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1.  Active immunization to tumor necrosis factor-α is effective in treating chronic established inflammatory disease: a long-term study in a transgenic model of arthritis 
Arthritis Research & Therapy  2009;11(6):R195.
Introduction
Passive blockade of tumor necrosis factor-alpha (TNF-α) has demonstrated high therapeutic efficiency in chronic inflammatory diseases, such as rheumatoid arthritis, although some concerns remain such as occurrence of resistance and high cost. These limitations prompted investigations of an alternative strategy to target TNF-α. This study sought to demonstrate a long-lasting therapeutic effect on established arthritis of an active immunotherapy to human (h) TNF-α and to evaluate the long-term consequences of an endogenous anti-TNF-α response.
Methods
hTNF-α transgenic mice, which spontaneously develop arthritides from 8 weeks of age, were immunized with a heterocomplex (TNF kinoid, or TNF-K) composed of hTNF-α and keyhole limpet hemocyanin after disease onset. We evaluated arthritides by clinical and histological assessment, and titers of neutralizing anti-hTNF-α antibody by enzyme-linked immunosorbent assay and L929 assay.
Results
Arthritides were dramatically improved compared to control mice at week 27. TNF-K-treated mice exhibited high levels of neutralizing anti-hTNF-α antibodies. Between weeks 27 and 45, all immunized mice exhibited symptoms of clinical deterioration and a parallel decrease in anti-hTNF-α neutralizing antibodies. A maintenance dose of TNF-K reversed the clinical deterioration and increased the anti-hTNF-α antibody titer. At 45 weeks, TNF-K long-term efficacy was confirmed by low clinical and mild histological scores for the TNF-K-treated mice. Injections of unmodified hTNF-α did not induce a recall response to hTNF-α in TNF-K immunized mice.
Conclusions
Anti-TNF-α immunotherapy with TNF-K has a sustained but reversible therapeutic efficacy in an established disease model, supporting the potential suitability of this approach in treating human disease.
doi:10.1186/ar2897
PMCID: PMC3003505  PMID: 20030816
2.  Expanded CD23+/CD21hi B cells in inflamed lymph nodes are associated with the onset of inflammatory-erosive arthritis in TNF-transgenic mice and are targets of anti-CD20 therapy1 
Anti-CD20 B cell depletion therapy (BCDT) is very effective for some patients with rheumatoid arthritis (RA), however the pathogenic role of B lymphocytes in RA and the primary targets of BCDT are unknown. The human TNF transgenic (hTNF-tg) mouse model of RA displays a chronic-progressive disease that spreads from distal to proximal joints, and is generally considered to be adaptive immune system-independent. We have previously reported that knee arthritis in hTNF-tg mice is accompanied by structural and functional changes of the adjoining popliteal lymph node (PLN), detectable by contrast-enhanced magnetic resonance imaging (CE-MRI). To better understand these changes, here we show that onset of knee synovitis and focal erosions are paralleled by PLN contraction and accumulation of large numbers of B cells in the lymphatic sinus spaces within the node. Flow cytometry from 2, 4-5, and 8-12 month old TNF-tg mice demonstrated that B cell accumulation in the PLN follows ankle arthritis, but commences before knee disease, and involves early expansion of CD21hi, CD23+, IgMhi, CD1d+, activation marker-negative, polyclonal B cells which are found to be specifically restricted to lymph nodes draining inflamed, arthritic joints. The same B cell population also accumulates in PLNs of K/BxN mice with autoantigen-dependent arthritis. Strikingly, we show that BCDT ameliorates hTNF-tg disease and clears follicular and CD21hi, CD23+ B cells from the PLNs. Based on these findings, we propose a model whereby B cells contribute to arthritis in mice, and possibly RA, by directly affecting the structure, composition and function of joint-draining lymph nodes.
doi:10.4049/jimmunol.0903489
PMCID: PMC2874087  PMID: 20435928
B-cells; Inflammation; Rheumatoid Arthritis; Lymph nodes; B cell depletion therapy
3.  Suppressive effect of secretory phospholipase A2 inhibitory peptide on interleukin-1β-induced matrix metalloproteinase production in rheumatoid synovial fibroblasts, and its antiarthritic activity in hTNFtg mice 
Arthritis Research & Therapy  2009;11(5):R138.
Introduction
Secretory phospholipase A2 (sPLA2) and matrix metalloproteinase (MMP) inhibitors are potent modulators of inflammation with therapeutic potential, but have limited efficacy in rheumatoid arthritis (RA). The objective of this study was to understand the inhibitory mechanism of phospholipase inhibitor from python (PIP)-18 peptide in cultured synovial fibroblasts (SF), and to evaluate its therapeutic potential in a human tumor necrosis factor (hTNF)-driven transgenic mouse (Tg197) model of arthritis.
Methods
Gene and protein expression of sPLA2-IIA, MMP-1, MMP-2, MMP-3, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 were analyzed by real time PCR and ELISA respectively, in interleukin (IL)-1β stimulated rheumatoid arthritis (RA) and osteoarthritis (OA) synovial fibroblasts cells treated with or without inhibitors of sPLA2 (PIP-18, LY315920) or MMPs (MMP Inhibitor II). Phosphorylation status of mitogen-activated protein kinase (MAPK) proteins was examined by cell-based ELISA. The effect of PIP-18 was compared with that of celecoxib, methotrexate, infliximab and antiflamin-2 in Tg197 mice after ip administration (thrice weekly for 5 weeks) at two doses (10, 30 mg/kg), and histologic analysis of ankle joints. Serum sPLA2 and cytokines (tumor necrosis factor (TNF)α, IL-6) were measured by Escherichia coli (E coli) assay and ELISA, respectively.
Results
PIP-18 inhibited sPLA2-IIA production and enzymatic activity, and suppressed production of MMPs in IL-1β-induced RA and OA SF cells. Treatment with PIP-18 blocked IL-1β-induced p38 MAPK phosphorylation and resulted in attenuation of sPLA2-IIA and MMP mRNA transcription in RA SF cells. The disease modifying effect of PIP-18 was evidenced by significant abrogation of synovitis, cartilage degradation and bone erosion in hTNF Tg197 mice.
Conclusions
Our results demonstrate the benefit that can be gained from using sPLA2 inhibitory peptide for RA treatment, and validate PIP-18 as a potential therapeutic in a clinically relevant animal model of human arthritis.
doi:10.1186/ar2810
PMCID: PMC2787297  PMID: 19765281
4.  The Protective Antibodies Induced by a Novel Epitope of Human TNF-α Could Suppress the Development of Collagen-Induced Arthritis 
PLoS ONE  2010;5(1):e8920.
Tumor necrosis factor alpha (TNF-α) is a major inflammatory mediator that exhibits actions leading to tissue destruction and hampering recovery from damage. At present, two antibodies against human TNF-α (hTNF-α) are available, which are widely used for the clinic treatment of certain inflammatory diseases. This work was undertaken to identify a novel functional epitope of hTNF-α. We performed screening peptide library against anti-hTNF-α antibodies, ELISA and competitive ELISA to obtain the epitope of hTNF-α. The key residues of the epitope were identified by means of combinatorial alanine scanning and site-specific mutagenesis. The N terminus (80–91 aa) of hTNF-α proved to be a novel epitope (YG1). The two amino acids of YG1, proline and valine, were identified as the key residues, which were important for hTNF-α biological function. Furthermore, the function of the epitope was addressed on an animal model of collagen-induced arthritis (CIA). CIA could be suppressed in an animal model by prevaccination with the derivative peptides of YG1. The antibodies of YG1 could also inhibit the cytotoxicity of hTNF-α. These results demonstrate that YG1 is a novel epitope associated with the biological function of hTNF-α and the antibodies against YG1 can inhibit the development of CIA in animal model, so it would be a potential target of new therapeutic antibodies.
doi:10.1371/journal.pone.0008920
PMCID: PMC2811745  PMID: 20111721
5.  Attenuation of inflammatory polyarthritis in TNF transgenic mice by diacerein: comparative analysis with dexamethasone, methotrexate and anti-TNF protocols 
Arthritis Research & Therapy  2003;6(1):R65-R72.
