Reduction of NOS2 overexpression in rheumatoid arthritis patients treated with anti-tumor necrosis factor alpha monoclonal antibody (cA2)

OBJECTIVE: Peripheral blood mononuclear cells (PBMC) from patients with rheumatoid arthritis (RA) have increased expression of nitric oxide synthase type 2 (NOS2) protein and enhanced formation of nitric oxide (NO) that correlate with disease activity. NO may play a role in the inflammation of RA. Treatment of RA patients with a chimeric monoclonal antibody against tumor necrosis factor alpha (TNFalpha; cA2) results in clinical improvement in the majority of patients. The present study was designed to determine if cA2 therapy decreases PBMC NOS2 protein expression and NOS enzyme activity in RA patients. METHODS: RA patients receiving background oral methotrexate participated in a double-blind, placebo-controlled clinical trial in which they were randomly assigned to receive a single infusion of either placebo or cA2 at 5, 10, or 20 mg/kg. NOS2 protein and NOS enzyme activity were measured in PBMC at baseline and 4 weeks following cA2 therapy. These results were compared with the degree of clinical change in disease activity. RESULTS: At baseline, elevated levels of NOS2 protein and NOS enzyme activity were more frequently detected in PBMC from RA patients than in those from healthy controls. Treatment of the RA patients with cA2 significantly reduced NOS2 protein expression and NOS enzyme activity. Changes in NOS activity following treatment correlated significantly with changes in the number of tender joints. CONCLUSION: These results indicate that TNFalpha likely plays an important role in enhancing NOS2 expression in RA, and that the antiinflammatory effects of cA2 treatment may be mediated by a reduction of NO overproduction.     

Therapeutic actions of cyclosporine and anti-tumor necrosis factor alpha in collagen-induced arthritis and the effect of combination therapy

OBJECTIVE: To define the mechanisms of action of 2 novel drugs, cyclosporine and anti-tumor necrosis factor alpha (TNFalpha), in collagen-induced arthritis and to determine the effect of combination therapy. METHODS: Type II collagen-immunized DBA/1 mice with established arthritis were treated with cyclosporine alone, anti-TNFalpha alone, cyclosporine plus anti-TNFalpha, or saline. RESULTS: Cyclosporine was found to ameliorate arthritis, suppress interferon-gamma (IFNgamma) production by CD4+ T cells, and reduce TNFalpha expression in arthritic joints. However, cyclosporine did not directly inhibit TNFalpha production by macrophages, indicating that the decrease in TNFalpha expression observed in vivo was probably an indirect consequence of the reduction in type 1 T helper cell activity. Anti-TNFalpha also reduced IFNgamma production by T cells, indicating that TNFalpha is involved in the cellular immune response to collagen. Combined treatment with cyclosporine plus anti-TNFalpha had an additive therapeutic effect. CONCLUSION: Although cyclosporine and anti-TNFalpha target different points in the inflammatory pathway, there is an overlap in the consequences of their actions in vivo.     

Adverse effects of low-dose methotrexate therapy in rheumatoid arthritis]

To analyze the possible adverse effects of low dose methotrexate (MTX) therapy, 276 patients with rheumatoid arthritis (RA) were examined retrospectively. One hundred and seven patients (39%) experienced 113 adverse events : 57 showed liver dysfunction, 24 gastrointestinal complaints, 13 cutaneous symptoms, 6 respiratory symptoms, and 6 malignancies. Interestingly, 3 patients developed a dry cough without infiltration nor interstitial shadow on chest X-ray. The cough was rapidly resolved by discontinuation of MTX, but it recurred in 1 patient when MTX was re-administered. This finding might suggest a close association between MTX administration and the occurrence of dry cough. Of the 6 patients with malignancies diagnosed during MTX therapy, 2 showed malignant lymphoma, 2 lung cancer, 1 breast cancer and 1 colon cancer. MTX might have an oncogenic potential in RA because the coincidence rate, especially with respect to lymphoma, was significantly higher than estimated in a normal population.     

