Utility of osteopontin in cerebrospinal fluid as a diagnostic marker for neuropsychiatric systemic lupus erythematosus.

The whole protein of osteopontin (OPN full) and its cleaved form (OPN N-half) are involved in the immune response and the migration of immune cells to an inflammatory lesion. We have reported that serum OPN full and urine OPN N-half are elevated in lupus nephritis (LN). Neuropsychiatric systemic lupus erythematosus (NPSLE) is a refractory complication of SLE. To investigate whether OPN full and OPN N-half could serve as diagnostic markers for NPSLE, and to elucidate their role in NPSLE pathogenesis, the concentrations of OPN full and OPN N-half in cerebrospinal fluid (CSF) were measured in NPSLE and non-NPSLE patients. We found that the concentration of OPN full in the CSF was significantly higher in NPSLE than in non-NPSLE, and it decreased after treatment. When the cutoff value of OPN full in CSF was set to 963.4 ng/ml, the sensitivity and specificity for the diagnosis of NPSLE were 70% and 100%, respectively. The correlation analysis of OPN full, OPN N-half and various cytokines/chemokines suggested that the cytokines/chemokines could be divided into two clusters: cluster A, which contains OPN full and cluster B, which contains interleukin-6. OPN full in CSF could be a novel diagnostic marker for NPSLE.

Upregulation of mouse CD14 expression in Kupffer cells by lipopolysaccharide.

Western blot analysis showed that a monoclonal antibody against recombinant mouse CD14 (mCD14), designated rmC5-3, specifically reacted with mouse macrophage cell line J774, but not myeloma cell line NS1. Fluorographic and immunocytochemical analysis demonstrated specific binding of rmC5-3 with mouse resident macrophages, inflammatory monocytes and neutrophils, and macrophage cell lines. Immunohistochemical staining using rmC5-3 showed that CD14-positive Kupffer cells (KC) were small in number in the liver in nonstimulated mice. The number of stained KC, which were rich in the midzonal and periportal regions, gradually increased with time after intraperitoneal injection of lipopolysaccharide (LPS), peaked 6 h after injection, and returned to normal by 20 h after injection. Staining intensity over time was proportional to the number of KC. A slight increase in mCD14 expression was observed in peritoneal macrophages 2 h after LPS administration in vivo using flow cytometric analysis. mCD14 mRNA became detectable at 1 h after the intraperitoneal injection of LPS (20 micrograms/mice), and the level dramatically increased with time, peaking at 3 h, and sharply dropped at 6 h. The resident peritoneal macrophages demonstrated a constitutively high mCD14 mRNA expression, which slightly increased 2 h after LPS (100 ng/ml) stimulation in vitro. The level of mCD14 expression in macrophages did not increase after intraperitoneal injection of LPS (20 micrograms/mice).

Detection of human T lymphotrophic virus type I (HTLV-I) proviral DNA and analysis of T cell receptor V beta CDR3 sequences in spinal cord lesions of HTLV-I-associated myelopathy/tropical spastic paraparesis.

Identification of the localization of human T lymphotrophic virus type I (HTLV-I) proviral DNA in the central nervous system (CNS) is crucial to the understanding of the pathogenesis of HTLV-I-associated myelopathy (HAM)/tropical spastic paraparesis (TSP) pathogenesis. We have developed a sensitive detection method, called two-step polymerase chain reaction (PCR) in situ hybridization, which enabled us to detect the HTLV-I proviral DNA in paraffin-embedded spinal cord tissue sections from HAM/TSP patients. HTLV-I proviral DNA was detected only in the nucleus of lymphocytes that had infiltrated into the spinal cord. However, no proviral DNA was amplified in any neuronal cells, including neurons and glial cells. This indicates that the demyelination of the spinal cord by HTLV-I as a result of viral infection of oligodendrocytes or neuronal cells is unlikely. The T cell receptor V beta gene sequence from lymphocytes in the spinal cord lesions taken from the same HAM/TSP autopsy cases revealed unique and restricted CDR3 motifs, CASSLXG(G) (one-letter amino acid. X is any amino acid), CASSPT(G), and CASSGRL which are similar to those described in T cells from brain lesions of multiple sclerosis (MS) and in a rat T cell clone derived from experimental allergic encephalomyelitis (EAE) lesions. The present results suggest that T cells containing restricted V beta CDR3 motifs, which are also found in MS and EAE, become activated upon HTLV-I infection and infiltrate into the spinal cord lesions of HAM/TSP patients.

