Localization of immunological complexes fixing beta1C (C3) in germinal centers of lymph nodes.

28 human and 60 experimentally stimulated rabbit lymph nodes were studied by means of light microscopy and immunofluorescence. 21 of the 28 human lymph nodes showed well-developed germinal centers. IgM, IgG, and the beta(1C) component of complement were found in the same distribution within germinal centers when examined in serial cryostat sections. 36 rabbits were stimulated with Brucella antigen, and 24 rabbits with BSA. A strikingly consistent correlation between distribution and appearance of specific staining for rabbit beta(1C), IgM, and IgG was observed; when lymph nodes were stimulated with BSA, antigen and specific antibody were present. Treatment of unfixed sections with citrate-buffered saline at low pH resulted in complete elution of immunoglobulins, beta(1C), and BSA from rabbit germinal centers, and in marked diminution of IgG and IgM in human germinal centers, while at the same time plasma cells remained strongly fluorescent. Specific selective fixation of heterologous (human) complement in rabbit germinal centers positive for beta(1C), IgG, IgM, and BSA was also obtained. These data present strong evidence for the existence within germinal centers of antigen-antibody complexes which fix at least the beta(1C) component of complement in vivo. The possibility of complete elution of immunoglobulins from rabbit germinal centers can be taken as evidence that, at least for 20 days after primary and secondary stimulation, a major component of the immunoglobulins present in germinal centers is not produced locally but accumulates at the surface of cells.

Failure of immunosuppression in a severe haemophilia B patient with specific antibody.

Prevention of a secondary response to factor IX by cyclophosphamide was attempted in an 11 year old patient with severe Christmas disease. An antibody to factor IX had been present for 4 years before immunosuppressive therapy was tried. Despite profound lymphopenia, synthesis of factor IX antibody was not depressed. The difficulties of modifying the anamnestic response to factor IX by chemical immunosuppression may be as real as has been reported for factor VIII in classical haemophilia.

Acquired haemophilia: functional study of antibodies to Factor VIII.

Factor VIII complex and its interaction with antibodies to factor VIII have been studied in 17 non-haemophilic patients with factor VIII inhibitor. Low VIII:C and high VIIIR:Ag levels were found in all patients. VIII:WF levels were 50% of those of VIIIR:Ag, possibly related to an increase of poorly aggregated and electrophoretically fast moving VIIIR:Ag oligomers. Antibody function has been characterized by kinetics of VIII:C inactivation, saturability by normal plasma and the slope of the affinity curve. Two major patterns were observed: 1) Antibodies from 6 patients behaved similarly to those from haemophiliacs by showing second order inhibition kinetics, easy saturability and steep affinity slope (greater than 1). 2) Antibodies from other patients, usually with lower titres, inactivated VIII:C according to complex order kinetics, were not saturable, and had a less steep affinity slope (less than 0.7). In native plasma, or after mixing with factor VIII concentrate, antibodies of the second group did not form immune complexes with the whole factor VIII molecular complex. However, dissociation procedures did release some antibodies from apparently low molecular weight complexes formed in vivo or in vitro. For appropriate management of non-haemophilic patients with factor VIII inhibitor, it is important to determine the functional properties of their antibodies to factor VIII.

Factor VIII procoagulant antigen in human tissues.

The localization of factor VIII procoagulant antigen (VIII:Ag) and factor VIII von Willebrand antigen (VWF:Ag) was investigated in human liver, lung, spleen, placenta and umbilical cord, by an immunoperoxidase technique using an avidin biotin complex (ABC). Positive staining for VIII:Ag was observed in the endothelial cells of liver sinusoids, veins and arteries, as well as in the endothelial cells of placenta, lung and spleen. VWF:Ag was detected in the vascular endothelial cells of all the organs explored. The staining intensity of both VIII:Ag and VWF:Ag varied in the different tissues and showed a distinctive pattern of distribution in the liver. VIII:Ag was also observed in the cytoplasm of dysplastic, foetal-like hepatocytes which infiltrated one liver specimen. Our results agree with the view that liver endothelial cells are a major site of Factor VIII (F VIII) storage and secondary release into the circulation. However, the bright staining intensity of VIII:Ag and VWF:Ag in the lung and placenta suggests that these two tissues might also be a substantial source of F VIII.

In vivo interactions of autoantibodies to factor VIII with the factor VIII complex.

