An assay for C1q biosynthesis in cultured human fibroblasts.

With a standard C1 haemolytic assay, cultured human fibroblasts were shown to synthesize and secrete haemolytically active C1 and C1q. An assay for detection and quantitation of intra-cellular biosynthesis and secretion of C1q was then developed, using Sepharose beads covalently coated with goat monospecific anti-C1q IgG. The molecular structure of fibroblast C1q was found to be unusual in 2 ways: firstly, it probably represented an enlarged pro-C1q, and secondly, the secreted molecule had a different structure from the cell associated molecule.

Mannan-binding protein in human liver.

Mannan-binding protein (MBP) is a Ca(2+)-dependent lectin which was first described in 1978 in rabbit liver, and subsequently in serum and liver tissue from humans and a range of animal species. MBP structurally resembles C1q, and may act both as a focus for complement activation on the surface of microorganisms and as an opsonin in its own right. Low serum levels of serum MBP have been described in a group of children known to suffer from severe recurrent infections. MBP has also been reported to behave as an acute phase reactant. This preliminary study has investigated the localisation of MBP in human tissues using material obtained both at post mortem and from diagnostic liver biopsies. Using the IgG fraction of rabbit anti-human MBP, immunoperoxidase staining showed no evidence of significant MBP in a wide range of normal tissues, including liver taken both at post mortem and needle biopsy. However, there was a significant degree of staining for MBP in liver biopsies showing a variety of different pathologies, in particular severely damaged alcoholic livers, and those harbouring metastatic tumour. Moderate degrees of staining were also seen in liver biopsies from patients suffering from chronic biliary disease. It is uncertain whether this localisation of MBP in abnormal liver is an acute phase response, or represents a more fundamental link with liver disease. This question could be the focus for future studies.

Evaluation of serologic disease markers in a population-based cohort of patients with ulcerative colitis and Crohn's disease.

BACKGROUND: The sensitivity of assays for antineutrophil cytoplasmic antibody (ANCA), anti-Saccharomyces cerevisiae antibody (ASCA), and antipancreatic antibody (PAB) in different laboratories is unknown. Likewise, the sensitivity and diagnostic usefulness of these assays in patients with inflammatory bowel disease (IBD) in the community is unknown. METHODS: An incidence cohort of 290 patients with IBD were offered participation in the study. Blood was obtained from 162 patients (56%) (83 with ulcerative colitis, 79 with Crohn's disease) who agreed to participate. ANCA was determined in five laboratories. ASCA in two laboratories, and PAB in one laboratory. RESULTS: In ulcerative colitis, the sensitivity of ANCA determined in five laboratories varied widely, ranging from 0-63%. In Crohn's disease, the sensitivity of ASCA determined in two laboratories did not vary significantly, ranging from 39-44%; and the sensitivity of PAB determined in one laboratory was 15%. The optimal diagnostic usefulness was obtained from one laboratory where the positive predictive values of a positive ANCA assay combined with a negative ASCA assay for ulcerative colitis, and a negative ANCA combined with a positive ASCA for Crohn's disease, were 75% and 86%, respectively. CONCLUSIONS: In patients with IBD, the sensitivity of ANCA varied widely in different laboratories, whereas the prevalence of ASCA was similar. The positive predictive values of the ANCA assay combined with the ASCA assay for ulcerative colitis and Crohn's disease are high enough to be clinically useful.

Viral respiratory infection and SIDS.

One of the many factors which have been implicated in the aetiology of SIDS is infection of the respiratory tract, particularly viral infection. This applies particularly to those infants who die from SIDS who are more than 3 months old. The evidence for this belief is based on both epidemiological and pathological factors. Among the epidemiological factors are the pronounced seasonal variation of SIDS (it being commoner in winter); the increased incidence of pre-existing illness, particularly upper respiratory infections, in the two weeks before death; and the increased occurrence of SIDS during epidemics of viral infection in the community. Not all of these factors are universally accepted, however, particularly when appropriate controls are investigated. The necropsy evidence includes the presence of lymphoid inflammatory infiltrates in the respiratory tract, particularly the upper respiratory tract. While these are present in many cases of SIDS, they are not present in all. Postmortem isolation of respiratory viruses has also given conflicting results: some authors show an apparent increase compared with controls, while others do not. No specific virus has been implicated. Part of the reason for these conflicting epidemiological and pathological results is failure to use proper controls. An additional explanation may be the technical difficulties involved in isolating viruses. Apart from the problems resulting from postmortem effects, culture, immunofluorescence, and ELISA tests are known to give significant false negative rates. Accordingly, newer, potentially more sensitive and robust techniques, such as molecular hybridisation, are being applied to cases of SIDS to determine whether viral infection is more common than is currently recognised. Whatever the outcome of these investigations, it is highly unlikely that viral infection per se is the cause of SIDS. One or more additional factors are also involved which may include an abnormal immune response, generation of thermal stress, precipitation of respiratory obstruction, bacterial overgrowth with toxin release, or suppression of the arousal response.

