Endothelins modulate inflammatory reaction in zymosan-induced arthritis: participation of LTB4, TNF-alpha, and CXCL-1

Endothelins (ETs) are involved in inflammatory events, including pain, fever, edema, and cell migration. ET-1 levels are increased in plasma and synovial membrane of rheumatoid arthritis (RA) patients, but the evidence that ETs participate in RA physiopathology is limited. The present study investigated the involvement of ETs in neutrophil accumulation and edema formation in the murine model of zymosan-induced arthritis. Intra-articular (i.a.) administration of selective ETA or ETB receptor antagonists (BQ-123 and BQ-788, respectively; 15 pmol/cavity) prior to i.a. zymosan injection (500 mu g/cavity) markedly reduced knee-joint edema formation and neutrophil influx to the synovial cavity 6 h and 24 h after stimulation. Histological analysis showed that ETA or ETB receptor blockade suppressed zymosan-induced neutrophil accumulation in articular tissue at 6 h. Likewise, dual blockade of ETA/ETB with bosentan (10 mg/kg, i.v.) also reduced edema formation and neutrophil counts 6 h after zymosan stimulation. Pretreatment with BQ-123 or BQ-788 (i.a.; 15 pmol/cavity) also decreased zymosan-induced TNF-alpha production within 6 h, keratinocyte-derived chemokine/CXCL1 production within 24 h, and leukotriene B-4 at both time-points. Consistent with the demonstration that ET receptor antagonists inhibit zymosan-induced inflammation, i.a. injection of ET-1 (1-30 pmol/cavity) or sarafotoxin S6c (0.1-30 pmol/cavity) also triggered edema formation and neutrophil accumulation within 6 h. Moreover, knee-joint synovial tissue expressed ETA and ETB receptors. These findings suggest that endogenous ETs contribute to knee-joint inflammation, acting through ETA and ETB receptors and modulating edema formation, neutrophil recruitment, and production of inflammatory mediators.

CXCR2-specific chemokines mediate leukotriene B-4-dependent recruitment of neutrophils to inflamed joints in mice with antigen-induced arthritis

Objective. To investigate the mechanism underlying neutrophil migration into the articular cavity in experimental arthritis and, by extension, human-inflammatory synovitis. Methods. Antigen-induced arthritis (AIA) was generated in mice with methylated bovine serum albumin (mBSA). Migration assays and histologic analysis were used to evaluate neutrophil recruitment to knee joints. Levels of inflammatory mediators were measured by enzyme-linked immunosorbent assay. Antibodies and pharmacologic inhibitors were used in vivo to determine the role of specific disease mediators. Samples of synovial tissue and synovial fluid from rheumatoid arthritis (RA) or osteoarthritis patients were evaluated for CXCL1 and CXCL5 expression. Results. High levels of CXCL1, CXCL5, and leukotriene B-4 (LTB4) were expressed in the joints of arthritic mice. Confirming their respective functional roles, repertaxin (a CXCR1/CXCR2 receptor antagonist), anti-CXCL1 antibody, anti-CXCL5 antibody, and MK886 (a leukotriene synthesis inhibitor) reduced mBSA-induced neutrophil migration to knee joints. Repertaxin reduced LTB4 production in joint tissue, and neutrophil recruitment induced by CXCL1 or CXCL5 was inhibited by MK886, suggesting a sequential mechanism. Levels of both CXCL1 and CXCL5 were elevated in synovial fluid and were released in vitro by RA synovial tissues. Moreover, RA synovial fluid neutrophils stimulated with CXCL1 or CXCL5 released significant amounts of LTB4. Conclusion. Our data implicate CXCL1, CXCL5, and LTB4, acting sequentially, in neutrophil migration in AIA. Elevated levels of CXCL1 and CXCL5 in the synovial compartment of RA patients provide robust comparative data indicating that this mechanism plays a role in inflammatory joint disease. Together, these results suggest that inhibition of. CXCL1, CXCL5, or LTB4 may represent a potential therapeutic strategy in RA.

