Divergent responses of different endothelial cell types to infection with Candida albicans and Staphylococcus aureus

Endothelial cells are important in the pathogenesis of bloodstream infections caused by Candida albicans and Staphylococcus aureus. Numerous investigations have used human umbilical vein endothelial cells (HUVECs) to study microbial-endothelial cell interactions in vitro. However, the use of HUVECs requires a constant supply of umbilical cords, and there are significant donor-to-donor variations in these endothelial cells. The use of an immortalized endothelial cell line would obviate such difficulties. One candidate in this regard is HMEC-1, an immortalized human dermal microvascular endothelial cell line. To determine if HMEC-1 cells are suitable for studying the interactions of C. albicans and S. aureus with endothelial cells in vitro, we compared the interactions of these organisms with HMEC-1 cells and HUVECs. We found that wild-type C. albicans had significantly reduced adherence to and invasion of HMEC-1 cells as compared to HUVECs. Although wild-type S. aureus adhered to and invaded HMEC-1 cells similarly to HUVECs, an agr mutant strain had significantly reduced invasion of HMEC-1 cells, but not HUVECs. Furthermore, HMEC-1 cells were less susceptible to damage induced by C. albicans, but more susceptible to damage caused by S. aureus. In addition, HMEC-1 cells secreted very little IL-8 in response to infection with either organism, whereas infection of HUVECs induced substantial IL-8 secretion. This weak IL-8 response was likely due to the anatomic site from which HMEC-1 cells were obtained because infection of primary human dermal microvascular endothelial cells with C. albicans and S. aureus also induced little increase in IL-8 production above basal levels. Thus, C. albicans and S. aureus interact with HMEC-1 cells in a substantially different manner than with HUVECs, and data obtained with one type of endothelial cell cannot necessarily be extrapolated to other types.     

Combination of gastrin-releasing peptide antagonist with cytotoxic agents produces synergistic inhibition of growth of human experimental colon cancers

We investigated the efficacy of a powerful antagonist of bombesin/gastrin-releasing peptide (BN/GRP) RC-3940-II administered as a single agent or in combination with cytotoxic agents on the growth of HT-29, HCT-116 and HCT-15 human colon cancer in vitro and in vivo. GRP-receptor mRNA and protein were found in all three cell lines tested. Exposure of HT-29 cells to 10 μM RC-3940-II led to an increase in the number of cells blocked in S phase and G₂/M and cells with lower G₀/G₁ DNA content. Similar changes on the cell cycle traverse of HT-29 cells could also be seen at lower concentrations of RC-3940-II (1 μM) after pretreatment with 100 nM GRP (14–27), indicating a dose-dependent mechanism of action based on the blockage of BN/GRP induced proliferation of tumor cells at lower concentrations. Daily in vivo treatment with BN/GRP antagonist RC-3940-II decreased the volume of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic nude mice by 25 to 67% (p < 0.005). Combined treatment with RC-3940-II and chemotherapeutic agents 5-FU and irinotecan resulted in a synergistic tumor growth suppression of HT-29, HCT-116 and HCT-15 xenografts by 43% to 78%. In HT-29 and HCT-116 xenografts the inhibition for the combinations of RC-3940-II and irinotecan vs. single substances (p < 0.05) was significantly greater. These findings support the use of RC-3940-II as an anticancer agent and may help to design clinical trials using RC-3940-II in combinations with cytotoxic agents.     

Somatic progenitor cell vulnerability to mitochondrial DNA mutagenesis underlies progeroid phenotypes in Polg mutator mice

Somatic stem cell (SSC) dysfunction is typical for different progeroid phenotypes in mice with genomic DNA repair defects. MtDNA mutagenesis in mice with defective Polg exonuclease activity also leads to progeroid symptoms, by an unknown mechanism. We found that Polg-Mutator mice had neural (NSC) and hematopoietic progenitor (HPC) dysfunction already from embryogenesis. NSC self-renewal was decreased in vitro, and quiescent NSC amounts were reduced in vivo. HPCs showed abnormal lineage differentiation leading to anemia and lymphopenia. N-acetyl-L-cysteine treatment rescued both NSC and HPC abnormalities, suggesting that subtle ROS/redox changes, induced by mtDNA mutagenesis, modulate SSC function. Our results show that mtDNA mutagenesis affected SSC function early but manifested as respiratory chain deficiency in nondividing tissues in old age. Deletor mice, having mtDNA deletions in postmitotic cells and no progeria, had normal SSCs. We propose that SSC compartment is sensitive to mtDNA mutagenesis, and that mitochondrial dysfunction in SSCs can underlie progeroid manifestations.     

