Hendrik Claeys

Evaluation of third-generation screening and confirmatory assays for HCV antibodies.

A third‐generation (gen.) screening and immunoblot assay (Ortho EIA‐3.0; Chiron RIBA‐3 prototype), using antigens derived from the capsid and different nonstructural regions (NS3, NS4 and NS5) of the hepatitis C virus viral genome, were evaluated in comparison with the corresponding second‐gen. assays (Ortho EIA‐2.0; revised Ortho EIA‐2.5; Chiron RIBA‐2). In 203 depository sera of blood donors, positive in EIA‐2.0, specificity of the screening assays was improved as shown by an increase in positive predictive value for viral carrier state from 0.23 (EIA‐2.0) to 0.37 (EIA‐2.5) and 0.52 (EIA‐3.0). Comparing the confirmation patterns on RIBA‐2 and RIBA‐3, this amelioration was mainly due to the specific elimination of false‐positive c22‐3 and c100‐3 reactions. Antibody response to the newly added NS5 antigen was not as prevalent as to the other antigens and had only a minor influence in sample allocation. In contrast, screening of 1,560 volunteer blood donors and 47 hemodialysis patients revealed 3 additional positive sera, only reacting with the NS5 antigen. However none of these isolated NS5 reactions could be confirmed on synthetic peptides [INNO‐LIA: NS5(p)] and none was PCR positive. A documented seroconversion, detected earlier with EIA‐3.0, was related to a better immunological response to the NS3 antigen and not to the additional NS5. From this pilot study third‐gen. assays appeared extremely useful in the reevaluation of HCV‐seropositive depository sera. However the additional value of the NS5 antigen in blood donor screening is still hypothetical and remains to be established in larger screening studies.

Interferon production and natural killer (NK) activity in leukocyte cultures from multiple sclerosis patients

Peripheral blood leukocyte (PBL) cultures from only 37% of MS patients produced detectable HuIFN-gamma in response to ConA as opposed to 85% of the cultures derived from normal blood donors. However, the yields in patient-derived cultures that were responsive, were not lower than those in cultures from controls. Production of HuIFN-alpha after stimulation with Sendai virus was not aberrant in cells taken from MS patients. The difference in HuIFN-gamma response rate between MS and normal donor-derived cells was more pronounced when DR2+ carriers were compared amongst each other than when DR2-k carriers were compared. Among the MS patients, the failure of PBLs to produce HuIFN-gamma in response to ConA was not correlated with age, sex, disease duration and type of disease. However, positive correlations were found with current disability indices and past disease progression rates. Unstimulated NK-activities of MS patient-derived PBLs were not different from those of normal donor-derived cells. the degree of augmentation of the activity by stimulation with ConA and interferon-alpha was also normal. Within the MS patients group, but not in the control group, there was a trend for DR2+ carriers to have lower spontaneous and stimulated NK-activities than DR2- individuals.

Confirmation of hepatitis C virus positive blood donors by immunoblotting and polymerase chain reaction.

In a series of 385 sera obtained from volunteer blood donors positive for the first‐generation hepatitis C virus assay (Ortho), the viral genome was detected by polymerase chain reaction (PCR) in 89 sera (23%). Most PCR‐positive sera were found positive with the c100‐3 neutralisation assay (Abbott) and by two second‐generation enzyme immunoassays (Abbott, Ortho). However overall specificity of these assays was rather low. By immunoblotting (Innogenetics and Chiron/Ortho) the specificity could be considerably improved and the best correlation with carrier state was obtained when analysing the results for lane‐specific reaction: all 89 viral carriers and only 9 other donors had antibodies against structural ‘core’ epitopes. From the present data we can conclude that in screening a volunteer blood donor population the confirmation of antibodies against ‘core’ epitopes by immunoblotting is strongly associated with viral carriage.

Activation of natural cytotoxicity of human peripheral blood mononuclear cells by interferon: a kinetic study and comparison of different interferon types

Summary. Overnight incubation of human peripheral blood mononuclear (PBMN) cells with leucocyte interferon (leucocyte IFN) resulted in a 2–5‐fold increase in natural cytotoxicity (NC) against the erythroleukaemic cell line K562. Fibroblast IFN, purified to homogeneity by zinc‐chelate chromatography, stimulated NC to the same extent, while partially purified immune IFN was about twice as active. Upon gel filtration of immune IFN, NC stimulating and antiviral activity co‐eluted. Treatment of PBMN cells with ammonium chloride buffer (AmCl) abrogated NC nearly completely. Incubation of AmCl‐treated cells with leucocyte IFN resulted in a partial regeneration of NC. Kinetic studies revealed that this regeneration required only a short exposure to IFN followed by a longer incubation period. The data are interpreted as indicating that in the process of NC activation IFN mainly acts as a trigger for precursor cells to mature into cells with NC.