Lipoxin A(4) attenuates zymosan-induced arthritis by modulating endothelin-1 and its effects

BACKGROUND AND PURPOSE Lipoxin A(4) (LXA(4)) is a lipid mediator involved in the resolution of inflammation. Increased levels of LXA(4) in synovial fluid and enhanced expression of the formyl peptide receptor 2/lipoxin A(4) receptor (FPR2/ALX) in the synovial tissues of rheumatoid arthritis patients have been reported. Endothelins (ETs) play a pivotal pro-inflammatory role in acute articular inflammatory responses. Here, we evaluated the anti-inflammatory role of LXA(4), during the acute phase of zymosan-induced arthritis, focusing on the modulation of ET-1 expression and its effects. EXPERIMENTAL APPROACH The anti-inflammatory effects of LXA(4), BML-111 (agonist of FPR2/ALX receptors) and acetylsalicylic acid (ASA) pre- and post-treatments were investigated in a murine model of zymosan-induced arthritis. Articular inflammation was assessed by examining knee joint oedema; neutrophil accumulation in synovial cavities; and levels of prepro-ET-1 mRNA, leukotriene (LT)B(4), tumour necrosis factor (TNF)-alpha and the chemokine KC/CXCL1, after stimulation. The direct effect of LXA(4) on ET-1-induced neutrophil activation and chemotaxis was evaluated by shape change and Boyden chamber assays respectively. KEY RESULTS LXA(4), BML-111 and ASA administered as pre- or post-treatment inhibited oedema and neutrophil influx induced by zymosan stimulation. Zymosan-induced preproET-1 mRNA, KC/CXCL1, LTB(4) and TNF-alpha levels were also decreased after LXA(4) pretreatment. In vitro, ET-1-induced neutrophil chemotaxis was inhibited by LXA4 pretreatment. LXA(4) treatment also inhibited ET-1-induced oedema formation and neutrophil influx into mouse knee joints. CONCLUSION AND IMPLICATION LXA(4) exerted anti-inflammatory effects on articular inflammation through a mechanism that involved the inhibition of ET-1 expression and its effects.

Comparison of clinical features and drug therapies among European and Latin American patients with juvenile dermatomyositis

Objective To compare the demographic features, presenting manifestations, diagnostic investigations, disease course, and drug therapies of children with juvenile dermatomyositis (JDM) followed in Europe and Latin America. Methods Patients were inception cohorts seen between 1980 and 2004 in 27 paediatric rheumatology centres. The following information was collected through the review of patient charts: sex; age at disease onset; date of disease onset and diagnosis; onset type; presenting clinical features; diagnostic investigations; course type; and medications received during disease course. Results Four hundred and ninety patients (65.5% females, mean onset age 7.0 years, mean disease duration 7.7 years) were included. Disease presentation was acute or insidious in 57.1% and 42.9% of the patients, respectively. The course type was monophasic in 41.3% of patients and chronic polycyclic or continuous in 58.6% of patients. The more common presenting manifestations were muscle weakness (84.9%), Gottron`s papules (72.9%), heliotrope rash (62%), and malar rash (56.7%). Overall, the demographic and clinical features of the 2 continental cohorts were comparable. European patients received more frequently high-dose intravenous methylprednisolone, cyclosporine, cyclophosphamide, and azathioprine, while methotrexate and antimalarials medications were used more commonly by Latin American physicians. Conclusion The demographic and clinical characteristics of JDM are similar in European and Latin American patients. We found, however, several differences in the use of medications between European and Latin American paediatric rheumatologists.

Safety and Immunogenicity of Varicella Vaccine in Patients With Juvenile Rheumatic Diseases Receiving Methotrexate and Corticosteroids

Objective. To evaluate the safety and immunogenicity of varicella vaccine (VV) in susceptible patients with juvenile rheumatic diseases receiving methotrexate and corticosteroids. Methods. Twenty-five patients with juvenile rheumatic diseases (ages 2-19 years) and 18 healthy children and adolescents (ages 3-18 years) received a single dose of VV. All 25 patients were receiving methotrexate; 13 were also receiving prednisone and 5 were also receiving other disease-modifying antirheumatic drugs. None of the vaccinated patients or controls had a previous history of varicella. Anti-varicella-zoster virus IgG antibody (anti-VZV-IgG) titers were measured by enzyme-linked immunosorbent assay immediately before, 4-6 weeks after, and 1 year after vaccination. The patients were monitored prospectively for adverse reactions related to the vaccine, exposure, and occurrence of varicella. Disease activity was assessed 3 months before and 3 months after VV. Results. Twenty patients and all of the controls had negative preimmunization titers of VZV-IgG, and 5 patients had equivocal levels. Positive VZV-IgG titers were detected in 10 (50%) of 20 seronegative patients and 13 (72.2%) of 18 controls 4-6 weeks after VV (P = 0.2). One year after vaccination, 8 of 10 patients maintained positive VZV-IgG titers. No overt varicella episodes and no severe adverse reactions were observed during the followup period. No worsening of clinical parameters and no flares of juvenile rheumatic diseases or changes in doses of medications used were detected after vaccination. In fact, the number of active joints in patients with juvenile idiopathic arthritis was significantly lower after VV (P = 0.009). Conclusion. VV appears to be safe in patients with juvenile rheumatic diseases receiving methotrexate, as long as continuous prospective vigilance for side effects is performed.

