Quantification of serum markers of hepatitis B (HBV) and Delta virus (HDV) infections in patients with chronic HDV infection.

The interplay between hepatitis B (HBV) and delta (HDV) viruses is complex and not always characterized during chronic HDV infection. We assessed the clinical usefulness of new quantitative assays for HBV and HDV serum markers in a retrospective cross-sectional study. Sera obtained from 122 HDV genotype 1 and HBV genotype D coinfected, anti-HIV-negative patients (71 males; median age 49.8 [21.7-66.9] years), recruited consecutively in two geographical areas (Italy 69 patients, Romania 53 patients) with different HBV and HDV epidemiology, were tested for HBsAg, HBV-DNA, HBcrAg, total anti-HBc, HDV-RNA, IgM and total anti-HDV using quantitative assays. Cirrhosis, which showed comparable prevalence in the two cohorts, was diagnosed in 97 of 122 (79.5%) patients. At multivariate analysis, cirrhosis was associated with lower total anti-HBc/IgM anti-HDV ratio (OR 0.990, 95% CI 0.981-0.999, P = .038), whereas disease activity was associated with higher total anti-HDV (OR 10.105, 95% CI 1.671-61.107, P = .012) and HDV-RNA levels (OR 2.366, 95% CI 1.456-3.844, P = .001). HDV-RNA serum levels showed a positive correlation with HBV-DNA (ρ = 0.276, P = .005), HBsAg (ρ = 0.404, P < .001) and HBcrAg (ρ = 0.332, P < .001). The combined quantitative profiling of HBV and HDV serum markers identifies specific patterns associated with activity and stage of chronic hepatitis D (CHD). HDV pathogenicity depends on the underlying active HBV infection in spite of the inhibition of its replication. HDV-RNA, IgM anti-HDV, total anti-HDV, total anti-HBc, HBsAg and HBcrAg serum levels qualify for prospective studies to predict progressive CHD and identify candidates to antiviral therapy.

Terminal glycosylation of bovine uroplakin III, one of the major integral-membrane glycoproteins of mammalian bladder.

Uroplakin III (UPIII) is one of the major transmembrane glycoproteins exposed at the luminal face of mammalian bladder. We investigated the terminal glycosylation of bovine UPIII in order to ascertain whether it contains the alpha 2,3-sialylated sequence thus potentially serving as a receptor for uropathogenic Escherichia coli expressing type S adhesins. We report the occurrence of sialic acid in alpha 2,3- and alpha 2,6-linkage to galactose in bovine UPIII glycans as evidenced by the sensitivity of UPIII to both Vibrio cholera and Newcastle disease virus neuraminidase and by the colocalization of UPIII antigen and material detected by lectins of Sambucus nigra and Maackia amurensis on the luminal face of the bladder. We also present evidence that UPIII glycans are capped by Gal-alpha 1,3-Gal epitope. Consistently, alpha 2,3- and alpha 2, 6-sialyltransferase, as well as alpha 1,3-galactosyltransferase were found to be present in the cells detached from the luminal side of bovine bladder, which are responsible for the UPIII biosynthesis. The putative role of UPIII sialylated glycans in enhancing the uropathogenicity of E. coli expressing type S adhesins is discussed.

Differentiation-dependent glycosylation of gp190, an oncofetal crypt cell antigen expressed by Caco-2 cells.

gp190 is a glycoprotein expressed on the cell surface of several human colon carcinoma cells in culture, on epithelial cells of fetal colon, but not on the normal mucosa of adult colon; thus it is referred to as an oncofetal crypt cell antigen. We report the characterisation of O-linked glycans carried by gp190 synthesised by [3H]glucosamine-labelled Caco-2 cells at the confluence (undifferentiated cells) and at three weeks of postconfluence (differentiated cells). By using a specific monoclonal antibody, gp190 was isolated and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The mobility of gp190 from differentiated cells was found to be lower than that from undifferentiated cells, suggesting a more extensive glycosylation process in the former glycoprotein. The major results of the glycan characterisation have been as follows: (i) gp190 carries mainly, if not exclusively, O-linked glycans with the core-2 structure; (ii) the elongation with N-acetyllactosamine units of the Gal beta1,4GlcNAc beta1,6(Gal beta1,3)GalNAc tetrasaccharide predominates in gp190 synthesised by differentiated cells, whereas the direct alpha2,3sialylation of the tetrasaccharide is prevalent in gp190 synthesised by undifferentiated cells. The increment in the core-2 beta1,6GlcNAc-transferase activity under the Caco-2 differentiation process may be relevant in producing the larger occurrence of polylactosaminoglycans in gp190 from differentiated cells. Since no change in the activity of the alpha2,3sialyltransferases upon cell differentiation was observed, we suggest that the lower alpha2,3sialylation in gp190 synthesised by polarised cells might be due to a changed transit-rate through the distal Golgi apparatus.

