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B Ziegler

Publications and source records attributed to B Ziegler.

At least 19 recordsLinked to original sources
PubMed2000

Ex vivo manipulation of hematopoietic stem and progenitor cells.

Ex vivo manipulations of hematopoietic stem and progenitor cells are Increasingly used in the context of autologous and allogeneic stem-cell transplantation. These manipulations include the positive selection of CD34+ cells for tumor-cell reduction and/or T-cell depletion, the ex vivo expansion of hematopoietic progenitor and stem cells under appropriate cytokine-stimulated culture conditions, and the ex vivo generation of myeloid or megakaryocytic postprogenitor cells and Immune effector cells. This article summarizes both the preclinical data on the ex vivo expansion of hematopoietic progenitor and stem cells from purified CD34+ cells and the Initial clinical studies with ex vivo-expanded stem and progenitor cells for hematopoietic support after high-dose chemotherapy.

Animals↗
PubMed2000

Inverse association between birth weight, birth length and serum total cholesterol in adulthood.

OBJECTIVES: To investigate whether impaired fetal growth, measured by low birth weight and short birth length, is linked with raised levels of serum lipids and increased risk and mortality of coronary heart disease. DESIGN: The association between birth length, birth weight, Ponderal Index and total serum cholesterol was examined in 545 Danish men and women aged 31 to 51 years who participated in the Ebeltoft Health Promotion Project in Denmark. RESULTS: No associations were found in women. For men, a negative association was found between birth weight and serum total cholesterol, with a fall in mean serum total cholesterol from 6.03 mmol/l at birth weight below 3300 g to 5.64 mmol/l at birth weight above 4000. A similar association was found between birth length and serum cholesterol, with a mean value of 6.23 mmol/l at birth length below 51 cm and a mean value of 5.56 mmol/l at birth length above 54 cm. No associations were found for Ponderal Index. Between 3% and 8% of the variance in serum total cholesterol could be explained by the statistical models used in this study. CONCLUSION: Our findings support the hypothesis of a negative association between birth weight, birth length and elevated serum cholesterol in adult life, but only in men.

Adult↗
PubMed1999

Karlsburg Type I diabetes risk study of a general population: frequencies and interactions of the four major Type I diabetes-associated autoantibodies studied in 9419 schoolchildren.

AIMS/HYPOTHESIS: The Karlsburg Type I (insulin-dependent) diabetes risk study on schoolchildren aims to evaluate the predictive diagnostic value of diabetes-associated autoantibodies in the general population. METHODS: We took capillary serum from 9419 schoolchildren, aged 6-17 years, for testing of autoantibodies (AAbs) to glutamic acid decarboxylase (GADA), protein tyrosine phosphatase (IA2A) and insulin (IAA) by 125I-antigen binding. We also tested for autoantibodies to cytoplasmic islet cell antigens (ICA) immunohistochemically. RESULTS: By testing of 9419 sera for the four AAbs at cut-off at or greater than the 98th centile for the radioassayed AAbs and at or greater than 10 Juvenile Diabetes Foundation (JDF) units for ICA, 8.1% of schoolchildren had at least one AAb. We found that 3.04, 2.97, 2.35, and 0.86% had IAA, GADA, IA2A or ICA, respectively. 7.3% had only one AAb and 0.8% (75) had two or more AAbs, reflecting a risk to develop diabetes. Thus, by primary screening by combined testing of GADA and IA2A, 98.7% (74/75) would be identified. At high AAb levels, cut-off at or greater than the 99.8th centile and at or greater than 40 JDF units for ICA, 0.23% (22/9419) of schoolchildren, similar to the disease prevalence of 0.3%, had two or more AAbs. Ten of 17 children tested had reduced (p < 0.001) first-phase insulin secretion by intravenous glucose tolerance test. Six of 22 subjects developed Type I diabetes within a follow-up of 19 +/- 10 months. CONCLUSION/INTERPRETATION: For children older than 5 years the combined anti-GAD/IA2 test with cut-off at or greater than the 98th centile should be used for primary screening followed by testing for IAA and ICA. Subjects at risk for diabetes have two or more AAbs at or greater than the 98th centile. Subjects at risk for rapid progression to Type I diabetes have two or more AAbs at or greater than the 99.8th centile.

