient digestibility were unaffected. Addition of sodium bicarbonate to the soyhull diet reduced milk production, but other production aspects were similar. As evaluated using body condition scoring, cows fed soyhulls with buffer appeared to lose less condition before the trial and to recondition earlier and more during the trial than did those fed soyhulls without buffer, which explains differences in milk production. Buffer did not increase digestibility of OM and NDF, perhaps because the high rate of passage of soyhulls limited digestibility more than did ruminal pH. Replacement of concentrate with soyhulls and fat tended to increase milk and FCM production, resulting in improved efficiency of milk production. However, fat fed to cows reduced the percentage of milk protein. As evaluated during the last 4 wk of a 6-wk posttreatment period, fat fed to cows had no residual effects on any production aspect measured.


Influence of buffer pH and raw corn starch addition on in vitro fiber digestion kinetics. The impact was studied of buffer pH (5.8, 6.2, and 6.8) on in vitro digestion kinetics of NDF from alfalfa hay, bromegrass hay, corn silage, and alfalfa and bromegrass hays with raw corn starch added to approximate a ration containing 30% NDF. Ash-free NDF was determined at 0, 6, 12, 18, 24, 30, 36, 48, 72, and 96 h of fermentation. Kinetic parameters were estimated by nonlinear regression using an iteratively reweighted least squares technique. Addition of raw corn starch decreased fiber digestion rate for alfalfa hay and lag for bromegrass hay. Both rate and lag of NDF digestion of all substrates were affected negatively below pH 6.2. Predicted ruminal NDF digestibilities (as percentage of that at pH 6.8 treatment) declined below pH 6.2 for all forages; addition of starch decreased predicted ruminal NDF digestibility by 23% for both alfalfa and bromegrass hays, even at pH 6.8. Results suggest that low pH decreases fiber digestion rate and increases lag and that starch accentuates this effect for some substrates.


The effect of repirinast on airway responsiveness to methacholine and allergen. Repirinast, a novel ingested antiallergic asthma medication from Japan, was compared versus placebo on airway responsiveness to methacholine and was compared versus placebo and cromolyn on airway responses to allergen. In 14 patients with mild, stable, atopic asthma, we performed a double-blind, double-dummy, random-order trial with ingested repirinast 300 mg twice daily for 7 days, inhaled cromolyn 40 mg spincaps single dose, and double placebo on allergen-induced early (EAR) and late (LAR) asthmatic responses and increased airway responsiveness. In the 14 subjects, no difference occurred in methacholine PC20 after 6 days of repirinast or 6 days of placebo. In the 13 subjects who completed the allergen study, single-dose cromolyn significantly reduced the EAR by 63% and the LAR by 65% versus placebo (p < 0.02); repirinast was not significantly different from placebo, both the EAR and LAR being reduced by less than 10%. Allergen-induced increase in methacholine responsiveness was borderline (p = 0.052), and no significant drug effects occurred. In these models, a 1-week treatment period with repirinast, like other oral antiallergic asthma medications (e.g., ketotifen, fumarate), provides no protection against airway responses to methacholine or allergen.


Platelet-activating factor enhances interleukin-6 production by alveolar macrophages. The production of the cytokine interleukin-6 (IL-6) by rat alveolar macrophages (AMs) was analyzed after their stimulation with muramyl dipeptide (1 microgram/ml), in the presence of graded concentrations of platelet-activating factor (PAF). Significantly enhanced production of IL-6 was observed at 10(-10) to 10(-8) mol/L PAF, with peak effect at 10(-10) mol/L. This enhancement was blocked by three structurally unrelated specific PAF receptor antagonists BN 52021, WEB 2170, and CV 3988. The biologically inactive PAF precursor/metabolite, lyso-PAF, and the enantiomer enantio-PAF failed to induce significant enhancement in IL-6 production. In parallel, addition of PAF to AM triggered leukotriene B4 (LTB4) release. Inhibition of 5-lipoxygenase pathway by AA-861 or MK 886 inhibited the PAF-induced augmentation of both IL-6 and LTB4 production, suggesting an implication of endogenous leukotrienes in this mechanism. Furthermore, addition of exogenous LTB4 to AMs could augment their IL-6 production, with peak activity at 10(-12) mol/L LTB4, and reverse the inhibitory effects of 5-lipoxygenase inhibitors. Taken together, these observations suggest that PAF can modulate lung immune and inflammatory responses by enhancing IL-6 production and that this activity may be dependent on secondary 5-lipoxygenase metabolites. This may have clinical relevance in PAF-mediated events in the lung, such as the cellular components of late-phase asthma.


Impaired beta-adrenoceptor function, increased leukocyte respiratory burst, and bronchial hyperresponsiveness. Inflammatory processes have potential importance in the pathogenesis of bronchial hyperresponsiveness and asthma. Because beta-adrenoceptor function may be impaired in asthma, we studied regulation of the leukocyte respiratory burst using blood samples from subjects with bronchial hyperresponsiveness to methacholine. Leukocytes from hyperresponsive subjects were less responsive to the beta-agonist isoproterenol than were leukocytes from healthy control subjects. The magnitude of the respiratory burst was increased in cells from hyperresponsive subjects and correlated with the degree of methacholine responsiveness. These results demonstrate that peripheral leukocytes reflect a functional impairment in beta-adrenergic responsiveness that parallels airway hyperresponsiveness. Because untreated subjects demonstrated a reduction in beta-adrenergic response, the impairment in beta-adrenoceptor function was not a result of drug therapy and may be associated with the pathogenesis of asthma.