Streptococcal preparation ok-432-induced interferon in human-leukocytes - purification and characterization

Interferon production was induced in leukocyte suspensions from human buffy coats after stimulation with the streptococcal preparation OK-432. At day 2-3 the induced interferon reached a maximal level of 0.9 units/1,000 cells. By a combination of batch adsorption/elution on silicic acid, batch adsorption to DEAE-Sephacel, affinity chromatography on concanavalin A-Sepharose and on poly(U)-Sepharose, this interferon could be purified to a specific activity of 10(7.5) units/mg protein. The antiviral activity was characterized as being solely due to gamma-type interferon by a variety of physicochemical, biochemical and serological criteria. Its molecular weight as determined by gel filtration amounted to 53,000 daltons, and its activity was completely neutralized by highly specific antisera to human gamma-type interferon (45K). The OK-432-induced interferon, as the crude supernatant of stimulated leukocytes, and at several stages of its purification, was found to stimulate the natural killer cell activity of fresh human lymphocytes.

Hepatitis C virus confirmation in blood donor screening.

A combination of different enzyme immunoassays (EIAs) was used for the serological confirmation of sera that were positive in a hepatitis C virus (HCV) second‐generation screening EIA. Different reaction patterns were related with the probability of the HCV‐carrier state as determined by polymerase chain reaction (PCR). Five hundred and eight sera of volunteer blood donors were send for confirmation and at first reexamined with both Abbott and Ortho second‐generation screening EIA. A group of 195 sera, positive in both assays, was further evaluated by the Abbott Supplemental Assay, the Monolisa anti‐HCV and an EIA with only the amino terminal part of the nucleocapsid protein as antigen. In addition PCR on the 5′‐noncoding region of the viral genome was performed. We observed that 75 of the 78 PCR‐positive sera were found in a group of 89 sera that were strongly positive in the four EIAs used. Moreover all but 1 PCR‐positive sera were reactive against the nucleocapsid protein of the virus. Hence we concluded that a genuine antibody response to the nucleocapsid protein is highly suggestive for the HCV‐carrier state.

Interferon therapy in multiple myeloma: Failure of human fibroblast interferon administration to affect the course of light chain disease

Abstract Fibroblast interferon at a dosage of 28 × 10 6 U/wk failed to influence disease progression in a preterminal case of therapy-resistant light chain myeloma. In a second case, that had not previously been treated, a first course of fibroblast interferon ( 30 × 10 6 U/wk ) associated with corticosteroids remained without effect. In this patient subsequent leukocyte interferon treatment was associated with a decrease in urinary light chain excretion and normalization of calcaemia, all other parameters remaining unaltered. A third patient with light chain disease was resistant to chemotherapy ab origine . None of the disease parameters responded to either fibroblast or leukocyte interferon therapy ( 21 × 10 6 U/wk ). During therapy a downward trend occurred in the relative number of lymphocytes characterizable as B and T cells. Mitogenic reactivity remained unchanged except for a downward trend, during fibroblast interferon therapy, in reactivity to PHA and Con A after 6 days culturing. Spontaneous (background) mitogenesis upon culture showed an upward trend.

Transfer factor therapy in multiple sclerosis A three‐year prospective double‐blind clinical trial

One hundred five patients with MS were divided into three groups matched for age, sex, and disability, and treated with either placebo, transfer factor prepared from leukocytes of random donors, or transfer factor from leukocytes of family members living with the patients. There were no differences in the three treatment groups for changes in disability, activities of daily living, or evoked potentials. Eighteen months of transfer factor therapy had no effect on gamma-interferon production or natural killer cell activities.

Interferon production and natural killer (NK) activity in multiple sclerosis

The purpose of the present study was to compare the ability of peripheral blood leukocytes (PBLs) of MS patients and of normal donors to respond to well-characterized inducers of HuIFN-α and HuIFN-γ. Both the production of interferons and the enhancement of NK activity were used as parameters of the response. A possible association of the responses with the DR2 HLA phenotype was examined by stratification of both patients and donors in DR2 + and DR2 -. The results of this study will be published in extenso elsewhere (J. Neurol. Sci., (1983), 60, 137–150).