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Biomedical subjects

J C Jensenius

Publications and source records attributed to J C Jensenius.

At least 109 records · Page 6Linked to original sources
PubMed1990

Hope for successful xenografting by immunosuppression with monoclonal antibody against CD4, total lymphoid irradiation and cyclosporine. Six months' survival of hamster heart transplanted into rat.

Hamster hearts were transplanted to rats, and the effects of combinations of total lymphoid irradiation (TLI), cyclophosphamide, cyclosporine A (CyA) and monoclonal antibodies (MAB) were investigated. Controls not immunosuppressed rejected their xenograft in 3 to 5 days, while combination immunosuppression including MABs against CD4 or IL-2-receptors extended graft survival significantly. In one case, the graft was still functioning 180 days after transplantation, which is the longest survival seen in this model. The use of specific MABs may open a new era for both xeno- and allo-transplantation.

Animals↗
PubMed1988

Curing human hybridomas infected with Mycoplasma hyorhinis.

Tiamuline and minocycline were evaluated for the treatment of an IgM producing human-human hybridoma cell line infected with Mycoplasma hyorhinis. Tiamuline was used at a concentration of 10 micrograms/ml culture medium and minocycline at a concentration of 5 micrograms/ml culture medium. Both antibiotics were found to eliminate mycoplasma infection over a treatment period of 3 weeks, and the hybridoma cell line remained mycoplasma-free for 6 months after treatment. Tiamuline had no effect on either cell growth or IgM secretion. Whereas treatment with minocycline alternated the cell proliferation and completely inhibited IgM secretion. This effect on cell function was found to be reversible since both cell growth and IgM secretion returned to normal 1 week after the minocycline had been removed. Tiamuline as well as minocycline may be recommended for the treatment of human hybridomas infected with mycoplasma.

Cell Division↗
PubMed1987

A method for the blocking of endogenous immunoglobulin on frozen tissue sections in the screening of human hybridoma antibody in culture supernatants.

Endogenous immunoglobulin in tissue sections pose a problem in immuno-histochemical techniques employing homologous antibody as primary reagents and enzyme-labelled anti-immunoglobulin for the development. A method for the blocking of endogenous immunoglobulin in human tissue sections by incubation with monomeric pepsin fragments (Fab') of rabbit anti-human immunoglobulin before applying monoclonal antibody was evaluated for the screening of human monoclonal antibody. It was initially demonstrated that Fab' rabbit anti-human IgM and anti-IgG could block endogenous immunoglobulin in human IgM and IgG producing tumors thereby abolishing the binding of subsequently applied peroxidase-labelled anti-IgM or anti-IgG. Frozen sections of human colo-rectal adenocarcinomas show a variable background staining caused by the endogenous immunoglobulin. The background completely disappeared in the IgM system by preincubation with Fab' anti-IgM while the background was clearly reduced but not abolished in the IgG system. A human hybridoma supernatant containing IgM reactive with colo-rectal adenocarcinoma could easily be screened on frozen sections using this method. This approach should be generally useful for the screening of human antibody on human tissue sections.

Antibodies, Monoclonal↗
PubMed1987

Demonstration in human plasma of a lectin activity analogous to that of bovine conglutinin.

Evidence of the existence in human plasma of an activity analogous to that of bovine conglutinin is presented. The human plasma component was characterized antigenically and functionally. Human plasma was shown to agglutinate complement-coated erythrocytes in the presence of Ca2+, and this conglutination was inhibited by EDTA. The molecule also binds to complement-reacted solid phase IgG and to zymosan in the presence of Ca2+. The binding to complement is not inhibited by N-acetyl-D-mannosamine, but is inhibited by N-acetyl-D-glucosamine, as previously shown for bovine conglutinin. Antibodies raised against bovine conglutinin cross-react with the human protein. The plasma concentration of the conglutinin-like protein showed a high inter-individual variation between apparently healthy donors. Unlike bovine conglutinin, the human conglutinin activity could not be demonstrated in serum but only in plasma. The activity was more stable in plasma containing metal-ion chelators like EDTA and citrate than in heparin or hirudin plasma.

Agglutination↗
PubMed1987

Characterization of a lectin in human plasma analogous to bovine conglutinin.