The impact of diacerein, an effective cartilage targeted therapy that is used in patients with osteoarthritis, on the development and progression of chronic inflammatory arthritis was evaluated in a tumor necrosis factor (TNF) transgenic mouse model (Tg197). The response to diacerein at 2, 20, or 60 mg/kg daily, as well as the comparative effects of other antiarthritis drugs including dexamethasone (0.5 mg/kg daily), methotrexate (1 mg/kg three times weekly) and an anti-TNF agent (5 mg/kg weekly), were assessed in the Tg197 mice. Treatment was initiated before the onset of arthritis and was continued for 5 weeks. A significant improvement in clinical symptoms was found in all three diacerein treated groups in comparison with untreated groups. Confirming these data, semiquantitative histopathologic analysis of the hind paws revealed a significant reduction not only in cartilage destruction but also in the extent of synovitis and bone erosion in diacerein treated groups in comparison with untreated groups. At the most effective dose tested (2 mg/kg daily), diacerein inhibited the onset of arthritis in 28% and attenuated the progression of arthritis in 35% of the Tg197 mice. Comparative analyses showed diacerein to be more potent than methotrexate but not as effective as dexamethasone or anti-TNF agents in suppressing the progression of the TNF mediated arthritis in this model. These results indicate that diacerein has a disease modifying effect on the onset and progression of TNF driven chronic inflammatory arthritis, suggesting that the prophylactic or therapeutic potential of diacerein in patients with RA should be further examined.
doi:10.1186/ar1028
PMCID: PMC400419  PMID: 14979939
arthritis; diacerein; inflammation; transgenic; tumor necrosis factor
6.  Genetic sphingosine kinase 1 deficiency significantly decreases synovial inflammation and joint erosions in murine TNFα-induced arthritis1 
SphK1 is an enzyme that converts sphingosine to bioactive sphingosine-1-phosphate (S1P). Recent in vitro data suggest a potential role of SphK1 in TNFα mediated inflammation. Our aims in this study were to determine the in vivo significance of SphK1 in TNFα mediated chronic inflammation and to define which pathogenic mechanisms induced by TNFα are SphK1 dependent. To pursue these aims, we studied the effect of SphK1 deficiency in an in vivo model of TNFα induced chronic inflammatory arthritis. Transgenic hTNFα mice, that develop spontaneous inflammatory erosive arthritis beginning at 14-16 weeks, were crossed with SphK1 null mice (SphK1-/-), on the C57BL6 genetic background. Beginning at 4 months of age, hTNF/SphK1-/- mice had significantly less severe clinically evident paw swelling and deformity, less synovial and periarticular inflammation and markedly decreased bone erosions as measured quantititatively through micro-CT images. Mechanistically, the mice lacking SphK1 had less articular COX-2 protein and fewer synovial Th17 cells than hTNF/SphK1+/+ littermates. Microarray analysis and real-time RT-PCR of the ankle synovial tissue demonstrated that hTNF/SphK1-/- mice had increased transcript levels of SOCS3 compared to hTNF/SphK1+/+ mice, likely also contributing to the decreased inflammation in the SphK1 deficient mice. Finally, significantly fewer mature osteoclasts were detected in the ankle joints of hTNF/SphK1-/- mice compared to hTNF/SphK1+/+ mice. These data indicate that SphK1 plays a key role in hTNFα induced inflammatory arthritis via impacting synovial inflammation and osteoclast number.
doi:10.4049/jimmunol.1000644
PMCID: PMC2942019  PMID: 20644167
7.  Protection against cartilage and bone destruction by systemic interleukin-4 treatment in established murine type II collagen-induced arthritis 
Arthritis Research  1999;1(1):81-91.
Destruction of cartilage and bone are hallmarks of human rheumatoid arthritis (RA), and controlling these erosive processes is the most challenging objective in the treatment of RA. Systemic interleukin-4 treatment of established murine collagen-induced arthritis suppressed disease activity and protected against cartilage and bone destruction. Reduced cartilage pathology was confirmed by both decreased serum cartilage oligomeric matrix protein (COMP) and histological examination. In addition, radiological analysis revealed that bone destruction was also partially prevented. Improved suppression of joint swelling was achieved when interleukin-4 treatment was combined with low-dose prednisolone treatment. Interestingly, synergistic reduction of both serum COMP and inflammatory parameters was noted when low-dose interleukin-4 was combined with prednisolone. Systemic treatment with interleukin-4 appeared to be a protective therapy for cartilage and bone in arthritis, and in combination with prednisolone at low dosages may offer an alternative therapy in RA.
Introduction:
Rheumatoid arthritis (RA) is associated with an increased production of a range of cytokines including tumour necrosis factor (TNF)-α and interleukin (IL)-1, which display potent proinflammatory actions that are thought to contribute to the pathogenesis of the disease. Although TNF-α seems to be the major cytokine in the inflammatory process, IL-1 is the key mediator with regard to cartilage and bone destruction. Apart from direct blockade of IL-1/TNF, regulation can be exerted at the level of modulatory cytokines such as IL-4 and IL-10. IL-4 is a pleiotropic T-cell derived cytokine that can exert either suppressive or stimulatory effects on different cell types, and was originally identified as a B-cell growth factor and regulator of humoral immune pathways. IL-4 is produced by activated CD4+ T cells and it promotes the maturation of Th2 cells. IL-4 stimulates proliferation, differentiation and activation of several cell types, including fibroblasts, endothelial cells and epithelial cells. IL-4 is also known to be a potent anti-inflammatory cytokine that acts by inhibiting the synthesis of proinflammatory cytokines such as IL-1, TNF-α, IL-6, IL-8 and IL-12 by macrophages and monocytes. Moreover, IL-4 stimulates the synthesis of several cytokine inhibitors such as interleukin-1 receptor antagonist (IL-1Ra), soluble IL-1-receptor type II and TNF receptors IL-4 suppresses metalloproteinase production and stimulates tissue inhibitor of metalloproteinase-1 production in human mononuclear phagocytes and cartilage explants, indicating a protective effect of IL-4 towards extracellular matrix degradation. Furthermore, IL-4 inhibits both osteoclast activity and survival, and thereby blocks bone resorption in vitro. Of great importance is that IL-4 could not be detected in synovial fluid or in tissues. This absence of IL-4 in the joint probably contributes to the disturbance in the Th1/Th2 balance in chronic RA.
Collagen-induced arthritis (CIA) is a widely used model of arthritis that displays several features of human RA. Recently it was demonstrated that the onset of CIA is under stringent control of IL-4 and IL-10. Furthermore, it was demonstrated that exposure to IL-4 during the immunization stage reduced onset and severity of CIA. However, after cessation of IL-4 treatment disease expression increased to control values.
Aims:
Because it was reported that IL-4 suppresses several proinflammatory cytokines and matrix degrading enzymes and upregulates inhibitors of both cytokines and catabolic enzymes, we investigated the tissue protective effect of systemic IL-4 treatment using established murine CIA as a model. Potential synergy of low dosages of anti-inflammatory glucocorticosteroids and IL-4 was also evaluated.
