Expression Of Interleukin-1beta Research Articles

Synthetic Prion Peptide 106-126 Resulted in an Increase Matrix Metalloproteinases and Inflammatory Cytokines from Rat Astrocytes and Microglial Cells

It has been shown that the accumulation of prion in the cytoplasm can result in neurodegenerative disorders. Synthetic prion peptide 106-126 (PrP) is a glycoprotein that is expressed predominantly by neurons and other cells, including glial cells. Prion-induced chronic neurodegeneration has a substantial inflammatory component, and an increase in the levels of matrix metalloproteinases (MMPs) may play an important role in neurodegenerative development and progression. However, the expression of MMPs in PrP induced rat astrocytes and microglia has not yet been compared. Thus, in this study, we examined the fluorescence intensity of CD11b positive microglia and Glial Fibrillary Acidic Protein (GFAP) positive astrocytes and found that the fluorescent intensity was increased following incubation with PrP at 24 hours in a dose-dependent manner. We also observed an increase in interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) protein expression, which are initial inflammatory cytokines, in both PrP induced astrocytes and microglia. Furthermore, an increase MMP-1, 3 and 11 expressions in PrP induced astrocytes and microglia was observed by real time PCR. Our results demonstrated PrP induced activation of astrocytes and microglia respectively, which resulted in an increase in inflammatory cytokines and MMPs expression. These results provide the insight into the different sensitivities of glial cells to PrP.

Krüppel-like factor 4 inhibits the expression of interleukin-1 beta in lipopolysaccharide-induced RAW264.7 macrophages

RAW264.7 macrophages and human peripheral blood mononuclear cells were treated with LPS to determine the expression of KLF4 and release of IL-1β. A full-length cDNA or short interference RNA of KLF4 was transfected into RAW264.7 macrophages; the expression and release of IL-1β were analyzed. The transcription and DNA binding activities of KLF4 to the IL-1β promoter were detected further. The results showed LPS treatment resulted in the increase of KLF4 level and IL-1β release; KLF4 overexpression decreased the expression of IL-1β, while KLF4 inhibition increased the expression of IL-1β; overexpression of KLF4 promoted the DNA binding activity of KLF4 to the IL-1β promoter and attenuated the transcription of IL-1β promoter, indicating an important role of KLF4 in regulating expression of IL-1β.

Regulation of inflammatory arthritis by the upstream kinase mitogen activated protein kinase kinase 7 in the c-Jun N-Terminal kinase pathway

IntroductionThe c-Jun N-terminal kinase (JNK) is a key regulator of matrix metalloproteinase (MMP) and cytokine production in rheumatoid arthritis (RA) and JNK deficiency markedly protects mice in animal models of arthritis. Cytokine-induced JNK activation is strictly dependent on the mitogen-activated protein kinase kinase 7 (MKK7) in fibroblast-like synoviocytes (FLS). Therefore, we evaluated whether targeting MKK7 using anti-sense oligonucleotides (ASO) would decrease JNK activation and severity in K/BxN serum transfer arthritis.MethodsThree 2'-O-methoxyethyl chimeric ASOs for MKK7 and control ASO were injected intravenously in normal C57BL/6 mice. PBS, control ASO or MKK7 ASO was injected from Day -8 to Day 10 in the passive K/BxN model. Ankle histology was evaluated using a semi-quantitative scoring system. Expression of MKK7 and JNK pathways was evaluated by quantitative PCR and Western blot analysis.ResultsMKK7 ASO decreased MKK7 mRNA and protein levels in ankles by about 40% in normal mice within three days. There was no effect of control ASO on MKK7 expression and MKK7 ASO did not affect MKK3, MKK4 or MKK6. Mice injected with MKK7 ASO had significantly less severe arthritis compared with control ASO (P < 0.01). Histologic evidence of synovial inflammation, bone erosion and cartilage damage was reduced in MKK7 ASO-treated mice (P < 0.01). MKK7 deficiency decreased phospho-JNK and phospho-c-Jun in ankle extracts (P < 0.05), but not phospho-MKK4. Interleukin-1beta (IL-1β), MMP3 and MMP13 gene expression in ankle joints were decreased by MKK7 ASO (P < 0.01).ConclusionsMKK7 plays a critical regulatory role in the JNK pathway in a murine model of arthritis. Targeting MKK7 rather than JNK could provide site and event specificity when treating synovitis.

