The effect of thalidomide and 2 analogs on collagen induced arthritis

OBJECTIVE: Thalidomide has been described as an inhibitor of both angiogenesis (which may account for its teratogenic effects on limb bud formation) and tumor necrosis factor-alpha (TNF-alpha) production. We evaluated its therapeutic potential in collagen induced arthritis (CIA), a rat model of rheumatoid arthritis (RA). METHODS: Rats were administered orally 200 mg/kg/day thalidomide (n = 10) or either of 2 analogs, EM-12 (n = 9) or supidimide (n = 9). An additional group was given thalidomide (n = 10) at 200 mg/kg twice daily, and a control group (n = 13) was given vehicle only. At completion of the protocols, serum levels of TNF-alpha and vascular endothelial growth factor (VEGF) were measured. RESULTS: Suppression of inflammatory synovitis by clinical and radiographic criteria was significantly lower in all experimental protocols except the lower dose thalidomide group. The EM-12 analog was the most efficacious, and twice daily thalidomide was better than once daily. The incidence of arthritis onset was comparable among all groups. Strong cell mediated and humoral responses to type II collagen, measured by a radiometric delayed type hypersensitivity assay and anti-type II collagen IgG ELISA, respectively, were similar in the experimental and control groups. TNF-alpha and VEGF levels were increased in all rats immunized with collagen compared to naive controls. CONCLUSION: Thalidomide and its analogs can suppress the clinical severity of rat CIA, but the mechanism of action is not a result of TNF-alpha or VEGF downregulation.     

Dual role of IL-12 in early and late stages of murine collagen type II arthritis

IL-12 can promote Th1 responses, and early administration of IL-12 during immunization was shown to enhance expression of autoimmune collagen-induced arthritis (CIA). We now studied the impact of IL-12 at the stage of disease expression and during established CIA in DBA-1 mice. Accelerated onset and enhanced severity were provoked when i.p. injections of 100 ng of murine IL-12 (mIL-12) were given around the time of arthritis onset. Moreover, the onset of CIA could be ameliorated with anti-mIL-12 Abs, indicating that IL-12 is a pivotal mediator in the expression of CIA. In addition, the effect of anti-mIL-12 treatment was analyzed in established CIA. Continued treatment did not suppress established arthritis. Instead, these mice showed an impressive exacerbation of arthritis shortly after cessation of anti-mIL-12 treatment, indicative of impairment of endogenous control. Exaggerated disease was characterized by massive granulocyte influx and enhanced expression of IL-1 beta and TNF-alpha mRNA in the synovial tissue. Subsequently, we treated established collagen arthritis with recombinant mIL-12 for 7 days. Profound suppression of the arthritis score was noted, including reduced influx of cells and diminished cartilage damage. Tenfold enhanced levels of IL-10 were detected in sera of mIL-12-treated mice, and up-regulated mRNA levels of IL-10, IFN-gamma, and IL-12 were measured in synovial tissue. Finally, the anti-inflammatory effect of IL-12 on CIA could be reversed by coadministration of anti-IL-10 Abs. This study indicates that IL-12 has a stimulatory role in early arthritis expression, whereas it has a suppressive role in the established phase of collagen arthritis.     

Synergistic protection against cartilage destruction by low dose prednisolone and interleukin-10 in established murine collagen arthritis

BACKGROUND: For simplification of blood cell transplantation, an automated apheresis system that exploits a dual-stage channel device for mononuclear cell (MNC) collection (AutoPBSC, designed for the COBE Spectra) was studied. STUDY DESIGN AND METHODS: The automated default software (AutoPBSC-Default) and three software modifications of the harvest frequency during leukapheresis, referred to as AutoPBSC-1.25, AutoPBSC-1.75, and AutoPBSC-2.75, were evaluated in comparison with the semiautomated Version 4.7 (V4.7) apheresis system in 119 leukapheresis procedures performed in 90 cancer patients treated with chemotherapy plus granulocyte-colony-stimulating factor. CD34+ cell and platelet collection efficiency (CE); volume and cell composition of the leukapheresis components; and patient platelet and red cell (RBC) loss during leukapheresis were measured. RESULTS: The majority of collection measures evaluated with the AutoPBSC compared favorably to those obtained with the V4.7. CD34+ cell CE increased from 55 percent with V4.7 to 68 percent with the AutoPBSC-Default (p = 0.05). The AutoPBSC provided lower platelet contamination in the collected component (1.18 x 10(11) vs. 2.26 x 10(11) with the V4.7; p<0.001). The volume of the AutoPBSC-Default component was significantly lower (67 vs. 180 mL with the V4.7; p<0.001). The MNC purity of the AutoPBSC component was greater (52 vs. 28% with the V4.7; p<0.001), and the RBC contamination lower (AutoPBSC, 0.53 x 10(11) vs. 1.04 x 10(11) with the V4.7; p<0.001). Modifications of the AutoPBSC to increase the harvest frequency by 1.25-, 1.75-, and 2.75-fold resulted in increased CD34+ cell CE (77%, 75%, and 83%, respectively; p<0.001 in all cases), but also in reduced numbers of circulating platelets, higher platelet contamination of the component, and lower MNC purity than were seen with the AutoPBSC-Default. CONCLUSION:The AutoPBSC offers the following advantages over the V4.7 system: a) better CE of CD34+ cells; b) reduced collection of platelets; c) reduced contamination of the leukapheresis component with granulocytes, platelets, and RBCs; d) reduced component volume; and e) automation.     