An anaerobic-aerobic sequential batch process with simultaneous methanogenesis and short-cut denitrification for the treatment of marine biofoulings.

Although combination of denitritation and methanogenesis for wastewater treatment has been widely investigated, an application of this technology to solid waste treatment has been rarely studied. This study investigated an anaerobic-aerobic batch system with simultaneous denitritation-methanogenesis as an effective treatment for marine biofoulings, which is a major source of intermittently discharged organic solid wastes. Preliminary NO2--exposed sludge was inoculated to achieve stable methanogenesis process without NO2- inhibition. Both high NH4+-N removal of 99.5% and high NO2--N accumulation of 96.4% were achieved on average during the nitritation step. Sufficient CH4 recovery of 101 L-CH4 kg-COD-1 was achieved, indicating that the use of NO2--exposed sludge is effective to avoid NO2- inhibition on methanogenesis. Methanogenesis was the main COD utilization pathway when the substrate solubilization occurred actively, while denitritation was the main when solubilization was limited because of substrate shortage. The results showed a high COD removal efficiency of 96.0% and a relatively low nitrogen removal efficiency of 64.4%. Fitting equations were developed to optimize the effluent exchange ratio. The estimated results showed that the increase of effluent exchange ratio during the active solubilization period increased the nitrogen removal efficiency but decreased CH4 content in biogas. An appropriate effluent exchange ratio with high anaerobic effluent quality below approximately 120 mg-N L-1 as well as sufficient CH4 gas quality which can be used as fuel for gas engine generator was achieved by daily effluent exchange of 80% during the first week and 5% during the subsequent 8 days.

Insertional activation of N-myc by endogenous Moloney-like murine retrovirus sequences in macrophage cell lines derived from myeloma cell line-macrophage hybrids.

Hybrids formed from a myeloma cell line, NS1, and macrophages initially show myeloma properties but later, after loss of the parental macrophage genome and consequent loss of myeloma characteristics, express macrophage properties. Molecular studies demonstrated that macrophage properties in the hybridomas originate from the NS1 parental cells (M. Setoguchi, S. Yoshida, Y. Higuchi, S. Akizuki, and S. Yamamoto, Somatic Cell Mol. Genet. 14:427-438, 1988). In such hybrids, N-myc was activated by insertion of endogenous Moloney-like retrovirus sequences into mouse N-myc exon 3 when the hybrids gained macrophage properties. Interestingly, expression of N-myc took place in all aged hybrids. These results suggest that such unique insertional mutagenesis occurs in a regionally specific manner and that expression of N-myc may play a role in hematopoietic lineage conversion.

Enhancement of c-fos expression is associated with activated macrophages.

Unique hybrids (HINS and CANS lines) between macrophages and a myeloma cell line, NS1 initially expressed myeloma functions but later expressed active macrophage functions together with constitutive expression of c-fos gene. Enhancement of c-fos transcription was also observed in activated mouse peritoneal macrophages, and a range of macrophage-stimulating substance was found to induce c-fos transcription kinetic unique to each stimulator including immediate, delayed and prolonged responses in aged HINS-B3 cells, which displayed low levels of steady-state c-fos transcription. It was also found that a significant enhancement of c-fos transcription followed restimulation with either interferon gamma (IFN gamma) or lipopolysaccharide (LPS) after an initial IFN gamma stimulation. Thus it appeared that enhanced c-fos expression was closely connected with macrophage activation. On the other hand, macrophage stimulators suppressed [3H]thymidine incorporation into aged HINS-B3 cells. These results may simply suggest that c-fos expression contributes to macrophase activation but not to cell proliferation. However, it is also possible to speculate that c-fos expression contributes to cell proliferation as a salvage system operating to overcome the suppression during the macrophage activation.

