Effects of Psychological Stress on Spontaneous Itch and Mechanical Alloknesis of Atopic Dermatitis.

Atopic dermatitis (AD), a chronic inflammatory skin disease, manifests as an intractable itch. Psychological stress has been suggested to play a role in the onset and worsening of AD symptoms. However, the pathophysiological relationships between psychological stressors and cutaneous manifestations remain unclear. To elucidate the mechanisms underlying the stress-related exacerbation of itch, we investigated the effects of water stress, restraint stress and repeated social defeat stress on itch-related scratching behaviour, mechanical alloknesis and dermatitis in male NC/Nga mice with AD-like symptoms induced by the repeated application of ointment containing Dermatophagoides farina body. NC/Nga mice with AD-like symptoms were subjected to water stress, restraint stress and repeated social defeat stress, and their scratching behaviour, sensitivity to mechanical stimuli (mechanical alloknesis) and severity of  dermatitis were evaluated. Social defeat stress+ Dermatophagoides farina body-treated mice exposed to stress showed slower improvements in or the exacerbation of AD-like symptoms, including dermatitis and itch. In the mechanical alloknesis assay, the mechanical alloknesis scores of social defeat stress+ Dermatophagoides farina body-treated mice exposed to stress were significantly higher than those of non-exposed social defeat stress+ Dermatophagoides farina body- and social defeat stress-treated mice. These results suggest that psychological stress delays improvements in dermatitis by exacerbating itch hypersensitivity in AD.

Sulfatide-selectin signaling in the spinal cord induces mechanical allodynia.

Sulfatide is a sulfated glycosphingolipid that is present abundantly in myelin sheaths of the brain and spinal cord. It is synthesized by a cerebroside sulfotransferase encoded by Gal3st1, which catalyzes the transfer of sulfate from 3'-phosphoadenylylsulfate to galactosylceramide. We previously reported that Gal3st1 gene expression in the spinal cord is up-regulated 1 day after intraplantar injection of complete Freund's adjuvant (CFA), indicating that sulfatide is involved in inflammatory pain. In the present study, we found that intrathecal injection of sulfatide led to mechanical allodynia. Sulfatide caused levels of glial fibrillary acidic protein (GFAP) and nitric oxide in the spinal cord to increase. Mechanical allodynia induced by intrathecal injection of sulfatide was blocked by nitric oxide synthase inhibitors and by suppression of astrocyte activation by L-α-aminoadipate. These results suggest that sulfatide-induced mechanical allodynia involved glial activation and nitric oxide production. Blocking selectin, a sulfatide-binding protein, with bimosiamose attenuated sulfatide-induced allodynia and ameliorated CFA-induced mechanical allodynia during inflammatory pain. Finally, elevated levels of sulfatide concentration in the spinal cord were observed during CFA-induced inflammatory pain. The elevated sulfatide levels enhanced selectin activation in the spinal cord, resulting in mechanical allodynia. Our data suggest that sulfatide-selectin interaction plays a key role in inflammatory pain.

Glycosphingolipid Biosynthesis Pathway in the Spinal Cord and Dorsal Root Ganglia During Inflammatory Pain: Early and Late Changes in Expression Patterns of Glycosyltransferase Genes.

Glycosphingolipids (GSLs) are abundant, ceramide-containing lipids in the nervous system that play key functional roles in pain and inflammation. We measured gene expression (Ugcg, St3gal5, St8sia1, B4galNT1, Ugt8a, and Gal3st1) of glycosyltransferases involved in GSL synthesis in murine dorsal root ganglion (DRG) and spinal cord after complete Freund's adjuvant (CFA)-induced unilateral hind-paw inflammation (1 day vs. 15 days). Chronic inflammation (15 days) sensitized both ipsilateral and contralateral paws to pain. One day of induced unilateral hind-paw inflammation (1d-IUHI) increased Ugcg, St8sia1, B4galnt1, and Gal3st1 expression in ipsilateral cord, suggesting that sulfatide and b-series gangliosides were also elevated. In addition, 1d-IUHI increased Ugcg, st3gal5 and Gal3st1 expression in contralateral cord, suggesting that sulfatide and a-/b-series gangliosides were elevated. By contrast, 1d-IUHI decreased Ugcg, St3gal5, and St8sia1 expression bilaterally in the DRG, suggesting that b-series gangliosides were depressed. Since intrathecal injection of b-series ganglioside induced mechanical allodynia in naïve mice, it seems reasonable that b-series gangliosides synthesized from upregulated St8sia1 in the ipsilateral spinal cord are involved in mechanical allodynia. By contrast, chronic inflammation led to a decrease of Ugcg, St3gal5, B4galnt1, and Gal3st1 expression in spinal cord bilaterally and an increase of St8sia1 expression in the ipsilateral DRG, suggesting that a-/b-series gangliosides in the spinal cord decreased and b-series gangliosides in ipsilateral DRG increased. These changes in glycosyltransferase gene expression in the DRG and the spinal cord may contribute to the modification of pain sensitivity in both inflamed and non-inflamed tissues and the transition from early to chronic inflammatory pain.