Long-Term Outcome and Prognostic Factors of Juvenile Dermatomyositis: A Multinational, Multicenter Study of 490 Patients

Objective. To investigate the long-term outcome and prognostic factors of juvenile dermatomyositis (DM) through a multinational, multicenter study. Methods. Patients consisted of inception cohorts seen between 1980 and 2004 in 27 centers in Europe and Latin America. Predictor variables were sex, continent, ethnicity, onset year, onset age, onset type, onset manifestations, course type, disease duration, and active disease duration. Outcomes were muscle strength/endurance, continued disease activity, cumulative damage, muscle damage, cutaneous damage, calcinosis, lipodystrophy, physical function, and health-related quality of life (HRQOL). Results. A total of 490 patients with a mean disease duration of 7.7 years were included. At the cross-sectional visit, 41.2-52.8% of patients, depending on the instrument used, had reduced muscle strength/endurance, but less than 10% had severe impairment. Persistently active disease was recorded in 41.2-60.5% of the patients, depending on the activity measure used. Sixty-nine percent of the patients had cumulative damage. The frequency of calcinosis and lipodystrophy was 23.6% and 9.7%, respectively. A total of 40.7% of the patients had decreased functional ability, but only 6.5% had major impairment. Only a small fraction had decreased HRQOL. A chronic course, either polycyclic or continuous, consistently predicted a poorer outcome. Mortality rate was 3.1%. Conclusion. This study confirms the marked improvement in functional outcome of juvenile DM when compared with earlier literature. However, many patients had continued disease activity and cumulative damage at followup. A chronic course was the strongest predictor of poor prognosis. These findings highlight the need for treatment strategies that enable a better control of disease activity over time and the reduction of nonreversible damage.

Systemic lupus erythematosus: Association with KIR and SLC11A1 polymorphisms, ethnic predisposition and influence in clinical manifestations at onset revealed by ancestry genetic markers in an urban Brazilian population

Systemic lupus erythematosus (SLE) is an autoimmune disorder of the connective tissue with a wide and heterogeneous spectrum of manifestations, with renal and neurological involvement usually related to worse prognosis. SLE more frequently affects females of reproductive age, and a high prevalence and renal manifestation seem to be associated with non-European ethnicity. The present study aims to investigate candidate loci to SLE predisposition and evaluate the influence of ethnic ancestry in the disease risk and clinical phenotypic heterogeneity of lupus at onset. Samples represented by 111 patients and 345 controls, originated from the city of Belem, located in the Northern Region of Brazil, were investigated for polymorphisms in HLA-G, HLA-C, SLC11A1, MTHFR, CASP8 and 15 KIR genes, in addition to 89 Amerindian samples genotyped for SLC11A1. We also investigated 48 insertion/deletion ancestry markers to characterize individual African, European and Amerindian ancestry proportions in the samples. Predisposition to SLE was associated with GTGT deletion at the SLC11A1 3`UTR, presence of KIR2DS2 +/KIR2DS5 +/KIR3DS1 + profile, increased number of stimulatory KIR genes, and European and Amerindian ancestries. The ancestry analysis ruled out ethnic differences between controls and patients as the source of the observed associations. Moreover, the African ancestry was associated with renal manifestations. Lupus (2011) 20, 265-273.