Effects of 15-Deoxy-Δ12,14-Prostaglandin J2 on the Cyclooxygenase-2 Expression in the Murine Lung in the Presence of Lipopolysaccharide

A previous study has demonstrated that 15-deoxy-delta12,14-prostaglandin J2 (15d-PG J2) enhanced acute lung injury induced by lipopolysaccharide (LPS) in mice. The enhancement in acute lung injury by 15d-PG J2 was concomitant with the enhanced expression of interleukin-1beta and chemokines in the lung. However, other underlying mechanisms of this enhancement remain to be elucidated. Cyclooxygenase (COX)-2 has been reported to be involved in enhanced pulmonary permeability during acute lung injury. This study investigated the effects of 15d-PG J2 on COXs expressions in the lung in the presence or absence of LPS. ICR mice were divided into 4 experimental groups that intratracheally received vehicle, lipopolysaccharide (LPS: 125 microg/kg), 15d-PG J2 (1 mg/kg), or 15d-PG J2 + LPS. The expression of mRNA for both COX-1 and -2 in the lung was evaluated 4 h after the intratracheal administration. 15d-PG J2 enhanced the COX-2 mRNA expression in the presence of LPS. In contrast, 15d-PG J2 did not affect the COX-1 expression. These results suggest that the enhancing effects of 15d-PG J2 on LPS-induced acute lung injury might be explained, at least in part, by those on the lung expression of COX-2.

Microglia and cyclooxygenase-2: Possible therapeutic targets of progesterone for stroke

Previous studies have demonstrated that progesterone (PROG) may be a pleiotropic neuroprotective agent. Although there have been reports about the neurotoxicity of activated microglia and cyclooxygenase-2 (COX-2) in animal models of ischemic stroke, the influence of PROG on the activation of microglia and the expression of COX-2 after stroke has not been examined in detail. In this investigation, we carried out research about the influence of PROG on cultured microglia by detection of the expression of tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β) in their supernatant fluid before and after induced with lipopolysaccharide (LPS) or influenced by PROG with Enzyme-Linked Immunosorbent Assay technique in vitro. Moreover, the expression of COX-2 and ionized calcium-binding adapter molecule 1 (Iba1) was also detected in the cortex of rats that underwent permanent middle cerebral artery occlusion and received PROG or vehicle treatment by immunohistochemistry and western blot technique. The results revealed that PROG significantly reduced the expression of TNF-α and IL-1β in cultured microglia after activated with LPS in vitro. In addition, PROG also valuably inhibited the expression of Iba1 and COX-2 after stroke in vivo. These observations raised the possibility that PROG can exert its neuroprotective effects by inhibiting the activation of microglia and the over expression of COX-2 after stroke.

Rehmannia glutinosa Ameliorates Scopolamine-Induced Learning and Memory Impairment in Rats

Many studies have shown that the steamed root of Rehmannia glutinosa (SRG), which is widely used in the treatment of various neurodegenerative diseases in the context of Korean traditional medicine, is effective for improving cognitive and memory impairments. The purpose of this study was to examine whether SRG extracts improved memory defects caused by administering scopolamine (SCO) into the brains of rats. The effects of SRG on the acetylcholinergic system and proinflammatory cytokines in the hippocampus were also investigated. Male rats were administered daily doses of SRG (50, 100, and 200 mg/kg, i.p.) for 14 days, 1 h before scopolamine injection (2 mg/kg, i.p.). After inducing cognitive impairment via scopolamine administration, we conducted a passive avoidance test (PAT) and the Morris water maze (MWM) test as behavioral assessments. Changes in cholinergic system reactivity were also examined by measuring the immunoreactive neurons of choline acetyltransferase (ChAT) and the reactivity of acetylcholinesterase (AchE) in the hippocampus. Daily administration of SRG improved memory impairment according to the PAT, and reduced the escape latency for finding the platform in the MWM. The administration of SRG consistently significantly alleviated memory-associated decreases in cholinergic immunoreactivity and decreased interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) mRNA expression in the hippocampus. The results demonstrated that SRG had a significant neuroprotective effect against the neuronal impairment and memory dysfunction caused by scopolamine in rats. These results suggest that SRG may be useful for improving cognitive functioning by stimulating cholinergic enzyme activities and alleviating inflammatory responses.

