Hepatitis B virus antigens in peripheral blood mononuclear cells during the course of viral infection.

We studied the expression of surface (HBsAg) and core (HBcAg) proteins of hepatitis B virus (HBV) on the surface of peripheral blood mononuclear cells (PBMC) from HBV-infected patients. A total of 122 patients with different liver viral diseases was analyzed by indirect immunofluorescence with monoclonal antibodies. The 35 patients with HBV chronic active hepatitis (CAH) and 38 of 60 patients with acute hepatitis B (63%) expressed HBsAg on the PBMC. No expression was detected on the cells from both normal and HBV-unrelated viral hepatitis control groups. Serial follow-up of patients with acute hepatitis B showed that HBsAg expression by PBMC tended to be undetectable 4 months after the onset of the disease and at the same time the clinical improvement was evident. Cell cultures of EBV-transformed B lymphocytes were established from PBMC of HBV-infected patients; immunoelectron microscopy demonstrated the HBsAg on the cellular membrane. One-third of HBV-infected patients who were studied showed the expression of HBcAg by PBMC. HBcAg was detected in patients with acute hepatitis B at the early stage of infection. The cells of these patients also expressed HBsAg in PBMC. In CAH patients, a positive association was observed between the expression of HBcAg and the presence of serum HBeAg.

Analysis of hepatitis B surface antigen expression on leucocyte subpopulations by two-color fluorescence with flow cytometry.

We have analyzed the expression of HBsAg on different subpopulations of peripheral blood cells from hepatitis B virus (HBV) infected patients using two-color immunofluorescence and flow cytometry. An average of 7.4 +/- 0.8% and 9.8 +/- 1.2% HBsAg + cells were found among cells from acute and chronic hepatitis B patients, respectively. When studied by two-color immunofluorescence, HBsAg was limited to cells expressing the B-cell restricted antigen CD19 (16.7 +/- 2.4%), whereas for the other subsets studied (CD3+, CD4+, CD8+, CD67+, CD68+) the percentage of positivity was lower than 4%. The expression of viral antigen on B cells might be relevant for a number of functional interactions between HBsAg+ B and T lymphocytes.

Analysis of hepatitis B surface antigen expression on leucocyte subpopulations by two-color fluorescence with flow cytometry.

We have analyzed the expression of HBsAg on different subpopulations of peripheral blood cells from hepatitis B virus (HBV) infected patients using two-color immunofluorescence and flow cytometry. An average of 7.4 +/- 0.8

and 9.8 +/- 1.2

HBsAg + cells were found among cells from acute and chronic hepatitis B patients, respectively. When studied by two-color immunofluorescence, HBsAg was limited to cells expressing the B-cell restricted antigen CD19 (16.7 +/- 2.4

), whereas for the other subsets studied (CD3+, CD4+, CD8+, CD67+, CD68+) the percentage of positivity was lower than 4

. The expression of viral antigen on B cells might be relevant for a number of functional interactions between HBsAg+ B and T lymphocytes.

Analysis of hepatitis B surface antigen expression on leucocyte subpopulations by two-color fluorescence with flow cytometry

En estudios previos hemos demostrado, empleando inmunofluorescencia con anticuerpos monoclonales (mAcs), que el antígeno de superficie (HBsAg) del virus de la hepatitis B (HBV) puede ser detectado en la membrana de las células mononucleares de pacientes infectados. En este trabajo hemos analizado la expresión del HBsAg en diferentes subpoplaciones leucocitarias de pacientes con heaptitis B aguda y crónica empleando citometría de flujo y doble fluorescencia. El HBsAg se detectó con un mAc anti-pre S2 + S conjugado con fluoresceína; las subpoblaciones celulares se caracterizaron con mAcs anti-CD3, CD4, CD8, CD19, CD67 y CD68 conjugados con ficoeritrina. El estudio por citometría de flujo confirmó la expresión del ABsAg por las células mononucleares de pacientes agudos y crónicos, con un porcentaje de células positivas de 7,4 ñ 0,8 por ciento y 9,8 ñ 1,2 por ciento respectivamente. Por análisis de diagramas de doble fluorescencia se seleccionaron las distintas poblaciones a fin de determinar el porcentaje de células que expresaban el HBsAg; el 16,7 ñ 2,4 por ciento de las células B presentaron el antígeno viral, mientras que los valores para las células T, monocitos y granulocitos fueron menores de 4 por ciento. Estos resultados confirman que la expresión del HBsAg en la membrana celular se halla restringida a los linfocitos B de pacientes infectados con el HBV (AU)

