Paul Schwarzenberger

Critical role of IL-17 receptor signaling in acute TNBS-induced colitis

Background Inflammatory bowel diseases (IBDs) such as Crohns disease and ulcerative colitis are characterized by recurrent inflammation in the gastrointestinal tract. Infiltration of CD4+ lymphocytes and neutrophils is one of the predominant features of IBD. Materials and Methods Recently, interleukin (IL)‐23 and the downstream T cell‐derived cytokine IL‐17 have been found to be elevated in intestinal tissue and serum of IBD patients. However, the role of IL‐17 and IL‐17R signaling in gut inflammation is unknown. To examine this role, we investigated gut inflammation in wild‐type or IL‐17R knockout mice. Results Using a model of acute trinitrobenzenesulfonic acid (TNBS)‐induced colitis, we found that IL‐17 was produced in colon tissue at 24 and 48 hours and that IL‐17R knockout mice were significantly protected against TNBS‐induced weight loss, IL‐6 production, colonic inflammation, and local macrophage inflammatory protein‐2 induction. This protection occurred in the presence of equivalent induction of local IL‐23 and higher levels of IL‐12p70 and interferon‐&ggr; in IL‐17R knockout mice compared with wild‐type mice. Moreover, IL‐17R knockout mice showed reduced tissue myeloperoxidase activity. Furthermore, overexpression of an IL‐17R IgG1 fusion protein significantly attenuated colonic inflammation after acute TNBS. Conclusions These results demonstrate that IL‐17R signaling plays a critical role in the development of TNBS‐induced colitis and may represent a target for therapeutic intervention for IBD.

IL-1-independent role of IL-17 in synovial inflammation and joint destruction during collagen-induced arthritis.

T cell IL-17 displays proinflammatory properties and is expressed in the synovium of patients with rheumatoid arthritis. Its contribution to the arthritic process has not been identified. Here, we show that blocking of endogenous IL-17 in the autoimmune collagen-induced arthritis model results in suppression of arthritis. Also, joint damage was significantly reduced. In contrast, overexpression of IL-17 enhanced collagen arthritis. Moreover, adenoviral IL-17 injected in the knee joint of type II collagen-immunized mice accelerated the onset and aggravated the synovial inflammation at the site. Radiographic and histologic analysis showed markedly increased joint destruction. Elevated levels of IL-1β protein were found in synovial tissue. Intriguingly, blocking of IL-1αβ with neutralizing Abs had no effect on the IL-17-induced inflammation and joint damage in the knee joint, implying an IL-1 independent pathway. This direct potency of IL-17 was underscored in the unabated IL-17-induced exaggeration of bacterial cell wall-induced arthritis in IL-1β−/− mice. In conclusion, this data shows that IL-17 contributes to joint destruction and identifies an IL-1-independent role of IL-17. These findings suggest IL-17 to be a novel target for the treatment of destructive arthritis and may have implications for tissue destruction in other autoimmune diseases.

IL-17 promotes bone erosion in murine collagen-induced arthritis through loss of the receptor activator of NF-kappa B ligand/osteoprotegerin balance.

IL-17 is a T cell-derived proinflammatory cytokine in experimental arthritis and is a stimulator of osteoclastogenesis in vitro. In this study, we report the effects of IL-17 overexpression (AdIL-17) in the knee joint of type II collagen-immunized mice on bone erosion and synovial receptor activator of NF-κB ligand (RANKL)/receptor activator of NF-κB/osteoprotegerin (OPG) expression. Local IL-17 promoted osteoclastic bone destruction, which was accompanied with marked tartrate-resistant acid phosphatase activity at sites of bone erosion in cortical, subchondral, and trabecular bone. Accelerated expression of RANKL and its receptor, receptor activator of NF-κB, was found in the synovial infiltrate and at sites of focal bone erosion, using specific immunohistochemistry. Interestingly, AdIL-17 not only enhanced RANKL expression but also strongly up-regulated the RANKL/OPG ratio in the synovium. Comparison of arthritic mice from the AdIL-17 collagen-induced arthritis group with full-blown collagen-arthritic mice having similar clinical scores for joint inflammation revealed lower RANKL/OPG ratio and tartrate-resistant acid phosphatase activity in the latter group. Interestingly, systemic OPG treatment prevented joint damage induced by local AdIL-17 gene transfer in type II collagen-immunized mice. These findings suggest T cell IL-17 to be an important inducer of RANKL expression leading to loss of the RANKL/OPG balance, stimulating osteoclastogenesis and bone erosion in arthritis.

