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Biomedical subjects

R von Baehr

Publications and source records attributed to R von Baehr.

At least 19 recordsLinked to original sources
PubMed1995

Introduction of the octanucleotide restriction site SwaI into the bicistronic vector pTiSDT for high level synthesis of proteins.

The octanucleotide recognition site for the endonuclease SwaI was introduced into the Escherichia coli bicistronic expression vector pTiSDT by mutating a single position in the coupling SD sequence between a truncated form of the cro-gene and the multicloning site. This mutation does not influence the expression rate. The introduction of this restriction site allows high level production of proteins, that are modified only by an N-terminal methionine incorporated as the start codon.

Base Sequence↗
PubMed1994

Cytomegalovirus infection in transplant recipients. The role of tumor necrosis factor.

Human cytomegalovirus (CMV) infection is an important cause of morbidity and mortality in transplant recipients. CMV infection commonly results from the reactivation of a latent infection. Using a set of monoclonal anti-CMV antibodies, we found CMV antigen expression in peripheral blood mononuclear cells (PBMNC), particularly in monocytes, in 312 of 816 samples from 190 allograft recipients. The detection of CMV-IE antigens and CMV-IE DNA in PBMNC indicates that positive cells may represent truly infected cells. The relation between increased cytokine plasma levels (particularly following treatment by pan-T cell antibodies) and the appearance of CMV antigens in PBMNC suggests that cytokines may play an important role in the reversal of CMV latency. This hypothesis is supported by our finding that tumor necrosis factor-alpha (TNF) is able to stimulate the activity of the CMV-IE enhancer/promoter region in the human monocytic cell line, HL-60. The interleukins 1, 2, 3, 4, 6, 8 and 10; transforming growth factor-beta; interferongamma; and granulocyte/macrophage colony-stimulating factor did not show any enhancing effect on the CMV promoter activity. Thus, TNF-alpha seems to play a key role in regulating the balance between latency and reactivation of CMV infection. Inhibition of TNF-alpha release or action may be an alternative strategy for preventing CMV-associated morbidity in allograft recipients.

Adult↗
PubMed1994

Cytomegalovirus reactivation and tumour necrosis factor.

Tumour necrosis factor-alpha (TNF) stimulates cytomegalovirus (CMV) activity in a transfected human monocytic cell line. We assessed whether this finding is relevant in vivo by evaluating the frequency of active CMV infection in patients with diseases that enhance plasma TNF. In septic disease, peripheral blood mononuclear cells of almost all patients studied were positive for CMV. Furthermore, CMV antigenaemia and enhanced plasma TNF occurred in many patients with liver cirrhosis, common variable immunodeficiency, and B-cell chronic lymphocytic leukaemia. Thus, TNF may have a central role in CMV reactivation.

Adolescent↗
PubMed1994

Expansion of a B-lymphocyte clone producing IgM auto-antibodies encoded by a somatically mutated VHI gene in the spleen of an autoimmune patient.

Seventy-six human B-cell hybridomas were obtained by fusing B lymphocytes from the spleen of a patient with chronic idiopathic thrombocytopenia (ITP). Two independent hybridoma clones producing IgM autoantibodies reacting with platelets and other antigens from both the internal and the external environments were established from this fusion experiment. The IgM autoantibodies produced by the two hybridoma clones were found to be encoded by identical VHDJH and VLJL genes. The comparison of the VHI gene expressed in both hybridomas with the germline equivalent cloned from the patient's DNA showed two somatic mutations in the complementarity-determining regions CDR1 and CDR2 resulting in amino acid replacements. These data suggest the selection and expansion of an autoantibody-producing B-cell clone in the spleen of an ITP patient, probably as a result of (auto)antigen-driven stimulation.

Adolescent↗
PubMed1994

Identification and characterization of a TNF alpha antagonist derived from a monoclonal antibody.

