Biomedical subjects
C Reiter
Publications and source records attributed to C Reiter.
A novel non-radioactive cellular cytotoxicity test based on the differential assessment of living and killed target and effector cells.
Monocyte/macrophage-mediated cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) are slow processes, requiring cocultivation of effector and target cells for up to several days. Because of the high spontaneous release and possible reutilization of isotopic labels, the conventional radioactive release assays are unsuited for measuring long term cytotoxicity. We developed a non-radioactive flow cytometric assay for the quantitative analysis of cell-mediated cytotoxicity. Because dead cells can dissolve and disappear during the incubation period (lysis, phagocytosis), we determined the absolute numbers of living cells in the well. Prior to incubation the effector cells are stained with the red lipophilic fluorescent dye PKH26 and the target cells with the green fluorescent dye PKH2. At the end of the incubation (1-6 days) a defined number of bright fluorescent cell standards and propidium iodide for staining of dead cells was added to each well. Using flow cytometric analysis, we determined the ratio of targets to standards and calculated the absolute target cell number by multiplication with the known number of standards added. The main advantages of the assay are the possibility of extended incubation periods, the avoidance of radioactivity and its potential applicability to autologous culture systems, where effector and tumor cells are derived from the same patient. The assay opens new avenues for preclinical testing of tumor therapeutics such as monoclonal antibodies and/or cytokines.
Restricted expression of the irreC-rst protein is required for normal axonal projections of columnar visual neurons.
The 104 kDa irreC-rst protein, a member of the immunoglobulin superfamily, mediates homophilic adhesion in cell cultures. In larval optic chiasms, the protein is found on recently formed axon bundles, not on older ones. In developing visual neuropils, it is present in all columnar domains of specific layers. The number of irreC-rst-positive neuropil stratifications increases until the midpupal stage. Immunoreactivity fades thereafter. The functional importance of the restricted expression pattern is demonstrated by the severe projection errors of axons in the first and second optic chiasms in loss of function mutants and in transformants that express the irreC-rst protein globally. Epigenesis of the phenotypes can be explained partially on the bases of homophilic irreC-rst interactions.
'Oral hygiene'--a method of killing in a medical setting.
'Oral hygiene' is a routine nursing care procedure used in the treatment of critically ill patients. In the course of their confessions the women accused of the homicides at the Lainz Hospital in Vienna admitted having applied another form of 'oral hygiene'. About 250 ml of tap water was administered per os into the airways of severely ill and comatose patients, and death occurred about one hour later. Pathophysiological investigations have shown that this procedure is a suitable method of killing comatose patients with pulmonary impairment. Possible findings and the problems of detection on the corpse are discussed.
Chimeric monoclonal CD4 antibody--a novel immunosuppressant for clinical heart transplantation.
The murine CD4 monoclonal antibody (mAb) M-T412 effectively downregulates T-helper-inducer function, while exhibiting high affinity and specificity for an epitope formed by the V1 and V2 domain of CD4. The antibody was chimerized by combining the murine VH and VL parts to the constant region of a human IgG1 kappa immunoglobulin. This chimeric CD4 monoclonal antibody (chim CD4 mAb) cM-T412 was used for adjunct immunosuppression in addition to standard triple-drug therapy for patients after orthotopic (n = 10) and heterotopic (n = 1) heart transplantation (HTx). cM-T412 was administered intraoperatively and postoperatively on days 1-7, 9, 11, 13, 17, and 21. A control group of similar composition (10 orthotopic, 1 heterotopic HTx) was conventionally treated in an adjunct fashion with antithymocyte globulin (ATG) until cyclosporine (CsA) in serum had reached therapeutic levels. Over the total observation time (mean: 600 days), the number of acute rejection episodes per 100 patient days was 0.26 in the cM-T412 group versus 0.41 in the control group, indicating a reduction of nearly 40%. Four of the 11 patients in the CD4 group have thus far not experienced any rejection crisis compared with two out of 11 in the control group. The mean time to the first rejection episode was 43.7 days in the CD4-treated patients versus 25.3 days in the control group. In addition the interval to the second rejection episode was longer in CD4 patients than in controls. Furthermore, patients treated with chim CD4 mAb had fewer episodes of infection during the first year after HTx (0.49 vs. 0.91 per 100 pt. days) and had a better overall survival rate (91% vs. 73%) than control group pts. No anaphylactic reaction was observed. The only adverse event probably related to cM-T412 infusion was a transient decrease of blood pressure in one patient. Although this study has only a limited number of patients, addition of cM-T412 to standard triple drug therapy appears to be an effective, specific, and well tolerated adjunct to current immunosuppression that offers a new approach for an improved immunomodulatory regimen after heart transplantation.