Detection of carbapenem resistance in clinical mucoid Pseudomonas aeruginosa isolates.

Of 19 isolates of mucoid Pseudomonas aeruginosa, 2 isolates showed imipenem resistance conferred by reduced OprD production. Imipenem resistance was detected by the MicroScan broth microdilution and Etest methods, but minimum inhibitory concentrations could not be determined by the Vitek system for an isolate. In cases where susceptibility cannot be determined by the broth microdilution methods, Etest results would be valuable for effective treatment.

The multiple promoter methylation profile of PR gene and ERalpha gene in tumor cell lines.

The changes of methylation status of various gene promoters are a common feature of malignant cells and these changes can occur early in the progression process. Therefore, abnormal methylation can be used as cancer marker. Such studies will first require the development of a panel of methylated markers that are methylated in cancer tissues but unmethylated in normal tissues or methylated status is different between cancer tissues and normal tissues. By using methylation-specific PCR (MSP) assay method, we observed alterations in DNA methylation at the double promoter regions of the progesterone receptor (PR) gene and estrogen receptor (ERalpha) gene in various tumor cell lines. Compared with normal white blood cell, the methylation status of PRA promoter in various cancer cell lines changed from unmethylation pattern to methylation pattern. That of PRB promoter changed from both unmethylated and methylated alleles to only methylated allele. The methylation status of ERalpha-A and ERalpha-B promoter in various cancer cell lines are cell -specific. This study indicates that PR promoter methylation may be a molecular marker in various cancer detections. And the methylation status of ERalpha-A and ERalpha-B is cell-specific.

Effect of basic amino acids on susceptibility to carbapenems in clinical Pseudomonas aeruginosa isolates.

We evaluated effects of medium composition, including basic amino acid content and pH, on susceptibility to carbapenems such as imipenem, panipenem and meropenem, in clinical isolates of Pseudomonas aeruginosa. Susceptibility to carbapenems was reduced by basic amino acids in the medium, while susceptibilities to ceftazidime and aztreonam were not. Among carbapenems, susceptibility to panipenem was most sharply reduced by addition of basic amino acids to 1:16 Mueller-Hinton agar (MHA). In 174 of 175 clinical isolates, MICs for carbapenems were affected to different degrees by medium composition. One isolate, in which MICs for carbapenems did not differ between MHA and 1:16 MHA, showed reduced production of porin (OprD). Our results suggest that susceptibility to individual carbapenems, especially panipenem, is difficult to evaluate based on MICs for other carbapenems determined on MHA. For a better prediction of antibiotic efficacy, it may be important to evaluate the susceptibility for each carbapenem individually.

Characterization of Pseudomonas aeruginosa isolates from patients with urinary tract infections during antibiotic therapy.

We characterized susceptibilities and genotypes in a series of Pseudomonas aeruginosa isolates from five cases of urinary tract infections (UTIs) to evaluate clonal shifts of carbapenem resistance. In one case, a series of isolates showed different susceptibility patterns for carbapenems but an identical genotype. In another case, genotypes varied among 4 P. aeruginosa isolates from recurrent UTIs over 9 months. Although the patient had been treated with no antibiotic immediately before isolation, the susceptibility patterns for carbapenems and ceftazidime varied. Further analysis in these two cases of outer membrane protein profiles showed that loss of OprD production resulted in reduced susceptibilities to carbapenems in all of the carbapenem-resistant isolates. Loss of OprD production was likely due to oprD gene inactivation in both of cases, since the carbapenem-resistant isolates showed no cross resistance to levofloxacin and chloramphenicol compared with the carbapenem-susceptible isolates. There was another case in which all isolates showed similar susceptibility patterns for carbapenems and ceftazidime, and an identical genotype during the intermittent use of antibiotics over 5 months. In two cases, a single course of antibiotic therapy resulted in eradication of P. aeruginosa. Our results suggest that clonal shifts of carbapenem resistance in P. aeruginosa may result from loss of OprD during antibiotic treatment. Therefore, it is important for clinicians to monitor susceptibilities to antibiotics, especially carbapenems, in P. aeruginosa isolated during therapy.

Detection of mutations in quinolone resistance-determining regions in levofloxacin- and methicillin-resistant Staphylococcus aureus: effects of the mutations on fluoroquinolone MICs.