Analysis of hepatitis B surface antigen expression on leucocyte subpopulations by two-color fluorescence with flow cytometry

En estudios previos hemos demostrado, empleando inmunofluorescencia con anticuerpos monoclonales (mAcs), que el antígeno de superficie (HBsAg) del virus de la hepatitis B (HBV) puede ser detectado en la membrana de las células mononucleares de pacientes infectados. En este trabajo hemos analizado la expresión del HBsAg en diferentes subpoplaciones leucocitarias de pacientes con heaptitis B aguda y crónica empleando citometría de flujo y doble fluorescencia. El HBsAg se detectó con un mAc anti-pre S2 + S conjugado con fluoresceína; las subpoblaciones celulares se caracterizaron con mAcs anti-CD3, CD4, CD8, CD19, CD67 y CD68 conjugados con ficoeritrina. El estudio por citometría de flujo confirmó la expresión del ABsAg por las células mononucleares de pacientes agudos y crónicos, con un porcentaje de células positivas de 7,4 ñ 0,8 por ciento y 9,8 ñ 1,2 por ciento respectivamente. Por análisis de diagramas de doble fluorescencia se seleccionaron las distintas poblaciones a fin de determinar el porcentaje de células que expresaban el HBsAg; el 16,7 ñ 2,4 por ciento de las células B presentaron el antígeno viral, mientras que los valores para las células T, monocitos y granulocitos fueron menores de 4 por ciento. Estos resultados confirman que la expresión del HBsAg en la membrana celular se halla restringida a los linfocitos B de pacientes infectados con el HBV

Analysis of CD1 molecules on haematological malignancies of myeloid and lymphoid origin. I. Cell surface antigen expression.

One hundred and ninety well-characterized acute and chronic leukaemias were studied for the expression of CD1a antigen by indirect immunofluorescence (IIF). CD1a was detected on 28 per cent of mature B cell lymphoproliferative disorders, 26 per cent of acute non-lymphoblastic leukaemias (ANLL), 21 per cent of chronic granulocytic leukaemias in blast crisis (CML-BC), 53 percent of T acute lymphocytic leukaemias (T-ALL) and in only one out of 35 common acute lymphoblastic leukaemias (c-ALL). In some cases the expression of the CD1a antigen on the surface of leukaemic cells showed a spontaneous fluctuation after a short period of incubation in vitro. CD1b and CD1c molecules were also detected on B cells and acute non-lymphoblastic leukaemias. The presence of CD1 antigens was confirmed using a dot blot assay (DBA) on the lysate of leukaemic cells.

Analysis of CD1 molecules on haematological malignancies of myeloid and lymphoid origin. II. Intracellular detection of CD1 antigens.

The surface and cytoplasmic expression of CD1a molecules was analysed by indirect immunofluorescence (IIF) and dot blot assay (DBA) in a panel of 40 acute and chronic leukaemias. Thirty-two per cent of the samples were positive by IIF but, surprisingly, 72 per cent of the patients were positive by DBA, suggesting the intracellular presence of these molecules, CD1b and CD1c were also detected by DBA at similar percentages. Immunocytochemical staining of cytocentrifuge preparations confirmed the intracellular presence of CD1a, CD1b, and CD1c in leukaemic cells of pre-B, B, T, and non-lymphoid lineages.

Expression of CD1 antigens by peripheral blood mononuclear cells from hepatitis B patients.

We have studied the expression of CD1 antigens on peripheral blood mononuclear cells (PBMC) from acute hepatitis B patients in order to analyse a possible role for CD1 antigens in hepatitis B virus (HBV) infection. Using immunofluorescence and the monoclonal antibodies which recognized CD1a, CD1b and CD1c molecules, we have shown that CD1 antigens were expressed on PBMC from acute hepatitis B patients but not from other acute and chronic liver disease. Dot blot analysis on nitrocellulose sheets of the lysates of the cells confirmed these observations. Cell fractionation and double-labelling experiments clearly demonstrated the CD1 antigens were expressed only on non-T cells. Furthermore, CD1 antigens were coexpressed with hepatitis B surface antigen (HBsAg) on the surface of Ig-positive cells. These results could indicate that CD1 expression may be associated with the lymphotropic effect of HBV.

