Erik Depla

Monoclonal antibodies against the human interferon-γ receptor(s)

Enriched human interferon-gamma (HuIFN-gamma) receptor preparations were obtained by affinity chromatography of non-ionic detergent solubilized COLO 205 cell membranes on immobilized recombinant HuIFN-gamma. The active fractions, identified by a competition ELISA, were used as the immunogen in a BALB/c mouse. Fusion of its splenocytes with myeloma cells yielded several hybrids secreting antibodies that inhibit the antiviral activity of HuIFN-gamma; the two most active ones were selected for further characterization. This blocking activity was restricted to both the human species and the gamma type of IFN. Affinity purification of cell membrane extracts on the immobilized monoclonal antibodies resulted in the visualization of a major protein band with an Mr of 90,000, which is in good agreement with the results obtained by other authors [Aguet M. and Merlin G. (1987) J. exp. Med. 165, 988-999; Novick D., Orchansky P., Revel M. and Rubinstein M. (1987) J. biol. Chem. 262, 8483-8487; Sheehan K. C. F., Calderon J. and Schreiber R. D. (1988) J. Immun. 140, 4231-4237].

Identification of human liver carboxylesterase as one of the proteins involved in Plasmodium falciparum malaria sporozoite invasion in primary cultures of human hepatocytes

BACKGROUND/AIMSnIn a previous study, we have demonstrated that primary human hepatocytes in culture are susceptible for Plasmodium falciparum sporozoite invasion and for development of parasites into exo-erythrocytic forms. In a separate study we demonstrated the involvement of two human liver plasma membrane proteins (55 kD and 20 kD) in the invasion of P. falciparum sporozoites in vitro. In this study, we have unravelled the nature of the 55 kD protein.nnnMETHODSnFor the identification of this protein, a 53-58 kD membrane protein fraction from human liver was isolated, radioactively labelled, incubated with sporozoites and cross-linked. After reduction of the cross-linker, the released proteins were mixed with unlabelled 53-58 kD protein fraction and separated on two-dimensional SDS-PAGE. Autoradiography showed a single spot corresponding to a protein of 55 kD and pI of 5.7-5.8.nnnRESULTSnAmino acid sequencing revealed the 55 kD protein as carboxylesterase. The biological activity of purified human liver carboxylesterase and of an antiserum against carboxylesterase on sporozoite invasion in vitro was evaluated. Human carboxylesterase as well as a rabbit antiserum against carboxylesterase inhibited the invasion of P. falciparum sporozoites into primary human hepatocytes in culture. A number of carboxylesterase cDNA clones were isolated from a human liver cDNA library. Sequence analysis revealed two different iso-types. Immunoaffinity purified recombinant human carboxylesterase was shown also to inhibit the invasion of sporozoites into primary human hepatocytes. Immunocytochemical analysis of the localisation of carboxylesterase in primary cultures of human hepatocytes using specific antibodies, showed its presence inside the hepatocytes and on the membrane.nnnCONCLUSIONSnCarboxylesterase plays a role in the invasion process of P. falciparum sporozoites into human hepatocytes in vitro. The implications of these findings are further discussed.

Anti-idiotypic and anti-anti-idiotypic antibodies as highly specific tools in epitope and active site studies on human interferon-γ

Immunization of rabbits with F(ab)2 fragments of different monoclonal antibodies directed against human interferon-gamma yielded antisera with anti-idiotypic characteristics. Isolation of the anti-idiotypic fraction, resulting in a highly specific antiserum, allowed us to prove that out of six competing monoclonal antibodies directed against human interferon-gamma, only two really recognize the same epitope. The other monoclonal antibodies compete on the basis of steric hindrance, which is not surprising, because of the large difference in Mr between interferon-gamma and an immunoglobulin. The anti-idiotypes provided us also with a tool to study isolated epitopes on the human interferon-gamma molecule, a task which was previously not practicable. Exploration of the biological properties of these anti-idiotypes allows us to determine whether the investigated epitopes are involved in receptor binding. The production of an anti-anti-idiotypic antiserum not only proved that we generated real internal images, but also that these images conserved all of their properties, although with a decreased affinity in comparison with the original monoclonal antibody. As the former is a polyclonal antiserum, directed against a single epitope of the human interferon-gamma molecule, competition experiments yielded additional information on the relative position of three epitopes recognized by inhibiting monoclonal antibodies. These antisera will possibly open new ways for the affinity purification of interferon-gamma and perhaps for the treatment of autoimmune diseases.

Concomitant findings of anti-HBc as a sole marker for hepatitis B virus infection and elevated serum transaminases are associated with a high prevalence of hepatitis C virus infection

Abstract From two hundred sixty five patients with liver disease and elevated serum transaminases who were admitted or referred to Atmajaya academic hospital in Jakarta, 37 patients were found to be anti-HBc positive as a sole marker of HBV infection. Nineteen out of these 37 patients were also anti-HCV positive, HCV-RNA was detected in 16 patients. From 1656 individuals with normal serum transaminases who underwent serological examination and consisted of 489 subjects from population based study in Kalianyar (an urban area in Jakarta), 258 students of Atmajaya Medical School, 541 women and 162 children from maternity-child clinic and 206 patients admitted to hospital without evidence of liver disease, anti-HBc as a sole marker for HBV infection was found in 213. However, HCV-RNA was detected only in 2 subjects. We conclude that concomitant findings of anti-HBc as a sole marker for HBV infection and elevated ALT are associated with a relative high prevalence of HCV infection. In a group of individuals without evidence of liver disease, HCV infection was uncommon, even when anti-HBc is present as a sole marker of HBV infection.