Previously, we demonstrated that spontaneous TNF-α production by macrophages in rheumatoid arthritis (RA) synovial tissue is largely driven by contact-dependent activation with T cells in that tissue. Whereas abundant IL-10 is present in these RA synovial cultures, it does not adequately control the production of TNF-α. In this study, we have compared the mechanisms involved in IL-10–mediated TNF-α regulation in LPS-stimulated macrophages with macrophages stimulated with activated T cells. We confirm that in LPS-stimulated macrophages the 3′ enhancer region of tnf is essential for tnf transcription, and its regulation by IL-10 is dominated by a STAT3-dependent pathway. However, in contrast, we have found that tnf transcription in macrophages stimulated by activated T cells or by RA synovial T cells does not require the 3′ enhancer region of tnf, and that its regulation by IL-10 is subsequently altered and clearly is not mediated by a dominant STAT3 pathway. These observations have very important implications for our understanding as to how IL-10 regulates TNF-α production at sites of chronic inflammation, such as the synovial tissue of patients with RA. Furthermore, these distinct IL-10 mechanisms will have bearing upon the identification of potential therapeutic targets in RA synovial macrophages where the activation stimulus is clearly not LPS.

ABSTRACT Herpes simplex virus 1 replication initiates angiogenesis and inflammation in the cornea. This can result in herpetic stromal keratitis (HSK), which is a leading cause of infection-induced corneal blindness. Host cellular factors mediate the progression of HSK, but little is known about these cellular factors and their mechanisms of action. We show here that the expression of the cellular transcription factor early growth response 1 (Egr-1) in HSV-1-infected mouse corneas was enhanced. Enhanced Egr-1 expression aggravated HSK by increasing viral replication and subsequent neovascularization with high levels of potent angiogenic factors, fibroblast growth factor 2, and vascular endothelial growth factor. Furthermore, Egr-1 deficiency due to a targeted disruption of the gene or knockdown of Egr-1 expression topically using a DNA-based enzyme significantly reduced HSK by decreasing both viral replication and the angiogenic response. The present study provides the first evidence that endogenous Egr-1 aggravates HSK and that blocking Egr-1 reduces corneal damage.

5 Hui-Wen Yao, Shih-Heng Chen, Ching Li, Yuk-Ying Tung, and 6 Shun-Hua Chen 7 8 9 Institute of Basic Medical Sciences,Department of Microbiology and Immunology, 10 and Infectious Disease and Signal Research Center, College of Medicine; and 11 Statistical Analysis Laboratory, Institute of Education, College of Social Sciences, 12 National Cheng Kung University, Tainan, Taiwan 701, Republic of China; 13 Department of Microbiology and Immunology, College of Life Sciences, National 14 Chiayi University, Chiayi, Taiwan 600, Republic of China 15 16 17 18 Running head: Egr-1 aggravates HSV-1-induced corneal disease 19 20 21 22 Address correspondence to Shun-Hua Chen, shunhua@mail.ncku.edu.tw 23 24 Copyright © 2012, American Society for Microbiology. All Rights Reserved. J. Virol. doi:10.1128/JVI.00505-12 JVI Accepts, published online ahead of print on 30 May 2012

Peripheral blood CD4(+) CD45RO(+) T cells activated in vitro are able to induce expression of tumour necrosis factor-α (TNF-α) in monocytes via a contact-dependent mechanism. Activation is achieved either with interleukin-2 (IL-2)/IL-6/TNF-α over an 8-day period or cross-linking CD3 using anti-CD3 antibody for 48 hr. In this paper, we show that the p38 mitogen-activated protein kinase (MAPK) signalling pathway played different roles in the generation of effector function in these two types of activated T cells. In anti-CD3 activated T cells, p38 MAPK is a negative regulator for anti-CD3 induced cell proliferation and has no significant effect on the acquisition of either the effector function (induction of monocyte-derived TNF-α) or production of T-cell cytokines. In contrast, the p38 MAPK signalling pathway is required for the acquisition of cytokine-induced effector function and promotes cell proliferation and cytokine production.

