Polymorphisms in Toll-like receptors 2, 4, and 9 are highly associated with hearing loss in survivors of bacterial meningitis

Genetic variation in innate immune response genes contributes to inter-individual differences in disease manifestation and degree of complications upon infection. We recently described an association of single nucleotide polymorphisms (SNPs) in TLR9 with susceptibility to meningococcal meningitis (MM). In this study, we investigate the association of SNPs in multiple pathogen recognition and immune response genes with clinical features that determine severity and outcome (especially hearing loss) of childhood MM and pneumococcal meningitis (PM). Eleven SNPs in seven genes (TLR2, TLR4, TLR9, NOD1, NOD2, CASP1, and TRAIL) were genotyped in 393 survivors of childhood bacterial meningitis (BM) (327 MM patients and 66 PM patients). Genotype distributions of single SNPs and combination of SNPs were compared between thirteen clinical characteristics associated with severity of BM. After correction for multiple testing, TLR4+896 mutant alleles were highly associated with post-meningitis hearing loss, especially MM (p=?0.001, OR 4.0 for BM, p=?0.0004, OR 6.2 for MM). In a multigene analysis, combined carriership of the TLR2+2477 wild type (WT) with TLR4+896 mutant alleles increases the risk of hearing loss (p<0.0001, OR 5.7 in BM and p=?0.0001, OR 7.6 in MM). Carriage of one or both mutant alleles in TLR4+896 and TLR9 -1237 increases the risk for hearing loss (p?=?0.0006, OR 4.1 in BM). SNPs in immune response genes contribute to differences in clinical severity and outcome of BM. The TLR system seems to play an important role in the immune response to BM and subsequent neuronal damage as well as in cochlear inflammation. Genetic markers may be used for identification of high-risk patients by creating prediction rules for post-meningitis hearing loss and other sequelae, and provide more insight in the complex immune response in the CNS possibly resulting in new therapeutic interventions.     

In vivo assessment of hepatic triglycerides in murine non-alcoholic fatty liver disease using magnetic resonance spectroscopy

In vivo¹H magnetic resonance spectroscopy (MRS) was used to examine the progression of fatty liver in two murine models of progressive hepatic steatosis: leptin-deficient obese (ob/ob) mice and mice maintained on a diet deficient in methionine and choline (MCDD). Ob/ob mice displayed high levels of intracellular hepatic triglycerides as early as 9 weeks after birth, as observed with MRS and histopathology. Single voxel spectra of ob/ob liver displayed strong resonances arising from saturated (1.3 ppm) and unsaturated (2.8 and 5.3 ppm) fatty acyl chains that could be resolved in the absence of water suppression. Hepatic inflammation, induced by lipopolysaccharide administration, led to a significant increase in unsaturated and polyunsaturated fatty acyl chain resonances (P < 0.05), indicating a change in the composition of hepatic triglycerides in lipid droplets. Mice maintained on the MCDD displayed histological evidence of hepatic steatosis as early as two weeks, progressing to macrovesicular steatohepatitis at 10 weeks. The histological changes were accompanied by significant increases in saturated and unsaturated fatty acyl chain resonances and a significant decrease in the lipid/(water + lipid) ratio (P < 0.05). These results indicate that in vivo¹H MRS may be a suitable method to monitor the progression of steatohepatitis.     

Increased epidermal growth factor receptor in multidrug-resistant human neuroblastoma cells

Multidrug-resistant human neuroblastoma cell lines obtained by selection with vincristine or actinomycin D from two independent clonal lines, SH-SY5Y and MC-IXC, have 3- to 30-fold more cell surface epidermal growth factor (EGF) receptors than the drug-sensitive parental cells as indicated by EGF binding assays and immunoprecipitation, affinity-labeling, and phosphorylation studies. Reversion to drug sensitivity in one line was accompanied by a return to the parental level of EGF receptor. SH-EP cells, a clone derived from the same neuroblastoma cell line as SH-SY5Y but which displays melanocyte rather than neuronal lineage markers, also express significantly more EGF receptor than SH-SY5Y cells. By nucleic acid hybridization analysis with a molecularly cloned probe, increased receptor level in multidrug-resistant cells was shown to be the result of higher levels of EGF receptor mRNA in drug-resistant than in drug-sensitive cells. The increased steady state amount of specific RNA did not result from amplification of receptor-encoding genes. A small difference was observed in the electrophoretic mobility under denaturing conditions of EGF receptor immunoprecipitated from drug-resistant and drug-sensitive cells. Quantitative and qualitative modulation of the EGF receptor might reflect alterations in the transformation and/or differentiation phenotype of the resistant cells or might result from unknown selective pressures associated with the development of multidrug resistance.     