Juvenile idiopathic arthritis with involvement of the temporomandibular joint: the role of image examinations

PURPOSE: Juvenile idiopathic arthritis (JIA) has unknown etiology, and the involvement of the temporomandibular joint (TMJ) is rare in the early phase of the disease. The present article describes the use of computed tomography (CT) and magnetic resonance (MRI) images for the diagnosis of affected TMJ in JIA. CASE DESCRIPTION: A 12-year-old, female, Caucasian patient, with systemic rheumathoid arthritis and involvement of multiple joints was referred to the Imaging Center for TMJ assessment. The patient reported TMJ pain and limited opening of the mouth. The helical CT examination of the TMJ region showed asymmetric mandibular condyles, erosion of the right condyle and osteophyte-like formation. The MRI examination showed erosion of the right mandibular condyle, osteophytes, displacement without reduction and disruption of the articular disc. CONCLUSION: The disorders of the TMJ as a consequence of JIA must be carefully assessed by modern imaging methods such as CT and MRI. CT is very useful for the evaluation of discrete bone changes, which are not identified by conventional radiographs in the early phase of JIA. MRI allows the evaluation of soft tissues, the identification of acute articular inflammation and the differentiation between pannus and synovial hypertrophy.

Infliximab Induces Increase in Triglyceride Levels in Psoriatic Arthritis Patients

Objectives. To evaluate lipid profile changes after anti-TNF therapy in patients with psoriatic arthritis (PsA). Methods. Fifteen PsA patients (eight polyarticular, four oligoarticular, two axial, and one mutilating) under infliximab were included. None had dyslipoproteinemia or previous statin use. Total cholesterol (TC) and its fractions, inflammatory markers, and prednisone use were evaluated. Results. The comparisons of lipid levels between baseline and after three months (3M) of anti-TNF therapy showed that there was a significant increase in mean triglycerides (117.8 +/- 49.7 versus 140.1 +/- 64.1 mg/dL, P = 0.028) and VLDL-c (23.6 +/- 10.5 versus 28.4 +/- 13.7 mg/dL, P = 0.019) levels. In contrast, there were no differences in the mean TC (P = 0.28), LDL-c (P = 0.42), and HDL-c (P = 0.26) levels. Analysis of the frequencies of each lipid alteration at baseline and at 3M were alike (P > 0.05). Positive correlations were found between VLDL-c and CRP (r = 0.647, P = 0.009) and between triglycerides and CRP (r = 0.604, P = 0.017) levels at 3M. ESR reduction was observed after 3M (P = 0.04). Mean prednisone dose remained stable at beginning and at 3M (P = 0.37). Conclusion. This study demonstrated that anti-TNF may increase TG and VLDL-c levels in PsA patients after three months.

A comparison of lower limb EMG and ground reaction forces between barefoot and shod gait in participants with diabetic neuropathic and healthy controls

Background: It is known that when barefoot, gait biomechanics of diabetic neuropathic patients differ from nondiabetic individuals. However, it is still unknown whether these biomechanical changes are also present during shod gait which is clinically advised for these patients. This study investigated the effect of the participants own shoes on gait biomechanics in diabetic neuropathic individuals compared to barefoot gait patterns and healthy controls. Methods: Ground reaction forces and lower limb EMG activities were analyzed in 21 non-diabetic adults (50.9 +/- 7.3 yr, 24.3 +/- 2.6 kg/m(2)) and 24 diabetic neuropathic participants (55.2 +/- 7.9 yr, 27.0 +/- 4.4 kg/m(2)). EMG patterns of vastus lateralis, lateral gastrocnemius and tibialis anterior, along with the vertical and antero-posterior ground reaction forces were studied during shod and barefoot gait. Results: Regardless of the disease, walking with shoes promoted an increase in the first peak vertical force and the peak horizontal propulsive force. Diabetic individuals had a delay in the lateral gastrocnemius EMG activity with no delay in the vastus lateralis. They also demonstrated a higher peak horizontal braking force walking with shoes compared to barefoot. Diabetic participants also had a smaller second peak vertical force in shod gait and a delay in the vastus lateralis EMG activity in barefoot gait compared to controls. Conclusions: The change in plantar sensory information that occurs when wearing shoes revealed a different motor strategy in diabetic individuals. Walking with shoes did not attenuate vertical forces in either group. Though changes in motor strategy were apparent, the biomechanical did not support the argument that the use of shoes contributes to altered motor responses during gait.

