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The p38 MAP kinase signal transduction pathway is an important regulator of proinflammatory cytokine production and inflammation. Defining the roles of the various p38 family members, specifically p38alpha and p38beta, in these processes has been difficult. Here we use a chemical genetics approach using knock-in mice in which either p38alpha or p38beta kinase has been rendered resistant to the effects of specific inhibitors along with p38beta knock-out mice to dissect the biological function of these specific kinase isoforms. Mice harboring a T106M mutation in p38alpha are resistant to pharmacological inhibition of LPS-induced TNF production and collagen antibody-induced arthritis, indicating that p38beta activity is not required for acute or chronic inflammatory responses. LPS-induced TNF production, however, is still completely sensitive to p38 inhibitors in mice with a T106M point mutation in p38beta. Similarly, p38beta knock-out mice respond normally to inflammatory stimuli. These results demonstrate conclusively that specific inhibition of the p38alpha isoform is necessary and sufficient for anti-inflammatory efficacy in vivo.

Background: Tacrolimus (FK506) is an immunosuppressive drug 50-100 times more potent than cyclosporine (CsA), the current mainstay of organ transplant rejection therapy. Despite being chemically unrelated, CsA and tacrolimus exert their immunosuppressive effects through the inhibition of calcineurin (CaN), a critical signaling molecule during T-lymphocyte activation. Although numerous clinical studies have proven the therapeutic efficacy of drugs within this class, tacrolimus and CsA also have a strikingly similar profile of unwanted side effects., Method: Our objective has been to identify a less toxic immunosuppressant through the modification of ascomycin (FK520). Quantitative in vitro immunosuppression and toxicity assays have demonstrated (see the accompanying article, p. 18) that we achieved our goal with L-732,531 (indolyl-ascomycin; indolyl-ASC), a 32-O-(1-hydroxyethylindol-5-yl) ascomycin derivative with an improved therapeutic index relative to tacrolimus., Results: We report that the attributes of indolyl-ASC may result from its distinctive biochemical properties. In contrast to tacrolimus, indolyl-ASC binds poorly to FK506 binding protein 12 (FKBP12), the major cytosolic receptor for tacrolimus and related compounds. However, the stability of the interaction between the FKBP12-indolyl-ASC complex and CaN is much greater than that of the FKBP12-tacrolimus complex. These distinguishing properties of indolyl-ASC result in the potent inhibition of CaN within T lymphocytes but may lower the accumulation of the drug at sites of toxicity., Conclusions: Indolyl-ASC may define those properties needed to increase the therapeutic efficacy of a macrolactam immunoregulant for treating both human autoimmune disease and organ transplant rejection.

Community-acquired pneumonia occurs 3 to 4 million times per year in the United States, accounting for about 500,000 hospitalizations annually. Empiric treatment is usually instituted because of a lack of early organism-specific diagnostic tests. This study compared empiric therapy with ofloxacin to standard antibiotic regimens (usually a beta-lactam with or without a macrolide) for patients hospitalized for community-acquired pneumonia. Therapy was administered to 298 patients (146 receiving ofloxacin and 152 receiving standard therapy); 227 patients (ofloxacin, 109; standard treatment, 118) were evaluable for treatment efficacy. The most common pyogenic respiratory pathogens were Haemophilus influenzae (30 isolates) and Streptococcus pneumoniae (24 isolates). There was evidence of infection with either Mycoplasma pneumoniae (38 patients), Chlamydia pneumoniae (40 patients), or a Legionella sp. (8 patients) in a total of 79 patients (35%). The clinical success rates were similar in both groups among evaluable patients (92%, ofloxacin; 87%, standard therapy) and among patients with atypical respiratory pathogens (88%, ofloxacin; 81%, standard therapy). The mean numbers (+/- the standard deviations) of intravenous doses of antibiotics were 7.5 +/- 8.0 in the ofloxacin group and 18.4 +/- 18.5 in the standard therapy group (P < 0.001); the mean number of oral doses of ofloxacin per patient was 19.7 +/- 11.2, compared with 30.2 +/- 16.0 oral antibiotic doses in the standard therapy group (P < 0.001). All treatments were well tolerated and associated with no significant clinical or laboratory abnormalities. The findings of this study indicate that ofloxacin is active against traditional bacterial pathogens as well as the major atypical respiratory pathogens. When given as monotherapy for the empiric treatment of community-acquired pneumonia, ofloxacin is as effective as standard antimicrobial therapy.

Calcineurin, a protein phosphatase found in eukaryotic cells, presents a challenging problem in heterologous protein expression because it is both heterodimeric and posttranslationally modified. In this paper, we describe the cloning of both subunits (catalytic A and regulatory B) of calcineurin from a human cDNA library and their expression at high levels in Escherichia coli. The calcineurin A subunit is expressed as an insoluble glutathione S-transferase fusion protein, while the calcineurin B subunit is soluble upon direct expression. Catalytically active holoenzyme is derived from the separately expressed subunits using a three-step refolding protocol. First, the fusion protein is solubilized, then it is cleaved at the fusion junction with thrombin, and, finally, a catalytically competent calcineurin A:calcineurin B:calmodulin complex is reconstituted by cofolding the separately purified components. In addition, we show that a similar refolding protocol can be applied to a C-terminally truncated form of calcineurin A, which lacks an autoinhibitory and calmodulin-binding domain.

