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20473350 Neutrophil adhesion and chemotaxis depend on substrate mechanics. 2010 May 19 Neutrophil adhesion to the vasculature and chemotaxis within tissues play critical roles in the inflammatory response to injury and pathogens. Unregulated neutrophil activity has been implicated in the progression of numerous chronic and acute diseases such as rheumatoid arthritis, asthma, and sepsis. Cell migration of anchorage-dependent cells is known to depend on both chemical and mechanical interactions. Although neutrophil responses to chemical cues have been well characterized, little is known about the effect of underlying tissue mechanics on neutrophil adhesion and migration. To address this question, we quantified neutrophil migration and traction stresses on compliant hydrogel substrates with varying elasticity in a micro-machined gradient chamber in which we could apply either a uniform concentration or a precise gradient of the bacterial chemoattractant fMLP. Neutrophils spread more extensively on substrates of greater stiffness. In addition, increasing the stiffness of the substrate leads to a significant increase in the chemotactic index for each fMLP gradient tested. As the substrate becomes stiffer, neutrophils generate higher traction forces without significant changes in cell speed. These forces are often displayed in pairs and focused in the uropod. Increases in the mean fMLP concentration beyond the K(D) of the receptor lead to a decrease in chemotactic index on all surfaces. Blocking with an antibody against beta(2)-integrins leads to a significant reduction but not an elimination of directed motility on stiff materials, but no change in motility on soft materials, suggesting neutrophils can display both integrin-dependent and integrin-independent motility. These findings are critical for understanding how neutrophil migration may change in different mechanical environments in vivo and can be used to guide the design of migration inhibitors that more efficiently target inflammation.
20462758 Synthesis and biological activity of alpha-L-fucosyl ceramides, analogues of the potent ag 2010 Jun 1 Several L-fucoglycolipids are associated with diseases such as cancer, cystic fibrosis and rheumatoid arthritis. Activation of iNKT cells is known to lead to the production of cytokines that can help alleviate or exacerbate these conditions. alpha-Galactosyl ceramide (alpha-GalCer) is a known agonist of iNKT cells and it is believed that its fucosyl counterpart might have similar immunogenic properties. We herein report the synthesis of alpha-L-fucosyl ceramide derivatives and describe their biological evaluation. The key challenge in the synthesis of the target molecules involved the stereoselective synthesis of the alpha-glycosidic linkage. Of the methods examined, the per-TMS-protected glycosyl iodide donor was completely alpha-selective, and could be scaled up to provide gram quantities of the azide precursor 11, from which a range of N-acylated alpha-L-fucosyl ceramides were readily obtained and evaluated for ex vivo expansion of human iNKT cells.
20398005 Estrogens interfere with leflunomide modulation of cytokine production by human activated 2010 Apr Rheumatoid arthritis (RA) prevalence is greater in females than in males, supporting estrogens as modulators of immune response. Leflunomide (LEF) is employed in the RA treatment. We studied the combinatory effects of LEF active metabolite A77 1726 (LEF-M) and 17beta-estradiol (E2) on inflammatory cytokine production by cultured macrophages obtained from activated human monocytes (THP-1 cells). Macrophages were cultured with LEF-M alone and in combination with E2. IL-6, TNF-alpha, and TGF-beta were evaluated by immunocytochemistry (ICC), Western blot (WB), and reverse transcriptase-polymerase chain reaction (RT-PCR). ICC, as well as WB and RT-PCR, showed that LEF-M, in respect to untreated cells, significantly downregulated the cytokine production (IL-6 P < 0.01, TNF-alphaP < 0.001, TGF-betaP < 0.01). On the contrary, E2 increased the cytokine production, a result that was significantly reversed when LEF-M was subsequently added (IL-6, TNF-alpha, TGF-betaP < 0.001 vs. E2). E2 seems to contrast the LEF-M activity. These results might support a more efficient therapeutical effect of LEF in male with respect to female RA patients.
