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Biomedical subjects

R Gruber

Publications and source records attributed to R Gruber.

At least 73 records · Page 4Linked to original sources
PubMed1997

Phenotypic heterogeneity of osteoblast-like MC3T3-E1 cells: changes of bradykinin-induced prostaglandin E2 production during osteoblast maturation.

We have examined clonal murine calvarial MC3T3-E1 cells obtained from different sources to compare their osteoblastic features (alkaline phosphatase [ALP], cyclic adenosine monophosphate [cAMP] response to parathyroid hormone, prostaglandin E2 (PGE2) and PGE1, bradykinin-induced production of PGE2). It was found that the sublines investigated showed large variation of the above-mentioned parameters, which may be attributed to distinct differentiated stages of osteoblast development. Increase of ALP activity was paralleled by an increase in cAMP accumulation in response to the above-mentioned agents. The most striking difference was observed with bradykinin-induced production of PGE2. Early stage cells (low ALP) produced high levels of PGE2, whereas cells with high ALP activity showed no bradykinin stimulation at all. This was consistent with the results of specific binding of 3H-bradykinin to its receptor and also correlated well with the bradykinin-induced signal transduction sequence (inositol triphosphate liberation and elevation of intracellular calcium levels). This was confirmed by Northern blot analysis of bradykinin receptor mRNA expression. These results indicate that the widely used osteoblast-like cell line MC3T3-E1 is synonymous for multiple sublines, representing different stages of osteoblast development. These sublines were most likely emerging from the early stage cell line due to the applied culture conditions. Moreover, distinct biochemical features are displayed in correlation to the differentiation stage, thus providing a useful model to study the molecular mechanism of osteoblast maturation.

3T3 Cells↗
PubMed1996

Multivalent antibody fragments with high functional affinity for a tumor-associated carbohydrate antigen.

We report in this work a human-derived self-assembling polypeptide based on the tetramerization domain of the human transcription factor p53, which can be fused to single-chain Fv Ab (scFv) fragments via a long and flexible hinge sequence of human origin, allowing exploitation of the functional affinity increase of binding to a ligand or cell surface with multimeric binding sites. We have demonstrated the use of this polypeptide by applying it to the construction of a tetrameric scFv against the tumor-associated carbohydrate Ag Lewis Y (Fuc alpha 1-->2Gal beta 1-->4[Fuc alpha 1-->3] GlcNAc beta 1-->3R). For comparison purposes, the corresponding scFv and dimeric mini-antibody, comprising the scFv fused via a flexible murine hinge to an artificial dimerization domain, were also created. The recombinant mini-antibody proteins were expressed in functional form in Escherichia coli and showed the expected m.w. of a dimer and tetramer, respectively. Analysis of Lewis Y-binding behavior by surface plasmon resonance revealed specific but very weak binding of the scFv fragment. In contrast, both dimeric and tetrameric scFv fusion proteins exhibited an enormous gain in functional affinity that was greatest in the case of the tetrameric mini-antibody.

Amino Acid Sequence↗
PubMed1996

Early renal graft dysfunction. The role of preformed antibodies to DR-typed lymphoblastoid cell lines.

Diverse pathogenetic factors may lead to the complex syndrome of early graft dysfunction, an important determinant of later renal graft outcome. That humoral factors could play a prominent role in the development of the syndrome was suggested by the capillary deposition of complement fragment C4d in about 50% of graft biopsies. This study investigates whether the presumed classical activation of complement is derived from preformed antibodies that would possibly react against endothelial HLA-class II molecules. Such antibodies were detectable by flow cytometry using a representative collection of 11 DR-typed lymphoblastoid cell lines (LCL) as targets. Simultaneous discrimination between complement-activating and -nonactivating antibodies was achieved by two-color FACS analysis. Using this method, 44 out of 86 pretransplant serum samples from recipients with early dysfunction showed reactivity against LCL (18 complement-activating, 14 nonactivating, 12 complement-activating non-IgG). Conventional panel-reactivity was observed in 20 sera only (14 also LCL-reactive). Evaluation of corresponding graft biopsies revealed that capillary C4d was associated with LCL (P = 0.018) and panel reactivity (P = 0.015) alone and in combination (P = 0.001; Pearson's chi-square test). Thirteen subsequent graft losses within one year were observed in the LCL-reactive group as compared with seven losses in the nonreactive group (panel-reactive: 7; nonreactive: 13). Thus, measurement of LCL-reactive antibodies in prospective transplant recipients improves the assessment of an individual immunological risk. The results further demonstrate that performed antibodies do not simply reflect the enhanced overall immune reactivity of certain recipients but rather act locally in vivo, thus emphasizing the role of humoral factors in the development of early graft dysfunction.