The immunosuppressant FK-506 (tacrolimus) forms a complex with a ubiquitous intracellular receptor, FK-506 binding protein (FKBP12), and this complex inhibits the heterodimeric Ca2+/calmodulin-dependent phosphatase, calcineurin, an essential component of the T-cell receptor signal transduction pathway. Using a series of truncated calcineurin catalytic subunits, we show here that a region within the catalytic subunit that regulates phosphatase activity, the autoinhibitory domain, also regulates the Ca(2+)-dependent interaction of calcineurin with the FK-506.FKBP12 complex. Deletion of this domain produces constitutive activation of the phosphatase as demonstrated by transient transfection experiments in which expression of the truncated protein permitted Ca(2+)-independent induction of interleukin-2 transcription. Thus, deletion of the autoinhibitory domain is necessary and sufficient to constitutively activate calcineurin (CaN). Furthermore, CaN A467-492, an inhibitory peptide based on the autoinhibitory domain from calcineurin (ITSFEEAKGLDRINERMPPRRDAMP), inhibited dephosphorylation of the RII peptide substrate competitively with a Ki = 4 microM, consistent with binding of the autoinhibitory domain at the active site of the enzyme. To assess the role of the autoinhibitory domain in regulating the interaction of CaN with the FK-506.FKBP12 complex, we reconstituted wild type and mutant phosphatase heterodimers using in vitro transcribed and translated subunits. Association of the reconstituted calcineurin heterodimers with FKBP12 was dependent on FK-506. In the case of the wild type heterodimer, association with the FK-506.FKBP12 complex was also dependent upon Ca2+; however, mutant catalytic subunits, in which the autoinhibitory domains were deleted, associated with the drug-binding protein complex in the presence of 10 mM EGTA. These results indicate that the conserved autoinhibitory domain regulates both Ca(2+)-dependent phosphatase activity and association with the FK-506.FKBP12 complex.

The macrolide FK-506, like the cyclic undecapeptide cyclosporin A (CsA), is a potent immunosuppressant that interferes with the transcriptional activation of several early-phase genes in T lymphocytes, including that for interleukin-2 (IL-2). We compared the effects of FK-506 and CsA on transcription from the 5' upstream activating sequences (UAS) of the human IL-2 gene and several cellular and viral UAS to define cis-acting sites which may be responsive to FK-506. The UAS surveyed included the human IL-2 receptor alpha-chain, human metallothionein II, simian virus 40 early, human cytomegalovirus immediate-early, adenovirus major late, and Rous sarcoma virus long terminal repeat UAS. In addition, we studied multimers of several defined promoter elements (NFIL-2A, NF-kappa B, or NF-AT1) which are found in the UAS of the human IL-2 gene and which have been reported to be responsive to CsA when linked to a minimal promoter element (TATA box and transcription start site). Each promoter-regulatory region was fused to the bacterial chloramphenicol acetyltransferase gene and used to transiently transfect Jurkat cells. Quantitative chloramphenicol acetyltransferase assay determinations indicated that the transcriptional activity of each UAS induced upon T-cell activation was (i) completely sensitive, (ii) partially sensitive, or (iii) resistant to inhibition by CsA and FK-506. The induced transcription driven by the IL-2 promoter elements NF-AT1 and NFIL-2A could be blocked completely by FK-506 or CsA. Gel mobility shift assays indicated that the binding activities of the factors specifically interacting with these sequences were detected in activated cells regardless of whether the cells were treated with FK-506 or CsA. The results suggest that FK-506 or CsA inhibits a transacting mechanism(s) without disrupting the binding activities of these transcription factors. The degree to which each UAS was resistant to FK-506 was consistent with the level of transcription induced by phorbol myristate acetate, while UAS which were sensitive to inhibition by FK-506 were dependent on the presence of both phorbol myristate acetate and ionomycin.

Human IL-1 alpha, IL-1 beta, and TNF-alpha mRNA expression was examined in peripheral blood monocytes (PBM) from normal individuals and in primary synoviocytes isolated from patients with rheumatoid arthritis by Northern blot and in situ hybridization. Cells cultured in the presence or absence of LPS were analyzed using in vitro synthesized 35S-labeled sense or antisense RNA probes to determine the relative abundance and the cell type expressing each of the mRNA for these potent inflammatory mediators. The results indicated that 72% of the LPS-stimulated PBM expressed detectable levels of IL-1 alpha mRNA, 89% IL-1 beta mRNA, and 10% TNF-alpha transcripts. Thus, the majority of activated PBM produced both IL-1 alpha and IL-1 beta. Experiments combining immunofluorescence for IL-1 beta protein with in situ hybridization for TNF-alpha mRNA demonstrated that monocytes expressing TNF-alpha mRNA also produced IL-1 beta. Primary synoviocytes from four patients with RA were also examined for the mRNA expression of each cytokine. Northern blot analyses of total RNA isolated from 0 to 72 h after LPS- or mock-stimulation showed that IL-1 beta mRNA was the most abundantly expressed, followed by TNF-alpha. In situ hybridization revealed that IL-1 beta and TNF-alpha transcripts were detected exclusively in synovial tissue macrophages. IL-1 alpha mRNA was not detected in these cultures by either method.

The pharmacokinetics of 1-oxa-beta-lactam (LY127935), a new semisynthetic beta-lactam antibiotic, was studied in four healthy adult volunteers (mean age of 27 years, mean body surface area +/- standard error [SE] of 1.87 +/- 0.08 m(2), and mean creatinine clearance +/- SE of 116 +/- 12 ml/min per 1.73 m(2)). Immediately after completion of a 1-g, 20-min intravenous (i.v.) infusion, the mean serum level +/- SE was 70.7 +/- 8.5 mug/ml. After a 1-g intramuscular (i.m.) injection, peak serum levels occurred from 30 min to 1 h, and the mean peak serum level +/- SE was 52.3 +/- 1.6 mug/ml. Beginning at 1 h, the serum concentrations after i.m. administration were higher than those after i.v. administration. At 8 h, the mean serum level +/- SE was 3.8 +/- 0.6 mug/ml after completion of the i.v. infusion and 4.8 +/- 0.7 mug/ml after the i.m. injection. The mean serum half-lives for the beta phase i.v. and i.m. administration were similar (2.3 +/- 0.7 h and 2.4 +/- 0.2 h, respectively). The mean apparent volume of distribution +/- SE was 16.6 +/- 1.9 liters per 1.73 m(2). The mean serum clearance +/- SE of LY127935 was 85.4 +/- 12.7 ml/min per 1.73 m(2), and the mean renal clearance +/- SE was 54.5 +/- 4.4 ml/min per 1.73 m.(2) Urine concentrations of LY127935 were at least 140 mug/ml in each volunteer during the first 12 h after i.m. or i.v. administration. The mean percentages of the dose recovered in the urine +/- SE within 2 h after i.v. or i.m. administration were similar (30 +/- 4 and 34 +/- 11, respectively). Only 67 +/- 3% and 75 +/- 13% were recovered in the urine within 24 h after i.v. and i.m. administration, respectively.