20374363 Rheumatology in Iran. 2009 Dec Rheumatology in Iran started in the 1960s by a group of eight rheumatologists trained in France and Switzerland. The Iranian Rheumatology Society was founded in 1973. Academic rheumatology started in the city of Teheran (capital of Iran), by the Tehran University and by the National University in the '60s. Later, Shiraz University and Mashhad University followed. After the creation of the subspecialty Board of Rheumatology in 1985, other universities gradually created their rheumatology sections with the help of newly trained rheumatologists. The first subspecialty rheumatology outpatient clinic was created in 1970, in Tehran. In 1973, rheumatology was recognized as an internal medicine subspecialty for the training of medical students. Thus, one-eighth of internal medicine training was allocated to rheumatology. The subspecialty Rheumatology National Board was created in 1985. Four medical universities were approved for the training of rheumatology fellows. They were Tehran University, Beheshti University, Iran University, and Shiraz University of Medical Sciences. From 1985 to 2008, more than 150 rheumatologists got their Board of Rheumatology subspecialty. Thirty-seven medical universities in Iran have a rheumatology section. The prevalence of musculoskeletal complaints in people aged 15 years and over, is 41.9% in urban areas and 66.6% in rural areas (P < 0.001). Two major ethnicities live in Iran; Caucasians (71.4%) and Turks (23.1%). Musculoskeletal complaints are more frequent in Turks than in Caucasians (46%vs.40.8%, P < 0.001). Men complained less than women in urban areas: 34.1% versus 50.2% (P < 0.001). The same was seen in rural areas: 51.6% versus 72.4% (P < 0.001). The most frequent complaints were (urban vs. rural): knee (25.5%vs. 39.2%), dorsolumbar spine (21.7%vs. 41.9%), shoulder (14.5%vs. 22.7%), and cervical spine (13.4%vs. 17.9%). The distribution of mechanical diseases was: low back pain (15.4%vs. 23.4%), osteoarthritis (16.6%vs. 20.5%), and soft tissue rheumatism (4.6%vs. 2.2%). The distribution of inflammatory diseases was: rheumatoid arthritis (0.33%vs. 0.19%), seronegative spondylarthropathies (0.23%vs. 1.1%), and systemic lupus erythematosus (0.04%vs. 0.06).
20356482 [Evaluating type I interferon-inducible gene expression in patients with systemic lupus er 2010 Jan OBJECTIVE: To explore the expression levels of interferon-inducible genes in patients with systemic lupus erythematosus (SLE), and to validate these gene expressions as potential biomarkers for the differentiation of disease flare and infection. METHODS: Peripheral blood was obtained from 48 SLE, 16 rheumatoid arthritis (RA) patients and 26 normal controls, and total RNA was extracted and reverse transcribed into complementary DNA. Real-time PCR technique was used to determine the gene expressions of MX1, OASL, OAS1, ISG15 and LY6E at transcription level. Univariate logistic regression analysis and multivariate conditional logistic regression model were applied to analyze 5 related factors for infection or activity. RESULTS: (1) The expression levels of MX1, OASL, OAS1, ISG15, and LY6E mRNA in SLE patients were significantly increased as compared with normal controls (P all < 0.01), while the expression levels of OASL, OAS1, ISG15 and LY6E mRNA in SLE patients were also higher than those in RA patients (P all < 0.05). (2) There were no significant difference between male and female patients of the 5 gene expression in SLE patients. (3) By logistic regression analysis, ISG15 and LY6E were independent risk factors for active SLE patients (P < 0.01), OASL expression was an independent risk factor for SLE patients with infection (P = 0.003). CONCLUSION: All the 5 interferon inducible genes are highly expressed in SLE patients, in which ISG15 and LY6E are independently associated with disease flare, while OASL may be helpful for the evaluation of infection in SLE patients.