The minimum inhibitory concentrations (MICs) of 18 antibiotics were determined for 66 clinical isolates of staphylococci. Genotypes, mutations in the quinolone resistance-determining regions (QRDRs), and effect of efflux were determined in the 18 levofloxacin-resistant isolates, for which the MICs of levofloxacin were high (> or =8 microg/ml). The increased levofloxacin resistance mainly resulted from some combinations of mutations in the QRDRs, although NorA-mediated efflux may play a minor role in resistance. A combination of mutations in GrlA (Ser80Phe), GrlB (Pro451Ser), and GyrA (Ser84Leu) was found in 4 methicillin-resistant Staphylococcus aureus (MRSA) isolates that were unrelated genotypically. The mutations in grlA QRDR varied in the isolates classified as being in an identical pulsed-field gel electrophoresis (PFGE) group, although the grlB, gyrA, and gyrB QRDRs were the same. These results suggest that the patterns of amino acid mutations in the QRDRs can provide distinct epidemiologic information from PFGE genotypes in fluoroquinolone-resistant MRSA. A combination of at least three mutations in GrlA, GrlB, and/or GyrA is required to increase the MICs of fluoroquinolones, although all of the levofloxacin-resistant MRSA retained the MICs of sitafloxacin in the range of 1 to 2 microg/ml.

Detection of macrolide resistance in Streptococcus pneumoniae.

We determined the susceptibilities of recent clinical isolates of Streptococcus pneumoniae to 19 antibiotics. The frequency of erythromycin nonsusceptibility was high, i.e. 8/13 (61.5%), 10/14 (71.4%) and 11/11 isolates (100%) from 13 penicillin-susceptible, 14 penicillin-intermediate and 11 penicillin-resistant S. pneumoniae, respectively. Macrolide resistance was detected by polymerase chain reaction (PCR) and disk diffusion methods. Of these erythromycin-nonsusceptible pneumococcal isolates, 13/29 (44.8%) isolates contained genomic copies of MEFA and showed non-'D'-shaped zones of inhibition observed around rokitamycin and/or clindamycin disks. Sixteen out of 29 isolates (55.2%) contained copies of ERMB and showed typical 'D'-shaped zones of inhibition, except one isolate. Although the macrolide resistance determinants, MEFA and ERMB, could be identified by PCR and disk diffusion methods, PCR methods were more reliable in elucidating these determinants. The susceptibility pattern to 14-, 15- and 16-membered macrolides and clindamycin differed between the MEFA+ and ERMB+ isolates. All isolates were susceptible to levofloxacin, sparfloxacin and vancomycin. The MICs of sitafloxacin were lowest among the fluoroquinolones examined for 38 isolates.

Effects of mupirocin at subinhibitory concentrations on flagella formation in Pseudomonas aeruginosa and Proteus mirabilis.

Colonization of Pseudomonas aeruginosa at trachea, nares and oropharynx can cause ventilator-acquired pneumonia. To identify beneficial effects of antibiotics on expression of virulence factors related to colonization by such pathogens, we evaluated the effect of mupirocin on flagella formation in P. aeruginosa and on motility and flagella formation in Proteus mirabilis. In P. aeruginosa, subinhibitory concentrations of mupirocin inhibited flagella formation, which was associated with reduced flagellin expression. In P. mirabilis, subinhibitory concentrations of mupirocin dose-dependently suppressed bacterial motility and flagella formation, again with reduced flagellin expression. Our results indicate that subinhibitory concentrations of mupirocin can suppress expression of virulence factors in P. aeruginosa and P. mirabilis.

Enhancement by alpha-tocopheryl hemisuccinate of nitric oxide production induced by lypopolysaccharide and interferon-gamma through the upregulation of protein kinase C in rat vascular smooth muscle cells.

The effect of alpha-tocopheryl hemisuccinate (TS) on lipopolysaccharide (LPS)/interferon-gamma (IFN)-induced nitric oxide production in rat vascular smooth muscle cells (VSMC) was examined. The LPS/IFN-induced NO production was enhanced by TS but not by the other alpha-tocopherol (alpha-T) derivatives alpha-tocopheryl acetate (TA) and alpha-tocopheryl nicotinate (TN), or alpha-T itself. alpha-T, TA and TN inhibited the enhancement by TS of LPS/IFN-induced NO production. The enhancing effect of TS was observed in the presence of LPS, but not IFN, suggesting that TS participates in the LPS-stimulated signal pathway leading to NO production. Protein kinase C (PKC) inhibitors, but not protein kinase A inhibitors, inhibited the enhancing effect of TS on LPS/IFN-induced NO production. Furthermore, TS enhanced the amount of PKCalpha in VSMC. From these results, we concluded that the enhancing effect of LPS/IFN-induced NO production was caused by upregulation of PKC in VSMC.