Cefazolin (CZ), cephalothin (CF), cefoxitin (CX), and cefamandole (CM) were evaluated in therapy of Staphylococcus aureus infection produced in perforated table tennis balls placed intraperitoneally in rabbits. Four weeks after placement of two balls in each rabbit, a beta-lactamase producing strain of S. aureus was injected into one of the balls. Twenty-four hours later therapy was initiated with 40 mg of CZ or 80 mg of CF, CX, or CM per kg intramuscularly every 6 h. After 24 h of treatment, the mean log(10) colony-forming units per ml were 7.1 for CZ, 6.7 for CF, 6.5 for CX, and 7.2 for CM. After 72 h the mean log(10) colony-forming units per ml were 5.0 for CZ, 4.1 for CF, 3.6 for CX, and 5.6 for CM. After 8 days, the titers were 1.6/ml for CZ, 1.0 for CF, 1.9 for CX, and 3.6 for CM. CZ serum levels were about double CF and CX levels and about two-thirds of CM levels. In sterile ball fluid CZ and CM levels were more than double CF or CX concentrations. Concentrations of all four antibiotics were lower in infected balls.

Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This virus does not only lead to pulmonary infection but can also infect other organs such as the gut, the kidney, or the liver. Recent studies confirmed that severe cases of COVID-19 are often associated with liver damage and liver failure, as well as the systemic upregulation of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNFa). However, the impact these immune mediators in the liver have on patient survival during SARS-CoV-2 infection is currently unknown. Here, by performing a post-mortem analysis of 45 patients that died from a SARS-CoV-2 infection, we find that an increased expression of TNFA in the liver is associated with elevated mortality. Using publicly available single-cell sequencing datasets, we determined that Kupffer cells and monocytes are the main sources of this TNFa production. Further analysis revealed that TNFa signaling led to the upregulation of pro-inflammatory genes that are associated with an unfavorable outcome. Moreover, high levels of TNFA in the liver were associated with lower levels of interferon alpha and interferon beta. Thus, TNFa signaling in the infected SARS-CoV-2 liver correlates with reduced interferon levels and overall survival time. [ABSTRACT FROM AUTHOR]

Purpose: Severe and fatal Capnocytophaga canimorsus infection has been described in immunocompromised patients. Data of C. canimorsus infection in immunocompetent and risk factors of severe courses are missing. Our aims were to describe the epidemiology of C. canimorsus infection and to identify potential risk factors of sepsis and fatal outcome. Methods: Observational study and systematic review of all cases reported in immunocompetent subjects between 2002 and 2019. Results: A total of 128 cases of C. canimorsus infection in immunocompetent individuals were reported. Male gender comprised 74.2%, the median age was 58 years and 47.7% were admitted with sepsis. Case-fatality rate was 29.7% and especially high in septic patients (55.7%). Transmission by bite (OR = 2.37, 95% CI: 1.05–6.52) and incubation time ≤3 d (OR = 7.98; 95% CI: 2.33–27.34) were identified as risk factors of sepsis on admission, and early wound cleansing as protective (OR = 0.42; 95% CI: 0.14–0.96). Sepsis (OR = 23.67; 95% CI: 2.85–197.89) and septic shock (OR = 45.50; 95% CI: 3.08–676.55) were risk factors of fatal outcome, whereas early wound cleansing (OR = 0.05; 95% CI: 0.01–0.72), initial penicillin therapy with beta-lactamase inhibitors (OR = 0.48; 95% CI: 0.16–0.92) and surgical removal of infectious focus (OR = 0.38; 95% CI: 0.06–0.95) were protective factors. Conclusions: Immunocompetent patients with C. canimorsus infection frequently develop sepsis. A shorter incubation period in cases of sepsis might be related to higher infectious dose. Fatal outcome may be prevented by early wound cleansing, initial use of penicillins in combination with beta-lactamase inhibitors and surgical removal of an infectious focus. [ABSTRACT FROM AUTHOR]

Tocilizumab (TCZ) is an interleukin-6 (IL-6) inhibitor used for the treatment of rheumatoid arthritis (RA). It was developed in 2008, and its effectiveness is supported by evidence from all over the world based on its first decade of use. Although the overall efficacy and safety profiles of TCZ are similar to those of tumor necrosis factor (TNF) inhibitors, TCZ displays certain differences. The most notable advantage of TCZ is its usefulness as a monotherapy. Additionally, TCZ is favorable in the improvement of systemic inflammatory symptoms such as anemia and fatigue. The low immunogenicity of TCZ contributes favorably to long-term drug retention. Due to frequent relapse after TCZ cessation, TCZ use should be tapered beyond remission. During TCZ therapy, C-reactive protein (CRP) is unable to recognize disease activity and the severity of infection. The most common adverse events (AEs) are infection and abnormalities in laboratory findings including dyslipidemia, neutropenia, thrombocytopenia, and abnormality of liver enzymes. TCZ obscures the symptoms of infection. Therefore, stealth infections without obvious CRP elevation can sometimes cause severe damage to patients. Lower intestinal perforation is an uncommon but serious AE in TCZ therapy. Further clinical investigations will continue to refine the IL-6 inhibitory strategy. [ABSTRACT FROM AUTHOR]

