Combined antiallodynic effects of Neurotropin®-tramadol and Neurotropin®-mirogabalin in rats with L5-spinal nerve ligation.

We aimed to examine the efficacy of combination therapies of Neurotropin® with tramadol and Neurotropin with mirogabalin for neuropathic pain management. A neuropathic pain model (L5 spinal nerve ligation model: L5-SNL) using male Wistar rats was generated through tight ligation of the left fifth lumbar nerve using silk sutures. Mechanical allodynia was assessed using the 50% paw withdrawal threshold. The combined antiallodynic effects were evaluated using isobolographic analyses. Small intestinal transit was evaluated using the charcoal meal test, and motor coordination using the rota-rod test. Neurotropin (50-200 NU/kg, p.o.), tramadol (7.5-60 mg/kg, p.o.), and mirogabalin (3-30 mg/kg, p.o.) showed a dose-dependent antiallodynic effect in L5-SNL rats. The combined antiallodynic effects of Neurotropin and tramadol were additive or synergistic, whereas those of Neurotropin and mirogabalin were additive. Neurotropin (100-400 NU/kg, p.o.) did not affect the small intestinal transit, whereas tramadol (30-100 mg/kg, p.o.) significantly inhibited it. Neurotropin (100-400 NU/kg, p.o.) did not affect the walking time, whereas mirogabalin (10-100 mg/kg, p.o.) significantly decreased it. Neurotropin dose-dependently ameliorated mechanical allodynia in rats, and combination therapy with Neurotropin-tramadol or Neurotropin-mirogabalin may alleviate neuropathic pain without aggravating the adverse effects of tramadol and mirogabalin.

Isolation of microorganisms from the feces of Kitasato Yakumo beef cattle as bioactive natural product producers.

We envisioned that the rumen of Kitasato Yakumo beef cattle would contain unique microorganisms which produce bioactive compounds as their defense response to the external environment. The variety of microorganisms were collected from the feces of Kitasato Yakumo beef cattle. We evaluated the biological activity of the culture broth of the isolated strains, proving the utility of our approach.

Adhesion of enterohemorrhagic Escherichia coli O157:H7 to the intestinal epithelia is essential for inducing secretory IgA antibody production in the intestine of mice.

We examined whether adherence of enterohemorrhagic Escherichia coli (EHEC) O157:H7 to intestinal epithelial cells contributed to the induction of secretory immunoglobulin A (IgA) antibody production in mice. Wild-type EHEC O157:H7 and its mutants deficient in the espA, sepL, tir and eae genes, encoding adherent factors on the locus of enterocyte effacement (LEE), were inoculated intragastrically into mice. Inoculation of wild-type EHEC induced fecal IgA antibodies specific to EHEC at 4 weeks after the inoculation, but that of espA- and sepL-deletion mutants did not. Furthermore, even 4 inoculations at weekly intervals with espA-deletion mutant, heat-killed wild-type EHEC and nonpathogenic E. coli did not induce fecal IgA antibodies, although these bacterial inoculations induced serum antibodies. Kanamycin (Km)-treated mice showed prolonged and similar fecal shedding of Km-resistant mutants of EHEC O157:H7 including A2-F6 having intact LEE, A6-E7 (sepL-insertion mutant), G1-E11 (tir-insertion mutant) and Δeae (eae-deletion mutant). In this case, A2-F6 induced fecal IgA antibodies, but the other mutants with defective LEE did not. In contrast to the fecal IgA antibodies, serum IgM and IgG antibodies were induced in mice inoculated with any of the LEE defective mutants as well as A2-F6. Thus, adhesion of EHEC to epithelial cells is essential for inducing the mucosal immune response in the intestine but not for inducing the systemic immune response.

Combined antiallodynic effect of Neurotropin® and pregabalin in rats with L5-spinal nerve ligation.

AIMS: In this study, we investigated the combined effect of Neurotropin® and pregabalin for L5-spinal nerve ligation (L5-SNL) model in rats and thiopental-induced sleep in mice. MAIN METHODS: The left fifth lumbar nerve of rats was tightly ligated with silk sutures under pentobarbital anesthesia. The hindpaw withdrawal threshold was measured by application of von Frey filaments. Thiopental sodium was intravenously administered in mice and sleeping time was measured. In L5-SNL rats, an isobolographic analysis was performed to clarify the combined antiallodynic effect of Neurotropin and pregabalin 14 days after ligation in rats. In isobolographic analysis and thiopental-induced sleep test, Neurotropin and pregabalin were orally administered to coincide with the timing of the peak effect of each drug. KEY FINDINGS: Neurotropin (50-200 NU/kg) and pregabalin (2.5-10mg/kg) showed a dose-dependent antiallodynic action in L5-SNL rats. The antiallodynic effect of pregabalin was reversed by intrathecal injection of yohimbine or ondansetron. Isobolographic analysis suggested that the combined antiallodynic effect of Neurotropin and pregabalin in L5-SNL rats may have been more than a mere additive effect. Neurotropin (50-400 NU/kg) had no effect on thiopental-induced sleeping time whereas pregabalin (30-100mg/kg) significantly prolonged it. When the dose of pregabalin was 30 mg/kg, Neurotropin (50-400 NU/kg) did not further exacerbate the prolongation effect of pregabalin on thiopental-induced sleep. SIGNIFICANCE: It was suggested that when Neurotropin was administered in combination with pregabalin, it might provide more effective pain relief than that obtained with each agent alone in neuropathic pain without aggravating adverse effects of pregabalin.

