Taxol (paclitaxel) involution of articular cartilage destruction in collagen induced arthritis: an ultrastructural demonstration of an increased superficial chondroprotective layer.

OBJECTIVE: To determine the ultastructural changes of Taxol (paclitaxel) involution of articular cartilage destruction in collagen induced arthritis (CIA) and to compare with articular cartilage from normal rats. METHODS: Forty-five Louvain rats were randomized to one of 3 protocols for structural analysis: (1) control group, (2) CIA group, and (3) Taxol treated CIA group. The latter group received 10 mg/kg body weight of Taxol at Days 10, 12, and 14 and 7.5 mg/kg body weight of Taxol on Days 16, 18, and 20 postimmunization with collagen type II. Eight days later, each group was examined by light microscopy and scanning and transmission electron microscopy. RESULTS: In Taxol treated rats, the morphology of the articular cartilage reverted to that observed in naive rats except for a striking increase in the thickness of the superficial amorphous layer covering the articular surface. CONCLUSION: The involution of CIA by Taxol suggests that this agent may be useful in the clinical treatment of RA.

Suppression of coronary vasculitis in a murine model of Kawasaki disease using an angiogenesis inhibitor.

Coronary arteritis can be induced in C57BL/6 mice with a single intraperitoneal (ip) injection of Lactobacillus casei cell fragments. Histologic sections resemble the vasculitis and aneurysms observed in the medium-sized coronary arteries of children with Kawasaki disease. Since endothelial cells could play an important role in the development of vasculitis, a recently described angiogenesis inhibitor that is not an immunosuppressive agent, AGM-1470 (derived from Aspergillus fumigatus), was used to evaluate its therapeutic potential in this model. A total of 32 mice were administered 0.5 mg of sterile L. casei preparation ip on day 0 and randomized to either a treatment (AGM-1470, 27mg/kg sc alternate days) or a control (vehicle only) protocol. Hearts were harvested on day 14 (early disease) or at the end of the study on day 28 (established disease). Histologic sections were scored blindly for vasculitis. Day 14 sections from both protocols manifested only minimal disease, indicating that the vasculitis had not yet matured. By day 28, the AGM-1470 group had significantly less coronary vasculitis than the control group (0.7 vs 2.6, p < 0.005, respectively). These studies suggest that endothelial cells may play an active role in this pathologic process and that angiogenesis inhibitors, such as AGM-1470, could be useful tools for the treatment and understanding of vasculitis.

Collagen induced arthritis: reversal by mercaptoethylguanidine, a novel antiinflammatory agent with a combined mechanism of action.

OBJECTIVE: We recently identified mercaptoethylguanidine (MEG) as an antiinflammatory agent with a combined mechanism of action. Its effects include inhibition of the inducible isoform of nitric oxide synthase (iNOS), scavenging peroxynitrite, a cytotoxic oxidant species produced from nitric oxide (NO) and superoxide, and inhibition of cyclooxygenase (COX). We investigate the effect of MEG in collagen induced arthritis (CIA). METHODS: Syngeneic LOU rats were immunized with native type II collagen on Day 0. After clinical signs of arthritis developed on Day 10, treatment with MEG was initiated (30 mg/kg ip tid) and continued until sacrifice on Day 28. Serum nitrite/nitrate was measured in control animals, at arthritis onset and 2 days after the start of MEG treatment. Clinical scores were obtained daily. At Day 28, radiographic scores were obtained, and joints were harvested for the measurement of mRNA for tumor necrosis factor-alpha (TNF-alpha), collagenase, and stromelysin. RESULTS: Serum nitrite/nitrate increased from 7.9+/-0.7 mM (baseline) to 13.5+/-2.6 at arthritis onset (p < 0.05). Within 48 h of MEG treatment, nitrite/nitrate levels fell to 7.2+/-1.1 (p < 0.05). By Day 28, clinical arthritis scores (measured on a scale of 0-8) were 7.1+/-0.6 in the vehicle group compared to 1.4+/-0.6 in the MEG treated group (p < 0.0001). Radiographic scores (scale 0-6) on Day 28 were reduced from 4.9+/-0.6 to 0.6+/-0.4 (p < 0.0002) by MEG treatment. MEG reduced the synovial expression of mRNA for TNF-alpha, collagenase, and stromelysin by 72, 67, and 52%, respectively. CONCLUSION: These data show that MEG has beneficial effects on established CIA. The mechanism of action may be related to inhibition of synovial iNOS expression or activity, inhibition of COX, scavenging of peroxynitrite, with subsequent inhibition of angiogenesis, metalloproteinase, and TNF-alpha expression.