The structural characteristics of a human plasma protein analogous to bovine conglutinin were studied. The protein was previously found to bind to complement-reacted IgG in a calcium-dependent and N-acetyl-D-glucosamine-inhibitable manner and it further shows cross-reactivity with anti-bovine conglutinin antibody. By gel permeation chromatography the conglutinin activity in human plasma was localized to fractions containing proteins of Mr at around 700,000. The conglutinin was localized by one ELISA for antigen determinants and by another for biological activity. When analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions these fractions were shown to contain proteins of about 300,000. When human conglutinin-like protein, partially purified by affinity chromatography, was analysed unreduced by SDS-PAGE followed by western blotting, the cross-reacting anti-bovine conglutinin antibody bound to a protein with an Mr of 330,000. When the sample was reduced and alkylated before electrophoresis a band of 66,000 was immunostained. The 330,000 and 66,000 proteins were shown to be collagenase sensitive. 125I-iC3b was seen to bind to the 330,000 band when incubated with western blots of partially purified human conglutinin.

Animals↗
PubMed1987

Human-human hybridomas generated with lymphocytes from patients with colorectal cancer.

Human-human hybridoma technology was used to produce human monoclonal antibodies with reactivity to colorectal cancer antigens. Two different B-lymphoma cell lines were fused with lymphocytes obtained from mesenteric lymph nodes from colorectal cancer patients. The fusion frequency was 11% with LICR-LON-HMy-2. Out of 294 growing hybridomas 26 secreted antibodies reacting with epitopes on cultured colon adenocarcinoma cells. Only one (D4213) was established and has now been in culture for 1.5 years. D4213 antibody shows a strong reaction with colon cancer tissue compared with normal colon epithelium. Using W1-L2-729-HF2 the fusion frequency was about 50%. Of 2,487 hybridomas 499 produced immunoglobulin and 44 of these reacted with colon cancer tissues or cultured cancer cells. One of the established hybridomas produces antibody reacting with cancer cell membrane antigens, and on immunoblotting a number of components were stained. The antibody from the other hybridomas reacts with cytoplasmatic antigens, and only one of these showed reactivity in immunoblotting where it bound to a component with Mr of about 60K.

Antibodies, Monoclonal↗
PubMed1986

Measurement of C3 conversion by ELISA estimation of neo-determinants on the C3d moiety.

An ELISA assay estimating neo-determinants on the C3d moiety is described. The C3d neo-determinants were detected by incubating the sample on F(ab')2 anti-C3d-coated plates followed by development with biotin-labelled anti-C3d and enzyme-labelled avidin. The assay was compared with rocket immunoelectrophoresis (IE) and crossed IE. Serum from a factor I deficient patient showed no detectable C3d when analysed by rocket IE, but activation was evident when analysing by crossed IE or ELISA. The results suggest that the neo-determinants detected by ELISA become exposed when C3 is split into C3a and C3b and this interpretation was supported by kinetic analysis of C3 activation by the three methods.

Complement Activation↗
PubMed1986

Human-human hybridomas for the study of anti-tumor immune response in patients with colorectal cancer.

Human-human hybridoma technology was evaluated for the study of humoral immune reactions of colorectal cancer patients against their own tumors. Six fusions were carried out with lymphocytes from mesenteric lymph nodes from patients with colorectal cancer, using the human B-lymphoma cell line LICR-LON-HMy-2 as fusion partner. A total of 294 wells with cell growth were obtained. Supernatants from 26 of these reacted in enzyme-linked immuno-sorbent assay (ELISA) with one or more colon cancer cell lines. Cells from only one of these wells (D 4213) could be cloned. The clone was shown to produce antibody which by immunocytochemical analysis reacted with a panel of colon cancer cell lines and melanoma cell lines but not with several other cancer cell lines or normal human leukocytes. By immunohistochemical analysis on formalin-fixed paraffin-embedded tissue this antibody reacted strongly with antigen expressed by autologous and allogeneic colorectal cancers. Faint staining could occasionally be observed on normal colon epithelium. D4213 is a hybrid cell line since it is tetraploid and produces kappa and lambda light chains as well as gamma and mu chains, whereas HMy-2 produces only kappa and gamma chains. The study produces only kappa and gamma chains. The study suggests that patients may possess B cells producing antibody reactive with their own malignant cells.

Adenocarcinoma↗
PubMed1986

Release of immune complexes bound to erythrocyte complement receptor (CR1), with particular reference to the role of factor I.