Increased adherence to Caco-2 cells caused by disruption of the yhiE and yhiF genes in enterohemorrhagic Escherichia coli O157:H7.

Adherence of enterohemorrhagic Escherichia coli (EHEC) to intestinal epithelium is essential for initiation of infections, including diarrhea, and expression of the genes of the locus of enterocyte effacement (LEE) is thought to be crucial for adherence. To identify genes involved in modulating the adherent capacity, bacteria collected from an EHEC O157:H7 strain (O157Sakai) mutagenized by mini-Tn5Km2 were screened for their ability to increase the number of microcolonies (MC) on Caco-2 cells and eight mutants with increased adherence were isolated. Analysis of the mini-Tn5Km2-flanked DNA sequences indicated that one possessed the insertion within an O157 antigen gene cluster, another possessed the insertion within the yhiF gene, and the remaining six mutants had their insertions in the yhiE gene. yhiE and yhiF products share amino acid homology (23% identity) to each other and with members of the LuxR family, which are known as transcriptional regulatory proteins. The mutant having the insertion within the O157 antigen gene cluster did not express the O157 side chain (as determined by agglutination test and immunoblotting with polyclonal O157-specific antiserum), unlike the other seven mutants. Importantly, the other mutants showed enhanced type III secretion. Levels of the related mRNAs of genes of the LEE, but not that of ler mRNA, were also increased compared with those in the wild type. Indeed, when we introduced an in-frame deletion into the yhiE or yhiF gene in O157Sakai, the capacity of the resultant mutants to adhere to Caco-2 cells was greatly increased. When one of the yhiE insertion mutants was orally inoculated into ICR mice, the number of bacteria shed into feces by day 14 was greater than that for the wild type. These results suggest that yhiE and yhiF are involved in the adherence of O157Sakai to epithelial cells as negative regulators for the expression of the genes required for the type III secretion system.

A murine model of enterohemorrhagic Escherichia coli O157:H7 infection to assess immunopotentiating activity of drugs on mucosal immunity: effect of drugs.

An enterohemorrhagic Escherichia coli (EHEC) O157 oral infection murine model was established to examine the potentiating activity of drugs on mucosal immune responses. Groups of ICR mice inoculated intragastrically with 10(11) CFU/kg EHEC O157 showed chronic intestinal infection with the pathogen that persisted over 3 weeks and resulted in the synthesis of relatively high levels of antigen specific fecal IgA antibody. Intraperitoneal administration of 80 NU/kg Neurotropin, an immunopotentiator, augmented the antigen specific mucosal immune responses to EHEC O157. On the other hand, FK506 clearly suppressed the response. To further document the augmenting effect of Neurotropin on mucosal immune responses, mice were immunized intranasally with a mixture of ovalbumin and cholera toxin. Co-administration of 80 NU/kg Neurotropin significantly potentiated the synthesis of fecal IgA and serum IgG antibodies. These results suggest that Neurotropin has potential as a mucosal adjuvant to promote secretory IgA antibody production and that the mice model of oral infection with EHEC O157 is useful for immunopharmacological studies of bacterial infection-defensive mucosal immune responses.

Adhesion and colonization of enterohemorrhagic Escherichia coli O157:H7 in cecum of mice.

Infectious diseases due to enterohemorrhagic Escherichia coli (EHEC) are characterized by diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. The adherence of EHEC on intestinal epithelial cells is a first step for developing these diseases. In the present study, we examined whether EHEC O157:H7 adhere to intestinal epithelial cells of mice and cause F-actin accumulation in the epithelial cells following the intragastric inoculation of the pathogen. Fecal shedding of the EHEC O157:H7 strain was observed in ICR mice up to 3 weeks. Fecal shedding periods of the type III secretion system-related gene (espA and sepL) deletion mutants were clearly shorter than that of the wild-type EHEC O157:H7 strain. The EHEC O157:H7 colonies were found on the epithelial surfaces of the ceca in association with F-actin accumulation beneath the attached bacteria.