Synaptophysin: A novel marker for human and rat hepatic stellate cells

Synaptophysin is a protein involved in neurotransmitter exocytosis and is a neuroendocrine marker. We studied synaptophysin immunohistochemical expression in 35 human liver specimens (normal and different pathological conditions), in rat models of galactosamine hepatitis and carbon tetrachloride-induced cirrhosis, and in freshly isolated rat stellate cells. Synaptophysin reactivity was present in perisinusoidal stellate cells in both human and rat normal liver biopsies. The number of synaptophysin-reactive perisinusoidal cells increased in pathological conditions. Double staining for alpha-smooth muscle actin and synaptophysin, detected by confocal laser scanning microscopy, unequivocally demonstrated colocalization of both markers in lobular stellate cells. In addition, freshly isolated rat stellate cells expressed synaptophysin mRNA (detected by polymerase chain reaction) and protein. Finally, electron microscopy showed the presence of small electron translucent vesicles, comparable to the synaptophysin-reactive synaptic vesicles in neurons, in stellate cell projections. We conclude that synaptophysin is a novel marker for quiescent as well as activated hepatic stellate cells. Together with the stellate cell's expression of neural cell adhesion molecule, glial fibrillary acidic protein, and nestin, this finding raises questions about its embryonic origin and its differentiation. In addition, the presence of synaptic vesicles in stellate cell processes suggests a hitherto unknown mechanism of interaction with neighboring cells.     

Squamous cell carcinoma antigen as an adjunct tumour marker in primary carcinoma of the lung.

AIMS--To determine (1) the detection rate of primary carcinoma of the lung by serological assay of CEA (carcinoembryonic antigen); and (2) whether addition of seroassay of squamous cell carcinoma related antigen before treatment improves detection sensitivity. METHODS--A prospective study spanning 27 months was conducted at the University Hospital, Kuala Lumpur. Serum CEA (Abbott IMx) and serum squamous cell carcinoma antigen (Abbott IMx) from patients clinically suspected of having primary carcinoma of the lung, were assayed using the microparticle enzyme immunoassay method. RESULTS--Thirty seven cases of histologically confirmed primary lung carcinoma were studied. Of these, 17 were squamous cell carcinomas, 10 adenocarcinomas, nine small cell carcinomas, and one large cell carcinoma. The patients' ages ranged from 34-82 years. The male:female ratio was 3.6:1. Squamous cell carcinoma antigen was raised above the cutoff value of 1.5 ng/ml in 94.1% of squamous cell carcinomas, 20.0% of adenocarcinomas, and 11.1% of small cell carcinomas. By comparison, CEA was raised above the cutoff value of 3.0 ng/ml in 70.6% of squamous cell carcinomas, 77.8% of small cell carcinomas, and 100% of adenocarcinomas. CEA and squamous cell carcinoma antigen were not raised in the patient with large cell carcinoma and in 14 healthy volunteers. None of 15 patients with a variety of benign lung diseases showed a rise of CEA, while two patients--a 25 year old Indian woman with pneumonia and a 64 year old Malay man with bronchial asthma--had raised squamous cell carcinoma antigen values above the cutoff. Serum CEA and squamous cell carcinoma antigen values did not seem to correlate with stage or degree of differentiation of the tumours. CONCLUSIONS--The findings suggest that CEA is a good general marker for carcinoma, particularly adenocarcinoma. In contrast, squamous cell carcinoma antigen is more specific for squamous carcinoma.     

Intracellular localisation of Fv1

Retroviral resistance mediated by the murine Fv1 gene is believed to result from a direct interaction between the Fv1 gene product and the viral capsid protein. To study the mechanism of Fv1 action, the expression and intracellular localisation of the Fv1 protein were examined. Only very low levels of protein expression seem necessary for virus restriction but the site of expression appears crucial. Active Fv1 was found in association with tubules of the trans-Golgi network, whereas an inactive form was localised in the endoplasmic reticulum. We hypothesize that Fv1 is compartmentalised in the cell on the pathway taken by virus en route to the nucleus, suggesting that incoming virus must pass the trans-Golgi network during its transit to the nucleus.     

