Radiologic search for embolized leaflets of prosthetic heart valves: A report of two cases

In a radiologic search for embolized leaflets of Edwards-Duromedics bileaflet valves in 2 patients, the embolized fragments were localized in the iliac vessels using computed tomography. Sonography was successful in one case and standard X-ray films of the abdomen were negative in both cases.In vitro investigations with Björk-Shiley and Edwards-Duromedics leaflets suggested that standard X-ray films of the abdomen and pelvis should be considered as the first investigational technique. If negative, computed tomography of the lower abdomen should be done.     

Mortality in patients with successful initial response to highly active antiretroviral therapy is still higher than in non-HIV-infected individuals

Mortality in HIV-infected patients has decreased dramatically since the introduction of highly active antiretroviral therapy (HAART). We analyzed progression to death in a population of 3678 antiretroviral treatment-naive patients from the ATHENA national observational cohort from 24 weeks after the start of HAART. Mortality was compared with that in the general population in the Netherlands matched by age and gender. Only log-transformed CD4 cell count (hazard ratio [HR] = 0.50, 95% confidence interval [CI]: 0.40 to 0.61 per unit increase) and plasma viral load (HR = 0.30, 95% CI: 0.15 to 0.60, HIV RNA level <100,000 vs. > or = 100,000 copies/mL) measured at 24 weeks and infection via intravenous drug use (IDU) (HR = 0.16, 95% CI: 0.10 to 0.26, non-IDU vs. IDU) were significantly associated with progression to death. For non-IDU patients with 600 x 10 CD4 cells/L and an HIV RNA level <100,000 copies/mL at 24 weeks, mortality was predicted to be 5.3 (95% CI: 3.5 to 8.4) and 10.4 (95% CI: 6.4 to 17.4) times higher than in the general population for 25-year-old men and women, respectively, and 1.15 (95% CI: 1.08 to 1.25) and 1.29 (95% CI: 1.16 to 1.50) times higher for 65-year-old men and women, respectively. Hence, mortality in HIV-infected patients with a good initial response to HAART is still higher than in the general population.     

Identification of a naturally processed cyclin D1 T-helper epitope by a novel combination of HLA class II targeting and differential mass spectrometry

T-helper (Th) cells play an important role in orchestrating the effector function of CTL in anti-tumor immunity. However, only a limited number of Th cell epitopes has been characterized. Here we describe a novel approach for identifying naturally processed and presented peptides derived from chosen antigens. This method combines a transfection step of antigen-presenting cells with a vector encoding a fusion protein between the Ii chain and the antigen of interest, elution of the HLA-bound peptides and identification of the antigen-derived peptides by mass spectrometric comparison to the non-transfected cells. In vitro-stimulated Th cells against the identified peptide of interest specifically recognize transfectants overexpressing the cognate antigen. Using this approach, we were able to identify the HLA-DR4-restricted Th cell epitope NPPSMVAAGSVVAAV derived from cyclin D1, which is frequently overexpressed in tumors. This method will help in identifying peptide candidates for vaccination studies for tumor immunotherapy.     

Long-range DNase I hypersensitivity mapping reveals the imprinted Igf2r and Air promoters share cis-regulatory elements

Epigenetic mechanisms restrict the expression of imprinted genes to one parental allele in diploid cells. At the Igf2r/Air imprinted cluster on mouse chromosome 17, paternal-specific expression of the Air noncoding RNA has been shown to silence three genes in cis: Igf2r, Slc22a2, and Slc22a3. By an unbiased mapping of DNase I hypersensitive sites (DHS) in a 192-kb region flanking Igf2r and Air, we identified 21 DHS, of which nine mapped to evolutionarily conserved sequences. Based on the hypothesis that silencing effects of Air would be directed towards cis regulatory elements used to activate genes, DHS are potential key players in the control of imprinted expression. However, in this 192-kb region only the two DHS mapping to the Igf2r and Air promoters show parental specificity. The remaining 19 DHS were present on both parental alleles and, thus, have the potential to activate Igf2r on the maternal allele and Air on the paternal allele. The possibility that the Igf2r and Air promoters share the same cis-acting regulatory elements, albeit on opposite parental chromosomes, was supported by the similar expression profiles of Igf2r and Air in vivo. These results refine our understanding of the onset of imprinted silencing at this cluster and indicate the Air noncoding RNA may specifically target silencing to the Igf2r promoter.     

Phenotypic characterization of human chondrocyte cell line C-20/A4: a comparison between monolayer and alginate suspension culture

DNA microarray analysis was used to investigate the molecular phenotype of one of the first human chondrocyte cell lines, C-20/A4, derived from juvenile costal chondrocytes by immortalization with origin-defective simian virus 40 large T antigen. Clontech Human Cancer Arrays 1.2 and quantitative PCR were used to examine gene expression profiles of C-20/A4 cells cultured in the presence of serum in monolayer and alginate beads. In monolayer cultures, genes involved in cell proliferation were strongly upregulated compared to those expressed by human adult articular chondrocytes in primary culture. Of the cell cycle-regulated genes, only two, the CDK regulatory subunit and histone H4, were downregulated after culture in alginate beads, consistent with the ability of these cells to proliferate in suspension culture. In contrast, the expression of several genes that are involved in pericellular matrix formation, including MMP-14, COL6A1, fibronectin, biglycan and decorin, was upregulated when the C-20/A4 cells were transferred to suspension culture in alginate. Also, nexin-1, vimentin, and IGFBP-3, which are known to be expressed by primary chondrocytes, were differentially expressed in our study. Consistent with the proliferative phenotype of this cell line, few genes involved in matrix synthesis and turnover were highly expressed in the presence of serum. These results indicate that immortalized chondrocyte cell lines, rather than substituting for primary chondrocytes, may serve as models for extending findings on chondrocyte function not achievable by the use of primary chondrocytes.     