Lack of immunosuppressive effect of low-dose oral methotrexate on lymphocytes in rheumatoid arthritis

Whether methotrexate (MTX) is effective in rheumatoid arthritis (RA) because of immunosuppressive and/or anti-inflammatory mechanisms of action is controversial. Many lines of investigation point to the latter. We evaluated DNA synthesis in peripheral blood lymphocytes (PBL) from 33 RA patients on oral MTX (7.5-15 mg/wk) and in 30 healthy controls by flow cytometric cell cycle analysis (CCA). DNA synthesis was also evaluated with a thymidilate synthetase activity assay (TSA) (3H-deoxyuridine incorporation) in 12 patients and 21 controls (12 on MTX and NSAID, and 9 healthy subjects). The patients had taken MTX for at least 3 months and were in different stages of clinical activity. There were no significant differences in TSA or in the cell cycle phase distributions (especially the S phase) between treated RA patients and controls. These data suggest that low-dose oral MTX does not inhibit DNA synthesis and therefore does not have an immunosuppressive effect on lymphocytes from patients with RA.     

Efficient adenoviral infection with IkappaB alpha reveals that macrophage tumor necrosis factor alpha production in rheumatoid arthritis is NF-kappaB dependent

Tumor necrosis factor (TNF) alpha has been shown to be a major therapeutic target in rheumatoid arthritis with the success of anti-TNFalpha antibody clinical trials. Although signaling pathways leading to TNFalpha expression have been studied in some detail, there is evidence for considerable differences between individual cell types. This prompted us to investigate the intracellular signaling pathways that result in increased TNFalpha synthesis from macrophages in the diseased synovial joint tissue. Using an adenoviral system in vitro we report the successful delivery of genes to more than 95% of normal human macrophages. This permitted us to show, by using adenoviral transfer of IkappaB alpha, the natural inhibitor of NF-kappaB, that induction of TNFalpha in normal human macrophages by lipopolysaccharide, but not by some other stimuli, was inhibited by 80%. Furthermore the spontaneous production of TNFalpha from human rheumatoid joint cell cultures was inhibited by 75%, indicating that the NF-kappaB pathway is an essential step for TNFalpha synthesis in synovial macrophages and demonstrating that NF-kappaB should be an effective therapeutic target in this disease.     

Modulation of angiogenic vascular endothelial growth factor by tumor necrosis factor alpha and interleukin-1 in rheumatoid arthritis

OBJECTIVE: To investigate the regulation of expression of the angiogenic cytokine vascular endothelial growth factor (VEGF) in rheumatoid arthritis (RA), in order to determine whether new blood vessel formation could be a potential therapeutic target in RA. METHODS: Dissociated RA synovial membrane cells were cultured in the presence of cytokine inhibitors, or under hypoxic conditions. Serum VEGF levels were serially measured in RA patients enrolled in clinical trials of anti-tumor necrosis factor alpha (anti-TNFalpha) monoclonal antibody treatment. RESULTS: Combined neutralization of TNFalpha and interleukin-1 (IL-1) in RA synovial membrane cultures reduced VEGF release by 45% (P < 0.05 versus control), although blockade of either TNFalpha or IL-1 activities alone resulted in only small inhibitory effects. In addition, release of VEGF from RA synovial membrane cells was selectively up-regulated by hypoxia. Serum VEGF levels were significantly elevated in RA patients relative to control subjects, and correlated with disease activity. Treatment of RA patients with anti-TNFalpha significantly decreased serum VEGF, and this effect was enhanced by cotreatment with methotrexate. CONCLUSION: Inhibition of TNFalpha and IL-1 activity in vivo could reduce the drive to new blood vessel formation, and hence pannus mass, adding to other therapeutic effects of anti-TNFalpha therapy in RA.     