Methods:
DBA-1J/Bom mice were immunized with bovine type II collagen and boosted at day 21. Mice with established CIA were selected at day 28 after immunization and treated for days with IL-4, prednisolone, or combinations of prednisolone and IL-4. Arthritis score was monitored visually. Joint pathology was evaluated by histology, radiology and serum cartilage oligomeric matrix protein (COMP). In addition, serum levels of IL-1Ra and anticollagen antibodies were determined.
Results:
Treatment of established CIA with IL-4 (1 μg/day) resulted in suppression of disease activity as depicted in Figure 1. Of great interest is that, although 1 μg/day IL-4 had only a moderate effect on the inflammatory component of the disease activity, it strongly reduced cartilage pathology, as determined by histological examination (Fig. 1). Moreover, serum COMP levels were significantly reduced, confirming decreased cartilage involvement. In addition, both histological and radiological analysis showed that bone destruction was prevented (Fig. 1). Systemic IL-4 administration increased serum IL-1Ra levels and reduced anticollagen type II antibody levels. Treatment with low-dose IL-4 (0.1 μg/day) was ineffective in suppressing disease score, serum COMP or joint destruction. Synergistic suppression of both arthritis severity and COMP levels was noted when low-dose IL-4 was combined with prednisolone (0.05 mg/kg/day), however, which in itself was not effective.
Discussion:
In the present study, we demonstrate that systemic IL-4 treatment ameliorates disease progression of established CIA. Although clinical disease progression was only arrested and not reversed, clear protection against cartilage and bone destruction was noted. This is in accord with findings in both human RA and animal models of RA that show that inflammation and tissue destruction sometimes are uncoupled processes. Of great importance is that, although inflammation was still present, strong reduction in serum COMP was found after exposure to IL-4. This indicated that serum COMP levels reflected cartilage damage, although a limited contribution of the inflamed synovium cannot be excluded.
Increased serum IL-1Ra level (twofold) was found after systemic treatment with IL-4, but it is not likely that this could explain the suppression of CIA. We and others have reported that high dosages of IL-1Ra are needed for marked suppression of CIA. As reported previously, lower dosages of IL-4 did not reduce clinical disease severity of established CIA. Of importance is that combined treatment of low dosages of IL-4 and IL-10 appeared to have more potent anti-inflammatory effects, and markedly protected against cartilage destruction. Improved anti-inflammatory effect was achieved with IL-4/prednisolone treatment. In addition, synergistic effects were found for the reduction of cartilage and bone destruction. This indicates that systemic IL-4/prednisolone treatment may provide a cartilage and bone protective therapy for human RA.
Effects in mice of treatment with interleukin-4 or control on disease activity, cartilage damage and bone destruction. Mice were treated intraperitoneally for 7 days with either vehicle (control) or 1 μg/day interleukin-4 (IL-4). CIA, collagen-induced arthritis. *P < 0.05, versus control, by Mann-Whitney U test.
PMCID: PMC17779  PMID: 11056663
bone destruction; cartilage oligomeric matrix protein levels; collagen-induced arthritis; interleukin-4; prednisolone
8.  Sclerostin inhibition reverses systemic, periarticular and local bone loss in arthritis 
Annals of the Rheumatic Diseases  2013;72(10):1732-1736.
Objective
To test whether inhibition of sclerostin by a targeted monoclonal antibody (Scl-Ab) protects from bone and cartilage damage in inflammatory arthritis. Sclerostin is a potent inhibitor of bone formation and may be responsible for the low level of bone repair in patients with rheumatoid arthritis.
Methods
Human tumour necrosis factor transgenic mice (hTNFtg mice) developing inflammatory arthritis and local and bone loss were administered either vehicle, anti-TNF antibody, Scl-Ab, or a combination of both agents. Inflammation, systemic and periarticular bone loss, bone erosion and cartilage damage were evaluated at baseline (week 8) and after 3 weeks of treatment by clinical assessment, micro-CT and histology.
Results
Scl-Ab did not affect joint swelling or synovitis. Systemic bone loss in the spine and periarticular bone loss in the proximal tibia were completely blocked and partially reversed by inhibition of sclerostin but not by inhibition of TNF. Moreover, Scl-Ab completely arrested the progression of bone erosion in hTNFtg mice and in combination with TNF inhibition even led to significant regression of cortical bone erosions. Protective effects of Scl-Ab were also observed for the articular cartilage.
Conclusions
These data suggest that sclerostin inhibition is a powerful tool to enhance bone repair in inflammatory arthritis.
doi:10.1136/annrheumdis-2013-203345
PMCID: PMC3786639  PMID: 23666928
Anti-TNF; Rheumatoid Arthritis; Inflammation; Bone Mineral Density
9.  Impact of Sphingosine Kinase 2 Deficiency on the Development of TNF alpha Induced Inflammatory Arthritis 
Rheumatology international  2012;33(10):2677-2681.
Sphingolipids are components of the plasma membrane whose metabolic manipulation is of interest as a potential therapeutic approach in a number of diseases. Sphingosine kinase 1 (SphK1), the major kinase that phosporylates sphingosine to sphingosine-1-phosphate (S1P), was previously shown by our group and others to modulate inflammation in murine models of inflammatory arthritis, inflammatory bowel disease and asthma. Sphingosine kinase 2’s (SphK2) impact on inflammation is less well known, as variable results were reported depending on the disease model. A specific SphK2 inhibitor inhibited inflammatory arthritis in one model, while siRNA knockdown of SphK2 worsened arthritis in another. We previously demonstrated that SphK1 deficient mice are protected against development of hTNF-α induced arthritis. To investigate the role of SphK2 in TNF-α induced arthritis, we developed SphK2 deficient hTNF-α overexpressing mice and separately treated hTNF-α mice with ABC294640, a SphK2 specific inhibitor. Our data show that genetic inhibition of SphK2 did not significantly impact the severity or progression of inflammatory arthritis, while pharmacologic inhibition of SphK2 led to significantly more severe arthritis. Compared to vehicle-treated mice, ABC294640 treated mice also had less S1P in whole blood and inflamed joint tissue, although the differences were not significant. ABC294640 treatment did not affect SphK1 activity in the inflamed joint while little SphK2 activity was detected in the joint. We conclude that the differences in the inflammatory phenotype in genetic inhibition vs. pharmacologic inhibition of SphK2 can be attributed to the amount of ABC294640 used in the experiments versus the impact of acute inhibition of SphK2 with ABC294640 vs. genetically-induced life-long SphK2 deficiency. Thus, inhibition of SphK2 appears to be proinflammatory in contrast to the clear anti-inflammatory effects of blocking SphK1. Therapies directed at this sphingosine kinase pathways will need to be specific in their targeting of sphingosine kinases.
doi:10.1007/s00296-012-2493-2
PMCID: PMC3784643  PMID: 23011090
Sphigosine kinase 2; inflammatory arthritis; sphingolipids; TNF
10.  Effect of phospholipase A2 inhibitory peptide on inflammatory arthritis in a TNF transgenic mouse model: a time-course ultrastructural study 
Arthritis Research & Therapy  2004;6(3):R282-R294.
