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Biomedical subjects

B O Boehm

Publications and source records attributed to B O Boehm.

At least 55 records · Page 3Linked to original sources
PubMed1999

Inadequate suspension of neutral protamine Hagendorn (NPH) insulin in pens.

BACKGROUND: Neutral protamine Hagedorn (NPH) insulin is one of the most commonly used insulins in insulin pens. NPH in pen cartridges is in a two-phase solution with either a solvent or a short-acting insulin, and needs adequate mixing for complete resuspension. We assessed whether NPH insulin is accurately resuspended by patients and the association of suspension errors with diabetes control. METHODS: 109 patients (39 with type 1 diabetes) who had received conventional diabetic education had the NPH content of their cartridges measured by an optical system; a control cartridge was designated as 100%. A questionnaire was used to assess clinical details and insulin suspension habits. After the information about residual insulin error was known, all 109 patients were instructed to resuspend their insulin by rolling and tipping the pen 20 times. 52 patients were randomly selected to have cartridges re-analysed 3 months or 6 months later and to complete another questionnaire. FINDINGS: Only 10 (9%) of 109 patients tipped and rolled their pen more than ten times. NPH insulin content ranged from 5% to 214% and varied by more than 20% in 71 (65%) of 109 cartridges. There was no relation between inadequate suspension and the frequency of hypoglycaemic episodes (r=0.2, p=0.08). For all patients, there was a correlation between the absolute error of NPH suspension and cycles of rolling and tipping the pen (r=-0.23, p<0.05). After education on resuspending the pen's contents, data were available from 44 of 52 patients; suspension errors decreased in 35 (80%), were unchanged in three (7%), and increased in six (13%). The 35 patients with improved NPH insulin suspension had fewer mean hypoglycaemic episodes per month compared with the previous period (0.4 [SD 0.1] vs 1.0 [0.3], p<0.05). Mean HbA1c values in patients with improved suspension quality did not differ from baseline (8.4% [0.3] vs 8.9% [0.4], p=0.07). Mixing of NPH insulin by a mechanical device showed that at least 20 cycles were necessary before complete resuspension was obtained. INTERPRETATION: Inadequate NPH insulin suspension is common. We recommended that patients tip pens that contain NPH insulin at least 20 times, since inadequate mixing may impair diabetes control.

Adult↗
PubMed1999

Primary structure, gene expression and chromosomal mapping of rodent homologs of the MEN1 tumor suppressor gene.

Mutations of the MEN1 tumor suppressor gene cause the multiple endocrine neoplasia type 1 (MEN1) syndrome in humans, and they are involved in a variety of sporadic human endocrine tumors. We here characterize the MEN1 gene homologs of the mouse and rat. cDNA was isolated from a mouse phage library, and two alternative MEN1 mRNA transcripts containing variant 5' untranslated regions were identified by RT-PCR in several mouse and rat tissues. When compared to the human molecule, mouse and rat MEN1 (611 and 610 amino acids, respectively) show an overall identity of 96.5% and 97.0% at the protein level, delimiting four conservational domains (A-D). Mouse and rat MEN1 mRNA, as studied by template-calibrated quantitative RT-PCR, is non-exclusively expressed in hematopoietic and endocrine cells, with similar expression patterns found in fetal and adult tissues. Fluorescent in situ hybridization maps the single murine MEN1 locus to chromosome 19, region B. No MEN1 gene mutations were identified in endocrine islet tumor cell lines RIN 5AH (rat) and NIT-1 (mouse) as compared to wild type cDNA. Our data define mouse and rat MEN1 as widely expressed and highly conserved homologs of the human MEN1 tumor suppressor gene whose role in biology and endocrine tumorigenesis is due for experimental study.

Animals↗
PubMed1999

Non-productive human TCR beta chain genes represent V-D-J diversity before selection upon function: insight into biased usage of TCRBD and TCRBJ genes and diversity of CDR3 region length.