Experience with the cytological demonstration of smoker cells in the identification of disaster victims illustrated by the findings concerning the Lauda-Air airliner crash near Bangkok.
On 26 May 1991, a Lauda-Air airliner crashed after take-off at Bangkok from an initial height of approximately 8000 m. The plane came down in inaccessible jungle terrain, so that the difficulties of locating and salvaging the victims were considerably increased by the tropical conditions and the looting that had occurred. Identification of the victims took place in extremely adverse working conditions in the pathological department of the Police Hospital in Bangkok. The technique developed by the first author for the identification of persons in the light of their smoking habits by determining the existence of the so-called smoker cells was used to subdivide the entire autopsy material into 'smokers', 'non-smokers' and 'incidental or passive smokers'. This allows the division of mass disaster victims into smaller specific groups and the application of further identification procedures within these groups in a shorter time. The findings of the cytological examination carried out locally were compared with the police investigations regarding the individual victim's smoking habits, and the method quick and simple to use even in 'field conditions', proved highly efficaceous.
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Treatment of rheumatoid arthritis with a chimeric CD4 monoclonal antibody (cM-T412): immunopharmacological aspects and mechanisms of action.
To investigate the mechanisms of action underlying the therapeutic effect of CD4 monoclonal antibody therapy in rheumatoid arthritis (RA), clinical responses were compared with several laboratory parameters. Twenty-nine RA patients received either 10 mg, 50 mg or 100 mg of cM-T412, a chimeric CD4 MoAb, for 7 days. The CD4 binding sites on circulating lymphocytes were saturated directly with cM-T412 and serum levels of unbound cM-T412 accumulated towards day 7 of treatment only in the patients treated with 50 and 100 mg. The treatment induced an instant and prolonged depression of the number of circulating CD4+ cells, similar for all dosages. Clinical improvement was observed predominantly in the patients treated with 50 or 100 mg cM-T412 daily and did not correlate with changes in counts of circulating leucocyte subsets nor with changes in serum cytokine levels. An antiglobulin response against cM-T412 developed in a majority of the patients. Side effects on the first day of treatment were correlated with an increase of serum IL-6 levels. This study indicates that a favourable clinical effect of cM-T412 administration was associated with the presence of unbound cM-T412 in the circulation of RA patients. Therefore penetration of unbound cM-T412 into the site of inflammation might determine the therapeutic effect in RA.
Treatment of endogenous uveitis with anti-CD4 monoclonal antibody: first report.
A patient with endogenous uveitis was treated with a chimeric monoclonal anti-CD4 antibody. This patient with long-standing therapy-refractive uveitis did not benefit immediately from antibody infusions, although the frequency of uveitis relapses was sharply reduced after this therapy. After treatment with the monoclonal antibody, this patient's response to conventional immunosuppression improved. The patient had very low CD4+ T-cell counts before the beginning of antibody therapy. In this case, peripheral T-cell counts slowly increased. Antigen-specific T-cell responses to retinal S-antigen in this patient were significantly elevated only just prior to a clinical relapse. The initially high level of spontaneous T-cell proliferation normalized after antibody infusions.
[Bristle end rounding on end-tuft, sulcus and orthodontic brushes].
A scanning electron microscopic study of bristle ends of two end-tuft, two sulcular and one orthodontic brush has shown differences in quality and quantity. One product had only 1.4% "acceptable" bristle ends. With the remaining brushes this part varied between 67.0% and 95.1%. There was no correlation between the bristle end shapes and the brush type.
Triple immunofluorescence flow cytometry, using whole blood, of CD4+ and CD8+ lymphocytes expressing CD45RO and CD45RA.