Adolescent↗
PubMed1998

Unilineage hematopoietic differentiation in bulk and single cell culture.

The rarity of hematopoietic stem and progenitor cells (HSCs, HPCs) has hampered the analysis of cellular and molecular mechanisms underlying early hematopoiesis. Methodology for HPC purification has partially offset this limitation. A further hurdle has been represented by the heterogeneity of the analyzed HPC/precursor populations: recently, development of unilineage HPC differentiation cultures has provided homogeneous populations of hematopoietic cells, particularly in the early differentiation state, i.e., populations pertaining to a single lineage and a restricted stage of differentiation/maturation, but sufficiently large for cellular/molecular analysis. This report focuses on the development and characterization of the unilineage HPC differentiation culture systems. A section is devoted to selected cellular and molecular mechanisms underlying hematopoiesis, which have been investigated by the HPC unilineage culture approach. Finally, recent advances in the development of HPC unilineage cultures at single cell level are discussed.

Animals↗
PubMed1998

A monoclonal antibody-based characterization of autoantibodies against glutamic acid decarboxylase in adults with latent autoimmune diabetes.

Autoantibodies to glutamic acid decarboxylase (GAD) are an important marker of the autoimmune-mediated beta-cell destruction in insulin-dependent (Type I) diabetes. However, these autoantibodies are also found in patients with Stiff-man syndrome (SMS) without onset of diabetes and some diabetic patients who initially present as non-insulin dependent (Type II) diabetes later becoming insulin-dependent, called as latent autoimmune diabetes in adults (LADA). To study the immune response to GAD in these LADA patients a competitive radiobinding assay based on murine monoclonal antibodies recognizing three different GAD regions was performed. The monoclonal antibodies against GAD recognize two different linear epitopes localized at the N- (amino acids 4-17) and C-terminus (amino acids 572-585) and one conformation-dependent epitope region (amino acids 221-442 IDDM-E1) known to be immunodominant for diabetes-associated autoantibodies. All LADA sera (20/20) reduced substantially the 125I-GAD binding of the monoclonal antibodies reactive with the conformation-dependent epitope region IDDM-E1 and only 20% of these sera additionally diminished the 125I-GAD65 binding by those monoclonals reactive with the both linear epitopes. The SMS sera completely abolished the GAD binding of all three monoclonals, reflecting a broader repertoire including an immune response against the IDDM-E1, a conformation-dependent GAD65 epitope region, also revealed if the SMS sera are diluted to equivalent antibody concentrations. In summary, our results show that diabetes-associated GAD autoantibodies even in adult patients with a late autoimmune process preferentially recognize a conformation-dependent middle GAD65 region. An immune response to all three GAD epitope regions is seldom in these LADA patients and only detectable in association with high antibody titres.

Adult↗
PubMed1997

Purging of peripheral blood progenitor cell autografts and treatment of minimal residual disease.

Clinical success of autologous peripheral blood progenitor cell (PBPC) transplantation is challenged by relapse of malignant disease which might at least in part be mediated by graft-contaminating tumor cells. Although the clinical efficacy of tumor cell depletion still remains to be demonstrated, multiple purging strategies are currently pursued in the context of autologous stem cell transplantation. This report discusses ex vivo manipulations of PBPC transplants with respect to purging of tumor cells, including positive selection of CD34+ cells with or without negative depletion of tumor cells as well as ex vivo expansion techniques. Moreover, strategies with an adoptive immunotherapy using ex vivo-generated autologous dendritic cells for the treatment of minimal residual disease after stem cell transplantation will be discussed here.

Antigens, CD34↗
PubMed1997

Sensitive monoclonal antibody-based sandwich ELISA for determination of the diabetes-associated autoantigen glutamic acid decarboxylase GAD65.