AP-1 and NF-kappaB regulation in rheumatoid arthritis and murine collagen-induced arthritis

Mandibular distraction was performed on 14 children, between September 1991 and December 1997. Their average age was 6.9 years, ranging from 1.5 to 13.5 years. All patients had severe hypoplastic mandibles with retromandibulism. Seven of the children (50%) had respiratory distress due to obstruction of the upper airway before distraction. This resolved in every case. Five patients underwent unilateral and nine bilateral distraction. A total of 23 distractors were used, 15 were applied extraorally and 8 endorally. The average latency time after operation was 2.8 days, but for the past 2 years, distraction was started beginning with the operation. The distraction was increased twice daily for an average of 5.5 weeks, by 0.4 or 0.5 mm each time, depending on the distractor. Computed tomography and ultrasound were used to follow the ossification process in the distraction gap and to measure the lengthening achieved. Subsequent retention time averaged 2.4 weeks. The mandibles were elongated by up to 18 mm (average 9.3 mm) and the respiratory distress symptoms resolved in all patients. Several minor complications which are reported occurred. Six patients were followed up for periods between 3 and 7 years. During this time further growth of the distracted mandibles was recorded.     

Unscheduled DNA synthesis in lymphocytes of rheumatoid arthritis patients and spleen cells of rats with experimentally induced arthritis

DNA repair is an important factor in the abolition or manifestation of mutations. Since intrinsic somatic mutations may be related to the occurrence of autoimmunity, DNA repair was investigated by measuring unscheduled DNA synthesis in lymphocytes of rheumatoid arthritis patients by autoradiography. Likewise, unscheduled DNA synthesis was investigated in mycoplasma- and Freund's adjuvant-induced arthritis in rats. Both in human rheumatoid arthritis and experimentally-induced arthritis the capacity for repairing lesions in DNA induced by gamma-irradiation of cells was reduced, while an increased rate of thymidine-incorporation into DNA was found after UV-irradiation. The results are discussed in relation to endonucleases of viral or mycoplasmal origin.     

The in vivo cytostatic effect induced by Propionibacterium granulosum on murine tumor cells

The cytostatic action of Propionibacterium granulosum was studied in a mouse sarcoma in vivo. Kinetic analysis of tumor cells 28 days after tumor implantation and systemic immunotherapy showed that the cell cycle time was identical in both treated and untreated tumors. P. granulosum treatment resulted in a marked prolongation of the S phase and shortening of the G1 phase of the cell cycle. A pronounced drop in the number of labeled interphases and the reduction of the growth fraction were observed in tumors obtained from mice given injection of P. granulosum. Cloning efficiency of tumor cells from P. granulosum treated animals was quantitatively similar to that of control animals and only differences in the size of lung colonies were observed.     

Effective treatment of mice with type II collagen induced arthritis with lethal irradiation and bone marrow transplantation

Ricin A-chain is delivered into macrophages via receptor-mediated endocytosis. We have found that following uptake via the mannose receptor, ricin A-chain is rapidly cleaved by endosomal proteases. Inhibition of endosomal proteases such as cathepsin D and B leads to the accumulation of toxin inside the cell. Inhibition of cathepsin D reduces ricin A-chain cytotoxicity, while blocking cathepsin B enhances cytotoxicity. Similar results were obtained using fibroblasts transfected with the mannose receptor. Our data strongly suggest that the activation or membrane translocation of ricin A-chain is dependent upon the action of specific proteases.     