Antibodies↗
PubMed1996

[In vitro cultivation of human chondrocytes using autologous human serum supplemented culture medium: minimizing possible risk of infection with pathogens of prion diseases].

BACKGROUND: The in vitro engineering of autologous transplant might play an important role in reconstructive surgery in the near future. The amplification of isolated cells is an important part of the engineering. Because of the use of fetal calf serum (fcs) in the cellculture, there is a potential risk of transmission of prion diseases. The aim of this study was to evaluate a way of amplifying chondrocytes with autologous human serum instead of fcs and to compare the rates of proliferation. METHODS: For this purpose the isolated chondrocytes were cultured using complete medium, with 10% fcs or 10% autologous human serum being added to the medium. RESULTS: The experiments show that a culture with complete medium and autologous human serum allows a 150 to 300fold increase in the number of cells after 40 days of culture and the proliferation is up to 3fold higher than in cultures using fcs. DISCUSSION: Chondrocytes can be stimulated by autologous human serum to proliferate. It is possible to avoid fcs in the culture of chondrocytes and to minimize the risk of an infection with prions.

Adult↗
PubMed1996

Determination of gamma/delta and other T-lymphocyte subsets in bronchoalveolar lavage fluid and peripheral blood from patients with sarcoidosis and idiopathic fibrosis of the lung.

We measured five different lymphocyte subpopulations, including gamma/delta-T cells, in peripheral blood and bronchoalveolar lavage (BAL) fluid from 48 consecutive patients undergoing diagnostic BAL (patients with sarcoidosis (n = 19), patients with idiopathic pulmonary fibrosis (IPF; n = 11) and patients with other diseases of the lung). Quantitative analysis of CD3+, CD4+, CD8+, CD16/56+ cells and gamma/delta-T cells was done by flow cytometry. The proportion of CD3+ and CD4+ cells in the peripheral blood of patients with sarcoidosis and IPF was significantly diminished, while CD8+ lymphocytes and NK cells were significantly elevated compared to healthy controls. There was no significant difference for the gamma/delta-T cell subpopulation between patients with sarcoidosis, IPF and control group. The proportion of CD3+ and CD4+ cells in BAL fluid was significantly elevated in sarcoidosis compared to IPF, while CD8+ cells were significantly diminished. Natural killer cells, defined as CD16/56+ CD3- cells, showed comparable low numbers in sarcoidosis and IPF. For gamma/delta-T cells no significant difference was found between patients with sarcoidosis and IPF.

Adult↗
PubMed1996

Increased antibody titers against mycobacterial heat-shock protein 65 in patients with vasculitis and arteriosclerosis.

Heat shock proteins (HSPs) are a group of highly conserved proteins that show extensive homology at the DNA and protein level among bacterial and mammalian species. Furthermore, bacterial HSPs induce specific cellular and humoral immune responses in mammals. Cross-reacting antibodies may therefore be induced in chronic infections. Recently, it has been claimed that patients with arteriosclerosis (AS) of the carotid arteries have significantly elevated antibody titers to mycobacterial HSPs. In this study, we extended the spectrum of vascular diseases and analyzed sera from patients with systemic vasculitis and systemic lupus erythematosus (SLE) for the presence of anti-HSP antibodies. Anti-HSP antibodies, tested in an ELISA with recombinant mycobacterial HSP 65, were significantly elevated in patients with vasculitis (n = 56; p < 0.01) and AS (n = 29; p < 0.0001), but only marginally in patients with SLE (n = 22; p > 0.05) compared to healthy controls (n = 90). These findings further support the concept of infection-induced immune reactions playing a pathogenic role in the development of both AS and vasculitis.

Adolescent↗
PubMed1995

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.

Antibodies, Monoclonal↗
PubMed1994

Randomised trial of monoclonal antibody for adjuvant therapy of resected Dukes' C colorectal carcinoma. German Cancer Aid 17-1A Study Group.

