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Biomedical subjects

E Kremmer

Publications and source records attributed to E Kremmer.

123 records · Page 7Linked to original sources
PubMed1993

Murine anti-mouse T cell monoclonal antibodies elicit anti-antibodies in mice: intra-species immunization model for estimating potential patient sensitization against humanized anti-T cell antibodies.

Humanization of immunosuppressive anti-T cell monoclonal antibodies (mAb) raises the question as to how completely it helps to avoid formation of neutralizing anti-antibodies (anti-Ab) in patients. To get more information on intra-species sensitization against anti-T cell mAb, we produced two immunosuppressive mouse IgG2a anti-mouse Thy-1.2 mAb (MmT1 and MmT5) in AKR/J mice and measured the potential of MmT1 to elicit inhibitory anti-Ab in AKR/J (H-2k), C57BL/6 (H-2b), congenic B10.BR (H-2k) and DBA/2 (H-2d) mice. After one injection once weekly for 4 weeks of 5 micrograms MmT1 (200 micrograms/kg) in C57BL/6 mice, without the use of any adjuvants, high concentrations of anti-Ab directed against MmT1 (300 micrograms/ml) and MmT5 (100 micrograms/ml) were measured by enzyme-linked immunosorbent assay. Similar concentrations of anti-Ab were found in immunized DBA/2 and less in B10.BR mice. No syngeneic anti-Ab could be produced in AKR/J. From the C57BL/6 mice, we raised anti-MmT1+, MmT5- idiotype (IDIO1) and anti-MmT1+, MmT5+ allotype (ALLO1) mAb. An in vivo test system was adapted to measure the inhibitory effects of circulating poly- or monoclonal anti-Ab. It revealed a reduction of in vivo depletion capacity not only of the sensitizing mAb (MmT1), but also of another anti-Thy-1.2 mAb (MmT5), with identical allotype but different idiotype. From this we conclude that intra-species immunization following injection of anti-T cell mAb can produce high titer inhibitory anti-idiotype and anti-allotype antibodies. Implications for hyperchimeric or fully human anti-T cell mAb are discussed.

Animals↗
PubMed1993

The Epstein-Barr virus nuclear antigen 2 interacts with an EBNA2 responsive cis-element of the terminal protein 1 gene promoter.

The Epstein-Barr virus protein EBNA2 acts as a transcriptional activator of cellular and viral genes and plays a crucial role in the immortalization of human primary B-cells by EBV. We have shown previously that EBNA2 transactivates the promoters of the latent membrane antigens LMP, TP1 and TP2. The promoter of the TP1 gene was chosen as a model system to study the molecular mechanism of EBNA2 mediated transactivation. To identify an EBNA2 dependent cis-acting element, various TP1 promoter-reporter gene constructs were transfected in the absence and presence of an EBNA2 expression vector into the established B-cell line BL41-P3HR1. We were able to delineate an 81 bp EBNA2 responsive region between -258 and -177 relative to the TP1 RNA start site. The element worked in either orientation and could mediate EBNA2 dependent transactivation on a heterologous promoter. Electrophoretic mobility shift assays revealed three specific protein-DNA complexes formed with sequences of the EBNA2 responsive element. Two of these were not cell type specific, but the third was detected only in EBNA2 positive cell extracts. Gel-shift analysis in the presence of EBNA2 specific monoclonal antibodies revealed that EBNA2 is a component of the third complex. Thus, these experiments demonstrate that EBNA2 interacts with an EBNA2 responsive cis-element of the TP1 promoter.

Antigens, Surface↗
PubMed1993

Immunological detection of proteins associated with the Epstein-Barr virus nuclear antigen 2A.