New fluorescent monoclonal antibody-dye conjugates permit three-color immunofluorescence analysis of leukocytes in whole blood using a single laser flow cytometer. The fluorochrome used in this study is a tandem conjugate of phycoerythrin (PE) and Cyan-5, which is excitable at 488 nm with a maximum in the emission spectrum at > 650 nm and it can be used together with PE and fluorescein isothiocyanate (FITC). The directly labelled monoclonal antibodies are incubated with unseparated anticoagulated blood and subsequently erythrocytes are lysed by a standardized automated procedure. The resulting leukocyte suspension can then be analyzed for three different surface markers in an individual sample of 100 microliters blood. When compared simultaneously with single-color analysis triple-color immunofluorescence yielded identical quantitative and qualitative results on various lymphocyte subpopulations. The efficacy of this method was evaluated by analyzing leukocytes of 42 healthy donors for the following markers: CD3, CD4, CD8, CD14, CD16, CD19, CD25, CD38, CD45RO, CD45RA, CD56, CD57, TCR-gamma/delta and HLA-DR. Of special interest was the finding that CD45RA and CD45RO are differently expressed in CD4 and CD8 cells. The reliability and convenience of this three-color analysis will make it possible to do more sophisticated examinations of subpopulations and their relevance in the monitoring of autoimmune diseases, immunodeficiency syndromes including AIDS and malignant disorders such as leukemias.
Chimeric CD4 monoclonal antibody cM-T412 as a therapeutic approach to rheumatoid arthritis.
OBJECTIVE: To investigate the effects of chimeric CD4 monoclonal antibody cM-T412 treatment in patients with rheumatoid arthritis (RA). METHODS: Thirty-two RA patients received daily doses of 10, 50, or 100 mg of cM-T412 intravenously for 7 days. RESULTS: There was a sustained decrease in the number of CD4+ T lymphocytes in all patients. Those who received 50 mg and 100 mg of the antibody experienced significant reductions in disease activity. CONCLUSION: Treatment with cM-T412 appears to have a dose-dependent beneficial effect in RA patients. The clinical effects of cM-T412 are independent of the depressed numbers of circulating CD4+ T cells.
Mhc-DRB and -DQA1 nucleotide sequences of three lowland gorillas. Implications for the evolution of primate Mhc class II haplotypes.
Mhc-DRB and -DQA1 second-exon and -DRB 3'-untranslated-region nucleotide sequences of three lowland gorillas with no known family relationship with each other and of two HLA homozygous typing cell lines were determined and compared with published primate Mhc-DRB and -DQA1 sequences. Eleven distinct MhcGogo-DRB second-exon sequences were found, which represent the gorilla counterparts of the HLA-DRB1*03, -DRB1*10, -DRB3, -DRB5, and -DRB6 allelic lineages. One Gogo-DRB second-exon sequence does not have an obvious human counterpart and is tentatively designated Gogo-DRBY*01. The gorilla equivalents of the HLA-DRB2 and -DRB8 loci were identified as judged on Mhc-DRB 3'-untranslated-region sequences. In addition, four different Gogo-DQA1 alleles belonging to three different allelic lineages were detected. The Mhc-DRB-DQA1 haplotypes of these gorillas were deduced based on the obtained Mhc-DRB and -DQA1 sequences and the two published Mhc-DRB haplotypes of the lowland gorilla Sylvia. All deduced Gogo-DRB-DQA1 haplotypes show gene constellations different from known HLA-DRB-DQA1 haplotypes, while some of the Gogo-DRB haplotypes presented here contain more DRB genes than the HLA-DRB haplotypes. Based on phylogenetic trees, bootstrap analyses, and the gorilla, chimpanzee, and human Mhc-DRB haplotypes described, we propose that at least two Mhc-DRB loci, here tentatively designated Mhc-DRBI and -DRBII, existed on an ancient primate Mhc-DRB haplotype. The Mhc-DRB1*01, -DRB1*02 (-DRB1*15 and -DRB1*16), -DRB1*03 (-DRB1*03, -DRB1*08, -DRB1*11, -DRB1*12, -DRB1*13, and DRB1*14), and -DRB1*10 allelic lineages and -DRB3 and -DRBY loci probably evolved from the hypothetical primate Mhc-DRBI locus, whereas the present primate Mhc-DRB2, -DRB4, and -DRB6 loci originate from the ancient Mhc-DRBII locus of this core primate Mhc-DRB haplotype.
Effects of isotype and Fc region on in vitro function of a mouse/human chimeric CD4 antibody.