Background: Cancers have become one of the most lethal diseases in elders. In traditional Chinese medicine, Tan-Chih-Hsiao-Yao-San (TCHYS) and Long-Daan-Shiah-Gan-Tang (LDSGT) are used to treat cancers. However, the growth-inhibitory effects and gene expression profiles of these drugs on cancer cells are still unclear.Methods: This study assessed the effects of TCHYS and LDSGT on viability of liver and bladder tumor cells, and bladder TCCSUP cells were further subjected to profile gene expression patterns with microarray technology for identifying gene candidates that may be involved in the tumorigenesis.Results: The results revealed that both drugs significantly eliminated the growth of Chang liver and three hepatoma cells. On the contrary, the embryonic liver WRL68 cells showed less response to the treatments, whereas the control agent genistein had much higher inhibitory effect in WRL68 cells than in the other hepatoma cells. Both TCHYS and LDSGT, as well as cisplatin and paclitaxel, exhibited dose-dependent suppression on the viability of all bladder cancer cells. To characterize the possible regulation for such effects, the profiling of gene expression was performed with complementary DNA chips. When bladder transitional cell carcinoma (TCC) TCCSUP cells were treated with TCHYS, 29 upregulated and 28 downregulated genes were detected; whereas 54 genes were upregulated in the same cells treated with LDSGT. Moreover, the detected gene expression patterns were also confirmed by using the reverse transcription-polymerase chain reaction assay.Conclusion: This study initiates the evaluations of drug efficacies and gene expression profiles of traditional Chinese medicines, which may provide important information and identify useful biomarkers for treating cancers. Copyright (C) 2011, Taiwan Society of Geriatric Emergency & Critical Care Medicine. Published by Elsevier Taiwan LLC. All rights reserved.

The Chinese medicinal herbs Radix Isatidis and Viola yedoensis Makino have been suggested to possess antiviral activity. This study tests whether these and other Chinese and Western herbal medicinal formulas can modulate the immune functions involving virus-suppression in BALB/c mouse. We first confirmed the extract from Viola yedoensis Makino, but not from Radix Isatidis, the traditional Chinese medicine (TCM) formula Chui-Uren-Chien (CUC), or a Western homeopathic medicinal drink Método Canova, could inhibit the replications of herpes simplex virus-1 and enterovirus 71 in the human neuroblastoma SK-N-SH cell line. Subsequently, the same herbal extracts and drink underwent toxicity and immunomodulatory tests on mice of 5–7 weeks old. After 8 weeks of feeding different herbal medicinal formulas, no hepatic or renal toxicity was noted in any tested animal; whereas among the immune function evaluations, only the mice treated with CUC extract were found to be associated with significant increases (p < 0.05) in both the level of plasma IgG and the percentage of monocyte in blood mononuclear cells as well as the activation of macrophage Raw264.7 cells for nitric oxide production, suggesting its role in modulating the non-specific immune response. Analyses using protein arrays showed CUC was the most potent herbal medicinal formula eliciting fluctuations in plasma cytokine and chemokine concentrations. Taking all experimental data together, we conclude Chui-Uren-Chien possesses immunomodulatory capability in mouse, but none of the herbal medicinal formulas tested here are involved in strengthening antiviral immunity.

Epstein-Barr virus is known to cause nasopharyngeal carcinoma. Although oral cavity is located close to the nasal pharynx, the pathogenetic role of Epstein-Barr virus (EBV) in oral cancers is unclear. This molecular epidemiology study uses EBV genomic microarray (EBV-chip) to simultaneously detect the prevalent rate and viral gene expression patterns in 57 oral squamous cell carcinoma biopsies (OSCC) collected from patients in Taiwan. The majority of the specimens (82.5%) were EBV-positive that probably expressed coincidently the genes for EBNAs, LMP2A and 2B, and certain structural proteins. Importantly, the genes fabricated at the spots 61 (BBRF1, BBRF2, and BBRF3) and 68 (BDLF4 and BDRF1) on EBV-chip were actively expressed in a significantly greater number of OSCC exhibiting exophytic morphology or ulceration than those tissues with deep invasive lesions (P = .0265 and .0141, resp.). The results may thus provide the lead information for understanding the role of EBV in oral cancer pathogenesis.

Abstract Chinese herbs Radix Isatidis and Viola yedoensis Makino have been implicated to possess antiviral activity. This study is to test whether these herbs are capable of modulating the immune function in Balb/cByJ mouse that supports such activity. We first confirmed that the extract from Viola yedoensis Makino, but not Radix Isatidis or a homeopathic medicinal drink Método Canova, could inhibit the replication of HSV-1 and enterovirus 71 in human neuroblastoma SK-N-SH cell line. Subsequently, the herbal extracts and Método Canova were subjected to the immunomodulatory tests with 120 6-8 weeks old mice. The animals were randomly divided into to 8 groups, of which 6 groups were fed with the half human-to-mouse-body-weight-adjusted doses of the herbal medicine and Método Canova for 9 weeks. At 8th week, these 6 groups and an unfed group of mice were injected peritoneally with one dose of the immune suppressant 5'-fluorouracil, and all 8 groups of animals were sacrificed a week later for immune function evaluations, which included the measurements of the WBC counts and the IgG levels, the numbers for B-, T-, and I-A/I-E-splenocytes, the proliferative rates for B- and T-splenocytes upon stimulation, and the serum cytokine and chemokine levels with protein arrays. Among the tests, we detected that serum BLC and MIP-2 levels were dramatically increased in mice fed with Radix Isatidis. This result indicates that taking the herb extract can improve the responsiveness of immune cells to inflammation, as these two chemokines possess the capability of inducing neutrophils, macrophages, and B-cells chemotaxis. However, no mouse in any group showed a clear improvement in cellular immunity that can suppress viral infections.

