Chronic musculoskeletal impairment is associated with alterations in brain regions responsible for the production and perception of movement.

KEY POINTS: Massive irreparable rotator cuff tear was used as a model to study the impact of chronic pain and motor impairment on the motor systems of the human brain using magnetic resonance imaging. Patients show markers of lower grey/white matter integrity and lower functional connectivity compared with control participants in regions responsible for movement and the perception of visual movement and body shape. An independent cohort of patients showed relative deficits in the perception of visual motion and hand laterality compared with an age-matched control group. These data support the hypothesis that the structure and function of the motor control system differs in patients who have experienced chronic motor impairment. This work also raises a new hypothesis, supported by neuroimaging and behaviour, that a loss of motor function could also be associated with off-target effects, namely a reduced ability to perceive motion and body form. ABSTRACT: Changes in the way we move can induce changes in the brain, yet we know little of such plasticity in relation to musculoskeletal diseases. Here we use massive irreparable rotator cuff tear as a model to study the impact of chronic motor impairment and pain on the human brain. Cuff tear destabilises the shoulder, impairing upper-limb function in overhead and load-bearing tasks. We used neuroimaging and behavioural testing to investigate how brain structure and function differed in cuff tear patients and controls (imaging: 21 patients, age 76.3 ± 7.68; 18 controls, age 74.9 ± 6.59; behaviour: 13 patients, age 75.5 ± 10.2; 11 controls, age 73.4 ± 5.01). We observed lower grey matter density and cortical thickness in cuff tear patients in the postcentral gyrus, inferior parietal lobule, temporal-parietal junction and the pulvinar - areas implicated in somatosensation, reach/grasp and body form perception. In patients we also observed lower functional connectivity between the motor network and the middle temporal visual cortex (MT), a region involved in visual motion perception. Lower white matter integrity was observed in patients in the inferior fronto-occipital/longitudinal fasciculi. We investigated the cognitive domains associated with the brain regions identified. Patients exhibited relative impairment in visual body judgements and the perception of biological/global motion. These data support our initial hypothesis that cuff tear is associated with differences in the brain's motor control regions in comparison with unaffected individuals. Moreover, our combination of neuroimaging and behavioural data raises a new hypothesis that chronic motor impairment is associated with an altered perception of visual motion and body form.

PASylation of IL-1 receptor antagonist (IL-1Ra) retains IL-1 blockade and extends its duration in mouse urate crystal-induced peritonitis.

Interleukin-1 (IL-1) is a key mediator of inflammation and immunity. Naturally-occurring IL-1 receptor antagonist (IL-1Ra) binds and blocks the IL-1 receptor-1 (IL-1R1), preventing signaling. Anakinra, a recombinant form of IL-1Ra, is used to treat a spectrum of inflammatory diseases. However, anakinra is rapidly cleared from the body and requires daily administration. To create a longer-lasting alternative, PASylated IL-1Ra (PAS-IL-1Ra) has been generated by in-frame fusion of a long, defined-length, N-terminal Pro/Ala/Ser (PAS) random-coil polypeptide with IL-1Ra. Here, we compared the efficacy of two PAS-IL-1Ra molecules, PAS600-IL-1Ra and PAS800-IL-1Ra (carrying 600 and 800 PAS residues, respectively), with that of anakinra in mice. PAS600-IL-1Ra displayed markedly extended blood plasma levels 3 days post-administration, whereas anakinra was undetectable after 24 h. We also studied PAS600-IL-1Ra and PAS800-IL-1Ra for efficacy in monosodium urate (MSU) crystal-induced peritonitis. 5 days post-administration, PAS800-IL-1Ra significantly reduced leukocyte influx and inflammatory markers in MSU-induced peritonitis, whereas equimolar anakinra administered 24 h before MSU challenge was ineffective. The 6-h pretreatment with equimolar anakinra or PAS800-IL-1Ra before MSU challenge similarly reduced inflammatory markers. In cultured A549 lung carcinoma cells, anakinra, PAS600-IL-1Ra, and PAS800-IL-Ra reduced IL-1α-induced IL-6 and IL-8 levels with comparable potency. In human peripheral blood mononuclear cells, these molecules suppressed Candida albicans-induced production of the cancer-promoting cytokine IL-22. Surface plasmon resonance analyses revealed significant binding between PAS-IL-1Ra and IL-1R1, although with a slightly lower affinity than anakinra. These results validate PAS-IL-1Ra as an active IL-1 antagonist with marked in vivo potency and a significantly extended half-life compared with anakinra.