Intracranial aneurysm risk locus 5q23.2 is associated with elevated systolic blood pressure

Although genome-wide association studies (GWAS) have identified hundreds of complex trait loci, the pathomechanisms of most remain elusive. Studying the genetics of risk factors predisposing to disease is an attractive approach to identify targets for functional studies. Intracranial aneurysms (IA) are rupture-prone pouches at cerebral artery branching sites. IA is a complex disease for which GWAS have identified five loci with strong association and a further 14 loci with suggestive association. To decipher potential underlying disease mechanisms, we tested whether there are IA loci that convey their effect through elevating blood pressure (BP), a strong risk factor of IA. We performed a meta-analysis of four population-based Finnish cohorts (n(FIN) = 11 266) not selected for IA, to assess the association of previously identified IA candidate loci (n = 19) with BP. We defined systolic BP (SBP), diastolic BP, mean arterial pressure, and pulse pressure as quantitative outcome variables. The most significant result was further tested for association in the ICBP-GWAS cohort of 200 000 individuals. We found that the suggestive IA locus at 5q23.2 in PRDM6 was significantly associated with SBP in individuals of European descent (p(FIN) = 3.01E-05, p(ICBP-GWAS) = 0.0007, p(ALL) = 8.13E-07). The risk allele of IA was associated with higher SBP. PRDM6 encodes a protein predominantly expressed in vascular smooth muscle cells. Our study connects a complex disease (IA) locus with a common risk factor for the disease (SBP). We hypothesize that common variants in PRDM6 can contribute to altered vascular wall structure, hence increasing SBP and predisposing to IA. True positive associations often fail to reach genome-wide significance in GWAS. Our findings show that analysis of traditional risk factors as intermediate phenotypes is an effective tool for deciphering hidden heritability. Further, we demonstrate that common disease loci identified in a population isolate may bear wider significance.     

Enhanced behavioral sensitivity to the competitive GABA agonist, gaboxadol, in transgenic mice over-expressing hippocampal extrasynaptic alpha6beta GABA(A) receptors

The behavioral and functional significance of the extrasynaptic inhibitory GABA(A) receptors in the brain is still poorly known. We used a transgenic mouse line expressing the GABA(A) receptor alpha6 subunit gene in the forebrain under the Thy-1.2 promoter (Thy1alpha6) mice ectopically expressing alpha6 subunits especially in the hippocampus to study how extrasynaptically enriched alphabeta(gamma2)-type receptors alter animal behavior and receptor responses. In these mice extrasynaptic alpha6beta receptors make up about 10% of the hippocampal GABA(A) receptors resulting in imbalance between synaptic and extrasynaptic inhibition. The synthetic GABA-site competitive agonist gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol; 3 mg/kg) induced remarkable anxiolytic-like response in the light : dark exploration and elevated plus-maze tests in Thy1alpha6 mice, while being almost inactive in wild-type mice. The transgenic mice also lost quicker and for longer time their righting reflex after 25 mg/kg gaboxadol than wild-type mice. In hippocampal sections of Thy1alpha6 mice, the alpha6beta receptors could be visualized autoradiographically by interactions between gaboxadol and GABA via [(35)S]TBPS binding to the GABA(A) receptor ionophore. Gaboxadol inhibition of the binding could be partially prevented by GABA. Electrophysiology of recombinant GABA(A) receptors revealed that GABA was a partial agonist at alpha6beta3 and alpha6beta3delta receptors, but a full agonist at alpha6beta3gamma2 receptors when compared with gaboxadol. The results suggest strong behavioral effects via selective pharmacological activation of enriched extrasynaptic alphabeta GABA(A) receptors, and the mouse model represents an example of the functional consequences of altered balance between extrasynaptic and synaptic inhibition.     

Exposure and mortality in a cohort of German nuclear power workers

A historical cohort study of German nuclear power workers was set up to investigate the overall and cancer mortality risk related to a long-term low-level exposure to ionising radiation. The cohort was part of an international collaborative study whose pooled analyses were carried out at the International Agency for Research on Cancer (IARC), Lyon, and published recently. Due to delays in data collection, data from the German cohort were not included in these analyses. This cohort includes 4,844 employees who worked in any of 10 nuclear power plants, between 1 January 1991 and 31 December 1997. Sixty-eight deaths among men were observed in 31,000 person years, and none among women. Standardized mortality ratios (SMRs) were computed for all causes of death, all cancers, cardiovascular diseases, external causes, and all other causes. Overall, a strong healthy worker effect was observed (all-cause SMR = 0.54, 95% CI 0.42–0.67), and no increase in total cancer mortality was seen (SMR = 0.66, 95% CI 0.43–0.95). These results are in line with and complement IARC results. Figures are yet too small for stable risk estimates and further work is therefore under way to include more power plants, and to extend the follow-up until the year 2005.     