This study aims to develop a sandwich ELISA system for the measurement of soluble folate receptor β (sFRβ) and evaluate whether base line levels of serum sFRβ are a biomarker for the activity of RA synovitis and the response to anti-TNF agents. Serum sFRβ from normal controls (41 samples), patients with OA (29 samples), and patients with RA (27 samples) and synovial fluid sFRβ from patients with RA (17 samples) were measured by sandwich ELISA, using anti-FRαβ and anti-FRβ antibodies as capture and detection antibodies, respectively. Baseline levels of serum sFRβ before therapy were evaluated in relation with DAS28-CRP or CRP and response to anti-TNF agents at 3-month follow-up. sFRβ levels in RA synovial fluids were higher than those in RA sera, and sFRβ levels in RA sera were higher than those in osteoarthritis and normal control sera. A significant relationship was observed between serum sFRβ levels and the DAS28-CRP scores or CRP values. The area under curve (AUC) values for receiver-operating characteristic curves defined using the serum sFRβ levels of RA patients before therapy had a higher predictive capacity than DAS28-CRP and CRP for the effective response of anti-TNF agents. The high serum sFRβ levels with a cutoff value of 8 ng/mL were 100% specificity for the effective response of anti-TNF agents. The findings support that the serum sFRβ levels in patients with RA act as a disease activation biomarker and that high serum sFRβ levels act as a predictive biomarker for the response to anti-TNF agents. [ABSTRACT FROM AUTHOR]

Background Activation of type IIB activin receptor (ActRIIB) in skeletal muscle leads to muscle atrophy because of increased muscle protein degradation. However, the intracellular signalling mechanism that mediates ActRIIB-activated muscle catabolism is poorly defined. Methods We investigated the role of p38β mitogen-activated protein kinases (MAPK) in mediating ActRIIB ligand activin A-activated muscle catabolic pathways in C2C12 myotubes and in mice with perturbation of this kinase pharmacologically and genetically. Results Treatment of C2C12 myotubes with activin A or myostatin rapidly activated p38 MAPK and its effector C/EBPβ within 1 h. Paradoxically, Akt was activated at the same time through a p38 MAPK-independent mechanism. These events were followed by up-regulation of ubiquitin ligases atrogin1 (MAFbx) and UBR2 (E3α-II), as well as increase in LC3-II, a marker of autophagosome formation, leading to myofibrillar protein loss and myotube atrophy. The catabolic effects of activin A were abolished by p38α/β MAPK inhibitor SB202190. Using small interfering RNA-mediated gene knockdown, we found that the catabolic activity of activin A was dependent on p38β MAPK specifically. Importantly, systemic administration of activin A to mice similarly activated the catabolic pathways in vivo, and this effect was blocked by SB202190. Further, activin A failed to activate the catabolic pathways in mice with muscle-specific knockout of p38β MAPK. Interestingly, activin A up-regulated MuRF1 in a p38 MAPK-independent manner, and MuRF1 did not appear responsible for activin A-induced myosin heavy chain loss and muscle atrophy. Conclusions ActRIIB-mediated activation of muscle catabolism is dependent on p38β MAPK-activated signalling. [ABSTRACT FROM AUTHOR]

Purpose: Fibrocytes (FC) are bone marrow-derived progenitor cells that are more abundant and infiltrate the thyroid and orbit in Graves orbitopathy (GO). FCs express high levels of thyrotropin receptor (TSHR) and insulin-like growth factor-1 receptor (IGF-1R). These receptors are physically and functionally associated, but their role in GO pathogenesis is not fully delineated. Treatment of FCs with thyroid stimulating hormone (TSH) or M22 (activating antibody to TSHR) induces the production of numerous cytokines, including tumor necrosis factor α (TNFα). Teprotumumab (TMB) is a human monoclonal IGF-1R blocking antibody currently in clinical trial for GO and inhibits TSHR-mediated actions in FCs. Aim: To characterize the molecular mechanisms underlying TSH-induced TNFα production by FCs, and the role of IGF-1R blockade by TMB. Design: FCs from healthy and GD patients were treated with combinations of TSH, M22, MG132 and AKTi (inhibitors of NF-κB and Akt, respectively), and TMB. TNFα protein production was measured by Luminex and flow cytometry. Messenger RNA expression was quantified by real time PCR. Results: Treatment with TSH/M22 induced TNFα protein and mRNA production by FCs, both of which were reduced when FCs were pretreated with MG132 and AKTi (p<0.0001). TMB decreased TSH-induced TNFα protein production in circulating FCs from mean fluorescent index (MFI) value of 2.92 to 1.91, and mRNA expression in cultured FCs from 141- to 52-fold expression (p<0.0001). TMB also decreased M22-induced TNFα protein production from MFI of 1.67 to 1.12, and mRNA expression from 6- to 3-fold expression (p<0.0001). Conclusion: TSH/M22 stimulates FC production of TNFα mRNA and protein. This process involves the transcription factor NF-κB and its regulator Akt. Blocking IGF-1R attenuates TSH/M22-induced TNFα production. This further delineates the interaction of TSHR and IGF1-R signaling pathways. By modulating the proinflammatory properties of FCs such as TNFα production, TMB may be a promising therapeutic agent for GO. [ABSTRACT FROM AUTHOR]

The p38α mitogen-activated protein kinase pathway not only regulates the production of inflammatory mediators, but also controls processes related to tissue homeostasis, such as cell proliferation, differentiation and survival, which are often disrupted during malignant transformation. The versatility of this signaling pathway allows for the regulation of many specific functions depending on the cell type and context. Here, we discuss mouse models that have been used to identify in vivo functions of p38α signaling in the pathogenesis of inflammatory diseases and cancer. Experiments using genetically modified mice and pharmacological inhibitors support that targeting the p38α pathway could be therapeutically useful for some inflammatory diseases and tumor types. [ABSTRACT FROM AUTHOR]

