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C Barth

Publications and source records attributed to C Barth.

At least 19 recordsLinked to original sources
PubMed1998

A rapid, small scale method for characterization of plasmid insertions in the Dictyostelium genome.

A rapid, simple method for characterization of plasmid insertions in the Dictyostelium discoideum genome was developed. It is based on the capability of linear plasmid multimers in the insertions to recircularize efficiently in Escherichia coli cells. This recombinational recircularization of plasmid multimers provides a highly sensitive and reliable tool for determining whether individual Dictyostelium transformants resulted from restriction enzyme-mediated integration (REMI) or from recombinational integration of plasmid (RIP). The method also reveals any rearrangements in RIP insertions and provides an estimate of the vector copy number in any particular transformant.

Animals↗
PubMed1998

Flow cytometric analysis of urine lymphocytes isolated from patients with renal transplants--purification of urine lymphocytes.

Early diagnosis of rejection is a pivotal problem in renal transplantation. Recent advances in urinary cell analysis using flow cytometry are still burdened with difficulties concerning urine lymphocyte (UL) isolation. The analysis of lymphocytes washed out with the urine from the kidney transplant offers a tool to monitor noninvasively the intragraft immune response. However, the demand for optimal isolation of UL with high viability and good separation of other cell types has not, as yet, been met. The present study was undertaken to evaluate the optimal conditions for harvesting UL in order to perform adequate UL analysis by flow cytometry. We found that UL viability is mainly dependent on the time of urine harvesting. Low UL viability was caused by high urine osmolality due to high concentrations of urea and glucose. In contrast, high protein concentrations protected UL viability. Hence, the following algorithm of adequate UL isolation for flow cytometric analysis was established: (1) Collection of morning urine directly onto foetal calf serum (FCS: 30% v/v): (2) UL isolation within 2 h; (3) Erythrocyte lysis with subsequent two-step density gradient isolation of UL from residual erythrocytes, granulocytes (Ficoll-Isopaque, 1.077 g/cm3) and from uroepithelial cells (30% methylglucamine 3,5-diacetomido-2,4,6-triiodobenzoicum, 1.085 g/cmn3); (4) Flow cytometric analysis of UL using the 'live gate' setting in the area of blood lymphocyte cluster. Adequate UL isolation and special settings of the flow cytometer may provide a useful tool for early diagnosis and the noninvasive monitoring of renal transplant rejection.

Cell Survival↗
PubMed1998

[Immunologic, alloantigen-dependent factors in chronic graft rejection].

BACKGROUND: The pathogenesis of chronic renal allograft rejection is still speculative. Amongst other factors immune-mediated graft injury is proposed. Since the allo-antigen is specifically recognized by the variable (V) alpha and beta chains of the T-cell receptor, a restricted T-cell repertoire might support the notion of allo-antigen involvement in chronic rejection. METHODS: By the means of semiquantitative polymerase chain reaction the V beta families 1-20 were assessed in allograft biopsies with histologically confirmed chronic and acute rejection. At the same time the V beta repertoire was analyzed in PBMC. RESULT: The intragraft V beta repertoire was limited to 1 to 3 dominant V beta families in chronic and acute rejection. The response was highly individual and did not correlate to the type or degree of HLA mismatches. The T-cell repertoire in PBMC was polyclonal and did not reflect the immune response in the graft. CONCLUSION: The finding of a restricted V beta repertoire in both forms of rejection might indicate an immunological basis not only for acute, but also for ongoing chronic rejection. Tailor-made antibodies against the dominant V beta clones might provide a tool for selective immunosuppression in both entities of rejection targeting only those T cells which were activated by allo-antigens.

Animals↗
PubMed1998

Co-insertional replication is responsible for tandem multimer formation during plasmid integration into the Dictyostelium genome.

We investigated the establishment of integrating transformation vectors in the genome of Dictyostelium discoideum to gain insight into the formation of the plasmid insertions and to investigate the conditions that determine the number of plasmid copies present in such insertions. Transformation vectors conferring resistance to neomycin and/or blasticidin were introduced into the cell as a calcium phosphate coprecipitate or by electroporation. The integration of the plasmid DNA was based on either recombinational integration of plasmids or restriction enzyme-mediated integration. The genomic DNA of the resulting transformants was examined by Southern blot analysis of pulsed-field gels and by the recently published method of direct electroporation into Escherichia coli. The number of insertion sites was found to be dependent on the transformation method used, and the minimum number of plasmid copies per insertion site required for resistance depended on the type and the concentration of the selective drug. Cotransformation studies revealed a strictly homogeneous composition of vector multimers from any given insertion site. This suggests that multimers arise by co-insertional replication of a single plasmid monomer, rather than by subsequent additional insertion events involving homologous recombination. The multimerization of the integrated vector only occurred when the insertion was established by homologous recombination. Moreover, the number of plasmid copies appeared to be random, was established at the time of the transformation, and did not change with subsequent alterations to the selection regime.