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.

Taxol involution of collagen-induced arthritis: ultrastructural correlation with the inhibition of synovitis and neovascularization.

Collagen-induced arthritis (CIA) is an animal model of rheumatoid arthritis (RA) that can be regressed with Taxol (paclitaxel), a chemotherapeutic agent. To identify structural changes that occur with involution, the synovium from naive, untreated CIA, and Taxol-treated CIA rats were evaluated by light microscopy plus transmission and scanning electron microscopy. Analysis included detailed images of vascular networks using polymeric corrosion casts. The CIA synovium was morphologically similar to human RA synovium. In CIA, the integrity of the intimal lining is lost by Type-B synoviocytes becoming highly elongated and polarized toward the joint space, resulting in non-overlapping cellular processes and the elimination of the basal lamina. In addition, the lining expanded from a width of 6-10 microns in naives to 200-250 microns in CIA due primarily to increased numbers of both Type-A and -B synoviocytes and more interstitial matrix. Vascular corrosion casts of CIA synovium illustrated a marked increase in blood vessel volume and an extensive interconnecting vascular architecture; neovascular arrays were observed to project toward the synovial surface. In Taxol-treated CIA, the synoviocyte and neovascular components reverted to the naive synovium morphology, suggesting that this agent might be useful in the therapy of RA.

Model studies directed toward the application of boron neutron capture therapy to rheumatoid arthritis: boron delivery by liposomes in rat collagen-induced arthritis.

The application of boron neutron capture therapy to rheumatoid arthritis requires the selective delivery of the boron-10 isotope to the synovitic tissue. The use of liposomes as a boron delivery method has been explored through the measurement of the time course biodistribution of boron in rats with collagen-induced arthritis (CIA). Small unilamellar vesicles were composed of a 1:1 mixture of distearoylphosphatidylcholine and cholesterol, incorporated K[nido-7-CH3(CH2)15-7,8-C2B9H11] as an addend in the lipid bilayer and encapsulated Na3[a2-B20H17NH2CH2CH2NH2] in the aqueous core. The tissue concentration of boron delivered by liposomes was determined by inductively coupled plasma-atomic emission spectroscopy after intravenous injection of liposome suspensions into Louvain rats with CIA. With the low injected doses of boron used [13-18 mg of boron per kg (body weight)], the peak boron concentration observed in arthritic synovium was 29 microg of boron per g of tissue. The highest synovium/blood boron ratio observed was 3.0, when the synovial boron concentration was 22 microg of boron per g of tissue. In an attempt to increase the synovium/blood boron ratio by lowering the blood boron concentration, a liposomal formulation characterized by a shorter blood clearance time was examined. Thus, the biodistribution of liposomes with additional K[nido-7-CH3(CH2)15-7, 8-C2B9H11] incorporated in the vesicle membrane not only demonstrated more rapid blood clearance and slightly higher synovium/blood boron ratios but also exhibited reduced boron uptake in synovial tissue. These studies with boron neutron capture therapy for CIA suggest that this form of therapy may be feasible in the treatment of rheumatoid arthritis.

Suppression of collagen-induced arthritis by an angiogenesis inhibitor, AGM-1470, in combination with cyclosporin: reduction of vascular endothelial growth factor (VEGF).