Peptides derived from the CDRs of the anti-TNF alpha monoclonal antibody Di62 were tested for inhibition of binding of Di62 to TNF alpha as well as of TNF alpha to its 55 and 75 kDa receptor. A peptide derived from the CDR1 of the light chain was shown to specifically inhibit Di62 binding to TNF alpha with markedly higher activity (Ki = 4 microM) than all other CDR-derived peptides. This peptide also significantly inhibited binding of TNF alpha to its 55 and 75 kDa receptor and protected L929 cells from the cytotoxic effect of TNF alpha (IC50 = 6 microM). The C-terminal region of this peptide, which is homologous to the 55 and 75 kDa TNF receptor, was found to be essential for activity.

Amino Acid Sequence↗
PubMed1994

Characterization of a B-CLL derived IgM-lambda antibody expressing typical features of a NPAB.

A human IgM-lambda hybridoma (CB-HB) was established from chronic lymphocytic leukaemia (CLL) B-cells. Immunochemical and molecular characterization of the monoclonal antibody (mab) produced by the CB-HB cells offered typical features of natural polyreactive antibodies (NPAPs) found in fetal and healthy adult organisms. In particular, the CB-HB mab reacted with different self and foreign (viral and bacterial) antigens when tested in three independent systems (solid- and fluid-phase ELISA, Western blot) showing binding constants in a range from 1.9 x 10(-7) to 7.5 x 10(-8) mol/l to four antigens chosen. In addition, the CB-HB mab binding could be inhibited by a rabbit polyclonal antiserum specific for a common idiotype (Id 102) on human polyreactive (auto)antibodies. The variable region of the CB-HB mab was found to be encoded by unmutated copies of germline genes. Interestingly, the VH-DP10 (51p1) segment, encoding for the autoantibody-associated G6-cross reactive idiotype frequently expressed on both fetal and malignant B-cells, was found to be used with a V segment (VLO11). Collectively these data imply that cells belonging to the natural polyreactive B-cell repertoire undergo malignant transformation. A stimulation by autoantigens or common foreign antigens may be involved.

Antibodies, Monoclonal↗
PubMed1994

Interleukin-6 and interleukin-8 concentrations as predictors of outcome in ventricular assist device patients before heart transplantation.

OBJECTIVE: To determine whether the serum concentrations of some circulating cytokines (as highly sensitive markers of inflammation) are of value in predicting the outcome of patients with cardiogenic shock and end-stage heart disease, who undergo ventricular assist device implantation until heart transplantation. DESIGN: Cohort study. SETTING: University teaching hospitals. PATIENTS: Twenty patients with cardiogenic shock or end-stage heart disease were consecutively selected for this study, if assist device implantation was performed as a bridge to heart transplantation. MEASUREMENTS AND MAIN RESULTS: The circulating concentrations of the cytokines interleukin (IL)-1 beta, IL-6, IL-8 and tumor necrosis factor (TNF)-alpha were monitored from the beginning to the end of assist device support two to three times a week, using commercial enzyme-linked immunosorbent assays (ELISA). In all patients, circulating IL-6 and IL-8 values were increased shortly after assist device implantation. In patients with uncomplicated courses, IL-6 and IL-8 concentrations decreased after an initial increase and were low at the time of transplantation, whereas serum cytokine concentrations increased and remained increased in the nonsurvivors (survivors vs. nonsurvivors, p < .001). Circulating IL-1 beta and TNF-alpha concentrations were rarely detectable. CONCLUSIONS: Monitoring of IL-6 and IL-8 values during ventricular assist device support provides a means of early identification of high-risk patients that may allow optimization of antimicrobial therapy and selection of the appropriate time for transplantation.

Adolescent↗
PubMed1994

A human monoclonal antibody with the capacity to neutralize Staphylococcus aureus alpha-toxin.