Over the past decade various clinical trials have used monoclonal antibodies as therapeutic agents against solid tumours. No consistent pattern of response or improved survival has yet emerged although antigenic heterogeneity and insufficient accessibility of cells in advanced tumours have been offered as explanations for these failures. We designed a study in which a monoclonal antibody was used to target minimal residual disease in an early stage of tumour cell dissemination in patients with colorectal cancer. Only patients in Dukes' stage C who had undergone curative surgery and were free of manifest residual tumour were admitted. 189 patients with colorectal cancer of stage Dukes' C were randomly assigned to an observation regimen or to postoperative treatment with 500 mg of 17-1A antibody, followed by four 100 mg infusions each month. A balance of risk factors in the two groups was achieved by dynamic randomisation procedure. After a median follow-up of 5 years, antibody treatment reduced the overall death rate by 30% (Cox's proportional hazard, p = 0.04, log-rank p = 0.05) and decreased the recurrence rate by 27% (p = 0.03, p = 0.05). The effect of antibody was most pronounced in patients who had distant metastasis as first sign of a relapse (p = 0.0014, p = 0.002), an effect that was not seen for local relapses (p = 0.74, p = 0.67). Toxic effects of 17-1A antibody were infrequent, consisting mainly of mild constitutional and gastrointestinal symptoms. During 371 infusions four anaphylactic reactions were seen, all controllable by intravenous steroids and none necessitated admission to hospital. Adjuvant therapy with 17-1A antibody extends life and prolongs remission in patients with colorectal cancer of Dukes' stage C.

Antibodies, Monoclonal↗
PubMed1994

Detection of autoantibodies against M2, LKM-1, and SLA in liver diseases by standardized uniform ELISA-techniques.

Antibodies directed against soluble liver antigen (SLA), liver kidney microsomal antigen (LKM-1-AG), and antimitochondrial antigen M2 (M2-AMA) are critical serological markers for the differential diagnosis of autoimmune chronic active hepatitis (AI-CAH) and primary biliary cirrhosis (PBC). The exact diagnosis of autoimmune hepatitis and PBC is of great clinical relevance, as it leads to different therapeutic strategies. In the present work, a simple and reliable ELISA test system is described, which applies the same test principle for the detection of three different species of autoantibodies important for the diagnosis of chronic liver disease. The ELISA assays are based on a competitive inhibition of binding of positive standard antibodies by patients sera containing antibodies of unknown specificity. The purified immunoglobulins of clinically and serologically clearly defined patients with SLA or LKM-1 positive AI-CAH and with M2 positive PBC were used as coating- and detection antibodies in the ELISAs. From homogenized rat liver the fractionated 100,000g supernatant was employed for the SLA ELISA, the microsomal preparation served as antigen for the LKM-1 ELISA and the mitochondrial preparation was used for the M2 ELISA. In 1,500 sera of patients with the differential diagnosis of a hepatobiliary disease, 17 gave a positive signal in the SLA-ELISA, 12 in the LKM-1-ELISA, and 72 in the M2-ELISA. The results of the ELISAs were compared with Western blotting and immunofluorescence staining pattern on cryostat sections and Hep2 cells. The antibody profiles of several patients are described in detail.

Adult↗
PubMed1993

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.

Adult↗
PubMed1993

Detection of antineutrophil cytoplasmatic antibodies (ANCA) and their association with other autoantibodies in patients with hepatobiliary disorders.

Sera from 35 out of 655 patients with suspected or confirmed hepatobiliary diseases were positive for anti-neutrophil antibodies with a perinuclear staining pattern (p-ANCA). Nineteen of these sera did not react with myeloperoxidase (MPO), Cathepsin G or Elastase, the three proposed antigens of p-ANCAs. No distinct antigen for these sera could be characterized by western blot techniques with fractionated granulocyte proteins.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed1991

Dual effect of the local anaesthetic penticainide on the Na+ current of guinea-pig ventricular myocytes.

1. The effect of the local anaesthetic penticainide (2-alkyl-(4-dialkylamino)-2-pyridyl-butyramide) on macroscopic and single-channel sodium current (INa) of guinea-pig ventricular myocytes was studied with the patch-clamp technique in the cell-attached and inside-out mode. 2. Penticainide (3-60 microM) affected the INa from the outside as well as from the cytoplasmic side. 3. Peak INa was reduced by penticainide at concentrations of 6, 30 and 60 microM, and this decrease of peak INa was more pronounced when the holding potential was more negative. Despite a reduction of peak INa, the time integral of the Na+ current was not changed (60 microM) or was even enhanced (6 microM), and this enhancement became more pronounced at less negative potentials. 4. At a concentration of 3 microM, penticainide increased both the time integral of the current and peak INa. 5. The shape of the steady-state current-voltage relationship and the steady-state inactivation curve were not influenced by penticainide. 6. In pronase-modified inside-out patches penticainide reduced INa at the beginning of a depolarizing pulse to the same extent as at the end (400 ms), indicating a very fast blockade of the bursting Na+ channel. The most prominent effects on pronase-modified single-channel INa were an increase of sweeps without activity, and a fast, repeatedly occurring block (flickering) of the bursting Na+ channel. 7. The amplitude of the unitary current was not altered. 8. It is concluded that penticainide blocks the open Na+ channel, and in addition shows the macroscopic inactivation.