In-plane and out-of-plane rotational motion of individual chain molecules in steady shear flow of polymer melts and solutions.

Recent nonequilibrium molecular dynamics (NEMD) simulations of mildly entangled C400H802 and moderately entangled C700H1402 linear polyethylene melts undergoing steady shear flow have revealed that several inconsistencies between theory and experiment could be rectified by consideration of the rotational motion of individual polymer chains that occurs at moderate to high flow strengths. In this study, we investigated the configurational dynamics of the individual molecular chains that allow these once-entangled, long-chain molecules to execute retraction/extension semi-periodic cycles in response to the imposed shear via NEMD simulations. Brownian dynamics simulations were also performed to extract dynamical and configurational information about the similar cycles of polymer chain behavior that occur in dilute solutions of macromolecular chain liquids dissolved in low molecular weight solvents. Results revealed that the configurational motions of the individual chains in both melt and solution were essentially the same and governed by a single timescale that scaled exponentially with the magnitude of the shear rate. This configurational motion contained both in-plane and out-of-plane components with respect to the flow-gradient plane, with the out-of-plane component playing a much larger role during the retraction phase of the cycle than during the extension phase. This was determined to be caused by the enhancement of the retraction motion by the out-of-plane entropic Brownian forces; however, these entropic forces were detrimental to the in-plane hydrodynamic diffusive forces during the extension phase of the cycle and were thus suppressed. Consequently, the configuration of a rotating chain was significantly more compact during the retraction stage than during the extension stage, wherein the latter phase most molecules were more preferentially distributed in the flow-gradient plane.

Amino Acid Block Copolymers with Broad Antimicrobial Activity and Barrier Properties.

Antimicrobial properties of a long-chain, synthetic, cationic, and hydrophobic amino acid block copolymer are reported. In 5 and 60 min time-kill assays, solutions of K100 L40 block copolymers (poly(l-lysine·hydrochloride)100 -b-poly(l-leucine)40 ) at concentrations of 10-100 µg mL-1 show multi-log reductions in colony forming units of Gram-positive and Gram-negative bacteria, as well as yeast, including multidrug-resistant strains. Driven by association of hydrophobic segments, K100 L40 copolymers form viscous solutions and self-supporting hydrogels in water at concentrations of 1 and 2 wt%, respectively. These K100 L40 preparations provide an effective barrier to microbial contamination of wounds, as measured by multi-log decreases of tissue-associated bacteria with deliberate inoculation of porcine skin explants, porcine open wounds, and rodent closed wounds with foreign body. Based on these findings, amino acid copolymers with the features of K100 L40 can combine potent, direct antimicrobial activity and barrier properties in one biopolymer for a new approach to prevention of wound infections.

Interleukin-10 deficiency impairs regulatory T cell-derived neuropilin-1 functions and promotes Th1 and Th17 immunity.

Regulatory T cells (Tregs) expand in peripheral lymphoid organs and can produce immunosuppressive cytokines to support tumor growth. IL-10 abrogation efficiently induces Treg formation but dampens tumoral neuropilin-1 (Nrp-1) Treg signaling, which simultaneously augments Th1 and Th17 immunity. These effects are associated with the plasticity and stability of Tregs and effector T cell functions that can limit tumorigenesis. Within the tumor microenvironment, there appears to be a "mutual antagonism" between immunoenhancement and immunosuppression mechanisms, eventually leading to decreased metastasis. In contrast, tumor progression is paralleled by a reduction in Nrp-1-producing Tregs controlled by the IL-10 and TGF-ß1 levels. However, Th1, Th17 and Treg immunity is primarily regulated by IL-10 or Nrp-1 and not TGF-ß1 except when combined with IL-10. These results emphasize the important implications for the therapeutic use of Tregs. The number of Treg cells must be maintained in a healthy and dynamic homeostatic range to prevent malignant diseases. Moreover, Treg-mediated immunosuppression can be limited by reducing tumor-derived Treg Nrp-1 levels.

