Treatment of adjuvant-induced arthritis with the combination of methotrexate and probiotic bacteria <Emphasis Type="Italic">Escherichia coli</Emphasis> O83 (Colinfant®)

A certain relationship was observed between the gastrointestinal system, arthritis and immune system. Patients with rheumatoid arthritis have an altered microflora composition and disturbed intestinal defensive barrier. Effect of probiotic bacteria (Colinfant®; COL) with known favorable effect on intestinal microflora was determined on the methotrexate (MTX) treatment of adjuvant arthritis. Rats with adjuvant arthritis were administered methotrexate 0.5 mg/kg body mass 2-times weekly per os, COL 1 mL/kg body mass every second day per os, and a combination of MTX+COL for a period of 28 d from the immunization. Levels of serum albumin, body mass, changes in hind paw swelling, and arthrogram score were estimated in rats as variables of inflammation and destructive arthritis-associated changes. Treatment with MTX, as well as with the combination treatment with MTX+COL significantly inhibited both inflammation and destructive arthritis-associated changes. The combination treatment inhibited both the hind paw swelling and arthrogram score more remarkably than MTX alone; on the other hand, the difference between combination treatment and MTX alone was not significant. Treatment with COL alone had no effect on adjuvant arthritis in rats. Colinfant® can increase the preventive effect of MTX treatment in rat adjuvant arthritis by improving its antiarthritic effects.     

Successful treatment of animal models of rheumatoid arthritis with small-molecule cyclin-dependent kinase inhibitors

Intraarticular gene transfer of cyclin-dependent kinase (CDK) inhibitors to suppress synovial cell cycling has shown efficacy in treating animal models of rheumatoid arthritis. Endogenous CDK inhibitors also modulate immune function via a CDK-independent pathway. Accordingly, systemic administration of small molecules that inhibit CDK may or may not ameliorate arthritis. To address this issue, alvocidib (flavopiridol), known to be tolerated clinically for treating cancers, and a newly synthesized CDK4/6-selective inhibitor were tested for antiarthritic effects. In vitro, they inhibited proliferation of human and mouse synovial fibroblasts without inducing apoptosis. In vivo, treatment of collagen-induced arthritis mice with alvocidib suppressed synovial hyperplasia and joint destruction, whereas serum concentrations of anti-collagen type II (CII) Abs and proliferative responses to CII were maintained. Treatment was effective even when therapeutically administered. Treated mice developed arthritis after termination of treatment. Thus, immune responses to CII were unimpaired. The same treatment ameliorated arthritis induced by K/BxN serum transfer to lymphocyte-deficient mice. Similarly, the CDK4/6-selective inhibitor suppressed collagen-induced arthritis. Both small-molecule CDK inhibitors were effective in treating animal models of rheumatoid arthritis not by suppressing lymphocyte function. Thus, the two small-molecule CDK inhibitors ameliorated arthritis models in a distinctive way, compared with other immunosuppressive drugs.     

Methotrexate Promotes Platelet Apoptosis via JNK-Mediated Mitochondrial Damage: Alleviation by N-Acetylcysteine and N-Acetylcysteine Amide

Thrombocytopenia in methotrexate (MTX)-treated cancer and rheumatoid arthritis (RA) patients connotes the interference of MTX with platelets. Hence, it seemed appealing to appraise the effect of MTX on platelets. Thereby, the mechanism of action of MTX on platelets was dissected. MTX (10 μM) induced activation of pro-apoptotic proteins Bid, Bax and Bad through JNK phosphorylation leading to ΔΨm dissipation, cytochrome c release and caspase activation, culminating in apoptosis. The use of specific inhibitor for JNK abrogates the MTX-induced activation of pro-apoptotic proteins and downstream events confirming JNK phosphorylation by MTX as a key event. We also demonstrate that platelet mitochondria as prime sources of ROS which plays a central role in MTX-induced apoptosis. Further, MTX induces oxidative stress by altering the levels of ROS and glutathione cycle. In parallel, the clinically approved thiol antioxidant N-acetylcysteine (NAC) and its derivative N-acetylcysteine amide (NACA) proficiently alleviate MTX-induced platelet apoptosis and oxidative damage. These findings underpin the dearth of research on interference of therapeutic drugs with platelets, despite their importance in human health and disease. Therefore, the use of antioxidants as supplementary therapy seems to be a safe bet in pathologies associated with altered platelet functions.     