The aim of the study was to assess the influence of constraints of V-D-J rearrangement on the nonrandom junctional diversity of productive T-cell receptor beta-chain genes in peripheral T-cells. Mature peripheral T lymphocytes are expected to display a biased repertoire of T cell receptors (TCRs), enriched for those that can recognize peptides presented by the major histocompatibility complex (MHC) molecules. Therefore, functional TCR rearrangements of peripheral T-cells are unsuitable to reveal the bias of the TCR repertoire, introduced by V-D-J rearrangement. To overcome this problem, we have studied nonfunctional TCR genes representing a repertoire of rearranged TCR gene sequences without any known post-rearrangement selection. Detailed molecular analysis of a database generated from more than 500 functional (TCRBV20S1) and nonfunctional (TCRBV10S1P and TCRBV19S1P) T-cell receptor genes from peripheral blood T-cells permitted a comparative analysis of recombination frequencies of each germline-encoded V, D, and J-segments, as well as exonucleolytic nibbling and addition of nucleotides in functional and nonfunctional transcripts. Our data demonstrate that V-D-J recombination generates a more diverse CDR3 length distribution than found among productive TCRBV genes, suggesting that selection constrains the CDR3 to an optimal junctional region length. Furthermore, the well established biased patterns of D- and J-usage in the rearranged TCRBV genes in human peripheral blood lymphocytes were also present in nonfuncrional transcripts. Therefore, V-D-J diversity is biased mainly by constraints of the rearrangement process rather than intrathymic T-cell selection and peripheral expansion of particular T-cell clones.

Adult↗
PubMed1999

Antibodies to human recombinant tissue transglutaminase measured by radioligand assay: evidence for high diagnostic sensitivity for celiac disease.

Celiac disease is associated with endomysial antibodies (EmA), which have recently been reported to be directed to tissue transglutaminase (tTG). To demonstrate binding of antibodies to recombinant tTG, human tTG was cloned, expressed by in vitro transcription/translation and used to develop novel radioligand assays for combined and single detection of immunoglobulin A (IgA) and G (IgG)-specific antibodies. IgA and IgG-tTGA were found in 43 (95.6%) of 45 patients with newly-diagnosed celiac disease verified by biopsy. In addition, all 30 sera from patients with gastrointestinal symptoms and positive EmA were positive for IgA-tTGA, and all but one serum (96.7%) had antibodies of the IgG class. Receiver-operating characteristic analysis including 574 sera from healthy controls revealed a specificity of 99.5%. By means of these new assays, we identified all patients with endomysial antibodies and achieved, at equal specificity, an even improved sensitivity (95.6%) as compared to EmA (91.1%) detected by the standard immunofluorescence test. Here, we have provided direct evidence that recombinant tTG is a major target of antibodies in celiac disease. Our data suggest that tTGA measured by radioligand assay have the power to overcome the limitations of the EmA-test. This new strategy may considerably facilitate large-scale screening for silent and latent celiac disease.

Adolescent↗
PubMed1999

Loss of wild-type MEN1 gene expression in multiple endocrine neoplasia type 1-associated parathyroid adenoma.

Multiple endocrine neoplasia type 1 (MEN1) is a human hereditary tumor syndrome characterized by the development of endocrine adenomas of the parathyroid, anterior pituitary, and enteropancreatic tissue. Several lines of evidence have implicated the recently identified MEN1 gene located on chromosome 11q13 as a recessive tumor suppressor gene. Here, we analyzed MEN1 wild-type gene expression in tumors from a large MEN1 kindred. A deletion of codons 227-228 (678del6) located in exon 4 was found in tumor and peripheral blood complementary DNA using a simplified single-strand conformational polymorphism (SSCP) approach well suited for clinical MEN1 mutation screening. The identified 678del6 cDNA mutation deletes a potential phosphorylation site (Tyr227) and corresponds to a germ line mutation co-segregating with disease phenotype in this MEN1 family. Loss of heterozygosity analysis by fluorescent microsatellite PCR showed an exclusive loss of the MEN1 wild-type (and retention of the mutated) allele detectable in DNA from microdissected parathyroid and pancreatic, but not in adrenal, adenomas. Our findings confirm the synergism between MEN1 gene mutations and subsequent MEN1 allelic losses in the tumorigenesis of MEN1-associated adenomas.

Adenoma↗
PubMed1998

Menin mutations in the diagnosis and prediction of multiple endocrine neoplasia type 1.

