Prevalence and distribution of HHV-8 in different subpopulations, with and without HIV infection, in Spain.

OBJECTIVE: To estimate the seroprevalence of HHV-8 in several Spanish subpopulations with different risk levels of acquiring HIV-1 infection and from different geographical regions. DESIGN: Cross-sectional seroprevalence study. METHODS: A total of 1699 serum samples from blood donors (613), children under the age of 12 years (100), injecting drug users (IDU) (382), heterosexuals attending a sexually transmitted disease (STD) clinic (273) and homosexual men attending a STD clinic or a HIV-based hospital unit (331) were analysed for anti-HHV-8 antibodies. The presence of antibodies against HHV-8 was tested with an indirect immunofluorescence assay (IFA). A subsample of HHV-8-positive samples was also tested for antibody titre against HHV-8. RESULTS: The overall seroprevalence of antibodies against HHV-8 for the blood donor population was 6.5% (7.0% in Andalusia, 8.0% in Catalonia and 4.5% in the Basque Country). None of the children tested positive for HHV-8. The HHV-8 prevalence was 86.7% in HIV-positive homosexual men and 28.0% in HIV-negative homosexual men (P < 0.001). Of heterosexual men attending STD clinics, 17.2% tested positive for HHV-8; 11.5% of IDU tested positive for HHV-8. HHV-8 antibody titres by groups parallel the distribution of HHV-8 prevalence. No association between HHV-8 antibody titres and CD4 cell count or HIV viral load was identified. CONCLUSIONS: The HHV-8 prevalence among blood donors in Spain is higher than in Northern Europe and the USA, but is similar to that in Northern Italy. The distribution of HHV-8 is compatible with a sexually transmitted agent. The distribution of HHV-8 correlates with that of Kaposi's sarcoma but factors other than HHV-8 seem to explain the Kaposi sarcoma distribution.

Immunohistochemistry of minor salivary gland biopsy specimens from patients with Sjögren's syndrome with and without hepatitis C virus infection.

OBJECTIVES: To characterise phenotypically the minor salivary glands of patients with clinical and histological features of Sjögren's syndrome (SS) infected with hepatitis C virus (HCV). PATIENTS AND METHODS: 75 consecutive patients with SS (31 primary SS, 44 secondary SS) diagnosed by preliminary European classification criteria. The presence of anti-HCV antibodies was detected by commercial third generation ELISA and by a second generation immunoblot assay. Presence of HCV genome in serum was determined by polymerase chain reaction analysis. Expression of CD3, CD4, CD8, CD20, HLA-DR, and CD25 molecules in lymphocytic and epithelial cells on minor salivary glands was detected by immunohistochemical assays. Expression of interferon gamma and interleukin 4 cytokines was determined by in situ hybridisation. RESULTS: Six of 31 primary SS (19%) and one of 44 secondary SS (2%) serum samples were positive for anti-HCV by ELISA. Three samples were positive, three indeterminate, and one sample corresponding to a secondary SS patient was negative by immunoblot. The three immunoblot positive serum samples were also HCV-RNA positive by PCR assay. The study of lymphocytic cells in the diffuse infiltrate of minor salivary glands showed a predominance of the CD3 lymphocytic population. A predominance of CD4 over CD8 T cells (ratio 2:1) was observed in HCV and non-HCV infected patients. The analysis of the lymphocytic focus showed that the HCV infected patients had a predominance of CD20 positive cells. Activation molecules (CD-25 and HLA-DR) were expressed in HCV and non-HCV infected patients in lymphocytic and epithelial cells, however epithelial cell expression of CD25 was low in HCV infected patients. As expected, a pronounced Th1 response was observed in the lymphocytic foci of HCV patients. CONCLUSIONS: HCV infected patients may develop an autoimmune sialadenitis, similar to that described in primary SS.

Characterization of the mucins produced by normal human colonocytes in primary culture.

Mature goblet cells filled with mucin ready for secretion represent about one third of the cells in primary cultures of human colonocytes. In the present study characterization of the mucins produced by cultured human colonocytes was made by histochemical methods by lectin and monoclonal antibody binding. Two monoclonal antibodies and three lectins (Dolichos biflorus (DBA), Helix pomatia (HPA) and Arachis hypogea (PNA) recognizing epitopes or sugar haptens characteristic of different stages of mucin glycoprotein maturation, were employed. The reactivity to these probes was tested both on cultured colonocytes and on tissue sections of the normal colon mucosa. The results show that the mucins produced in culture are glycosylated to the mature form, as they show the same reactivity to lectins and antibodies of the mucins expressed in tissue sections of the normal colon mucosa. In addition, it is demonstrated that cultured human colonocytes do not express mucins reactive to PNA, which are characteristic of tumors. Since the cultured colonocytes maintain the expression of differentiated functions for at least three days, they may offer a useful model to study metabolism, function and regulation of colon mucins in health and disease.

Differential apomucin expression in normal and neoplastic human gastrointestinal tissues.