PKs transfer a phosphate from ATP to the side-chain hydroxyl group of a serine, threonine or tyrosine residue of a substrate protein. This in turn can alter that protein's function; modulating fundamental cellular processes including, metabolism, transcription, growth, division, differentiation, motility and survival. PKs are subdivided into families based on homology. One such group are the stress-activated kinases, which as the name suggests, are activated in response to cellular stresses such as toxins, cytokines, mechanical deformation and osmotic stress. Members include the p38 MAPK family, which is composed of α, β, γ and δ, isoforms which are encoded by separate genes. These kinases transduce extracellular signals and coordinate the cellular responses needed for adaptation and survival. However, in cardiovascular and other disease states, these same systems can trigger maladaptive responses that aggravate, rather than alleviate, the disease. This situation is analogous to adrenergic, angiotensin and aldosterone signalling in heart failure, where inhibition is beneficial despite the importance of these hormones to homeostasis. The question is whether similar benefits could accrue from p38 inhibition? In this review, we will discuss the structure and function of p38, the history of p38 inhibitors and their use in preclinical studies. Finally, we will summarize the results of recent cardiovascular clinical trials with p38 inhibitors. Linked Articles This article is part of a themed section on Conditioning the Heart - Pathways to Translation. To view the other articles in this section visit [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis is a chronic crippling disease, where protein-based tumor necrosis factor-alpha (TNF-α) inhibitors show significant relief, but with potentially fatal side effects. A need for a safe, oral, cost-effective small molecule or phyto-pharmaceutical is warranted. BV-9238 is an Ayurvedic poly-herbal formulation containing specialized standardized extracts of Withania somnifera, Boswellia serrata, Zingiber officinale and Curcuma longa. The anti-inflammatory and anti-arthritic effects of BV-9238 were evaluated for inhibition of TNF-α and nitric oxide (NO) production, in lipopolysaccharide-stimulated, RAW 264.7, mouse macrophage cell line. BV-9238 reduced TNF-α and NO production, without any cytotoxic effects. Subsequently, the formulation was tested in adjuvant-induced arthritis (AIA) and carrageenan-induced paw edema (CPE) rat animal models. AIA was induced in rats by injecting Freund's complete adjuvant intra-dermally in the paw, and BV-9238 and controls were administered orally for 21 days. Arthritic scores in AIA study and inflamed paw volume in CPE study were significantly reduced upon treatment with BV-9238. These results suggest that the anti-inflammatory and anti-arthritic effects of BV-9238 are due to its inhibition of TNF-α, and NO, and this formulation shows promise as an alternate therapy for inflammatory disorders where TNF-α and NO play important roles. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The protein kinase, p38α MAPK, is a key intracellular transducer of stressor-induced neuroinflammatory responses and, as such, is of high interest as a potential therapeutic target. We recently reported the synthesis and evaluation of first-in-class CNS-penetrant and highly specific p38 MAPK inhibitors that avoid target crossover issues seen in popular small molecule p38 MAPK inhibitors used in hundreds of previous reports. The novel p38 MAPK inhibitors, represented in this study by MW181, are efficacious in vivo. Pharmacodynamic actions include attenuation of stressor-induced increases in brain proinflammatory cytokine levels. We report here more detailed analyses of MW181 target engagement and specific linkage to the downstream increase in glia proinflammatory cytokine production. In vivo validation included demonstration that oral administration of MW181 suppresses lipopolysaccharide-induced increases in mouse brain IL-1β, TNFα, IL-6, IL-10, and CXCL1 but not in a drug-resistant p38α MAPK mutant mouse. [ABSTRACT FROM AUTHOR]

Background: Various parts of Margaritaria discoidea find use in traditional medicine in the treatment of pain and oedema. This study evaluated the anti-allergic, anti-inflammatory and anti-arthritic effects of a 70% (v/v) aqueous ethanol extract of the stem bark of Margaritaria discoidea, MDE in rodents. Materials and Methods: Systemic anaphylaxis was induced by the injection of compound 48/80 into mice and their survival rate was monitored to evaluate the anti-allergic action of the extract. The effect of MDE assessed on the maximal and total oedema responses in the mouse carrageenan-induced paw oedema was used to evaluate the anti-inflammatory action of the extract while the Freund's adjuvant-induced arthritis model was employed to study the anti-arthritic effects of MDE. Results: MDE dose-dependently increased the time for compound 48/80-induced mortality in mice. MDE suppressed the mean maximal swelling and the total paw swellings induced over 6 h in the carrageenan-induced paw oedema when administered either prophylactically or therapeutically. MDE caused a reduction in serum levels of TNFα and IL-6 and significantly suppressed Freund's adjuvant-induced arthritis. Conclusion: Margaritaria discoidea suppresses allergy and exhibits anti-inflammatory activity in mice. In addition it attenuates Freund's adjuvant-induced arthritis through a reduction in serum levels of TNFα and IL-6 in rats. [ABSTRACT FROM AUTHOR]

A series of novel 3-hydroxy-2-(2,4,5-trimethoxyphenyl)-4H-chromen-4-one (flavonol) derivatives ( 2a- u) of biological interest have been prepared via CLAISEN-SCHMIDT condensation followed by ALGAR-FLYNN-OYAMADA reaction and to search for the potent nonsteroidal anti-inflammatory agents from this novel series. All the synthesized compounds have been screened for their in vitro proinflammatory cytokines tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) inhibitory activity along with antimicrobial activity. As many as three compounds viz. 2h, 2l, and 2q from this novel series were found to be potent TNF-α and IL-6 inhibitor (up to 72-81 % TNF-α and 86-92 % IL-6 inhibitory activity) but at 10 μM concentration as compared with the standard dexamethasone (71 % TNF-α and 84 % IL-6 inhibitory activities at 1 μM concentration). While the compounds 2d, 2m, 2n, and 2s were found to be potent antimicrobial agent showing even 2-2.5-fold more potency than that of standard ciprofloxacin and miconazole at the same MIC value of 10 μg/mL. [ABSTRACT FROM AUTHOR]