A systematic review with procedural assessments and meta-analysis of Low Level Laser Therapy in lateral elbow tendinopathy (tennis elbow)

Background: Recent reviews have indicated that low level level laser therapy (LLLT) is ineffective in lateral elbow tendinopathy (LET) without assessing validity of treatment procedures and doses or the influence of prior steroid injections. Methods: Systematic review with meta-analysis, with primary outcome measures of pain relief and/or global improvement and subgroup analyses of methodological quality, wavelengths and treatment procedures. Results: 18 randomised placebo-controlled trials (RCTs) were identified with 13 RCTs (730 patients) meeting the criteria for meta-analysis. 12 RCTs satisfied half or more of the methodological criteria. Publication bias was detected by Egger's graphical test, which showed a negative direction of bias. Ten of the trials included patients with poor prognosis caused by failed steroid injections or other treatment failures, or long symptom duration or severe baseline pain. The weighted mean difference (WMD) for pain relief was 10.2 mm [95% CI: 3.0 to 17.5] and the RR for global improvement was 1.36 [1.16 to 1.60]. Trials which targeted acupuncture points reported negative results, as did trials with wavelengths 820, 830 and 1064 nm. In a subgroup of five trials with 904 nm lasers and one trial with 632 nm wavelength where the lateral elbow tendon insertions were directly irradiated, WMD for pain relief was 17.2 mm [95% CI: 8.5 to 25.9] and 14.0 mm [95% CI: 7.4 to 20.6] respectively, while RR for global pain improvement was only reported for 904 nm at 1.53 [95% CI: 1.28 to 1.83]. LLLT doses in this subgroup ranged between 0.5 and 7.2 Joules. Secondary outcome measures of painfree grip strength, pain pressure threshold, sick leave and follow-up data from 3 to 8 weeks after the end of treatment, showed consistently significant results in favour of the same LLLT subgroup (p < 0.02). No serious side-effects were reported. Conclusion: LLLT administered with optimal doses of 904 nm and possibly 632 nm wavelengths directly to the lateral elbow tendon insertions, seem to offer short-term pain relief and less disability in LET, both alone and in conjunction with an exercise regimen. This finding contradicts the conclusions of previous reviews which failed to assess treatment procedures, wavelengths and optimal doses.

Aerobic exercise is safe and effective in systemic sclerosis

Several studies have established that systemic sclerosis patients have a reduced exercise capacity when compared to healthy individuals. It is relevant to evaluate whether aerobic exercise in systemic sclerosis patients is a safe and effective intervention to improve aerobic capacity. Seven patients without pulmonary impairment and seven healthy controls were enrolled in an 8-week program consisting of moderate intensity aerobic exercise. Patients and controls had a significant improvement in peak oxygen consumption (19.72 +/- 3.51 vs. 22.27 +/- 2.53 and 22.94 +/- 4.70 vs. 24.55 +/- 3.00, respectively, p = 0.006), but difference between groups was not statistically significant (p = 0.149). This finding was reinforced by the fact that at the end of the study both groups were able to perform a significantly higher exercise intensity when compared to baseline, as measured by peak blood lactate (1.43 +/- 0.51 vs. 1.84 +/- 0.33 and 1.11 +/- 0.45 vs. 1.59 +/- 0.25, respectively, p = 0.01). Patients improved the peak exercise oxygen saturation comparing to the baseline (84.14 +/- 9.86 vs. 90.29 +/- 5.09, p = 0.048). Rodnan score was similar before and after the intervention(15.84 +/- 7.84 vs. 12.71 +/- 4.31, p = 0.0855). Digital ulcers and Raynaud`s phenomenon remained stable. Our data support the notion that improving aerobic capacity is a feasible goal in systemic sclerosis management. The long term benefit of this intervention needs to be determined in large prospective studies.