Animals↗
PubMed1998

Intraluminal ultrasound imaging of the fallopian tube wall: results of standardized in vitro investigations of pig and human tubal specimens.

OBJECTIVE: To investigate to what extent anatomic structures of the tubal wall can be identified reproducibly and whether altered areas can be detected and delimited by intraluminal ultrasound. DESIGN: Standardized in vitro experiment with descriptive evaluation of findings, comparative analysis of apparative and morphologic data, and determination of interobserver variability (video documentation, blinded reviewer). SETTING: Department of Gynecology and Obstetrics, University of Heidelberg, Germany. SPECIMEN(S): Seventy-two human and pig fallopian tubes. INTERVENTION(S): Catheterization with a 2.9F or 3.2F ultrasound catheter and sonographic depiction of the fallopian tube, with either simultaneous manual and sonographic wall-thickness measurement or coagulation of the tubal wall. MAIN OUTCOME MEASURE(S): A correlation coefficient of r = 0.76 for manual and sonographic tubal wall measurements and K = 0.88 (with 95% confidence interval of 0.74-1.0) for interobserver variability in recognizing coagulated areas. RESULT(S): Tubal wall anatomy and artificially altered (coagulated) areas were displayed reproducibly with intraluminal ultrasound, thus giving a characteristic, recognizable pattern of the tubal wall. CONCLUSION(S): These in vitro experiments provide evidence that intraluminal ultrasound may expand the current diagnostic possibilities in cases of tubal pathology, providing nonsurgical access to the tubal wall.

Animals↗
PubMed1998

Effect of Pseudomonas aeruginosa exotoxin A on CD3-induced human T-cell activation.

The effect of Pseudomonas aeruginosa (PA) exotoxin A (P-ExA) on CD3-induced T-cell activation was studied on the level of T-cells (proliferation, synthesis of interleukin (IL)-2, expression of IL-2R complex, ICAM-1,2 and LFA-1 molecules), and on the level of monocytes (expression of ICAM-1,2, LFA-1 molecules, as well as FcRI and CD14 receptors). We found that: (1) P-ExA blocked T-cell proliferation and this effect was totally reversed by intact monocytes, and partially by IL-2 or TPA but not by costimulatory cytokines (IL-1alpha, IL-1beta, TNF-alpha or IL-6); (2) P-ExA transiently, in short-term cultures (48 h), inhibited synthesis of IL-2; (3) prolonged stimulation (96 h) of peripheral blood mononuclear cells (PBMC) or CD4 + T-cells with P-ExA in high or low doses (100 and 10 ng/ml, respectively), enhanced the level of IL-2 in the cultures; (4) P-ExA at low dose, combined with IL-1beta, TNF-alpha or IL-6, up-regulated synthesis of IL-2; and (5) stimulation of T-cells with anti-CD3 monoclonal antibody (mAb) and P-ExA at high dose diminished the expression of the p55 chain but not of the p75 chain of IL-2R complex and slightly affected the expression of CD3 complex, ICAM-1,2 and LFA-1 molecules. Hence, P-ExA can regulate the level of IL-2 in cultures of CD3-induced T-cells either by inhibition of IL-2 consumption (when P-ExA is applied in high dose), or by induction of IL-2 production (a costimulatory effect exerted by P-ExA in low dose in combination with monokines). Action of P-ExA on monocytes resulted in: (1) inhibition of the expression of ICAM-1,2 molecules and their ligand LFA-1 molecule; (2) low expression of FcRI receptor (a ligand for Fc part of CD3 mAb); and (3) inhibition (over 90%) of the expression of CD14 molecule. In conclusion, P-ExA-induced anergy of T-cells depends on: (a) decrease in the affinity of IL-2R complex on activated T-cells; and (b) inhibition of the accessory activities of monocytes.

