Rüdiger Von Baehr

CYTOMEGALOVIRUS INFECTION IN TRANSPLANT RECIPIENTS : THE ROLE OF TUMOR NECROSIS FACTOR

Human cytomegalovirns (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-β: 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.

The high efficiency, human B cell immortalizing heteromyeloma CB-F7. Production of human monoclonal antibodies to human immunodeficiency virus.

This paper describes the construction of a new heteromyeloma cell line designated CB-F7. The cell line was derived from xenogeneic somatic cell hybridization between normal human B lymphocytes and the murine HAT-sensitive P3X63Ag8/653 cell line. CB-F7 cells were characterized by rapid cell growth (doubling time about 16 h) and high cloning efficiencies in culture medium supplemented with 10% or 5% fetal calf serum, respectively. The karyotype of the cells consists of about 75-78 chromosomes as well as two chromosomal fragments. Fusions of the cells with human peripheral blood cells resulted in approximately 2-6 clones per 10(5) seeded lymphocytes. Furthermore, the cells are ouabain resistant and therefore suitable for fusions with EBV-transformed lymphoblastoid cell lines. Using CB-F7 as the parental cell line a number of specific human mAb producing hybrids were established. For the first time, we describe here the generation of hybrids secreting human monoclonal antibodies to human immunodeficiency virus (HIV). Two monoclonal antibodies of IgG type and one of IgM type reacted with the major core protein p25 and one IgG antibody reacted with the transmembrane protein gp41.

Identification and characterization of a TNFα antagonist derived from a monoclonal antibody

Abstract Peptides derived from the CDRs of the anti-TNFα monoclonal antibody Di62 were tested for inhibition of binding of Di62 to TNFα as well as of TNFα 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 TNFα with markedly higher activity (Ki = 4βM) than all other CDR-derived peptides. This peptide also significantly inhibited binding of TNFα to its 55 and 75 kDa receptor and protected L929 cells from the cytotoxic effect of TNFα (IC50 = 6 μM). 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.

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

ObjectiveTo 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. DesignCohort study. SettingUniversity teaching hospitals. PatientsTwenty 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 ResultsThe circulating concentrations of the cytokines inter-leukin (IL)-1 β, IL-6, IL-8 and tumor necrosis factor (TNF)-α 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-1β and TNF-α concentrations were rarely detectable. ConclusionsMonitoring of IL-6 and IL-8 values during ventricular assist device support provides a means of early identification of highrisk patients that may allow optimization of antimicrobial therapy and selection of the appropriate time for transplantation. (Crit Care Med 1994; 22:448–454)

Immunologic effector mechanisms of a standardized mistletoe extract on the function of human monocytes and lymphocytes in vitro, ex vivo, and in vivo.

Even though mistletoe extracts have been in clinical use for centuries their exact mode of action is still unknown. Currently, the application scheme for registered preparations is a dose-escalating scheme to thus reduce side effects. In this study, healthy controls and patients were evaluated for their immunologic response to treatment with a standardized mistletoe extract (Iscador). It shows a strong effect as adjuvant that induces TNF-α and IL-12, which was partly mediated via CD14. Desensitization of the TNF-α response could be shown after repeated application in vitro and in vivo. Furthermore, Iscador induces a specific lymphocyte sensitization upon multiple injections and production of IgG1- and IgG3-mistletoe antibodies. Remarkably, a systemic bystander effect (heterologous immunity against other recall antigens) was observed after long-term treatment. In conclusion, dose-escalation reduces the monocyte-related clinical side effects. A T-lymphocyte sensitization stimulates mainly a specific Th1 response. The most interesting clinical long-term effect is the bystander stimulation of various memory T cells that might mediate in vivo antitumor and antiinfectious T-cell response under mistletoe-extract immunization.