Murine CD4 mAbs have shown potential for the treatment of allograft rejection and autoimmune disorders including rheumatoid arthritis. Clinical usefulness of the murine mAbs has been limited by immunogenicity and a short circulating half-life. Mouse/human chimeric antibodies have been constructed, composed of the variable region of M-T412 (a murine G2a mAb specific for the human CD4 molecule) and human G1 (cM-T412 G1) or G4 (cM-T412 G4) Fc regions. F(ab')2 and F(ab) fragments of the murine G2a and chimeric G1 mAbs were generated by enzymatic digestion. The chimeric mAbs and all fragments retained the avidity and specificity of the murine M-T412 and were evaluated in in vitro assays measuring Ig production by pokeweed mitogen (PWM)-stimulated peripheral blood mononuclear cells (PBMC), sIL-2R produced by phytohemagglutinin-stimulated PBMC, and proliferation in response to tetanus toxoid, CD3 mAb plus IL-2, and mixed lymphocyte response (MLR). When PBMC were stimulated with tetanus toxoid, 10 ng/ml of cM-T412 G1 inhibited proliferation by 90%, while neither the cM-T412 G4, M-T412 G2a, nor any mAb fragment produced > 65% inhibition, even at 1000-fold higher concentrations. A similar pattern of inhibition was observed in MLR assays. In contrast, the F(ab')2 fragment of the cM-T412 G1 was as effective as the whole antibody in inhibiting PWM-stimulated IgM synthesis and PBMC proliferation in response to stimulation by a CD3 mAb plus IL-2.(ABSTRACT TRUNCATED AT 250 WORDS)
Selection and chimerization of a monoclonal CD4 antibody for heart transplantation.
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Human-murine chimeric CD4 monoclonal antibodies. A modern panacea for autoimmune disease?
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Combinatorial functions of two chimeric antibodies directed to human CD4 and one directed to the alpha-chain of the human interleukin-2 receptor.
The general feasibility of chimerization of monoclonal antibodies (mAbs) has already been shown for a large number of them. In order to evaluate in vitro parameters relevant to immunosuppressive therapy, we have chimerized and synthesized two anti-CD4 mAbs recognizing two different epitopes on the human T-lymphocyte antigen, CD4. The chimerized mAbs are produced at levels corresponding to those of the original hybridoma cell lines. With respect to activation of human complement, the individual Abs are negative; however, when used in combination, complement activation was performed. When applied in combination, they were found to modulate the CD4 antigen, whereas the individual mAb do not display this property. Individually they mediate an up to 60% inhibition of the mixed lymphocyte reaction (MLR). However, by combination of an anti-CD4 mAb with one directed against the alpha-chain of the human IL2 receptor, nearly 100% inhibition of the MLR was achieved, even with reduced dosage of the mAbs. Our data suggest that the combination of an anti-CD4 mAb and an anti-IL2R alpha chain mAb is more effective with respect to immunosuppression than each mAb by itself, indicating that this mAb cocktail could be a new strategy for immunosuppressive therapy.
The monoclonal CD4 antibody M-T413 inhibits cellular infection with human immunodeficiency virus after viral attachment to the cell membrane: an approach to postexposure prophylaxis.
Infectious cellular uptake of human immunodeficiency virus (HIV) is initiated by a complex sequence of interactions between the viral envelope gp120/gp41 complex and the cellular CD4 receptor resulting in the exposure of a hydrophobic region of gp41 that mediates the irreversible fusion of the virus with the cell membrane. Here we show that viral penetration into a susceptible cell can be inhibited by the high-affinity monoclonal CD4 antibody (CD4 mAb) M-T413 even when it is added as late as 30-120 min after the initial contact of virus with the cell membrane. Inhibition of infection was assessed by monitoring cultures for 34 days after exposure to virus using four different methods simultaneously, including detection of viral DNA by PCR. The interval during which HIV remains sensitive to postbinding neutralization by CD4 mAb depends on strain of virus and type of target cell. Preparations of recombinant soluble CD4 (and the immunoadhesin CD4-IgG1) were much less efficient when compared with mAb M-T413, particularly in blocking infection by fresh HIV-1 isolates. Also cellular transmission of HIV, as determined by syncytia formation within 24 hr, was prevented by mAb M-T413 when added within 45 min of contact of infected H9 cells with uninfected C8166 cells. Together with the favorable clinical experience obtained with CD4 mAbs as immunomodulatory drugs, these data suggest that infusion of CD4 mAb M-T413 may be a therapeutic modus for immediate prophylactic intervention after occupational exposure to HIV and for prevention of intrapartum mother-to-infant HIV transmission.