We evaluated the therapeutic effect of secretory phospholipase A2 (sPLA2)-inhibitory peptide at a cellular level on joint erosion, cartilage destruction, and synovitis in the human tumor necrosis factor (TNF) transgenic mouse model of arthritis. Tg197 mice (N = 18) or wild-type (N = 10) mice at 4 weeks of age were given intraperitoneal doses (7.5 mg/kg) of a selective sPLA2 inhibitory peptide, P-NT.II, or a scrambled P-NT.II (negative control), three times a week for 4 weeks. Untreated Tg197 mice (N = 10) were included as controls. Pathogenesis was monitored weekly for 4 weeks by use of an arthritis score and histologic examinations. Histopathologic analysis revealed a significant reduction after P-NT.II treatment in synovitis, bone erosion, and cartilage destruction in particular. Conspicuous ultrastructural alterations seen in articular chondrocytes (vacuolated cytoplasm and loss of nuclei) and synoviocytes (disintegrating nuclei and vacuoles, synovial adhesions) of untreated or scrambled-P-NT.II-treated Tg197 mice were absent in the P-NT.II-treated Tg197 group. Histologic scoring and ultrastructural evidence suggest that the chondrocyte appears to be the target cell mainly protected by the peptide during arthritis progression in the TNF transgenic mouse model. This is the first time ultrastructural evaluation of this model has been presented. High levels of circulating sPLA2 detected in untreated Tg197 mice at age 8 weeks of age were reduced to basal levels by the peptide treatment. Attenuation of lipopolysaccharide- and TNF-induced release of prostaglandin E2 from cultured macrophage cells by P-NT.II suggests that the peptide may influence the prostaglandin-mediated inflammatory response in rheumatoid arthritis by limiting the bioavailability of arachidonic acid through sPLA2 inhibition.
doi:10.1186/ar1179
PMCID: PMC416452  PMID: 15142275
peptide; secretory phospholipase A2 inhibition; rheumatoid arthritis; TNF transgenic mouse model; ultrastructural alterations
11.  Two doses of NGR-hTNF in combination with capecitabine plus oxaliplatin in colorectal cancer patients failing standard therapies 
Annals of Oncology  2010;22(4):973-978.
Background: asparagine-glycine-arginine-human tumour necrosis factor (NGR-hTNF), an agent selectively damaging the tumour vasculature, showed a biphasic dose–response curve in preclinical models. Previous phase I trials of NGR-hTNF indicated 0.8 and 45 μg/m2 as optimal biological and maximum-tolerated dose, respectively.
Patients and methods: Two sequential cohorts of 12 colorectal cancer (CRC) patients who had failed standard therapies received NGR-hTNF 0.8 or 45 μg/m2 in combination with capecitabine–oxaliplatin (XELOX).
Results: Median number of prior treatment lines was 3 in the low-dose and 2 in the high-dose cohort. Overall, 21 patients had been pretreated with oxaliplatin-based regimens. No grade 3–4 NGR-hTNF-related toxicities were observed. Grade 1–2 chills were reported in 43% and 40% of cycles in the low-dose and high-dose cohorts, respectively. In the low-dose cohort, one patient achieved a partial response and five had stable disease for a median of 4.6 months. In the high-dose cohort, six patients had stable disease for a median of 3.6 months. Three-month progression-free survival (PFS) rates were 50% and 33% in the low-dose and high-dose cohort, respectively. Three patients in low-dose cohort experienced PFS longer than PFS on last prior therapy.
Conclusions: Both NGR-hTNF doses were safely combined with XELOX in pretreated CRC patients. Hint of activity was apparent only with low-dose NGR-hTNF.
doi:10.1093/annonc/mdq436
PMCID: PMC3065876  PMID: 20855468
colorectal cancer; NGR-hTNF; vascular targeting agent
12.  Gene therapy for established murine collagen-induced arthritis by local and systemic adenovirus-mediated delivery of interleukin-4 
Arthritis Research  2000;2(4):293-302.
To determine whether IL-4 is therapeutic in treating established experimental arthritis, a recombinant adenovirus carrying the gene that encodes murine IL-4 (Ad-mIL-4) was used for periarticular injection into the ankle joints into mice with established collagen-induced arthritis (CIA). Periarticular injection of Ad-mIL-4 resulted in a reduction in the severity of arthritis and joint swelling compared with saline- and adenoviral control groups. Local expression of IL-4 also reduced macroscopic signs of joint inflammation and bone erosion. Moreover, injection of Ad-mIL-4 into the hind ankle joints resulted in a decrease in disease severity in the untreated front paws. Systemic delivery of murine IL-4 by intravenous injection of Ad-mIL-4 resulted in a significant reduction in the severity of early-stage arthritis.
Introduction:
Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that is characterized by joint inflammation, and progressive cartilage and bone erosion. Recent research has identified certain biologic agents that appear more able than conventional therapies to halt effectively the progression of disease, as well as ameliorate disease symptoms. One potential problem with the use of biologic agents for arthritis therapy is the need for daily or weekly repeat dosing. The transfer of genes directly to the synovial lining can theoretically circumvent the need for repeat dosing and reduce potential systemic side effects [1,2]. However, although many genes have been effective in treating murine CIA if administrated at a time before disease onset, local intra-articular or periarticular gene transfer has not been highly effective in halting the progression of established disease. IL-4, similar to tumor necrosis factor (TNF)-α and IL-1 inhibitors, has been shown be therapeutic for the treatment of murine CIA when administered intravenously as a recombinant protein, either alone or in combination with IL-10. IL-4 can downregulate the production of proinflammatory and T-helper (Th)1-type cytokines by inducing mRNA degradation and upregulating the expression of inhibitors of proinflammatory cytokines such as IL-1 receptor antagonist (IL-1Ra) [3,4]. IL-4 is able to inhibit IL-2 and IFN-γ production by Th1 cells, resulting in suppression of macrophage activation and the production of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNF-α by monocytes and macrophages [4,5,6,7,8,9].
Objective:
In order to examine the therapeutic effects of local and systemic IL-4 expression in established CIA, an adenoviral vector carrying the gene for murine IL-4 (Ad-mIL-4) was generated. The ability of Ad-mIL-4 to treat established CIA was evaluated by local periarticular and systemic intravenous injection of Ad-mIL-4 into mice at various times after disease onset.
Materials and methods:
Male DBA/1 lacJ (H-2q) mice, aged 7-8 weeks, were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The mice were immunized intradermally at the base of tail with 100 μ g bovine type II collagen. On day 21 after priming, mice received a boost injection (intradermally) with 100 μ g type II collagen in incomplete adjuvant. For the synchronous onset of arthritis, 40 μ g lipopolysaccharide (Sigma, St Louis, MO, USA) was injected intraperitoneally on day 28. Ad-mIL-4 was injected periarticularly into the hind ankle joints of mice on day 32 or intravenously by tail vein injection on day 29. Disease severity was monitored every other day using an established macroscopic scoring system ranging from 0 to 4: 0, normal; 1, detectable arthritis with erythma; 2, significant swelling and redness; 3, severe swelling and redness from joint to digit; and 4, maximal swelling with ankylosis. The average of macroscopic score was expressed as a cumulative value for all paws, with a maximum possible score of 16 per mouse. Cytokine production by joint tissue or serum were assessed using enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN, USA).
Results:
To examine the therapeutic effects of IL-4 gene transfer in a murine model of arthritis, 5×108 particles of Ad-mIL-4 and enhanced green fluorescent protein (Ad-eGFP) were administered by periarticular injection into the ankle joints of mice with established disease 4 days after lipopolysaccharide injection. All mice had established disease at time of injection. As shown in Figure 1, the severity of arthritis (Fig. 1a), paw thickness (Fig. 1b), and the number of arthritic paws (Fig. 1c) were all significantly reduced in the Ad-mIL-4 group, compared with the saline- and Ad-eGFP-treated groups. Analysis of the bones in the ankle joints of control arthritic mice showed evidence of erosion with an associated monocytic infiltrate around the joint space compared with the Ad-mIL-4-treated and nonarthritic control joints. In addition, injection of the ankle joints in the hind legs resulted in a therapeutic effect in the front paws. A similar contralateral effect has been observed with adenoviral-mediated delivery of viral (v)-IL-10. Interestingly, a high level of murine IL-10 also was detected from the joint lysates of Ad-mIL-4-treated naïve and arthritic mice, with the production of endogenous IL-10 correlating with the dose of Ad-mIL-4. The administration of recombinant IL-4 protein systemically has been shown to be therapeutic in murine CIA models if given before disease onset. To examine the effect of systemic IL-4 delivered by gene transfer, 1×109 particles of Ad-mIL-4 were injected via the tail vein of collagen-immunized mice the day after lipopolysaccharide injection. Whereas the immunized control mice, injected with Ad-eGFP, showed disease onset on day 3 after lipopolysaccharide injection, Ad-mIL-4-treated mice showed a delay in disease onset and as a reduction in the total number of arthritic paws. Also, systemic injection of Ad-mIL-4 suppressed the severity of arthritis in CIA mice according to arthritis index.