Alpha-1-antitrypsin and the liver: a routine immunohistological screen.

One hundred and eighty five consecutive liver biopsies were immunostained using anti-alpha-1-antitrypsin to assess the use of routine immunohistochemistry in the diagnosis of alpha-1-antitrypsin (AAT) deficiency. About half the livers showed staining of hepatocytes for alpha-1-antitrypsin, but most of these livers showed a panlobular pattern, possibly indicating increased synthesis of AAT. Only ten contained periportal granules, said to be typical of AAT deficiency. In cases in which serum was also available for quantitation and phenotyping there was no absolute relation between staining pattern, phenotype, and serum concentrations: the immunohistological screening technique, therefore, has limitations in the diagnosis of AAT deficiency in liver biopsy specimens.

Alcohol induced liver disease.

Alcohol induces a variety of changes in the liver: fatty change, hepatitis, fibrosis, and cirrhosis. The histopathological appearances of these conditions are discussed, with special attention to differential diagnosis. Many forms of alcoholic liver disease are associated with Mallory body formation and fibrosis. Mallory bodies are formed, at least in part, from intermediate filaments. Associated changes in intermediate filament organisation in alcoholic liver disease also occur. Their significance in the pathogenesis of hepatocyte death may be related to abnormalities in messenger RNA function. The mechanisms underlying hepatic fibrogenesis are also discussed.

Removal of inhibitor(s) of the polymerase chain reaction from formalin fixed, paraffin wax embedded tissues.

A problem associated with use of the polymerase chain reaction to amplify specific DNA fragments from formalin fixed, paraffin wax embedded tissues is the not infrequent failure of amplification. One possible reason for this could be the presence of inhibitor(s), which interfere with the activity of the reaction. It has been shown that such inhibitor(s) exist when amplifying the human beta globin gene (which exists in human genomic DNA as a single copy gene) from routine clinical samples. A variety of methods to remove such inhibitor(s) were investigated. The results indicate that inhibitor(s) are removed by proteinase K digestion, followed by purification with phenol/chloroform, and centrifugation through a Centricon-30 membrane (30,000 molecular weight cut off). Other factors, including the length and concentration of the DNA sequence to be amplified, can also affect amplification.

In vitro amplification of hepatitis B virus sequences from liver tumour DNA and from paraffin wax embedded tissues using the polymerase chain reaction.

A 185 base pair fragment from the core-polymerase overlap region of the hepatitis B virus (HBV) genome was amplified using the polymerase chain reaction (PCR). The results were compared with those of Southern blotting on extracted DNA from eight hepatocellular carcinomata. The data agreed with those of Southern blotting in six cases (two positive, four negative) but in two other positive cases PCR failed to amplify HBV sequences. This suggests deletion or mutation, or both, of this viral region in these cases. PCR was also used to amplify HBV sequences from formalin fixed, paraffin wax embedded tissue. Tissue inhibition of PCR occurred which increased with the number of tissue sections. It was present in tissues from different organs and species and fixed by different procedures, thus highlighting the need for a positive control during amplification. Use of formalin fixed Alexander cells, however, showed a sensitivity of one viral copy per 5000 cells. Confirmation of the identity of the PCR products was carried out using PCR-generated biotinylated probes, and suggested the insertion of extra nucleotide sequences or infection with an HBV variant in one case.

Tissue distribution of autoantigen specific for primary sclerosing cholangitis.