L-carnitine and Zinc supplementation impedes intestinal damage in methotrexate-treated adjuvant-induced arthritis rats: Reinstating enterocyte proliferation and trace elements

BackgroundMethotrexate (MTX), a folic acid analogue, is used as a first-line treatment for rheumatoid arthritis (RA) since it has more therapeutic mechanisms than any other drug. Being an undeniable drug for the treatment of arthritis, even low-dose MTX provokes intestinal toxicity as a primary adverse effect and does not revive an anti-inflammatory element. Thus, our study aims to elucidate the anti-arthritic and prophylactic activity of supplements L-carnitine (L) and zinc (Z) against MTX-mediated intestinal damage in arthritis rats.MethodsThe rats were assessed for arthritic parameters such as body weight, paw volume, x-ray scan, and serum trace elements level. To analyze the toxic effects of MTX in the rats, intestine pH, mucosal weight, digestive enzymes, myeloperoxidase, histopathological, and immunohistochemical analysis were performed.ResultsOur study demonstrated that the arthritic parameters have shown that MTX has an ameliorative effect on arthritic rats. Besides, our findings showed that low-dose MTX (2.5 mg/kg b.w.) given once a week for two weeks during arthritis treatment had toxic effects in the rat's intestine, as evidenced by changes in intestine pH and mucosal weight, decreased digestive enzymes, increased MPO, and degenerative changes in histopathological analysis. Concurrent therapy of LZ with MTX, on the other hand, restored the modifications in these parameters.ConclusionMTX in combination with LZ effectively manages arthritis than monotherapy and significantly prevents MTX-induced intestinal damage in arthritis rats. Thus, LZ could be used as an improved therapeutic and safety for MTX-instigated intestinal damage during arthritis treatments. Therefore, our combination of L-carnitine and zinc with MTX would be promising prophylactic activity for arthritis patients.     

Wild chamomile (Matricaria recutita L.) mouthwashes in methotrexate-induced oral mucositis

Oral mucositis is a known complication of methotrexate (MTX) therapy, but a single efficacious intervention or agent for prophylaxis or management of this side effect has not yet been identified. We report a case of MTX-induced oral mucositis in a patient with rheumatoid arthritis, who was successfully treated with Wild chamomile mouthwashes.     

Intervention of α-lipoic acid ameliorates methotrexate-induced oxidative stress and genotoxicity: A study in rat intestine

Methotrexate (MTX) is an anti-metabolite, widely used in the cancer chemotherapy and rheumatoid arthritis. However, its long-term clinical use is restricted on account of its severe intestinal toxicity. The present study was aimed to investigate the intestinal toxicity of MTX and the possible protective effect of α-lipoic acid (LA) on Sprague–Dawley rats. MTX-induced intestinal toxicity was evaluated at the dose of 2.5 mg/kg for short-term (5 days treatment) and 1 mg/kg for long-term (5 days in a week for four consecutive weeks treatment) study. The possible protective effect of LA was evaluated in both short- as well as long-term study in a dose-dependent manner. MTX treatment induced diarrhoea and mortality in rats, indicating its severe toxicity in the target organ of investigation, i.e., intestine. Further, the intestinal toxicity of MTX was assessed by evaluating different parameters of oxidative stress, DNA damage, cytotoxicity as well as histological changes. Immunostaining for p53 revealed higher genotoxic assault in the intestinal cells due to MTX treatment. Pretreatment of rats with LA led to significant decrease in the oxidative stress, DNA damage, cellular damage, inflammatory changes and apoptosis as determined by malondialdehyde level, glutathione level, comet assay parameters, histological evaluation, immunostaining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. In the present investigation, we report that LA pretreatment ameliorates MTX-induced intestinal toxicity in rat as evident from the protection against oxidative stress, decrease in DNA damage and protection of cellular morphology as well as improvement in the stool consistency and animal survival rate.     