Discussion:
Gene therapy represents a novel approach for delivery of therapeutic agents to joints in order to treat the pathologies associated with RA and osteoarthritis, as well as other disorders of the joints. In the present study we examined the ability of local periarticular and systemic gene transfer of IL-4 to treat established and early-stage murine CIA, respectively. We have demonstrated that both local and systemic administration of Ad-mIL-4 resulted in a reduction in the severity of arthritis, as well as in the number of arthritic paws. In addition, the local gene transfer of IL-4 reduced histologic signs of inflammation and of bone erosion. Interestingly, local delivery of Ad-mIL-4 was able to confer a therapeutic effect to the untreated, front paws through a currently unknown mechanism. In addition, both local and systemic expression of IL-4 resulted in an increase in the level of endogenous IL-10, as well as of IL-1Ra (data not shown). Previous experiments have shown that gene transfer of IL-10 and IL-1 and TNF inhibitors at the time of disease initiation (day 28) is therapeutic. However, delivery of these agents after disease onset appeared to have only limited therapeutic effect. In contrast, the present results demonstrate that IL-4, resulting from local periarticular and systemic injection of Ad-mIL-4, was able partially to reverse progression of established and early-stage disease, respectively. These results, as well as those of others, support the potential application of IL-4 gene therapy for the clinical treatment of RA.
PMCID: PMC17812  PMID: 11056670
adenoviral vectors; collagen-induced arthritis; gene therapy; IL-4; IL-10; rheumatoid arthritis
13.  Influence of antiTNF-alpha antibody treatment on fracture healing under chronic inflammation 
Background
The overexpression of tumor necrosis factor (TNF)-α leads to systemic as well as local loss of bone and cartilage and is also an important regulator during fracture healing. In this study, we investigate how TNF-α inhibition using a targeted monoclonal antibody affects fracture healing in a TNF-α driven animal model of human rheumatoid arthritis (RA) and elucidate the question whether enduring the anti TNF-α therapy after trauma is beneficial or not.
Methods
A standardized femur fracture was applied to wild type and human TNF-α transgenic mice (hTNFtg mice), which develop an RA-like chronic polyarthritis. hTNFtg animals were treated with anti-TNF antibody (Infliximab) during the fracture repair. Untreated animals served as controls. Fracture healing was evaluated after 14 and 28 days of treatment by clinical assessment, biomechanical testing and histomorphometry.
Results
High levels of TNF-α influence fracture healing negatively, lead to reduced cartilage and more soft tissue in the callus as well as decreased biomechanical bone stability. Blocking TNF-α in hTNFtg mice lead to similar biomechanical and histomorphometrical properties as in wild type.
Conclusions
High levels of TNF-α during chronic inflammation have a negative impact on fracture healing. Our data suggest that TNF-α inhibition by an anti-TNF antibody does not interfere with fracture healing.
doi:10.1186/1471-2474-15-184
PMCID: PMC4059090  PMID: 24885217
Anti-TNFα; Inflammation; Fracture healing; Rheumatoid arthritis; Treatment
14.  Role of TNF alpha in the induction of antigen induced arthritis in the rabbit and the anti-arthritic effect of species specific TNF alpha neutralising monoclonal antibodies. 
Annals of the Rheumatic Diseases  1995;54(5):366-374.
OBJECTIVE--To investigate the role of tumour necrosis factor alpha (TNF alpha) in the development of antigen induced arthritis (AIA) in rabbits. METHODS--Monoclonal antibodies to rabbit TNF alpha were developed in rats and were used to detect TNF alpha in synovial fluid by enzyme linked immunosorbent assay and to localise it in tissue sections of synovium and cartilage from rabbits up to 21 days after induction of AIA. An antibody which neutralised TNF alpha activity in vitro was injected into rabbits to block TNF alpha action in vivo in AIA. Joint swelling, leucocyte infiltration into synovium and proteoglycan loss from cartilage were measured and compared with a control group, which were injected with sterile saline. RESULTS--Monoclonal antibodies to purified rabbit TNF alpha were prepared in rats and two were selected which were able to neutralise rabbit TNF alpha in a cytotoxicity bioassay. TNF alpha was detected in significant concentrations (21.7 (SE 0.5) pg/ml) in the arthritic joint fluid of rabbits with AIA only at one day after induction and it was then also sparsely localised in cells of the synovium, but from day 3 onwards it was localised more strongly in the deep zone of articular cartilage. Injection of anti-TNF monoclonal antibody R6 over three days into rabbits with AIA reduced joint swelling and leucocyte infiltration into joint fluid and decreased the expression of CD11b and CD18 on cells in the joint fluid. However, there was no significant reduction in the loss of proteoglycan from articular cartilage, although the joint fluid at three days contained a lower glycosaminoglycan content. The antibody R6 gave most effect at a dose of 0.6 mg/kg and there was no increase in its effectiveness at a fivefold greater dose (3.0 mg/kg). Treatment over 10 days gave a more complete suppression of joint swelling, but did not result in any less proteoglycan loss from cartilage. Treatment for five days with a 16 day follow up gave a significant reduction in swelling for several days beyond the treatment, but the swelling then slowly returned, until by day 21 there was no significant difference in joint swelling and there was also no recovery of cartilage proteoglycan content. A rabbit anti-rat immunoglobulin response was detected at 21 days, which may have limited the long term effectiveness of the antibody. CONCLUSIONS--In AIA in rabbits, TNF alpha was only detected in synovial fluid at one day after induction and there was only limited cellular localisation of TNF alpha in synovium and cartilage from three days. However, neutralising TNF alpha with a monoclonal antibody was effective in suppressing inflammatory changes in the joint during the acute onset of AIA, but it had little effect on the loss of proteoglycan from cartilage. The results suggest that blocking inflammation and synovitis with anti-TNF alpha may be more easily achieved than preventing damage to articular cartilage.
Images
PMCID: PMC1005596  PMID: 7794042
15.  Osteoclasts are essential for TNF-α–mediated joint destruction 
The Journal of Clinical Investigation  2002;110(10):1419-1427.
The detailed cellular and molecular mechanisms leading to joint destruction in rheumatoid arthritis, a disease driven by proinflammatory cytokines, are still unknown. To address the question of whether osteoclasts play a pivotal role in this process, transgenic mice that express human TNF (hTNFtg) and that develop a severe and destructive arthritis were crossed with osteopetrotic, c-fos–deficient mice (c-fos–/–) completely lacking osteoclasts. The resulting mutant mice (c-fos–/–hTNFtg) developed a TNF-dependent arthritis in the absence of osteoclasts. All clinical features of arthritis, such as paw swelling and reduction of grip strength, progressed equally in both groups. Histological evaluation of joint sections revealed no difference in the extent of synovial inflammation, its cellular composition (except for the lack of osteoclasts), and the expression of matrix metalloprotein-ase-3 (MMP-3) and MMP-13. In addition, cartilage damage, proteoglycan loss, and MMP-3, -9, and -13 expression in chondrocytes were similar in hTNFtg and c-fos–/–hTNFtg mice. However, despite the presence of severe inflammatory changes, c-fos–/–hTNFtg mice were fully protected against bone destruction. These data reveal that TNF-dependent bone erosion is mediated by osteoclasts and that the absence of osteoclasts alters TNF-mediated arthritis from a destructive to a nondestructive arthritis. Therefore, in addition to the use of anti-inflammatory therapies, osteoclast inhibition could be beneficial for the treatment of rheumatoid arthritis.
doi:10.1172/JCI15582
PMCID: PMC151809  PMID: 12438440
16.  Antagonistic effect of TNF-alpha and insulin on uncoupling protein 2 (UCP-2) expression and vascular damage 
Cardiovascular Diabetology  2014;13(1):108.