Anesthetics, Local↗
PubMed1991

Quantitative analysis of CD6, CD4 and CD8 cell surface molecules compared to the absolute numbers of CD6+, CD4+ and CD8+ T-cells in peripheral blood in patients with HIV-infection.

T lymphocyte subsets were determined on blood samples from 16 HIV-seropositive patients with manifest AIDS (CDC IV), 24 HIV-seropositive patients with lymphadenopathy syndrome (LAS, CDC III), 16 HIV-seropositive clinical healthy persons (CDC II) and 11 HIV-seronegative homosexuals as control group. Absolute numbers of T-cells (CD6+), T-helper/inducer-cells (CD4+) and T-suppressor/cytotoxic-cells (CD8+), obtained by immunofluorescence staining were compared with the absolute amount of subset specific surface molecules, obtained by a T-cell-ELISA. With both, indirect immunofluorescence technique and ELISA technique a highly significant decrease of the absolute numbers of CD4+ cells and the absolute amount of CD4 surface molecules, respectively, was found in asymptomatic HIV-infection, LAS and in manifest AIDS. In all HIV-seropositive groups the relative decrease of CD4 surface molecules was significantly greater than the decline of CD4+ cells. This phenomenon however was not seen in HIV-seronegative homosexuals. The absolute number of CD6+ cells and the amount of CD6 surface molecules were found significantly lowered in AIDS compared to HIV-seronegative homosexuals. No significant changes were found for CD8+ cell numbers and CD8 surface molecule in the progression of the HIV-infection.

Antigens, CD↗
PubMed1989

The activation gate of the sodium channel controls blockade and deblockade by disopyramide in rabbit Purkinje fibres.

1. The effect of disopyramide on the maximum upstroke velocity (Vmax) and the sodium current of rabbit cardiac Purkinje fibres was studied with the two-microelectrode voltage-clamp technique. 2. In the absence of stimulation the drug did not cause block at membrane potentials ranging from -100 to -65 mV. Use-dependent block of Vmax was most pronounced at -75 mV. At hyperpolarized membrane potentials development of use-dependent block was faster than at depolarized membrane potentials. The time course of development of use-dependent block was not significantly influenced by the duration of the depolarizing pulse. These results strongly suggest that disopyramide predominantly blocks activated sodium channels. 3. The relative decrease of the sodium current at the beginning of a 2 s depolarizing clamp to -45 mV was almost the same as at the end, implying a rapid blockade of activated sodium channels. The Hill plots were linear with slopes ranging from 0.978 to 1.08 indicating a first order reaction; the dissociation constant for activated channels was 70 microM. 4. Recovery of Vmax from use-dependent block during rest was strongly voltage-dependent, the time constant of recovery increasing upon hyperpolarization. When the fraction of charged molecules was reduced by changing the pH of the external solution, the voltage-dependence of recovery was decreased. In contrast, recovery of Vmax for a change in holding potential from -80 to -95 mV was very fast during repetitive stimulation. 5. It is concluded that disopyramide blocks the sodium channel during activation and is trapped in the channel when the activation gate closes.

Animals↗
PubMed1988

The prognosis of cranioplasty following large craniectomy in children.

The experience acquired during 10 years treatment of 33 children having undergone a large craniectomy with an average follow-up time of 5.3 years is presented. Different methods of primary and delayed closure of the skull defect are documented and discussed. Whenever possible the deep frozen conserved skull flap was reimplanted. The problem of skull flap resorption and insufficient spontaneous ossification and the alternative of a heterologous cranioplasty with methylmethacrylate (MMA) in cases of insufficient reossification is considered. The decision to use MMA as a secondary skull defect graft should be delayed at least one year after craniectomy, since a spontaneous reossification of the defect is possible until adolescence.

Adolescent↗
PubMed1988

Graft-versus-host disease in the central nervous system. A real entity?

Graft-versus-host disease (GVHD) classically involves the skin, intestines, liver, esophagus, and tongue. clinically apparent disease involving the heart, lungs, kidneys, and central nervous system (CNS) is frequently secondary to other complicating factors. This report describes a case of an infant with severe combined immune deficiency (SCID) who developed unusual manifestations of GVHD following a bone marrow transplant (BMT). These were complete heart block and respiratory insufficiency in the absence of significant pulmonary disease. He lived 133 days post-transplantation. At autopsy, the brain showed focal lymphohistiocytic aggregates which may represent a hitherto unreported lesion of GVHD.

Acute Disease↗