Overexpression of IL-17 in the knee joint of collagen type II immunized mice promotes collagen arthritis and aggravates joint destruction

IL17 is a new cytokine secreted by CD4+ activated memory T cells which is produced in the RA synovium [1-3] . IL-17 stimulated the production o f ILl f l and T N F a by human macrophages [2] and increased the effect o f IL-1 and TNFot on synoviocytes [3], indicating that it may play an important role in the pro-inf lammatory response. The pattern o f cellular responses induced by IL-17 is similar to that o f lL-1 , suggesting that IL-17 may also contribute to joint destruction. The potency o f IL-17 as a stimulator o f osteoclastogenesis has been shown in vitro [4] and IL-17 expression was found in the synovium of collagen induced arthritis mice [5]. In addition, IL-17 inhibits the chondrocyte metabol ism in murine intact articular cartilage through an NO mediated pathway [6], indicating direct involvement o f this T cell derived cytokine in cartilage damage. Although IL-17 is expressed in the RA synovium its in vivo role is still not clear. In this study, we investigated the effect o f local IL17 application, introduced by a recombinant adenoviral vector, in the knee joint o f collagen type II immunized mice. The data we summarized here shows that local IL-17 overexpression promotes collagen arthritis and indicates that IL-17-induced inflammation and joint damage is independent o f IL1.

Requirement of IL-17 Receptor Signaling in Radiation-Resistant Cells in the Joint for Full Progression of Destructive Synovitis

IL-17 is a proinflammatory cytokine suspected to be involved in inflammatory and autoimmune diseases such as rheumatoid arthritis. In the present study, we report that IL-17R signaling is required in radiation-resistant cells in the joint for full progression of chronic synovitis and bone erosion. Repeated injections of Gram-positive bacterial cell wall fragments (streptococcal cell wall) directly into the knee joint of naive IL-17R-deficient (IL-17R−/−) mice had no effect on the acute phase of arthritis but prevented progression to chronic destructive synovitis as was noted in wild-type (wt) mice. Microarray analysis revealed significant down-regulation of leukocyte-specific chemokines, selectins, cytokines, and collagenase-3 in the synovium of IL-17R−/− mice. Bone marrow (BM) chimeric mice revealed the need for IL-17R expression on radiation-resistant joint cells for destructive inflammation. Chimeric mice of host wt and donor IL-17R−/− BM cells developed destructive synovitis in this chronic reactivated streptococcal cell wall arthritis model similar to wt→wt chimeras. In contrast, chimeric mice of host IL-17R−/− and donor wt BM cells were protected from chronic destructive arthritis similar as IL-17R−/−→IL-17R−/− chimeras. These data strongly indicate that IL-17R signaling in radiation-resistant cells in the joint is required for turning an acute macrophage-mediated inflammation into a chronic destructive synovitis.

CD4+ T cell–independent DNA vaccination against opportunistic infections

Depletion or dysfunction of CD4+ T lymphocytes profoundly perturbs host defenses and impairs immunogenicity of vaccines. Here, we show that plasmid DNA vaccination with a cassette encoding antigen (OVA) and a second cassette encoding full-length CD40 ligand (CD40L), a molecule expressed on activated CD4+ T lymphocytes and critical for T cell helper function, can elicit significant titers of antigen-specific immunoglobulins in serum and Tc1 CD8+ T cell responses in CD4-deficient mice. To investigate whether this approach leads to CD4+ T cell-independent vaccine protection against a prototypic AIDS-defining infection, Pneumocystis (PC) pneumonia, we used serum from mice vaccinated with PC-pulsed, CD40L-modified DCs to immunoprecipitate PC antigens. Kexin, a PC antigen identified by this approach, was used in a similar DNA vaccine strategy with or without CD40L. CD4-deficient mice receiving DNA vaccines encoding Kexin and CD40L showed significantly higher anti-PC IgG titers as well as opsonic killing of PC compared with those vaccinated with Kexin alone. Moreover, CD4-depleted, Kexin-vaccinated mice showed a 3-log greater protection in a PC challenge model. Adoptive transfer of CD19+ cells or IgG to SCID mice conferred protection against PC challenge, indicating a role of humoral immunity in the protection. The results of these studies show promise for CD4-independent vaccination against HIV-related or other opportunistic pathogens.

Clinical Protocol The Treatment of Malignant Mesothelioma with a Gene Modified Cancer Cell Line: A Phase I Study

1.1 To evaluate the safety and side effects of treatment with the gene-modified ovarian cancer cell line PA1-STK, which is administered intrapleurally and activated with ganciclovir. 1.2 To determine a maximum treatment dose (MTD) and the dose limiting side effects of this treatment. 1.3 To evaluate the immunologic response to this treatment. 1.4 To measure intrapleural pharmakokinetics of the drug ganciclovir. 1.5 To observe for clinical effects on the residual malignant mesothelioma.