Folic acid inhibits lipopolysaccharide-induced inflammatory response in RAW264.7 macrophages by suppressing MAPKs and NF-κB activation

The aim of the present study was to investigate the effects of folic acid on the inflammatory responses to lipopolysaccharide (LPS) in RAW264.7 cells and the signal transduction pathways involved. RAW264.7 macrophages were used. The production of nitric oxide (NO) was determined by the Griess test. The protein levels and mRNA expression of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) were determined by ELISA and RT-PCR, respectively. The phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear nuclear factor-kappa B (NF-κB) p65 protein were analyzed by Western blotting. Folic acid inhibited LPS-induced production of NO, TNF-α and IL-1β with decreased mRNAs of inducible nitric oxide synthase (iNOS), TNF-α and IL-1β. Further study showed that folic acid inhibited the LPS-induced phosphorylation of MAPKs and the nuclear translocation of NF-κB. Folic acid inhibits the inflammatory response of RAW 264.7 cells to LPS through inhibiting the MAPKs and NF-κB pathways.

Abstract 2030: A novel role for the 185delAG BRCA1 mutant protein, BRAT, in ovarian cancer pathology

Abstract Objective: Familial history is the strongest risk factor for developing ovarian cancer (OC) and is associated with BRCA1 truncation mutations, such as the founder mutation 185delAG. Studying these risk-associated mutations may reveal signaling pathways important in OC etiology. Normal human ovarian surface epithelial (HOSE) cells expressing the 185delAG mutant, BRAT, exhibit phenotypic changes due, in part, to up-regulation of maspin, a tumor suppressor important in apoptosis, invasion, and metastasis that exhibits unique up-regulation in some cancer types. In the current study, we determined BRAT's impact on expression of Interleukin-1beta (IL1β), another key player in invasion and metastasis. Methods: HOSE cells transiently and stably expressing the 185delAG mutation and HOSE cells with endogenous 185delAG were analyzed for differential target gene expression by semi-quantitative, real time PCR, western blot; as well as, IL1β bioactivity. Western blot was used to identify cell signaling targets of BRAT mediated IL1β expression via MAPK pathways. Breast epithelial and breast cancer cells transiently and stably expressing BRAT were evaluated by real time PCR to determine whether BRAT induced IL1β is tissue specific. Results: BRAT-expressing HOSE cells exhibited enhanced IL1β mRNA expression. Higher levels of pro-IL1β protein were detected by Western blotting of cell lysate and conditioned media. Increased phosphorylation of MAPK pathway constituents were seen in BRAT-expressing HOSE cells as detected by Western blotting of cell lysate. However, expression of BRAT in normal breast and breast cancer cell lines failed to significantly alter IL1β expression levels. Conclusion: Normal HOSE cells expressing the BRAT mutation exhibit increased expression of IL1β. Further investigation is warranted to elucidate the mechanism of tissue-specific BRAT-mediated IL1β expression since increased IL1β expression may represent an early step of malignant transformation contributing to ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2030. doi:10.1158/1538-7445.AM2011-2030

Effect of spearmint oil on lipopolysaccharide induced emphysema-like changes and expression of matrix metalloproteinase-9