Protective effect of ellagic acid and pumpkin seed oil against methotrexate-induced small intestine damage in rats

Gastrointestinal toxicity is one of the most serious side effects in the methotrexate (MTX) treatment. This study was designed to investigate whether ellagic acid (EA) and/or pumpkin seed oil (PSO) had a protective effect on MTX-induced small intestine damage. Forty albino rats were randomized into five groups of 8 rats each. Group I served as a normal control group. In Group II, MTX was administered as a single dose (20 mg/kg) intraperitoneally. Groups III, IV and V were pre-treated respectively with either PSO (40 mg/kg), EA (10 mg/kg) or 0.2% DMSO (vehicle control) orally every day by gavage for 5 days and then they received MTX. All animals were sacrificed 5 days after the intraperitoneal injection of MTX for histopathological examination, estimation of serum prostaglandin E2 (PGE2) level, assay of tissue malondialdehyde (MDA), reduced glutathione (GSH) and nitric oxide (NO) levels and myloperoxidase (MPO), xanthine oxidase (XO) and adenosine deaminase (AD) activities. Administration of EA and/or PSO decreased the intestinal damage, PGE2, MDA and NO levels and MPO, XO and AD activities and increased GSH level. These results suggest that EA and PSO protect the small intestine of rats from MTX-induced damage through their antioxidant and anti-inflammatory effects and thus have potential as a promising drug in the prevention of undesired side effects of MTX.     

Pomegranate reverses methotrexate-induced oxidative stress and apoptosis in hepatocytes by modulating Nrf2-NF-κB pathways

The clinical efficacy of the widely used chemotherapeutic drug methotrexate (MTX) is limited due to its associated hepatotoxicity. Pomegranate polyphenols are of huge health benefits and known to possess remarkable antioxidant properties capable of protecting normal cells from various stimuli-induced oxidative stress and cell death. In this study, we explored the protective role of pomegranate fruit extract (PFE) in ameliorating MTX-induced hepatic damage. Male Swiss albino mice exposed to MTX (20 mg/kg body weight) exhibited distinct markers of toxicity such as increased activities of enzymes alanine transaminase, aspartate transaminase, lactate dehydrogenase and alkaline phosphatase and also increased oxidative stress in liver evidenced by increased ROS generation and lipid peroxidation. Decrease in reduced glutathione levels, superoxide dismutase, catalase, hepatic heme oxygenase 1 and NQO-1 activities were also observed. Tracing the signal transduction pathways, it was seen that MTX exposure significantly increased nuclear translocation of NF-κB coupled with increase in phosphorylated Iκ-B and down-regulation of NF-kappaB-dependent antiapoptotic protein Bcl-2. Treatment with MTX increased the expression of the apoptotic enhancer Rho/Cdc42 as well as the phosphorylation of SAPK/JNK. A shift in the Bax/Bcl-2 ratio towards apoptosis and increase in the caspase 3 level was also evident. Administration of PFE for 7 consecutive days before and after MTX challenge suppressed MTX-induced cell death, mitigated the injurious effects of MTX and offered protection against apoptosis. PFE was shown to reduce ROS generation in hepatocytes by activating the Nrf2-ARE pathway and inhibiting NF-κB as a consequence of which the antioxidant defense mechanism in the liver was up-regulated, thereby conferring protection against MTX-induced hepatotoxicity and apoptosis.     