Mouse and human CD14 (myeloid cell-specific leucine-rich glycoprotein) primary structure deduced from cDNA clones.

cDNA clones complementary to MS7-4 (Setoguchi et al. (1988) Somat. Cell Mol. Genet. 14, 427-438) from a mouse macrophage cDNA library were separated. Sequence analysis of these clones demonstrated that the longest cDNA clone, MS7X, had a 1366 bp insert and high homology with that of the human CD14 gene (Ferrero and Goyert (1988) Nucleic Acids Res. 16, 4173). Using the MS7X cDNA probe, cDNA clones were separated from cDNA libraries constructed from a human macrophage cell line and macrophages. The total cDNA sequence was 1364 bp in length, with an open reading frame of 1125 nucleotides matching that of the human CD14 gene except for one nucleotide difference. The amino-acid sequence (mouse CD14), deduced from the nucleotide sequence of the MS7X insert consisted of 351 amino-acid residues with a high leucine content (17.66%) and five putative N-glycosylation sites, and in vitro translation predicted a protein of molecular mass of 37.5 kDa. Human CD14 had 356 amino-acid residues, with high leucine content (15.5%), and contained four putative N-glycosylation sites. Mouse CD14 showed 13 building blocks, of which internal nine blocks have a conserved leucine motif and significant homology with human leucine-rich alpha 2-glycoprotein.

Influence of nifedipine on collateral blood flow during acute ischemia in the dog.

Vasodilators have been found effective in increasing blood flow in the lateral border surrounding a central zone of infarction, but any change in blood flow to this border zone may be to the normal tissue in this zone, rather than to the ischemic tissue. In this study of the effects of nifedipine on collateral blood flow, 31 open chest dogs underwent coronary occlusion followed by nifedipine infusion, either 3 or 1 microgram/kg per min. A balloon perfusion microsphere labeling device was used to separate the influence of normally perfused tissue overlapping with ischemic tissue in the lateral border zone. Nifedipine increased blood flow in the border zone, but this increase could be accounted for by the effect of nifedipine on admixed normal tissue. In the central ischemic zone, nifedipine administration resulted in a decrease in collateral blood flow. Thus, to fully understand the effect of a vasodilator on ischemic zone blood flow, it is necessary to account for flow in overlapping normal tissue.

Infarct size limitation by the xanthine oxidase inhibitor, allopurinol, in closed-chest dogs with small infarcts.

The present study was designed to evaluate the ability of allopurinol to limit infarct size following permanent coronary occlusion in the greyhound. Coronary occlusion was produced by injecting 2.5 mm plastic beads into the coronary artery of the closed chest dog. Non-perfused myocardium, the area at risk, was visualised by autoradiography of 141Cerium labelled microspheres which were infused immediately following coronary embolization. The treated dogs (n = 12) received 400 mg of allopurinol orally one day before surgery. A 25 mg . kg-1 bolus was administered (iv) immediately before occlusion, and repeated every 8 h. 11 dogs served as controls. After 24 h, the dogs were killed and the hearts were sliced into 5.0 mm transverse sections. The infarcted myocardium was visualised by triphenyl tetrazolium chloride staining. The percentage of the risk zone which evolved to infarct was calculated. This percentage was 18.1 +/- 3.95% in the allopurinol group vs 58.4 +/- 2.81% in the control group (p less than 0.001). We conclude that allopurinol is a potent drug for the limitation of infarct size in the dog with permanent coronary occlusion.

Influence of nitroglycerin on collateral blood flow during acute ischaemia in the dog.

To investigate the effects of nitroglycerin on collateral blood flow 10 open chest dogs underwent coronary occlusion followed by nitroglycerin infusion (80 to 300 micrograms . min-1) to lower mean systemic blood pressure by 20 mmHg, followed by phenylephrine infusion (10 to 40 micrograms . min-1) to restore blood pressure to the pre-nitroglycerin level. Myocardial blood flow was measured with microspheres. The contribution of overlapping normal zone tissue in the ischaemic zone was evaluated with the balloon perfusion technique. Collateral flow was measured with microspheres in the most ischaemic tissue. In addition "load line" analysis was used to calculate collateral flow from retrograde flow. Nitroglycerin lowered blood flow to non-ischaemic tissue, and tended to lower blood flow to ischaemic tissue. Phenylephrine restored blood flow to the value after coronary occlusion. Load line analysis data was similar to data on myocardial blood flow from the microspheres. Collateral resistance changed little during the experiment. Th effects of nitroglycerin on collateral blood flow are, thus, minimal. While it is possible that under special circumstances there may be some decrease in collateral resistance, the bulk of data from this study and others do not support the idea that systemic infusion of nitroglycerin in the setting of an acute myocardial infarction will affect collateral flow.