The biological function and clinical utilization of CD147 in human diseases: a review of the current scientific literature.

CD147 or EMMPRIN is a member of the immunoglobulin superfamily in humans. It is widely expressed in human tumors and plays a central role in the progression of many cancers by stimulating the secretion of matrix metalloproteinases (MMPs) and cytokines. CD147 regulates cell proliferation, apoptosis, and tumor cell migration, metastasis and differentiation, especially under hypoxic conditions. CD147 is also important to many organ systems. This review will provide a detailed overview of the discovery, characterization, molecular structure, diverse biological functions and regulatory mechanisms of CD147 in human physiological and pathological processes. In particular, recent studies have demonstrated the potential application of CD147 not only as a phenotypic marker of activated regulatory T cells but also as a potential diagnostic marker for early-stage disease. Moreover, CD147 is recognized as an effective therapeutic target for hepatocellular carcinoma (HCC) and other cancers, and exciting clinical progress has been made in HCC treatment using CD147-directed monoclonal antibodies.

Recent development of poly(ethylene glycol)-cholesterol conjugates as drug delivery systems.

Poly(ethylene glycol)-cholesterol (PEG-Chol) conjugates are composed of "hydrophilically-flexible" PEG and "hydrophobically-rigid" Chol molecules. PEG-Chol conjugates are capable of forming micelles through molecular self-assembly and they are also used extensively for the PEGylation of drug delivery systems (DDS). The PEGylated DDS have been shown to display optimized physical stability properties in vitro and longer half-lives in vivo when compared with non-PEGylated DDS. Cell uptake studies have indicated that PEG-Chol conjugates are internalized via clathrin-independent pathways into endosomes and Golgi apparatus. Acid-labile PEG-Chol conjugates are also able to promote the content release of PEGylated DDS when triggered by dePEGylation at acidic conditions. More importantly, biodegradable PEG-Chol molecules have been shown to decrease the "accelerated blood clearance" phenomenon of PEG-DSPE. Ligands, peptides or antibodies which have been modified with PEG-Chols are oftentimes used to formulate active targeting DDS, which have been shown in many systems recently to enhance the efficacy and lower the adverse effects of drugs. Production of PEG-Chol is simple and efficient, and production costs are relatively low. In conclusion, PEG-Chol conjugates appear to be very promising multifunctional biomaterials for many uses in the biomedical sciences and pharmaceutical industries.

IL-37 ameliorates the inflammatory process in psoriasis by suppressing proinflammatory cytokine production.

IL-37 is a potent inhibitor of innate immunity by shifting the cytokine equilibrium away from excessive inflammation. Psoriasis is thought to be initiated by abnormal interactions between the cutaneous keratinocytes and systemic immune cells, triggering keratinocyte hyperproliferation. In the current study, we assessed IL-37 in two well-known psoriasis models: a human keratinocyte cell line (HaCaT) and the keratin 14 VEGF-A-transgenic mouse model. First, we used the HaCaT cell line, which was transiently transfected with an overexpressing IL-37 vector, and tested the effect of IL-37 on these cells using a mixture of five proinflammatory cytokines. IL-37 was effective in suppressing the production of CXCL8, IL-6, and S100A7, which were highly upregulated by the mixture of five proinflammatory cytokines. Keratin 14 VEGF-A-transgenic mice were treated with plasmid coding human IL-37 sequence-formulated cationic liposomes, and we observed potent immunosuppressive effects over the 18-d period. In this model, we observed reduced systemic IL-10 levels, local IFN-γ gene transcripts, as well as mild mast cell infiltration into the psoriatic lesions of the mice. Immunohistochemical analysis indicated that IL-37 was expressed by effector memory T cells, as well as macrophages, in human psoriatic plaques. In conclusion, our studies strongly indicate that IL-37 plays a potent immunosuppressive role in the pathogenesis of both experimental psoriasis models in vitro and in vivo by downregulating proinflammatory cytokines. Importantly, our findings highlight new therapeutic strategies that can be designed to use this immunosuppressive anti-inflammatory cytokine in psoriasis and other inflammatory cutaneous diseases.