[The dose dependence of the development of compensatory-restorative body reactions to irradiation. The EPR method]. The study deals with the mechanism of organism's adaptive responses to the effect of radiation in widely ranging dose. Post-irradiation metabolic changes were evaluated in canine blood as well as in murine blood, spleen, bone marrow and liver using the EPR spectroscopy. It was shown that the dynamics of changes in transferrin and ceruloplasmin pools and ribonucleotide reductase activity were phase-dependent with the maxima at the 2nd, 6th and 10-12th days after irradiation. Such dynamics was observed at various irradiation doses applied. The data allow us to suggest that the nonspecific compensatory--adaptive reactions of organisms develop as the response to irradiation. The dose-response function of the reaction intensity was found to be linear. The shape of the dose-response curve indicates that the minimum response of organism depends on the dose linearly up to 3.2 Gy (for dogs) as well as the maximum one. However, in the case of low-dose irradiation (0.25 or 0.5 Gy) there were deviations of maximum responses from the linearity, i.e. the amplification of the amplitude of compensatory adaptive reactions. These effect were shown to be dependent upon initial individual characteristics of animal blood and to be related to the "depressed" or "activated" state of organism prior to irradiation. The ribonucleotide reductase activity was measured in bone marrow and spleen of animals by the EPR method. The nature of non-repairable DNA damage is discussed in view of the inactivation of ribonucleotide reductase.


Bovine inositol monophosphatase: development of a continuous fluorescence assay of enzyme activity. This paper describes a continuous assay for the enzyme inositol monophosphatase which has been developed using a new substrate, the fluorescent compound 4-methylumbelliferyl phosphate. The hydrolysis of the phosphate group from this compound can be readily detected by a resultant large red shift in the emission spectrum from 390-450 nm. The kinetic constants for the enzyme using this new substrate are described.


Chronic central administration of enalaprilat lowers blood pressure in stroke-prone spontaneously hypertensive rats. Earlier studies on the cardiovascular effects of intracerebroventricular (i.c.v.) administration of angiotensin converting enzyme (ACE) inhibitors implicate angiotensin II (AII) present in the central nervous system in the pathogenesis of hypertension. We have now examined whether central AII contributes to the maintenance of established hypertension in adult stroke-prone spontaneously hypertensive rats (SHRSP). The ACE inhibitor, enalaprilat, was infused i.c.v. for two weeks at a rate of 5 micrograms/h via osmotic minipumps. Control rats were either untreated or infused with saline. Mean arterial pressure (MAP), measured via an indwelling catheter, fell within 24 h in the enalaprilat-treated rats and remained at least 30 mmHg lower than in controls. This difference persisted after intravenous (i.v.) administration of a vasopressin (AVP) antagonist but was eliminated by subsequent ganglion blockade with i.v. pentolinium. Without prior administration of the AVP antagonist, however, the reductions of MAP after pentolinium were smaller. The reduction was still attenuated in treated rats compared with controls but there was a significant difference in the residual MAP. Circulating catecholamine levels were reduced by central ACE inhibition. However, pressor responsiveness to i.v. phenylephrine was unaffected. The results suggest that, in SHRSP, central ACE inhibition lowers blood pressure by reducing sympathetic outflow, implying that central AII has a tonic sympathoexcitatory effect in this strain.


Limited clinical usefulness of plasma corticotropin-releasing hormone, adrenocorticotropin and beta-endorphin measurements as markers of lung cancer. We measured plasma corticotropin-releasing hormone (CRH), ACTH, beta-endorphin (beta-EP), and cortisol levels as possible tumor markers in a sequence of 103, randomly selected, patients with lung cancer but without the ectopic Cushing's syndrome and in 72 age- and sex-matched controls. Plasma CRH levels of cancer patients were similar to those of controls both in patients sampled in the morning or in the afternoon. On the other hand, plasma ACTH levels of cancer patients were significantly higher than control patients both in the morning and in the afternoon and showed a preserved circadian rhythm. However, about 35% of cancer patients sampled in the morning and about 60% of those sampled in the afternoon had ACTH levels within the 95% confidence interval (CI) of controls. Also plasma beta-EP levels were more elevated in cancer patients than controls in the morning but about 33% of them and about 80% of those sampled in the afternoon had beta-EP levels within the 95% CI of controls. Despite the higher plasma ACTH levels, cancer patients had cortisol plasma levels similar to controls with preserved circadian rhythm. In conclusion, although mean plasma ACTH and beta-EP were higher in patients affected by lung cancer, their measurements, as well as those of CRH, have practically no diagnostic value. Perhaps measurement of ACTH levels in the bronchial lavage may be more helpful.


Renal beta-adrenoceptors in thyroxine-treated rats. The number of beta-adrenoceptors was measured in cardiac and renal cortex membranes from controls and thyroxine-treated rats. Two methods of membrane preparation (sucrose- and KCl-extraction) were compared. KCl-extraction yielded a higher number of beta-adrenoceptors, accompanied by increased activity of the plasma membrane enzyme marker 5'-nucleotidase. Thyroxine increased the number of beta-adrenoceptors in both cardiac and renal cortex membranes prepared with both methods. These results suggest that: i) Enrichment of the membrane fraction may explain the higher number of beta-adrenoceptors found with KCl, ii) Thyroid hormone plays a role in the regulation of beta-adrenoceptors in the renal cortex.


The membrane receptor for sex steroid binding protein is not ubiquitous. The tissue distribution of the membrane receptor for the Sex Steroid Binding Protein (SBP) has been studied, either in estrogen/androgen dependent tissues and in tissues not strictly sex steroid dependent. A specific interaction of SBP with cell membranes has been observed to occur only in estrogen/androgen dependent tissues, some of them had been previously shown by our laboratory and by other authors to possess a specific receptor for the protein. Thus, the sex steroid dependence of the tissue is likely to be determinant for the expression of the membrane receptor for Sex Steroid Binding Protein.