To investigate the effect of spearmint oil on emphysema-like changes and the expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta(IL-1beta), matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-9) in lipopolysaccharide (LPS) treated rats. Emphysematous changes model was induced by intratracheal instillation of LPS once a week for up to 8 weeks in rats. Rats were divided into control, dexamethasone (0.3 mg x kg(-1)), and spearmint oil (10, 30,100 mg x kg(-1)) groups. Each group was treated with saline, dexamethasone, and spearmint of oil respectively for 4 weeks. Then total and different white blood cell counts in bronchoalveolar lavage fluid(BALF) were carried out. The pathologic changes of lung tissue such as alveolar structure, airway inflammation, and goblet cell metaplasia were observed by HE and AB-PAS staining. Expression of TNF-alpha, IL-1beta, TIMP-1 and MMP-9 were measured. Both spearmint and dexamethasone decreased the destruction of pulmonary alveolus. The total and different white blood cell counts in BALF including neutrophile and lymphocyte of spearmint oil 100 mg x kg(-1) and dexamethasone group were significantly reduced, and the goblet cell metaplasia was also inhibited. Dexamethasone had inhibitory effect on the expression of TNF-alpha, IL-1beta, TIMP-1 and MMP-9. Spearmint oil 30, 100 mg x kg(-1) significantly reduced TNF-alpha and IL-1beta respectively. Spearmint oil 10, 30 and 100 mg x kg(-1) had no effect on the expression of TIMP-1, but could decrease the expression of MMP-9 significantly in lung tissues. Spearmint oil has protective effect on rats with emphysematous changes, since it improves alveolar destruction, pulmonary inflammation, and goblet cell metaplasia. The mechanism may include reducing TNF-alpha, IL-1beta content and inhibiting overexpression of matrix metalloproteinase-9 in lung tissues.

Abstract 5533: A RT-PCR study of cytokine expression with macrophages and tumors

Abstract Macrophages (MØ) are very important in initiating and directing an immune response. However, it has been noted that when MØ are in the presence of a tumor, they often undergo a change in phenotype. In this environment, MØ will be converted from an aggressive phenotype (M1) to one that promotes tissue remodeling and repair (M2) through a process that is not fully understood. The purpose of this study was to identify which cytokines, in both MØ and cancer cells, show altered expression when the two are in close proximity. To accomplish this, MCF-7 (breast cancer), MDA-MB-231 (breast cancer), MDA-MB-435 (melanoma), and HT29 (colon cancer) cell cultures were grown in prescribed media followed by a 48-hour co-incubation with whole blood MØ. Lipopolysaccharide (LPS) was used as a positive control of an M1-type stimulation. The cytokine levels of both the cancer cells and MØ were then analyzed by quantitative PCR (qPRC) for expression of interleukin-1beta (IL-1β), macrophage migration inhibition factor (MIF), and tumor necrosis factor-alpha (TNF-α). Mathematical calculations were analyzed using a Step One® comparative quantification program and the cytokine expression equation 2⁁-(Δct1- Δct2). MØ IL-1β showed a fold change of 2.76, 1.58, 0.95, 3.09, 10.11 when exposed to MCF-7, MDA-MB-231, MDA-MB-435, and HT29 cells and LPS respectively. These expressions correlated with the aggressiveness of the cancer. Aggressive MDA-MB-435's down regulated IL-1β. Fold change in MIF expression was 3.14, 1.33, 7.56, and 1.22 in MCF-7, MDA-MB-231, MDA-MB-435, and HT29 cells respectively. HT29's had the lowest expression levels suggesting one reason why it's been reported that infiltration of MØ happens properly in colon cancers. Fold expression in MØ expression of TNF-α was 1.34, 1.65, 1.10, and 0.85 with MCF-7, MDA-MB-231, MDA-MB-435, and HT29 cells respectively. An interesting finding that warrants further investigation was in regards to the colon cancer. We observed with interest that the colon cancer line, HT29, caused a decrease in expression of MØ TNF-α. These findings show that colon cancer HT29 decreases TNF-α expression whereas other tested cancer lines did not. This suggests that colon cancers might evade the immune system by regulating MØ TNF-α, an important factor in initiating the caspase-8 regulated apoptotic pathway. These findings suggest that each cancer has an individual method in managing it's microenvironment in respect to MØ. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5533. doi:10.1158/1538-7445.AM2011-5533

Temporal profiles of age-dependent changes in cytokine mRNA expression and glial cell activation after status epilepticus in postnatal rat hippocampus