Pyrvinium targets autophagy addiction to promote cancer cell death.

Autophagy is a cellular catabolic process by which long-lived proteins and damaged organelles are degradated by lysosomes. Activation of autophagy is an important survival mechanism that protects cancer cells from various stresses, including anticancer agents. Recent studies indicate that pyrvinium pamoate, an FDA-approved antihelminthic drug, exhibits wide-ranging anticancer activity. Here we demonstrate that pyrvinium inhibits autophagy both in vitro and in vivo. We further demonstrate that the inhibition of autophagy is mammalian target of rapamycin independent but depends on the transcriptional inhibition of autophagy genes. Moreover, the combination of pyrvinium with autophagy stimuli improves its toxicity against cancer cells, and pretreatment of cells with 3-MA or siBeclin1 partially protects cells from pyrvinium-induced cell death under glucose starvation, suggesting that targeted autophagy addiction is involved in pyrvinium-mediated cytotoxicity. Finally, in vivo studies show that the combination therapy of pyrvinium with the anticancer and autophagy stimulus agent, 2-deoxy-D-glucose (2-DG), is significantly more effective in inhibiting tumor growth than pyrvinium or 2-DG alone. This study supports a novel cancer therapeutic strategy based on targeting autophagy addiction and implicates using pyrvinium as an autophagy inhibitor in combination with chemotherapeutic agents to improve their therapeutic efficacy.

Combined anti-tumor necrosis factor-α therapy and DMARD therapy in rheumatoid arthritis patients reduces inflammatory gene expression in whole blood compared to DMARD therapy alone.

Periodic assessment of gene expression for diagnosis and monitoring in rheumatoid arthritis (RA) may provide a readily available and useful method to detect subclinical disease progression and follow responses to therapy with disease modifying anti-rheumatic agents (DMARDs) or anti-TNF-α therapy. We used quantitative real-time PCR to compare peripheral blood gene expression profiles in active ("unstable") RA patients on DMARDs, stable RA patients on DMARDs, and stable RA patients treated with a combination of a disease-modifying anti-rheumatoid drug (DMARD) and an anti-TNF-α agent (infliximab or etanercept) to healthy human controls. The expression of 48 inflammatory genes were compared between healthy controls (N = 122), unstable DMARD patients (N = 18), stable DMARD patients (N = 26), and stable patients on combination therapy (N = 20). Expression of 13 genes was very low or undetectable in all study groups. Compared to healthy controls, patients with unstable RA on DMARDs exhibited increased expression of 25 genes, stable DMARD patients exhibited increased expression of 14 genes and decreased expression of five genes, and combined therapy patients exhibited increased expression of six genes and decreased expression of 10 genes. These findings demonstrate that active RA is associated with increased expression of circulating inflammatory markers whereas increases in inflammatory gene expression are diminished in patients with stable disease on either DMARD or anti-TNF-α therapy. Furthermore, combination DMARD and anti-TNF-α therapy is associated with greater reductions in circulating inflammatory gene expression compared to DMARD therapy alone. These results suggest that assessment of peripheral blood gene expression may prove useful to monitor disease progression and response to therapy.

Transcriptome profiling of whole blood cells identifies PLEK2 and C1QB in human melanoma.

BACKGROUND: Developing analytical methodologies to identify biomarkers in easily accessible body fluids is highly valuable for the early diagnosis and management of cancer patients. Peripheral whole blood is a "nucleic acid-rich" and "inflammatory cell-rich" information reservoir and represents systemic processes altered by the presence of cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: We conducted transcriptome profiling of whole blood cells from melanoma patients. To overcome challenges associated with blood-based transcriptome analysis, we used a PAXgene™ tube and NuGEN Ovation™ globin reduction system. The combined use of these systems in microarray resulted in the identification of 78 unique genes differentially expressed in the blood of melanoma patients. Of these, 68 genes were further analyzed by quantitative reverse transcriptase PCR using blood samples from 45 newly diagnosed melanoma patients (stage I to IV) and 50 healthy control individuals. Thirty-nine genes were verified to be differentially expressed in blood samples from melanoma patients. A stepwise logit analysis selected eighteen 2-gene signatures that distinguish melanoma from healthy controls. Of these, a 2-gene signature consisting of PLEK2 and C1QB led to the best result that correctly classified 93.3% melanoma patients and 90% healthy controls. Both genes were upregulated in blood samples of melanoma patients from all stages. Further analysis using blood fractionation showed that CD45(-) and CD45(+) populations were responsible for the altered expression levels of PLEK2 and C1QB, respectively. CONCLUSIONS/SIGNIFICANCE: The current study provides the first analysis of whole blood-based transcriptome biomarkers for malignant melanoma. The expression of PLEK2, the strongest gene to classify melanoma patients, in CD45(-) subsets illustrates the importance of analyzing whole blood cells for biomarker studies. The study suggests that transcriptome profiling of blood cells could be used for both early detection of melanoma and monitoring of patients for residual disease.