Background
It has been reported that increased expression of UCP-2 in the vasculature may prevent the development of atherosclerosis in patients with increased production of reactive oxygen species, as in the diabetes, obesity or hypertension. Thus, a greater understanding in the modulation of UCP-2 could improve the atherosclerotic process. However, the effect of TNF-α or insulin modulating UCP-2 in the vascular wall is completely unknown. In this context, we propose to study new molecular mechanisms that help to explain whether the moderate hyperinsulinemia or lowering TNF-α levels might have a protective role against vascular damage mediated by UCP-2 expression levels.
Methods
We analyzed the effect of insulin or oleic acid in presence or not of TNF-α on UCP-2 expression in murine endothelial and vascular smooth muscle cells. At this step, we wondered if some mechanisms studied in vitro could be of any relevance in vivo. We used the following experimental models: ApoE−/− mice under Western type diet for 2, 6, 12 or 18 weeks, BATIRKO mice under high-fat diet for 16 weeks and 52-week-old BATIRKO mice with o without anti-TNF-α antibody pre-treatment.
Results
Firstly, we found that TNF-α pre-treatment reduced UCP-2 expression induced by insulin in vascular cells. Secondly, we observed a progressive reduction of UCP-2 levels together with an increase of lipid depots and lesion area in aorta from ApoE−/− mice. In vivo, we also observed that moderate hyperinsulinemic obese BATIRKO mice have lower TNF-α and ROS levels and increased UCP-2 expression levels within the aorta, lower lipid accumulation, vascular dysfunction and macrovascular damage. We also observed that the anti-TNF-α antibody pre-treatment impaired the loss of UCP-2 expression within the aorta and relieved vascular damage observed in 52-week-old BATIRKO mice. Finally, we observed that the pretreatment with iNOS inhibitor prevented UCP-2 reduction induced by TNF-α in vascular cells. Moreover, iNOS levels are augmented in aorta from mice with lower UCP-2 levels and higher TNF-α levels.
Conclusions
Our data suggest that moderate hyperinsulinemia in response to insulin resistance or lowering of TNF-α levels within the aorta attenuates vascular damage, this protective effect being mediated by UCP-2 expression levels through iNOS.
Electronic supplementary material
The online version of this article (doi:10.1186/s12933-014-0108-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s12933-014-0108-9
PMCID: PMC4149264  PMID: 25077985
TNF-α; Insulin; UCP-2; Atherogenesis
17.  Treatment of Established Lung Metastases with Tumor-Infiltrating Lymphocytes Derived from a Poorly Immunogenic Tumor Engineered to Secrete Human TNF-α 
The growth of a poorly immunogenic methylcholanthrene (MCA)-induced murine (m) sarcoma genetically engineered to secrete human (h) TNF-α (MCA-102-hTNF) was studied. MCA-102-hTNF tumor cells were implanted in animals bearing three- or 7-day pulmonary metastases established with the parental line MCA-102-WT (wild type). This model approximates the clinical situation in which patients with metastatic cancer would be vaccinated with autologous tumor genetically modified to stimulate the host immune response. Reduction in the number of pulmonary metastases was occasionally seen but was not consistently reproducible. Other cytokine-producing tumors had either no effect on distant pulmonary metastases (mIL-4, IFN-γ) or a mild, inconclusive effect similar to hTNF-α (mTNF-α). Significant growth inhibition of MCA-102-hTNF was noted in animals bearing pulmonary metastases. This inhibition was: 1) tumor specific (regression occurred only in animals bearing pulmonary metastases from the same parental line), 2) TNF specific (it was inhibited by in vivo administration of anti hTNF mAbs), 3) dependent on cellular immunity (immune-depletion with anti-CD4 or CD8 mAbs permitted growth). Tumor-infiltrating lymphocytes (TIL) could not be grown from MCA-102-WT or MCA-102-hTNF tumors nor from MCA-102-WT subcutaneous implants in mice bearing MCA-102-WT pulmonary metastases. However, TIL could be grown from hTNF-secreting tumors implanted in mice bearing MCA-102-WT metastases. These TIL were therapeutic against established lung metastases from the parental tumor in adoptive immunotherapy models. These studies suggest a strategy for using gene modified tumors for the therapy of established cancer.
PMCID: PMC2248454  PMID: 8144931
18.  A proinflammatory role for Fas in joints of mice with collagen-induced arthritis 
Arthritis Research & Therapy  2004;6(5):R404-R414.
Collagen-induced arthritis (CIA) is a chronic inflammatory disease bearing all the hallmarks of rheumatoid arthritis, e.g. polyarthritis, synovitis, and subsequent cartilage/bone erosions. One feature of the disease contributing to joint damage is synovial hyperplasia. The factors responsible for the hyperplasia are unknown; however, an imbalance between rates of cell proliferation and cell death (apoptosis) has been suggested. To evaluate the role of a major pathway of cell death – Fas (CD95)/FasL – in the pathogenesis of CIA, DBA/1J mice with a mutation of the Fas gene (lpr) were generated. The susceptibility of the mutant DBA-lpr/lpr mice to arthritis induced by collagen type II was evaluated. Contrary to expectations, the DBA-lpr/lpr mice developed significantly milder disease than the control littermates. The incidence of disease was also significantly lower in the lpr/lpr mice than in the controls (40% versus 81%; P < 0.05). However DBA-lpr/lpr mice mounted a robust immune response to collagen, and the expression of local proinflammatory cytokines such as, e.g., tumor necrosis factor α (TNF-α) and IL-6 were increased at the onset of disease. Since the contribution of synovial fibroblasts to inflammation and joint destruction is crucial, the potential activating effect of Fas on mouse fibroblast cell line NIH3T3 was investigated. On treatment with anti-Fas in vitro, the cell death of NIH3T3 fibroblasts was reduced and the expression of proinflammatory cytokines TNF-α and IL-6 was increased. These findings suggest that impairment of immune tolerance by increased T-cell reactivity does not lead to enhanced susceptibility to CIA and point to a role of Fas in joint destruction.
doi:10.1186/ar1205
PMCID: PMC546278  PMID: 15380040
apoptosis; Fas; rheumatoid arthritis; tolerance
19.  Chronic Tumor Necrosis Factor Alters T Cell Responses by Attenuating T Cell Receptor Signaling 
The Journal of Experimental Medicine  1997;185(9):1573-1584.
