The phosphatidylinositol 3-kinase/protein kinase B signaling pathway is activated by lipoteichoic acid and plays a role in Kupffer cell production of interleukin-6 (IL-6) and IL-10

Sepsis caused by gram-positive bacteria lacking lipopolysaccharide (LPS) has become a major and increasing cause of mortality in intensive-care units. We have recently demonstrated that the gram-positive-specific bacterial cell wall component lipoteichoic acid (LTA) stimulates the release of the proinflammatory cytokines in Kupffer cells in culture. In the present study, we have started to assess the signal transduction events by which LTA induces the production of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and the anti-inflammatory cytokine IL-10 in rat Kupffer cells. LTA was found to trigger phosphorylation of mitogen-activated protein kinases (MAPK) (p38 MAPK and ERK 1/2) and protein kinase B (PKB). Compared to LPS, LTA was more potent in inducing PKB phosphorylation after 40 min, although we found that the cytokine responses were similar. For both bacterial molecules, blocking phosphatidylinositol 3-kinase (PI3-K; Ly294002) or Janus kinase 2 (JAK-2; AG490) particularly affected the induction of IL-6 and IL-10 release, whereas TNF-alpha levels were strongly reduced by inhibition of Src family tyrosine kinases (PP2). All three cytokines were reduced by inhibition of p38 MAPK (SB202190) or the broad-range tyrosine kinase inhibitor genistein, whereas IL-6 release was particularly blocked by inhibition of ERK 1/2 (PD98059). Divergences in the regulatory pathways controlling TNF-alpha, IL-10, and IL-6 production in Kupffer cells following LPS or LTA stimulation may create a basis for understanding how the balance between pro- and anti-inflammatory cytokines is regulated in the liver following infections by gram-positive or gram-negative bacteria.     

Fertilization and early embryology: Effects of persistent chlorinated hydrocarbons on fertility and embryonic development in the rabbit

The commercial polychlorinated biphenyl (PCB) formulation Aroclor1260 (4 mg/kg body weight), technical grade dichlorodiphenyltrichloroethane(DDT; 3 mg) and Lindane (-hexachlorocyclohexane; 0.8 mg) wereadministered orally, either separately or in combination, tosexually mature female rabbits three times per week for 12–15weeks. Oviductal and uterine luminal fluid, cleavage stage embryos(day 1 post coitum), blastocysts (day 6), fetuses, exocoelicfluid and placentae (day 11) were analysed, firstly for chlorinatedhydrocarbon residues, and secondly for embryonic and fetal development.The doses applied were well tolerated by the treated animals.PCB and DDT accumulated in uterine secretions (day 6) but notin oviductal luminal fluid (day 1). Both chlorinated hydrocarbonswere found in preimplantation blastocysts. Residues in day 11fetuses were 16- (DDT) or 18-fold (PCB) higher than in day 6blastocysts. Significant amounts were also detected in placentaltissue and in exocoelic fluid. A specific accumulation of thehighly chlorinated biphenyl congener no. 180 was noted in fetuses,placentae and exocoelic fluid. The clear accumulation of thechlorinated hydrocarbon compounds in luminal fluid and embryonictissue is contrasted by rather weak effects on fertility. Nostatistically significant differences between treated animalsand controls were observed for fertilization rate and pre- andpost-implantation (up to day 11 post coitum) losses. However,in females exposed to PCB, a 20% higher loss of blastocystswas noticed, as compared with controls (P > 0.05). This effectwas shown on day 6 of embryonic development and may be due tothe embryotoxic activities of PCB.     