Synovial tissue rank ligand expression and radiographic progression in rheumatoid arthritis: observations from a proof-of-concept randomized clinical trial of cytokine blockade.

The objective of the study was to evaluate synovial tissue receptor activator of nuclear factor-κß ligand (RANKL) and osteoprotegerin (OPG) as biomarkers of disease activity, progressive joint damage, and therapeutic response, during cytokine blockade in rheumatoid arthritis (RA). Patients with active RA entered a randomized open-label 12-month study of anakinra 100 mg/day, administered as monotherapy or in combination with pegsunercept 800 µg/kg twice weekly. Arthroscopic synovial tissue biopsies were obtained at baseline, at 4 weeks and at the final time point. Following immunohistochemical staining, RANKL and OPG expression was quantified using digital image analysis. Radiographic damage was evaluated using the van der Heijde modification of the Sharp scoring system. Twenty-two patients were randomized. Baseline expression of RANKL, but not OPG, correlated significantly with baseline CRP levels (r = 0.61, P < 0.01). While a significant reduction in OPG expression following treatment was observed in clinical responders at the final time point (P < 0.05 vs. baseline), RANKL levels did not change, and the RANKL:OPG ratio remained unaltered, even at the highest levels of clinical response. When potential predictors of radiographic outcome were evaluated, baseline RANKL expression correlated with erosive progression at 1 year (r = 0.71, P < 0.01). Distinct, though related, pathophysiologic processes mediate joint inflammation and destruction in RA. Elevated synovial tissue RANKL expression is associated with progressive joint erosion, and may be independent of the clinical response to targeted therapy. The potential therapeutic importance of modulating RANKL in RA is highlighted, if radiographic arrest is to be achieved.

Generation of a prophylactic melanoma vaccine using whole recombinant yeast expressing MART-1.

Malignant melanoma is a potentially deadly form of skin cancer and people at high-risk of developing melanoma will benefit from effective preventive intervention. Yeast can be used as an efficient vehicle of antigen loading and immunostimulation. Saccharomyces cerevisiae is not pathogenic to humans and can be easily engineered to express specific antigens. In this study, we have developed a melanoma vaccine using a yeast-based platform expressing a full-length melanocyte/melanoma protein to investigate its utility as a prophylactic melanoma vaccine in a transplantable mouse melanoma model. Yeast was engineered and expanded in vitro without technical difficulties, administered easily with subcutaneous injection, and did not show adverse effects, indicating its practical applicability and favourable safety profile. Despite the lack of knowledge of dominant epitopes of the protein recognized by mouse MHC-class I, the vaccine protected mice from tumor development and induced efficient immune responses, suggesting that the precise knowledge of epitopic sequences and the matched HLA type is not required when delivering a full-length protein using the yeast platform. In addition, the vaccine stimulated both CD4(+) T cells and CD8(+) T cells simultaneously. This study provides a 'proof of principle' that recombinant yeast can be utilized as an effective prophylactic vaccine to target patients at high-risk for melanoma.

Tumor necrosis factor (TNF) protects resistant C57BL/6 mice against herpes simplex virus-induced encephalitis independently of signaling via TNF receptor 1 or 2.