The release of 125I-bovine serum albumin (BSA)-anti-BSA immune complexes (IC) bound to human erythrocyte complement receptors (E-CR1) was studied. IC were complement-solubilized in normal human serum (NHS), and reacted with human erythrocytes at conditions optimal for binding of the IC to E-CR1. E-CR1-bound IC could be released by the addition of NHS or purified factor I. Factor I-deficient or I-depleted serum mediated no release, and addition of purified factor I restored the release. Factor H was not required for the release of IC. The kinetics of IC release was influenced by the NHS concentration, the presence of EDTA, and the time of prior storage of the erythrocytes at 4 degrees C. NHS (1:5 to 1:10) in the presence of EDTA caused nearly maximal release within 10-20 min at 37 degrees C. In the absence of EDTA the NHS-induced IC release was markedly slower. IC released within the first 30 min showed significant rebinding to new E. The release of IC was not associated with loss of the IC binding activity of E-CR1. The NHS-mediated release of IC could be inhibited by rabbit anti-CR1 and by a mixture of protease inhibitors. Release induced by purified factor I was also inhibited by protease inhibitors. The affinity of IC binding to E-CR1 was reduced after cleavage of CR1-bound C3b-IC to iC3b-IC by factor I.

Antigen-Antibody Complex↗
PubMed1986

Passage of undegraded dietary antigen into the blood of healthy adults. Further characterization of the kinetics of uptake and the size distribution of the antigen.

The molecular weight distribution of ovalbumin (OA) absorbed into the blood in eight healthy adults after a test meal was analysed by high pressure liquid gel permeation chromatography (HPLC) followed by ELISA for OA. OA was found either as free OA or as aggregates of a size mainly below 700 kD. The addition of OA in vitro to an antibody-containing serum resulted in OA-containing immune complexes (OA-IC) of similar size. The size distribution of serum OA-IC was more heterogeneous late than early in the 7-h observation period. Free OA disappeared at an equal or higher rate than OA-IC when both entities were present in the same individual. The presence of OA-IC was related to the serum IgG antibody levels. IgG, but not IgA or IgM, could be detected in OA-IC (in three individuals) by ELISA. The kinetics of OA appearance was followed for 48 h after a test meal in three individuals. In one person, serum OA reached peak values at 24 h and detectable amounts persisted for 48 h after the test meal. In the other test persons the OA levels peaked earlier. The present study indicates that in healthy adults dietary antigens are absorbed and circulate regularly in minute amounts apparently as native protein and/or as small immune complexes, mostly containing IgG antibodies.

Adult↗
PubMed1986

The attachment of serum- and plasma-derived C3 to solid-phase immune aggregates and its relation to complement-mediated solubilization of immune complexes.

The interaction between immune aggregates and complement (C) was investigated. Solid-phase immune aggregates were prepared by coating microwells with heat-aggregated bovine serum albumin (BSA) followed by rabbit anti-BSA antibody. The immune aggregates were reacted with human serum or citrated plasma at 37 degrees C. The binding of C3 components was investigated with biotinylated F(ab')2 antibodies to C3c and C3d and avidin-coupled alkaline phosphatase. The form of the incorporated C3, whether C3b-iC3b or C3dg, can be deduced from the response with these two antibodies. The maximal binding of C3b-iC3b to the immune aggregates was observed within 5 min of incubation with serum or citrated plasma. The conversion to C3dg was evident by a decrease in bound anti-C3c concomitant with increasing anti-C3d reactivity within about 10 min of incubation. When the classical C pathway activation was inhibited, the binding of C3b-iC3b was delayed by 20-30 min, whereas stopping of the alternative pathway did not influence the initial kinetics of the reaction. The addition of human red blood cells had no measurable influence on the degradation of bound C3b-iC3b. 125I-labelled anti-BSA antibody bound to the solid-phase BSA was not released during the C3 incorporation. The incorporation of C3b into the immune aggregates was mediated equally well by serum and by citrated plasma. The incorporation of C3b-iC3b into immune complexes (IC) is thought to be responsible for the C-mediated solubilization (CMS) of IC. Citrated plasma, however, exerted no CMS capacity when measured by a radiometric assay. The CMS capacity of serum was inhibited by citrate, but could then be restored by adding Ca2+ and Mg2+, whereas no CMS could be demonstrated with citrated plasma to which divalent metal ions were added.

Antibodies↗
PubMed1985

ELISA quantitation of IgG subclass antibodies to dietary antigens.

The IgG subclasses of human antibodies against 2 dietary antigens, ovalbumin (OA) and beta-lactoglobulin (BLG), were studied by ELISA using monoclonal anti-human IgG subclass antibodies. Under the assay conditions used, the anti-IgG subclass antibodies were subclass specific. Quantitative estimates of the subclass antibodies were obtained by reference to a 'capture' assay using F(ab')2 anti-light chain antibody as ligand and IgG myelomas as standards. The validity of these estimates was supported by antibody quantitation using the Farr assay. In healthy adults with serum anti-OA or anti-BLG antibodies, anti-OA antibodies were found mainly in the IgG1 (9/11) and IgG4 (6/11) subclasses, whereas 5 sera showed high levels of IgG2 antibodies. In contrast, the IgG subclass distribution of anti-BLG antibodies was predominantly IgG4 (10/10).