Experimental therapy of rheumatoid arthritis with antibodies to tumor necrosis factor

Two trials were carried out to compare the nutritional values of two Zambian indigenous plant protein sources--soy bean cake (SBC) and sunflower meal (SFM) in the diets of broilers and growing pigs. In trial 1, 120 one week old chickens (Abbor acre strain) were used. There were no differences (P > 0.05) between chickens on SBC and SFM in voluntary feed intake. Average daily gain of SBC chickens differed (P < 0.05) from those of SFM. The protein source had an effect (P < 0.05) on N retained [g/day]. Carcasses dry matter and crude protein were higher (P < 0.05) in SBC chickens, but ash, ether extract, Ca and P were the same as SFM. In trial 2, 12 Large white x Landrace growing barrows 1-2 months old were used. In this trial, SBC diet was consumed more than SFM. Pigs on SBC and SFM gained 0.526 and 0.284 g/head/day, respectively (P < 0.05). Nutrient digestibility was higher (P < 0.05) in SBC diet. Trial 1, demonstrated that SBC and SFM could be used for broilers without adverse effect on growth rate and body conformation. However, for growing pigs SBC is a better protein source than SFM in the tropical environment of Zambia. Finally, results obtained seem to suggest that SBC and SFM can be used as plant protein sources, but SFM is not an ideal plant protein source for growing pigs.     

Serum zinc and copper in active rheumatoid arthritis: correlation with interleukin 1 beta and tumour necrosis factor alpha

Serum zinc and copper levels and serum interleukin 1 beta (IL1 beta) and tumour necrosis factor alpha (TNF alpha) levels were evaluated in 57 female patients with active rheumatoid arthritis (RA) to investigate a possible role of IL1 beta and TNF alpha on zinc and copper homeostasis in RA. Serum zinc levels were significantly lower and serum copper levels significantly higher in RA patients when compared with osteoarthritis or asymmetrical psoriatic oligoarthritis patients and with normal controls. No differences were observed in serum IgM rheumatoid factor positive and serum IgM rheumatoid factor negative patients as regards serum zinc and copper concentration. In RA patients the erythrocyte sedimentation rate and acute-phase proteins correlated negatively with serum zinc and positively with serum copper. IL1 beta and TNF alpha were found to correlate negatively with zinc and positively with copper in RA patients. Lower levels of zinc may be due to an accumulation of zinc-containing proteins in the liver and in the inflamed joints in RA. Elevated serum copper levels seem to be linked to the increased synthesis of ceruloplasmin by the liver.     

Failure of prophylactic and therapeutic use of a murine anti-tumor necrosis factor monoclonal antibody in Escherichia coli sepsis in the rabbit

OBJECTIVE: To determine the efficacy of a murine anti-tumor necrosis factor (TNF) monoclonal antibody in the treatment of Escherichia coli peritonitis and sepsis in the rabbit. DESIGN: Prospective, paired, randomized, blinded, controlled animal trial. SETTING: Animal research laboratory. SUBJECTS: Male New Zealand white rabbits. INTERVENTIONS: Anesthetized rabbits were cannulated with indwelling femoral arterial and venous catheters. Peritonitis and sepsis were induced by intraperitoneal challenge using live E. coli O18ac bacteria. All animals were treated with gentamicin and ceftriaxone 1 hr after challenge. One group (prophylaxis experiment) consisting of ten rabbit pairs (the prophylaxis group), was treated with either murine anti-TNF monoclonal antibody or an equivalent volume of 5% albumin 3 hrs before E. coli challenge. A second group (therapeutic experiment) of 17 rabbit pairs, the treatment group, was also treated with murine anti-TNF monoclonal antibody or albumin control 1 hr after E. coli challenge. MEASUREMENTS AND MAIN RESULTS: All animals were bacteremic 1 hr after challenge. Physiologic measures of sepsis (heart rate, mean arterial pressure, serum bicarbonate, and arterial pH) did not differ between control, prophylaxis, and treatment groups. Peak serum TNF concentration was significantly (p < .01) lower in animals receiving anti-TNF monoclonal antibody, in both the prophylaxis and treatment groups, than in control animals. The survival rate was not improved significantly in either the prophylaxis or treatment group. CONCLUSIONS: Prophylactic and therapeutic use of anti-TNF monoclonal antibody in a rabbit model of E. coli peritonitis and sepsis significantly lowers TNF concentrations but does not ameliorate the physiologic effects of sepsis and does not significantly improve survival.     