The Epstein-Barr virus nuclear antigen 2A (EBNA-2A) has been strongly implicated in the EBV-mediated B-cell transformation process. Since EBNA-2A might exert this function through interaction with proteins of the infected cell, we studied the association of EBNA-2A with cellular proteins. Immunoprecipitation of EBNA-2A from 32P-labeled cell extracts separated by sucrose gradient centrifugation revealed the presence of phosphoproteins complexed with the two forms of the EBNA-2A sedimenting at 13 S and 34 S. Prominent bands were observed at 250, 170, 120, 110, 105, and 95 kDa with minor species at 78, 52, 45, 31, 26, 22 and 18 kDa. By "West-Western" or "Far-Western" blotting using EBNA-2A protein from insect cells as a probe we detected binding to proteins migrating with apparent molecular masses of about 200, 130, 110, 105, 95, and 31 kDa with minor species detectable at 90, 68, 50-55, 40, and 17 kDa. The protein with an apparent molecular mass of 31 kDa was identified by competition experiments as histone H1. Some of the EBNA-2A-complexed phosphoproteins, notably the proteins of 110 and 95 kDa, comigrated with the proteins detectable by "West-Western" analysis. The binding of EBNA-2A to the 130-kDa protein was stable against up to 1.5 M NaCl and could not be competed with histone H1. In a similar experiment, the less transforming EBNA-2B which is encoded by the subtype 2 virus bound to most of the proteins detected with EBNA-2A but with strongly reduced efficiency to the protein of 130 kDa indicating that this protein might be a target for EBNA-2 during EBV-mediated transformation.

Animals↗
PubMed1993

Identification and characterization of a mouse monoclonal antibody (M10) directed against canine (dog) CD8+ lymphocytes.

Monoclonal antibodies (mAb) were produced by immunizing BALB/c mice with non-adherent dog lymphocytes. M10 was specific for a subset of dog lymphocytes. M10 belonged to the IgG1 subclass and reacted with 26% of dog peripheral blood lymphocytes, 24% of spleen lymphocytes, 81% of thymus cells, 1.2% of bone marrow cells (5.8% of bone marrow lymphocytes) and 23% of PHA-stimulated lymphocytes. Immunohistology of snap-frozen thymus and spleen showed that the spleen B-cell area stained negative, whereas the spleen T-cell area and the thymus medulla exhibited positive reaction in 20-30%. The thymus cortex was strongly positive. M10 diminished cell lysis by 58% in cell mediated lysis assays (CML). Immunoblot assays revealed that M10 recognized an antigen with a molecular weight of 76 kD under non-reducing and 33 kD under reducing conditions. Finally, M10 bound to a canine CD8 alpha transfected rat T-cell line (NB2). These findings characterize M10 as an antibody directed against the dog CD8 antigen.

Animals↗
PubMed1993

Differences in the activity of the alternative pathway of complement in BALB/c and C57Bl/6 mice.

The deposition of C3b on the surface of zymosan was assessed in inbred strains of mice. Mg(2+)-EGTA plasma from C57Bl/6 and BALB/c mice was incubated with zymosan and deposited C3b was eluted by methylamine. Eluted C3b was detected and quantified by Western blot and an enzyme-linked immunosorbent assay. 3- to 4-fold more C3b was deposited on zymosan incubated with C57Bl/6 plasma than on zymosan incubated with BALB/c plasma (p < 0.01). Data on the kinetics of deposition of C3b suggest a faster and greater activity of the alternative pathway of complement (ACP) in C57Bl/6 than in BALB/c. Higher C3b deposition in C57Bl/6 plasma occurs in the presence of higher plasma factor H concentration (1.3-fold of BALB/c concentration; p < 0.01) and is accompanied by higher factor H deposition on zymosan (3- to 10-fold of BALB/c concentration; p < 0.01). Demonstrable differences in the activity of the proteins of the ACP may provide a biological basis for the genetic variation in innate resistance to infectious organisms observed in inbred strains of mice.

Animals↗
PubMed1992

Determination of monoclonal antibody specificity by immunoadsorption and western blotting.

A rapid and simple method has been developed for identifying the specificity of monoclonal antibodies at an early stage in the production of hybridomas. The technique is a micro-method utilizing biotinylated crude antigen and the surface of microtiter plates as an immunoaffinity matrix. The monoclonal antibodies to be tested are adsorbed to the microtiter wells and incubated with the labeled antigen preparation. Non-specific binding can be reduced by blocking and repeated washing steps. The specific antigen is then eluted by SDS-containing buffers, subjected to SDS-PAGE, blotted onto nitrocellulose and detected by enzyme-labeled avidin in a Western blot assay. The amount of bound and removed antigen can be quantitated by developing eluted and non-eluted control wells by ELISA techniques. Since this ELISA can be performed rapidly, only samples which contain sufficient specific material can be selected for electrophoresis and blotting. The major advantages of the technique are (i) the use of a non-radioactive label resulting in an easy and time-saving procedure, (ii) the possibility of quantitating the amount of captured and detached antigen by ELISA, (iii) the need for only a minimal amount of antigen, (iv) the use of unpurified antibodies of all isotypes, (v) a high signal-to-noise ratio, and (vi) as with all immunoprecipitation techniques, the possibility of detecting SDS-sensitive epitopes and of using crude antigen preparations. Using this method we were able to characterize monoclonal antibodies against both soluble proteins (mouse and human C1q) and membrane determinants (human pan T cell CD5 and CD7).