Repeated injections of adult mice with recombinant murine TNF prolong the survival of NZB/W F1 mice, and suppress type I insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice. To determine whether repeated TNF injections suppress T cell function in adult mice, we studied the responses of influenza hemagglutinin-specific T cells derived from T cell receptor (HNT-TCR) transgenic mice. Treatment of adult mice with murine TNF for 3 wk suppressed a broad range of T cell responses, including proliferation and cytokine production. Furthermore, T cell responses of HNT-TCR transgenic mice also expressing the human TNF-globin transgene were markedly reduced compared to HNT-TCR single transgenic littermates, indicating that sustained p55 TNF-R signaling is sufficient to suppress T cell function in vivo. Using a model of chronic TNF exposure in vitro, we demonstrate that (a) chronic TNF effects are dose and time dependent, (b) TNF suppresses the responses of both Th1 and Th2 T helper subsets, (c) the suppressive effects of endogenous TNF produced in T cell cultures could be reversed with neutralizing monoclonal antibodies to TNF, and (d) prolonged TNF exposure attenuates T cell receptor signaling. The finding that anti-TNF treatment in vivo enhances T cell proliferative responses and cytokine production provides evidence for a novel regulatory effect of TNF on T cells in healthy laboratory mice. These effects are more pronounced in chronic inflammatory disease. In addition, our data provide a mechanism through which prolonged TNF exposure suppresses disease in animal models of autoimmunity.
PMCID: PMC2196294  PMID: 9151895
20.  Influence of tumor necrosis factor α in rheumatoid arthritis  
Objective
Rheumatoid arthritis (RA) is the most prevalent inflammatory rheumatic disorder. It is a chronic and incurable disease that leads to painful inflammation, often irreversible joint damage, and eventually to functional loss.
Conventional treatment is based on unspecific immunosuppressive agents, e.g. Methotrexate, Azathioprin or Gold. However, the longterm outcomes of these approaches have been poor with frequently ongoing inflammatory disease activity, functional decline, and temporary or permanent work disability. More recently, antagonists of the human cytokine Tumor Necrosis Factor α (TNF-α) have been introduced that are potent suppressors of inflammatory processes. Infliximab is a chimeric antibody against TNF-α. Etanercept is a soluble human TNF-α receptor.
The report assesses the efficacy of TNF-α-antagonists to down-regulate inflammation, improve functional status and prevent joint damage in RA with particular regard to the following indications: Treatment of severe, refractory and ongoing disease activity despite adequate use of conventional antirheumatic agents; and treatment of early RA before conventional treatment failure has been demonstrated.
Methods
A systematic review of the literature is been performed using established electronic databases. The literature search is supplemented by a hand search of journals and publications relevant to RA, reviews of websites of national and international rheumatologic expert societies, as well as contacts to manufacturers. A priori defined inclusion and exclusion criteria are used for literature selection. Analysis and evaluation of included publications are based on standardised criteria sets and checklists of the German Scientific Working Group for Technology Assessment in Health Care.
Results
Health Technology Assessment reports and metaanalyses cannot be identified. A total of 12 clinical trials are analysed, as well as national and international expert recommendations and practice guidelines. Numerous non-systematic reviews are found and analysed for additional sources of information that is not identified through the systematic search. Case reports and safety assessements are considered as well. A total of 137 publications is included.
The primary outcome measures in clinical trials are suppression of inflammatory disease activity and slowing of structural joint damage. Clinical response is usually measured by standardised response criteria that allow a semi-quantitative classification of improvement from baseline by 20%, 50%, or 70%.
In patients with RA refractory to conventional treatment, TNF-α-antagonists are unequivocally superior to Methotrexate with regard to disease activity, functional status and prevention of structural damage. In patients with early RA, TNF-α-antagonists show a more rapid onset of anti-inflammatory effects than Methotrexate. However, differences in clinical response rates and radiologic progression disappear after a few months of treatment and are no longer statistically significant. Serious adverse events are rare in clinical trials and do not occur significantly more often than in the control groups. However, case reports and surveillance registries show an increased risk for serious infectious complications, particularly tuberculosis. Expert panels recommend the use of TNF-α-antagonists in patients with active refractory RA after failure of conventional treatment. Studies that compare Infliximab and Etanercept are lacking.
There are no pharmacoeconomic studies although decision analytic models of TNF-α-antagonists for the treatment of RA exist. Based on the results of the models, a combination therapy with Hydroxychloroquin (HCQ), Sulfaslazin (SASP) and Methotrexate as well as Etanercept/Methotrexate can be considered a cost-effective treatment for Methotrexate-resistant RA.
Conclusions
TNF-α-antagonists are clearly effective in RA patients with no or incomplete response to Methotrexate and superior to continuous use of Methotrexate. It refers to both, reduction of inflammatory disease activity including pain relief and improved functional status, and prevention of structural joint damage. Therefore, TNF-α-antagonism is an important new approach in the treatment of RA. There is still insufficient evidence that early use of TNF-α-antagonists in RA prior to standard agents is beneficial and further studies have to be awaited.
An analytic model suggests that TNF-α-antagonists are, due to their clinical effectiveness in patients with no or incomplete response to Methotrexate, a cost-effective alternative to common therapies chosen in the subpopulations of patients. Nevertheless, it has to be borne in mind that the acquisition costs of TNF-α-antagonists lead to high incremental costs and C/E ratios, which exceed the common frame of assessing the cost-effectiveness of medical methods and technologies. Hence, society's willingness-to-pay is the critical determinant in the question whether TNF-α-antagonists shall be reimbursed or not, or to define criteria for reimbursement. Changes in the quality of life attributable to the use of TNF-α-antagonists in RA have not yet been assessed which might assist the decision making.
With respect of the questions mentioned above and the potential financial effect of a systematic use of TNF-α-antagonists in the treatment of RA, we come to the conclusion that TNF-α-antagonists should not introduced as a standard benefit reimbursed by the statutory health insurers in Germany.
PMCID: PMC3011313  PMID: 21289933
health economics; tumor necrosis factor; TNF-alpha; treatment; rheumatoid arthritis; cost-effectiveness
21.  Generation and Characterization of Human Heme Oxygenase-1 Transgenic Pigs 
PLoS ONE  2012;7(10):e46646.
Xenotransplantation using transgenic pigs as an organ source is a promising strategy to overcome shortage of human organ for transplantation. Various genetic modifications have been tried to ameliorate xenograft rejection. In the present study we assessed effect of transgenic expression of human heme oxygenase-1 (hHO-1), an inducible protein capable of cytoprotection by scavenging reactive oxygen species and preventing apoptosis caused by cellular stress during inflammatory processes, in neonatal porcine islet-like cluster cells (NPCCs). Transduction of NPCCs with adenovirus containing hHO-1 gene significantly reduced apoptosis compared with the GFP-expressing adenovirus control after treatment with either hydrogen peroxide or hTNF-α and cycloheximide. These protective effects were diminished by co-treatment of hHO-1 antagonist, Zinc protoporphyrin IX. We also generated transgenic pigs expressing hHO-1 and analyzed expression and function of the transgene. Human HO-1 was expressed in most tissues, including the heart, kidney, lung, pancreas, spleen and skin, however, expression levels and patterns of the hHO-1 gene are not consistent in each organ. We isolate fibroblast from transgenic pigs to analyze protective effect of the hHO-1. As expected, fibroblasts derived from the hHO-1 transgenic pigs were significantly resistant to both hydrogen peroxide damage and hTNF-α and cycloheximide-mediated apoptosis when compared with wild-type fibroblasts. Furthermore, induction of RANTES in response to hTNF-α or LPS was significantly decreased in fibroblasts obtained from the hHO-1 transgenic pigs. These findings suggest that transgenic expression of hHO-1 can protect xenografts when exposed to oxidative stresses, especially from ischemia/reperfusion injury, and/or acute rejection mediated by cytokines. Accordingly, hHO-1 could be an important candidate molecule in a multi-transgenic pig strategy for xenotransplantation.
doi:10.1371/journal.pone.0046646
PMCID: PMC3465346  PMID: 23071605
22.  Therapeutic effect of neutralizing endogenous IL-18 activity in the collagen-induced model of arthritis 
Journal of Clinical Investigation  2001;108(12):1825-1832.
