Dynamics of MMP­9, MMP­2 and TIMP­1 in a rat model of brain injury combined with traumatic heterotopic ossification.

The present study aimed to detect early changes in the concentration of matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase­2 (MMP­2) and tissue inhibitor of metalloproteinase­1 (TIMP­1) in a rat model of brain injury combined with traumatic heterotopic ossification (HO). A total of 132 male Sprague­Dawley rats were used to establish the experimental and control groups. Anatomy and sample collection were conducted on postoperative days 1, 2, 3, 4, 5, 6 and 7. Hematoxylin and eosin and immunohistochemical staining were performed for local tissues. MMP­9, MMP­2 and TIMP­1 levels and gene expression level were measured by ELISA and reverse transcription­quantitative polymerase chain reaction. Radiological investigation of the rat lower limbs was conducted at weeks 5 and 10 following modeling to observe the occurrence of HO. The incidence of HO for rats in the experimental group was higher compared with the control group. The serum MMP­9 levels of the experimental group were notably higher on postoperative days 5­7 compared with the control group. The MMP­9 gene expression of the experimental group was higher on postoperative days 3­7 compared with the control group. The TIMP­1 gene expression levels were markedly higher compared with the control group at each time point. Thus, an increase in inflammatory response is closely associated with brain injury, in addition to an increase in the number of inflammatory cells with the incidence of HO. The pathological elevation of MMP­9 and the altered dynamic equilibrium between MMP­9 and TIMP­1 contributed to the degradation, remodeling and calcification of the extracellular matrix, resulting in the induction of osteoblast precursor cells in HO. MMP­9 is a predictive marker of HO.

Improvement in T helper 1-related immune responses in BALB/c mice immunized with an HIV-1 gag plasmid combined with a chimeric plasmid encoding interleukin-18 and flagellin.

Both flagellin (fliC) and IL-18 (INF-γ-inducing factor) have been developed as adjuvants for improving immunogenicity in DNA-vaccinated hosts. An HIV-1 gag plasmid encodes a protein harboring broad epitopes for cytotoxic T-lymphocytes. In this study, the immunogenicity of BALB/c mice immunized with an HIV-1 gag plasmid (pVAX/gag) combined with a chimeric plasmid encoding IL-18 fused to flagellin (pcDNA3/IL-18_fliC) or a single plasmid encoding IL-18 (pcDNA3/IL-18) and/or flagellin (pcDNA3/fliC) was assessed. Through in vitro transcription and translation, it was demonstrated that both mRNA and protein were appropriately expressed by each construct. The IL-18 and flagellin fusion protein, which could be detected in supernatants from transfected cells, was effective in inducing IFN-γ by lymphocytes. Following i.m. immunization, expressions of flagellin or IL-18 were detected in muscle cells by immunohistochemistry analysis from 72 hr. At 12 weeks post-immunization, both gag-specific IgG in sera and spleen cell proliferation were high in all murine groups. However, the IgG2a/IgG1 ratio, Th1 cytokine (IL-2 and IFN-γ) production and proportion of gag-specific CD3(+) CD8(+) IFN-γ-secreting cells were significantly higher in the murine group co-immunized with pVAX/gag plasmid and pcDNA3/IL-18_fliC than in the mice immunized with pVAX/gag plasmid combined with either pcDNA3/fliC or pcDNA3/IL-18 plasmid or both. These findings suggest that a chimeric plasmid encoding IL-18 fused to flagellin can be used as an adjuvant-like plasmid to improve the Th1 immune response, particularly for induction of CD3(+) CD8(+) IFN-γ-secreting cells in gag plasmid-vaccinated mice.

Induction of mouse melioidosis with meningitis by CD11b+ phagocytic cells harboring intracellular B. pseudomallei as a Trojan horse.