20353751 Pharmacokinetic comparison and bioequivalence of two leflunomide formulations in humans: a 2010 Apr BACKGROUND AND AIMS: Leflunomide is a disease-modifying antirheumatic drug (DMARD) with comparable efficacy to methotrexate in the treatment of rheumatoid arthritis. We compared the pharmacokinetic characteristics of two leflunomide formulations in healthy subjects and assessed whether these formulations were bioequivalent. SUBJECTS AND METHODS: A randomized, two-way, crossover study was conducted in 24 healthy male volunteers to compare the pharmacokinetics of two leflunomide formulations after administration of a single 20 mg dose of each drug with a 7 week washout period. Blood samples for the analysis of A77 1726, the main active metabolite of leflunomide, were obtained 624 h after drug administration. RESULTS: After administering a single dose of 20 mg of each leflunomide formulation, the mean AUC(0-t) and Cmax values of A771726 were 487.3 +/- 167.6 microg*h/ml and 2.24 +/- 0.85 microg/ml for the reference formulation and 468.5 +/- 148.6 microg*h/ml and 1.98 +/- 0.45 microg/ml for the test formulation, respectively. The 90% confidence intervals of the test/reference mean ratios for AUC(0-t), AUC(0-inf), and Cmax fell within the predetermined equivalence range of 0.8 - 1.25. No serious adverse events occurred during the study period. CONCLUSIONS: The two leflunomide formulations showed similar pharmacokinetic profiles in terms of A77 1726, and the test formulation was found to be bioequivalent to the reference formulation with respect to the rate and extent of leflunomide absorption.
20339312 Serum concentrations of selected endogenous estrogen and estrogen metabolites in pre- and 2010 Oct The purpose of this study was to investigate whether serum levels of selected endogenous estrogens and their metabolites are involved in the pathogenesis of osteoarthritis in pre- and post-menopausal women with osteoarthritis. Sixty-four patients with osteoarthritis (OA) of the knee, 48 patients with rheumatoid arthritis (RA) of the knee, and 48 healthy women were included in this study. Serum concentrations of estradiol and estrogen metabolites, such as 2- hydroxyestrone, 2-hydroxyestradiol, and 16α-hydroxyestrone, were measured by high performance liquid chromatography-mass spectrometry. Our results show that the serum concentrations of free estradiol and total 2-hydroxyestrone were significantly lower in pre-menopausal women with OA compared to the levels detected in the control groups (RA and healthy women). While serum concentrations of free and total estradiol in post-menopausal women with OA was significantly decreased compared to those of the control groups, the level of total 2-hydroxyestradiol significantly increased in postmenopausal women. Furthermore, the total 2-hydroxyestrone concentration positively correlated with the total estradiol level in pre-menopausal women with OA. In addition, the total 2- hydroxyestradiol level positively correlated with free and total estradiol levels in post-menopausal women with OA. In conclusion, estradiol and estrogen metabolites, including 2-hydroxyestrone and 2-hydroxyestradiol, were found in the sera of pre- and post-menopausal women with OA. Except for free and total estradiol deficiency, a decreased serum level of total 2- hydroxyestrone in pre-menopausal women and an increased total 2-hydroxyestradiol level in post-menopausal women with OA may also correlate with the pathogenesis of female OA.
20224826 [Selective inhibitors of cyclooxygenase-2 (COX-2), celecoxib and parecoxib: a systematic r 2010 Feb Cyclooxygenase (COX) enzymes mediate prostaglandin generation. COX-1 is expressed in all cells, producing prostaglandins that maintain cellular homeostasis, and COX-2 is an inducible enzyme that generates inflammatory prostaglandins at sites of inflammation and healing. Nonsteroidal antiinflammatory drugs (NSAIDs) that nonselectively inhibit COX-1 and COX-2 continue to be an important option for the management of pain. However, despite the potential advantages of NSAIDs, including their opioid-sparing effect and reduced opioid-related side effects, improved analgesia, and attenuation of the inflammatory pain response, several side effects limit their use. NSAIDs predispose to ulcer formation and upper gastrointestinal bleeding, impaired coagulation, cardiovascular effects and renal dysfunction. Selective cyclooxygenase-2 (COX-2) inhibitors were designed based on the hypothesis that selective inhibition of the COX-2 isoform should reduce pain and inflammation without compromising the integrity of the gastric mucosa. Celecoxib and parecoxib are two COX-2 inhibitors (coxibs) that are approved for the relief of acute postoperative pain and symptoms of chronic inflammatory conditions such as osteoarthritis and rheumatoid arthritis. They have similar pharmacological properties but a slightly improved gastrointestinal safety profile compared with traditional NSAIDs. Celecoxib is an orally administered coxib. Agents such as celecoxib, which are highly COX-2 specific and have shown excellent efficacy in relieving inflammation and associated pain, unfortunately exhibit only modest aqueous solubility, thus restricting dosing options. Parecoxib is the sulfonamide-based prodrug of valdecoxib and is the only parenterally administered coxib available to date. There is no evidence demonstrating any greater degree of pain relief between these two coxibs. However, parenteral preparations may be especially useful in the immediate postoperative period, when patients are unable to take oral medication or are experiencing nausea and vomiting.