Herpes simplex virus (HSV)Early growth response protein 1 (EGR1)Studies in mice suggest that inhibiting host EGR1 may be useful for treating or preventing HSV-1 infection.In mice with corneal HSV-1 infection, Egr1 knockout lowered viral load and levels of inflammatory cell infiltration in CNS tissue compared with what was seen in wild-type controls.Also in infected mice, Egr1 knockdown by DNAzyme increased survival compared with survival of controls that received a mock nucleotide oligomer.Next steps could include identifying and evaluating compounds that block EGR1 activity in preclinical models of HSV-1 infection.Valtrex valacyclovir and Zovirax acyclovir, two nucleoside analogs from GlaxoSmithKline plc, are marketed to treat HSV infection.Famvir famciclovir, a synthetic acyclic guanine derivative prodrug of penciclovir from Novartis AG, is marketed for the same indication.At least 20 other companies have compounds in Phase III development or earlier to treat HSV infection.

Progress into the understanding of immunopathology in rheumatoid arthritis is reviewed in the present article with regard to pro-inflammatory cytokine production, cell activation and recruitment, and osteoclastogenesis. Studies highlight the potential importance of T helper 17 cells and regulatory T cells in driving and suppressing inflammation in rheumatoid arthritis, respectively, and highlight other potential T-cell therapeutic targets. The genetic associations of the HLA shared epitope alleles with antibodies to citrullinated peptides in rheumatoid arthritis patients indicate that T cells are providing help to B cells to produce autoantibodies, and there is increasing evidence that these autoantibodies are pathogenic in rheumatoid arthritis.

BACKGROUND:Previously we described a system whereby human peripheral blood T cells stimulated for 8 days in a cytokine cocktail acquired effector function for contact-dependent induction of proinflammatory cytokines from monocytes. We termed these cells cytokine-activated (Tck) cells and found that the signalling pathways elicited in the responding monocytes were identical whether they were placed in contact with Tck cells or with T cells isolated from rheumatoid arthritis (RA) synovial tissue.METHODS:Here, using magnetic beads and fluorescence-activated cell sorting, we extensively phenotype the Tck effector cells and conclude that effector function resides within the CD4+CD45RO+, CCR7-, CD49dhigh population, and that these cells are derived from the effector memory CD4+ T cells in resting blood.RESULTS:After stimulation in culture, these cells produce a wide range of T-cell cytokines, undergo proliferation and differentiate to acquire an extensively activated phenotype resembling RA synovial T cells. Blocking antibodies against CD69, CD18, or CD49d resulted in a reduction of tumour necrosis factor-alpha production from monocytes stimulated with CD4+CD45RO+ Tck cells in the co-culture assay. Moreover, blockade of these ligands also resulted in inhibition of spontaneous tumour necrosis factor-alpha production in RA synovial mononuclear cell cultures.CONCLUSION:Taken together, these data strengthen our understanding of T-cell effector function, highlight the multiple involvement of different cell surface ligands in cell-cell contact and, provide novel insights into the pathogenesis of inflammatory RA disease.

P38α is a protein kinase that regulates the expression of inflammatory cytokines, suggesting a role in the pathogenesis of diseases such as rheumatoid arthritis (RA) or systemic lupus erythematosus. Here, we describe the preclinical pharmacology of pamapimod, a novel p38 mitogen-activated protein kinase inhibitor. Pamapimod inhibited p38α and p38β enzymatic activity, with IC50 values of 0.014 ± 0.002 and 0.48 ± 0.04 μM, respectively. There was no activity against p38δ or p38γ isoforms. When profiled across 350 kinases, pamapimod bound only to four kinases in addition to p38. Cellular potency was assessed using phosphorylation of heat shock protein-27 and c-Jun as selective readouts for p38 and c-Jun NH2-terminal kinase (JNK), respectively. Pamapimod inhibited p38 (IC50, 0.06 μM), but inhibition of JNK was not detected. Pamapimod also inhibited lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF) α production by monocytes, interleukin (IL)-1β production in human whole blood, and spontaneous TNFα production by synovial explants from RA patients. LPS- and TNFα-stimulated production of TNFα and IL-6 in rodents also was inhibited by pamapimod. In murine collagen-induced arthritis, pamapimod reduced clinical signs of inflammation and bone loss at 50 mg/kg or greater. In a rat model of hyperalgesia, pamapimod increased tolerance to pressure in a dose-dependent manner, suggesting an important role of p38 in pain associated with inflammation. Finally, an analog of pamapimod that has equivalent potency and selectivity inhibited renal disease in lupus-prone MRL/lpr mice. Our study demonstrates that pamapimod is a potent, selective inhibitor of p38α with the ability to inhibit the signs and symptoms of RA and other autoimmune diseases.

