Strain-Specific Identification and In Vivo Immunomodulatory Activity of Heat-Killed Latilactobacillus sakei K040706.

We previously reported that the immunostimulatory activity of heat-killed Latilactobacillus sakei K040706 in macrophages and cyclophosphamide (CTX)-treated mice. However, identification of heat-killed L. sakei K040706 (heat-killed LS06) using a validated method is not yet reported. Further, the underlying molecular mechanisms for its immunostimulatory effects in CTX-induced immunosuppressed mice remain unknown. In this study, we developed strain-specific genetic markers to detect heat-killed L. sakei LS06. The lower detection limit of the validated primer set was 2.1 × 105 colony forming units (CFU)/mL for the heat-killed LS06 assay. Moreover, oral administration of heat-killed LS06 (108 or 109 CFU/day, p.o.) effectively improved the body loss, thymus index, natural killer cell activity, granzyme B production, and T and B cell proliferation in CTX-treated mice. In addition, heat-killed LS06 enhanced CTX-reduced immune-related cytokine (interferon-γ, interleukin (IL)-2, and IL-12) production and mRNA expression. Heat-killed LS06 also recovered CTX-altered microbiota composition, including the phylum levels of Bacteroidetes, Firmicutes, and Proteobacteria and the family levels of Muribaculaceae, Prevotellaceae, Tannerellaceae, Christensenellaceae, Gracilibacteraceae, and Hungateiclostridiaceae. In conclusion, since heat-killed L. sakei K040706 ameliorated CTX-induced immunosuppression and modulated gut microbiota composition, they have the potential to be used in functional foods for immune regulation.

Age-related change of Iba-1 immunoreactivity in the adult and aged gerbil spinal cord.

In the present study, we examined change of ionized calcium-binding adapter molecule 1 (Iba-1) in the adult and aged gerbil spinal cords. Significant change of morphological feature and neuronal cell loss were not observed in both adult and aged spinal cords of gerbil after NeuN immunohistochemistry and Fluoro-Jade B histofluoresce staining. Iba-1-immunoreactive microglia broadly distributed in the spinal cord. Most of Iba-1-immunoreactive microglia showed ramified forms in the adult gerbil cervical and lumbar spinal cords. However, morphological changes of Iba-1-immunoreactive microglia were observed in the cervical and lumbar regions of the aged gerbil spinal cord. These microglia were showed a hypertrophied body with shortened swollen processes which was characteristic of activated microglia. In addition, Iba-1 protein level significantly higher in aged cervical and lumbar spinal cords than those in the adult gerbil. The present study showed an increase of activated forms of Iba-1-immunoreactive microglia and its protein level without marked changes in morphological features and neuronal loss in the aged spinal cord compared to those in the adult gerbil spinal cord. This result suggests that the increase of Iba-1 expression in the aged spinal cord may be closely associated with age-related changes in aged gerbil spinal cord.

SP, CGRP changes in pyridoxine induced neuropathic dogs with nerve growth factor gene therapy.

BACKGROUND: Nerve growth factor (NGF) is known not only as a major factor for neuronal plasticity but also as a pain stimulator. Although there have been several trials with NGF for its application in the regeneration or protection of the nervous system, the pain induced by NGF remains a challenge to be overcome. In this study, the pain induced by NGF gene therapy was evaluated. RESULTS: Vehicle or recombinant dog NGF plasmid was administered into the intrathecal space of dogs. Twenty-four hours after the vehicle or NGF plasmid inoculation, dogs were subcutaneously treated with 150 mg/kg pyridoxine every day for 7 days. For pain assessment, physical examination and electrophysiological recording were performed. Only in the vehicle-treated group, weight loss occurred, while NGF plasmid inoculation significantly improved this physical abnormalities. In the vehicle-treated group, electrophysiological recordings showed that H-reflex disappeared at 24 h after the last pyridoxine treatment. However, in the NGF plasmid inoculated group, the H-reflex were normal. In the results of immunohistochemistry, the NGF plasmid administration efficiently expressed in the dorsal root ganglia and significantly increased the pyridoxine-induced reduction of calcitonin gene-related peptide (CGRP) immunoreactive neurons, but not in substance P immunoreactive neurons, in the dorsal root ganglia. CONCLUSIONS: Given these results, we reason that NGF gene therapy in pyridoxine induced neuropathic dogs does not induce neuropathic pain with this dosage, even with increasing the expression of CGRP.