Propylthiouracil reduces xenograft tumor growth in an athymic nude mouse prostate cancer model

METHODS Several anecdotal reports indicate that cancer may occasionally remain in a dormant state for prolonged periods in patients with hypothyroidism. Once the hypothyroid state is recognized and supplementation therapy with thyroid hormones is initiated, disease progression occurs. In this experiment, 6-n-propyl-2-thiouracil (PTU) was added to the water of athymic nude mice. The animals were subsequently inoculated with cells from a human prostate cancer cell line. RESULTS The growth rate of subcutaneously implanted prostate xenografts was significantly slower in mice treated with PTU compared with mice that did not receive PTU. In a separate experiment, tritiated thymidine incorporation assays were performed in DU145 and PC3 human prostate cancer cells with and without PTU. No significant differences were observed, indicating that PTU did not exert any antitumor effect in vitro. CONCLUSIONS Our study demonstrates that PTU inhibits the growth of human prostate tumors in nude mice via an indirect effect. This antitumor effect may be caused by hypothyroidism. This is the first in vivo study suggesting potential therapeutic applications for thyroid hormone manipulations in human cancer of the prostate. Further studies will determine growth kinetics of xenotransplanted prostate cancer in vitro and in vivo. PTU-induced hypothyroidism may be further explored in conjunction with other antineoplastic therapy.

A Phase 2 Randomized Trial of Paclitaxel and Carboplatin with or without Panitumumab for First-Line Treatment of Advanced Non–Small-Cell Lung Cancer

Introduction: This two-part phase 2 study evaluated the efficacy and safety of panitumumab, a fully human anti–epidermal growth factor receptor monoclonal antibody, combined with carboplatin/paclitaxel in patients with previously untreated advanced non–small-cell lung cancer. Methods: In part 1, patients were sequentially enrolled to receive paclitaxel 200 mg/m2 and carboplatin (area under the concentration-versus-time curve, 6 mg/min/ml) plus panitumumab (1.0, 2.0, or 2.5 mg/kg). In part 2, patients were randomized 2:1 to receive paclitaxel/carboplatin with (arm A) or without (arm B) the maximum tolerated dose of panitumumab identified in part 1. Primary endpoints in parts 1 and 2 were the incidence of dose-limiting toxicities and time to progression (TTP), respectively. Results: In part 1, four of 19 patients had dose-limiting toxicities: three at 2.0 mg/kg (fatigue, pain in extremity, dyspepsia) and one at 2.5 mg/kg (rash). The maximum tolerated dose was not reached; panitumumab 2.5 mg/kg was selected for part 2. In part 2, TTP was 18.1 weeks (95% confidence interval [CI], 13.6−23.3) in arm A and 23.0 weeks (95% CI, 15.9−24.1) in arm B (hazard ratio, 0.9; 90% CI, 0.66−1.21; p = 0.555). Progression-free survival in arms A and B was 17.6 weeks and 18.3 weeks, respectively, and the objective response rate was 15.2% and 11.1%. Adverse events occurring more frequently in arm A than in arm B included skin toxicity, diarrhea, stomatitis, vomiting, and dizziness. Exploratory analyses did not demonstrate associations between potential biomarkers and outcomes. Conclusion: Although toxicity was predictable and manageable, the addition of panitumumab to paclitaxel/carboplatin did not improve TTP in patients with previously untreated advanced non–small-cell lung cancer.

Marrow Stem Cells, Mesenchymal Progenitor Cells, and Stromal Progeny

During development, embryonic stem cells (ES) have been thought traditionally to give rise to organ-restricted stem cells. This process is believed to occur as a continuum through adult life since these stem cells also give rise to lineage committed progenitor cells, which retain a certain degree of multipotentiality in adult tissues. An example is the CFU-GEMM of the hematopoietic system which renews the formed elements of the blood. Tissue restricted stem cells and progenitor cells have been described for all adult tissues including bone marrow, liver, and brain. Quiescent stem cells that are retained in adult hematopoietic and non-hematopoietic tissues may be recruited from the quiescent state (G0) and mobilized when tissues are injured or depleted. They eventually form committed progenitor cells which differentiate to mature (terminal) cells that express genes that are functionally tissue specific. Differences exist between the hematopoietic and nonhematopoietic regenerative processes. Injury to or depletion of hematopoietic cells provokes replacement of blood or immune elements by newly generated cells derived from committed progenitor cells in the marrow microenvironment. In contrast, repair mechanisms associated with non-hematopoietic tissues may involve cells which appear mature, but which have retained some degree of proliferation and differentiation potential. Examples are hepatocytes, skin fibroblasts, endothelial cells, and smooth muscle cells. Previously, the differentiation potential of mesenchymal progenitor cells (MPC) or stromal progenitor cells in the bone marrow has been thought to be limited to the generation of hematopoietic supportive stromal cells of