Activation of mitogen-activated protein kinase (MAPK) enzymes in nociceptive plasticity has been extensively studied. P38 MAPK enzyme, which can be activated by cytokines, acts as a crucial intracellular regulator of environmental changes. The aim of this study was to elucidate the cellular events during arthritis-induced hyperalgesia that are mediated by interleukin-6 and p38 MAPK, and their effects on the expression of spinal mu-opioid receptors (MORs), in different stages of arthritis in male Wistar rats. Complete Freund's adjuvant (CFA)-induced arthritis (AA) was caused by subcutaneous injection of CFA into the rats' hindpaw. Anti-IL-6 antibody and p38 MAPK phosphorylation inhibitor were administered during 21 days of study. Spinal MOR, p38, and phosphorylated-p38 (pp38) proteins expressions were detected by Western blotting. Daily treatment with anti-IL-6 antibody and p38 MAPK phosphorylation inhibitor, SB203580, significantly decreased paw edema in AA group. Daily anti-IL-6 and SB203580 administration caused a significant reduction in hyperalgesia in the first week of the study, but increased hyperalgesia in the next 2 weeks in experimental groups compared to the AA control group. Expression of pp38 MAPK protein significantly decreased on the 3, 7, 14, and 21 days in AA+SB203580 and AA+anti-IL6 groups compared to AA group. Additionally, daily treatment with anti-IL6 antibody and SB203580 in AA group caused significantly decrease in spinal MOR expression compared to AA control group. The results of our study can confirm that activated spinal p38 MAPK enzyme may play an important role in cellular IL-6 signaling pathways in hyperalgesia variation during different stages of AA inflammation. Also, it can be suggested that at least a part of p38 MAPK effects on hyperalgesia is mediated by spinal MOR expression variation. [ABSTRACT FROM AUTHOR]

Background: The p38 mitogen-activated protein kinases (MAPK) family plays pivotal roles in skeletal muscle metabolism. Recent evidence revealed that p38α and p38β exert paradoxical effects on muscle protein homeostasis. However, it is unknown why p38β, but not p38α, is capable of mediating muscle catabolism via selective activation of the C/EBPβ that upregulates atrogin1/MAFbx. Methods: Tryptic phosphopeptide mapping was carried out to identify p38α- and p38β-mediated phosphorylation sites in C/EBPβ. Chromosome immunoprecipitation (ChIP) assay was used to evaluate p38α and p38β effect on C/EBPβ binding to the atrogin1/MAFbx promoter. Overexpression or siRNA-mediated gene knockdown of p38α and p38β, and site-directed mutagenesis or knockout of C/EBPβ, were used to analyze the roles of these kinases in muscle catabolism in C2C12 myotubes and mice. Results: Cellular expression of constitutively active p38α or p38β resulted in phosphorylation of C/EBPβ at multiple serine and threonine residues; however, only p38β phosphorylated Thr-188, which had been known to be critical to the DNA-binding activity of C/EBPβ. Only p38β, but not p38α, activated C/EBPβ-binding to the atrogin1/MAFbx promoter. A C/EBPβ mutant in which Thr-188 was replaced by alanine acted as a dominant-negative inhibitor of atrogin1/MAFbx upregulation induced by either p38β or Lewis lung carcinoma (LLC) cell-conditioned medium (LCM). In addition, knockdown of p38β specifically inhibited C/EBPβ activation and atrogin1/MAFbx upregulation induced by LCM. Finally, expression of active p38β in mouse tibialis anterior specifically induced C/EBPβ phosphorylation at Thr-188, atrogin1/MAFbx upregulation and muscle mass loss, which were blocked in C/EBPβ-null mice. Conclusions: The α and β isoforms of p38 MAPK are capable of recognizing distinct phosphorylation sites in a substrate. The unique capacity of p38β in mediating muscle catabolism is due to its capability in phosphorylating Thr-188 of C/EBPβ. [ABSTRACT FROM AUTHOR]

Upregulation of ubiquitin ligase atrogin1/MAFbx and muscle wasting are hallmarks of cancer cachexia; however, the underlying mechanism is undefined. Here, we describe a novel signalling pathway through which Lewis lung carcinoma (LLC) induces atrogin1/MAFbx upregulation and muscle wasting. C2C12 myotubes treated with LLC-conditioned medium (LCM) rapidly activates p38 MAPK and AKT while inactivating FoxO1/3, resulting in atrogin1/MAFbx upregulation, myosin heavy chain loss, and myotube atrophy. The p38?/? MAPK inhibitor SB202190 blocks the catabolic effects. Upon activation, p38 associates with C/EBP? resulting in its phosphorylation and binding to a C/EBP?-responsive cis-element in the atrogin1/MAFbx gene promoter. The promoter activity is stimulated by LCM via p38?-mediated activation of the C/EBP?-responsive cis-element, independent of the adjacent FoxO1/3-responsive cis-elements in the promoter. In addition, p38 activation is observed in the muscle of LLC tumour-bearing mice, and SB202190 administration blocks atrogin1/MAFbx upregulation and muscle protein loss. Furthermore, C/EBP??/? mice are resistant to LLC tumour-induced atrogin1/MAFbx upregulation and muscle wasting. Therefore, activation of the p38? MAPK-C/EBP? signalling pathway appears a key component of the pathogenesis of LLC tumour-induced cachexia. [ABSTRACT FROM AUTHOR]

p38 mitogen-activated protein kinase plays a pivotal role in skin inflammation. The purpose of this study was to investigate the role of the various p38 isoforms. p38β/δ-knockout-C57BL/6 mice were generated, studied in a 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced skin inflammation model and compared with wild-type mice. The inflammatory response was determined by ear thickness, myeloperoxidase activity and histology. mRNA and protein expression of interleukin (IL)-1β and IL-6 was determined by quantitative real-time reverse transcription PCR and enzyme-linked immunoassay. In both groups application of TPA resulted in a significant increase in inflammation, and pretreatment with the p38α/β inhibitor, SB202190 resulted in a significant inhibition. A significantly slower onset but prolonged duration of the response was seen in p38β/δ knockout mice. This was paralleled by a significant, but transient, lower IL-1β and IL-6 protein expression in p38β/δ knockout mice. Although the p38α isoform is important, our data also demonstrate an important role of the p38β and/or δ isoforms in the regulation of TPA-induced skin inflammation. [ABSTRACT FROM AUTHOR]

T-helper 17 (Th17) cells are a newly discovered CD4+ helper T-cell subset that produces interleukin-17A (IL-17A) and IL-17F. IL-17A plays important roles in allergic responses such as delayed-type hypersensitivity, contact hypersensitivity, and allergic airway inflammation. IL-17A promotes inflammation by inducing various proinflammatory cytokines and chemokines, recruiting neutrophils, enhancing antibody production, and activating T cells. IL-17A expression is also augmented in autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. Using mouse models of these diseases, we found that IL-17A plays a central role in their development. IL-6 is required for the development of Th17 cells and tumor necrosis factor functions downstream of IL-17A during the effector phase. IL-1 is important both for developing Th17 cells and eliciting inflammation. Th17 cells, like Th1 and Th2 cells, are involved in host defense against infections, but the contribution of these Th subsets to defense mechanisms differs among pathogens. The roles of IL-17F remain largely unknown. In this review, we introduce how IL-17A/IL-17F are involved in inflammatory immune responses and host defense mechanisms and discuss their relationship with other cytokines in the development of inflammatory and infectious diseases. [ABSTRACT FROM AUTHOR]