Local removal of phagocytic synovial lining cells by clodronate-liposomes decreases cartilage destruction during collagen type II arthritis

OBJECTIVE: To investigate whether local removal of phagocytic synovial lining cells (SLCs) from the knee joint before onset of collagen type II arthritis has an effect on development of cartilage destruction. METHODS: Phagocytic SLCs were selectively depleted by a single injection of clodronate laden liposomes in the knee joint seven days before induction of collagen type II arthritis (CIA). Clodronate laden liposomes were given in one knee joint either alone or in combination with a short-term oral treatment of dexamethasone. Cartilage damage including proteoglycan depletion and chondrocyte death was measured in total knee joints sections stained with safranin-o or haematoxylin. RESULTS: Local removal of phagocytic SLCs, seven days before arthritis onset, prevented cell influx for the larger part. Chondrocyte death was significantly decreased in the SLC depleted arthritic joint both at an early (6 days) and late (12 days) time point after CIA induction. However, depletion of proteoglycans from femoral and patellar cartilage layers was not prevented. If the mild acute inflammation caused by a single clodronate laden liposome injection in the left knee joint, was blocked by a short-term (on consecutive days 9, 8, 7, 6, 5 before CIA onset) oral treatment with dexamethasone, cell influx, but also proteoglycan depletion was almost completely blocked. In the contralateral control right knee joint prominent cell influx and severe cartilage damage was observed, indicating that there was no effect of dexamethasone anymore at the onset of CIA. CONCLUSIONS: This study shows that removal of phagocytic lining cells before CIA induction, particularly in the presence of a short-term treatment with dexamethasone, decreases cartilage destruction.     

A peptide sequence from platelet factor 4 (CT-112) is effective in the treatment of type II collagen induced arthritis in mice

OBJECTIVE: Platelet factor 4 (PF-4) is a critical alpha chemokine in inflammation and injury responses, with multiple effects upon cellular activities. Discrete peptide sequences of the PF-4 molecule have been shown to retain biological activity. Our aim was to examine the influence of the PF-4 derived octapeptide (CT-112; TTSQVRPR) on type II collagen induced arthritis in mice, to determine if this peptide exhibited antiinflammatory properties. METHODS: DBA/1 mice were treated with CT-112 from either the time of immunization with type II collagen or from the initial onset of arthritis. RESULTS: CT-112 both prevented the development of arthritis in mice treated prophylactically and reduced progression of disease in animals treated therapeutically, and was active when delivered by either subcutaneous injection or oral gavage. No marked immunosuppressive effects were observed during CT-112 treatment, with only moderate decrease in antibody levels and mitogen responses. A significant reduction of the circulating levels of IL-1 was a consistent finding in mice treated therapeutically with CT-112. CONCLUSION: These data suggest PF-4 derived octapeptide exerts antiinflammatory effects of experimental arthritis in mice.     

Differential display analysis of murine collagen-induced arthritis: cloning of the cDNA-encoding murine ATPase inhibitor

Perforin-deficient (-/-) mice were used as T-cell donors for infusion into irradiated major histocompatibility complex (MHC)-disparate recipients to investigate the requirement for perforin-mediated cytolysis during graft-versus-host disease (GVHD) generation. Administration of 5x10(6) C57BL/6 (H2b) perforin -/- splenocytes was significantly less effective in inducing GVHD lethality when given to MHC class I + II disparate B10.BR (H2k) recipients, as compared with wild-type (+/+) controls. Perforin expression by donor T cells was not required for GVHD induction because recipients given fivefold higher numbers of perforin -/- donor splenocytes uniformly succumbed to lethal GVHD. Because both CD4+ and CD8+ donor T cells are required for optimal GVHD lethality in this strain combination, to discern the relative contribution of perforin-mediated cytolysis by CD4+ and CD8+ T cells, additional studies were performed. For these latter studies, we used a sensitive assay involving the infusion of highly purified CD4+ or CD8+ T cells into sublethally irradiated MHC class II or I disparate recipients, respectively. As compared with recipients of perforin +/+ T cells, recipients of either CD4+ or CD8+ perforin -/- T-cell subsets had a significant reduction in GVHD-mediated lethality at T-cell doses that were uniformly lethal. T-cell dose titration studies established that GVHD lethality in recipients of perforin -/- CD4+ or CD8+ T cells was reduced by approximately threefold. These data are the first to indicate that approaches to limit perforin-mediated cytolysis should be similarly effective in situations in which CD4+ or CD8+ T cells dominate the GVHD response.     