[Demons-Meigs' syndrome. A case of thecoma and ovarian fibroma]. The authors report two cases of Demons-Meigs' syndrome, one following an ovarian fibrothecoma and the other an ovarian fibroma. There was a striking rise in the serum CA 125 level which was higher than 200 UI/ml, suggestive of cancer of the ovary in both cases. With the help of the literature data, the clinicopathological features of this rare condition and the different hypotheses concerning its aetiology are commented.


The double opposing palmar flaps in complex syndactyly. Double opposing flaps on the palmar surface were designed to create nail folds and to cover bony defects of the distal phalanges in the treatment of complex syndactyly. This is a two-stage procedure, and the flaps were used on 10 hands in 8 patients. There were no failures, and the results were satisfactory after a mean follow-up time of 2 1/2 years. The two stages were performed 2 weeks apart, and the surgery can be done as an outpatient procedure.


Paroxetine: a pharmacological review.

Paroxetine: a pharmacological review. Paroxetine has been characterized as a highly potent and selective 5-hydroxytryptamine (serotonin) reuptake inhibitor. This selectivity has been demonstrated not only for monoamine uptake mechanisms but also for neurotransmitter receptor systems. The pharmacokinetic studies indicate that paroxetine may be administered once a day, and that on this regimen steady state will be achieved in 7-14 days. Furthermore, studies indicate that the metabolites of paroxetine do not contribute to its clinical effects. This profile supports the evidence from clinical studies that paroxetine is an effective and well-tolerated antidepressant with substantial advantages over the tricyclic antidepressants and other selective serotonin reuptake inhibitors.


Antiviral activity of tumor necrosis factor is signaled through the 55-kDa type I TNF receptor [corrected]. Agonist antibodies (Ab) to the two TNF receptors, TNF-R1 (55 kDa) and TNF-R2 (75 kDa), have been shown to signal many of the distinct functions induced by TNF-alpha. We have found that anti-TNF-R1, but not anti-TNF-R2, Ab trigger antiviral activity in human hepatoma Hep-G2 cells and enhance the antiviral activity of IFN-gamma in human lung fibroblast A549 cells. Likewise, anti-human-TNF-R1 Ab had antiviral enhancing activity on murine L929 cells engineered to express human TNF-R1. However, L929 cells that express human TNF-R1 lacking most of the intracellular domain fail to respond to anti-human-TNF-R1 Ab. This demonstrates that the intracellular domain of TNF-R1 is necessary to generate antiviral activity. TNF-R1 but not TNF-R2 also signals killing of virus-infected cells by TNF-alpha. Thus, all the known antiviral activities of TNF-alpha are mediated through TNF-R1.


A novel post-translational incorporation of tyrosine into multiple proteins in activated human neutrophils. Correlation with phagocytosis and activation of the NADPH oxidase-mediated respiratory burst. Activation of human neutrophils by PMA causes a post-translational incorporation of 14C-labeled tyrosine into multiple neutrophil (PMN) proteins, that is distinctly different from the enzymatic tyrosinolation of tubulin in FMLP-stimulated PMN. Post-translational incorporation of other radiolabeled amino acids, including the structurally similar amino acid phenylalanine, does not occur under identical conditions of neutrophil activation, suggesting an involvement of the phenolic hydroxyl group of tyrosine in the PMA-mediated reaction. Similar to the stimulation of PMN tubulin tyrosinolation by FMLP, the PMA-induced incorporation of tyrosine into multiple PMN proteins is closely associated with activation of the NADPH oxidase-mediated respiratory burst in stimulated PMN and can be inhibited by a variety of reducing agents, inhibitors of peroxidase-mediated reactions, and intracellular scavengers of oxygen radicals. Moreover, the PMA-induced post-translational incorporation of tyrosine does not occur in PMN from patients with chronic granulomatous disease and is significantly reduced (50%) in PMN of an individual with myeloperoxidase deficiency. A similar stimulus-induced incorporation of tyrosine into multiple PMN proteins is also observed in PMN exposed to various phagocytic stimuli, and the incorporated radioactivity in cells undergoing phagocytosis is substantially enriched (40- to 50-fold) in isolated PMN phagolysosomes. Consistent with this latter observation, HPLC fractionation of stimulated PMN proteins and analysis of the incorporated radioactivity reveal that the 14C label is primarily associated with PMN membrane proteins. Furthermore, this post-translational incorporation of tyrosine, like that associated with PMA stimulation, is associated with production of oxygen radicals and the generation of protein carbonyl derivatives, which are indicative of oxidative protein modifications via mixed function oxidases. Our findings indicate that tyrosine incorporation into membrane proteins of stimulated PMN is functionally relevant to the physiologic host-defense responses of human neutrophils undergoing phagocytosis.


Characterization of the Mlsf system. I. A novel "polymorphism" of endogenous superantigens. In addition to Mlsa (Mls-1a) and Mlsc (Mls-2a, Mls-3a), we and others have recently described a third set of stimulatory minor lymphocyte stimulating (Mls) determinants, which are ligands for "I-E related" V beta, V beta 5, V beta 11, and V beta 12. Although all V beta associated with the recognition of the conventional Mls determinants are, in general, uniformly deleted in those animals expressing relevant Mls, expression of Mlsf-related V beta reveals various deletion patterns among different strains. Here we describe extensive genetic studies to evaluate the relationship among the self-Ag responsible for clonal deletion of T cells bearing Mlsf-related V beta by using antibodies specific for TCR V beta chain. In addition, a panel of T cell clones specific for the Mlsf determinant were generated and employed to analyze the determinant specificity, which is recognized by Mlsf-reactive T cells in vitro as well as the role of class II molecules in T cell recognition of the Mlsf determinants. The results of these two independent approaches provide evidence that the Mlsf system is composed of a set of gene products that reveal a unique polymorphism in the induction of clonal deletion in vivo and in T cell activation in vitro. One of these gene products causes almost complete deletion of the self-Mlsf reactive T cell repertoire in vivo and elicits a strong proliferative response to Mlsf-specific T cell clones. Expression of the other gene products results in the clonal deletion of only part of the Mlsf-reactive T cell repertoire. Furthermore, the response pattern of Mlsf-specific clones to intra-MHC recombinant inbred strains and the inhibition pattern of these clones by anti-class II antibody suggested that although expression of the I-E molecule is essential for T cell recognition of Mlsf determinants, the A beta gene may also contribute to the efficient presentation of Mlsf determinants by forming unique class II E alpha A beta molecules.