Comparative effects of nitroglycerin and nifedipine on myocardial blood flow and contraction during flow-limiting coronary stenosis in the dog.

Both nifedipine and nitroglycerin are used to treat angina pectoris. The comparative effects of these agents on myocardial blood flow and contraction in the setting of flow-limiting coronary stenosis are poorly understood. Thus 24 open chest dogs underwent carotid to left anterior descending coronary arterial perfusion with coronary flow probe and perfusion pressure monitoring. Segment length was measured with ultrasonic crystals in the subendocardial ischemic and nonischemic zones. Myocardial blood flow was measured with radioactive microspheres. Partial coronary occlusion was performed to attain a diastolic perfusion pressure of 40 mm Hg. Twelve dogs received intravenous nifedipine, 3 micrograms/kg per min, and 12 received intravenous nitroglycerin to reduce aortic pressure by 20 mm Hg. Partial occlusion resulted in a slight but significant decrease in segment shortening in the ischemic zone. Neither nitroglycerin nor nifedipine affected shortening in the ischemic zone. After occlusion, blood flow decreased in the subendocardial ischemic zone but was unchanged in the subepicardium. Nifedipine increased subendocardial blood flow in the nonischemic zone and decreased it in the ischemic zone but caused no change in subepicardial flow in the ischemic zone. In contrast, nitroglycerin decreased subendocardial and subepicardial blood flow in both the ischemic and nonischemic zones. In the setting of coronary stenosis, different classes of vasodilators may have varying effects on myocardial blood flow, suggesting different sites and mechanisms of action. In addition, segment function may not always reflect changes in myocardial blood flow.

An autopsy case of human T-lymphotropic virus type I-associated myelopathy.

This report describes the first autopsy case of human T-lymphotropic virus type I (HTLV-I)-associated myelopathy (HAM). The disease mainly affected the spinal cord, particularly the lateral and anterior columns, where loss of myelin and axon was observed. The changes were bilateral and occurred mainly along the tract. Perivascular and parenchymal infiltration with lymphocytes and macrophages, as well as astrocytosis, were observed in the white and grey matters of the spinal cord. Blood vessels in the spinal cord and in the subarachnoid space of the spinal cord showed hyalinoid thickening of media and adventitia associated with infiltration of lymphocytes. These findings are similar to those of tropical spastic paraparesis (TSP).

Disseminated infection of herpes simplex virus with fulminant hepatitis in a healthy adult. A case report.

An autopsy case of fulminant hepatitis caused by herpes simplex virus type 1 in a healthy adult is presented. The clinical course was characterized by hepatic failure, disseminated intravascular coagulation and acute renal failure. Many small ulcerations were present in the tongue and tonsils, and there were foci of hemorrhagic necrosis in the liver. Herpes simplex viral antigen was identified in the liver, tonsils, spleen, tongue, pharynx, larynx, esophagus, stomach, intestine, adrenal glands, and lymph nodes with immunohistochemical staining using antibodies to herpes simplex virus type 1. The electron microscopic examination revealed many virions in the hepatocytes. Herpes simplex virus was isolated from the liver, and viral DNA, which had some distinctive features of herpes simplex virus type 1, was examined. We discuss possible reasons for this opportunistic infection occurring in a healthy adult.