Although various methods for the detection of autoantibodies against glutamic acid decarboxylase (GAD65-AAb) are known, no sensitive method for the quantification of GAD65 as autoantigen is available. We describe a sandwich ELISA based on monoclonal GAD65 antibodies (Mc-GAD65-Ab) of different epitope specificities to quantify GAD65 in pancreatic islets and in different organ/cell extracts and during the preparation of GAD from brain extracts. GAD65 was captured via solid phase coated Mc-GAD65-Ab and detected via a second biotin-labelled Mc-GAD65-Ab recognizing a NH2-terminal epitope of the molecule. The detection limit was estimated to be 0.03 ng GAD65/ml using alkaline phosphatase (AP)-conjugated streptavidin. GAD65 contents in islets of neonatal BB/OK rats and Lewis rats amounted to 37.4 and 43.7 pg/islet, respectively. Furthermore, GAD65 was quantified in brain extracts of pig (55.1 ng/mg protein), mouse (39.5 ng/mg), rat (243.8 ng/mg) and pig cerebellum (514.8 ng/mg) and in different organ extracts of Lewis rat.

Animals↗
PubMed1997

Immunohistochemical differentiation of monoclonal GAD antibodies recognizing linear or conformational epitope regions.

GAD65 is targeted by different patterns of autoantibodies [glutamic acid decarboxylase (GAD)-AAbs] in insulin-dependent diabetes mellitus (IDDM) and stiff-man syndrome (SMS). To study differentiation of the GAD-AAb pattern by immunohistochemistry, we examined the immunostaining of 15 monoclonal GAD antibodies (mc-GAD-Abs), which recognized different epitope regions of the antigen, on human pancreatic sections that were unfixed or fixed with different fixatives. By a competitive sandwich enzyme-linked immunosorbent assay (ELISA), three binding patterns of mc-GAD-Abs were identified: 5 of 15 mc-GAD-Abs recognized a linear N-terminal epitope (p1), 5 of 15 were reactive with a conformational GAD65 epitope region (p2), and 5 of 15 were cross-reactive with GAD67 (p3). These patterns of mc-GAD-Abs were tested for islet cell binding by indirect immunofluorescence on pancreatic sections treated with either (1) Bouin's solution, (2) Zamboni's solution, or (3) phosphate-buffered formaldehyde for 0.5, 1, 2, and 18 h at 4 degrees C. After fixation for up to 2 h no differentiation of immunoreactivity of patterns was observed using the three fixatives. mc-GAD-Abs recognizing conformational epitope regions (p2) revealed a marked reduced immunoreactivity on pancreatic sections fixed for 18 h with 4% formaldehyde, while mc-GAD-Abs reactive with linear epitopes (p1, p3) were detectable with strong binding. This fixation procedure was used to compare the immunoreactivity of GAD-AAb+ or GAD-AAb- islet cell cytoplasmic antibody-positive (ICA+) sera of IDDM (n = 27) and SMS patients (n = 3). The three SMS sera were reactive with GAD on fixed islets but showed a reduced titer, whereas the majority of IDDM sera (22/27; 81.5%) were not detectable; 70.6% (12/ 17) of GAD-AAb+ IDDM sera were not detectable on fixed islets. Furthermore, all 10 GAD-AAb- IDDM sera tested failed to react with fixed pancreas, which also suggested an alteration of non-GAD-ICA antigens. In conclusion, the fixation of human pancreatic sections with formaldehyde for 18 h allows the differentiation of GAD-AAbs recognizing linear and conformational epitope regions.

Animals↗
PubMed1996

Evidence for somatic mutation and affinity maturation of diabetes associated human autoantibodies to glutamate decarboxylase.

The processes that lead to the production of islet cell autoantibodies in insulin-dependent (type 1) diabetes mellitus (IDDM) are largely unknown. Humoral autoimmunity may be the result of an antigen-independent polyclonal B cell activation, or a consequence of an antigen driven B cell activation and selection for the antigen. We have analysed the gene elements encoding the immunoglobulin variable regions of seven human monoclonal islet cell antibodies (MICA) 1-7 directed to the major islet autoantigen glutamate decarboxylase (GAD65). These autoantibodies were derived from two patients with newly diagnosed IDDM. The variable gene regions of the MICA revealed different sequences, and no relation between V gene usage and shared epitope recognition of the MICA was evident. An elevated usage of VH 1, VH 4 and Vlambda 2 gene segments was observed. The underrepresentation of VH 3 family members in the MICA discriminated them from most autoantibodies. The high relative avidities for GAD65 of MICA 1, 3, 4 and 6 and their high, nonrandom ratio of replacement versus silent mutations in the antigen binding regions indicated that the humoral response to GAD65 is driven by the antigen. MICA 2, 5 and 7 showed as well an excess of replacement mutations in the antigen binding regions, but revealed lower relative avidities for their antigen. Since these clones accumulated many somatic mutations in their variable gene regions, they may be characteristic for later stages of the autoimmune disease. The results suggest that, in humans, an antigen driven B cell activation and affinity maturation process may contribute to the production of GAD65-autoantibodies found in patients with IDDM.