Quantitative gait analysis as a method to assess mechanical hyperalgesia modulated by disease-modifying antirheumatoid drugs in the adjuvant-induced arthritic rat

In the present study, azothioprine, chloroquine, D-penicillamine, methotrexate and sodium aurothiomalate (gold salt) were evaluated for possible disease-modifying effects in the adjuvant-induced arthritis model of human rheumatoid arthritis in rats. Gait analysis was used to examine the role of disease-modifying antirheumatic drugs in the development of pain. Body weights were also measured to monitor the progression of disease and the systemic antiarthritic effects of the test compounds used in this study, as well as their systemic toxicity. Our results showed that azothioprine (5 mg/kg/day), chloroquine (12.5 mg/kg/day), sodium aurothiomalate (2.5 mg/kg/day) and methotrexate (1 mg/kg/week) not only inhibited the macroscopic changes such as erythema and swelling of limbs, but also exhibited significant reversal of gait deficits seen in the untreated or saline-treated arthritic rats. No reduction in the body weights were observed in the arthritic rats treated with azothioprine, chloroquine, sodium aurothiomalate and methotrexate. D-Penicillamine (12.5 mg/kg/day), however, showed a significant reduction (P < 0.03) in the body weights of the arthritic rats over a period of 22 days; furthermore, it was unable to show any reduction in arthritic score (P < 0.1). In earlier experiments, chloroquine and methotrexate failed to suppress carageenan-induced edema, suggesting that the mode of antiarthritic action may be different from those of nonsteroidal anti-inflammatory agents. Since these disease-modifying antirheumatic drugs are reported to have an immunomodulatory role, especially the gold salt, which influences the monocyte–macrophage system, it is suggested that the observed antiarthritic effects of disease-modifying antirheumatic drugs may be partly attributed to their immunomodulatory activity.     

Amelioration of methotrexate-induced enteritis by melatonin in rats

The anti-tumour drug methotrexate (MTX) induces intestinal mucosa injury resulting in malabsorption and diarrhoea. The purpose of this study was to investigate whether exogenous melatonin could protect the gut from MTX-induced damage in rats. A single dose of MTX (20 mg kg(-1), i.p.) was followed by i.p. saline or melatonin injections (10 mg kg(-1), MTX + Mel) for the next 5 days. On the fifth day, intestinal transit was assessed using charcoal propagation. Rats were decapitated and small intestinal segments were fixed for light (LM) and scanning electron microscope (SEM) examinations. Other intestinal segments were stored to measure glutathione (GSH) and malondialdehyde (MDA) levels, myeloperoxidase (MPO) and ATPase activity. MTX led to loss of more than 10% of the initial body weight (p < 0.01). Conversely, weight loss was markedly less in the melatonin-treated MTX group (p < 0.05). Bowel motility was increased in MTX-treated rats, while the transit index in the MTX-Mel group was not different from the control group. MTX caused decreases in GSH levels and ATPase activity, with increases in MDA levels and MPO activity. These changes were reversed in MTX-Mel-treated rats (p < 0.05-p < 0.001). LM and SEM in the MTX group revealed desquamation of surface epithelium and glandular degeneration, while the epithelium was slightly damaged in the MTX-Mel group. In conclusion, the present study demonstrates that melatonin is capable of reversing MTX-induced intestinal dysfunctions, indicating that it may be beneficial in ameliorating the symptoms of chemotherapy-induced enteritis.     

Differential MHC class II-mediated presentation of rheumatoid arthritis autoantigens by human dendritic cells and macrophages

Rheumatoid arthritis is characterized by synovial joint infiltration of activated CD4(+) T cells and MHC class II(+) APC, and is linked to specific HLA-DR alleles. Candidate autoantigens in synovial fluid and cartilage include type II collagen (CII) and cartilage gp39 (HCgp39). Using preparations of native Ag and T cells derived from Ag-immunized DR4-transgenic mice, we determined that human ex vivo differentiated DR4(+) dendritic cells (DC) and macrophages (Mphi) can mediate MHC class II presentation of CII or HCgp39 epitopes. The form of the Ag (soluble, partially degraded, or particulate) delivered to the APC influenced its presentation by DC and Mphi. DC efficiently presented partially degraded, but not native CII alpha-chains, while Mphi presentation was most efficient after phagocytosis of bead-conjugated CII. Both DC and Mphi presented soluble HCgp39, and activated Mphi from some donors presented epitopes derived from endogenously synthesized HCgp39. When synovial fluid from rheumatoid arthritis patients was used as a source of Ag, DC presentation of HCgp39 and CII epitopes was efficient, indicating that synovial fluid contains soluble forms of CII and HCgp39 amenable to internalization, processing, and presentation. These data support the hypothesis that CII and HCgp39 are autoantigens and that their class II-mediated presentation by DC and Mphi to T cells in vivo has a critical role in the pathogenesis of human rheumatoid arthritis.     