Human X-chromosomal lineages in Europe reveal Middle Eastern and Asiatic contacts

Within Europe, classical genetic markers, nuclear autosomal and Y-chromosome DNA polymorphisms display an east-west frequency gradient. This has been taken as evidence for the westward migration of Neolithic farmers from the Middle East. In contrast, most studies of mtDNA variation in Europe and the Middle East have not revealed clinal distributions. Here we report an analysis of dys44 haplotypes, consisting of 35 polymorphisms on an 8 kb segment of the dystrophin gene on Xp21, in a sample of 1203 Eurasian chromosomes. Our results do not show a significant genetic structure in Europe, though when Middle Eastern samples are included a very low but significant genetic structure, rooted in Middle Eastern heterogeneity, is observed. This structure was not correlated to either geography or language, indicating that neither of these factors are a barrier to gene flow within Europe and/or the Middle East. Spatial autocorrelation analysis did not show clinal variation from the Middle East to Europe, though an underlying and ancient east-west cline across the Eurasian continent was detected. Clines provide a strong signal of ancient major population migration(s), and we suggest that the observed cline likely resulted from an ancient, bifurcating migration out of Africa that influenced the colonizing of Europe, Asia and the Americas. Our study reveals that, in addition to settlements from the Near East, Europe has been influenced by other major population movements, such as expansion(s) from Asia, as well as by recent gene flow from within Europe and the Middle East.     

Hepatitis C virus and its genotypes in patients suffering from chronic hepatitis C with or without a cryoglobulinemia-related syndrome

Recently, evidence has been presented for a possible association between hepatitis C virus (HCV) infection and essential mixed cryoglobulinemia (EMC). Eleven consecutive patients with EMC and two with cryoglobulinemia type I were examined for the presence of markers of HCV infection. Eleven of 13 patients (10 with EMC and 1 with type I cryoglobulinemia) had anti-HCV antibodies (as determined by a second generation anti-HCV assay) and HCV-RNA in plasma or serum. HCV-RNA was also detected in liver biopsies of five patients. Genotyping showed that HCV genotype 1 was found in 10 of 11 patients with HCV-RNA (9 genotype 1b and 1 genotype 1a) and only one patient had HCV genotype 2. However, a similar high prevalence of genotype 1b (100%) was found in a group of 14 consecutive patients with chronic hepatitis C, who had no clinical evidence of cryoglobulinemia. Concomitant infection was present in three patients with genotypes 2, 3 and 4, respectively. These findings stress the high prevalence of HCV infection in patients with EMC and further study shows that a difference in genotype prevalence was not found between HCV-related EMC and chronic hepatitis C without clinical manifestations of EMC. © 1994 Wiley-Liss, Inc.     

The matrix metalloproteinase-3 (MMP-3) 5A/6A promoter polymorphism is not associated with ischaemic heart disease: analysis employing a family based approach

Matrix metalloproteinase-3 (MMP-3) has been proposed as an important mediator of the atherosclerotic process. The possible role of the functional -1612 5A/6A polymorphism of the MMP-3 gene in the susceptibility to ischaemic heart disease (IHD) was investigated in a well-defined Irish population using two recently described family based tests of association. One thousand and twelve individuals from 386 families with at least one member prematurely affected with IHD were genotyped. Using the combined transmission disequilibrium test (TDT)/sib-TDT and the pedigree disequilibrium test (PDT), no association between the MMP-3 -1612 5A/6A polymorphism and IHD was found. Our data demonstrate that, in an Irish population, the MMP-3 -1612 5A/6A polymorphism is not associated with IHD.     

Molecular defects in the beta-globin gene identified in different ethnic groups/populations during prenatal diagnosis for beta-thalassemia: a Malaysian experience

