Time dependency of CSF cell count, lactate and blood-CSF barrier dysfunction after epileptic seizures and status epilepticus.

BACKGROUND: This retrospective observational study was conducted to examine the temporal relationship between increased cell count, lactate concentration in cerebrospinal fluid (CSF) and blood-CSF barrier dysfunction and the onset of a seizure event. METHODS: Patients with a seizure event who underwent lumbar puncture for CSF analysis during diagnostic work-up (interindividual analysis) and those with at least one follow-up CSF analysis (intraindividual analysis) were studied. Pathologically altered parameters, such as cell count, lactate concentration, and blood-CSF barrier dysfunction as indicated by the albumin quotient (QAlb=CSF albumin/serum albumin), were examined with regard to the changes over time after seizure onset. RESULTS: An increased CSF cell count (>4/µl) was shown in 3% of our patients, whereas pathological lactate concentrations were found in 24% after single seizures and 28% after status epilepticus (SE)/recurring seizures. However, lactate levels showed a marked decrease with increasing time after an isolated seizure (p<0.0001) but not after SE/recurring seizures. Lactate levels were most frequently and significantly elevated within the first six hours after a single seizure (p<0.0001). Blood-CSF barrier dysfunction was detected in 34% after isolated seizures and in 47% after SE/recurrent seizures. Blood-CSF barrier dysfunction showed no association with latency between seizure onset and time of CSF collection. CONCLUSIONS: Changes in lactate and CSF protein concentrations are common after epileptic seizures. In contrast, CSF pleocytosis is uncommon and should prompt careful investigation for the presence of intrathecal infection or autoimmune CNS disease. Elevated lactate levels more than 6 h after the seizure event may indicate ongoing epileptic activity.

Performance of induction module cavity ring-down spectroscopy (IM-CRDS) for measuring δ18 O and δ2 H values of soil, stem, and leaf waters.

RATIONALE: Induction module cavity ring-down spectroscopy (IM-CRDS) has been proposed as a rapid and cost-effective alternative to cryogenic vacuum distillation (CVD) and isotope ratio mass spectrometry (IRMS) for the measurement of δ18 O and δ2 H values in matrix-bound waters. In the current study, we characterized the performance of IM-CRDS relative to CVD and IRMS and investigated the mechanisms responsible for differences between the methods. METHODS: We collected a set of 75 soil, stem, and leaf water samples, and measured the δ18 O and δ2 H values of each sample with four techniques: CVD and IRMS, CVD and CRDS, CVD and IM-CRDS, and IM-CRDS alone. We then calculated the isotopic errors for each of the three CRDS methods relative to CVD and IRMS, and analyzed the relationships among these errors and suites of diagnostic spectral parameters that are indicative of organic contamination. RESULTS: The IM-CRDS technique accurately assessed the δ18 O and δ2 H values of pure waters, but exhibited progressively increasing errors for soil waters, stem waters, and leaf waters. For soils, the errors were attributable to subsampling of isotopically heterogeneous source material, whereas for stems and leaves, they were attributable to spectral interference. Unexpectedly, the magnitude of spectral interference was higher for the solid samples analyzed directly via IM-CRDS than for those originally extracted via CVD and then analyzed by IM-CRDS. CONCLUSIONS: There are many types of matrix-bound water samples for which IM-CRDS measurements include significant errors from spectral interference. As a result, spectral analysis and validation should be incorporated into IM-CRDS post-processing procedures. In the future, IM-CRDS performance could be improved through: (i) identification of the compounds that cause spectral interference, and either (ii) modification of the combustion step to completely oxidize these compounds to CO2 , and/or (iii) incorporation of corrections for these compounds into the spectral fitting models used by the CRDS analyzers. Copyright © 2016 John Wiley & Sons, Ltd.

A micro RNA-146a polymorphism is associated with coronary restenosis.

The association of the miRNA-146a polymorphism rs2910164 with atherosclerosis and restenosis was investigated. We found no association with atherosclerosis; however, we found a negative association for the G/C (P = 0.007) and a positive association for the C/C genotype with the risk of restenosis, which is the main drawback for cardiac surgery.

Association of vitamin D receptor gene polymorphisms with severe atopic dermatitis in adults.