The naturally occurring flavonoid nobiletin reverses methotrexate resistance via inhibition of P-glycoprotein synthesis

Methotrexate (MTX) is the first-line treatment for rheumatoid arthritis (RA). However, after long-term treatment, some patients develop resistance. P-glycoprotein (P-gp), as an indispensable drug transporter, is essential for mediating this MTX resistance. In addition, nobiletin (NOB), a naturally occurring polymethoxylated flavonoid, has also been shown to reverse P-gp–mediated MTX resistance in RA groups; however, the precise role of NOB in this process is still unclear. Here, we administered MTX and NOB alone or in combination to collagen II-induced arthritic (CIA) mice and evaluated disease severity using the arthritis index, synovial histopathological changes, immunohistochemistry, and P-gp expression. In addition, we used conventional RNA-seq to identify targets and possible pathways through which NOB reverses MTX-induced drug resistance. We found that NOB in combination with MTX could enhance its performance in synovial tissue and decrease P-gp expression in CIA mice compared to MTX treatment alone. In vitro, in MTX-resistant fibroblast-like synoviocytes from CIA cells (CIA-FLS/MTX), we show that NOB treatment downregulated the PI3K/AKT/HIF-1α pathway, thereby reducing the synthesis of the P-gp protein. In addition, NOB significantly inhibited glycolysis and metabolic activity of CIA-FLS/MTX cells, which could reduce the production of ATP and block P-gp, ultimately decreasing the efflux of MTX and maintaining its anti-RA effects. In conclusion, this study shows that NOB overcomes MTX resistance in CIA-FLS/MTX cells through the PI3K/AKT/HIF-1α pathway, simultaneously influencing metabolic processes and inhibiting P-gp–induced drug efflux.     

Therapeutic vaccination of active arthritis with a glycosylated collagen type II peptide in complex with MHC class II molecules

In both collagen-induced arthritis (CIA) and rheumatoid arthritis, T cells recognize a galactosylated peptide from type II collagen (CII). In this study, we demonstrate that the CII259-273 peptide, galactosylated at lysine 264, in complex with Aq molecules prevented development of CIA in mice and ameliorated chronic relapsing disease. In contrast, nonglycosylated CII259-273/Aq complexes had no such effect. CIA dependent on other MHC class II molecules (Ar/Er) was also down-regulated, indicating a bystander vaccination effect. T cells could transfer the amelioration of CIA, showing that the protection is an active process. Thus, a complex between MHC class II molecules and a posttranslationally modified peptide offers a new possibility for treatment of chronically active autoimmune inflammation such as rheumatoid arthritis.     

A combination of methotrexate and zoledronic acid prevents bone erosions and systemic bone mass loss in collagen induced arthritis

Introduction  

Osteoclasts play a key role in the pathogenesis of bone erosion and systemic bone mass loss during rheumatoid arthritis (RA). In this study, we aimed to determine the effect of methotrexate (MTX) and zoledronic acid (ZA), used alone or in combination, on osteoclast-mediated bone erosions and systemic bone mass loss in a rat model of collagen induced arthritis (CIA). We hypothesized that MTX and ZA could have an additive effect to prevent both bone erosion and systemic bone loss.     

Application of surface roughness analysis on micro-computed tomographic images of bone erosion: examples using a rodent model of rheumatoid arthritis

Quantifying the bone erosion in preclinical models of rheumatoid arthritis is valuable for the evaluation of drug treatments. This study introduces a three-dimensional method for bone surface roughness measurement from micro-computed tomographic data obtained from rats subjected to collagen-induced arthritis (CIA), in which the degree of bone erosion is related to the severity and the duration of the disease. In two studies of rat CIA, the surface roughness of the talus bone following 21 days of disease increased 559% and 486% from the control group. At 41 days following disease induction, the roughness of the bone surface increased 857% above baseline. The roughness of the control samples was similar from each study (less than 4% different), demonstrating the robustness of the algorithm. Treatment with methotrexate at 0.1 mg/kg daily demonstrated significant protection from bone erosion, whereas the 0.05 mg/kg daily dose was not efficacious (98% versus 22% inhibition of roughness-measured bone erosion). The main advantage of such an algorithm is demonstrated in the preclinical drug study of rat CIA with methotrexate treatment, indicating the immediate utility of this approach in drug development studies.     

