Genotypic and phenotypic analysis of Borrelia burgdorferi isolates from The Netherlands.

Sixty-three Borrelia burgdorferi isolates recovered from Ixodes ricinus ticks collected in 17 locations in The Netherlands and three Dutch human skin isolates were characterized by rRNA gene restriction fragment length polymorphism, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting (immunoblotting). All three human isolates belonged to B. burgdorferi group VS461. Of the tick isolates, 29 (46%) were B. burgdorferi sensu stricto, 2 (3%) were group VS461, 19 (30%) were Borrelia garinii, and 13 (21%) were different from any previously described genomic species. On the basis of the criteria described, 12 isolates formed a distinct genomic group, designated M19. rRNA gene restriction patterns of the group M19 isolates resembled but were not identical to the B. garinii patterns. Hybridization of digested DNA with a flagellin probe confirmed the separation of group M19 from the B. garinii isolates. One isolate, M63, was different from all the others. In conclusion, the occurrence of B. burgdorferi sensu stricto, B. garinii, and B. burgdorferi group VS461 in ticks from The Netherlands corresponds with the occurrence of these genomic species among tick isolates from other European countries. However, our findings suggest that B. burgdorferi sensu lato probably contains more than three genomic species.     

Amplified region of chromosome band 11q13 in breast and squamous cell carcinomas encompasses three CpG islands telomeric of FGF3, including the expressed gene EMS1

DNA markers that map within the karyotypically defined band q13 on human chromosome 11 are amplified in a subset of mammary and squamous cell carcinomas. It is assumed that the amplified DNA includes a critical gene (or genes) whose overexpression provides a selective force in the development of the tumor. To help identify such genes, we have begun to construct a physical map of CpG islands in the region, making use of a squamous cell carcinoma cell line (UMSCC2) in which the 11q13 region is amplified 11-fold. We previously described the proximal end of this amplicon and the order of markers extending ～800 kb centromeric of the FGF3 locus (formerly INT2). We now report the use of chromosome jumping techniques to define additional CpG islands that lie distal to FGF3. These map within the amplified region in UMSCC2 cells and the most telomeric corresponds to the EMS1 gene. The data imply that the amplified DNA in UMSCC2 cells extends for over 1,500 kb and includes at least 7 potential genes. EMS1 and CCND1 (formerly PRAD1), the best candidates for the key gene on the 11q13 amplicon, are ≥800 kb apart. © 1993 Wiley-Liss, Inc.     

Chronic obstructive pulmonary disease is associated with enhanced bronchial expression of FGF-1, FGF-2, and FGFR-1

An important feature of chronic obstructive pulmonary disease (COPD) is airway remodelling, the molecular mechanisms of which are poorly understood. In this study, the role of fibroblast growth factors (FGF-1 and FGF-2) and their receptor, FGFR-1, was assessed in bronchial airway wall remodelling in patients with COPD (FEV1 < 75%; n = 15) and without COPD (FEV1 > 85%; n = 16). FGF-1 and FGFR-1 were immunolocalized in bronchial epithelium, airway smooth muscle (ASM), submucosal glandular epithelium, and vascular smooth muscle. Quantitative digital image analysis revealed increased cytoplasmic expression of FGF-2 in bronchial epithelium (0.35 +/- 0.03 vs 0.20 +/- 0.04, p < 0.008) and nuclear localization in ASM (p < 0.0001) in COPD patients compared with controls. Elevated levels of FGFR-1 in ASM (p < 0.005) and of FGF-1 (p < 0.04) and FGFR-1 (p < 0.001) in bronchial epithelium were observed. In cultured human ASM cells, FGF-1 and/or FGF-2 (10 ng/ml) induced cellular proliferation, as shown by [3H]thymidine incorporation and by cell number counts. Steady-state mRNA levels of FGFR-1 were elevated in human ASM cells treated with either FGF-1 or FGF-2. The increased bronchial expression of fibroblast growth factors and their receptor in patients with COPD, and the mitogenic response of human ASM cells to FGFs in vitro suggest a potential role for the FGF/FGFR-1 system in the remodelling of bronchial airways in COPD.     

Insulin inhibits amyloid beta-induced cell death in cultured human brain pericytes

Amyloid-beta (Abeta) deposition in the cerebral arterial and capillary walls is one of the characteristics of Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type. In vitro, Abeta1-40, carrying the "Dutch" mutation (DAbeta1-40), induced reproducible degeneration of cultured human brain pericytes (HBP), by forming fibrils at the cell surface. Thus, this culture system provides an useful model to study the vascular pathology seen in Alzheimer's disease. In this study, we used this model to investigate the effects of insulin on Abeta-induced degeneration of HBP, as it has been mentioned previously that insulin is able to protect neurons against Abeta-induced cell-death. The toxic effect of DAbeta1-40 on HBP was inhibited by insulin in a dose-dependent matter. Insulin interacted with Abeta and inhibited fibril formation of Abeta in a cell-free assay, as well as at the cell surface of HBP. Our data indicate that the formation of a fibril network is essential for Abeta-induced cell death in HBP. Additionally, insulin may be involved in the regulation of Abeta fibrillization in AD.     