Distinct sites of action of clostridial neurotoxins revealed by double-poisoning of mouse motor nerve terminals

(1) We investigated the effects of single- and double-poisoning with tetanus toxin (TeTx), botulinum neurotoxin type A (BoTx A) and botulinum neurotoxin type B (BoTx B) on spontaneous and nerve-evoked quantal transmitter release at motor endplates of the triangularis sterni preparation of the mouse. (2) Inhibitory effects of TeTx and BoTx B on spontaneous and nerve-evoked transmitter release were very similar, except that the action of BoTx B required 500-fold lower concentrations and was less dependent on temperature. BoTx A caused stronger inhibition of quantal release than TeTx or BoTx B, but was comparatively much easier counteracted by 4-aminopyridine (4-AP). (3) In contrast to BoTx A, with TeTx or BoTx B the increase of transmitter release following onset of 50 Hz nerve stimulation was delayed for a few seconds and synaptic latencies of quanta showed large variations. This release pattern was also evident in all double-poisoning experiments, regardless of intoxication sequence. (4) Inhibition of evoked release was found to be slightly stronger with TeTx than with BoTx B, so the amount of nerve-evoked quanta released after double-poisoning with any sequence of these toxins always approached that of TeTx. In no case supraadditive actions were observed. (5) A strong reduction of evoked quanta was observed when BoTx A was applied in addition to either of the two other toxins. With reversed poisoning sequences (BoTx A-TeTx or BoTx A-BoTx B) the resulting values remained at the extremely low level of BoTx A. (6) In the presence of 4-AP double-poisoning with any combination between BoTx A and TeTx or BoTx B (regardless of intoxication sequence) revealed supra-additive effects, since the number of quanta released was considerably lower than that obtained with any of the toxins alone (in the presence of 4-AP). (7) Our results indicate that tetanus toxin and botulinum toxin type B have a common site of action which is different and independent from that of botulinum toxin type A.This is part of the thesis of M. G. to be presented to the Fachbereich Humanmedizin, Justus-Liebig-Universität Gießen     

Magnetic immuno-PCR assay with inhibitor removal for direct detection of Helicobacter pylori in human feces

A PCR protocol was developed to detect Helicobacter pylori in human stool specimens. This protocol was based on the association of a magnetic immuno-PCR assay with a technique to remove inhibitors (agarose-embedded DNA preparation). Of the 47 H. pylori-positive and 57 H. pylori-negative patients included in this study, 38 were positive and 66 were negative by this new protocol. The sensitivity, specificity, and predictive values for a positive or a negative result were 80.9% (95% confidence interval [CI], 66.3 to 90.4), 100% (95% CI, 92.1 to 100), 100% (95% CI, 88.6 to 100), and 86.4% (95% CI, 75.2 to 93.2), respectively.     

Construction and Characterization of an Isogenic slt-ii Deletion Mutant of Enterohemorrhagic Escherichia coli

Enterohemorrhagic Escherichia coli (EHEC) produces Shiga-like toxins (SLT), potent protein synthesis inhibitors. To further dissect the role of SLT-II in the course of disease, we have constructed E. coli TUV86-2, an isogenic SLT-II-negative mutant of EHEC strain 86-24. The slt-ii gene was inactivated by suicide vector mutagenesis. We also isolated derivatives of strain 86-24 that were cured of the phage carrying the toxin genes.     

Norepinephrine transporter (NET) promoter and 5'-UTR polymorphisms: association analysis in panic disorder

Several biochemical and pharmacological studies suggest that the catecholaminergic system involving the norepinephrine transporter (NET) is relevant for the pathogenesis of panic disorder. Three single nucleotide polymorphisms in the promoter or untranslated 5' region of the NET gene were investigated by means of RFLP analysis in a sample of 115 German patients with panic disorder and 115 matched controls. Statistical analysis failed to show association with the overall diagnosis of panic disorder. In the subgroup of patients with panic disorder without agoraphobia, however, two polymorphisms were found to be associated with the disease (G/C (rs2397771): p < 0.05; T/C (rs2242446): p < 0.01). While our data do not support a major function of the NET gene in the development of panic disorder, it may play a role in the subgroup of panic disorder without agoraphobia.     

Sulfonylurea-sensitive K+ channels and their probable role for the membrane potential of mouse motor nerve endings

We studied the effect of the K_ATP channel blockers tolbutamide and glibenclamide on presynaptic membrane currents in the mouse M. triangularis sterni preparation using the perineural recording technique. Both sulfonylureas blocked part of the fast K⁺ component within 2 min after application. The block was much more pronounced under glucose-free conditions and was completelyreversible by washing. Addition of glucose to glucose-free bath solution also reduced the K⁺ component. A further effect of the sulfonylureas was observed under glucose-free conditions. With a delay of 5 to 10 min, the nodal Na⁺ component began to diminish and disappeared within 30 min. This was associated with a dramatic increase in spontaneous quantal transmitter release suggesting that the block of sulfonylurea-sensitive K⁺ channels causes depolarization of motor nerve terminals and fibres thus inactivating Na⁺ channels. Tetraethylammonium (TEA) which blocks ATP-dependent K⁺ channels in high concentrations caused, under glucose-free conditions, the same delayed effect as the sulfonylureas. This delayed effect was fully reversible by washing with glucose-containing, but not with glucose-free solution. Our findings strongly suggest that K_ATP channels exist in mammalian motor nerve endings and that under hypoglycemic conditions these channels open and become essential for the maintenance of the membrane potential.