Animals↗
PubMed1990

Monoclonal antibodies to complement components without the need of their prior purification. II. Antibodies to mouse C3 and C4.

Rat monoclonal antibodies (MAbs) to mouse complement components C3 and C4 were produced by immunizing rats with cell-bound C3 and C4. This principle involves: a) using mouse thymocytes coated with syngeneic rat antibody isotypes that show high affinity to C1q, b) the intercalation of C1q from serum and c) the subsequent activation of the classical complement pathway leading to deposition of cell-bound complement components. Screening for anti-complement antibodies was performed on antibody coating microtiter plates with mouse serum as source of complement. The reactivity of the MAbs was determined by variations of the ELISA screening system using EDTA-serum to inhibit complement activation by C1 dissociation, serum rendered deficient of functionally active C3 by treatment with cobra venom factor (CVF) or serum of genetically C5-deficient mice. The specificity of the MAbs was confirmed by affinity chromatography followed by SDS-PAGE and immunoblotting. We were able to establish a panel of anti-C3 and anti-C4 MAbs of various isotypes.

Animals↗
PubMed1989

Monoclonal antibodies to complement components without the need of their prior purification. I. Antibodies to mouse C1q.

Rat monoclonal antibodies (MAbs) reactive with mouse complement subcomponent C1q were raised applying a principle that requires minute amounts of serum and circumvents purification of serum-derived C1q. The principle involves a) using the high affinity of certain cell-bound antibody isotypes for intercalating C1q from serum of various species, b) selecting such antibodies as are syngeneic to the immunized animal species, thus avoiding the production of antibodies against the intercalating antibodies and c) screening for the anti-C1q MAb in microtiter plates coated with C1q-intercalating MAb isotypes that are heterogeneic to the immunized animal species. We could establish 3 MAbs of IgM subclass, whose reactivity to mouse C1q was shown by ELISA techniques. One of them (RmC13C9) crossreacted with human C1q and was shown by SDS-PAGE and subsequent immunoblotting to recognize the C-chain of mouse and human C1q. The other two MAbs are directed against SDS-sensitive epitopes on mouse C1q and were, therefore, further characterized by native agarose gel electrophoresis and immunohistochemical studies.

Animals↗
PubMed1989

Neutralization of immunosuppression by antibodies against variable as well as constant regions of monoclonal anti-Thy-1 xenoantibodies and their ability to be suppressed by initial T cell depletion.

Timing, magnitude, and effect of the murine antibody response to rat pan-T-cell antibodies were studied in a bone marrow (C57BL/6-to-CBA mice) transplantation model. Prospective C57BL/6 marrow donor mice were sensitized against pan-T-cell (Thy-1, Thy-1.2, Lyt-1) monoclonal antibodies of various rat isotypes or against polyclonal rat antimouse-thymocytes (rat ATG) antibodies. Three days prior to transfer of spleen and bone marrow cells, the sensitized C57BL/6 donors received a dose of anti-Thy-1 mAb (RmT1) known to abolish graft-versus-host reactivity of unsensitized donors. The injected mAb provoked anti-antibodies reacting with RmT1. The anti-antibodies inhibited immunosuppression of the rat mAb RmT1 even if they bound only to nonidiotypic epitopes on RmT1. Avoiding cell-binding of the sensitizing rat anti-Thy-1.2 mAb by its injection into Thy-1.1 mice induced only low-titer and delayed anti-antibodies. This indicated the enhanced immunization potential of anti-Thy-1 when bound to cells. Finally, sensitization leading to the mouse antirat anti-antibodies and reversion of immunosuppression was prevented or reduced considerably by T cell depletion with a mouse IgG2a anti-Thy-1.2 mAb or high-dose cyclophosphamide or by rabbit ATG, provided it was initiated before starting the sensitizing injections of the rat antimouse T cell antibodies.

Animals↗