Modulating parameters of excitability during and after transcranial direct current stimulation of the human motor cortex

Weak transcranial direct current stimulation (tDCS) of the human motor cortex results in excitability shifts which occur during and after stimulation. These excitability shifts are polarity-specific with anodal tDCS enhancing excitability, and cathodal reducing it. To explore the origin of this excitability modulation in more detail, we measured the input–output curve and motor thresholds as global parameters of cortico-spinal excitability, and determined intracortical inhibition and facilitation, as well as facilitatory indirect wave (I-wave) interactions. Measurements were performed during short-term tDCS, which elicits no after-effects, and during other tDCS protocols which do elicit short- and long-lasting after-effects. Resting and active motor thresholds remained stable during and after tDCS. The slope of the input–output curve was increased by anodal tDCS and decreased by cathodal tDCS. Anodal tDCS of the primary motor cortex reduced intracortical inhibition and enhanced facilitation after tDCS but not during tDCS. Cathodal tDCS reduced facilitation during, and additionally increased inhibition after its administration. During tDCS, I-wave facilitation was not influenced but, for the after-effects, anodal tDCS increased I-wave facilitation, while cathodal tDCS had only minor effects. These results suggest that the effect of tDCS on cortico-spinal excitability during a short period of stimulation (which does not induce after-effects) primarily depends on subthreshold resting membrane potential changes, which are able to modulate the input-output curve, but not motor thresholds. In contrast, the after-effects of tDCS are due to shifts in intracortical inhibition and facilitation, and at least partly also to facilitatory I-wave interaction, which is controlled by synaptic activity.     

Clara cell protein 16 (CC16) gene polymorphism influences the degree of airway responsiveness in asthmatic children

BACKGROUND: Several studies have indicated linkage of chromosome 11q12-13 to asthma and associated traits. Among other candidate genes, the Clara cell protein 16 (CC16) gene maps to this region. CC16 is expressed in the bronchial epithelium and exhibits potent anti-inflammatory properties. A single-nucleotide polymorphism (SNP) in the CC16 gene (A38G) was previously associated with asthma. OBJECTIVE: We evaluated the role of the CC16 SNP in pediatric asthma and asthma severity in 2 German study populations. METHODS: The German Multicenter Allergy Study (MAS) cohort (n = 872, 94 asthmatic patients) and 112 allergic asthmatic children recruited in Freiburg, Germany, were included in the present study. Histamine provocations were performed at the age of 7 years in the MAS cohort to determine bronchial hyperreactivity; in the Freiburg study population a standardized exercise-induced decrease in FEV1 was evaluated. For genotyping, melting-curve analysis and restriction enzyme digestion were applied. RESULTS: No association of the CC16*38A allele with asthma could be observed in either study population. However, in asthmatic subjects (MAS cohort) PC(20)FEV(1) values were significantly lower in individuals homozygous or heterozygous for the CC16*38A allele compared with those in subjects with the CC16*38GG genotype (P <.05 and P <.03, respectively). Similarly, allergic asthmatic patients in the Freiburg cohort showed a significantly greater decrease in FEV1 after exercise when homozygous for the CC16*38A allele compared with that seen in asthmatic patients with the *38AG or *38GG genotype (P <.04 and P =.006, respectively). CONCLUSION: We conclude that the CC16*A38G SNP influences bronchial hyperreactivity and might be a genetic determinant of asthma severity in German children.     

Laser microdissection and pressure catapulting (LMPC) in paraffin sections mounted on glass slides. A methodological report

The technique of laser microdissection together with laser pressure catapulting (LMPC) is demonstrated in paraffin sections obtained from surgical specimens of brain tumors mounted on glass slides. A sufficient and precise application of microdissection techniques in tissue on glass slides is worthwhile, since it offers the possibility of a retrospective analysis of archived paraffin sections in histopathology. We could demonstrate a precise dissection of areas in tissues of different thicknesses (4 microm and 20 microm). Areas of tissue mounted directly on glass need to be dissected in a scanning mode in order to remove the total region in form of small tissue fragments row by row. This mode provided a precise microdissection of tissue areas of different sizes and shapes. A successful molecular biological analysis of the microdissected regions could be demonstrated. As an example for such an analysis, differential-PCR for detecting an amplification of the gene for the epidermal growth factor receptor (EGFR) was performed.