Tumor necrosis factor (TNF) is a multifunctional cytokine that has a role in induction and regulation of host innate and adaptive immune responses. The importance of TNF antiviral mechanisms is reflected by the diverse strategies adopted by different viruses, particularly members of the herpesvirus family, to block TNF responses. TNF binds and signals through two receptors, Tnfrsf1a (TNF receptor 1 [TNFR1], or p55) and Tnfrsf1b (TNFR2, or p75). We report here that herpes simplex virus 1 (HSV-1) infection of TNF-/- mice on the resistant C57BL/6 genetic background results in significantly increased susceptibility (P < 0.0001, log rank test) to fatal HSV encephalitis (HSE) and prolonged persistence of elevated levels of virus in neural tissues. In contrast, although virus titers in neural tissues of p55-/- N13 mice were elevated to levels comparable to what was found for the TNF-/- mice, the p55-/- N13 mice were as resistant as control C57BL/6 mice (P > 0.05). The incidence of fatal HSE was significantly increased by in vivo neutralization of TNF using soluble TNFR1 (sTNFR1) or depletion of macrophages in C57BL/6 mice (P = 0.0038 and P = 0.0071, respectively). Strikingly, in vivo neutralization of TNF in HSV-1-infected p55-/- p75-/- mice by use of three independent approaches (treatment with soluble p55 receptor, anti-TNF monoclonal antibody, or in vivo small interfering RNA against TNF) resulted in significantly increased mortality rates (P = 0.005), comparable in magnitude to those for C57BL/6 mice treated with sTNFR1 (P = 0.0018). Overall, these results indicate that while TNF is required for resistance to fatal HSE, both p55 and p75 receptors are dispensable. Precisely how TNF mediates protection against HSV-1 mortality in p55-/- p75-/- mice remains to be determined.

Soluble human p55 and p75 tumor necrosis factor receptors reverse spontaneous arthritis in transgenic mice expressing transmembrane tumor necrosis factor alpha.

OBJECTIVE: The roles of the transmembrane and secreted forms of tumor necrosis factor alpha (TNFalpha) in rheumatoid arthritis (RA) remain unclear. Agents used to inhibit TNFalpha have shown varying efficacy in RA patients, suggesting that anti-TNFalpha agents possess dissimilar mechanisms of action, including the ability to neutralize transmembrane (tmTNFalpha) and secreted TNFalpha. In this study, TNFalpha-knockout (TNFalpha-KO) mice that were genetically altered to express elevated levels of tmTNFalpha were constructed to further understand the roles of the 17-kd secreted, trimeric, and 26-kd transmembrane forms of TNFalpha. METHODS: A speed-congenic mating scheme was used to generate 3 unique strains of mice: 1) transgenic tmTgA86 mice overexpressing 26-kd tmTNFalpha and also secreting 17-kd trimeric TNFalpha (tmTNFalpha-transgenic), 2) TNFalpha-/- mice (TNFalpha-KO), and 3) transgenic mice overexpressing tmTNFalpha backcrossed to TNFalpha-KO mice (tmTNFalpha-transgenic/TNFalpha-KO). Mice were treated with phosphate buffered saline (as vehicle control), dexamethasone (as positive control), or modified recombinant human soluble TNF receptor (sTNFR) p55 or p75, and were assessed clinically and histopathologically for signs of inflammation and development of arthritis. RESULTS: The tmTNFalpha-transgenic/TNFalpha-KO mice were born with crinkled tails and spinal deformities similar to those in ankylosing spondylitis. By 2-4 weeks, these mice developed symmetric inflammatory arthritis, characterized by tissue swelling, pannus formation, and bone deformities. The tmTNFalpha-transgenic mice also developed spontaneous-onset arthritis, but at a slower rate (100% incidence by 10-12 weeks). Clinical and histologic progression of arthritis in the tmTNFalpha-transgenic/TNFalpha-KO mice was reduced by treatment with dexamethasone or with the p55 or p75 sTNFR (69% and 63% reduction in total histologic score, respectively). CONCLUSION: These data show that arthritis is sufficiently initiated and maintained in tmTNFalpha-transgenic/TNFalpha-KO mice, and that it can be neutralized by recombinant human p55 or p75 sTNFR, resulting in amelioration of the biologic and subsequent histologic destructive effects of tmTNFalpha.