Repeated administration of a F(ab')2 fragment of an anti-tumor necrosis factor alpha monoclonal antibody in patients with severe sepsis: effects on the cardiovascular system and cytokine levels

In an uncontrolled clinical trial the effects of repeated administration of the F(ab')2 fragment of a murine monoclonal anti-tumor necrosis factor alpha (TNF alpha)-antibody (MAK 195F) on cytokine levels and the cardiovascular system were studied in 20 patients with severe sepsis. Patients were treated with a total of 11 single dosages of the anti-TNF alpha-antibody intravenously over 5 days using either 1 mg/kg (n = 10) or 3 mg/kg (n = 10). The anti-TNF alpha-antibody was well tolerated in all patients without signs of toxicity and without development of anti-murine antibodies. As assessed by cytokine levels (TNF alpha, Interleukin-6) and hemodynamics there was no evidence that the higher dosage of the anti-TNF alpha-antibody (3 mg/kg per dose) was more effective than the lower dosage (1 mg/kg per dose). Comparison of our data with recent data from phase I or II trials using a complete murine monoclonal anti-TNF alpha-antibody suggest that the F(ab')2 fragments of the murine monoclonal anti-TNF alpha-antibody may be of similar efficacy. Definitive conclusions, however, with respect to improvement of mortality and improvement of the cardiovascular system, await the results of larger ongoing placebo-controlled trials.     

Production of tumor necrosis factor alpha and interleukin-6 by alveolar macrophages from patients with rheumatoid arthritis and interstitial pulmonary disease

The aim of this study was to measure the production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) by alveolar macrophages in patients with rheumatoid arthritis and interstitial lung disease (ILD). Rheumatoid arthritis patients diagnosed by ACR criteria (n = 8) with associated ILD documented by pulmonary function tests and high resolution computerized tomography scanning, and 12 healthy volunteers (6 smokers and 6 nonsmokers). We determined the spontaneous and induced production of bacterial lipopolysaccharides (LSP), TNF-alpha and IL-6 by alveolar macrophages obtained by bronchoalveolar lavage. The macrophages were isolated by Ficoll-Hypaque gradient centrifugation and plastic adherence and cultured in serum-containing medium (low endotoxin) in the presence and absence of LPS (100 ng/ml). TNF-alpha and IL-6 levels contents were determined in supernatants by ELISA. In the patient group both spontaneous and induced production of TNF-alpha were significantly higher than in controls (p < 0.01). Macrophages stimulated with LPS in patients with rheumatoid arthritis and ILD also released greater amounts of IL-6 than did those of the healthy controls. IL-6 spontaneous and induced production was significantly lower in smokers than in nonsmokers. TNF-alpha and IL-6 production in patients with rheumatoid arthritis and ILD, studied in bronchoalveolar lavage specimens reveals that alveolar macrophages are hyperreactive in these patients, who are possibly sensitized as a consequence of the inflammatory lung process inherent to the disease. Further study is needed to define the pathogenic role of these mediators.     