20188319 Thrombotic risk and immobility in residents of long-term care facilities. 2010 Mar BACKGROUND: Hospitalized patients and residents of long-term care (LTC) facilities account for about 60% of all cases of venous thromboembolism (VTE), as incidence is correlated with increasing age, immobility, and underlying medical conditions. The primary aim of the study was to develop an evidence-based VTE risk stratification tool and definition of immobility for residents in LTC facilities. METHODS: Using the Delphi process, a panel of vascular thrombotic and geriatric experts reviewed and ranked statements of VTE risk and immobility derived from randomized controlled trials, meta-analyses, cohort trials, case-control trials, and case series to arrive at consensus for the importance of each statement. Rating was conducted before and during an on-site meeting following discussion. Statements rated high and very high were used to develop a VTE risk stratification and immobility tool. RESULTS: A total of 1165 publications related to VTE risk were identified from which 137 (12%) pertained to subjects with a median age of 60 years or older; 42 (31%) met study criteria. Eight (29.6%) of 79 publications pertaining to immobility met study criteria. There were 4 studies related to VTE risk and 1 to immobility that were rated as high quality. VTE risk factors were age older than 60 years, active cancer, acute infectious disease, catheter in a central vein, chronic obstructive pulmonary disease, dehydration, history of VTE, having a first-degree relative with VTE, heart failure, hypercoagulable state, immobility, inflammatory bowel disease, obesity, rheumatoid arthritis and treatment with erythroid stimulating agents to a hemoglobin value greater than 12 g/dL, aromatase inhibitor, hormone replacement therapy, megestrol acetate, or selective estrogen receptor modulators. Immobility was defined as the presence of at least 1 of the following: being bedridden or bedridden except for bathroom privileges, unable to walk at least 10 feet, recent reduction in ability to walk at least 10 feet for at least 72 hours, and having a lower limb cast. CONCLUSIONS: A risk stratification tool for VTE and immobility was developed to assist clinicians in caring for residents of LTC facilities. A prospective trial is needed to validate the tool.
20179326 Selection of a novel and highly specific tumor necrosis factor alpha (TNFalpha) antagonist 2010 Apr 16 Inhibition of tumor necrosis factor alpha (TNFalpha) is a favorable way of treating several important diseases such as rheumatoid arthritis, Crohn disease, and psoriasis. Therefore, an extensive range of TNFalpha inhibitory proteins, most of them based upon an antibody scaffold, has been developed and used with variable success as therapeutics. We have developed a novel technology platform using C-type lectins as a vehicle for the creation of novel trimeric therapeutic proteins with increased avidity and unique properties as compared with current protein therapeutics. We chose human TNFalpha as a test target to validate this new technology because of the extensive experience available with protein-based TNFalpha antagonists. Here, we present a novel and highly specific TNFalpha antagonist developed using this technology. Furthermore, we have solved the three-dimensional structure of the antagonist-TNFalpha complex by x-ray crystallography, and this structure is presented here. The structure has given us a unique insight into how the selection procedure works at a molecular level. Surprisingly little change is observed in the C-type lectin-like domain structure outside of the randomized regions, whereas a substantial change is observed within the randomized loops. Thus, the overall integrity of the C-type lectin-like domain is maintained, whereas specificity and binding affinity are changed by the introduction of a number of specific contacts with TNFalpha.