Crohn's disease (CD) and ulcerative colitis (UC) are subforms of inflammatory bowel diseases (IBD). Genetic and environmental factors influencing the onset and course of the diseases have been recently identified. This study uses a two-step approach to detect genes involved in the pathogenesis of IBD by microarray analysis and real-time PCR (RT-PCR). In a first step, microarray expression screening was used to obtain tumour necrosis factor-α (TNF-α) induction profiles of two human cell lines to represent the tissue cell types involved in IBD. In a second step, a subset of differentially expressed genes was examined by real-time PCR in intestinal biopsy samples of normal controls (NC) compared with UC and CD patients, as well as to a cohort of patients suffering from intestinal diseases other than IBD. Data were obtained from 88 CD, 88 UC, 53 non-IBD patients (inflammatory control), DC and 45 NC individuals. The experimental design enabled the identification of disease-specific expressed genes. DnaJ (Hsp40) homologue, subfamily B, member 5 (DNAJB5) was downregulated in intestinal biopsy samples of the UC cohort compared with NC. A difference in JUNB expression levels was observed by comparing biopsy samples from inflamed and non-inflamed areas of UC patients. Transcript expression differences between IBD and control cohorts were found by examining histamine N-methyltransferase (HNMT), interleukin-1A (IL-1A) and proplatelet basic protein (PPBP) expression. The experimental procedure represents an approach to identify disease-relevant genes, which is applicable to any disease where appropriate model systems are available.Genes and Immunity (2008) 9, 240–248; doi:10.1038/gene.2008.11; published online 13 March 2008 [ABSTRACT FROM AUTHOR]

Protein kinases are critical modulators of a variety of cellular signal transduction pathways, and abnormal phosphorylation events can be a cause or contributor to disease progression in a variety of disorders. This has led to the emergence of protein kinases as an important new class of drug targets for small molecule therapeutics. A serine/threonine protein kinase, p38α mitogen-activated protein kinase (MAPK), is an established therapeutic target for peripheral inflammatory disorders because of its critical role in regulation of proinflammatory cytokine production. There is increasing evidence that p38α MAPK is also an important regulator of proinflammatory cytokine levels in the central nervous system, raising the possibility that the kinase may be a drug discovery target for central nervous system disorders where cytokine overproduction contributes to disease progression. Development of bioavailable, central nervous system-penetrant p38α MAPK inhibitors provides the required foundation for drug discovery campaigns targeting p38α MAPK in neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Recently, two excellent methods have been used for the diagnosis of Legionnaires’ disease: urinary antigen detection and PCR. The purpose of the present study is to analyze and evaluate the sensitivity and specificity of three different urinary antigen detection kits as well as PCR. A total of 148 samples were collected from 33 patients between 1993 and 2004. These consisted of 73 urine samples obtained from 33 patients, 57 serum samples provided by 29 patients, and 18 respiratory tract specimens from 13 patients. Three commercially available kits were used to detect urinary antigen. For the 5S PCR reaction, primers L5SL2 and L5SR84 were used. Positive results were shown in all patients’ urine (representing 79.5% of total samples) using the Binax EIA kit, in 93.9% patients (representing 75.3% samples) using the Binax NOW immunochromatographic kit, and in 90.9% (representing 72.6% samples) using the Biotest EIA kit. Urine samples from 12.1% patients (representing 6.8% of total samples), serum samples from 41.4% patients (representing 35.1% of total samples), and respiratory samples from 84.6% patients (representing 88.9% of total samples) showed positive results with PCR. In testing urine of legionellosis patients, it was suggested that three kits were all valuable tools for diagnosis of legionellosis. Since over one-third of patients’ serum samples and most respiratory specimens showed positive results with PCR, the addition of PCR for testing of these samples might be useful, particularly in cases of culture negative and serum antibody negative patients. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis, juvenile idiopathic arthritis, the seronegative spondyloarthropathies including psoriatic arthritis, and systemic lupus erythematosus are all examples of rheumatic diseases in which inflammation is associated with skeletal pathology. Although some of the mechanisms of skeletal remodeling are shared among these diseases, each disease has a unique impact on articular bone or on the axial or appendicular skeleton. Studies in human disease and in animal models of arthritis have identified the osteoclast as the predominant cell type mediating bone loss in arthritis. Many of the cytokines and growth factors implicated in the inflammatory processes in rheumatic diseases have also been demonstrated to impact osteoclast differentiation and function either directly, by acting on cells of the osteoclast-lineage, or indirectly, by acting on other cell types to modulate expression of the key osteoclastogenic factor receptor activator of nuclear factor (NF) κB ligand (RANKL) and/or its inhibitor osteoprotegerin (OPG). Further elucidation of the mechanisms responsible for inflammation-induced bone loss will potentially lead to the identification of novel therapeutic strategies for the prevention of bone loss in these diseases. In this review, we provide an overview of the cell types, inflammatory mediators, and mechanisms that are implicated in bone loss and new bone formation in inflammatory joint diseases. [ABSTRACT FROM AUTHOR]