AIM: To investigate the tissue distribution of the autoantigen specific for primary sclerosing cholangitis. METHODS: A range of normal frozen tissues including nervous system, muscle, uterus, ovary, prostate, pancreas, thyroid, salivary gland, adrenal gland, colon, gall bladder, stomach, jejunum, aorta, skin, kidney, liver, spleen and thymus was sectioned, fixed with acetone, and air-dried. Normal bone marrow and HL60, K562, and U937 cells were cytocentrifuged on to slides, air-dried, and alcohol fixed. Four sera from primary sclerosing cholangitis with high titre antibody (> 1/100) were used to screen the tissues using either two-step or APAAP immunohistochemistry. Normal sera were used as controls. RESULTS: Positive signal was detected in neutrophils in spleen with three out of four primary sclerosing cholangitis sera while one out of four primary sclerosing cholangitis sera stained spindle cells in the liver. All four sera stained mature neutrophils of the normal bone marrow. Some bone marrow neutrophil precursors (metamyelocytes and myelocytes) were also positive. All other tissues, including HL60, K562, and U937 cells, were negative. Normal sera were negative on all tissues. CONCLUSION: Antigen specific for primary sclerosing cholangitis seems to be unique to neutrophil polymorphs and is present only after myeloblast differentiation of the myeloid cell line. The antigen may be within the secondary granule of the neutrophil polymorph.

Biliary expression of heat shock protein: a non-specific feature of chronic cholestatic liver diseases.

AIM: To analyse the expression of heat shock protein (HSP) 60 in biliary epithelium in auto-immune liver conditions and also in chronic cholestatic and other liver diseases. METHODS: Hepatic expression of HSP-60 in frozen liver biopsy specimens from patients with primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), auto-immune hepatitis (AIH), obstructive jaundice (LDO), alcoholic liver disease (ALD), and from normal controls was studied by immunohistochemistry using the APAAP technique and confocal laser scanning microscopy. RESULTS: Increased expression of HSP-60 was demonstrated in the biliary epithelium of patients with PBC, LDO and, to a lesser extent, with PSC. Focal, weaker, biliary epithelial expression of HSP-60 was observed in AIH, ALD and normal liver tissue. Increased expression was also seen on Kupffer cells in LDO and in hepatocytes in areas of piecemeal necrosis in AIH. CONCLUSION: Enhanced biliary expression of HSP-60 is a common feature of chronic biliary disease irrespective of aetiology and is not specific to auto-immune diseases.

Combined non-isotopic in situ hybridisation and immunohistochemistry on routine paraffin wax embedded tissue: identification of cell type infected by human parvovirus and demonstration of cytomegalovirus DNA and antigen in renal infection.

A method for combined immunohistochemistry using alkaline phosphatase antialkaline phosphatase (APAAP) and in situ hybridisation using biotinylated probes was developed. The method requires no change to either technique and no additional procedures between them. The procedure was able to show the cell types involved in parvovirus infection of the fetus. The efficiency of immunohistochemistry and in situ hybridisation for the detection of cytomegalovirus in kidney were also compared: occasional cells contained cytomegalovirus DNA but not antigen. The method is rapid, straightforward, and has wide applications in the study of viral infections, genes, and gene products in tissue sections because it permits the combined demonstration of antigen and nucleic acid on the same section in routine clinical material.

Comparison between liver and serum concentrations of mannan binding protein.

AIMS: To investigate staining patterns for mannan binding protein (MBP) by immunocytochemistry in liver biopsy specimens from patients with various hepatic disorders; to measure the serum MBP concentration in the patients at the time of biopsy; and to compare these to define further the role of MBP in disease. METHODS: Fifty seven consecutive patients with a variety of types of liver disease were studied. Fresh liver biopsy specimens were immunostained with anti-MBP and graded for intensity of staining. Serum MBP concentrations were measured on samples obtained on the day of biopsy, as were a full range of liver blood tests. RESULTS: MBP was only detectable in liver biopsy specimens from patients with morphological evidence of liver disease. MBP was most prominent in the livers of patients with severe alcoholic liver disease; livers harbouring metastases or showing biliary disease had moderate concentrations. Patients with liver disease were more likely to have raised serum MBP concentrations, but there was no correlation between these values and those found in the biopsy specimens. There was also no significant correlation between either of these concentrations and liver blood test abnormalities. CONCLUSIONS: Patients with liver disease tend to have raised MBP concentrations in both the liver and serum, but the exact relation between the two is as yet undefined.

Detection of reactivation and size variation in the regulatory region of JC virus in brain tissue.