BACKGROUND: Vitamin D mediates immunomodulatory functions, and beneficial functions in allergic diseases have been suggested. Vitamin D receptor gene (VDR) polymorphisms are known but have not been studied in patients with atopic dermatitis (AD). OBJECTIVES: To investigate the frequency of four common VDR gene polymorphisms in patients with AD, and their potential functional relevance. METHODS: In this case-control study, 265 patients with AD [n=142 severe AD, Scoring AD index (SCORAD) > 40; n=123 moderate AD, SCORAD 15-40] and 265 healthy controls were genotyped for four common VDR gene polymorphisms by restriction fragment length polymorphism analysis. The VDR haplotype sequences were analysed in silico. Baseline and activation-induced gene expression of VDR and the vitamin D metabolizing enzyme CYP24A1 were analysed in monocytes of homozygous VDR haplotype carriers by quantitative reverse transcription-polymerase chain reaction. RESULTS: In patients with severe AD, the VDR BsmI (rs1544410) G allele, ApaI (rs7975232) C allele and TaqI (rs731236) T alleles were over-represented compared with healthy controls. These single nucleotide polymorphisms (SNP) were tightly linked, and the VDR haplotype GCT was correlated with severe AD and complementary AAC with protection from AD. The VDR haplotype GCT region is evolutionarily conserved. The VDR FokI (rs2228570) SNP was not associated with AD. Baseline VDR expression in monocytes and short-term activation were haplotype independent. CONCLUSION: A specific VDR haplotype is more frequent in patients with severe AD. These data indicate that VDR contributes to the control of AD, e.g. by regulation of the epidermal barrier function and/or local immune response.

Association of the toll-like receptor 2 A-16934T promoter polymorphism with severe atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease with a multifactorial pathogenesis and increasing incidence in the Western world. A genetically determined defective function of pattern recognition receptors such as toll-like receptors (TLRs) has been proposed as a candidate mechanism in the pathogenesis of AD. AIM: To study the impact of genetic predisposition of five genes encoding for pattern recognition-related molecules for the phenotype of AD. METHODS: We examined nine different single-nucleotide polymorphism (SNP) frequencies in the genes encoding TLR1, -2, -4, -9 and the adapter molecule TIRAP by PCR with subsequent melting curve analysis in a case/control cohort of 136 adult AD patients and 129 age and gender matched non-atopic, healthy individuals. TLR2-expression and -function in cells from genotyped individuals were analysed. RESULTS: For the SNPs examined, similar genotype frequencies were found in both groups. In a subgroup of patients suffering from severe AD (SCORAD >50), a significantly increased representation of the A-allele in position -16934 of the tlr2 gene was present (P = 0.004). Constitutive tlr2 mRNA expression in peripheral monocytes was independent of this tlr2 promoter SNP. Stimulation assays indicated that IL-6, but not TNF-alpha secretion following TLR2 stimulation is reduced in homozygous tlr2-16934-A allele carriers. CONCLUSION: These data indicate that TLR2 is relevant for the phenotype of severe AD in adults.

Toll-like receptor (TLR) polymorphisms in African children: common TLR-4 variants predispose to severe malaria.

Genetic host factors play a substantial role in susceptibility to and severity of malaria, which continues to cause at least one million deaths per year. Recently, members of the toll-like receptor (TLR) family have been shown to be involved in recognition of the etiologic organism Plasmodium falciparum: The glycosylphosphatidylinisitol anchor induces signaling in host cells via TLR-2 and -4, while hemozoin-induced immune activation involves TLR-9. Binding of microbial ligands to the respective TLRs triggers the release of pro-inflammatory cytokines via the TLR/IL-1 receptor (TIR) domain and may contribute to the host response, including pro-inflammatory cytokine induction and malarial fever. In a case-control study among 870 Ghanaian children, we examined the influence of TLR-2, -4, and -9 polymorphisms in susceptibility to severe malaria. TLR-2 variants common in Caucasians and Asians were completely absent. However, we found a new, rare mutation (Leu658Pro), which impairs signaling via TLR-2. We failed to detect any polymorphisms within the TLR-9/interleukin-1 receptor domain. Two frequent TLR-9 promoter polymorphisms did not show a clear association with malaria severity. In contrast, the TLR-4-Asp299Gly variant occurred at a high rate of 17.6% in healthy controls, and was even more frequent in severe malaria patients (24.1%, p<0.05). Likewise, TLR-4-Thr399Ile was seen in 2.4% of healthy children and in 6.2% of patients (p=0.02). TLR-4-Asp299Gly and TLR-4-Thr399Ile conferred an 1.5- and 2.6-fold increased risk of severe malaria, respectively. These findings suggest TLR4-mediated responses to malaria in vivo and TLR-4 polymorphisms to be associated with disease manifestation. However some gray areas also suggest the scope for further improvements.