ADP Ribose Transferases↗
PubMed1997

Limited T-cell repertoire in renal allograft and allogeneic melanoma transmitted by the graft.

In a patient with metastatic melanoma transmitted by the renal allograft, HLA serves as an alloantigen per se and is associated with tumor antigens at the same time. The influence of this antigeneic pattern on the Vbeta T-cell repertoire in an allogeneic melanoma, allograft, and peripheral blood mononuclear cells (PBMC) was assessed by polymerase chain reaction. Vbeta13.1 and 19 were found in both the melanoma and the graft. Vbeta14 was detected only in the melanoma and Vbeta6 was detected only in the kidney. PBMC revealed an unrestricted Vbeta pattern. Markers for cytotoxic activity of T cells--granzyme B and perforin--were not expressed during immunosuppressive therapy as clinically reflected in a nonrejecting allograft and in a progressing melanoma. In vitro PBMC proliferated to recombinant interleukin-2, whereas recombinant interferon-gamma did not augment this response. Initiation of immune therapy, in addition to discontinuation of immunosuppression, might support the rejection of the allogeneic tumor by dominant Vbeta T cells.

Aged↗
PubMed1997

Mitochondrial mutations impair signal transduction in Dictyostelium discoideum slugs.

Subpopulations of mutant mitochondria appear to play important roles in degenerative processes associated with aging and are characteristic of many mitochondrial diseases. We have generated mutants carrying plasmid insertions in the Dictyostelium discoideum mitochondrial genome and have shown that phototaxis and thermotaxis in these mutants is more sensitive than growth and division to the presence of a subpopulation of defective mitochondria. This could result from direct impairment of a mitochondrial role in signal transduction, or indirectly from the effects of energy depletion. Either way, signal transduction may be the first cellular activity to be compromised by the accumulation of defective mitochondria in age-related tissue dysfunction and in mitochondrial disease.

Animals↗
PubMed1997

Genetic regulation of the impaired immune response to hepatitis-B vaccine associated with low TCR density in end stage renal disease patients: contribution of complement C4 and factor B alleles.

We have studied the relationship between T-cell receptor (TCR) density, genetic factors and the specific immune response in 153 end stage renal disease (ESRD) patients on haemodialysis immunised with HBsAg vaccine. One-hundred and nineteen patients raised a protective (> 10 U/ml) antibody response to hepatitis-B vaccination (responder, R), while 34 patients were found to be non-responders (NR). The density of the T-cell receptors was determined by flow cytometry. Proliferation of the T-cells induced by autologous monocytes presenting HBsAg was also measured and expressed as a stimulation index (SI). MHC class I, II and III alleles of the patients were also determined. The densities of TCR/CD3 receptors in NR patients were found to be significantly decreased as compared to the R patients (189 +/- 22 vs. 282 +/- 58 arbitrary units, P = 1.3 x 10(-7). TCR/CD3 receptor densities were found to be strongly associated (Spearman correlation coefficient: 0.84, P < 0.000001) with the SI values. Both parameters were found to be under dual genetic control: (a) very low density of the TCR/CD3 receptors and very low SI were found mainly in NR patients carrying HLA-A1, HLA-B8 and HLA-DR3 alleles; and (b) TCR/CD3 densities and function in R group were found to be significantly lower in carriers than in non-carriers of two MHC class III complement protein alleles: C4A*6, and Bf*F. Non-responsiveness to hepatitis-B vaccination was found to be associated with extremely increased neopterin levels. These findings indicate that both genetic and acquired factors contribute to the hepatitis-B vaccination failure in ESRD patients.

Alleles↗
PubMed1997

A new look at dietary carbohydrate: chemistry, physiology and health. Paris Carbohydrate Group.

The current view of dietary carbohydrates as simply providing us with energy is outdated. Because of their varied chemistry and physical form the rate and extent to which the different types are digested in and absorbed from the small intestine varies. This in turn leads to affects on satiety, blood glucose and insulin, protein glycosylation, lipids and bile acids. Some carbohydrates reach the colon where they are fermented and affect many aspects of large bowel function, colonocyte and hepatic metabolism. A new framework for classifying and measuring food carbohydrates is needed to allow a greater understanding of the role of individual species in health and to inform the public of their importance. A classification based primarily on molecular size (degree of polymerisation) into sugars, oligosaccharides and polysaccharides, is suggested, with sub-groups identified by the nature of the monosaccharides. Greater knowledge of the chemical and physical properties of carbohydrates allow a more precise relation with physiology and health to be drawn. The Carbohydrate Group met in Paris in December 1995 at the invitation of Gerard Pascal, Director of CNERNA. Financial support for the meeting was provided by CNERNA.