Phagocytic synovial lining cells regulate acute and chronic joint inflammation after antigenic exacerbation of smouldering experimental murine arthritis

Vascular permeability factor/vascular endothelial cell growth factor (VPF/VEGF) can both potently enhance vascular permeability and induce proliferation of vascular endothelial cells. We report here that mouse or human mast cells can produce and secrete VPF/VEGF. Mouse mast cells release VPF/VEGF upon stimulation through Fcepsilon receptor I (FcepsilonRI) or c-kit, or after challenge with the protein kinase C activator, phorbol myristate acetate, or the calcium ionophore, A23187; such mast cells can rapidly release VPF/VEGF, apparently from a preformed pool, and can then sustain release by secreting newly synthesized protein. Notably, the Fc epsilonRI-dependent secretion of VPF/VEGF by either mouse or human mast cells can be significantly increased in cells which have undergone upregulation of Fc epsilonRI surface expression by a 4-d preincubation with immunoglobulin E. These findings establish that at least one cell type, the mast cell, can be stimulated to secrete VPF/VEGF upon immunologically specific activation via a member of the multichain immune recognition receptor family. Our observations also identify a new mechanism by which mast cells can contribute to enhanced vascular permeability and/or angiogenesis, in both allergic diseases and other settings.     

Collagen-induced arthritis in nonhuman primates: multiple epitopes of type II collagen can induce autoimmune-mediated arthritis in outbred cynomolgus monkeys

A key event in signal transduction during chemotaxis of Salmonella typhimurium and related bacterial species is the interaction between the phosphorylated form of the response regulator CheY (CheY approximately P) and the switch of the flagellar motor, located at its base. The consequence of this interaction is a shift in the direction of flagellar rotation from the default, counterclockwise, to clockwise. The docking site of CheY approximately P at the switch is the protein FliM. The purpose of this study was to identify the CheY-binding domain of FliM. We cloned 17 fliM mutants, each defective in switching and having a point mutation at a different location, and then overexpressed and purified their products. The CheY-binding ability of each of the FliM mutant proteins was determined by chemical crosslinking. All the mutant proteins with an amino acid substitution at the N terminus, FliM6LI, FliM7SY and FliM10EG, bound CheY approximately P to a much lesser extent than did wild-type FliM. CheY approximately P-binding of the other mutant proteins was similar to wild-type FliM. To investigate whether the FliM domain that includes these three mutations is indeed the CheY-binding domain, we synthesized a peptide composed of the first 16 amino acid residues of FliM, including a highly conserved region of FliM (residues 6 to 15). The peptide bound CheY and, to a larger extent, CheY approximately P. It also competed with full-length FliM on CheY approximately P. These results indicate that the CheY-binding domain of FliM is located at the N terminus, within residues 1 to 16, and suggest that FliM monomers can form a complete site for CheY binding.     

N-ras mutations in radiation-induced murine leukemic cells

N-ras mutations were examined in DNA samples extracted from the spleen of CBA/Ca mice that developed myeloid leukemia (ML) following exposure to radiations of different qualities. A total of 17 ML cases, i.e. 5 cases of neutron-induced and 12 cases of photon- (3 gamma-ray and 9 x-ray) induced ML were included in the study along with 12 DNA samples from the bone marrow cells of control mice. Polymerase chain reaction-single strand conformational polymorphisms (PCR-SSCP) and the direct sequencing of PCR products were used to analyze three regions of the N-ras gene: (i) a 120 base-pair (bp) long portion of exon I (codons 2-37); (ii) a 103 bp long portion of exon II (codons 48-82); and (iii) a 107 bp long portion of exon III (codons 118-150). PCR-SSCP mobility shifts indicated mutations within only exon II of the N-ras gene. Such mutations were more prevalent in samples from mice exposed to fast neutrons. The exact type and location of these mutations were then determined by direct DNA sequencing. Silent point mutations, i.e. base transitions at the third base of codons 57 (GAC-->GAT), 62 (CAA-->CAC), or 70 (CAG-->CAA) were present only in mice that developed ML after exposure to fast neutrons. A base transversion at the third base of codon 61 (CAA-->CAC) was also observed in some ML cases. DNA sequencing demonstrated that ML samples contained normal as well as mutated DNA sequences. The higher frequency of N-ras mutations in neutron-induced ML suggested that fast neutrons are more effective in inducing genomic instability at the N-ras region of the genome. More importantly, N-ras mutations are not the initiating event in radiation leukemogenesis. This conclusion was supported by the finding that N-ras mutations were detected only in mice with an overt leukemic phenotype but not in mice with minimal tissue infiltration of leukemic cells, suggesting that the disease may be present prior to the presence of N-ras mutations. Alternatively, N-ras may be present in these mice but a large number of normal spleen cells in these mice interferes with the detection of mutation in a small population of leukemic cells.     