AIMS: To develop a sensitive and specific polymerase chain reaction (PCR) based system for detecting genomic variation in JC virus. To apply this system to formalin fixed, paraffin wax embedded brain tissue from patients with and without progressive multifocal leucoencephalopathy (PML). METHODS: A pair of primers (JC1 and JC2) were designed to be complementary to the early and late regions of JC and BK polyomaviruses, respectively. A third primer (JC3), internal to JC1 and JC2, was designed to be specific for JC virus. The specificity of JC3 was investigated by amplifying plasmids with BK or JC virus genomes. Sensitivity was estimated by titration of a plasmid containing JC virus genome. Seven brains from patients with PML (PMLB) and 30 from patients without PML (non-PMLB) were amplified using JC1 and JC2, followed by JC1 and JC3. Amplification of the beta globin gene was used as an amplification control. RESULTS: Amplification with JC1 and JC2 was common for JC and BK viruses, but with JC1 and JC3 it was specific for JC virus. The sensitivity of the system was 25 copies of JC plasmid per 10 microliters of digested tissue. Five out of seven PMLB and 28 of the 30 non-PMLB amplified for beta globin, but only the PMLB gave a signal with polyoma primers. Hypervariation of the length of the regulatory region of the JC isolates in the PML tissues was consistent with the presence of multiple strains of JC. CONCLUSIONS: Variation in the regulatory region of JC virus can be specifically and sensitively detected from routinely processed, paraffin wax embedded brain tissue. Variation in the regulatory region is common in PML derived JC strains, but JC virus was not detectable in non-PMLB tissue.

Detection of low copy human papilloma virus DNA and mRNA in routine paraffin sections of cervix by non-isotopic in situ hybridisation.

In analysing human papilloma virus (HPV) infection of the cervix in formalin fixed paraffin sections by non-isotopic in situ hybridisation two main problems were found: detachment of sections from the glass during hybridisation and probe detection; inadequate sensitivity and inability to assess sensitivity of the in situ procedure. The first problem was investigated by assessing the efficiency of various tissue adhesives individually and in combination. The second problem was addressed by optimising conditions for DNA unmasking, hybridisation, and biotinylated probe detection. Sensitivity of the final in situ procedure developed was assessed by using the detection of pHY2.1 repeats as a built-in control. Extrapolation of data showed that less than 10 copies of HPV DNA can be visualised by these procedures. HPV nucleic acid, mainly in the form of DNA, was detected not only in koilocytic nuclei but also in suprabasal cells in condylomas and CIN lesions. HPV mRNA was also visualised in the cytoplasm (and probably also nuclei) of the same cell types. These non-isotopic in situ procedures give results comparable to those obtained with radiolabelled probes, but they are less time consuming and provide better morphological resolution.

Non-isotopic in situ hybridisation and immunophenotyping of infected cells in the investigation of human fetal parvovirus infection.

AIMS: To compare the use of biotinylated and digoxigenin labelled probes for diagnosis of human fetal parvovirus B19 infection in formalin fixed, paraffin wax embedded tissues; and to assess the cellular distribution of the virus in positive cases. METHODS: Sections of lung tissue from 23 cases of anatomically normal non-immune fetal hydrops presenting between 1984 and 1989, and from 13 control cases of hydrops due to chromosomal abnormality were probed for B19 DNA by in situ hybridisation using both biotinylated and digoxigenin labelled probes. The distribution of the virus was then investigated in all cases of fetal B19 infection confirmed in this laboratory to date (n = 11) by combining in situ hybridisation for viral DNA (using the digoxigenin system) with immunohistological labelling for a range of cellular antigens. RESULTS: Five unequivocal cases of B19 infection were identified among the 23 fetuses with unexplained hydrops using both probe labels. When combined with data from previous studies of the period 1974-1983, the results indicate that B19 infection was responsible for 27% of cases of anatomically normal non-immune hydrops and 8% of all cases, of non-immune hydrops presenting to this hospital over 15 years. False positive signal was seen in an additional three cases, using biotinylated probes. Digoxigenin labelled probes gave greater specificity and permitted detailed investigation of tissues high in endogenous biotin. Though most cells containing B19 DNA colabelled as erythroid precursors, viral DNA was frequently detected within mononuclear-phagocytic cells. In three cases viral signal was also found within occasional myocardial cells labelled by antibody to desmin. CONCLUSIONS: A relatively high proportion of cases of anatomically normal, non-immune hydrops are caused by B19 infection. Digoxigenin is a more reliable probe label than biotin for in situ hybridisation in archival fetal tissues. Double labelling for cellular antigens and viral nucleic acid is a powerful technique for investigating virus-host cell interactions, and provides evidence that cell types other than those of erythroid lineage may have a role in human fetal parvovirus infection.