A coding mutation within the first exon of the human MD-2 gene results in decreased lipopolysaccharide-induced signaling.

MD-2 is an accessory protein of the Toll-like receptor (TLR)-4, necessary for assembling a receptor complex to sense low quantities of lipopolysaccharide in order to subsequently trigger innate immune responses. MD-2 and TLR-4 are expressed on a variety of immunocompetent cells. Mutations within the TLR-4 gene have been shown to attenuate immune responses against lipopolysaccharide in mice. In humans, a TLR-4 polymorphism has been associated with a higher risk for developing severe Gram-negative sepsis and with a lower risk for atherosclerosis. Since MD-2 is an essential part of the lipopolysaccharide receptor complex, we screened 20 patients that underwent surgical cancer therapy for novel MD-2 mutations by a single-strand conformation polymorphism technique. In one patient we found an A --> G substitution at position 103, resulting in an amino-acid exchange from Thr 35 to Ala. Reporter gene assays revealed that this mutation resulted in a reduced lipopolysaccharide-induced signaling. The patient displayed an uneventful postoperative course, with the exception of slightly decreased TNF-alpha levels after in vitro stimulation with LPS as compared to wt patients. Genotyping of a further 41 patients by a newly developed Lightcycler/FRET method failed to detect any additional polymorphism carriers, indicating that this is a rare mutation.

Decay-accelerating factor (DAF/CD55) is a functional active element of the LPS receptor complex.

Previously, we identified an 80 kDa membrane protein (LMP80) that is capable of binding to LPS and lipid A in the presence of LBP and sCD14. LMP80 could also be detected after immuno-coprecipitation of cell membranes with LPS and lipid A, indicating a physical contact of LMP80 and LPS/lipid A. Further analysis and peptide sequencing revealed that LMP80 is identical to CD55 (decay accelerating factor, DAF), a regulatory molecule of the complement cascade. Transfection of LPS-hyporesponsive Chinese hamster ovary (CHO) cells with human CD55 resulted in the translocation of NF-B upon stimulation with LPS or lipid A. Our results demonstrate a new functional role of CD55 as a molecule able to mediate LPS-induced activation of cells that may be part of a multimeric LPS receptor complex.

Ventilation-induced chemokine and cytokine release is associated with activation of nuclear factor-kappaB and is blocked by steroids.

Recent clinical trials have shown that the survival of patients with acute respiratory distress syndrome (ARDS) is improved by ventilation with reduced volumes. These studies suggested that overinflation of the lungs causes overactivation of the immune system. The present study investigated the hypothesis that ventilation with increased tidal volumes results in early responses similar to those caused by stimulation with one of the major risk factors for ARDS: bacterial lipopolysaccharide (LPS). We therefore compared the effects of ventilation (-10 cm H2O or -25 cm H2O end-inspiratory pressure) and LPS (50 microg/ml) on nuclear factor (NF)-kappaB activation, chemokine release, and cytokine release in isolated perfused lungs obtained from BALB/C mice. We found that both LPS and ventilation with -25 cm H2O (overventilation; OV) caused translocation of NF-kappaB, which was abolished by pretreatment with the steroid dexamethasone. Furthermore, both treatments resulted in similar increases in perfusate levels of alpha-chemokines (macrophage inflammatory protein; [MIP]-2; KC), beta-chemokines (macrophage chemotactic protein-1; MIP-1alpha), and cytokines (tumor necrosis factor-alpha, interleukin-6), which were largely prevented by dexamethasone pretreatment. In LPS-resistant C3H/HeJ mice, only OV, and not LPS, caused translocation of NF-kappaB and release of MIP-2. We conclude that OV evokes early inflammatory responses similar to those evoked by LPS (i.e., NF-kappaB translocation and release of proinflammatory mediators). The NF-kappaB translocation elicited by OV appears to be independent of Toll-like receptor 4 and not due to LPS contamination introduced by the ventilator. Our data further suggest that steroids might be considered as a subsidiary treatment during artificial mechanical ventilation.