Randomised comparison of combined step-down prednisolone, methotrexate and sulphasalazine with sulphasalazine alone in early rheumatoid arthritis

BACKGROUND: The value of intensive combination therapy in early rheumatoid arthritis is unproven. In a multicentre, double-blind, randomised trial (COBRA), we compared the combination of sulphasalazine (2 g/day), methotrexate (7.5 mg/week), and prednisolone (initially 60 mg/day, tapered in 6 weekly steps to 7.5 mg/day) with sulphasalazine alone. METHODS: 155 patients with early rheumatoid arthritis (median duration 4 months) were randomly assigned combined treatment (76) or sulphasalazine alone (79). Prednisolone and methotrexate were tapered and stopped after 28 and 40 weeks, respectively. The main outcomes were the pooled index (a weighted change score of five disease activity measures) and the Sharp/Van der Heijde radiographic damage score in hands and feet. Independent health-care professionals assessed the main outcomes without knowledge of treatment allocation. FINDINGS: At week 28, the mean pooled index was 1.4 (95% CI 1.2-1.6) in the combined treatment group and 0.8 (0.6-1.0) in the sulphasalazine group (p < 0.0001). At this time, 55 (72%) and 39 (49%) patients, respectively, were improved according to American College of Rheumatology criteria. The clinical difference between the groups decreased and was no longer significant after prednisolone was stopped, and there were no further changes after methotrexate was stopped. At 28 weeks, the radiographic damage score had increased by a median of 1 (range 0-28) in the combined-therapy group and 4 (0-44) in the sulphasalazine group (p < 0.0001). The increases at week 56 (2 [0-43] vs 6 [0-54], p = 0.004), and at week 80 (4 [0-80] vs 12 [0-72], p = 0.01) were also significant. Further analysis suggests that combined therapy immediately suppressed damage progression, whereas sulphasalazine did so less effectively and with a lag of 6 to 12 months. There were fewer withdrawals in the combined therapy than the sulphasalazine group (6 [8%] vs 23 [29%]), and they occurred later. INTERPRETATION: This combined-therapy regimen offers additional disease control over and above that of sulphasalazine alone that persists for up to a year after corticosteroids are stopped. Although confirmatory studies and long-term follow-up are needed, this approach may prove useful in the treatment of early rheumatoid arthritis.     

Pulse therapy with prospidin and methotrexate: comparative efficacy in rheumatoid arthritis (12-month controlled study)

Efficiency of pulse-therapy with prospidin (500 mg for 5 days in hospital, 500-1000 mg for a month as maintenance) and methotrexate (30 mg a week i.v. in hospital, 7-10 mg a week as maintenance) was investigated in 93 patients with severe RA. The response to prospidin and methotrexate arose quickly (within 10-14 and 4-5 weeks, respectively) and occurred in 73 and 70% of patients, respectively. Withdrawal of the drug was caused by side effects of methotrexate (19.3%) and resistance to prospidin (23.2%).     

Safety and efficacy of hydroxychloroquine as maintenance therapy for rheumatoid arthritis after combination therapy with methotrexate and hydroxychloroquine

OBJECTIVE: To evaluate the ability of hydroxychloroquine sulfate (HCQ) to extend the response to combination therapy with HCQ and methotrexate (MTX) and the safety of longterm HCQ maintenance therapy in patients with active rheumatoid arthritis (RA). METHODS: Two-part study consisting of an open label segment evaluating combination HCQ/MTX therapy followed by a double blind segment evaluating maintenance therapy for a total of 60 weeks. First, all patients were treated with HCQ 400 mg/day and MTX 7.5 to 15 mg/week for 24 weeks. Then, responders were randomized into 3 groups: (1) HCQ with MTX as needed for disease flare (n = 40), (2) HCQ 400 mg/day (n = 41), or (3) placebo with MTX as needed for disease flare (n = 40), each for 36 weeks. RESULTS: Clinical disease and laboratory variables improved significantly during initial combination therapy with HCQ and MTX. After MTX withdrawal, HCQ-containing maintenance regimens delayed the onset of disease flare (p = 0.023). There were no unexpected adverse events at any time or between-group differences in the distribution of adverse events during the double blind segment. CONCLUSION: Combination of HCQ and MTX appeared to be effective and well tolerated for 24 weeks. After withdrawal of MTX, HCQ extended the response seen with combination therapy and was well tolerated for 36 weeks. Initial therapy with HCQ and MTX, followed by maintenance HCQ, may be a useful alternative for the treatment of RA.