Two distinct IL-18 neutralizing strategies, i.e. a rabbit polyclonal anti-mouse IL-18 IgG and a recombinant human IL-18 binding protein (rhIL-18BP), were used to treat collagen-induced–arthritic DBA/1 mice after clinical onset of disease. The therapeutic efficacy of neutralizing endogenous IL-18 was assessed using different pathological parameters of disease progression. The clinical severity in mice undergoing collagen-induced arthritis was significantly reduced after treatment with both IL-18 neutralizing agents compared to placebo treated mice. Attenuation of the disease was associated with reduced cartilage erosion evident on histology. The decreased cartilage degradation was further documented by a significant reduction in the levels of circulating cartilage oligomeric matrix protein (an indicator of cartilage turnover). Both strategies efficiently slowed disease progression, but only anti–IL-18 IgG treatment significantly decreased an established synovitis. Serum levels of IL-6 were significantly reduced with both neutralizing strategies. In vitro, neutralizing IL-18 resulted in a significant inhibition of TNF-α, IL-6, and IFN-γ secretion by macrophages. These results demonstrate that neutralizing endogenous IL-18 is therapeutically efficacious in the murine model of collagen-induced arthritis. IL-18 neutralizing antibody or rhIL-18BP could therefore represent new disease-modifying anti-rheumatic drugs that warrant testing in clinical trials in patients with rheumatoid arthritis.
PMCID: PMC209462  PMID: 11748266
23.  Activity and safety of NGR-hTNF, a selective vascular-targeting agent, in previously treated patients with advanced hepatocellular carcinoma 
British Journal of Cancer  2010;103(6):837-844.
Background:
Hepatocellular carcinoma (HCC) is a highly vascularised and poor-prognosis tumour. NGR-hTNF is a vascular-targeting agent consisting of human tumour necrosis factor-alpha fused to the tumour-homing peptide NGR, which is able to selectively bind an aminopeptidase N overexpressed on tumour blood vessels.
Methods:
Twenty-seven patients with advanced-stage disease resistant to either locoregional (59% range, 1–3), systemic treatments (52% range, 1–3) or both (33%) received NGR-hTNF 0.8 μg m−2 once every 3 weeks. The primary aim of the study was progression-free survival (PFS).
Results:
No grade 3–4 treatment-related toxicities were noted. Common toxicity included mild-to-moderate, short-lived chills (63%). Median PFS was 2.3 months (95% CI: 1.7–2.9). A complete response ongoing after 20 months was observed in a sorafenib-refractory patient and a partial response in a Child-Pugh class-B patient, yielding a response rate of 7%. Six patients (22%) experienced stable disease. The disease control rate (DCR) was 30% and was maintained for a median PFS time of 4.3 months. Median survival was 8.9 months (95% CI: 7.5–10.2). In a subset of 12 sorafenib-resistant patients, the response rate was 8% and the median survival was 9.5 months.
Conclusion:
NGR-hTNF was well tolerated and showed single-agent activity in HCC. Further investigation in HCC is of interest.
doi:10.1038/sj.bjc.6605858
PMCID: PMC2966632  PMID: 20717115
NGR-hTNF; vascular-targeting agent; hepatocellular carcinoma
24.  Functional characterization of the human tumor necrosis factor receptor p75 in a transfected rat/mouse T cell hybridoma 
The Journal of Experimental Medicine  1992;176(4):1015-1024.
We investigated the biological role of the human tumor necrosis factor p75 (hTNF-R75), making use of the species specificity of TNF responses in murine (m) T cell lines. Several TNF-mediated activities on mouse T cells, such as cytokine induction or proliferation, showed a 100-500- fold difference in specific biological activity between mTNF and hTNF. After transfection of hTNF-R75 cDNA in a rat/mouse T cell hybridoma (PC60), however, the 100-fold lower specific biological activity of hTNF was converted to the same specific biological activity as mTNF. The TNF-mediated induction of granulocyte/macrophage colony-stimulating factor was strongly synergized by the addition of interleukin 1. In the presence of the latter cytokine, ligand-competing monoclonal antibodies against hTNF-R75 (utr-1, utr-2, utr-3) were agonistic on transfected PC60 cells. This agonistic activity was further enhanced by crosslinking with sheep anti-murine immunoglobulin antibodies. These data provide direct evidence for a functional role of TNF-R75, without ligand-dependent TNF-R55 involvement, in the induction of cytokine secretion in T cells.
PMCID: PMC2119388  PMID: 1328463
25.  Mesothelin confers pancreatic cancer cell resistance to TNF-α-induced apoptosis through Akt/PI3K/NF-κB activation and IL-6/Mcl-1 overexpression 
Molecular Cancer  2011;10:106.
Background
Previous studies showed that mesothelin (MSLN) plays important roles in survival of pancreatic cancer (PC) cells under anchorage dependent/independent conditions as well as resistance to chemotherapy. The recent success of intratumorally-injected adeno-encoded, chemo/radiation-inducible-promoter driven hTNF-α, (TNFerade) + gemcitabine in pre-clinical models of PC have renewed interest in use of TNF-α as a therapeutic component. To help find additional factors which might affect the therapy, we examined the resistance of MSLN-overexpressing pancreatic cancer cell lines to TNF-α-induced growth inhibition/apoptosis.
Methods
Stable MSLN overexpressing MIA PaCa-2 cells (MIA-MSLN), stable MSLN-silenced AsPC-1 cells (AsPC-shMSLN) and other pancreatic cells (MIA-PaCa2, Panc 28, Capan-1, BxPC3, PL 45, Hs 766T, AsPC-1, Capan-2, Panc 48) were used. NF-κB activation was examined by western blots and luciferase reporter assay. TNF-α induced growth inhibition/apoptosis was measured by MTT, TUNEL assay and caspase activation. IL-6 was measured using luminex based assay.
Results
Compared to low endogenous MSLN-expressing MIA PaCa-2 and Panc 28 cells, high endogenous MSLN-expressing Capan-1, BxPC3, PL 45, Hs 766T, AsPC-1, Capan-2, Panc 48 cells were resistant to TNF-α induced growth inhibition. Stable MSLN overexpressing MIA-PaCa2 cells (MIA-MSLN) were resistant to TNF-α-induced apoptosis while stable MSLN-silenced AsPC1 cells (AsPC-shMSLN) were sensitive. Interestingly, TNF-α-treated MIA-MSLN cells showed increased cell cycle progression and cyclin A induction, both of which were reversed by caspase inhibition. We further found that MIA-MSLN cells showed increased expression of anti-apoptotic Bcl-XL and Mcl-1; deactivated (p-Ser75) BAD, and activated (p-Ser70) Bcl-2. Constitutively activated NF-κB and Akt were evident in MIA-MSLN cells that could be suppressed by MSLN siRNA with a resultant increase in sensitivity of TNF-α induced apoptosis. Blocking NF-κB using IKK inhibitor wedelolactone also increased sensitivity to TNF-α-mediated cytotoxicity with concomitant decrease in Mcl-1. Blocking Akt using PI3K inhibitor also had a likewise effect presumably affecting cell cycle. MIA-MSLN cells produced increased IL-6 and were increased furthermore by TNF-α treatment. SiRNA-silencing of IL-6 increased TNF-α sensitivity of MIA-MSLN cells.
Conclusions
Our study delineates a MSLN-Akt-NF-κB-IL-6-Mcl-1 survival axis that may be operative in PC cells, and might help cancer cells' survival in the highly inflammatory milieu evident in PC. Further, for the success of TNFerade + gemcitabine to be successful, we feel the simultaneous inhibition of components of this axis is also essential.
doi:10.1186/1476-4598-10-106
PMCID: PMC3175472  PMID: 21880146
Pancreatic cancer; Mesothelin; TNF-α; Apoptosis

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