BackgroundStatus epilepticus (SE) is proposed to lead to an age-dependent acute activation of a repertoire of inflammatory processes, which may contribute to neuronal damage in the hippocampus. The extent and temporal profiles of activation of these processes are well known in the adult brain, but less so in the developing brain. We have now further elucidated to what extent inflammation is activated by SE by investigating the acute expression of several cytokines and subacute glial reactivity in the postnatal rat hippocampus.MethodsSE was induced by an intraperitoneal (i.p.) injection of kainic acid (KA) in 9- and 21-day-old (P9 and P21) rats. The mRNA expression of interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), matrix metalloproteinase-9 (MMP-9), glial-derived neurotrophic factor (GDNF), interferon gamma (IFN-γ), and transforming growth factor-beta 1 (TGF-β1) were measured from 4 h up to 3 days after KA injection with real-time quantitative PCR (qPCR). IL-1β protein expression was studied with ELISA, GFAP expression with western blotting, and microglial and astrocyte morphology with immunohistochemistry 3 days after SE.ResultsSE increased mRNA expression of IL-1β, TNF-α and IL-10 mRNA in hippocampus of both P9 and P21 rats, their induction being more rapid and pronounced in P21 than in P9 rats. MMP-9 expression was augmented similarly in both age groups and GDNF expression augmented only in P21 rats, whereas neither IFN-γ nor TGF-β1 expression was induced in either age group. Microglia and astrocytes exhibited activated morphology in the hippocampus of P21 rats, but not in P9 rats 3 d after SE. Microglial activation was most pronounced in the CA1 region and also detected in the basomedial amygdala.ConclusionOur results suggest that SE provokes an age-specific cytokine expression in the acute phase, and age-specific glial cell activation in the subacute phase as verified now in the postnatal rat hippocampus. In the juvenile hippocampus, transient increases in cytokine mRNA expression after SE, in contrast to prolonged glial reactivity and region-specific microglial activity after SE, suggest that the inflammatory response is changed from a fulminant and general initial phase to a more moderate and specific subacute response.

Neuronal NR2B-containing NMDA receptor mediates spinal astrocytic c-Jun N-terminal kinase activation in a rat model of neuropathic pain

Spinal N-methyl d-aspartate receptor (NMDAR) plays a pivotal role in nerve injury-induced central sensitization. Recent studies suggest that NMDAR also contributes to neuron-astrocyte signaling. c-Jun N-terminal kinase (JNK) is persistently and specifically activated (indicated by phosphorylation) in spinal cord astrocytes after nerve injury and thus it is considered as a dependable indicator of pain-related astrocytic activation. NMDAR-mediated JNK activation in spinal dorsal horn might be an important form of neuron-astrocyte signaling in neuropathic pain. In the present study, we observed that intrathecal injection of MK-801, a noncompetitive NMDA receptor antagonist, or Ro25-6981 and ifenprodil, which are selective antagonists of NR2B-containing NMDAR each significantly reduced nerve injury-induced JNK activation. Double immunostaining showed that NR2B was highly expressed in neurons, indicating the effect of NMDAR antagonists on JNK activation was indirect. We further observed that intrathecal injection of NMDA (twice a day for 3 days) significantly increased spinal JNK phosphorylation. Besides, NMDAR-related JNK activation could be blocked by a neuronal nitric oxide synthase (nNOS) selective inhibitor (7-nitroindazole sodium salt) but not by a nNOS sensitive guanylyl cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). Finally, real-time RT-PCR and immunostaining showed that nerve injury-induced interleukin-1beta expression was dependent on astrocytic JNK activation. Treatments targeting NMDAR-nNOS pathway also influenced interleukin-1beta expression, which further confirmed our hypothesis. Taken together, our results suggest that neuronal NMDAR-nNOS pathway could activate astrocytic JNK pathway. Excitatory neuronal transmission initiates astrocytic activation-induced neuroinflammation in this way, which contributes to nerve injury-induced neuropathic pain.