Antiviral effects of Lactobacillus ruminis SPM0211 and Bifidobacterium longum SPM1205 and SPM1206 on rotavirus-infected Caco-2 cells and a neonatal mouse model.

Rotavirus is worldwide cause of severe gastroenteritis including severe diarrhea and fatal dehydration in infants and young children. There is an available vaccination program for preventing rotavirus infection, but it has limits and restrictions. Probiotics therapy could be an alternative method of antiviral prevention and modulation against rotavirus infection. In this study, we screened the antiviral activity of probiotic bacteria such as 3 Lactobacillus spp. and 14 Bifidobacterium spp. isolated from young Korean. Three of the bacteria, Lactobacillus ruminis SPM0211, Bifidobacterium longum SPM1205, and SPM1206, inhibited human strain Wa rotavirus infection in Caco-2 cells. Furthermore, these bacterial strains inhibited rotavirus replication in a rotavirus-infected neonatal mouse model. To clarify the mechanism of inhibition, we investigated gene expression of Interferon (IFN)-signaling components and IFN-inducible antiviral effectors. All 3 probiotics increased IFN-α and IFN-ß levels compared with the control. Gene expression of IFNsignaling components and IFN-inducible antiviral effectors also increased. Overall, these results indicate that L. ruminis SPM0211, B. longum SPM1205 and 1206 efficiently inhibit rotavirus replication in vitro and in vivo. Especially, the antiviral effect of Lactobacillus ruminis SPM0211 is worthy of notice. This is the first report of L. ruminis with antiviral activity. Anti-rotaviral effects of the 3 probiotics are likely due to their modulation of the immune response through promoting type I IFNs, which are key regulators in IFN signaling pathway.

Effects of 1,7-substituted methylxanthine derivatives on LPS-stimulated expression of cytokines and chemokines in Raw 264.7 and HK-2 cells.

Chronic kidney diseases are based on uncontrolled immunological and inflammatory responses to pathophysiological renal circumstances such as glomerulonephritis, which is caused by immunological mechanisms of glomerular inflammation with increased production of renal pro-inflammatory cytokines. Pentoxifylline (PTX) exhibits anti-inflammatory properties by inhibiting cytokine and chemokine production through aggregation of erythrocytes and thrombocytes. We synthesized a series of 1,7-substituted methylxanthine derivatives by the Traube purine reaction, and the formation of purine ring was completed through nitrosation, a reduction of the nitroso to the amine by catalytic hydrogenation as derivatives of PTX. Then we studied biological activities such as renal anti-inflammatory effects of the synthesized compounds in the production of cytokines such as nitric oxide (NO), interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α (TNF-α) and of chemokines such as monocyte chemoattractant protein-1 and IL-8 in Raw 264.7 and HK-2 cells. Renal antiinflammatory activities of this novel series of N-1 and N-7-substituted methylxanthine showed that the N-7 methyl-group-substituted analogs (S7b) showed selective 61% and 77% inhibition of the production of NO and IL-8. The other replacement of the N-1-(CH2)4COCH3 group, as in the case of compound S6c, also showed an effective 50% and 77% inhibition of TNF-α and IL-8 production in LPS-stimulated Raw 264.7 and HK-2 cells.

Antiviral activity of Bifidobacterium adolescentis SPM0212 against Hepatitis B virus.

Bifidobacteria are considered one of the most beneficial probiotics and have been widely studied for their effects in preventing and treating specific pathological conditions. The present study explored the antiviral activity of Bifidobacterium adolescentis SPM0212 isolated from healthy Koreans against hepatitis B virus (HBV) and its mechanism of action. To determine the effect of B. adolescentis SPM0212 against HBV, the level of HBV surface antigen (HBsAg) in the culture medium and the levels of viral transcripts in HepG2.2.15 cells were measured by enzyme-linked immunosorbent assay and reverse transcription-quantitative PCR (RT-qPCR), respectively. To clarify the mechanism, we performed RT-qPCR using specific primers for genes encoding Interferon (IFN)-signaling components and IFN-inducible antiviral effectors. The cell extract of B. adolescentis SPM0212 dose-dependently decreased the extracellular HBsAg level by up to 50 %. Its gene expression in HepG2.2.15 cells was also inhibited by 40 %. This extract significantly increased the expression level of myxovirus resistance A, which is an IFN-inducible antiviral effector. Furthermore, the antiviral activity was observed in the fraction of compound(s) with molecular weights under 30 kDa. Thus, the cell extract of B. adolescentis SPM0212 inhibits HBV and its antiviral mechanism is associated with the Mx GTPase pathway.