Carbohydrates↗
PubMed1997

[Thoracoscopy is a reliable alternative to thoracotomy in peripheral lung coin lesion].

Video-assisted thoracoscopy (VATS) is useful for improving preoperative diagnosis and staging in patients with peripheral pulmonary lesions. Additional information obtained by VATS had a major influence on the therapeutic strategy in 34% of the patients, and in 39% of the patients in the conventional surgery group thoracotomy could possibly have been avoided if VATS had been available.

Diagnosis, Differential↗
PubMed1996

Replicon rescue: a novel strategy to clone the genomic DNA flanking insertions of integrating shuttle vector DNA.

A novel cloning strategy, replicon rescue, was developed for cloning genes disrupted by plasmid insertions. After ligation to a tetracycline resistance cassette, fragments containing a bacterial origin of replication from the insertion are recovered in Escherichia coli because they replicate autonomously. Restriction enzymes for cloning are so chosen that the only legitimate two fragment ligation yielding TetR clones involves a fragment spanning the boundary of the insertion. Replicon rescue was used successfully firstly in a test system to clone the chromosomal orl from a Klebsiella aerogenes strain, and secondly to recover a disrupted gene from a phototaxis-deficient mutant of Dictyostelium.

Animals↗
PubMed1996

Restricted TCR V beta repertoire in the human mixed lymphocyte reaction is determined by the TCR on the responder cell and not by the stimulating antigen on the stimulator cell.

The use of a restricted TCR repertoire has been reported in antigen-stimulated T cells. We have examined by flow cytometry and polymerase chain reaction the changes in the TCR variable beta (V beta) repertoire as they occur in the mixed leukocyte reaction. The HLA class I-negative lymphoblastoid B cell line, Daudi, an HLA class I-positive variant of the Daudi cell line, and the homozygous typing cell line HTC 9062 were used to stimulate HLA-mismatched responder cells from 8 individuals. The magnitude of HLA incompatibility in various combinations ranged from single DR10 and DR13 differences to full 6-antigen differences at the HLA-A, -B, and -DR loci. Using a panel of 19 V beta-specific oligonucleotide primers, changes in the level of TCR V beta mRNA were assessed. The observed V beta repertoire was not specific for the stimulator cell antigens used. We did not find a correlation between the number of mismatched HLA antigens and the number of V beta elements involved. It rather appeared that individual responder cells displayed a uniform V beta pattern following exposure to single and multiple HLA antigens. We conclude that the V beta repertoire was largely determined by factors associated with the TCR on the responder cells, and not as obviously influenced by the HLA antigen(s) on the stimulator cells.

Antigen Presentation↗
PubMed1996

Efficient circularization in Escherichia coli of linear plasmid multimers from Dictyostelium discoideum genomic DNA.

Transformation of Escherichia coli with Dictyostelium discoideum genomic DNA containing integrated shuttle vectors in multicopy, tandemly duplicated format resulted in the establishment of the linear plasmid molecules as circular monomeric replicons. The transformation efficiencies were comparable to those obtained with circular plasmid DNA and the recovered plasmids were free of deletions and rearrangements. Digestion of the genomic DNA prior to the transformation using restriction enzymes that cut within the inserted plasmids reduced the transformation efficiency dramatically and a high proportion of the recovered plasmids carried deletions. Our results provide evidence that the linear plasmid multimers cyclize in E. coli by homologous recombination in order to be established as autonomously replicated plasmids. The efficiency of recircularization was found to be independent of the recA gene product but dramatically reduced in the absence of recB recC or sbcB gene products. However, the paradoxically high efficiency of transformation with plasmid multimers of a recB recC sbcB mutant indicated the presence of an additional pathway for recombinational recircularization independent of these gene products. Unlike previous studies using as a DNA source linearized plasmid monomers and dimers that were created in vitro, the use of linear plasmid multimers integrated into the D. discoideum genome ensured that none of the E. coli transformants we obtained could be attributed to low levels of uncut circular plasmid molecules. The efficient recovery of the plasmid monomers faithfully reflects the structure of the insertion and thus provides a useful tool in the characterization of such plasmid insertions in the genome of D. discoideum.