Pharmacokinetics and adverse events following 5-day repeated administration of lenograstim, a recombinant human granulocyte colony-stimulating factor, in healthy subjects.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) (lenograstim) was administered to healthy subjects at doses of 2, 5 and 10 micrograms/kg/day for 5 days (twice a day subcutaneously) to examine the optimal dose and schedule of lenograstim in mobilizing peripheral blood progenitor cells (PBSC) for allogeneic transplantation. Lenograstim administration significantly increased CD34+ cells in a dose-related manner. A significant correlation was observed between the maximal post-dosing counts and the pre-dosing baseline counts of CD34+ cells. Peripheral neutrophils increased markedly by seven to 13 times from the baseline to a peak of approximately 40,000/microliter on day 5 for the 5 and 10 micrograms/kg/day doses. After peak serum concentration (Cmax) was attained 4 h following administration, serum G-CSF declined with time in a log-linear fashion. The Cmax and 12 h area-under-the-curve increased dose dependently, but minimum drug level increased up to day 2 and then decreased until day 5. Clearance decreased with increasing dosage at the first dose, and increased significantly at the last dose. We found a highly significant correlation between absolute neutrophil counts and clearance for each dose. Adverse events most frequently occurred on day 6, with increases of alkaline phosphatase and lactate dehydrogenase and onset of bone pain. Increases of aspartate aminotransferase and alanine aminotransferase occurred as delayed events. Platelet count gradually decreased after the end of drug administration to 57% of the pre-dosing count on day 10, but was still within the normal range. These preliminary results suggest that repeated doses of lenograstim induce mobilization of PBSC in a dose-dependent manner and the pre-dosing baseline count of PBSC may predict the post-dosing maximal mobilization. The drug treatment may cause delayed-onset moderate thrombocytopenia and increased transaminase, and the drug clearance changes in a complex manner during repeated dosing.

Structure of the osteopontin gene and its promoter.

We cloned the hOPN gene and its 5' upstream region, and analyzed its exon-intron structure and potential regulatory sequences of the promoter region in comparison with those of mouse and porcine homologues. The hOPN gene consists of 7 exons that are similar to those of the mouse gene, although the hOPN gene is longer than the mouse homologue. This difference is attributable to an insertion of about 1750 bp immediately before exon 4 in the hOPN gene. A region of approximately 285 bp immediately upstream of the hOPN transcription initiation site was highly conserved and contained a number of potential cis regulatory consensus sequences. CAT analysis using SCC-3 cells demonstrated that nucleotides at positions -439 to -270, -124 to -80, and -55 to -39 contained cis-acting enhancing elements, in which the -124 to -80 element was much more active than the others. Deletion of the sequences between -474 and -270 localized the cis elements to the sequence at position -439 to -410, whereas the deletion between -124 to -80 localized it to -124 to -115, and -94 to -80 (data not shown). Gel shift analysis using synthesized double-stranded oligonucleotides corresponding to the 30 bp at position -439 to -410 (data not shown), and 10 and 15 bp regions at positions -124 to -115 and -94 to -80, respectively, as probes revealed that each probe formed one or two bands complexed with a nuclear protein prepared from SCC-3 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinicopathologic characteristics of the lungs of patients with human T cell lymphotropic virus type 1-associated myelopathy.

Lung autopsy specimens were evaluated histologically in the six patients with human T cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy (HAM). The results revealed two histologic changes. First, lymphoid infiltrates were distributed widely in peribronchiolar and perivascular regions, subpleural regions, and the alveolus. Lymphoid infiltrates were also observed in bronchial mucosal glands in relatively large bronchi, in which the acinar epithelium was sometimes degenerated. Second, chronic inflammatory changes, such as smooth muscle hypertrophy, fibrosis, or squamous cell metaplasia, were increased significantly in the membranous bronchioles of HAM patients compared with specimens from lung cancer control patients. Such histologic changes were subclinical in most cases, but one case had an abnormal chest shadow, and two cases had recurrent pneumonia. In HAM patients, high levels of HTLV-1-specific cytotoxic T lymphocytes are believed to attack the HTLV-1-bearing cells in the lung, resulting in inflammatory reactions.

Relationship between impairment of prenatal brain growth and family history of psychosis in schizophrenia.