INTRODUCTION: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by the development of multiple endocrine adenomas, typically in the pancreas, anterior pituitary, and parathyroid glands. The disease is associated with germ-line mutations of the menin gene, a putative tumor-suppressor gene located on human chromosome 11q13. METHODS: To facilitate the diagnosis and prediction of MEN1 in patients and their relatives, we developed a molecular two-step strategy to screen for menin gene mutations. DNA fragments covering the entire menin coding sequence are generated from patient cDNA by polymerase reaction (PCR) and subsequently analyzed by single-strand conformational polymorphism electrophoresis (SSCP). Fragments with aberrant SSCP migration are DNA-sequenced to directly characterize menin mutations. In a second diagnostic step, genomic DNA of healthy relatives of the corresponding MEN1 index patient is analyzed by PCR, with only the specific exon amplified harboring the family-specific mutation. Mutation-specific restriction enzyme digestion of this PCR product finally allows the identification of mutation carriers through pathological restriction fragment patterns. RESULTS: Using this approach, we identified an in-frame deletion mutation (delta Tyr Met) located in menin exon 4 (codon 227-228) that co-segregates with the disease phenotype in a large MEN1 family from Southern Germany. CONCLUSION: It is likely that the direct molecular analysis of menin gene mutations will replace the genetic and biochemical screening tests currently used in the clinical management of MEN1 families. In addition, these studies may provide clues to the tumor biology of both sporadic and MEN1-associated endocrine adenomas.

Adult↗
PubMed1998

DNA vaccination with glutamic acid decarboxylase (GAD) generates a strong humoral immune response in BALB/c, C57BL/6, and in diabetes-prone NOD mice.

The technique of DNA-based vaccination was used to generate a T-cell-dependent antibody response to glutamic acid decarboxylase (GAD) in BALB/c, C57BL/6, and non-obese diabetic (NOD) mice. Plasmids were constructed in which the expression of the rat GAD65 (rGAD65) or the rat GAD67 (rGAD67) gene was driven by the immediate early region promoter of the human cytomegalovirus (pCMV). This "naked" plasmid DNA was then injected into the regenerating muscles of the studied mice. In the vaccinated animals, antibody responses to GAD65 or to GAD67 were induced. Epitope recognition of GAD was studied by protein footprinting, a technique which makes use of a limited proteolysis of antibody-bound antigen. Different epitope recognition patterns were found, corresponding to strain-specific patterns. Mild trypsin treatment generated 50 kD, 46 kD, 40 kD, 30 kD, and 21 kD proteolytic fragments. In NOD mice, 50, 46 and 40 kD bands were the most prominent signals. In non-diabetes prone BALB/c mice, a faint 40 kD band appeared suggesting a rather weak protection of GAD from tryptic lysis. The pattern observed in C57BL/6 mice was more comparable to the NOD mice pattern with prominent 40 kD and 30 kD signals and a faint 21 kD fragment. Diabetes incidence was unchanged in NOD mice, and no diabetes was observed in C57BL/6 and BALB/c mice, respectively. The data demonstrate that genetic immunization is a suitable novel tool to stimulate and to manipulate an immune response against the diabetes-associated protein glutamic acid decarboxylase. Interestingly, our results indicate that, by genetic vaccination, distinct B-cell epitopes were generated in the various studied mouse strains.

Animals↗
PubMed1997

Analysis of the alpha/beta T-cell receptor repertoire by competitive and quantitative family-specific PCR with exogenous standards and high resolution fluorescence based CDR3 size imaging.

The characterization of the human T-cell receptor (TCR) repertoire in various physiological and pathological conditions has become an important tool in studies of the immune response. Therefore, a number of PCR based strategies for the semiquantitative analysis of the TCR repertoire have been described. Family specific amplification of TCR cDNA has been employed in a number of studies often with contradictory results. We have developed a strategy utilizing exogenous standards with homologous primer binding sites for the quantitative analysis of the alpha/beta T-cell receptor repertoire. This system allows the detection of even minute differences in T-cell populations based on quantitative PCR (Q-PCR) and competitive PCR (C-PCR). Results presented here demonstrate that expansions of T-cell subsets as defined by the specificity of the variable gene segments can be readily monitored when exceeding 1% of the total repertoire. In addition, the proposed method reveals direct information of CDR3 size heterogeneity and can be used to estimate the T-cell repertoire complexity and monitor clonal expansions. We discuss variables such as cell number and experimental conditions influencing accuracy and reproducibility of the analyses. We have used this protocol based on non-radioactive techniques for characterization of the fine specificity of the T-cell repertoire in peripheral and organ-infiltrating T-lymphocytes. The analyses revealed information about polyclonal or clonal expansion of T-cells in vivo and in vitro following various stimuli such as superantigenic stimulation of T-cell subsets as well as antigen-driven shaping of the alpha/beta T-cell repertoire in autoimmune and infectious diseases.