BACKGROUND/AIMS: The cloning of genes encoding human mucins is the basis for the study of their normal tissue distribution and the alterations associated with cancer. The aim of this study was to determine the normal and tumor tissue expression of MUC1, MUC2, MUC5B, and MUC5C. METHODS: The reactivity of apomucin-specific antibodies with fresh normal and tumor tissues was analyzed using immunohistochemical techniques. RESULTS: Anti-MUC1 antibodies reacted with most glandular epithelia. Anti-MUC2 antibody was mainly reactive with intestinal goblet cells and cervical mucous cells. Anti-MUC5B was reactive with a wide range of epithelial tissues whereas anti-MUC5C was reactive with stomach, trachea, and endocervix. Double-labeling experiments showed coexpression of MUC1/MUC2 and MUC2/MUC5C in colonic tissue. Multiple apomucins were detected in colon cancers, but no relationship to histochemical mucus stains was observed. CONCLUSIONS: It is concluded that (1) each apomucin shows a distinct tissue expression pattern; (2) multiple apomucins are present in a single tissue and at the single cell level; and (3) altered apomucin expression takes place in pathological colonic tissue.

Altered expression of MUC2, MUC4, and MUC5 mucin genes in pancreas tissues and cancer cell lines.

BACKGROUND/AIMS: Neoplastic transformation of epithelial cells is commonly associated with altered synthesis and structure of mucin glycoproteins. The aim of the study was to determine if altered mucin gene expression takes place in pancreas cancer. METHODS: To examine mucin gene expression in normal pancreas and pancreas cancer, antibodies detecting the MUC1, MUC2, MUC5B, and MUC5C apomucins were used in immunohistochemical techniques and complementary DNA probes specific for the MUC1-MUC5 genes were used in Northern blots. RESULTS: MUC1 is the major apomucin expressed in normal pancreas, whereas MUC2-MUC5 are weakly expressed or undetectable. In pancreas cancer tissues and cell lines, increased expression of MUC2, MUC4, and MUC5C is shown. The cytoplasmic expression of MUC2 and MUC5C in tumor cells suggests that these apomucins are underglycosylated and abnormally compartmentalized. CONCLUSIONS: Enhanced expression of MUC2, MUC4, and MUC5C genes is a frequent event in pancreas cancer and may contribute to the alterations in the biochemical structure of pancreas cancer mucins.

cis-Golgi resident proteins and O-glycans are abnormally compartmentalized in the RER of colon cancer cells.

Neoplastic transformation is commonly associated with altered glycosylation of proteins and lipids. To understand the basis for altered mucin glycosylation, we have examined the distribution of RER markers, a cis-Golgi resident protein, and the GalNAc alpha-O-Ser/Thr epitope (Tn) in human colon cancer cells and in normal colon. In cultured mucin-producing colon cancer cells, Gal-NAc alpha-O-Ser/Thr was found in mucin droplets and in RER cisternae. In addition, the Golgi apparatus was disorganized in a proportion of cells and a 130 kDa cis-Golgi resident protein was also abnormally redistributed to the RER. The distribution of the MUC2 intestinal apomucin, protein disulphide isomerase, Gal-NAc alpha-O-Ser/Thr, and the 130 kDa cis-Golgi resident protein was analysed in normal colon and in colon cancer tissues. In normal colon, MUC2 apomucin and protein disulphide isomerase were located in the RER, whereas the cis-Golgi resident protein and GalNAc alpha-O-Ser/Thr were detected only in the cis-Golgi compartment. In contrast, the two Golgi markers colocalized with the MUC2 apomucin and protein disulphide isomerase in the RER of colon cancer cells. On the basis of these results, we propose that in colon cancer cells a redistribution of molecules normally present in the Golgi apparatus takes place; this alteration may contribute to the abnormal glycosylation of proteins and lipids associated with neoplastic transformation.

Detection of the MUC2 apomucin tandem repeat with a mouse monoclonal antibody.

BACKGROUND: The MUC2 intestinal mucin gene contains tandem repeats of 23 amino acid length that are rich in threonine. METHODS: Mouse monoclonal antibody LDQ10 was raised against chemically deglycosylated mucin isolated from LS174T colon cancer nude mouse xenografts. RESULTS: LDQ10 reacts with deglycosylated colon cancer mucin and with a synthetic peptide encompassing the MUC2 tandem repeat sequence. In immunohistochemical assays, strong reactivity with goblet cells in colon, small bowel, and stomach is observed; weaker reactivity with mucin-producing cells in other epithelial tissues is shown. The epitope recognized by LDQ10 is localized in the rough endoplasmic reticulum of normal colonic goblet cells. LDQ10 also shows strong reactivity with colorectal and stomach cancers and weaker reactivity with pancreas, breast, and bladder cancers. CONCLUSIONS: Antibody LDQ10 detects a peptide epitope of MUC2 that becomes cryptic on glycosylation. Altered synthesis of the MUC2 apomucin takes place in a variety of epithelial cancers.