Adult↗
PubMed1985

Increased non-specific binding of heat-treated proteins to plastic surfaces analyzed by ELISA and HPLC-fractionation.

Heat-treatment of proteins from human sera and bovine serum albumin caused an increase in their binding to microplates in a temperature-dependent manner. Quantitative data were obtained for 125I-labelled human IgG, with binding of up to 400 ng of 56 degrees-treated IgG per untreated well. However, only a minor increase in available antibody activity of adsorbed rabbit antibody was found upon pretreatment at 56 degrees. The increased binding to microplates as a result of pre-incubation at raised temperatures was by high pressure liquid gel permeation chromatography demonstrated to be accompanied by polymerization. Inhibition of the direct binding of the proteins to plastic surface was achieved most efficiently by coating with non-interfering proteins followed by non-ionic detergent (Tween 20), which should also be present in the wash buffer.

Animals↗
PubMed1985

Passage of undegraded dietary antigen into the blood of healthy adults. Quantification, estimation of size distribution, and relation of uptake to levels of specific antibodies.

The absorption into the blood of ovalbumin (OA) and beta-lactoglobulin (BLG) was investigated in eight healthy adults. Enzyme immunoassays showed up to 10.5 ng OA/ml serum in seven out of eight individuals 2-3 h after a test meal, whereas no BLG was demonstrated. The apparent size distribution of the absorbed OA was investigated by high-pressure liquid gel permeation chromatography fractionation, followed by enzyme-linked immunosorbent assay, which in the fractionated sera showed OA in all eight individuals. OA was found at the elution volume of intact OA and, in addition, was present in high molecular weight fractions, as part of the immune complexes. The serum concentrations of OA could not be clearly correlated to levels of serum and secretory anti-OA antibodies. No significant alterations of the levels of circulating immune complexes could be demonstrated by two antigen-nonspecific assays. The presented data indicate that, for certain dietary proteins, low-grade absorption of apparently undegraded protein into the blood occurs regularly in healthy adults.

Absorption↗
PubMed1985

Analysis of rheumatoid factors by a biotin-avidin based isotype-specific ELISA.

A one day enzyme immunoassay for the detection of rheumatoid factors of the IgG, IgM, and IgA class is described. The assay utilizes rabbit IgG as solid-phase reactant and the biotin-avidin interaction for the coupling of enzyme to indicator antibody. Three different indicator antibodies discriminated effectively between rheumatoid arthritis patients and normal subjects. F(ab')2 fragments of goat antibodies were found best suited for the test. Rheumatoid factor activity was expressed in U/ml by comparing samples to an internal standard, which was related to the WHO international reference serum for rheumatoid arthritis. Rheumatoid factor activity (U/ml) in the IgM-specific assay showed a close correlation to the latex agglutination titer. Avidity indices estimated from the slopes of the dose response curves of test sera were significantly higher for rheumatoid arthritis patients than for a group of healthy persons, indicating a higher avidity of the rheumatoid factors in the patients' sera.

Adult↗
PubMed1985

Humoral immunity to dietary antigens in healthy adults. Occurrence, isotype and IgG subclass distribution of serum antibodies to protein antigens.

The occurrence of antibodies to five dietary protein antigens in the sera from 21 healthy adults was investigated by a modified Farr assay. Antibody to ovalbumin (OA) occurred most frequently (90%) whereas only 24% had antibodies to alpha-lactalbumin (ALA). No correlation was noted between the titer of antibodies against bovine serum albumin (BSA) and OA in the single individual. The avidity constants (10(8)-10(9) l/mol) and cross-reactivities against other albumins of anti-BSA antibodies in two human sera were comparable to that of the antibodies in pooled hyperimmune rabbit antiserum. Crossed radioimmunoelectrophoresis showed serum anti-BSA and anti-OA antibodies to be predominantly of the IgG class (13/13, 10/10), occasionally of the IgA- (6/13, 1/10) and rarely of the IgM class (1/13, 0/10). Analysis by radioelectroimmunoassay (rocket immunoelectrophoresis) of the IgG subclass distribution of anti-BSA and anti-OA antibodies showed total absence of IgG3. In contrast, antibodies of the IgG4 subclass were frequently present even in sera with very low levels of total IgG4.

Adult↗