Characterization of the Mlsf system. II. Identification of mouse mammary tumor virus proviruses involved in the clonal deletion of self-Mlsf-reactive T cells. The genetic linkage of loci encoding stimulatory Mlsa and Mlsc determinants with proviruses of mouse mammary tumour viruses (MMTV) has been shown. We previously have reported that the ligand(s) for V beta 5, V beta 11, and V beta 12 behaves as a novel minor lymphocyte-stimulating (Mls) determinant(s), Mlsf, to induce the strong proliferation of unprimed T cells, and that this ligand(s) also functions as a self-Ag for the clonal deletion of self-reactive T cells. In the accompanying paper (Part I), a unique polymorphism characteristic of the Mlsf gene product is presented. In order to determine the genetic basis for this novel Mls system, we examined the progeny of multiple genetic crosses to identify the MMTV proviral loci involved in the clonal deletion of self-Mlsf-reactive T cells. Results from these investigations indicated that at least three known MMTV proviruses, Mtv-8, Mtv-9, and Mtv-11 are involved in the expression of Mlsf gene products. Presence of Mtv-9 results in the complete deletion of V beta 5, V beta 11, and V beta 12; Mtv-8 is associated with the complete deletion of V beta 12, but only a partial deletion of V beta 11 (primarily CD4-positive T cell subset) with little or no deletion of V beta 5; and Mtv-11 induces the complete deletion of V beta 11 and V beta 12, but no deletion of V beta 5. Given the significant sequence homology in the C-terminal portion of the open reading frame (ORF) region among these three MMTV and the almost equivalent effect of these three MMTV provirus upon the V beta 12 repertoire, their apparent hierarchic effect upon the V beta 5 and V beta 11 repertoires suggests that affinity differences in recognition of the same determinant by different TCR V beta may play a significant role in the clonal deletion of self-reactive T cells.


Intraoperative ultrasound of hepatic tumors. Intraoperative ultrasound (IOUS) scanning of liver masses was compared with those of preoperative computed tomography (CT) and intraoperative palpation. Between March 1989 and May 1991, 24 patients underwent 25 IOUS procedures during laparotomy. Intraoperative ultrasound provided more information than the other modalities in 10 patients (40%) and affected operative management in eight patients (32%). It was concluded that IOUS, when used in conjunction with CT and palpation, is an important technique in the surgical management of patients with hepatic neoplasms.


A stimulatory Mls-1 superantigen is destroyed by ultraviolet light while other Mtv-7 antigens remain intact. Significance for Mls-1 unresponsiveness. Accessory cells present Ag together with costimulatory signals as immunogens and without costimulatory signals as tolerogens. Responsiveness and unresponsiveness are thus alternatives of T cell immune reactions to Ag. Superantigens appear to make an exception; being presented by accessory cells capable of providing costimulatory signals, these Ag induce a strong T cell response but leave T cells unresponsive to a secondary challenge (anergy). We show here that T cell anergy is not a mandatory consequence of superantigen-induced activation. Mls-1- BALB/c recipients of DBA/2 spleen cells mount vigorous Mls-1 responses in vivo but their T cells retain the ability to respond to a subsequent Mls-1 challenge in vitro. We tested the possibility that the inability of DBA/2 spleen cells to inactivate Mls-1-reactive BALB/c T cells was the result of excessive costimulatory activity provided by Mls-1+ DBA/2 B cells. Costimulatory accessory cell activity has been reported to be destroyed by UV light. We exposed superantigen-presenting cells to UV radiation and found that they had lost the ability to stimulate an Mls-1 response without, however, gaining the capacity to render Mls-1-specific T cells anergic. Despite their inability to noticeably stimulate Mls-1-reactive T cells, UV-treated Mls-1+ lymphocytes induced an absolute unresponsiveness in Mls-1- recipients to a second challenge with the superantigen. Our data are in agreement with previous evidence, confirmed here, that BALB/c mice establish immunity against Mls-1+ cells, which causes the accelerated rejection of superantigen-bearing lymphocytes. Thus, our data imply that, whereas it takes stimulatory superantigenic Mtv-7 gene products to induce the activation of superantigen-reactive T cells, nonsuperantigenic Mtv-7 gene products may induce an immune response leading to the elimination of Mtv-7+ lymphoid cells.


Negative selection of thymocytes. A novel polymerase chain reaction-based molecular analysis detects requirements for macromolecular synthesis. Self-tolerance is mainly established through clonal deletion of autoreactive T cells during thymic differentiation. The mechanisms by which deletion is achieved are poorly understood. Here we use a specific polymerase chain reaction-based system to characterize DNA fragmentation and show that after in vivo treatment of neonatal mice with staphylococcus enterotoxin B, selective apoptosis of V beta 8+ thymocytes occurs. This process precedes detectable deletion of V beta 8+ cells as determined by phenotypic analysis. Moreover, in vivo administration of cycloheximide and, to a lesser extent, actinomycin D, inhibits apoptosis of staphylococcus enterotoxin B specific thymocytes. Thus, macromolecular synthesis is a requirement for negative selection.