CCK increases the rate of net protein synthesis in rat pancreatic acini by activating initiation and elongation factors required for translation. The immunosuppressant FK506 inhibits the Ca2+-calmodulin-dependent phosphatase calcineurin in pancreatic acinar cells and blocks pancreatic growth induced by chronic CCK treatment. To test a requirement for calcineurin in the activation of the translational machinery stimulated by CCK, we evaluated the effects of FK506 on protein synthesis and on regulatory initiation and elongation factors in rat pancreatic acini in vitro. CCK acutely increased protein synthesis in acini from normal rats with a maximum increase at 100 pM CCK to 170 ± 11% of control. The immunosuppressant FK506 dosedependently inhibited CCK-stimulated protein synthesis over the same concentration range that blocked calcineurin activity, as assessed by dephosphorylation of the calcineurin substrate calciumregulated heat-stable protein of 24 kDa. Another immunosuppmssant, cyclosporin A, inhibited protein synthesis, but its effects appeared more complex. FK506 also inhibited protein synthesis stimulated by bombesin and carbachol. FK506 did not significantly affect the activity of the initiation factor-2B, or the phosphorylation of the initiation factor-2α, ribosomal protein protein S6, or the mRNA cap binding protein eukaryotic initiation factor (elF) 4E. Instead, blockade of calcineurin with FK506 reduced the phosphorylation of the elF4E binding protein, reduced the formation of the eIF4F complex, and increased the phosphorylation of eukaryotic elongation factor 2. From these results, we conclude that calcineurin activity is required for protein synthesis, and this action may be related to an effect on the formation of the mRNA cap binding complex and the elongation processes. [ABSTRACT FROM AUTHOR]

The expression level of tumor necrosis factor (TNF)-α is elevated in idiopathic inflammatory myopathies and Duchenne muscular dystrophy (DMD), but the precise role of TNF-α is unknown. To elucidate the possible role of TNF-α, we investigated the expression of TNF-α and its receptor in polymyositis (PM), dermatomyositis (DM), and DMD using in situ hybridization (ISH) and immunohistochemistry. We showed that TNF-α mRNA and protein were present in muscle fibers. TNF-α-positive fibers were observed in all cases of PM, DM and DMD, but were rare or absent in neurogenic disorders and normal controls. The proportion of TNF-α-positive fiber showed a significant positive correlation with the proportion of regenerating fibers that were positive for the developmental form of myosin heavy chain (MHC-d). The number of TNF receptor-positive fibers was small. Some muscle fibers expressed both TNF-α and its receptor simultaneously. Our results indicate that TNF-α is produced and expressed by muscle fibers and associated with muscle regeneration. [ABSTRACT FROM AUTHOR]

The purpose of this study is to investigate the expression of tumor necrosis factor-α converting enzyme (TACE) in the synovium and subchondral bone region of patients with rheumatoid arthritis (RA) and to determine the contribution of the enzyme to the pathogenesis of RA. Joint tissues were obtained during total knee arthroplasty from patients with RA and osteoarthritis (OA). The expression of TACE and TNF-α mRNA was detected by in situ hybridization. Characterization of TACE expressing cells was performed by immunohistochemistry using serial sections. We found that TACE mRNA was expressed in both synovium and subchondral bone region and co-localized with TNF-α mRNA in RA. On the other hand, TACE mRNA expression was scarcely detectable in OA samples. TACE was expressed in mononuclear cells, such as CD3 and CD14 positive cells in RA samples. In conclusion, the expression of TACE is up-regulated in the rheumatoid synovium and subchondral bone region, and the results in this study demonstrate that TACE may be involved and play a role in the pathogenesis of RA. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance is an inescapable consequence of the overuse and abuse of antibiotics. In both the hospital and community environments, resistance problems are evolving through the selection of resistant subpopulations during therapy and through the increased prevalence of pathogens exhibiting resistance through intrinsic or acquired mechanisms. As resistance mechanisms continue to evolve and spread at an alarming rate, clinicians worldwide are faced with the challenge of treating bacteria which have become resistant to virtually all available antibiotics. It is critical that steps be taken to slow the progression of antibiotic resistance among bacteria. Strategies must include both the judicious and optimal use of available drugs, as well as the development of new agents which are active against multiply-resistant bacteria. The fluoroquinolones are one class of antibiotics which are currently being developed to increase potency against multidrugresistant gram-positive pathogens. This review will focus on the advances which have been gained with newer fluoroquinolones against the multiply- resistant enterococci, staphylococci, pneumococci, and viridans streptococci. [ABSTRACT FROM PUBLISHER]

We evaluated the expression of tumor necrosis factor-α (TNF-α) mRNA in muscle biopsy specimens from patients with polymyositis (PM) and dermatomyositis (DM) to clarify its role in the pathogenesis of PM and DM. We performed non-radioactive in situ hybridization studies for TNF-α combined with immunohistochemistry for cell type-specific markers on muscles from ten PM and five DM patients. TNF-α-positive infiltrating cells present in the endomysium and perimysium were found in all PM and DM muscles. The frequency of TNF-α-positive cells against total infiltrating cells was similar among PM and DM (27.1 ± 7.4% in PM and 28.5 ± 13.6% in DM). However, TNF-α/CD8-positive lymphocytes and TNF-α-positive macrophages invading the non-necrotic muscle fiber were observed only in PM but not in DM. TNF-α was more highly expressed in PM and DM than was previously thought, and it was suggested that TNF-α plays a role in muscle fiber degeneration in PM. [ABSTRACT FROM AUTHOR]

Some studies have suggested that for trisomies, some genes are expressed far in excess of the expected 150% level and that this “dysregulation” is one of the mechanisms for the pathogenesis of trisomies. In an attempt to generalize this result to a monosomy, we examined mRNA isolated from lymphoblastoid cell lines derived from patients with 18q– syndrome, a deletion syndrome involving loss of the distal long arm of chromosome 18. Expression levels of ten chromosome 18 genes were compared between cell lines from eight patients with 18q– syndrome and four diploid controls. Gene expression was investigated by a quantitative reverse-transcription polymerase chain reaction (RT-PCR) method. With the exception of the transcription factor NFATC1, which shows a tendency towards gene dosage compensation (the expression pattern correlates with IgA deficiency), all of the other genes were expressed at a level proportional to their gene copy number. This was true regardless of mRNA abundance or different patterns of gene expression (ubiquitous versus tissue-specific gene expression). These results indicate that, unlike dysregulated gene expression apparent in some trisomies, this monosomic syndrome is largely due to consequences of reduced gene expression. [ABSTRACT FROM AUTHOR]