Recently McNeil et al. (1993b), showed that schizophrenics had smaller head circumference (HC) at birth than controls. This small head size at birth was observed more commonly among schizophrenics without a family history of psychosis than among familial schizophrenics, suggesting that some prenatal environmental factors, rather than genetic factors, are related to the impaired brain growth in utero. We attempted to replicate this finding in 100 Japanese schizophrenics (DSM-III-R), using contemporaneous data on body measures at birth. Conversely, in the current study, HC at birth was found to be significantly smaller in schizophrenics with a family history of psychosis (N = 19) than those without (N = 81). A multiple regression analysis, controlling for gender, gestational age, maternal age, birth order and year of birth, yielded an overall reduction of about 1 cm in HC at birth among familial schizophrenics compared with non-familial schizophrenics. When HC at birth in family history positive and negative groups was compared separately with the local population norms with adjustment for gender and gestational age, familial and non-familial schizophrenics were both found to have significantly smaller HC at birth, although the difference was less marked for the latter. These results suggest that schizophrenics have delayed cerebral development in utero, and that genes which operate on prenatal neurodevelopment may play an important role in the aetiology of schizophrenia, although it is possible that some environmental factors may also be involved in the impaired brain growth.

Three different point mutations in the butyrylcholinesterase gene of three Japanese subjects with a silent phenotype: possible Japanese type alleles.

OBJECTIVE: To investigate genetic mutations in three Japanese subjects homozygous for silent butyrylcholinesterase mutations. METHODS AND RESULTS: One of them was compound heterozygous for two mutations; GGA(Gly) to CGA(Arg) at codon 365 (G365R) and CAA(Gln) to TAA(Ter) at codon 119 (Q119X). The other two subjects were homozygous for different missense mutations: CGT(Arg) to TGT(Cys) at codon 515 (R515C) and G365R, respectively. Simple identification methods for all of the mutations were developed and applied for family analysis and to control individuals. Two mutations, G365R and R515C, have been reported in the Japanese population, while the nonsense mutation Q119X was discovered in the present study. Genetic heterogeneity between human populations with regard to the butyrylcholinesterase gene was suggested. CONCLUSIONS: Among the three mutations found in this investigation, one was novel, and none of these mutations have been reported outside Japan.

A novel in-frame deletion mutation in a case of lactate dehydrogenase (LD) H subunit deficiency showing an atypical LD isoenzyme pattern in serum and erythrocytes.

OBJECTIVE: We report a case showing an atypical lactate dehydrogenase (LD) isoenzyme pattern involving deficiency only of LD-1 and LD-2 in serum and erythrocytes. LD activity in serum from this patient was extremely low, similar to complete LD-H deficiency, and also that in erythrocytes was low. DESIGN: The DNA fragment containing exon 1 through 7 of the LD-H gene were amplified by PCR and directly sequenced. Total RNA was prepared from venous blood and the proportion of LD-H cDNA to total LD cDNA was semiquantified. RESULTS: Genetic analysis by DNA sequencing detected a three base deletion (AAT) at codon 220 of exon 5, which caused a deletion of one asparagine. The present case did not show reduced LD-H expression at the mRNA level in whole blood. Residue 220 is involved in turning beta-J to alpha1-G and is not buried in the interior of the protein. The novel homozygous in-frame deletion mutation at codon 220 may cause a three-dimensional change of the subunit-binding domain.

Tensile properties of human knee joint cartilage. II. Correlations between weight bearing and tissue pathology and the kinetics of swelling.

The nonequilibrium or kinetic swelling behavior of normal, fibrillated, and osteoarthritic (OA) (removed from total knee joint replacements) human knee joint cartilage has been measured using our isometric tensile apparatus (ITA). We found that large local variations exist in the manner with which human knee joint cartilage swells, including anisotropic effects, inhomogeneities, and dependence on local biochemical composition and pathological condition. The ITA provides three convenient biomechanical parameters--peak stress (sigma p), stress relaxation (sigma R), and diffusion coefficient (D)--to quantify the kinetics of swelling. We used these parameters to quantify and differentiate the kinetic swelling behavior of normal, fibrillated, and osteoarthritic cartilage, as well as the swelling behavior of cartilage from high and low weight-bearing areas. Also, these kinetic swelling parameters correlated very well, though by varying degrees, with such biochemical measures as collagen/proteoglycan ratio, hexosamine content/wet weight, and hydroxyproline content/dry weight, providing important insight into the mechanisms and processes involved during the course of swelling. Hence, the kinetic swelling behavior of cartilage should be used to provide important information not obtainable from equilibrium swelling studies.