Sequence motifs important for peptide binding to the human MHC class I molecule, HLA-A2. Previous studies have indicated that most HLA-A2-binding peptides are 9 amino acid (aa) residues long, with a Leu at position 2 (P2), and a Val or Leu at P9. We compared the binding properties of different peptides by measuring the rate of dissociation of beta 2-microglobulin from peptide-specific HLA-A2 complexes. The simplest peptide that we identified that could form HLA-A2 complexes had the sequence (in single letter aa code) GLFGGGGGV, indicating that three nonglycine aa are sufficient for binding to HLA-A2. To determine whether most nonapeptides that contained Leu at P2 and Val or Leu at P9 could bind to HLA-A2, we tested the binding of nonapeptides selected from published HIV and melanoma protein sequences, and found that six of seven tested formed stable HLA-A2 complexes. We identified an optimal antigenic undecapeptide from the cytomegalovirus gB protein that could form stable HLA-A2 complexes that contained apparent anchor residues at P2 and P11 (sequence FIAGN-SAYEYV), indicating that the spacing between anchor residues can be somewhat variable. Finally, we tested the importance of every aa in the influenza A matrix peptide 58-66 (sequence GILGFVFTL) for binding to HLA-A2, by using Ala-substituted and Lys-substituted peptides. We found that multiple positions were important for stable binding, including P2, P3, P5-P7, and P9. We conclude that the P2 and P9 anchor residues are of prime importance for peptide binding to HLA-A2. However, other peptide side chains (especially at P3) contribute to the stability of the interaction. In certain cases, the optimal length for peptide binding can be longer than 9 residues.


Emerging human B cell repertoire. Influence of developmental stage and interindividual variation. In B cell precursors developing in fetal lymphopoietic tissue, the selection of VH, DH, and JH gene segments for initial H chain gene assembly is biased. The present study was designed to determine whether these biases persist in fully developed human fetal B cells and to examine specificities encoded by the favored elements. B cells were prepared from two sites representing different stages of development, i.e., fetal liver as a source of cells newly generated in that lymphopoietic environment and fetal spleen as a source of more mature cells, potentially subject to selective environmental factors. The expressed repertoires were sampled by two methods. EBV transformation so binding and structure could be examined simultaneously and generation of cDNA from individual, sorted, unstimulated B cells. We found that mature B cells in liver and secondary lymphoid tissue exhibit the same degree of bias in VH use we previously reported in lymphopoietic tissue of the same gestational age. However, the pattern of DH and JH use more nearly resembled that of the adult, suggesting that some constraints imposed by the rearrangement process are normalized rapidly. Sequences recovered from EBV-transformed clones were not distinguishable from transcripts recovered from single cells by direct amplification. Among antibodies expressed by the EBV clones, binding to self-Ag was common, binding profiles varied, and, in contrast to mice, there was little relationship between specificity and VH element. Interestingly, the two individuals studied differed in the VH element most commonly used. One resembled previously studied fetal repertoires in that VH56p1 encoded about 20% of expressed antibodies, whereas the other did not express VH56p1 and used VH26 in 25% of expressed antibodies. This was found to reflect a lack of the genomic VH56p1 allele, suggesting that genetic variation at the VH locus may significantly influence the emerging human antibody repertoire.


Superoxide production by normal and chronic granulomatous disease (CGD) patient-derived EBV-transformed B cell lines measured by chemiluminescence-based assays. We have compared assays for products of the neutrophil respiratory burst in normal EBV-transformed B cell lines stimulated with agonists of protein kinase C. Those measuring O2- directly or its immediate product, H2O2, were successful. Of these, the most sensitive were the lucigenin- and luminol-based chemiluminescence assays for O2- and H2O2 respectively. Cell lines from CGD patients, with X-linked or autosomal recessive genetic defects in the neutrophil NADPH oxidase, did not respond in these assays, indicative of their inability to produce O2-. The defects in the lines studied encompass both proteins forming the cytochrome b-245 membrane component, and the 47 kDa cytosolic component of the NADPH oxidase. The possession of the disease associated phenotype by these cell lines provides evidence that in the normal situation both neutrophils and B cells produce O2- via the same system.


A rapid method for isolating murine intestine intraepithelial lymphocytes with high yield and purity. Because murine intestine intraepithelial lymphocytes (IEL) are dispersed throughout the intestine epithelium, it is important that IEL extraction procedures result in lymphocyte preparations of sufficient purity for use in in vitro and in vivo experimental systems. Here, we describe an improved technique for isolating murine IEL consisting of a single 30 min extraction followed by multiple nylon wool filtrations and centrifugation through Percoll. This procedure yields a preparation of IEL with high overall recovery and purity yet takes only 2-2.5 h. Evaluation of individual steps in the extraction process indicated that nylon wool filtration, in particular multiple filtrations, and Percoll fractionation both were important for achieving highly-enriched IEL populations by removal of enterocytes and cellular debris, and demonstrated that multiple nylon wool filtration improved the overall IEL recovery. This procedure has several advantages for studies of murine IEL in that the resultant IEL population is ideal for phenotypic, functional, or molecular analyses. Moreover, this technique is effective for isolating IEL on a single-animal basis, thereby permitting analyses of IEL from individual mice rather than as pooled IEL obtained from several animals.


Homogeneous phase pyrophosphate (PPi) measurement (H3PIM). A non-radioactive, quantitative detection system for nucleic acid specific hybridization methodologies including gene amplification. A one-step, non-radioactive, homogeneous phase revelation system designed to detect and quantify nucleic acid hybridization is described. The principle of the procedure, termed homogeneous phase pyrophosphate (PPi) measurement (H3PIM), is to detect and quantify the release of PPi from nucleotides, which occurs stoichiometrically when nucleic acids are synthesized. The method does not require any special reagents before the H3PIM revelation step. H3PIM is particularly adapted to quantitative measurement of gene amplification or cDNA gene expression using PCR, but can also be used following random priming or simultaneous multi-step nucleic acid amplification. This rapid, sensitive, liquid phase procedure permits the design of low-cost, fully automated devices for gene detection and quantification.