BACKGROUND: Approximately 3-5% of patients with melioidosis manifest CNS symptoms; however, the clinical data regarding neurological melioidosis are limited. METHODS AND FINDINGS: We established a mouse model of melioidosis with meningitis characterized by neutrophil infiltration into the meninges histologically and B. pseudomallei in the cerebrospinal fluid (CSF) by bacteriological culturing methods. As the disease progresses, the bacteria successively colonize the spleen, liver, bone marrow (BM) and brain and invade splenic and BM cells by days 2 and 6 post-infection, respectively. The predominant cell types intracellularly infected with B. pseudomallei were splenic and BM CD11b(+) populations. The CD11b(+)Ly6C(high) inflamed monocytes, CD11b(+)Ly6C(low) resident monocytes, CD11b(+)Ly6G(+) neutrophils, CD11b(+)F4/80(+) macrophages and CD11b(+)CD19(+) B cells were expanded in the spleen and BM during the progression of melioidosis. After adoptive transfer of CD11b populations harboring B. pseudomallei, the infected CD11b(+) cells induced bacterial colonization in the brain, whereas CD11b(-) cells only partially induced colonization; extracellular (free) B. pseudomallei were unable to colonize the brain. CD62L (selectin) was absent on splenic CD11b(+) cells on day 4 but was expressed on day 10 post-infection. Adoptive transfer of CD11b(+) cells expressing CD62L (harvested on day 10 post-infection) resulted in meningitis in the recipients, but transfer of CD11b(+) CD62L-negative cells did not. CONCLUSIONS/SIGNIFICANCE: We suggest that B. pseudomallei-infected CD11b(+) selectin-expressing cells act as a Trojan horse and are able to transmigrate across endothelial cells, resulting in melioidosis with meningitis.

An experimental study of artificial murine bladder reflex arc established by abdominal reflex.

BACKGROUND: The neurogenic bladder dysfunction caused by spinal cord injury is difficult to treat clinically. The aim of this research was to establish an artificial bladder reflex arc in rats through abdominal reflex pathway above the level of spinal cord injury, reinnervate the neurogenic bladder and restore bladder micturition. METHODS: The outcome was achieved by intradural microanastomosis of the right T13 ventral root to S2 ventral root with autogenous nerve grafting, leaving the right T13 dorsal root intact. Long-term function of the reflex arc was assessed from nerve electrophysiological data and intravesical pressure tests during 8 months postoperation. Horseradish peroxidase (HRP) tracing was performed to observe the effectiveness of the artificial reflex. RESULTS: Single stimulus (3 mA, 0.3 ms pulses, 20 Hz, 5-second duration) on the right T13 dorsal root resulted in evoked action potentials, raised intravesical pressures and bladder smooth muscle, compound action potential recorded from the right vesical plexus before and after the spinal cord transaction injury between L5 and S4 segmental in 12 Sprague-Dawley rats. There were HRP labelled cells in T13 ventral horn on the experimental side and in the intermediolateral nucleus on both sides of the L6-S4 segments after HRP injection. There was no HRP labelled cell in T13 ventral horn on the control side. CONCLUSION: Using the surviving somatic reflex above the level of spinal cord injury to reconstruct the bladder autonomous reflex arc by intradural microanastomosis of ventral root with a segment of autologous nerve grafting is practical in rats and may have clinical applications for humans.

Elevated expression of NF-kappaB in oral submucous fibrosis--evidence for NF-kappaB induction by safrole in human buccal mucosal fibroblasts.

Nuclear factor-kappa B (NF-kappaB) is considered to be important in many inflammatory and immune responses. The aim of this study was to compare NF-kappaB expression in normal human buccal mucosa and oral submucous fibrosis (OSF) specimens and further explore the potential mechanism that may lead to induction of NF-kappaB expression. Seventeen OSF and six normal buccal mucosa specimens were examined by immunohistochemistry. Primary human buccal mucosal fibroblasts (BMFs) were established and challenged with safrole, a major polyphenolic compound in the influorescence of Piper betel, by cytotoxicity and western blot assays. Furthermore, glutathione precursor N-acetyl-L-cysteine (NAC), extracellular signal-regulated protein kinase (ERK) inhibitor PD98059, cyclooxygenase-2 (COX-2) inhibitor NS-398, dexamethasone, and cyclosporin A were added to find the possible mechanism. NF-kappaB expression was significantly higher in OSF specimens and expressed mainly by fibroblasts, endothelial cells, and inflammatory cells. Safrole was cytotoxic to BMFs in a dose-dependent manner (p<0.05). Western blot demonstrated highly elevated NF-kappaB protein expression in BMFs stimulated by safrole (p<0.05). In addition, pretreatment with pharmacological agents markedly inhibited the safrole induced-NF-kappaB expression (p<0.05). The result suggests that chewing areca quid may activate NF-kappaB expression that may be involved in the pathogenesis of OSF. NF-kappaB expression induced by safrole in fibroblasts may be mediated by ERK activation and COX-2 signal transduction pathway.