DNA Primers↗
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

HLA class II gene frequencies in Crohn's disease: a population based analysis in Germany.

BACKGROUND: Ulcerative colitis is the only known inflammatory bowel disease associated with particular HLA alleles. Whereas the association with the HLA-DRB1*15 allele has been described in several independent studies for ulcerative colitis, no contribution of HLA alleles to susceptibility in Crohn's disease has yet been shown. AIM: This study was designed to study the strength of association of HLA class II alleles as risk markers for Crohn's disease in a homogenous population in Germany. PATIENTS: A total of 4251 randomly selected control subjects, and 162 unrelated subjects with Crohn's disease were studied. Subjects were studied for their HLA-DRB1, HLA-DQA1, and HLA-DQB1 alleles. METHOD: HLA DNA typing was performed after locus specific amplification with the polymerase chain reaction and reverse dot blot hybridisation. RESULTS: The HLA-DRB1*07 was the only HLA class II allele found to be significantly associated with Crohn's disease (relative risk (RR) = 1.9, 95% CI: 1.66 to 2.14; p = 0.0001). This association remained significant after correction for the number of DRB1 alleles compared. In patients with disease onset before 35 years the RR for the disease in HLA-DRB1*07 positive subjects was found to be higher (RR = 3.1, 95% CI: 2.44 to 3.76). The HLA-DRB1*03 was significantly decreased in frequency in Crohn's disease (RR = 0.5, 95% CI: 0.39 to 0.61; p = 0.0028). CONCLUSION: The HLA-DRB1*07 allele provides risk for the disease especially in patients with younger ages of onset. These data also provide indirect evidence for an immunogenetically based heterogeneity of the disease.

Adolescent↗
PubMed1995

Immunoglobulin variable gene analysis of human autoantibodies reveals antigen-driven immune response to glutamate decarboxylase in type 1 diabetes mellitus.

Insulin-dependent (type 1) diabetes mellitus is an organ-specific autoimmune disease frequently associated with an islet-specific humoral autoimmune response. The role of islet cell autoantibodies in the disease process is unclear; in particular, it is not known whether they are a non-specific side effect of islet cell destruction or play a role in the autoimmune network leading to type 1 diabetes. Here we report the immunoglobulin gene usage and somatic mutation rates of a panel of seven human monoclonal islet cell autoantibodies (MICA 1-7) directed towards the major islet cell autoantigen glutamate decarboxylase (GAD). These autoantibodies were produced from cells from two patients with newly diagnosed type 1 diabetes. VH1, VH4 and V lambda 2 gene segments were frequently used in the MICA, but no correlation between V gene usage and epitope recognition was found. The nonrandom ratio of replacement versus silent mutations in the variable gene region, an accumulation of replacement mutations in the complementarity determining regions, which confer antigen binding, and the high relative avidity for GAD observed for MICA 1, 3, 4, and 6, suggested that the immune response to GAD is driven by the antigen. In contrast, MICA 2, 5, and 7, revealing a lower affinity for antigen, have accumulated a large number of silent mutations. These latter antibodies may, therefore, be characteristic for later stages of the chronic autoimmune disease. Our results argue in favor of an antigen-driven autoantibody response to islets in human type 1 diabetes. They suggest that GAD is an important target of autoimmunity associated with type 1 diabetes.

Amino Acid Sequence↗
PubMed1995

Long-term adherence to intensified conventional insulin therapy.