Pannus formation characterized by neovascularization is a prominent pathologic finding in both rheumatoid arthritis (RA) and rat collagen-induced arthritis (CIA). CIA is a T-cell-dependent process induced by immunization of inbred LOU rats with native type II collagen in incomplete Freund's adjuvant. AGM-1470 is a highly specific inhibitor of new blood vessel formation by its effects on endothelial cell migration, endothelial cell proliferation, and capillary tube formation. Cyclosporin A (CSA) is an immunomodulating agent that inhibits IL-2 and other cytokine production involved in early antigen activation of T-cells. In this study the effects of single and combination therapy with AGM-1470 (27 mg/kg alternate days) and low-dose CSA (4 mg/kg/day continuous infusion via osmotic pump) on established CIA (total n = 62) were examined. At Day 18 post arthritis onset, clinical arthritis was significantly reduced in rats treated with single-agent AGM-1470 (1.88 +/- 0.33) or combination therapy (1.13 +/- 0.32) (P < 0.00001 and 0.000001, respectively) versus control. Single-agent CSA-treated rats, even if given CSA beginning on the day of immunization, did not attenuate arthritis severity. THe longitudinal mean arthritis score of combination-treated rats was significantly lower than that of rats receiving AGM-1470 (P < 0.0001), reflecting a more moderate early disease course in combination-treated rats. Disease severity in rats treated with single-agent CSA was not significantly different from control rats. Mean WBC counts, differentials, and delayed-type hypersensitivity responses were similar in all groups. CII antibody levels were lower in AGM-1470 protocols compared to CSA or controls. Flow cytometry of peripheral blood, spleen, and lymph nodes demonstrated decreased levels of CD4+ cells in rats given CSA. TNF-alpha levels remained elevated, even in treated rats, while vascular endothelial growth factor levels were reduced in rats receiving AGM-1470 compared to both arthritic controls and naive rats. Both single-agent and combination therapies were well tolerated. This is the first study to examine the effects of AGM-1470 together with CSA. Combination therapy was more effective than single-agent therapy. The results suggest that the use of interventions with distinct mechanisms of action may be efficacious in the treatment of RA.

A novel angiogenesis inhibitor suppresses rat adjuvant arthritis.

Rat adjuvant arthritis (AA) is an animal model of rheumatoid arthritis in which pannus formation and destruction of joints occur after immunization with complete Freund's adjuvant. Neovascularization is present within the synovium and may be critical for pannus growth. In this study the effects of a novel angiogenesis inhibitor, AGM-1470, on AA were evaluated. Lewis rats were immunized with CFA to induce arthritis. AGM-1470 treatment was initiated prior to arthritis onset (preventative protocol) or administered to rats with established disease (suppressive protocol). The severity of synovitis and the immunologic status of all rats were then evaluated. Using clinical and radiographic criteria, AGM-1470 significantly reduced arthritis incidence (preventative protocol) (P < 0.01) and disease severity (both protocols, P < 0.001, compared to controls) without affecting T cell function in vitro or phenotype in vivo. Additionally, histologic sections from control rats revealed marked pannus formation, destruction of bone/cartilage, and neovascularization. These findings were absent in AGM-1470-treated rats. AGM-1470 may offer a new treatment option for rheumatoid arthritis and other angiogenesis-dependent diseases.

Suppression of collagen-induced arthritis using an angiogenesis inhibitor, AGM-1470, and a microtubule stabilizer, taxol.