Antibodies, Monoclonal↗
PubMed1996

Murine monoclonal glutamic acid decarboxylase (GAD)65 antibodies recognize autoimmune-associated GAD epitope regions targeted in patients with type 1 diabetes mellitus and stiff-man syndrome.

To study the immune response to glutamic acid decarboxylase (GAD) in insulin-dependent diabetes mellitus, monoclonal GAD antibodies after fusion of splenocytes from a nondiabetes-susceptible BALB/c mouse immunized with human recombinant GAD65 were generated. Of the 44 monoclonals, 35 are specific for the GAD65 isoform, whereas 9 also react with GAD67. Some 37 monoclonals, including all GAD65/67 reactive antibodies, react with GAD by Western blot analysis. The remaining 7 GAD65 monoclonals bind GAD only in an immunoprecipitation assay, which implies that they target epitopes dependent on the conformation of the GAD molecule. The 125I-GAD binding of the GAD65 monoclonals reactive on Western blotting was significantly diminished by all 3 sera from Stiff-man syndrome patients but only by 3/30 (10%) sera from type 1 diabetic patients. In contrast, the 7 monoclonal antibodies reactive with a conformation-dependent GAD epitope were competitive with 83% of GAD-autoantibody-positive sera from these diabetic patients. Using chimeric GAD65/67 proteins, the epitope region targeted by these monoclonals was mapped to the middle of GAD65 (amino acids 221-442). This central conformation-dependent GAD region was also targeted by sera from patients with type 1 diabetes. In conclusion, our data show that even after common immunization of a nondiabetes-susceptible mouse strain, monoclonal were obtained which preferentially react with the GAD65 linear amino-terminus (amino acids 4-17) and a conformation-dependent region located in the middle of GAD targeted by autoantibodies, indicating that this GAD region is not restricted to the autoimmune response associated with the Stiff-man syndrome and the beta-cell destruction in type 1 diabetes mellitus.

Animals↗
PubMed1996

Comparison of the islet cell antibody pattern of monoclonal glutamic acid decarboxylase antibodies recognizing linear and conformational epitopes.

In order to compare the reactivity of glutamic acid decarboxylase (GAD) antibodies recognizing linear and conformational epitopes as islet cell cytoplasmic antibodies (ICA), monoclonal antibodies were generated. An ELISA displacement test using two biotinylated monoclonals recognizing a linear (M61/7E11) or a conformational GAD65 epitope (M65/6B12) was performed to identify epitope regions recognized by monoclonal GAD antibodies. The GAD binding by monoclonal GAD antibodies was tested by immunofluorescence on fixed and unfixed pancreatic sections of human, rat, and mouse, and by Dot-blot experiments. 16/23 (69.6%) of the monoclonals were specifically reactive with GAD65 and 7/23 (30.4%) were reactive with both GAD isoforms. 8/16 (50%) of monoclonal GAD65 antibodies recognized a linear GAD epitope located at the N-terminus (pattern 1). 5/16 (31.3%) displaced M65/6B12, indicating the recognition of a conformational GAD epitope (pattern 2). Monoclonals belonging to patterns 1 and 2 showed strong ICA binding. 3/16 (18.8%) of monoclonals specific for GAD65 with weak or no immunostaining of pancreatic islets (pattern 3) did not inhibit the binding of both biotinylated antibodies in the displacement test, indicating other epitope specificities. In conclusion, GAD antibodies recognizing both conformational and linear epitopes of the GAD65 molecule are involved in ICA binding with strong reactivity. Furthermore, results obtained with monoclonals of pattern 3 suggest the occurrence of GAD65 epitopes partly inaccessible on cryosections, which may result in an ICA-negative test of GAD65 autoantibody positive sera.