The interaction of human peripheral blood eosinophils with bacterial lipopolysaccharide is CD14 dependent.

Bacterial lipopolysaccharide (LPS, endotoxin) is a ubiquitous component of dust and air pollution and is suspected to contribute after inhalation to an activation of eosinophils in bronchial tissues of asthmatic patients, provoking inflammatory and allergic processes. We were therefore interested in the interaction of eosinophil granulocytes with LPS and have examined the activation of and uptake to human peripheral blood eosinophils by LPS. Eosinophils were stimulated by LPS and the endotoxic component lipid A and the release of tumor necrosis factor alpha (TNF-alpha) and of the eosinophil-specific granule protein eosinophil cationic protein (ECP) was estimated. The results show induction of TNF-alpha and ECP-release by LPS and lipid A in a dose-dependent manner. Anti-CD14 monoclonal antibody (moAb) (clone MEM-18) and the synthetic lipid A partial structure 406 blocked the release of TNF-alpha and ECP by LPS-stimulated eosinophils. Studies with radioactively labeled LPS showed dose-dependent uptake of (3)H-LPS to eosinophils. The (3)H-LPS uptake was found to be specific because preincubation with unlabeled LPS, compound 406 and also anti-CD14 antibodies inhibited uptake of (3)H-LPS to eosinophil granulocytes. By flow cytometry using anti-CD14 moAb and by reverse transcriptase-polymerase chain reaction (RT-PCR) technique, CD14 expression was detectable. Furthermore, messenger RNA (mRNA) expression of Toll-like receptors (TLR) 2 and TLR 4 was detected, indicating the presence of these CD14 coreceptors. The results indicate that eosinophils can take up LPS and can be stimulated by LPS in a CD14-dependent manner. Hence, in addition to allergens, eosinophils interact with endotoxin, a process that possibly exacerbates ongoing inflammatory and allergic processes.

Effects of isosteric pyridone replacements in androgen receptor antagonists based on 1,2-dihydro- and 1,2,3,4-tetrahydro-2,2-dimethyl-6-trifluoromethyl-8-pyridono[5,6-g]quin olines.

A series of nonsteroidal human androgen receptor (hAR) antagonists based on 8-substituted 1,2-dihydro- and 1,2,3,4-tetrahydro-2,2-dimethyl-6-trifluoromethylpyrido[3,2-g]quin olines was synthesized. Compounds in this series were tested for the ability to bind to hAR and inhibit hAR-dependent transcription in a mammalian cellular background.

Binding of lipopolysaccharide (LPS) to CHO cells does not correlate with LPS-induced NF-kappaB activation.

Activation of myeloid cells by lipopolysaccharide (LPS) is a key event in the development of gram-negative sepsis. One crucial step within this process is the binding of LPS to CD14. CD14 is a glycosylphosphatidylinositol (GPI)-anchored membrane protein requiring at least one additional membrane-spanning molecule for signal transduction. It is not clear whether the function of CD14 is to merely catalyze LPS binding, followed by the interaction of LPS with the signal transducer, or whether CD14 has a more specific function and may be a part of the signaling complex. To address this question we generated Chinese hamster ovary (CHO) cells expressing a human GPI-anchored form of LPS-binding protein (mLBP) to substitute for CD14 as LPS acceptor molecule. By comparison of CHO / mLBP with CHO / vector and CHO / CD14 cells we found that expression of GPI-linked LBP results in an enhanced binding of LPS but not in an increase in cell activation as determined by translocation of NF-kappaB. Furthermore, excess of recombinant soluble LBP resulted also in increased LPS binding without affecting NF-kappaB translocation. These data show that LPS binding alone is not sufficient to induce signaling. We conclude that CD14 is more than a catalyst for LPS binding: it seems to be directly involved in LPS signaling and thus appears to be an essential part of the signaling complex.

Myofascial pain unresponsive to standard treatment: successful use of a strain and counterstrain technique with physical therapy.