Collagen-induced arthritis (CIA) is a T-cell-dependent rat model of rheumatoid arthritis (RA) that is induced by injection of collagen type II in incomplete Freund's adjuvant. Neovascularization within the synovium is a prominent feature of CIA and RA. The novel angiogenesis inhibitor AGM-1470 and the microtubule-stabilizing agent Taxol represent two new classes of agents with specific mechanisms of action. AGM-1470 inhibits fibroblast growth factor-induced stimulation of endothelial cell migration, endothelial cell proliferation, and capillary tube formation, resulting in effective suppression of new blood vessel formation. By enhancing microtubule polymerization, Taxol interferes with normal microtubule function in cell mitosis, migration, chemotaxis, and intracellular transport. Using a suppression protocol in established CIA, the effects of AGM-1470 and Taxol as single agents and in combination were evaluated. Combination therapy significantly reduced clinical arthritis compared to control rats (P < 0.00001). The combination therapy group also experienced earlier and significantly greater reduction of clinical arthritis compared to either single agent-treated groups (P < 0.05). Blinded radiographic scores at the end of the study demonstrated less soft tissue swelling and joint destruction using combination therapy than either single agent. This is the first use of AGM-1470 and Taxol in combination therapy. Further study of agents with distinct mechanisms of action may lead to more effective treatment options in chronic inflammatory arthritis and to a better understanding of the pathophysiologic processes of pannus formation.

Regression of collagen-induced arthritis with taxol, a microtubule stabilizer.

OBJECTIVE: To investigate the capacity of taxol, a microtubule stabilizer, to inhibit collagen-induced arthritis (CIA), a model of rheumatoid arthritis. METHODS: Louvain rats were immunized with type II collagen (day 0) to induce arthritis. Taxol was administered beginning on day 2 (prevention protocol) or at arthritis onset on day 9 (in either a high-dose or low-dose suppression protocol). Rats were assessed clinically and radiographically for arthritis severity. Cellular and humoral immune responses to type II collagen were also evaluated. RESULTS: Institution of taxol prior to arthritis onset completely precluded the development of CIA (P < 0.0001 versus controls). It also suppressed established clinical disease (high-dose protocol P < 0.0000001; low-dose protocol P < 0.0001) and radiographic erosions (high-dose protocol P < 0.00001; low-dose protocol P < 0.001) compared with controls. Levels of IgG antibodies, but not delayed-type hypersensitivity, to type II collagen were reduced after taxol administration. CONCLUSION: Taxol completely prevented the induction of CIA and caused significant regression of existing arthritis.

Prevention of experimental autoimmune arthritis with a peptide fragment of type II collagen.

Collagen arthritis is induced in inbred rats with the injection of native type II collagen. The pathogenesis of this experimental autoimmune disease is T cell dependent. This study demonstrates that collagen-specific T cells, derived from pathogenic and nonpathogenic rat T cell lines, both recognize the same peptide epitope. The epitope, consisting of amino acids 58-73 of cyanogen bromide fragment 11 of type II collagen, was as effective as whole collagen in stimulating a panel of collagen-specific rat/mouse T cell hybridomas. This peptide may, therefore, constitute a dominant epitope for CD4+ rat T cells in their response to type II collagen. Administration of the peptide to either neonatal or adult rats prevented the subsequent induction of experimental arthritis with whole collagen, demonstrating that the in vivo response to this dominant epitope is, therefore, relevant in the pathogenesis of arthritis. Despite its ability to prevent collagen-induced arthritis, administration of this peptide in incomplete Freund's adjuvant intradermally did not induce disease.

Effects of tumor necrosis factor alpha (TNF-alpha) on collagen arthritis.

Louvain rats were administered tumor necrosis factor alpha (TNF-alpha) via a continuous osmotic infusion pump. These rats were then immunized with native type II collagen (CII) to determine the effects of exogenous TNF-alpha on collagen-induced arthritis (CIA), an animal model of rheumatoid arthritis (RA). In this highly susceptible strain, 100% of experimental and control rats developed arthritis although TNF-alpha-treated rats had more severe disease as judged by both clinical and blinded radiographic parameters. Humoral responses to collagen were high in both groups, but cellular responses to CII were augmented by TNF-alpha. Serum IL-6 levels were significantly increased in all arthritic rats. This study suggests that TNF-alpha is a proinflammatory cytokine in CIA and that future studies targeting TNF-alpha might be therapeutic.