Hapten-induced colitis associated with maintained Th1 and inflammatory responses in IFN-gamma receptor-deficient mice

IFN-gamma is a potent pro-inflammatory cytokine thought to be involved in the pathogenesis of Crohn's disease. To further define the role of IFN-gamma in intestinal inflammation, we studied the effects of intra-colonic 2,4,6-trinitrobenzene sulfonic acid (TNBS) instillation in mice with a functionally inactivated IFN-gamma receptor 1 (IFN-gammaR1(- / -)). Our results indicate that IFN-gamma is not necessary for the induction of hapten-induced colitis: after TNBS administration both wild-type and IFN-gammaR1(- / -) mice lost body weight, and the histological features of TNBS-induced colitis were comparable. Colons of IFN-gammaR1(- / -) mice contained a greater number of cells, represented by macrophages and CD4(+) T cells; caudal lymph node cells produced more IFN-gamma and TNF-alpha upon stimulation in vitro. Moreover, IL-18 and IL-12 p40 RNA levels were comparably up-regulated after TNBS treatment in IFN-gammaR1(- / -) wild-type mice. These findings demonstrate that IFN-gamma is dispensable for the development of TNBS-induced colitis. Importantly, the production of Th1 cytokines (e. g. IFN-gamma and TNF-alpha) by caudal lymph node T lymphocytes was enhanced rather than decreased in IFNgammaR1(- / -) mice with no evidence for default Th2 development.     

Different sympathovagal modulation of heart rate during social and nonsocial stress episodes in wild-type rats

The acute consequences of a social aversive stimulus (defeat) on the autonomic control upon the electrical activity of the heart were measured and compared to those observed in three nonsocial stress paradigms, namely restraint, shock-probe test, and swimming. Electrocardiograms were recorded from rats via radiotelemetry, and the autonomic neural control of the heart was evaluated via measures of heart rate and heart rate variability, such as the average R-R interval (RR), the standard deviation of RR (SD), the coefficient of variance (SD/RR), and the root-mean-square of successive R-R interval differences (r-MSSD). Although all stressors induced significant reductions of average R-R interval, the effect of defeat was significantly larger (p < 0.05). The social stimulus also determined a significant decrease in the variability indexes (p < 0.01 for all), whereas in the other stress conditions they were either unchanged or increased (SD/RR during restraint, p < 0.05; SD and SD/RR during swimming, p < 0.05 and p < 0.01). Cardiac arrhythmias (mostly ventricular premature beats, VPBs) were far more frequent during defeat than during the other challenging situations (p < 0.01), with an average of 33.5 +/- 6.5 VPBs per 15-min test recording. These data suggest that during defeat autonomic control was shifted toward a sympathetic dominance, whereas in rats exposed to nonsocial stressors, although significant heart rate accelerations were also found, sympathovagal balance was substantially maintained. These differences in autonomic stress responsivity explain the different susceptibility to ventricular arrhythmias and indicate that a social challenge can be far more detrimental for cardiac electrical stability than other nonsocial aversive stimuli.     

Inheritance of mitochondrial and chloroplast genome markers in backcrosses of Chlamydomonas eugametos x Chlamydomonas moewusii hybrids

Summary Southern blot analysis of AvaI-digested total cellular DNA from the interfertile species Chlamydomonas eugametos and Chlamydomonas moewusii with a coxI mitochondrial gene probe from Chlamydomonas reinhardtii revealed single hybridizing fragments of 5.0 and 3.5 kb, respectively. The transmission of these mitochondrial DNA physical markers along with that of chloroplast genetic markers for resistance to streptomycin and resistance to erythromycin was studied in the fourth backcrosses of F₁ hybrids to one or the other parent. Viability in these backcrosses is high in contrast to the cross C. eugametos x C. moewusii and its reciprocal which are associated with considerable meiotic product lethality. The resulting zygospores were found to transmit the mitochondrial and chloroplast genome markers uniparentally or preferentially from the mating-type-plus parent. Thus the species pair C. eugametos and C. moewusii differs from the pair Chlamydomonas reinhardtii and Chlamydomonas smithii in which mitochondrial genome markers are transmitted uniparentally by the mating-type minus parent, while the chloroplast genome markers are transmitted uniparentally by the opposite parental mating-type (Boynton et al. 1987).     

Pharmacokinetic analysis of pegylated megakaryocyte growth and development factor in humans

Phase I studies with pegylated megakaryocyte growth and development factor (PEG-rHuMGDF), a c-Mpl ligand that stimulates megakaryopoiesis, have demonstrated that PEG-rHuMGDF is biologically active alone and causes a dose-related enhancement of platelet recovery when administered after chemotherapy. Here we report the dose-ranging pharmacokinetics of PEG-rHuMGDF. Pre-injection blood samples were drawn daily for pharmacokinetic studies on 43 patients. An ELISA, established using PEG-rHuMGDF as the standard, was able to quantitate Mpl ligand at concentrations > 0.02 ng/mL. Over the dose range 0.03 to 5.0 microg/kg/day, subcutaneous administration produced linear increases in steady-state serum levels. Maximum levels of PEG-rHuMGDF attained after 5.0 microg/kg/day were 5.88 to 10.9 ng/mL. After discontinuation of PEG-rHuMGDF, concentrations of Mpl ligand returned to baseline within 5 days. The pharmacokinetics were best described by a one-compartment model with first-order absorption, an absorption delay, and non linear clearance over the first 48 hours. The mean terminal half-life was 33.3 + 16.7 hours, and the average apparent at steady state was 27.7 + 14.0 mL/h/kg; both were independent of administered dose. The apparent clearance of PEG-rHuMGDF was not predicted by platelet count. Administration of chemotherapy and Filgrastim did not alter the pharmacokinetics of PEG-rHuMGDF.