A human monoclonal antibody, CB STL-1, against staphylococcal alpha-toxin has been established by hybridoma technology. It is of IgG1 subclass with lambda light chain and possesses a dissociation constant of 8 x 10(-10) mol/l. 1 mg of purified antibody neutralizes the hemolytic activity of 800 micrograms/ml alpha-toxin in an in vitro hemolysis assay using rabbit erythrocytes. The antibody does not bind to overlapping (7 residues) decapeptides spanning the sequence of alpha toxin, thus it might bind to a conformational epitope. The epitope recognized by the antibody is not accessible in oligomeric toxin. The antibody binds both to the hydrophilic and amphipathic forms of the monomeric toxin Fab fragments of the antibody are stable and show no significant loss of activity. CB STL-1 was able to protect mice in vivo from i.p. challenge with alpha toxin. Thus, the antibody is a candidate for passive immunotherapy. The variable regions of the antibody secreted by CB STL-1 were sequenced and found to be encoded by a VH gene segment belonging to the VH1 family, and a Vlambda segment most likely belonging to the VlambdaIII subgroup. Further analysis concerning the third complementarity determining region (CDR3) of the heavy chain is presented.

Amino Acid Sequence↗
PubMed1994

VH/VL gene expression in polyreactive-antibody-producing human hybridomas from the fetal B cell repertoire.

Among a panel of nearly 3,000 IgM-producing hybridomas obtained from 22 independent fusions of human fetal lymphocytes (liver/spleen; 15th-36th gestational week) a high number (5-10%) produced autoantibodies, independently of the gestational age. A significant portion of these autoantibodies was found to be polyreactive, i.e. capable of binding to more than two antigens, when tested against a set of five antigens of the internal (ssDNA, thrombocytes, keratin) and external (lipid A, tetanus toxoid) environment. Analyzing the IgVH genes utilized in eight polyreactive and two putatively nonpolyreactive hybridomas, members of the VHI, III, IV and VI families were found once, seven times, once and once, respectively, mostly with germline identity. All but one of the utilized gene elements could be related to the biased VH gene repertoire said to be expressed during the early ontogeny of the human immune system. We also noted a bias for the utilization of DN1 (3/10), DHQ52 (3/10), JH2 (4/10) and JH6 (4/10) elements, whereas all heavy-chain CDR3 regions manifest a diversity by addition of N nucleotides and/or exonuclease activity on coding segments. In addition, VL segments which belong to different subgroups of both isotypes were found to be used. The molecular basis of polyreactive immunoglobulin specificities in human fetuses is discussed.

Antibodies, Bispecific↗
PubMed1994

Monitoring of the cellular immune system in patients with biventricular assist devices awaiting cardiac transplantation.

Lack of objective parameters to predict the clinical course and outcome are a major problem in managing the patients selected for BVAD-support as a bridge to heart transplantation. This study was intended to assess whether cellular immune parameters have a predictive value for the clinical result of VAD-support. Various cellular immune markers were monitored by multiparameter cytofluorometry in 30 patients who received a VAD system (Berlin Heart). We did not find significant differences in preoperative values of immune parameters between groups of survivors (n = 14) and non-survivors (n = 16). All 9 patients who died of septic multiple organ failure (MOF) had shown increased levels of T-cell activation (CD 71, CD 25, HLA-DR) as well as leukocytosis and 7 patients who died of noninfectious complications (mostly hemorrhage or cerebral complications) had exhibited T-lymphopenia. Seven of 9 patients who died of septic MOF had extremely decreased levels of HLA-DR+ monocytes (< 30%) while all 14 survivors and all 7 patients who died of noninfectious complications showed almost normal monocytic HLA-DR antigen expression, antigen-presenting capacity and cytokine secretion. These observations point to the reduced antimicrobial immunity ("immunoparalysis") in the non-survivors and may explain the fatal course of infection in these individuals. The in vitro results of restitution experiments call for new therapeutic strategies to improve the survival of VAD-patients.

Adolescent↗
PubMed1993

An anti-lipid A antibody obtained from the human fetal repertoire is encoded by VH6-V lambda 1 genes.