20150813 Actual status of antiinterleukin-1 therapies in rheumatic diseases. 2010 May PURPOSE OF REVIEW: Several studies have evaluated the efficacy and safety of novel therapeutic options targeting interleukin-1 (IL-1), which not only plays a significant role in rheumatoid arthritis, but also in other rheumatic diseases, for which only limited therapeutical options exist. RECENT FINDINGS: Three different strategies have been pursued and evaluated in the past years: preventing IL-1 binding by occupying IL-1 receptors with anakinra, an imitation of the naturally occurring IL-1 receptor antagonist, anakinra; development of the fully human monoclonal anti-IL-1beta antibody canakinumab; and synthesis of the dimeric fusion protein rilonacept, consisting of the ligand-binding domain of interleukin-1 receptor type I and its accessory protein, bound to human IgG1. Each of these three anti-IL-1 agents proved efficacy in distinct clinical situations and disease entities. SUMMARY: Owing to the observation that IL-1 is not only involved in signaling processes resulting in autoimmune and crystal-induced joint destruction but also in several hereditary autoinflammatory syndromes, its value both in pathophysiology as well as for novel advances in medication has significantly improved in the past years leading to an enrichment of the current therapeutic armamentarium for the affected patients.
20108252 IL-1alpha and IL-1beta have different effects on formation and activity of large osteoclas 2010 Apr 1 Interleukin 1 (IL-1) is a proinflammatory cytokine upregulated in conditions such as rheumatoid arthritis and periodontal disease. Both isoforms, IL-1alpha and IL-1beta, have been shown to activate osteoclasts (OCs), the cells responsible for resorbing bone. Inflammatory conditions are also characterized by increased bone loss and by the presence of large OCs (10+ nuclei). We and others have previously shown that large OCs are more likely to be resorbing compared to small OCs (2-5 nuclei). Moreover, large OCs express higher levels of the IL-1 activating receptor IL-1RI, integrins alphav and beta3, RANK, and TNFR1, while small OCs have higher levels of the decoy receptor IL-1RII. We hypothesized that IL-1 would have different effects on large and small OCs due to these distinct receptor expression patterns. To test this hypothesis, RAW 264.7 cells were differentiated into populations of small and large OCs and treated with IL-1alpha or IL-1beta (1 and 10 ng/ml). In the presence of sRANKL, both IL-1alpha and IL-1beta increased total OC number and resorptive activity of large OCs. IL-1alpha stimulated formation of large OCs and increased the number of resorption pits, while IL-1beta changed the morphology of large OCs and integrin-beta3 phosphorylation. No effects were seen in small OCs in response to either IL-1 isoform. These results demonstrate that IL-1 predominantly affects large OCs. The dissimilarity of responses to IL-1alpha and IL-1beta suggests that these isoforms activate different signaling pathways within the two OC populations.
20030635 The TRAF1/C5 locus confers risk for familial and severe alopecia areata. 2010 Apr BACKGROUND: Alopecia areata (AA) is a common hair loss disorder with a complex mode of inheritance. Autoimmune mechanisms are presumed to be crucial aetiologically. It is plausible that a number of autoimmune disorders may share a common genetic background. This phenomenon has been demonstrated in previous studies, which have shown an overlap of susceptibility alleles between AA and other autoimmune disorders. Recent studies have shown that genetic variants on the TRAF1/C5 (tumor necrosis factor receptor-associated factor 1, complement component 5) locus confer susceptibility to rheumatoid arthritis (RA). OBJECTIVES: To examine the role of the TRAF1/C5 locus in the development of AA using a large sample of 1,195 patients with AA and 1280 controls. METHODS: We genotyped the two most significant single nucleotide polymorphisms (SNPs) (rs10818488, rs2416808) from a former RA candidate gene study. After having obtained evidence for association, we performed a fine-mapping study and genotyped the locus with an additional 27 SNPs. RESULTS: While no significant result was obtained for the overall sample, rs2416808 showed significant associations in the analysis of the subgroups with severe AA and with a positive family history. The most significant P-value for rs2416808 was in familial cases (P = 0.004, P(corr) = 0.026). The fine mapping revealed significant associations for four additional SNPs in the analysis of subgroups, with rs2416808 remaining the most significant marker. CONCLUSIONS: Our results point to the involvement of the TRAF1/C5 locus in the aetiology of familial and severe AA, and provide further support for a shared aetiology between AA and other autoimmune disorders.