Phorbol ester- and protein kinase C-mediated phosphorylation of the cellular Kirsten ras gene product

The effect of phorbol 12-myristate 13-acetate on the phosphorylation of the ras p21 protein was studied by metabolically labeling cultured cells with [32P]orthophosphate and using a monoclonal antibody to immunoprecipitate the protein. Phorbol 12-myristate 13-acetate (100 nM) induced phosphorylation of cKi-ras p21 in a mouse adrenocortical cell line (Yl) expressing high levels of cKi-ras with exon 4B. Phosphorylation was detected at 10 min and was maximal at 2 h. The ras protein was not phosphorylated in response to phorbol 12-myristate 13-acetate in NIH 3T3 cells expressing activated cHa-ras or vHa-ras. In vitro, protein kinase C phosphorylated cKi-ras in a phosphatidylserine and diolein-dependent manner. Both in intact cells and in vitro the amino acid phosphorylated was serine. Analysis of p21 from NIH 3T3 cells expressing a variety of ras proteins indicated that phosphorylation occurs within a domain encoded by exon 4B of cKi-ras. Phosphorylation affected neither the binding nor the GTPase activity of the ras protein. We conclude that cKi-ras is a substrate for protein kinase C and that the site of phosphorylation is likely to be serine 181 encoded by exon 4B.     

Estimation of the membrane potential of cultured macrophages from the fast potential transient upon microelectrode entry

Analysis of membrane potential recordings upon microelectrode impalement of four types of macrophages (cell lines P388D1 and PU5-1.8, cultured mouse peritoneal macrophages, and cultured human monocytes) reveals that these cells have membrane potentials at least two times more negative than sustained potential values (E(s)) frequently reported. Upon microelectrode entry into the cell (P388D1), the recorded potential drops to a peak value (E(p)) (mean -37 mV for 50 cells, range -15 to -70 mV) within 2 ms, after which it decays to a depolarized potential (E(n)) (mean -12 mV) in about 20 ms. Thereafter, the membrane develops one or a series of slow hyperpolarizations before a final sustained membrane potential (E(s)) (mean -14 mV, range -5 to -40) is established. The mean value of the peak of the first hyperpolarization (E(h)) is -30 mV (range -10 to -55 mV). The initial fast peak transient, measured upon microelectrode entry, was first described and analyzed by Lassen et al. (Lassen, U.V., A.M. T. Nielson, L. Pape, and L. O. Simonsen, 1971, J. Membr. Biol. 6:269-288 for other change in the membrane potential from its real value before impalement to a sustained depolarized value. This was shown to be true for macrophages by two-electrode impalements of single cells. Values of E(p), E(n), E(h), E(s), and membrane resistance (R(m)) measured for the other macrophages were similar to those of P388D1. From these results we conclude that E(p) is a better estimate of the true membrane potential of macrophages than E(s), and that the slow hyperpolarizations upon impalement should be regarded as transient repolarizations back to the original membrane potentials. Thus, analysis of the initial fast impalement transient can be a valuable aid in the estimation of the membrane potential of various sorts of small isolated cells by microelectrodes.     

Aquatic entomofauna of a Dead Sea oasis

A survey of the aquatic and semi-aquatic insect fauna of a semi-tropical, arid zone oasis on the western Dead Sea coast, was conducted primarily in the summer and winter seasons of 1980/81. A table is given of the aquatic entomofauna, including their zoogeographical affinities, Israeli distribution and presence in En Gedi and in each of the two canyons there. Zoogeographical analysis reveals a predominance of tropical and arid African affinity (Ethiopian = 31%, Saharo-Arabian = 19%), with a major affinity also to the Mediterranean (21%). The entomofaunal community was divided among seven ecological biotopes: fast or slow flowing streams and pools; eddies; bedrock or deep pools; standing or stagnant pools. A community analysis table of the occurrence of the major faunal elements in each of seven biotopes is presented. Some rather stenotopic taxa were indicative of biotopes. A comparison is made of summer and winter seasonality including the effects of flash floods and the relation of these phenomena to emigration/imigration and life cycles of the entomofauna. The effects of agriculture and tourism are discussed and proposals made for conservation of the biotope communities. These biotopes and their entomofaunal communities are presumed to represent most of the habitats of Middle East arid zone springs.