Src signaling involvement in Japanese encephalitis virus-induced cytokine production in microglia

Numerous studies have demonstrated that the disease pathogenesis of Japanese encephalitis involves cytokine-mediated bystander damage. The mechanisms involved in the regulation of Japanese encephalitis virus (JEV)-induced cytokine expression are not well defined but rely mainly on the tight regulation of transcription factor NF-κB. The Src-family tyrosine kinases participate in diversity of cellular signaling and have been demonstrated in JEV-infected cells. A direct link leading from Src activation to NF-κB activation in JEV-induced cytokine expression is incompletely understood. Here, we report that Src-related Ras/Raf/extracellular signal-regulated kinase (ERK) cascades participate in NF-κB activation and consequent tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) expression in JEV-infected microglia. Central microglia were capable of producing TNF-α and IL-1β after JEV infection. However, JEV infection had a negligible effect on triggering TNF-α and IL-1β production by neurons and astrocytes. The expression of TNF-α and IL-1β caused by JEV was accompanied by increased Src phosphorylation, Ras membrane association, Raf serine-338 as well as tyrosine-340 phosphorylation, ERK phosphorylation, NF-κB DNA binding activity, and decreased Raf serine-259 phosphorylation. Pharmacological studies revealed that the integrity of lipid raft and the activation of Src, Ras, Raf, ERK, and NF-κB all contributed to JEV-induced TNF-α and IL-1β expression. Pharmacological and biochemical studies further suggested that Src, upon activation, might transmit signals to the Raf/ERK cascades via Ras-dependent and -independent mechanisms that in turn might lead to NF-κB activation. Overall, our results show that the lipid raft might play a role in mediating JEV-initiated Src/Ras/Raf/ERK/NF-κB signaling and TNF-α/IL-1β expression in microglia.

Optogenetic Stimulation Enhances Interleukin-1beta Levels in Cultured Neurons

The pro-inflammatory cytokine interleukin-1beta (IL-1) has a role in physiological regulation, e.g. sleep, and in pathology including the chronic inflammation associated sleep disorders and in the neuropathology of brain trauma, epilepsy and stroke. Sleep and inflammation are affected by cell activity. However, a mechanistic connection between specific brain activity at the cellular level and IL-1 expression remains to be established. We have recently developed an investigative tool based on an in vitro culture of neurons and glial cells forming a network.

Spinal Cord Injury Causes Sustained Disruption of the Blood-Testis Barrier in the Rat

There is a high incidence of infertility in males following traumatic spinal cord injury (SCI). Quality of semen is frequently poor in these patients, but the pathophysiological mechanism(s) causing this are not known. Blood-testis barrier (BTB) integrity following SCI has not previously been examined. The objective of this study was to characterize the effects of spinal contusion injury on the BTB in the rat. 63 adult, male Sprague Dawley rats received SCI (n = 28), laminectomy only (n = 7) or served as uninjured, age-matched controls (n = 28). Using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), BTB permeability to the vascular contrast agent gadopentate dimeglumine (Gd) was assessed at either 72 hours-, or 10 months post-SCI. DCE-MRI data revealed that BTB permeability to Gd was greater than controls at both 72 h and 10 mo post-SCI. Histological evaluation of testis tissue showed increased BTB permeability to immunoglobulin G at both 72 hours- and 10 months post-SCI, compared to age-matched sham-operated and uninjured controls. Tight junctional integrity within the seminiferous epithelium was assessed; at 72 hours post-SCI, decreased expression of the tight junction protein occludin was observed. Presence of inflammation in the testes was also examined. High expression of the proinflammatory cytokine interleukin-1 beta was detected in testis tissue. CD68+ immune cell infiltrate and mast cells were also detected within the seminiferous epithelium of both acute and chronic SCI groups but not in controls. In addition, extensive germ cell apoptosis was observed at 72 h post-SCI. Based on these results, we conclude that SCI is followed by compromised BTB integrity by as early as 72 hours post-injury in rats and is accompanied by a substantial immune response within the testis. Furthermore, our results indicate that the BTB remains compromised and testis immune cell infiltration persists for months after the initial injury.