19880027 Clinical experiences with bisphosphonate-associated osteonecrosis of the jaws: analysis of 2009 Nov PURPOSE: The aim of the present study was to analyze the clinical presentation, risk factors, radiologic features, histopathologic and microbiological findings, treatment, and evolution of bisphosphonate-associated osteonecrosis of the jaws (BONJ). METHODS: This study made a retrospective review of 21 patients who underwent treatment and diagnosis of BONJ during 2004 to 2007 in a tertiary health care center reference for 1,100,000 inhabitants. RESULTS: The mean patient age at the time of presentation was 65.1 years. Of the 21 patients observed, 19 (90.4%) were receiving intravenous zoledronate. Of the 21 patients, 15 were treated with bisphosphonates for bone metastasis (71.4%), 5 for multiple myeloma (23.8%), and 1 for rheumatoid arthritis (4.7%). In 17 patients, the lesions occurred in the mandible. Fifteen patients had previous tooth extractions at the same site of bone necrosis. CONCLUSION: In our series, most patients improved with conservative surgical debridement. Prospective clinical trials would enable clinicians to make accurate judgments about risk, treatment, and outcome for patients with BONJ.
19721346 [Examination of availability of the criteria for protective therapy against Pneumocystis p 2009 Aug Twenty patients with collagen diseases complicated with Pneumocystis pneumonia (PCP) were retrospectively examined in reference to the criteria for its protective therapy provided by the Ministry of Health Labor and Welfare. The breakdown of 20 patients was rheumatoid arthritis (RA) in 5 cases, systemic lupus erythematosus (SLE) in 5, dermatomyositis (DM) in 2, systemic scleroderma (SSc) in 1, mixed connective tissue disease (MCTD) in 1, Sjögren syndrome (SjS) in 1, polyarteritis nodosa (PN) in 3, rapidly progressive glomerulonephritis (RPGN) in 1, Schönlein-Henoch purpura in 1. Patients having interstitial pneumonia (IP) or renal dysfunction before acquiring PCP showed poor prognosis. High level of beta-D glucan was observed in all patients, and elevated levels of LDH and KL-6 were also characteristic of PCP. For the treatment of their own collagen diseases, high dose steroids had been given in 11 patients (55%), and immunosuppressive agents in 12 (60%), resulting in severe suppression of immune function in these patients. They were treated with Sulfamethoxazole/trimethoprim (ST) after Pneumocystis infection, however, 10 patients died and 8 of them died of respiratory failure in spite of high dose steroids. Nine patients fulfilled the criteria for PCP protective therapy provided by Ministry of Health Labor and Welfare, and 7 of them died of respiratory failure. The frequency of PCP remarkably decreased in our hospital after we had started the protective therapy with ST using the criteria, suggesting that it is effective for the protection of PCP. However, some patients who do not fulfill the criteria may acquire severe PCP.
19683883 Long-term results of periarterial sympathectomy. 2009 Oct PURPOSE: To compare long-term results (minimum follow-up of 23 months) of periarterial sympathectomy for patients with digital vasospasm secondary to either an autoimmune disease or generalized atherosclerotic disease. Patients with posttraumatic or localized occlusive disease and vasospasm were not evaluated. METHODS: Twenty-eight patients had periarterial sympathectomy at 1 hospital by 1 senior surgeon. Periarterial sympathectomy was targeted to the areas of ulceration. Twenty patients (with 24 involved extremities and 42 ulcerated digits) had a documented autoimmune disease; 17 patients had scleroderma or an undifferentiated mixed connective tissue disorder, 2 had systemic lupus erythematosus, and 1 had rheumatoid arthritis. Eight patients (with 9 involved extremities and 17 ulcerated digits) had atherosclerotic disease. The primary outcomes were complete healing of all ulcers, a decrease in the number of ulcers, and need for amputation by the end of follow-up. Statistical analysis was done using the Fischer exact t-test. RESULTS: The average follow-up for all patients was 96 months (90 months for the autoimmune group and 113 months for the atherosclerotic group). Fifteen of the 20 patients (28 of 42 digits) in the autoimmune group had complete healing or decrease in ulcer number. Conversely, only 1 of the 8 patients (2 of 17 digits) in the atherosclerotic group had complete healing or decrease in ulcer number. Eleven of the 42 (26%) digits treated in the autoimmune group required amputation. In contrast, 10 of the 17 (59%) digits treated in the atherosclerotic group ultimately required amputation. CONCLUSIONS: Periarterial sympathectomy can lead to complete healing and decrease in ulcer number in autoimmune disease patients with digital ischemia from vasospasm. However, our data suggest that periarterial sympathectomy may be of little or no benefit in patients with chronic digital ischemia and vasospasm secondary to severe atherosclerotic disease. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic III.