Efficacy and safety of concurrent training in systemic sclerosis

Pinto, ALS, Oliveira, NC, Gualano, B, Christmann, RB, Painelli, VS, Artioli, GG, Prado, DML, and Lima, FR. Efficacy and safety of concurrent training in systemic sclerosis. J Strength Cond Res 25(5): 1423-1428, 2011-The optimal training model for patients with systemic sclerosis (SSc) is unknown. In this study, we aimed to investigate the effects of a 12-week combined resistance and aerobic training program (concurrent training) in SSc patients. Eleven patients with no evidence of pulmonary involvement were recruited for the exercise program. Lower and upper limb dynamic strengths (assessed by 1 repetition maximum [1RM] of a leg press and bench press, respectively), isometric strength (assessed by back pull and handgrip tests), balance and mobility (assessed by the timed up-and-go test), muscle function (assessed by the timed-stands test), Rodnan score, digital ulcers, Rayland`s phenomenon, and blood markers of muscle inflammation (creatine kinase and aldolase) were assessed at baseline and after the 12-week program. Exercise training significantly enhanced the 1RM leg press (41%) and 1RM bench press (13%) values and back pull (24%) and handgrip strength (11%). Muscle function was also improved (15%), but balance and mobility were not significantly changed. The time-to-exhaustion was increased (46.5%, p = 0.0004), the heart rate at rest condition was significantly reduced, and the workload and time of exercise at ventilatory thresholds and peak of exercise were increased. However, maximal and submaximal (V)over dotO(2) were unaltered (p > 0.05). The Rodnan score was unchanged, and muscle enzymes remained within normal levels. No change was observed in digital ulcers and Raynaud`s phenomenon. This is the first study to demonstrate that a 12-week concurrent training program is safe and substantially improves muscle strength, function, and aerobic capacity in SSc patients.

Pharmacokinetics of tetracycline in plasma, synovial fluid and milk using single intravenous and single intravenous regional doses in dairy cattle with papillomatous digital dermatitis

The purpose of this study was to compare the pharmacokinetics of tetracycline in plasma, synovial fluid, and milk following either a single systemic intravenous (i.v.) injection or a single i.v. regional antibiosis (IVRA) administration of tetracycline hydrochloride to dairy cattle with papillomatous digital dermatitis (PDD). To this end, plasma and synovial fluid tetracycline concentrations were compared with the minimal inhibitory concentration (MIC) values of the major bacteria, which are known to cause digital diseases and thus assess its efficacy in PDD. Residual tetracycline concentrations in milk from cows treated by both methods were also determined. Twelve Holstein cows with various stages of PDD were randomly assigned to two groups of six animals. Group 1 received a single systemic i.v. injection of 10 mg/kg of tetracycline hydrochloride. Group 2 received 1000 mg of tetracycline hydrochloride by IVRA of the affected limb. Blood, synovial fluid and milk samples were taken prior to tetracycline administration (time 0 control), and then at 22, 45 and 82 min, and 2, 3, 4, 6, 8, 12, 24, 48, 72, 96, and 120 h following drug administration. Tetracycline concentrations were determined by high-performance liquid chromatography. Mean tetracycline plasma and milk concentrations in Group 1 were higher than Group 2. The opposite was observed for synovial fluid concentrations. Group 2 synovial fluid concentrations were higher than the MIC value over 24 h for the bacteria most frequently responsible for claw disease. Compared with i.v. administration, IVRA administration of tetracycline produced very high synovial fluid and low plasma and milk concentrations.

Refolding and purification of the human secreted group IID phospholipase A(2) expressed as inclusion bodies in Escherichia coli

The secreted phospholipases A(2) (sPLA(2)s) are water-soluble enzymes that bind to the surface of both artificial and biological lipid bilayers and hydrolyze the membrane phospholipids. The tissue expression pattern of the human group IID secretory phospholipase A(2) (hsPLA(2)-IID) suggests that the enzyme is involved in the regulation of the immune and inflammatory responses. With an aim to establish an expression system for the hsPLA(2)-IID in Escherichia coli, the DNA-coding sequence for hsPLA(2)-IID was subcloned into the vector pET3a, and expressed as inclusion bodies in E. coli (BL21). A protocol has been developed to refold the recombinant protein in the presence of guanidinium hydrochloride, using a size-exclusion chromatography matrix followed by dilution and dialysis to remove the excess denaturant. After purification by cation-exchange chromatography, far ultraviolet circular dichroism spectra of the recombinant hsPLA(2)-IID indicated protein secondary structure content similar to the homologous human group IIA secretory phospholipase A(2). The refolded recombinant hsPLA(2)-IID demonstrated Ca(2+)-dependent hydrolytic activity, as measuring the release free fatty acid from phospholipid liposomes. This protein expression and purification system may be useful for site-directed mutagenesis experiments of the hsPLA(2)-IID which will advance our understanding of the structure-function relationship and biological effects of the protein. (C) 2009 Elsevier Inc. All rights reserved.