Mechanism of action differences in the antitumor effects of transmembrane and secretory tumor necrosis factor-alpha in vitro and in vivo.

The proinflammatory cytokine tumor necrosis factor-alpha (TNFalpha) exists naturally in two forms, a 26 kDa transmembrane form (TM-TNFalpha), and a 17 kDa secretory form (S-TNFalpha). The biological roles for each of these forms of TNFalpha in tumor killing have not been completely elucidated. Therefore, in this study, three different recombinant retroviral vectors, wild-type TNFalpha, solely secretable TNFalpha mutant, and uncleavable transmembrane TNFalpha mutant, were constructed by molecular techniques and stably transfected into a murine hepatic carcinoma cell line (H22). TNFalpha, either secreted in cell culture supernatants by secretable TNFalpha mutant- or wild-type TNFalpha-producing tumor cells, or as a treansmembrane form expressed on the cell surface of uncleavable TNFalpha mutant- or wild-type TNFalpha-synthesizing tumor cells, was demonstrated to be cytotoxic against the TNF sensitive L929 cell line. The H22 cells transfected with the three different forms of TNFalpha were shown to kill parental H22 cells in an in vitro cytotoxicity assay [effect/target (E/T) ratio-dependent manner], and their maximal killing rates were approximately 38-43% at E/T ratio of 5:1. The injection of total 2.5 x 10(5) mixed cells containing transfected and parental H22 tumor cells at different ratios into syngeneic mice resulted in the inhibition of tumor growth with a maximal inhibition rates of approximately 57 approximately 72% at E/T ratio of 5:1. A transient weight loss was found in mice bearing solely secretable TNFalpha mutant producing tumors, whereas no obvious side effects were seen in mice bearing uncleavable TNFalpha mutant or wild-type TNFalpha expressing tumors. Finally, we demonstrate that tumors secreting S-TNFalpha promoted the subsequent infiltration of CD4(+) T cells, and to a lesser extent CD8(+) T cells, to the tumor site. The TM-TNFalpha expressing tumors up-regulated Fas (CD95) expression and inhibited the expression of tumor metastasis associated molecule CD44v3. These results suggest that S-TNFalpha and TM-TNFalpha kill cancer cells in vivo through different mechanisms of action. We conclude that the non-secreted form of TNFalpha may be an ideal candidate for cancer gene therapy due to its therapeutic potential and lowered side effect profile.

Mouse splenic B lymphocyte activation using different activation stimuli induces in vitro splicing of tumor necrosis factor-alpha nuclear pre-mRNA.

The pleiotropic functions of tumor necrosis factor-alpha (TNFalpha) have brought considerable attention in the past decade to its physiological and pathological roles in inflammatory and autoimmune diseases. However, little is known about how the production of TNFalpha is regulated at the transcriptional and translational levels in immune cells such as T and B lymphocytes. Our previous study demonstrated that unspliced "pre-mRNA" of TNFalpha is present in resting T cells. Initiation of splicing of TNFalpha pre-mRNA to mature mRNA requires T cell activation, which is unique and necessary for TNFalpha production when compared to its production in mononuclear phagocytes, including different lineages of macrophages (Mvarphi) and dendritic cells (DC). In this study, we further demonstrate that resting mouse B cells also contain pre-existing TNFalpha mRNA. The physiological process of B cell activation induced by (1) either the cross-linking of the B cell receptor (BCR) or CD40, (2) treatment with LPS, or PMA plus ionomycin, induces TNFalpha mRNA splicing in vitro. The kinetic response of TNFalpha splicing in B cells is much slower when compared to that in activated T cells. Studies using well-known kinase inhibitors demonstrated that MAP kinase kinase (MEK) and protein kinase C (PKC) are required for TNFalpha splicing upon stimulation through the BCR. These studies demonstrate that the production of TNFalpha in activated B cells is regulated differently than in activated T cells, and these differences may allow for the selective inhibition of TNFalpha in various autoimmune diseases depending on the mechanism of action of the selected anti-TNFalpha therapy.

A phase 2 dose-finding study of PEGylated recombinant methionyl human soluble tumor necrosis factor type I in patients with rheumatoid arthritis.