Animals↗
PubMed1996

Glutamate decarboxylase (GAD) is not detectable on the surface of rat islet cells examined by cytofluorometry and complement-dependent antibody-mediated cytotoxicity of monoclonal GAD antibodies.

After fusion of splenocytes from a Balb/c mouse which received three injections of human recombinant GAD65 44 hybridomas producing monoclonal GAD antibodies (mc-GAD Ab) could be established. 35 out of the 44 mc-GAD Ab specifically recognized the GAD65 isoform, whereas 9 showed a cross-reactivity with GAD67. All antibodies belong to the IgG class. The mc-GAD Ab were reactive with recombinant as well as natural GAD tested in enzyme-linked immunosorbent assay. Twenty-one antibodies stained islet cells very well in cryosections of human, monkey, rat, and pig pancreas. However, the mc-GAD Ab failed to bind on the surface of viable rat islet cells, although they reveal a striking binding on permeabilized cells examined by cytofluorometry. Furthermore, the mc-GAD Ab were analyzed for complement-dependent antibody-mediated cytoxicity (C'AMC) to rat islet cells. Whereas our monoclonal Beta-cell specific surface antibody K14D10 caused a high C'AMC measured as a 51Cr-release of 56.5 +/- 4.6%, n = 10, only 4/44 mc-GAD Ab provoked a moderate increase of 51Cr-release ranging from 7.1-10.5% (cut-off 7.0%). From these findings it is suggested that GAD is not detectable on the islet-cell surface by binding and cytotoxicity test.

Animals↗
PubMed1996

Detection of Borna disease virus (BDV) antibodies and BDV RNA in psychiatric patients: evidence for high sequence conservation of human blood-derived BDV RNA.

In several vertebrate species, Borna disease virus (BDV), the prototype of a new group of animal viruses, causes central nervous system disease accompanied by diverse behavioral abnormalities. Seroepidemiological data indicate that BDV may contribute to the pathophysiology of certain human mental disorders. This hypothesis is further supported by the detection of both BDV antigens and BDV RNA in peripheral blood mononuclear cells (PBMCs) of patients with psychiatric disorders and the isolation of BDV from such PBMCs. Here we describe serological and molecular epidemiological studies on psychiatric patients and healthy individuals from the area of Homburg, Germany. Using a novel Western blot (immunoblot) assay, we found a BDV seroprevalence of 9.6% among 416 neuropsychiatric patients, which is significantly higher than the 1.4% found among 203 healthy control individuals. Human sera displayed a prominent immunoreactivity against the virus nucleoprotein, the p40 antigen. Reverse transcriptase-mediated PCR analysis of RNA extracted from PBMCs of a subset of 26 of the neuropsychiatric patients revealed that 50% were BDV RNA positive. Three of the 13 BDV RNA-positive patients also had BDV-positive serology, whereas one patient with serum antibodies to BDV p40 antigen did not harbor detectable BDV RNA in PBMCs. BDV p40 and p24 sequences derived from human PBMCs exhibited both a high degree of inter- and intrapatient conservation and a close genetic relationship to animal-derived BDV sequences.

Adolescent↗
PubMed1995

Unilineage megakaryocytic proliferation and differentiation of purified hematopoietic progenitors in serum-free liquid culture.