Characterization of crystals induced arthritis and periarthritis

Subfoveal choroidal neovascular membrane is a leading cause of legal blindness. Photocoagulation has been effective, but photocoagulation of the fovea causes a decrease in vision immediately after treatment. Surgical removal of the choroidal neovascular membrane is effective for choroidal neovascular membrane in some cases, but it restores useful vision for reading (20/40 or better) in a small number of cases of choroidal neovascular membrane not due to presumed ocular histoplasmosis syndrome. A new treatment for subfoveal choroidal neovascular membrane, foveal translocation, is an innovative procedure in which the fovea is translocated onto healthier retinal pigment epithelium. Three techniques have been developed to relocate the retina, 2 of which (retinotomy and scleral shortening) we performed in 5 cases each. Preliminary results in these 10 cases indicate that foveal translocation provides improvement of visual acuity in 40% of eyes and final visual acuities useful for reading (better than 20/40) in 20% of eyes undergoing translocation with either technique. Further study is essential to refine the amount of translocation needed and to decrease complications of the 2 techniques, including retinal detachment, proliferative vitreoretinopathy, macular pucker, corneal astigmatism, and constricted visual field.     

Adenovirus-mediated transfer of viral IL-10 gene inhibits murine collagen-induced arthritis

IL-10 is a potent anti-inflammatory cytokine that has received growing attention for its therapeutic potential. We examined the efficiency of adenoviral-mediated gene transfer of IL-10 on the incidence and severity of murine collagen-induced arthritis (CIA). Male DBA1 mice immunized with collagen II were treated by systemic administration of 10(9) plaque-forming units of replication-defective adenoviral vector expressing viral IL-10 (vIL-10) on day 30, when clinical symptoms of arthritis start. The transgene was shown to inhibit the onset of CIA, to decrease severity, and profoundly suppress the overall joint histopathology of the experimental arthritis. Significant IL-10 concentrations were obtained in the serum of injected animals for 7 days. Inhibition of arthritis was enhanced by administration of increasing doses of adenovirus-vIL-10. In addition, the local immunosuppressive effect of gene-delivered vIL-10 could be neutralized by a monoclonal anti-vIL-10 Ab. The CIA symptoms in the group treated with the same construct expressing inactive vIL-10 (vIL-10 mut) were similar to those in untreated animals. Our data indicate that a single systemic administration of an adenoviral vector encoding vIL-10 may be a good candidate to suppress arthritis.     

Fluoride therapy in prevention of rheumatoid arthritis induced bone loss

OBJECTIVE: To determine the efficacy of sodium fluoride (40 mg/day) in preventing rheumatoid arthritis (RA) induced bone loss, which may lead to osteoporosis. METHODS: We conducted an 18 month, randomized, double blind, placebo controlled trial in 38 patients with RA. The primary outcome measure was the difference in the percentage change between groups in lumbar spine bone mineral density (BMD) from baseline values after 18 months of therapy. The secondary outcome measures were the differences in the percentage change between groups in femoral neck, Ward's triangle, trochanter, and total body BMD from baseline after 18 months of therapy. RESULTS: There was a significant percentage difference (SD) between groups of 6.2% (7.3%) (p = 0.0005) in lumbar spine BMD after 18 months of treatment in favor of the fluoride group. The fluoride group experienced a 5.2% (8.4%) (p = 0.0125) increase, whereas the placebo group showed a 1.0% (4.8%) (p = 0.8015) decrease in lumbar spine BMD after treatment. No significant differences were found for the femoral neck, Ward's triangle, trochanter, and total body BMD in terms of the percentage changes from baseline within each treatment group or in the differences in the degree of change between groups after therapy. Lumbar spine BMD increased in about 80% of patients treated with fluoride (responders) compared to 44% of patients treated with placebo. CONCLUSION: The results showed that fluoride therapy was well tolerated and increased vertebral bone mass in patients with RA.