OBJECTIVE: In a phase 2 study, to assess the efficacy and safety of pegsunercept, a soluble tumor necrosis factor receptor type I, for the treatment of rheumatoid arthritis (RA). METHODS: Patients were randomized to receive weekly subcutaneous injections of placebo (n = 61) or active drug [400 microg/kg (n = 67) or 800 microg/kg (n = 66)] for 12 weeks. The primary efficacy endpoint was American College of Rheumatology 20% response (ACR20) at Week 12. Secondary efficacy measures included ACR50 and ACR70 responses, and changes in individual ACR components at Week 12. Safety assessments included summaries of adverse events including infectious episodes. RESULTS: Treatment with pegsunercept resulted in a significantly higher ACR20 response at Week 12 in the 800 microg/kg group (45%) compared with the placebo group (26%; p = 0.020). The treatment effect of pegsunercept (both doses) over the study period showed statistically significant improvement for most ACR components and health related quality of life, with the 800 microg/kg group showing greater clinical improvements in efficacy measures. The overall incidence of adverse events and infectious episodes was similar among the treatment and placebo groups. CONCLUSION: In this 12 week dose-finding study of 194 patients, weekly subcutaneous dosing with pegsunercept showed beneficial effects in improving the signs and symptoms of RA. It appeared to be safe and well tolerated in this small number of patients. Significant clinical improvements were seen in patients in the 800 microg/kg group; however, this dose may be suboptimal, and further evaluation of this product with higher doses or a more frequent dosing regimen is warranted.

Differential maturation of murine bone-marrow derived dendritic cells with lipopolysaccharide and tumor necrosis factor-alpha.

Dendritic cells (DCs) play a key role in the interface between the innate and acquired immune systems. In response to both exogenous as well as endogenous signals, DCs undergo a programmed maturation to become an efficient, antigen-presenting cell. Yet little is known regarding the differential responses by endogenous versus exogenous stimuli on DC maturation. In the present report, we have compared the phenotypic, functional, and genome-wide expression responses associated with maturation by bone marrow derived DCs to either an endogenous danger signal, tumor necrosis factor-alpha (TNF-alpha), or a microbial product, bacterial lipopolysaccharide (LPS). Examination of the cell surface expression of DCs as well as cytokine production demonstrated that patterns of DC maturation varied dramatically depending upon the stimulus. Whereas LPS was highly effective in terms of inducing phenotypic and functional maturation, TNF-alpha exposure produced a phenotypically distinct DC. Gene expression patterns in DCs 6 and 24 h after LPS and TNF-alpha exposure revealed that these activation signals produce fundamentally different genomic responses. Supervised analysis revealed that the expression of 929 probe sets discriminated among the treatment groups, and the patterns of gene expression in TNF-alpha stimulated DCs were more similar to unstimulated cells at both 6 and 24 h post-stimulation than to LPS-stimulated cells at the same time points. These findings reveal that DCs are capable of a varying phenotypic response to different antigens and endogenous signals.

Drug discovery and development for inflammatory diseases.

Numerous recent investigations have pointed to a key role of the pro-inflammatory, pleotropic cytokines TNF-alpha and IL-1 in host defence and inflammatory disease processes. TNF and IL-1 overexpression has been found in disease target tissue and in the circulation of patients with acute and chronic inflammatory diseases, and it was suggested early on in this field of basic medical research that TNF-alpha and IL-1 were crucial in these diseases. Over the last 10 years, several approaches to inhibit TNF-alpha and, in one case, IL-1 activity, have been developed by the biotechnology and pharmaceutical industries. These include neutralising antibodies to TNF-alpha as well as soluble TNF-alpha receptors with characteristic properties designed to bind the 17 kDa soluble trimeric TNF-alpha and the 26 kDa membrane-bound form of TNF-alpha. Clinical trials have demonstrated significant effects with these agents and it is likely that blocking TNF-alpha will become an important standard therapeutic option for clinicians. The data available from these trials contribute to the further understanding of inflammation by demonstrating the major role of these cytokines. This research and clinical background have provided the basis for a variety of lectures on novel drug discovery targets, the current status of previously identified targets and technologies to develop new therapeutics in inflammation. At this meeting, emphasis was placed upon drug targets and their validation in animal models and early stage clinical trials.