Glial‐derived neurotrophic factor regulates intestinal epithelial barrier function and inflammation and is therapeutic for murine colitis

Although enteric glial cells (EGCs) have been demonstrated to play a key role in maintaining intestinal epithelial barrier integrity, it is not known how EGCs regulate this integrity. We therefore hypothesized that glial-derived neurotrophic factor (GDNF) produced by EGCs might be involved in this regulation. Here we investigated the role of GDNF in regulating epithelial barrier function in vivo. Recombinant adenoviral vectors encoding GDNF (Ad-GDNF) were administered intracolonically in experimental colitis induced by dextran sulphate sodium (DSS). The disease activity index (DAI) and histological score were measured. Epithelial permeability was assayed using Evans blue dye. The anti-apoptotic potency of GDNF in vivo was evaluated. The expression of tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and myeloperoxidase (MPO) activity were measured by ELISA assay and/or RT-PCR. The expression of ZO-1, Akt, caspase-3, and NF-kappaB p65 was analysed by western blot assay. Our results showed that GDNF resulted in a significant reduction in enhanced permeability, inhibited MPO activity, IL-1beta and TNF-alpha expression, and increased ZO-1 and Akt expression. Moreover, GDNF strongly prevented apoptosis in vivo and significantly ameliorated experimental colitis. Our findings indicate that GDNF participates directly in restoring epithelial barrier function in vivo via reduction of increased epithelial permeability and inhibition of mucosal inflammatory response, and is efficacious in DSS-induced colitis. These findings support the notion that EGCs are able to regulate intestinal epithelial barrier integrity indirectly via their release of GDNF in vivo. GDNF is namely an important mediator of the cross-talk between EGCs and mucosal epithelial cells. GDNF may be a useful therapeutic approach to the treatment of inflammatory bowel disease.

Inhibition of titanium particle‐induced inflammatory osteolysis through inactivation of cannabinoid receptor 2 by AM630

Wear particle could induce inflammatory osteolysis and is the primary pathological factor for aseptic loosening. Although it is known that cannabinoid receptor 2 (CB2) inhibits osteoclast differentiation, the effect on inflammatory osteolysis induced by wear particles remains unclear. This study examined the effect of CB2 in the regulation of osteoclast differentiation in a murine macrophage cell line (RAW264.7), which has been shown to be stimulated by titanium (Ti) particles and receptor activator of the NF-kappaB ligand (RANKL). Results showed that CB2 expression in RAW cells cultured with Ti particles and RANKL. CB2 inactivation by AM630, a CB2 selective antagonist, effectively inhibited osteoclastogenesis in the differentiation medium system. AM630 treatment (> or =100 nM) significantly reduced the number of tartrate-resistant acid phosphatase-positive cells when compared with the control. Real-time reverse transcription polymerase chain reaction analysis revealed that AM630 (100 nM) inhibited mRNA expression of RANK and cathepsin K in RAW cells stimulated by Ti particles and RANKL. Moreover, enzyme-linked immunosorbent assay showed that AM630 (100 nM) reduced protein expression of interleukin-1beta and tumor necrosis factor-alpha in RAW cells cultured with Ti particles. In addition, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide revealed that AM630 had no toxic effect on RAW cells. These results suggested that CB2 inactivation by AM630 could provide a promising therapeutic target for treating or preventing aseptic loosening.

Apelin plays a catabolic role on articular cartilage: In vivo and in vitro studies

Adipokines play key roles in the regulation of bone growth, obesity, diabetes mellitus type 2, and HIV infection. As a newly discovered hormone in the adipokine family, the precise role of apelin on articular cartilage metabolism is not yet clear. The aim of this study was to evaluate the role of apelin on articular cartilage. In vitro, we examined the effects of apelin on normal chondrocyte proliferation and gene expression of metalloproteinases (MMPs) and interleukin-1beta (IL-1beta). In vivo, by intra-articular injection with apelin, we examined MMP-3, -9, collagen II and IL-1beta at both gene and protein levels. Furthermore, we measured the messenger RNA (mRNA) expression of ADAMTS-4 and -5 (a disintegrin and metalloproteinase with thrombospondin motifs 4 and 5) and the proteoglycan content in articular cartilage. Apelin stimulated the proliferation of chondrocytes and significantly increased mRNA levels of MMP-1, -3, -9 and IL-1beta in vitro. Intra-articular injection with apelin in vivo up-regulated the expression of MMP-3, -9, and IL-1beta as well as decreased the level of collagen II. Additionally, after treatment with apelin, mRNA levels of ADAMTS-4 and -5 markedly increased and depletion of proteoglycan in articular cartilage was found by histological assessment. These findings suggest that apelin plays a catabolic role in cartilage metabolism and is a risk factor in the pathophysiology of osteoarthritis.