Methotrexate in rheumatoid arthritis is frequently effective, even if re-employed after a previous failure

Effectiveness of therapy with individual disease-modifying antirheumatic drugs (DMARDs) in rheumatoid arthritis (RA) is limited, and the number of available DMARDs is finite. Therefore, at some stage during the lengthy course of RA, institution of traditional DMARDs that have previously been applied may have to be reconsidered. In the present study we investigated the effectiveness of re-employed methotrexate in patients with a history of previous methotrexate failure (original course). A total of 1,490 RA patients (80% female, 59% rheumatoid factor positive) were followed from their first presentation, yielding a total of 6,470 patient-years of observation. We identified patients in whom methotrexate was re-employed after at least one intermittent course of a different DMARD. We compared reasons for discontinuation, improvement in acute phase reactants, and cumulative retention rates of methotrexate therapy between the original course of methotrexate and its re-employment. Similar analyses were peformed for other DMARDs. Methotrexate was re-employed in 86 patients. Compared with the original courses, re-employment was associated with a reduced risk for treatment termination because of ineffectiveness (P = 0.02, by McNemar test), especially if the maximum methotrexate dose of the original course had been low (<12.5 mg/week; P = 0.02, by logistic regression). In a Cox regression model, re-employed MTX was associated with a significantly reduced hazard of treatment termination compared with the original course of methotrexate, adjusting for dose and year of employment (hazard ratio 0.64, 95% confidence interval 0.42-0.97; P = 0.04). These findings were not recapitulated in analyses of re-employment of other DMARDs. Re-employment of MTX despite prior inefficacy, but not re-employment of other DMARDs, is an effective therapeutic option, especially in those patients in whom the methotrexate dose of the original course was low.     

In vivo quantitative measurement of arthritis activity based on hydrophobically modified glycol chitosan in inflammatory arthritis: more active than passive accumulation

We demonstrated that arthritis could be visualized noninvasively using hydrophobically modified glycol chitosan nanoparticles labeled with Cy5.5 (HGC-Cy5.5) and an optical imaging system. Activated macrophages expressing Mac-1 molecules effectively phagocytosed HGC-Cy5.5, which formed spherical nanoparticles under physiologic conditions. We estimated the applicability of HGC-Cy5.5 to quantitative analysis of arthritis development and progression. Near-infrared fluorescence images, captured after HGC-Cy5.5 injection in mice with collagen-induced arthritis, showed stronger fluorescence intensity in the active arthritis group than in the nonarthritis group. According to the progression of arthritis in both collagen-induced arthritis and collagen antibody-induced arthritis models, total photon counts (TPCs) increased in parallel with the clinical arthritis index. Quantitative analysis of fluorescence after treatment with methotrexate showed a significant decrease in TPC in a dose-dependent manner. Histologic evaluation confirmed that the mechanism underlying selective accumulation of HGC-Cy5.5 within synovitis tissues included enhanced phagocytosis of the probe by Mac-1-expressing macrophages as well as enhanced permeability through leaky vessels. These results suggest that optical imaging of arthritis using HGC-Cy5.5 can provide an objective measurement of disease activity and, at the same time, therapeutic responses in rheumatoid arthritis.     

