Association of interacting genes in the toll-like receptor signaling pathway and the antibody response to pertussis vaccination

Background

Activation of the Toll-like receptor (TLR) signaling pathway through TLR4 may be important in the induction of protective immunity against Bordetella pertussis with TLR4-mediated activation of dendritic and B cells, induction of cytokine expression, and reversal of tolerance as crucial steps. We examined whether single nucleotide polymorphisms (SNPs) in genes of the TLR4 pathway and their interaction are associated with the response to whole-cell vaccine (WCV) pertussis vaccination in 490 one-year-old children.

Methodology/Principal Findings

We analyzed associations of 75 haplotype-tagging SNPs in genes in the TLR4 signaling pathway with pertussis toxin (PT)-IgG titers. We found significant associations between the PT-IgG titer and SNPs in CD14, TLR4, TOLLIP, TIRAP, IRAK3, IRAK4, TICAM1, and TNFRSF4 in one or more of the analyses. The strongest evidence for association was found for two SNPs (rs5744034 and rs5743894) in TOLLIP that were almost completely in linkage disequilibrium, provided statistically significant associations in all tests with the lowest p-values, and displayed a dominant mode of inheritance. However, none of these single gene associations would withstand correction for multiple testing. In addition, Multifactor Dimensionality Reduction Analysis, an approach that does not need correction for multiple testing, showed significant and strong two and three locus interactions between SNPs in TOLLIP (rs4963060), TLR4 (rs6478317) and IRAK1 (rs1059703).

Conclusions/Significance

We have identified significant interactions between genes in the TLR pathway in the induction of vaccine-induced immunity. These interactions underline that these genes are functionally related and together form a true biological relationship in a protein-protein interaction network. Practically all our findings may be explained by genetic variation in directly or indirectly interacting proteins at the extra- and intracytoplasmic sites of the cell membrane of antigen-presenting cells, B cells, or both. Fine tuning of interacting proteins in the TLR pathway appears important for the induction of an optimal vaccine response.     

Long-term effects of graduated compression stockings on cardiorespiratory performance

The use of graduated compression stockings (GCS) in sport has been increasing in the last years due to their potential positive effects for athletes. However, there is little evidence to support whether these types of garments actually improve cardiorespiratory performance. The aim of this study was to examine the cardiorespiratory responses of GCS during running after three weeks of regular use. Twenty recreational runners performed three tests on different days: test 1) – a 5-min maximal effort run in order to determine the participants’ maximal aerobic speed; and tests 2) and 3) – a fatigue running test of 30 minutes at 80% of their maximal aerobic speed with either GCS or PLACEBO stockings at random. Cardiorespiratory parameters (minute ventilation, heart rate, relative oxygen consumption, relative carbon dioxide production, ventilatory equivalents for oxygen and carbon dioxide, and oxygen pulse) were measured. Before each test in the laboratory, the participants trained with the randomly assigned stockings (GCS or PLACEBO) for three weeks. No significant differences between GCS and PLACEBO were found in any of the cardiorespiratory parameters. In conclusion, the present study provides evidence that running with GCS for three weeks does not influence cardiorespiratory parameters in recreational runners.     

1,25-Dihydroxyvitamin D3 inhibits ultraviolet B-induced apoptosis,Jun kinase activation,and interleukin-6 production in primary human keratinocytes

We investigated the capacity of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] to protect human keratinocytes against the hazardous effects of ultraviolet B (UVB)-irradiation, recognized as the most important etiological factor in the development of skin cancer. Cytoprotective effects of 1,25(OH)(2)D(3) on UVB-irradiated keratinocytes were seen morphologically and quantified using a colorimetric survival assay. Moreover, 1,25(OH)(2)D(3) suppressed UVB-induced apoptotic cell death. An ELISA, detecting DNA-fragmentation, demonstrated that pretreatment of keratinocytes with 1,25(OH)(2)D(3) 1 microM for 24 h reduced UVB-stimulated apoptosis by 55-70%. This suppression required pharmacological concentrations 1,25(OH)(2)D(3) and a preincubation period of several hours. In addition, 1,25(OH)(2)D(3) also inhibited mitochondrial cytochrome c release (90%), a hallmark event of UVB-induced apoptosis. Furthermore, we demonstrated that 1,25(OH)(2)D(3) reduced two important mediators of the UV-response, namely, c-Jun-NH(2)-terminal kinase (JNK) activation and interleukin-6 (IL-6) production. As shown by Western blotting, pretreatment of keratinocytes with 1,25(OH)(2)D(3) 1 microM diminished UVB-stimulated JNK activation with more than 30%. 1,25(OH)(2)D(3) treatment (1 microM) reduced UVB-induced IL-6 mRNA expression and secretion with 75-90%. Taken together, these findings suggest the existence of a photoprotective effect of active vitamin D(3) and create new perspectives for the pharmacological use of active vitamin D compounds in the prevention of UVB-induced skin damage and carcinogenesis.     