Angiogenesis inhibition suppresses collagen arthritis.

Neovascularization is observed in a spectrum of diseases such as solid tumors, diabetic retinopathy, and rheumatoid arthritis. It is also evident in rat collage-induced arthritis (CIA), an animal model with histologic, clinical, and radiographic manifestations resembling rheumatoid arthritis. To evaluate the effects of angioinhibition in CIA, Louvain rats were immunized with type II collagen to induce arthritis and then administered an angiogenesis inhibitor, AGM-1470, in an attempt to either prevent arthritis or suppress established disease. Using clinical and radiographic criteria, AGM-1470 prevented CIA and significantly suppressed established disease without evidence of immunosuppression. Histologic sections from control ankle joints manifested pannus and neovascularization, which were absent in experimental animals. This is the first study to investigate this novel agent in an autoimmune disease, and additional evaluation of this promising compound in other diseases that are potentially angiogenesis dependent, such as rheumatoid arthritis, might be warranted.

Suppression of collagen arthritis with antibodies to an arthritogenic, oligoclonal T cell line.

Rats immunized with type II collagen (CII) develop an immunologically mediated polyarthritis. T cells have been implicated in the pathogenesis of this model since they can adoptively transfer the disease. A CII-specific T cell line (VA), consisting of three distinct clones by Southern blot analysis, has been shown to be arthritogenic. Antibodies specific for this line were generated by immunizing rabbits. In an attempt to prevent collagen-induced arthritis (CIA), Louvain rats were injected with 1 ml of anti-VA ip on Days -1, +1, +3 and 0.5 ml on Day +5 (early treatment). To evaluate its effect on existing disease, rats received anti-VA on the day of arthritis onset and subsequently on 4 successive alternate days using the same dosage protocol (late treatment). Control rats received no therapeutic injections or were administered normal rabbit serum. All rats were immunized with CII on Day 0 to induce CIA. Rats administered antibodies using the early anti-VA treatment protocol had a significantly diminished incidence of arthritis compared to controls. Established arthritis was significantly diminished compared to controls in rats given the late anti-VA treatment. In both protocols, radiographic evidence of joint destruction was significantly reduced compared to controls. T cell phenotyping using flow cytometry analysis demonstrated that the anti-VA antibody therapy selectively eliminated a small subset of T cells since there was little difference in total T cell counts in the experimental versus control groups. Delayed type hypersensitivity and IgG antibody titers to CII were minimally decreased in the experimental versus control group. These results suggest that antibodies raised to an oligoclonal arthritogenic T cell line can suppress collagen arthritis. This may have implications with respect to 1) the size of the T cell receptor repertoire involved in the pathogenesis of collagen arthritis and 2) immunospecific protocols for CIA and other autoimmune diseases.

Suppression of collagen-induced arthritis by combination cyclosporin A and methotrexate therapy.

Louvain (LOU) rats were administered either methotrexate (MTX; 0.3 mg/kg/week or 0.8 mg/kg/week intraperitoneally), cyclosporin A (CSA; 4 mg/kg/day or 10 mg/kg/day continuous infusion via osmotic pump), or a combination of both agents. The rats were immunized with native type II collagen (CII) to determine the effects of these agents on collagen-induced arthritis, an animal model of rheumatoid arthritis. A significant decrease in the incidence (P less than 0.01) and severity of arthritis by clinical (P less than 0.05) and radiographic assessments (P less than 0.05) was found in recipients of combination therapy, compared with controls. Delayed-type hypersensitivity reactions to CII were measured on day 26, and production of IgG antibody to CII was measured on days 7, 14, and 26. IgG antibody was evident by day 7, and titers were near-maximal by day 14. Both delayed-type hypersensitivity and antibodies to CII were reduced in animals that received the higher dosage of CSA. Liver, kidney, and spleen specimens obtained from rats treated with CSA and MTX demonstrated no histologic abnormalities on light microscopy, compared with controls. These studies indicate that CSA and MTX in combination is a safe and effective therapy for collagen-induced arthritis and may be useful in the treatment of rheumatoid arthritis.