A 15-year surveillance study of antibodies to herpes simplex virus types 1 and 2 in a cohort of young girls. A cohort of 839 young girls at the ages of 14 and 15 years was screened for total antibodies to herpes simplex virus (HSV) and, if positive, for specific antibodies to HSV-2, by means of a sensitive, enzyme-linked immunosorbent assay (ELISA). The cohort was followed from 1972-1987. Blood samples were obtained on six occasions during these 16 years. In total, 2270 blood samples were taken. The number of sero-converting girls was studied in relation to calendar time. Two methods were constructed for the statistical analyses. The first of these gave an estimate of the sero-prevalence at different points in time. This analysis showed that the sero-prevalence which was 23% against HSV-1 in 1972 had increased to 36% in 1976. At the end of the study in 1987, 50% of the cohort had sero-converted against HSV-1. The proportion of girls who had sero-converted against HSV-2 was 0.4% in the 14-15-year-olds and had reached 22% by the end of the study. The second statistical method used all the available information implicit in the observations so as to obtain a maximum-likelihood (ML) estimate of the prevalence. The ML estimates were slightly more precise, but the two estimates did not differ significantly. The observations were further analysed by the Mantel-Haenszel test in order to see if there was any dependence between positivity to HSV-1 and HSV-2 respectively but none was found.


Increased level of intracellular MHC class II molecules in murine Langerhans cells following in vivo and in vitro administration of contact allergens. Treatment of murine Langerhans cells (LC) with contact allergens results in increased internalization of cell membrane constituents and therefore in depressed cell-surface expression of major histocompatibility complex (MHC) class II molecules during the first hours after haptenization. In this presentation we show that this downregulation of cell-surface-expressed Ia-antigens is accompanied by an augmentation of the intracellular pool of MHC class II molecules. Rat MoAb 2G9 was developed, which recognizes IA and IE molecules of the d-haplotype. This MoAb competes with the murine MoAb MK-D6 for binding sites to IAd-molecules. After blocking the cell-surface-expressed molecules with 2G9 and permeabilizing the cell membranes this allowed us to measure selectively the intracellular amount of IA molecules by double immunofluorescence staining and flow cytometric analysis. Cell-surface expression of IA molecules was found to be depressed but their internal pool was significantly elevated following in vivo treatment with the contact allergens DNFB, DNCB, oxazolone, and K2Cr2O7 for 3 h. In vitro culture of freshly prepared LC in the presence of 1 microgram/ml DNFB yielded similar results. Blocking of protein biosynthesis with cycloheximide did not prevent this intracellular class II accumulation. An augmented representation of internalized class II molecules in haptenized LC might play an important role in the presentation of contact allergens.


T-cell receptor V beta-family usage in primary cutaneous and primary nodal T-cell non-Hodgkin's lymphomas. To evaluate whether the expression of T-cell receptor (TCR) V beta families in eight cases of malignant T-cell lymphomas took place in a preferential manner, we analyzed four cases of mycosis fungoides (MF), the most common form of primary cutaneous T-cell non-Hodgkin's lymphomas (NHL), and four cases of primary nodal T-cell NHL. The usage of V beta families in T-cell populations was investigated on mRNA that was transcribed to cDNA using a C beta primer and reverse transcriptase. Subsequently, the specific usage of the families was analyzed by polymerase chain reaction (PCR) using combinations of the selected C beta-oligonucleotide primer and one of the family-specific V beta primers. Peripheral blood lymphocytes from four healthy volunteers and 1 "reactive" lymph node served as a control and expressed all 20 V beta families tested for. In T-cell lines, with restricted V beta expression, and in three patients with advanced MF, only one or two V beta families were expressed at the mRNA level. In an early MF lesion this monoclonal expression was absent: several V beta families were expressed with a weak intensity. This may indicate either a polyclonal origin of MF, or that too few monoclonal neoplastic cells were present in the tissue specimen. In the four nodal T-cell NHL, only one family could be clearly distinguished, whereas some of the other V beta families showed only a weak expression. These latter families represent the reactive T-cell component in the nodal T-cell NHL. Both in nodal T-cell NHL and in MF there was no preferential expression of a particular V beta family. There was a good correlation between PCR data and the expression of V beta-family protein products observed by immunohistochemistry on tissue sections of the T-cell lymphomas. All T-cell lines, three cases of MF, and three cases of nodal T-cell NHL showed a rearrangement of the TCR beta chain on DNA level.


Genotypes and sequence variants of human papillomavirus DNAs from human immunodeficiency virus type 1-infected women with cervical intraepithelial neoplasia. Human papillomavirus (HPV) DNA was found in cervicovaginal lavage fluids from 9 of 11 human immunodeficiency virus type 1 (HIV-1)-seropositive female prostitutes with cervical intraepithelial neoplasia (CIN) in Kinshasa, Zaire. Since 7 yielded complex nucleic acid hybridization results consistent with mixed HPV infections, limited sequencing of HPV DNA was used to identify the HPVs present. Three of HPV 16 and 1 each of HPV 18, 31, 33, and 56 and ME180-HPV were identified by sequencing in 8 samples. Each of these genotypes has been found in specimens from HIV-1-seronegative women with CIN. Some DNAs had nucleic acid and amino acid sequence variations relative to the reference HPVs, but the variants were closely related to variants that have been found in HIV-1-seronegative women. Variant amino acids were found predominantly at three positions in one 40-amino-acid segment of the L1 open reading frame sequenced. The predominant HPV 16 variant observed has been found rarely in other countries.