Improving the in vitro antigen specific T cell proliferation assay: the use of interferon-alpha to elicit antigen specific stimulation and decrease bystander proliferation

The measurement of the proliferative response of primed T cells to an antigenic stimulus (lymphocyte transformation assay: LTT) is commonly used for determining T cell immune responsiveness. However, the ratio between the spontaneous and the antigen-triggered response (stimulation index) is frequently quite low (<3-5) making the interpretation difficult. We modified the assay by the addition of interferon-alpha and the use of fresh autologous serum instead of human AB pool serum. These measures significantly enhanced the stimulation index following stimulation with tetanus toxoid, Candida albicans and tick-borne encephalitis (TBE) viral antigen in studies of sensitized patients. There was no concomitant increase in false positive results. Kinetic studies showed a reduced nonspecific background proliferation of non-stimulated cultures particularly between days 4 and 6 of culture. Furthermore, the positive effect of interferon-alpha were confirmed in studies of patients with contact allergy to nickel and gold. We conclude that this modified form of proliferation assay significantly increases the signal to noise ratio which can be attained. This may be of particular value when looking at T cell responses in immunocompromised patients or in diagnostic attempts to detect very low frequencies of antigen-specific T cells.

The Lymphocyte Transformation Test for Borrelia Detects Active Lyme Borreliosis and Verifies Effective Antibiotic Treatment

Borrelia-specific antibodies are not detectable until several weeks after infection and even if they are present, they are no proof of an active infection. Since the sensitivity of culture and PCR for the diagnosis or exclusion of borreliosis is too low, a method is required that detects an active Borrelia infection as early as possible. For this purpose, a lymphocyte transformation test (LTT) using lysate antigens of Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii and recombinant OspC was developed and validated through investigations of seronegative and seropositive healthy individuals as well as of seropositive patients with clinically manifested borreliosis. The sensitivity of the LTT in clinical borreliosis before antibiotic treatment was determined as 89,4% while the specificity was 98,7%. In 1480 patients with clinically suspected borreliosis, results from serology and LTT were comparable in 79.8% of cases. 18% were serologically positive and LTT-negative. These were mainly patients with borreliosis after antibiotic therapy. 2.2% showed a negative serology and a positive LTT result. Half of them had an early erythema migrans. Following antibiotic treatment, the LTT became negative or borderline in patients with early manifestations of borreliosis, whereas in patients with late symptoms, it showed a regression while still remaining positive. Therefore, we propose the follow-up monitoring of dis-seminated Borrelia infections as the main indication for the Borrelia-LTT.

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.

Application of a human monoclonal antibody in a rapid competitive anti-HIV ELISA

The ELISA is the established screening technique for the detection of antibodies directed against HIV. The first generation assays, mostly based on the sandwich principle, employed purified virus from cell culture and gave both false-positive and false-negative results. Sandwich-type assays preferentially detect IgG antibodies, require a high serum dilution and are two-step procedures. In order to detect an immune response as early as possible after infection anti-HIV antibodies of the IgM class should also be measured. To this end a competitive ELISA has been developed using a solid phase-adsorbed recombinant HIV envelope protein and an enzyme-labelled human monoclonal antibody. This detects both IgM and IgG antibodies, the results are available within 1 h and a serum predilution is not necessary.

Binding of natural human IgM auto-antibodies to human tumor cell lines and stimulated normal T lymphocytes.

In a recent publication we described the binding of natural IgM antibodies derived from the human fetal B cell repertoire to the cell surface of some human tumor cells including colon carcinoma, small-cell lung cancer and B lymphoma lines [1]. Further analyses showed that a similar molecule was bound by the respective monoclonal human antibodies on the cell surface of polyclonally stimulated human CD3+ T cells, but is absent from unstimulated MNC. Both CD4+ and CD8+ stimulated cells were recognized. The molecule was found to be expressed together with lymphocyte activation markers (4F2, CD72, CD25). The membrane antigen expressed on both the activated T lymphocytes and tumor cells was characterized in a 2-D electrophoresis system: molecular weight 55-60 kDa, pI-approximately 6.0. Whereas the proliferation capacity of tumor cells was detected to be decreased significantly in the presence of the binding antibodies, no influence on [3H]thymidine uptake into stimulated T cells was found, suggesting different functional consequences of binding the respective antigen on malignant and normal cells. An interesting finding is the enhanced expression of major histocompatibility complex class I molecules on tumor cells incubated with human natural antibodies.