Lethal and sublethal costs of autotomy and predator presence in damselfly larvae

We studied the costs of lamellae autotomy with respect to growth and survival of Lestes sponsa damselfly larvae in field experiments. We manipulated predation risk by Aeshna cyanea dragonfly larvae and lamellae status of L. sponsa larvae in field enclosures and compared differences in numbers, size and mass of survivors among treatments. In the absence of a free-ranging A. cyanea larva, about 29% of the L. sponsa larvae died. This was probably due to cannibalism. The presence of a free-ranging A. cyanea reduced larval survival by 68% compared to treatments in which it was absent or not permitted to forage on L. sponsa damselflies. Across all predator treatments, lamellae autotomy reduced survival by about 20%. The mean head width and mass of survivors was lower in the enclosures with a free-ranging A. cyanea compared to the other two predator treatments. This suggested that larvae grew less in the presence of a free-ranging predator, indicating that increased antipredator behaviours were more important in shaping growth responses than reduced population density. Mass, but not head width, of survivors was also reduced after autotomy. The fitness consequences of these effects for the adults may be pronounced. In general, these field data strongly suggest that lamellae autotomy affects population regulation of damselflies. Received: 1 February 1999 / Accepted: 22 March 1999     

Synthesis, characterization and inhibitory potency of two oxono and thiono analogues of phosphoramidate compounds on acetylcholinesterase

Two novel structurally related phosphoramidate compounds, 1 and 2, with likely beta-diketone system were synthesized and characterized by 1H, 13C, 31P NMR, IR spectroscopy and elemental analysis. Compound 2 exhibited a 31P NMR signal which was significantly shielded (8 ppm) relative to compound 1. Determination of human erythrocyte acetylcholinesterase (hAChE) inhibitory activity was carried out according to Ellman's modified kinetic method and the IC50 values of compounds 1 and 2 were 1.567 and 2.986 mM, respectively. The k(i) values of 1 and 2 were 1.39 to 2.65 min(-1) respectively. A comparison of the bimolecular rate constant (k(i)) and IC50 values for the irreversible inhibitors 1 and 2 revealed that the oxono analogue has greater affinity for hAChE than the thiono compound. Furthermore effects of two conventional oximes paralidoxime (A) and obidoxime (B) on reactivation of the inhibited hAChE were studied but low reactivity was shown by both the oximes.     

Adaptation of Mycobacterium smegmatis to Stationary Phase

Mycobacterium tuberculosis can persist for many years within host lung tissue without causing clinical disease. Little is known about the state in which the bacilli survive, although it is frequently referred to as dormancy. Some evidence suggests that cells survive in nutrient-deprived stationary phase. Therefore, we are studying stationary-phase survival of Mycobacterium smegmatis as a model for mycobacterial persistence. M. smegmatis cultures could survive 650 days of either carbon, nitrogen, or phosphorus starvation. In carbon-limited medium, cells entered stationary phase before the carbon source (glycerol) had been completely depleted and glycerol uptake from the medium continued during the early stages of stationary phase. These results suggest that the cells are able to sense when the glycerol is approaching limiting concentrations and initiate a shutdown into stationary phase, which involves the uptake of the remaining glycerol from the medium. During early stationary phase, cells underwent reductive cell division and became more resistant to osmotic and acid stress and pool mRNA stabilized. Stationary-phase cells were also more resistant to oxidative stress, but this resistance was induced during late exponential phase in a cell-density-dependent manner. Upon recovery in fresh medium, stationary-phase cultures showed an immediate increase in protein synthesis irrespective of culture age. Colony morphology variants accumulated in stationary-phase cultures. A flat colony variant was seen in 75% of all long-term-stationary-phase cultures and frequently took over the whole population. Cryo scanning electron microscopy showed that the colony organization was different in flat colony strains, flat colonies appearing less well organized than wild-type colonies. Competition experiments with an exponential-phase-adapted wild-type strain showed that the flat strain had a competitive advantage in stationary phase, as well a providing evidence that growth and cell division occur in stationary-phase cultures of M. smegmatis. These results argue against stationary-phase M. smegmatis cultures entering a quiescent state akin to dormancy but support the idea that they are a dynamic population of cells.     

Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30 kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.     

AP-3 Mediates Tyrosinase but Not TRP-1 Trafficking in Human Melanocytes

Patients with Hermansky-Pudlak syndrome type 2 (HPS-2) have mutations in the beta 3A subunit of adaptor complex-3 (AP-3) and functional deficiency of this complex. AP-3 serves as a coat protein in the formation of new vesicles, including, apparently, the platelet's dense body and the melanocyte's melanosome. We used HPS-2 melanocytes in culture to determine the role of AP-3 in the trafficking of the melanogenic proteins tyrosinase and tyrosinase-related protein-1 (TRP-1). TRP-1 displayed a typical melanosomal pattern in both normal and HPS-2 melanocytes. In contrast, tyrosinase exhibited a melanosomal (i.e., perinuclear and dendritic) pattern in normal cells but only a perinuclear pattern in the HPS-2 melanocytes. In addition, tyrosinase exhibited a normal pattern of expression in HPS-2 melanocytes transfected with a cDNA encoding the beta 3A subunit of the AP-3 complex. This suggests a role for AP-3 in the normal trafficking of tyrosinase to premelanosomes, consistent with the presence of a dileucine recognition signal in the C-terminal portion of the tyrosinase molecule. In the AP-3-deficient cells, tyrosinase was also present in structures resembling late endosomes or multivesicular bodies; these vesicles contained exvaginations devoid of tyrosinase. This suggests that, under normal circumstances, AP-3 may act on multivesicular bodies to form tyrosinase-containing vesicles destined to fuse with premelanosomes. Finally, our studies demonstrate that tyrosinase and TRP-1 use different mechanisms to reach their premelanosomal destination.