Human cytomegalovirus infection of the major leukocyte subpopulations and evidence for initial viral replication in polymorphonuclear leukocytes from viremic patients. Fourteen immunocompromised patients were examined for viremia, pp65 and p72 antigenemia, and presence of viral DNA in leukocyte fractions of polymorphonuclear leukocytes (PMNL), monocytes/macrophages (M/M), and B and T lymphocytes after purification by fluorescence-activated cell sorting. Nearly all PMNL and M/M fractions were positive for DNA and pp65 antigenemia, while p72 antigenemia was detected in 73% and 62%, respectively. The virus isolation rate was 45% from PMNL and 17% from M/M. T lymphocytes were positive for DNA in 50% of cases and for pp65 and p72 antigenemia in only 11%, while B lymphocytes were DNA-positive in 43% of samples and consistently negative for antigenemia; neither T nor B lymphocytes had virus isolated. Immediate-early (IE)1 RNA was present in 23 (85.2%) of 27 dextran-enriched DNA-positive p72-positive PMNL samples and, in sequential PMNL samples from two heart-transplanted patients, was detected during peak infection in association with p72. Thus, PMNL and M/M are the subpopulations primarily involved in HCMV infection; PMNL may undergo IE replicative events and are not merely passive carriers of phagocytized viral material.


Evidence that protection against rotavirus diarrhea after natural infection is not dependent on serotype-specific neutralizing antibody. This case-control study sought to determine whether protection against clinically significant rotavirus diarrhea in children aged 4-35 months correlated with titers of serum neutralizing antibody and, if so, whether this protection was serotype-specific. Titers of acute-phase sera from 156 cases of treated rotavirus diarrhea in rural Bangladesh were contrasted with titers from 312 contemporaneously selected, age-matched controls. Analyses of the culture-adapted rotaviruses from the cases revealed that 24%, 15%, 43%, and 17% belonged to serotypes 1-4, respectively. Titers of both homologous and heterologous neutralizing antibody in acute blood specimens of cases were significantly lower than those of matched controls. However, multivariate logistic regression models demonstrated that only antibody titers to heterotypic rotaviruses were independently associated with protection against rotavirus disease. These data, which indicate that the correlation of protection with neutralizing antibody titers is not serotype-specific, suggest that immunity to rotavirus disease may be mediated by other factors.


Interaction of lactoferrin and lipopolysaccharide (LPS): effects on the antioxidant property of lactoferrin and the ability of LPS to prime human neutrophils for enhanced superoxide formation. Lactoferrin is a 77-kDa iron-binding protein to which a wide variety of divergent biologic functions have been ascribed. It has recently been reported that lactoferrin interacts with bacterial lipopolysaccharide (LPS) in such a fashion as to affect the binding of lactoferrin to myeloid cells. Two other potential interactions of LPS and lactoferrin were explored. Lactoferrin prevents hydroxyl radical formation by binding iron, even at low pH. Lactoferrin inhibited iron-catalyzed formation of hydroxyl radical in the presence of LPS at pH 7.4 and 4.5. Low concentrations of LPS can be used to "prime" neutrophils toward enhanced function, such as formation of stimulated superoxide anion. Lactoferrin inhibited LPS priming of neutrophils if LPS contamination of the protein (provided by commercial suppliers) was first reduced. Inhibition of LPS priming was observed whether apolactoferrin or iron-saturated lactoferrin was used. Similar inhibition of LPS priming was observed when neutrophils were incubated with other serum proteins (e.g., albumin, apotransferrin, or iron-saturated transferrin). These results show that LPS should not be expected to affect the free radical biology of lactoferrin, which is a crucial physiologic function of this protein. However, lactoferrin inhibits LPS priming, and this effect requires consideration in experimental models of inflammation.


Enhanced inhibition of herpes simplex virus type 1 growth in human corneal fibroblasts by combinations of interferon-alpha and -gamma. Trials testing the topical treatment of herpes simplex virus (HSV) ocular infections with single interferons (IFN) have provided mixed results. To determine if combination therapy with IFN may be more effective, the ability of combinations of IFN-alpha and IFN-gamma to inhibit HSV growth in human corneal fibroblasts (HCF) was assessed. Virus yields were reduced 282-fold and 37-fold, respectively, in HCF treated with either IFN-alpha or IFN-gamma (10(3) units/mL each). In cells treated with a combination of IFN-alpha and IFN-gamma (10(3) units/mL each), an average reduction of 5.1 x 10(5)-fold in the yield of infectious virus was achieved. Combinations of IFN-alpha and IFN-gamma considerably enhanced the antiviral effect in HCF, suggesting that combination treatment may be efficacious against ocular HSV infections; these findings provide a possible explanation at the cellular level for the poor results achieved in previous clinical trials.


Aging and reactivation of latent murine cytomegalovirus. BALB/c mice were experimentally infected with murine cytomegalovirus (MCMV) to discover whether latent MCMV persisted in aging mice and to examine the effect of aging on MCMV reactivation. Latently infected mice received saline, cyclophosphamide, or allogeneic blood at 6 and 18 months of age. MCMV DNA was detected by polymerase chain reaction in submaxillary salivary gland biopsy specimens from saline-treated young and old mice. Evidence of MCMV reactivation was sought by culture of biopsy specimens and by MCMV IgG ELISA of pre- and posttreatment sera from all animals. Very few cyclophosphamide- or saline-treated mice reactivated MCMV at either age, but young transfused mice reactivated MCMV significantly more often than did old transfused mice. These experiments indicate that MCMV DNA persists in the salivary gland of aging mice but that the likelihood of MCMV reactivation does not increase with age.