Chronic pain disorders, including fibromyalgia and myofascial pain syndrome often do not respond adequately to standard therapy. The cases reviewed herein suggest the strain and counterstrain (SCS) technique, described in 1981 by Jones, may be helpful in reducing pain and improving function in patients with localized myofascial pain syndromes. This was a case study and retrospective review of 20 patients who had had chronic pain for an average of 2.7 years and were treated with SCS for pain relief. For all these patients, prior medical treatment had failed to provide pain relief or return of function. The procedure is a fairly common osteopathic and chiropractic technique, which to our knowledge has not received any systematic evaluation. Areduction in pain and an increase in function of 50%-100% occurred in 19 of 20 patients immediately after SCS therapy. Partial improvement was maintained for 6 months in 11 of 20 patients, and 4 were still pain free. We believe that SCS techniques should be considered and evaluated further as adjunctive therapy for patients previously unresponsive to standard treatment for myofascial pain syndrome.

A DNA sequence corresponding to the gene encoding cysteine proteinase 5 in Entamoeba histolytica is present and positionally conserved but highly degenerated in Entamoeba dispar.

Cysteine proteinases of Entamoeba histolytica are considered to be one of the most important classes of molecules responsible for the parasite's ability to destroy human tissues. Interestingly, one particular cysteine proteinase, located on the surface of E. histolytica trophozoites and designated cysteine proteinase 5 (CP5), is not expressed in the closely related but nonpathogenic species Entamoeba dispar. By comparing the E. histolytica and E. dispar genomic loci containing the gene for CP5 (cp5), it was found that the position of cp5 within the genomic context is conserved between the two organisms, but that the gene is highly degenerated in E. dispar, as it contains numerous nucleotide exchanges, insertions, and deletions, resulting in multiple stop codons within the cp5 reading frame. An alignment of all available orthologous E. histolytica and E. dispar DNA sequences suggested that cp5 started to degenerate in E. dispar coincidently when the two organisms began to diverge from a common ancestor.

4-Alkyl- and 3,4-dialkyl-1,2,3,4-tetrahydro-8-pyridono[5,6-g]quinolines: potent, nonsteroidal androgen receptor agonists.

A series of human androgen receptor (hAR) agonists based on 4-alkyl-; 4,4-dialkyl-; and 3,4-dialkyl-1,2,3,4-tetrahydro-8-pyridono[5,6-g]quinoline was synthesized and evaluated in competitive receptor binding assays and an androgen receptor cotransfection assay in a mammalian cell background. A number of compounds in this series demonstrated activity equal to or better than dihydrotestosterone in both assays and represent a novel class of compounds for use in androgen replacement therapy.

Nonsteroidal androgen receptor agonists based on 4-(trifluoromethyl)-2H-pyrano[3,2-g]quinolin-2-one.

A series of 2H-pyrano[3,2-g]quinolin-2-ones was prepared and tested for the ability to modulate the transcriptional activity of the human androgen receptor (hAR). The parent compound, 4-(trifluoromethyl)-2H-pyrano[3,2-g]quinolin-2-one, displayed moderate interaction with hAR, but substituted analogues were potent hAR modulators in vitro as measured by an hAR cotransfection assay in CV-1 cells and bound to hAR with high affinity in a whole cell assay. Several analogues were able to activate hAR-mediated gene transcription more potently and efficaciously than dihydrotestosterone.

Identification of the 80-kDa LPS-binding protein (LMP80) as decay-accelerating factor (DAF, CD55).

The activation of immunocompetent cells by lipopolysaccharide (LPS) during severe Gram-negative infections is responsible for the pathophysiological reactions, possibly resulting in the clinical picture of sepsis. Monocytes recognize LPS mainly through the LPS receptor CD14, however, other cellular binding structures have been assumed to exist. In previous studies, we have described an 80-kDa LPS-binding membrane protein (LMP80), which is present on human monocytes as well as endothelial cells. Here we demonstrate that LMP80 is widely distributed and that it forms complexes together with LPS and sCD14. Furthermore, we report on the biochemical purification of LMP80 and its identification as decay-accelerating factor, CD55, by amino acid sequencing and cloning techniques. Our results imply a new feature of CD55 as a molecule which interacts with LPS/sCD14 complexes. However, the involvement of CD55 in LPS-induced signaling remains to be elucidated.