Elimination of clonogenic malignant human T cells using monoclonal antibodies in combination with 2'-deoxycoformycin.

2'Deoxycoformycin (dCF) specifically inhibits adenosine deaminase (ADA) and causes selective cytotoxicity of normal and malignant T cells. In clinical trials, dCF caused rapid lysis of malignant T lymphoblasts. Although dCF has been associated with dose-limiting nonhematopoietic toxicities, myelosuppression has not been observed. Since dCF is relatively nontoxic to hematopoietic stem cells, we tested dCF for utility in the ex vivo purging of malignant T lymphoblasts from remission leukemic bone marrow for autologous bone marrow transplantation. We found that T lymphoblast cell lines were sensitive to dCF (plus deoxyadenosine [dAdo]) under conditions that did not ablate human hematopoietic colony-forming cells. Moreover, combined pharmacologic (dCF plus dAdo) and immunologic (anti-T cell monoclonal antibodies [McAb] plus complement) purging resulted in additive reduction in clonogenic T lymphoblasts. These results provide the basis for a clinical trial of bone marrow transplantation using combined pharmacologic/immunologic purging of T lymphoblasts from patients' harvested autologous marrow.

Antigenic analysis of hematopoiesis. VI. Flow cytometric characterization of My-10-positive progenitor cells in normal human bone marrow.

We have previously shown [1] that the anti-My-10 murine monoclonal antibody detected an epitope of a 115-kDa glycoprotein expressed specifically on KG-1a leukemia cells and a small subset of normal human bone marrow cells. This My-10+ marrow cell subset was shown to contain a highly enriched population of morphologic blast cells and hematopoietic colony-forming cells [1]. In this report, My-10+ cells were characterized, by flow cytometry, as an approximately 1% subpopulation of normal human bone marrow cells. My-10+ cells were slightly larger than lymphocytes and agranular, as determined by their fluorescence-activating cell sorting (-er) (FACS) light-scattering properties. In two-color immunofluorescence experiments, My-10+ cells coexpressed the HLA-DR antigen. However, there was no detectable cellular coexpression of My-10 with either the Leu 1-5, 7, 9, 11, 15, M3, or My-18 antigens. There was an average of approximately 50,000 My-10 molecules per My-10+ marrow cell. This provides further evidence that the My-10 molecule is expressed, at relatively low levels, selectively on early human marrow cells but not on mature lymphohematopoietic cells.

Rapid, efficient cloning of murine hybridoma cells in low gelation temperature agarose.

A rapid, efficient method is described for cloning of lymphocyte hybridoma cells. The method utilizes ultra-low gelation temperature agarose.

My-1, new myeloid-specific antigen identified by a mouse monoclonal antibody.

Antibody-secreting hybridomas were produced by fusion of P3-NS1/1Ag--4-1 mouse plasmacytoma cells with splenocytes from a mouse immunized with HL-60 human promyelocytic leukemia cells. A cloned hybridoma cell line was identified that secreted antibody against a cell surface antigen expressed on all normal human peripheral blood granulocytes, on bone marrow granulocytic precursor cells, and on blast cells from 3 of 15 patients with nonlymphoid leukemia. However, the antibody did not react with normal peripheral blood lymphocytes, monocytes, platelets, or red cells, or with blast cells from 20 patients with acute lymphoblastic leukemia or from 5 patients with lymphoma. This monoclonal antibody identified a granulopoietic differentiation antigen (designated My-1) and may prove useful in the subclassification on nonlymphoid leukemia and in the investigation of hematopoiesis.