All diabetic patients of the outpatient clinic of the University of Frankfurt/Main, who started intensified conventional insulin therapy (ICT) between 1980 and 1991, and who could be followed for at least one year (n = 141) were evaluated retrospectively. Fourteen patients changed from ICT to continuous subcutaneous insulin infusion (CSII). No patient changed back permanently to conventional insulin therapy. Mean glycosylated hemoglobin-levels (HbA1) decreased significantly in the first year from 9.3% to 8.5% and remained at a near normal level in the following years. HbA1-levels were found not to be associated with age, age at diagnosis, weight gain, frequency of visits to the outpatient clinic, number of consultations with the dietician as well as the frequency of attendance at special seminars for ICT. These results demonstrate that ICT lowered blood glucose levels permanently, that patients were highly compliant, and that ICT was practicable and safe for long-term treatment under routine conditions without initial hospitalization and with an acceptable expenditure of time for patients and medical staff.

Adult↗
PubMed1995

Expression of a glutamate transporter cDNA in human pancreatic islets.

A number of neuron-specific molecules have been found to be also expressed in pancreatic islets. Some of them might play a major role in the pathogenesis of an autoimmune reaction against the insulin-secreting beta-cells resulting in beta-cell destruction and the manifestation of insulin-dependent diabetes mellitus (IDDM). In ongoing studies our goal was to determine the role of other neuron-specific molecules in beta-cells function and in insulitis. We cloned a L-glutamate transporter from a highly purified fraction of human islet cells. The expression of that specific mRNA was marked in various regions of the brain; weak expression was detected in human islet cells, while no expression in any other tissue was observed. Sequence analysis revealed identity to the transcripts isolated from human brain. The cloned novel cDNA from human islets encodes a key molecule of the excitatory neurotransmission pathway. The techniques used provide a model for the cloning and expression of molecules expressed both in endocrine and neuronal tissue. With characterization of its structure the possible role of the L-glutamate transporter as autoantigen in IDDM is now under investigation.

ATP-Binding Cassette Transporters↗
PubMed1995

In vitro stimulation with glutamic acid decarboxylase (GAD65) leads to an oligoclonal response of peripheral T-cells in an IDDM patient.

The enzyme glutamic acid decarboxylase (GAD65) is a major autoantigen in insulin-dependent diabetes mellitus (IDDM). To study T-cell reactivity towards GAD, peripheral blood leucocytes from seven patients with IDDM and five control subjects were stimulated in vitro with recombinant GAD. All diabetics studied were heterozygous for diabetes-associated HLA alleles, i.e. HLA-DRB1*03,*04-DQB1 *0302,*0201. A single IDDM subject (no. GAD65.05) revealed a strong response against GAD65. After stimulation, his T-cell receptor beta (TCRBV) usage was found to be oligoclonal. The sequence analysis of the putative peptide binding region of the T-cell receptor (CDR3 region) of 37 GAD-reactive T-cell clones revealed no common CDR3 motif. The stimulation of GAD-reactive T-cells could be inhibited with anti-class II monoclonal antibodies, indicating a class II restricted T-cell response. In addition, GAD65-responsive T-cells revealed a Th1 cytokine response pattern. The author's data suggest that GAD-reactive T-cells of Th1 phenotype can be obtained after in vitro stimulation of peripheral blood leucocytes from an HLA-DRB1*03/*04 heterozygous IDDM patient. The lack of a common CDR3 motif suggests the absence of an immunodominant T-cell epitope in that patient, or may indicate receptor repertoire spreading of peripheral T-lymphocytes.

Amino Acid Sequence↗
PubMed1994

Cloning and characterization of a glutamate transporter cDNA from human brain and pancreas.

L-Glutamate is the major excitatory neurotransmitter in the brain. Sufficient removal from the synaptic cleft after neurotransmission by the L-glutamate transport system is essential to prevent excitotoxicity and neurotoxicity. We isolated mRNA from human brain and pancreatic islet cells and screened for sequences of high homology to a previously characterized rat brain glutamate transporter. An isolated sequence (GLTR) shows a 87.5% and a 92.5% sequence similarity at the nucleotide and amino acid level, respectively, with a rat brain specific L-glutamate transporter but only a 65% homology to the recently cloned human glutamate/aspartate transporter. The human mRNA is differentially expressed in brain and to a lesser degree in pancreas and in fetal liver. The gene encoding for the newly identified cDNA is located on chromosome 5.

Amino Acid Sequence↗