Intracellular transport, cell-surface exposure and release of recombinant Tamm-Horsfall glycoprotein.

Human Tamm-Horsfall glycoprotein (T-H), first described as the major urinary glycoprotein, is a glycosylphosphatidyl-inositol (GPI)-anchored membrane protein which mainly resides at the luminal face of cells of the thick ascending limb of Henle's loop (TAL) and early distal convoluted tubules of nephron. Since no human renal cell-line producing T-H is available, T-H cDNA was transfected in HeLa cells and a cell line was selected in which 95% of the cells stably expressed T-H, in order to elucidate the biosynthesis, mechanisms regulating the transport of T-H along the exocytic pathway, exposure at the cell surface and release in soluble form. Treatment of cells with an exogenous reducing agent results in a drastic delay in the conversion from precursor to mature T-H. Since the accumulating T-H-precursor carries glycans not yet processed by Golgi-mannosidases, we propose that the formation of a correct set of intrachain disulphide bonds is required for T-H exit out the endoplasmic reticulum. Even the treatment of cells with an inhibitor of GPI-anchor biosynthesis results in an intracellular accumulation of T-H precursor, loss of T-H localization into Golgi apparatus and reduced surface exposure. These results indicate that the GPI-anchor addition is necessary for T-H delivery to the cell-surface. The release rate of new synthesized T-H shows an initial lag time very likely depending on the time required for T-H surface exposure. A portion of released T-H appears to contain ethanolamine, a component of GPI anchor, indicating that, at least in HeLa cells, a GPI-specific phospholipase contributes to the T-H release. Exposure of cells to monensin and brefeldin A results in a loss of accumulation of T-H in the Golgi perinuclear region and a reduced delivery to the cell surface. Under monensin treatment an intermediate T-H form non-exposed at the cell surface is released in the medium, indicating that a soluble T-H may be produced inside the cell under conditions that alter the Golgi apparatus. If such an event occurs in polarized kidney cells, a T-H release from the basolateral face may be postulated, inasmuch as the GPI-anchor is an apical sorting signal. Since T-H is a powerful autoantigen, the accumulation of soluble T-H in the interstitium of TAL may cause the formation of immunocomplexes.

Mechanism of release of urinary Tamm-Horsfall glycoprotein from the kidney GPI-anchored counterpart.

Human Tamm-Horsfall glycoprotein (THP) is synthesised in the thick ascending limb of Henle and convoluted distal tubules, inserted into luminal cell-surface by the glycosyl-phosphatidylinositol (GPI)-anchor and excreted in urine at a rate of 50-100 mg per day. Up to date there is no indication on the way in which THP is excreted into the urinary fluid. In this study, we examined by Western blotting THP from human kidney in comparison to urinary THP. As expected for a GPI-anchored protein, THP was recovered from the kidney lysate in a Triton X-100 insoluble form, which moved in a sucrose gradient to a zone of low density. The apparent molecular weight of kidney THP appeared greater than that of urinary THP, but no difference in the electrophoretic mobility was observed when the former was subjected to GPI-specific phospholipase-C treatment, strongly suggesting that a proteolytic cleavage at the juxtamembrane-ectodomain of kidney THP is responsible for the urinary excretion.

Binding of human neutrophils to cell-surface anchored Tamm-Horsfall glycoprotein in tubulointerstitial nephritis.

BACKGROUND: Human Tamm-Horsfall glycoprotein (T-H) is a glycosylphosphatidylinositol-anchored protein exposed at the surface of distal nephron cells, and urinary T-H is the released soluble counterpart. The latter has been implicated in tubulointerstitial nephritis, and the proinflammatory potential has been related to its ability to bind in vitro human neutrophils (PMNs). We have examined the conditions required for the binding of neutrophils to cell-surface anchored T-H and the consequent effects. METHODS: A HeLa cell-line derivative permanently transformed with human T-H cDNA and expressing T-H at the cell surface was used throughout the study. The adhesion of PMNs to cells expressing T-H was analyzed by immunofluorescence microscopy before and after the opsonization of cells with anti-T-H antibodies. The oxidative burst induced by adhesion of PMNs to the cells was determined by the activation of myeloperoxidase. Quantitative and qualitative changes in the release of T-H under the adhesion of activated PMNs were determined by dot-blot and Western blot analysis. RESULTS: No binding of neutrophils to cell-surface-anchored T-H was observed. On the contrary, the opsonization of cells with anti-T-H antibodies resulted in a dramatic adhesion of neutrophils. Such an adhesion induced the oxidative burst of PMNs and a large increment in the release of T-H, as well as the release of the slightly faster migrating T-H form, which is normally retained intracellularly. CONCLUSIONS: These results support the notion that, after the autoimmune response, the adhesion of neutrophils to cell-surface T-H contributes to the pathogenesis of tubulointerstitial nephritis, favoring a further accumulation of T-H in the interstitium and inducing the loss of cell integrity via reactive oxygen metabolites generated by activated neutrophils.