Detection of hepatitis B pre-core mutant by allele specific polymerase chain reaction.

AIM: Development of a specific polymerase chain reaction (PCR) assay for detection of the pre-core, stop codon, mutant of hepatitis B virus (HBV). METHODS: PCR primers, specific at the 3'-end for nucleotide 1896 of either the pre-core, stop codon, mutant or wild type HBV, were synthesised using published sequence data. Positive control templates for both types of virus were synthesised by the PCR, incorporating sequences specific for each virus type at the appropriate position. These templates were used to optimise the specificity of the procedure. Formalin fixed, paraffin wax embedded human tissue from acute or fulminant HBV hepatitis from Hong Kong or Oxford was then investigated for presence of mutant or wild type virus. The HBV DNA was amplified from this tissue using a two step procedure, with an initial amplification phase followed by a second diagnostic phase on optimally diluted target DNA. RESULTS: Specific detection of mutant or wild type HBV was achieved. An important factor in determining specificity was the temperature of annealing, 70 degrees C proving to be highly specific. To overcome the inherent variation of target copy number in clinical samples and to provide an intrinsic positive control, it was important to generate and standardise the amount of target HBV used for the specific PCR. Two cases of fulminant hepatitis and four cases of acute hepatitis from Hong Kong, and one case of fulminant hepatitis from Oxford, contained only wild type HBV, with no evidence of a mutant virus. CONCLUSION: This method can be applied to FFPE tissues. It is rapid, non-radioactive, and specific for the stop codon mutation at nucleotide 1896 of HBV. Preliminary investigation of a small number of cases of fulminant hepatitis from Oxford and Hong Kong showed only wild type virus. The result differs from results published from Japan and Israel.

Parvovirus as a cause of hydrops fetalis: detection by in situ DNA hybridisation.

Lung tissue from 13 cases of unexplained non-immunological hydrops fetalis was examined by in situ hybridisation to detect parvovirus. Four specimens contained parvovirus DNA in cells in the blood vessel lumina and alveoli. Twenty six control cases were negative for parvovirus DNA. As there was no known epidemic of parvovirus infection during the study period, this suggests that parvovirus is a relatively common cause of non-immunological hydrops fetalis. In situ hybridisation may have a role in clinical medicine, particularly for retrospective investigations.

Distribution of HLA class 1 antigens in normal human tissue and in mammary cancer.

With a monoclonal antibody which reacts with all HLA class 1 antigens it was found that these antigens are not uniformly distributed in all nucleated cells. Rather HLA class 1 antigens are restricted in their distribution to lymphoid cells, endothelial cells of small vessels, and certain epithelia including mammary duct cells. These antigens were not detected on hepatocytes, specialised cells of the central nervous system, or on the tumour cells of 8 out of 17 human mammary cancers. Given the hypothesis that T cells only respond to foreign antigens on cells which share a common major histocompatibility antigen, these results imply that the T cell responses to viral infections of hepatocytes--for example, hepatitis B virus and the CNS--for example, subacute sclerosing encephalitis, are mediated through an antigen system other than HLA class 1. The absence of HLA class 1 antigen on many mammary cancer cells may be of prognostic significance if T cell modulation of tumour growth is mediated through this class of antigens.

Sensitive system for visualising biotinylated DNA probes hybridised in situ: rapid sex determination of intact cells.

A fast and sensitive method for detecting biotinylated deoxyribonucleic acid (DNA) probes was used for sex determination of cells and tissues by in situ hybridisation of a probe "specific" for the Y chromosome (pHY 2.1). Within 24 hours this procedure visualizes the Y chromosome in fetal and adult cells and tissue, without background noise. This procedure should facilitate antenatal determination of sex on small numbers of uncultured cells. The sensitivity of the procedure also permits the chromosomal assignment of genes present in low copy number.

Epithelial membrane antigen and cytokeratin expression by meningiomas: an immunohistological study.

Thirteen meningiomas of varying light microscopic features were studied immunohistologically using a panel of monoclonal antibodies directed against epithelial, mesenchymal, and neural components. All 13 meningiomas showed expression of epithelial membrane antigen, vimentin, and S100 protein, as did normal meninges. Five of the 13 meningiomas also showed focal expression of cytokeratins, with double labelling showing expression of cytokeratins and vimentin by different cells. The cytokeratin expression was especially noticeable in cells surrounding the hyaline bodies of meningiomas. These results provide further evidence that meningiomas have features of both mesenchymal and epithelial tissues.