L-Carnitine ameliorates methotrexate-induced oxidative organ injury and inhibits leukocyte death

Methotrexate (MTX), a folic acid antagonist widely used for the treatment of a variety of tumors and inflammatory diseases, affects normal tissues that have a high rate of proliferation, including the hematopoietic cells of the bone marrow and the gastrointestinal mucosal cells. To elucidate the role of free radicals and leukocytes in MTX-induced oxidative organ damage and the putative protective effect of L-carnitine (L-Car), Wistar albino rats were administered a single dose of MTX (20 mg/kg) followed by either saline or L-Car (500 mg/kg) for 5 days. After decapitation of the rats, trunk blood was obtained, and the ileum, liver, and kidney were removed for histological examination and for the measurement of malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) activity, and collagen content. Our results showed that MTX administration increased the MDA and MPO activities and collagen content and decreased GSH levels in all tissues, while these alterations were reversed in L-Car-treated group. The elevated serum TNF-α level observed following MTX treatment was depressed with L-Car. The oxidative burst of neutrophils stimulated by Annexin V was reduced in the saline-treated MTX group, while L-Car abolished this inhibition. Similarly, flow cytometric measurements revealed that leukocyte apoptosis was increased in MTX-treated animals, while L-Car reversed these effects. Severe degeneration of the intestinal mucosa, liver parenchyma, and glomerular and tubular epithelium observed in the saline-treated MTX group was improved by L-Car treatment. These results suggest that L-Car, possibly via its free radical scavenging and antioxidant properties, ameliorates MTX-induced oxidative organ injury and inhibits leukocyte apoptosis. Thus, supplementation with L-Carnitine as an adjuvant therapy may be promising in alleviating the systemic side-effects of chemotherapeutics.     

Tracking of proinflammatory collagen-specific T cells in early and late collagen-induced arthritis in humanized mice

Rheumatoid arthritis is a chronic inflammatory disease associated with certain HLA-DR4 subtypes. The target autoantigen(s) is unknown, but type II collagen (CII) is a candidate, with a single immunodominant DR4-restricted 261-273 T cell epitope (CII(261-273)). In the present study, we have prepared HLA-DR4:CII(261-273) tetramers and analyzed peripheral blood, lymph node, and synovial fluid cells from DR4-transgenic mice with early and late collagen-induced arthritis to draw a fuller picture of the role of CII-reactive Th cells in disease development. Their frequencies increased approximately 20-fold in blood 1-2 wk postimmunization, and even more in acutely arthritic joints. Our data strongly suggest that CII-specific Th cells are necessary, but not sufficient for collagen-induced arthritis. The CII-specific Th cells displayed an activated proinflammatory Th1 phenotype, and their expansion correlated with onset and severity of arthritis and also with anti-CII Ab levels. Surprisingly, shortly after the first clinical signs of arthritis, activated HLA-DR4:CII tetramer(+) cells became undetectable in the synovial fluid and rare in the blood, but persisted in lymph nodes. Consequently, future human studies should focus on patients with early arthritis, and on their synovial cells, to re-evaluate the occurrence and pathogenic importance of CII-specific or other Th cells in rheumatoid arthritis.     

Nimesulide Improves the Symptomatic and Disease Modifying Effects of Leflunomide in Collagen Induced Arthritis

Nimesulide is a COX-2 inhibitor used for symptomatic relief of rheumatoid arthritis. Leflunomide is an anti-pyrimidine used to manage the progression of rheumatoid arthritis. Herein we studied the influence of nimesulide and leflunomide combination in terms of disease symptoms and progression using collagen-induced arthritis model in mice, as a model for rheumatoid arthritis. Collagen induced arthritis was induced by immunization with type II collagen. Assessment of joint stiffness and articular hyperalgesia were evaluated using a locomotor activity cage and the Hargreaves method, respectively. Disease progression was assessed via arthritic index scoring, X-ray imaging, myeloperoxidase enzyme activity and histopathologic examination. Nimesulide induced only transient symptomatic alleviation on the top of decreased leucocytic infiltration compared to arthritis group. However, nimesulide alone failed to induce any significant improvement in the radiological or pathological disease progression. Leflunomide alone moderately alleviates the symptoms of arthritis and moderately retarded the radiological and pathological disease progression. Combination of nimesulide and leflunomide significantly improved symptomatic (analgesia and joint stiffness) and arthritic disease progression (radiological, pathological and Myeloperoxidase enzyme activity) in collagen induced arthritis animal model.