19631193 Photolabeling identifies transmembrane domain 4 of CXCR4 as a T140 binding site. 2009 Dec 1 CXCR4, a G-protein-coupled receptor, which binds the chemokine stromal cell-derived factor 1 alpha (SDF-1alpha, CXCL12), is one of two co-receptors most frequently used by HIV-1 to infect CD4+ lymphocytes. The SDF-1alpha/CXCR4 axis is also involved in angiogenesis, in stem cell homing to bone marrow, in rheumatoid arthritis and in cancer. Here, we directly determined the binding site of the inverse agonist T140 on CXCR4 using photoaffinity labeling. Two T140 photoanalogs were synthesized containing the photoreactive amino acid p-benzoyl-l-phenylalanine (Bpa) in positions 5 or 10, yielding [Bpa(5)]T140 and [Bpa(10)]T140. Binding experiments on HEK293 cells stably expressing the wild-type CXCR4 receptor using 125I-SDF-1alpha demonstrated that T140 and both photoanalogs had affinities in the nanomolar range, similar to SDF-1alpha. Photolabeling led to the formation of specific, covalent 42 kDa T140-CXCR4 complexes. V8 protease digestion of both CXCR4/125I-[Bpa(5)]T140 and CXCR4/125I-[Bpa(10)]T140 adducts generated a fragment of 6kDa suggesting that the T140 photoanalogs labeled a fragment corresponding to Lys(154)-Glu(179) of the receptor's 4th transmembrane domain. Further digestion of this 6kDa fragment with endo Asp-N led to the generation of a shorter fragment validating the photolabeled region. Our results demonstrate that T140 interacts with residues of the fourth transmembrane domain of the CXCR4 receptor and provide new structural constraints enabling us to model the complex between T140 and CXCR4.
19519463 T-lymphocytes: a target for stimulatory and inhibitory effects of zinc ions. 2009 Jun The trace element zinc is a crucial cofactor for many proteins involved in cellular processes like differentiation, proliferation and apoptosis. Zinc homeostasis is tightly regulated and disturbance of this homeostasis due to genetic defects, zinc deficiency, or supplementation influences the development and the progression of various infectious and autoimmune diseases. The immune system is strongly impaired during zinc deficiency, predominantly the cell-mediated response by T-lymphocytes. During zinc deprivation T-lymphocyte development, polarization into effector cells, and function are impaired. This leads to reduced T-cell numbers, a decreased ratio of type 1 to type 2 T-helper cells with reduced production of T-helper type 1 cytokines like interferon-gamma, and compromised T-cell mediated immune defense. Accordingly, disturbed zinc homeostasis increases the risk for infections, and zinc supplementation restores normal immune function. Furthermore, several disorders, like mycobacterial infections, asthma, diabetes, and rheumatoid arthritis are accompanied by decreased zinc levels and in some cases disease progression can be affected by zinc supplementation. On the molecular level, apoptosis of T-cell precursors is influenced by zinc via the Bcl-2/Bax ratio, and zinc ions inhibit caspases-3, -6, -7, and -8. In mature T-cells, zinc interacts with kinases involved in T-cell activation, like protein kinase C and the lymphocyte protein tyrosine kinase (Lck), while higher zinc concentrations are inhibitory, reducing the activities of the interleukin-1 receptor-associated kinase (IRAK) and calcineurin. Taken together, zinc homeostasis influences T-lymphocytes via several molecular targets, leading to a modulation of T-cell-dependent immune responses.