Abstract   β-thalassemia is the most-common genetic disorder of hemoglobin synthesis in Malaysia, and about 4.5% of the population are heterozygous carriers of the disorder. Prenatal diagnosis was performed for 96 couples using the Amplification Refractory Mutation System and Gap-Polymerase Chain Reaction. We identified 17 β-globin defects-initiation codon for translation (T-G), -29 (A-G), -28 (A-G), CAP +1 (A-C), CD 8/9 (+G), CD 15 (G-A), CD 17 (A-T), CD 19 (A-G), Hb E (G-A), IVS1-1 (G-T), IVS1-5 (G-C), CD 41/42 (-CTTT), CD 71–72 (+A), IVS2-654 (CT), poly A(A-G), 100-kb ^Gγ(^Aγδβ)° and 45-kb Filipino deletions. The 192 β-alleles studied comprised Chinese (151 patients), Malay (21), Orang Asli from East Malaysia (15), Filipino (1), Indian (1), Indonesian Chinese (2), and Thai (1). In the Chinese, 2 β-globin defects at CD 41/42 and IVS2-654 were responsible for 74% of β-thalassemia. β-mutations at CD 19, IVS1-1 (G-T), IVS1-5, poly A, and hemoglobin E caused 76% of the hemoglobin disorders in the Malays. The Filipino 45-kb deletion caused 73.3% of bthalassemia in the Orang Asli. Using genomic sequencing, the rare Chinese β-mutation at CD 43 (G-T) was confirmed in 2 Chinese, and the Mediterranean mutation IVS1-1 (G-A) was observed in a Malay β-thalassemia carrier. The β-globin mutations confirmed in this prenatal diagnosis study were heterogenous and 65 (68%) couples showed a different globin defect from each other. The use of specific molecular protocols has allowed rapid and successful prenatal diagnosis of β-thalassemia in Malaysia.     

Reexpression of cytokeratin-19 in a primary human hepatocyte culture

Isolated human hepatocytes cultured in liver biomatrix express cytokeratins 8 and 18. By the end of the first and during the second week in culture, hepatocytes, express these cytokeratins and react with antibodies to cytokeratin-19. Cytokeratin-7 was found in individual cholangiocytes contaminating the culture. The presence of cytokeratin-19 in hepatocytes during the second week of culturing can be regarded as its reexpression. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 7, pp. 118–120, July, 1998     

Development of a slide latex agglutination test for rotavirus antigen detection

The aim of this study was to optimize the conditions for the passive adsorption of polyclonal antibody onto plain surface polystyrene latex particles and its performance in a slide latex agglutination test for rotavirus antigen detection. Cleaning of latex particles by washing through repetitive centrifuging, decanting and resuspending in distilled water was adequate in removing surfactants from the particles' surfaces to enable coating. A study of antibody concentration, incubation temperature and buffer pH revealed that optimum coating was achieved with a 3-fold excess of antibody to the calculated total particle surface capacity for the antibody in a glycine-saline buffer of pH 9.2 at 40 degrees C for 4 hours. The ionic strength and pH of the latex suspending buffer and the sample buffer were critical factors determining the sensitivity of the test and the appearance of non-specific agglutination. Ultrasonication, addition of glycerol and Tween 20, either individually or in combination, were able to suppress non-specific agglutination in some batches of latex reagents. Polyethylene glycol 6000 enhanced the quality of agglutination as well as reduced the time of its appearance, especially in reagents that produced poor agglutination.     

A novel inhibitor of the alternative complement pathway prevents antiphospholipid antibody-induced pregnancy loss in mice

Studies in gene-targeted mice have demonstrated that factor B of the alternative complement pathway plays an important role in several disease models, but an exogenous inhibitor of factor B has not previously been available. We have developed an inhibitory monoclonal antibody directed against a critical epitope on mouse factor B and have tested it in a model of antiphospholipid (aPL) antibody (Ab)-induced fetal loss. Gene-targeted factor B-deficient mice (fB-/-) were injected with a fusion protein comprised of the second and third short consensus repeat (SCR) domains of mouse factor B linked to a mouse IgG1 Fc domain. Hybridomas were made from splenocytes of the immunized mouse. One mAb, designated 1379, produced an IgG1 antibody that inhibited alternative pathway activation in vitro and in vivo by preventing formation of the C3bBb complex. Strikingly, this mAb inhibited alternative pathway activation in serum from mice, rats, humans, monkeys, pigs and horses. Fab fragments made from this mAb also inhibited alternative pathway activation. Epitope mapping demonstrated that this antibody binds to factor B within the third SCR domain. When mAb 1379 was administered to mice that also received human IgG containing antiphospholipid antibodies, it provided significant protection from antiphospholipid antibody-induced complement activation and fetal loss. Thus, this mAb to factor B has broad species reactivity and effectively inhibits alternative pathway activation. The mAb protects mice in an in vivo model of antiphospholipid antibody syndrome, demonstrating the therapeutic potential for the inhibition of factor B in this disease.