Animals↗
PubMed1996

Restricted T cell V beta repertoire in renal allografts during acute and chronic rejection.

Allo-MHC specific antigen recognition might not only be involved in acute, but also in chronic rejection. The clonotypic specificity of the T-cell receptor to recognize all-MHC is located in the variable (V) alpha and beta chain. A restricted T-cell receptor repertoire could support an immunological basis for chronic rejection. The novel feature of this study is that V beta repertoire was assessed in ongoing chronic rejection before end-stage renal failure and in acute rejection. V beta s 1 to 20 were quantitated by PCR in PBMC and biopsies of rejecting renal allografts. The V beta pattern in PBMC demonstrated a polyclonal distribution. However, the intragraft V beta repertoire was restricted to 1 to 3 dominant V betas and highly individual in 9 of 12 patients. Number and type of the HLA mismatch and the time interval between transplantation and biopsy did not correlate to the V beta distribution. The individual response is attributed to genetic predisposition factors of the recipient. Therefore, the restriction of the V beta repertoire indicates allo-MHC dependent immune processes not only in acute, but also in ongoing chronic rejection. Tailor-made antibodies against dominant V betas might offer specific individual immunosuppression in treating both acute and ongoing chronic rejection.

Graft Rejection↗
PubMed1996

Comparison of in-vitro falloposcopy with tubal histology in the diagnosis of fallopian tube pathology.

In order to assess the diagnostic quality of falloposcopy in relation to pathomorphology, a consecutive series of 30 Fallopian tubes obtained from surgical salpingectomy cases were prospectively examined by in-vitro falloposcopy and histology. Falloposcopy was performed using an over-the-wire catheterization system and a 0.5 mm falloposcope with 3000 pixels. Assessment of the specimens included the description of lumen geometry, intraluminal changes and status of the mucosal surface. Falloposcopy classified 14 tubes as normal and 16 pathological. Histology resulted in 17 normal versus 13 pathological tubes. Pathologies included lumen obstructions and dilatations, intraluminal synechiae and mucosal damage. Sensitivity and specificity of falloposcopy were calculated to be 0.85 and 0.71; positive and negative predictive values were 0.69 and 0.86. It was concluded that falloposcopic findings indeed reflect and successfully differentiate normal and pathological conditions allowing adequate and reproducible image interpretation. However, variations of the diagnostic accuracy with the type of pathology and the tubal segment have to be taken into account before clinical consequences are drawn from a falloposcopic investigation.

Endoscopy↗
PubMed1996

A proteoglycan that activates fibroblast growth factors during early neuronal development is a perlecan variant.

Cells in the early embryonic vertebrate nervous system are dependent on members of the fibroblast growth factor (FGF) family for their proliferation and subsequent differentiation. These growth factors will only bind to their specific high affinity cell surface receptors after formation of a ternary complex with the glycosaminoglycan heparan sulfate. Such specific heparan sulfates are secreted as proteoglycans from neural precursor cells and localise to their surfaces. One such proteoglycan, HSPG-PRM (Perlecan-related molecule), was isolated through its ability to potentiate neural cell responses to either FGF-1 or FGF-2. In this study, we have verified the relative molecular mass of the core protein of PRM as 45,000 and obtained partial amino acid sequence from it. The sequences bore significant homology to native perlecan. A probe generated by reverse transcriptase polymerase chain reaction using oligonucleotides designed from the protein sequence used on northern blots of RNA from a neuroepithelial cell line detected perlecan at 12.6 kilobases, as well as novel transcripts at 6.5 and 3.5 kilobases. The latter species appears by virtue of its size and abundance to be the novel PRM transcript. PRM appears to be encoded by the same gene as perlecan, as genomic Southern blotting only detected a single gene. Polyclonal antibodies raised against the PRM molecule detected a single proteoglycan species at 290x10(3) with a core protein of 45x10(3). Polyclonal anti-perlecan antibodies cross-reacted with PRM confirming their relatedness, although immunohistochemical studies revealed a differential staining pattern for PRM as compared to perlecan within the developing nervous system. The PRM molecule was shown to be localised to several different tissues of the developing embryo, indicating that it plays a broad role. We conclude that PRM is a variant of perlecan that is differentially glycosylated in a manner that confers highly specific functions at critical stages of neural development and tissue growth.

Alternative Splicing↗