Are Polyommatus hispana and Polyommatus slovacus bivoltine Polyommatus coridon (Lepidoptera: Lycaenidae)? The discriminatory value of genetics in taxonomy

Taxonomic confusion exists in several sibling species groups. The Polyommatus coridon species complex (Chalk Hill Blues) serves as a model group of sibling species in which genetic analyses provide suitable means for taxonomic clarification. We studied the allozyme patterns of the two described bivoltine species of this complex, Polyommatus hispana and Polyommatus slovacus, and compared them to the two genetic lineages of the univoltine P. coridon. P. hispana is well distinguished from P. coridon (genetic distance: 0.081), and most probably is a sibling species that has evolved during glacial isolation on the Iberian Peninsula. P. slovacus is genetically indistinguishable from the eastern, Pontic–Mediterranean lineage of P. coridon; therefore we suggest that it represents a local bivoltine population only. Since the spring generation of P. slovacus was much less common than the summer generation and showed less genetic diversity, it is probable that uni-/bivoltinism is a dimorphism affecting only part of the whole population. We suggest that the higher genetic diversity of the second generation may be a consequence of gene flow from adjacent single-brooded populations.     

Substantial Rise in the Prevalence of Lyme Borreliosis Spirochetes in a Region of Western Germany over a 10-Year Period

More than a decade after a study on the transmission cycle of Borrelia burgdorferi sensu lato in the Siebengebirge, a nature reserve near Bonn, Germany, questing nymphal and adult Ixodes ricinus ticks were collected again in three selected areas of the same low mountain range and examined for infection with B. burgdorferi sensu lato. Between May and October 2001, a total of 1,754 ticks were collected by blanket dragging; 374 ticks were analyzed for B. burgdorferi sensu lato by both an immunofluorescence assay (IFA) and at least two different PCR tests, whereas 171 ticks were analyzed by PCR only. By combining all assays, an average of 14% of the ticks tested positive for B. burgdorferi sensu lato, 5.5, 15.8, and 21.8% in the three collection areas. Of the nymphs and adults examined, 12.9 and 21.1%, respectively, were found to be spirochete infected. A lower total infection prevalence was obtained by IFA (14.4%) than by a nested PCR approach (16.5%), but both were higher than that obtained by a simple PCR approach (11.9%). Compared with data collected over a decade ago, the mean infection prevalence of B. burgdorferi sensu lato in the ticks was significantly higher for all three biotopes, whereas a similar pattern of habitat-specific infection prevalence was observed. Genotyping of B. burgdorferi sensu lato revealed high relative prevalences of B. valaisiana (identified in 43.1% of infected ticks) and B. garinii (32.3%), whereas B. afzelii (12.3%) and B. burgdorferi sensu stricto (1.5%) were relatively rare. We conclude that B. burgdorferi sensu lato infection has increased in this region over the last 15 years due to presently unknown changes in ecological conditions, perhaps related to climate change or wildlife management.     

Water-borne seed transport and seed deposition during flooding in a small river-valley in Northern Germany

Water-borne seed transport and seed deposition during flooding were studied in the Upper Eider river (N-Germany) by direct sampling of the rivers seed content with aquatic seed traps and by analysing the number of deposited seeds on sedimentation mats which were exposed near the river on the soil surface during a flooding period of approx. three weeks.The number of seeds which were transported at the surface of the river Eider was continuously analysed by four aquatic seed traps for a period of 20 weeks (July–December 2000). To test the capture rate of these traps, a recapture experiment with colour marked seeds of Helianthus annuus L. was carried out. During the investigation period approx. 9000 seeds of 76 species were captured by the four aquatic seed traps. The number of trapped seeds varied both spatially (across the river profile) and temporally. Considering this variation and the capture rate of the traps, the water-borne seed transport was estimated to be 3139 seeds per week and meter of the river profile.The seed deposition during a flood in early spring 2002 was analysed by using 20 sedimentation mats. To distinguish effects of seed dispersal into patches from outside from seed rearrangement within patches, the water-borne seed transport was excluded from one half of the mats by fencing them with a woven fabric which was permeable for water but not for floating seeds. Outside of the exclosures 152 viable seeds of 26 species were deposited on the sedimentation mats while only one single seedling was found on mats from which water-borne seed transport was excluded.The results demonstrate that hydrochorous dispersal processes might play an important role in connecting otherwise fragmented populations in periodically flooded habitats along rivers.