Detection of hepatitis B surface antigen (HBsAg) in peripheral blood mononuclear cells of patients with acute and chronic active hepatitis B.

Seventy five patients with acute and chronic active hepatitis (CAH) were studied by indirect immunofluorescence with monoclonal antibodies for the presence of hepatitis B surface antigen (HBsAg) on peripheral blood mononuclear cells (PBMC). The viral surface antigen was detected in the PBMC of all the patients with hepatitis B virus (HBV)-induced CAH and in acute patients with more than 2 months of evolution. No HBsAg was detected in the samples obtained from 12 normal controls or from 14 non-A, non-B CAH patients. Analysis of PBMC subsets revealed that HBsAg was present in non-T cells; dual fluorescence studies showed HBsAg on surface Ig-positive lymphocytes. The binding of anti-HBs monoclonal antibodies was higher than that of a goat anti-HBs serum, and the highest reactivity was observed with an antibody against the pre-S(2)-region sequence. Both HBsAg and hepatitis B core antigen (HBcAg) were also detected in lysates of PBMC by dot blot analysis.

Superoxide radical production by human leukocytes exposed to immune complexes: inhibitory action of gold compounds.

Superoxide radicals produced by phagocytic cells are considered to be important mediators in the rheumatoid inflammation. The effect of the gold compounds auranofin (AF) and gold sodium thiomalate (GST) on superoxide production by human leukocytes was investigated in two models of immunologic injury: immune-complex phagocytosis and frustrated phagocytosis. In both systems, AF (0.5-1.0 micrograms Au/ml) showed a potent inhibitory activity on superoxide generation, quantitated by ferricytochrome c and NBT reduction. GST showed only modest inhibition at higher concentrations (100 microM). The thiol protecting agent dithiothreitol, 1 mM, completely blocks the inhibitory effect of AF. The inhibition of the oxy radical generation by AF may play an important role in the control of rheumatoid inflammation; it is suggested that this action might be mediated through sulfhydryl-AF interaction at the cellular membrane level.

Oral chrysotherapy in rheumatoid arthritis: minimum effective dose.

We studied the dose-response to a new oral gold compound in 28 patients with definite rheumatoid arthritis, divided in 4 groups of 7 patients, each treated with different doses of auranofin for 3 months. Clinical and laboratory parameters were recorded weekly, and blood gold levels (BGL) measured by atomic absorption spectroscopy. Six and 9 mg daily doses of auranofin were most effective based on clinical and laboratory results. Correlation studies between BGL and percent decrease of humoral measurements, within the 3 months were statistically were statistically significant. Mean BGL, associated with clinical improvement, reached 0.73 microgram/ml, and was accompanied by a 17.6% decrease from initial value of IgG, 17.1% of alpha 2-globulin, 48.9% of RF titer and 25.9% of ESR.

Effect of auranofin, a new antiarthritic agent, on immune complex-induced release of lysosomal enzymes from human leukocytes.

Auranofin, an oral chrysotherapeutic agent effective in the treatment of rheumatoid arthritis (RA), was found to be a potent, noncytotoxic inhibitor of IgG-RF immune complex-induced lysosomal enzyme release (LER) from human leukocytes. At a concentration of 1 microg Au/ml (5 microM), auranofin produced a marked reduction in beta-glucuronidase (100%), acid phosphatase (88%), and lysozyme (72%) release. In contrast, gold sodium thiosulfate (GST, an injectable gold compound) had no inhibitory activity on LER at equivalent gold concentrations (i.e., 1 microg Au/ml) and only modest activity (less than 36% inhibition) at concentrations as high as 40 microg Au/ml. The 50% inhibitory dose (LD50) of auranofin on LER was calculated to be 3-4 microM (0.6-0.8 microg Au/ml). Blood gold levels in auranofin-treated RA patients were found to be within the range required for in vitro inhibition of LER, and correlated with decreases in IgG, RF titers, and IgG-RF immune-complex formation in vitro. These results suggest that the therapeutic action of auranofin may be caused, at least in part, by inhibition of LER and/or decreases in immune-complex formation.