The human hybridoma cell line CB-201 has been obtained from a fusion of fetal spleen lymphocytes (31st gestational week) with heteromyeloma cells. The IgM (lambda) secreted was found to bind to lipid A, whereas other endogenous and exogenous antigens were not recognized. The CB-201 antibody is encoded by the unmutated VH6 gene recombined with DN4 and JH3 elements and a V lambda subgroup 1 gene. Therefore, a VH gene, which was previously described to be over-represented in the fetal repertoire and to be expressed in autoantibody-producing B cells, may encode an anti-bacterial specificity.

Antibodies, Monoclonal↗
PubMed1993

Immune restoration in children after partial splenectomy.

Splenectomy (SE) is recognized to be a therapeutical approach in treating children with severe autoimmune diseases (chronic idiopathic thrombocytopenia; hemolytic anemia) or hypersplenism because of portal hypertension. Nevertheless, removal of a main immune organ results in elevated infection risk for these patients. Partial splenectomy (PSE) was developed as a therapeutical compromise to retain immunologically active spleen tissue. Here, we document the analysis of immune parameters obtained from children after both partial and total splenectomy, which have been followed up for a period of more than 6 years: (i) Lymphocytes from both groups of patients failed to produce IgG in response to pokeweed mitogen in vitro. This was observed in 11/20 splenectomized patients even 10 years after operation, whereas in PSE patients a restoration of this parameter after 1-2 years was seen. (ii) In patients after PSE, but not in splenectomized persons, an elevated number of HLA-class II positive cells had been detected suggesting a different situation of immune regulation following this operation. However, in parallel with an improvement of B cell in vitro activity this parameter was found to achieve normal values. Our findings indicate that partial splenectomy may be a therapeutical alternative, if the therapeutic goal can be achieved by this procedure.

Adolescent↗
PubMed1992

Inhibition of HIV-1 infection in vitro by murine monoclonal anti-p24 antibodies.

Murine monoclonal antibodies (Mabs) to the major core protein p24 of the human immunodeficiency virus type 1 (HIV-1) were tested for their ability to inhibit the replication and spread of the virus in permanent cell cultures (Molt4/8, K37, H9) and in the culture of II-2 stimulated T cells of healthy donors. After addition of ascitic fluid containing monoclonal anti-p24 antibodies or purified anti-p24 antibodies or the respective control to co-cultures of infected and non-infected cells, HIV-1 replication was evaluated by determining the percentage of infected cells and the activity of reverse transcriptase (RT) in cell-free supernatant. In addition, the supernatant's infectivity was determined. FACS analysis demonstrated p24 antigen in about 40% of unfixed HIV-1 infected cells at the cell membrane. Monoclonal anti-p24 antibodies of different epitope specificity added to the cells but not to the virus delayed the spread of HIV-1 infection in permanent cell culture. Furthermore, anti-p24 Mabs inhibited the release of RT-active virus particles by HIV-1 infected cell lines or II-2 stimulated T-lymphocytes, respectively, up to 60%. The mode of action of anti-p24 antibodies after HIV-1 infection is discussed on the basis of the data obtained.

Animals↗
PubMed1992

Immunogenicity of recombinant core particles of hepatitis B virus containing epitopes of human immunodeficiency virus 1 core antigen.

A Gag protein segment of human immunodeficiency virus 1 (HIV-1) has been fused to a C terminally truncated core antigen of hepatitis B virus (HBcAg) using an E. coli expression system. Fusion of 90 amino acids of HIV-1 Gag protein to HBcAg still allowed the formation of capsids presenting on their surface epitopes of HIV-1 core protein, whereas fusion of 317, 189, or 100 amino acids of Gag prevented self-assembly of chimeric particles. Mice immunized with recombinant particles emulsified with Freund's complete adjuvant (CFA) or aluminium hydroxide developed high anti-HBcAg titers. However, anti-HIVp24 antibodies were detected only in mice inoculated with immunogen emulsified with CFA.

AIDS Vaccines↗