Herpes simplex virus type 1 (HSV-1) infection is the most common cause of sporadic, fatal encephalitis, but current understanding of how the virus interacts with cellular factors to regulate disease progression is limited. Here, we show that HSV-1 infection induced the expression of the cellular transcription factor early growth response 1 (Egr-1) in a human neuronal cell line. Egr-1 increased viral replication by activating promoters of viral productive cycle genes through binding to its corresponding sequences in the viral promoters. Mouse studies confirmed that Egr-1 expression was enhanced in HSV-1-infected brains and that Egr-1 functions to promote viral replication in embryonic fibroblasts. Furthermore, Egr-1 deficiency or knockdown of Egr-1 by a DNA-based enzyme greatly reduced the mortality of HSV-1-infected mice by decreasing viral loads in tissues. This study provides what we believe is the first evidence that Egr-1 increases the mortality of HSV-1 encephalitis by enhancing viral replication. Moreover, blocking this cellular machinery exploited by the virus could prevent host mortality.

A mutant library of 249 mutants with mutations that span the entire Epstein-Barr virus (EBV) genome was generated by transposition with EZ : : TN and insertion with an apramycin resistance gene by a PCR-targeting method. This study also demonstrates the feasibility of generating deletions and site-specific mutations in the BRLF1 promoter on the EBV genome to determine the regions in the promoter that are crucial to transcription. Analysing BZLF1 and BRLF1 mutants by microarray analysis revealed that these two genes regulate the transcription of EBV lytic genes differently. A BZLF1 mutation affects global expression of EBV lytic genes; almost no lytic gene is expressed by the mutant after lytic induction. However, although a BRLF1 mutant still transcribes most lytic genes, the expression of these lytic genes is inefficient. Furthermore, this study shows that the proximal Zta-response element in the BRLF1 promoter is crucial to BRLF1 transcription from the EBV genome, despite the fact that another work demonstrated that this site was unimportant in transient transfection analysis. Furthermore, mutants with a mutation in BDLF1 and BORF1 cannot assemble viral capsids. Results of this study demonstrate the usefulness of a comprehensive mutant library in genetic analyses of EBV.

BACKGROUND AND PURPOSE Agaricus blazei Murill has been reported to possess biological effects that include immunomodulatory and tumoricidal activities, but its potential effects have not been systematically analyzed in vivo. We evaluated the immunomodulatory effects of A. blazei in Balb/cByJ mice. METHODS 160 male Balb/cByJ mice were divided into four groups and treated with various quantities of intragastric A. blazei extract or distilled water for 8 to 10 weeks. Nine parameters, relating to general immune function or adaptive immunity against immunogen chicken oval albumin, were determined. RESULTS The results revealed that mice receiving A. blazei extract exhibited significantly greater serum immunoglobulin G levels, increased T-cell numbers in spleen, and elevated phagocytic capability compared with controls. Consumption of A. blazei was also associated with significant increases in oval albumin-specific serum immunoglobulin G level, delayed-type hypersensitivity, splenocyte proliferation rate, and tumor necrosis factor-alpha secretion by splenocytes. CONCLUSIONS Consumption of A. blazei extract was associated with significant enhancement of seven out of nine immune functions tested. We conclude that A. blazei Murill possesses a wide range of immunomodulatory effects in vivo.

Although allergic asthma and allergic rhinitis have recently been considered to be a single disease, many questions remain unanswered. Why do some atopic patients develop asthma symptoms and others develop allergic rhinitis symptoms? Which factors play a role in the development of different allergic phenotypes? We hypothesized that angiotensin-converting enzyme (ACE) gene polymorphism might play a role in the development of asthma phenotypes in children with allergic rhinitis. The study sample included 106 children with allergic rhinitis, but no asthma, and 105 age- and gender-matched children with allergic rhinitis and asthma. Subjects of both groups exhibited the same systemic immunologic changes and allergen sensitivities. Controls consisted of 102 healthy children. The ACE genotype was determined by polymerase chain reaction. The serum total immunoglobulin E (IgE) level, allergen-specific IgE sensitivity, and eosinophil count were also measured. The frequencies of the DD genotype were significantly higher in the children with both allergic rhinitis and asthma than in the children with allergic rhinitis but no asthma [p = 0.018; odds ratio (OR) = 3.257; (1.222-8.680)]. Results of this study suggest that ACE gene polymorphism DD genotype might play a role in the development of the asthma phenotype in children with allergic rhinitis.