Two non-haemophilic elderly patients who had developed autoantibodies to factor VIII were studied over a period of 9 months to 5 years. Sequential measurements of antibody to factor VIII (anti-VII:C), factor VIII coagulant activity (VIII:C), factor VIII coagulant antigen (VIII:CAg), factor VIII-related antigen (VIIIR:Ag), and factor VIII ristocetin cofactor (VIII:WF) were performed. Before treatment, low VIII:C, normal or increased VIII:CAg and high VIIIR:Ag levels were found and were indicative of the presence of circulating immune complexes. Immunosuppressive therapy induced progressive correction of VIII:C and VIIIR:Ag values. High levels of VIII:CAg subsided in the patient who relapsed. It is suggested that antibodies to factor VIII bind and remove VIII:C from the circulation thereby inducing an increased synthesis of VIII:CAg which may be associated with an augmented release or production of VIIIR:Ag.

HLA antigens and factor VIII antibody in classic hemophilia. European study group of factor VIII antibody.

Possible interrelations between the immune response factor VIII and the major histocompatibility system were investigated in 57 multi-transfused hemophilic brothers belonging to 26 families. Linkage appears very unlikely although formal proof of independence cannot be offered. The HLA system, therefore, does not provide markers predictive for the development of antibodies to factor VIII in severe hemophilia A.

A survey of antibodies to hepatitis C virus in Ethiopia.

Antibodies to hepatitis C virus (HCV) were measured in 1,580 Ethiopian subjects representing urban and rural populations. Sera found positive by a repeated second generation enzyme immunoassay (EIA) were subjected to three additional confirmatory tests. The overall confirmed seroprevalence was 2.0%. Less than 1% were confirmed to be seropositive in rural communities, with 1.4% positive among blood donors, 1.6% positive among patients with dermatologic disorders, 3.6% among leprosy patients, and 6.0% among patients attending a University Hospital clinic for neurologic disorders. The patients in the groups with leprosy and neurologic disorders have most likely been in ill health for many years and have sought relief by traditional healers or treatment at poorly equipped clinics. This group of patients demonstrated a high prevalence of antibodies to HCV. In Ethiopia, especially in small clinics, there is a shortage of syringes and needles and they have to be reused many times often with inadequate sterilization. Therefore, these syringes and needles may be contaminated, thus being a risk factor for HCV and HIV infection.

High affinity human monoclonal antibodies directed against hepatitis B surface antigen.

Peripheral blood mononuclear cells from donors immunized with hepatitis B vaccine (Pasteur Hevac B) were transformed with Epstein-Barr virus. Two polyclonal cell lines, producing antibodies to hepatitis B surface antigen were established and cloned. Seven clones were isolated; they secreted between 10 and 20 micrograms/ml of HBs specific IgG1 kappa or lambda antibody with anti-HBs titer of 300-800 IU/ml. These human antibodies expressed the anti 'a' specificities and had high affinity and avidity; their potential use as reagents for hepatitis B virus detection and for passive immunotherapy is under study.

The use of DNA hybridization for the detection of Leishmania aethiopica in naturally infected sandfly vectors.

Hybridization with kinetoplast deoxyribonucleic acid (kDNA) probes was used to detect Leishmania aethiopica in naturally infected sandflies in south-west Ethiopia, an endemic area for cutaneous leishmaniasis. 396 sandflies were dissected; microscopy revealed flagellates in the midgut of 5 Phlebotomus pedifer. The infecting flagellates were confirmed as L. aethiopica by isoenzyme typing. Gut specimens for all dissected sandflies were hybridized with total L. aethiopica kDNA as well as with a cloned kDNA probe specific for L. aethiopica. Samples from sandflies which were found to be infected microscopically also hybridized with the L. aethiopica kDNA probes. One additional sandfly hybridized but was not shown to be infected by microscopical examination. Hybridization experiments with 65 whole squash-blotted sandflies gave results that correlated very well with results obtained using microscopy. Our results indicate that DNA probing is a useful method to detect Leishmania infection in sandfly midguts as well as in whole squash-blotted sandflies, and can be used to follow changes of infection rate. DNA probing is therefore an alternative to microscopy in large-scale epidemiological studies as well as monitoring control programmes aimed at human leishmaniasis.

Vaccination and treatment trials against murine leishmaniasis with semi-purified Leishmania antigens.

Antigens with molecular weight ranges of 94-67 kDa (LiF2), 30-20 kDa (LiF5), or below 20 kDa (LiF6), isolated from lysates of Leishmania infantum promastigotes by electroelution from polyacrylamide gels were injected into mice which were genetically either partially resistant (C57BL/6) or susceptible (BALB/c) to Leishmania infection. One month after the completion of the intravenous (C57BL/6) or subcutaneous (BALB/c) schedules, the mice were challenged with 1 x 10(3) L. major promastigotes. All mice immunized with LiF2, LiF5 and LiF6 were completely resistant. Furthermore, the C57BL/6 mice immunized with LiF2 resisted a second challenge with 1 x 10(4) L. major amastigotes. 5 months later, LiF2 antigen was used for immunotherapy of L. major leishmaniasis; parasites disappeared from the treated skin lesions, although ensuing systemic infection could not be averted.