19473188 Sulphasalazine accelerates apoptosis in neutrophils exposed to immune complex: Role of cas 2009 Nov 1. Neutrophils release several histotoxic molecules that cause tissue injury. Neutrophil apoptosis is a crucial process that governs the persistence of inflammatory disorders and tissue damage. Thus, in the present study, we investigated whether the anti-inflammatory drug sulphasalazine (SSZ) affects neutrophil apoptosis in the presence of insoluble immune complex (IC). 2. Neutrophils were obtained from healthy donors. Neutrophils were resuspended in incubation medium and incubated for 2-12 h with or without 10, 30 or 100 micromol/L SSZ and 25 microg/mL IC. In some experiments, cells were co-incubated with 20 micromol/L Z-IETD-fmk (a caspase 8 inhibitor) or 20 micromol/L Z-LEHD-fmk (a caspase 9 inhibitor). Apoptosis was evaluated morphologically on cytological preparations stained with May-Grünwald-Giemsa as well as by flow cytometry analysis of annexin V and propidium iodide staining. Caspase 3 activity was determined spectrophotometrically. 3. At 100 micromol/L, SSZ significantly accelerated IC-induced neutrophil apoptosis. Treatment of neutrophils with 20 micromol/L of the caspase 8 or 9 inhibitors Z-IETD-fmk or Z-LEHD-fmk, respectively, demonstrated that the SSZ-induced pro-apoptotic effect was mediated by a caspase 8- but not caspase 9-dependent pathway. The caspase 3 activity assay showed that treatment with 100 micromol/L SSZ increased caspase 3 activation. 4. In conclusion, the results of the present study indicate that it is possible that the molecular mechanism underlying SSZ protection against neutrophil-mediated tissue injury inflammatory disorders, such as rheumatoid arthritis and inflammatory bowel diseases, involves a caspase 8-dependent pathway.
19357114 Reliability of high-resolution ultrasonography in the assessment of Achilles tendon enthes 2009 Dec OBJECTIVE: The present study was mainly aimed at investigating the interobserver and intraobserver reproducibility of ultrasound (US) results in the assessment of Achilles tendon enthesopathy in patients with seronegative spondyloarthropathies (SpA). METHODS: A total of 28 patients with a diagnosis of SpA according to the European Spondyloarthropathy Study Group criteria were included. The patient female/male ratio was 1.8 (18/10), mean age was 42 (range 25-75) years and mean disease duration was 9 (range 1-35) years. Mean (SD) Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and Bath Ankylosing Spondylitis Functional Index (BASFI) scores were 32.4 (14.5) and 26.3 (9.2), respectively. Bilateral Achilles tendon US examinations were carried out independently by three investigators using a MyLab70 XVG (Esaote Biomedica, Genoa, Italy), equipped with a broadband 6-18 MHz linear probe. Each Achilles tendon was scanned for assessing the presence/absence of US findings indicative of enthesopathy according to the Outcome Measures in Rheumatoid Arthritis Clinical Trials (OMERACT) preliminary definition. The same findings were also scored on a 3-grade semiquantitative scoring system on which investigators reached a consensus prior to the study. Total additive scores per Achilles tendon were calculated. RESULTS: Moderate to excellent interobserver and intraobserver agreements were found for most of the US findings indicative of enthesopathy. Similar results were obtained using semiquantitative assessments, with weighted kappa values estimating the interobserver and intraobserver agreements for soft tissue inflammation of 0.696 and 0.816, respectively and for tissue damage 0.711 and 0.901, respectively. CONCLUSION: US assessment of Achilles tendon enthesopathy in patients with SpA, using the OMERACT preliminary definition, was found to be reliable. Bone irregularity and entheseal hypoechogenicity were the most difficult abnormalities to reach agreement on.