Cellular and molecular analysis of megakaryocytopoiesis has been hampered thus far by the lack of pure and abundant megakaryocyte (MK) cell populations. In this study, hematopoietic progenitor cells (HPCs), stringently purified from peripheral blood, were induced to megakaryocytic differentiation/maturation in serum-free liquid suspension culture treated with a growth factor cocktail (interleukin-3 [IL-3], c-kit ligand, and IL-6) and/or recombinant mpl ligand (mpIL). In particular, (1) the growth factor cocktail induced the growth of a 40% MK population, ie, 4 x 10(4) cells at day 0 generated 2 x 10(5) MK at terminal maturation; (2) further addition of mpIL increased the MK purity level to 80% with a final yield of 4 x 10(5) MKs; (3) treatment with mpIL alone resulted in a 97% to 99% MK population, with a mild increase of cell number (to 1.5 x 10(5) cells). In mpIL-supplemented culture, morphological evaluation indicated the presence of putative mononuclear MK precursors and then of mature polynucleated platelet-forming MKs, peaking at days 5 and 12, respectively. Membrane phenotype analysis showed a gradual decrease of CD34+ HPCs, coupled with an inverse increase of MK-specific antigens (eg, CD61/62/42b) starting before mature MK detection by morphology analysis. In situ hybridization showed the expression of MK-specific von Willebrand gene in both MK precursors and mature MKs. Furthermore, MKs synthesize and secrete low but significant amounts of both IL-6 and granulocyte-macrophage colony-stimulating factor. Comparative culture studies were performed on purified bone marrow CD34+/38hi or CD34+/38lo cells stimulated by mpIL alone. Both populations generated a highly enriched MK progeny (62% and 93% MKs at day 12 of culture, respectively) but showed either little or no proliferation. In conclusion, the purified peripheral blood HPC differentiation culture system allows for growth of a relatively large number of highly purified or "pure" megakaryocytic precursors and then mature MKs, thus providing an in vitro experimental tool to dissect the cellular and molecular basis of megakaryocytopoiesis.

Adult↗
PubMed1995

Impact of metabolic activity of beta cells on cytokine-induced damage and recovery of rat pancreatic islets.

The influence of beta cell activity on cytokine-induced functional and structural impairments as well as the ability of those damaged cells to recover were investigated. Rat islets cultured for 4 days in the presence of 5, 10, and 30 mmol/l glucose were exposed to interferon-gamma (IFN, 500 U/ml) and tumor necrosis factor-alpha (TNF, 250 U/ml) for the last 24 h. After cytokine removal islets were allowed to recover spontaneously in culture medium containing 10 mmol/l glucose for a further 7 days. Cytokines significantly inhibited insulin release into culture medium, insulin storage, glucose-stimulated insulin secretion, protein, and DNA synthesis. In the presence of cytokines there was a six- to eightfold increase in nitrite production by the islets. The functional impairments were more pronounced in metabolically stimulated beta cells. In addition, cytokines caused membrane alterations as indicated by increased spontaneous chromium-51 release. The cytokines specifically induced the synthesis of two proteins (72 and 88 kDa, respectively). By immunoblotting, the 72-kDa protein was identified as heat shock protein. After a 1-week recovery period, insulin storage and stimulated insulin secretion of cytokine-treated islets were still significantly diminished. However, protein and DNA synthesis of cytokine-exposed islets returned to pre-exposure levels. In conclusion, high beta cell activity increases islet susceptibility to TNF+IFN. Cytokine-induced, long-lasting, inhibitory effects are primarily directed to beta-cell-specific functions, while general vital cell functions clearly recover after cytokine removal. The induction of certain proteins and the increased protein synthesis and replication rate after cytokine removal might reflect activated repair processes.

Animals↗
PubMed1994

Antibody response in rats against non-toxic glucose sensor membranes tested in cell culture.

Several glucose sensors have been developed, but none are commercially available. The most urgent in vivo problem is the drift of glucose sensor output with time, which may be caused by leakage or denaturation of glucose oxidase, and events at the body-sensor interface such as protein coating, encapsulation with cells, toxicity of the device and inflammation. In the present study, a specific immune response against the outer cellulose acetate membrane of a glucose sensor implanted into rats has also been proved. The immune response against polymeric membranes can be confirmed by detection of specific immunoglobulin G antibodies to cellulose acetate, polyurethane and regenerated cellulose after implantation of the respective membrane. The individually different antibody formation against polymers in rats was amplified by one application of complete Freund's adjuvant in combination with the first implantation. The cell culture results using the fibroblast cell line L-929 showed only a minor toxicity of regenerated cellulose, whereas the other polymers had no effect on cell growth and viability. From the results of this study, it is proposed to integrate immunogenicity as a further parameter for evaluation of the biocompatibility and biosafety of materials or medical devices which are provided for implantation.

Animals↗