A Cytokine-Inducing Hemagglutinin from Small Taros

A 22.4-kDa dimeric hemagglutinin was isolated from tubers of Colocasia esculenta cv. 'Small Taro' by employing a purification protocol that involved ion exchange chromatography on Q-Sepharose, fast protein liquid chromatography (FPLC)-ion exchange chromatography on Mono Q, and FPLC-gel filtration on Superdex 75. The hemagglutinin was isolated from the fraction of the taro extract adsorbed on Q-Sepharose and subsequently adsorbed on Mono Q. The major absorbance peak from the Superdex 75 column constituted purified hemagglutinin. Its hemagglutinating activity could not be inhibited by simple sugars, and was stable after exposure for 30 minutes to temperatures up to 40 degrees C and to ambient pH in the range of pH 2 to pH 13. The activity decreased progressively when the ambient temperature was raised from 40 degrees C to 100 degrees C. Negligible activity was detected at 100 degrees C. The activity plummeted, with about 40% and 10% remaining, 4 minutes and 20 minutes after exposure to 100 degrees C, respectively. About half of the activity remained at pH 0 and pH 1 whereas the activity was completely abolished at pH 14. The hemagglutinin exhibited slight anti-tumor activity toward hepatoma HepG2 cells, and weak mitogenic activity toward murine splenocytes. It induced expression of the cytokines interleukin-1 beta, interleukin-2, interferon-gamma and tumor necrosis factor-alpha. However, it was devoid of anti-fungal activity toward a number of fungal species.

Profiling lipoxygenase metabolism in specific steps of colorectal tumorigenesis.

Lipoxygenases (LOX) are key enzymes for the oxidative metabolism of polyunsaturated fatty acids into biologically active products. Clinical data on comparative levels of various LOX products in tumorigenesis are lacking. Therefore, we examined the profiles of several LOX products (5-LOX, 12-LOX, 15-LOX-1, and 15-LOX-2) by liquid chromatography/tandem mass spectrometry in the major steps of colorectal tumorigenesis (normal, polyp, and cancer) in a clinical study of 125 subjects (49 with normal colon, 36 with colorectal polyps, and 40 with colorectal cancer) who underwent prospective colorectal biopsies to control for various potential confounding factors (e.g., diet, medications). Mean 13-hydroxyoctadecadienoic acid (13-HODE) levels were significantly higher in normal colon [mean, 36.11 ng/mg protein; 95% confidence interval (95% CI), 31.56-40.67] than in paired colorectal cancer mucosa (mean, 27.01 ng/mg protein; 95% CI, 22.00-32.02; P = 0.0002), and in normal colon (mean, 37.15 ng/mg protein; 95% CI, 31.95-42.34) than in paired colorectal polyp mucosa (mean, 28.07 ng/mg protein; 95% CI, 23.66-32.48; P < 0.001). Mean 13-HODE levels, however, were similar between the left (mean, 37.15 ng/mg protein; 95% CI, 31.95-42.35) and the right normal colon (mean, 32.46 ng/mg protein; 95% CI, 27.95-36.98; P = 0.09). No significant differences with regard to 12- or 15-hydroxyeicosatetraenoic acid or leukotriene B(4) levels were detected between normal, polyp, and cancer mucosae. 15-LOX-1 inhibited interleukin-1beta expression. This study establishes that reduced 13-HODE levels are a specific alteration in the LOX product profile associated with human colorectal tumorigenesis.