Cytomegalovirus infection of the central nervous system in patients with AIDS: diagnosis by DNA amplification from cerebrospinal fluid. A nested polymerase chain reaction (PCR) was evaluated for the detection of cytomegalovirus (CMV) DNA in cerebrospinal fluid (CSF). CSF and serum samples from 19 AIDS patients with intracerebral CMV infection diagnosed at autopsy were retrospectively examined. As controls, CSF and serum samples from 15 AIDS patients with only extracerebral CMV involvement at autopsy, from 10 AIDS patients without CMV infection at autopsy, and from 10 anti-human immunodeficiency virus-negative patients without ongoing CMV infection, were studied. CMV DNA was detected from patients with intracerebral CMV infection in 9 of 9, 5 of 6, and 1 of 4 CSF samples collected, respectively, 1-30, 30-90, and 90-300 days before death. Twelve of 13 sera from these patients were CMV PCR-positive. None of the control patients had CMV DNA in CSF. PCR was positive in 6 of 8 sera from AIDS patients with only extracerebral CMV infection and in serum from 1 AIDS patient without CMV involvement at autopsy. CMV PCR on CSF is highly sensitive and specific. It should be considered a rapid and reliable diagnostic method for CMV infection of the central nervous system.


Diagnosis of human cytomegalovirus central nervous system disease in AIDS patients by DNA amplification from cerebrospinal fluid. The utility of amplification of human cytomegalovirus (HCMV) DNA in cerebrospinal fluid (CSF) for the diagnosis of HCMV central nervous system (CNS) disease in AIDS patients was studied. CSF specimens from 30 patients with neurologic dysfunction were assayed by polymerase chain reaction (PCR), and the results were correlated with histopathologic findings, CSF culture, and clinical manifestations. PCR was positive in all 11 patients who had histopathologic evidence of HCMV CNS disease, including 4 who were CSF culture-negative. Three patients with HCMV polyradiculopathy had CSF positive by PCR. Nine patients negative for HCMV by neuropathologic study and an additional 7 patients with HCMV-unrelated clinical diagnosis were all CSF PCR-negative, despite concomitant systemic HCMV infection in 7. In addition, 24 asymptomatic human immunodeficiency virus-infected individuals were CSF PCR-negative. CSF PCR appears to be a sensitive and specific diagnostic method for detection of HCMV CNS disease in AIDS patients.


Rapid detection of cytomegalovirus DNA in cerebrospinal fluid of AIDS patients with neurologic disorders. A polymerase chain reaction (PCR)-based method was used to detect cytomegalovirus (CMV) DNA in 82 cerebrospinal fluid (CSF) samples from 67 patients infected by human immunodeficiency virus (HIV). The test was positive for 14 patients, 8 of whom had CMV-related neurologic disease proven by viral culture of CSF or histologic examination. Encephalitis was the most frequent manifestation in patients with positive PCR results, but CMV DNA was also present in some patients with peripheral neuropathy or polyradiculomyelitis. All patients with proven CMV neurologic disease were positive by PCR. In contrast, viral culture was negative for 4 of the 8 patients and pathologic studies were available only for 5. The specificity of the PCR-based assay could not be assessed precisely because of the lack of a reference standard, but the results correlated well with clinical course and results of the other methods. These findings suggest that the PCR-based method may be a useful noninvasive tool for the rapid diagnosis of CMV-related neurologic disease.


Silent mother-to-child transmission of hepatitis C virus through two generations determined by comparative nucleotide sequence analysis of the viral cDNA. To investigate the routes of transmission of hepatitis C virus (HCV), a family in which HCV was considered to be transmitted from mother to child through two generations was studied. By the polymerase chain reaction method, HCV cDNA was isolated from the serum of a female baby with self-limited hepatitis C. HCV cDNA was also obtained from her mother and grandmother, who are healthy carriers of HCV, as well as from her uncle suffering from chronic persistent hepatitis C. The nucleotide sequence of the HCV cDNA fragment obtained from the baby was identical to that of the mother and was much closer to those of the grandmother and the uncle than to HCV cDNA isolates previously obtained from other Japanese patients or carriers. These results indicate the presence of mother-to-child transmission of HCV and suggest a role of this transmission route in establishing HCV carriers and maintaining a high incidence of HCV infection.


Cytoskeletal modulation of plasma membrane events induced by interferon-alpha. Cytochalasin B, a drug that alters microfilament structure, was found to modulate interferon-alpha (IFN-alpha)-induced changes in ion fluxes, in motional freedom of spin probes, and lateral diffusion of surface antigens. These changes occur in Daudi cells inherently sensitive to the antiproliferative signal of IFN-alpha, but not in insensitive cells, and were associated with the antiproliferative signal previously. The biophysical effects of cytochalasin B were detected by flow cytometric quantitation of membrane potential using an oxonol dye, by electron spin resonance (ESR) spectrometry, and by measurements of fluorescence recovery after photobleaching (FRAP) of surface antigens using a laser-interactive cell imaging system. Cytochalasin B treatment increased an IFN-alpha-induced membrane potential shift by -5 mV. The motional freedom of 5-doxyl-stearic acid changed from 0.67 to 0.63, as expressed by the order parameter, S, with IFN-alpha treatment and was prevented by cytochalasin B. Changes in the lateral diffusion of surface antigens induced by IFN-alpha treatment, D = 5.3 x 10(-10) without treatment and D = 7.8 x 10(-10) cm2/s with treatment, was blocked by cytochalasin B. In contrast, the microtubule stabilizers taxol and D2O and the microtubule depolymerizing colcemid were ineffective at dose levels sufficient to cause the characteristic cell physiological alterations of these agents. These results implicate microfilaments but not the microtubule system in transduction of the antiproliferative signal by IFN-alpha in Daudi cells.