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Biologic therapy for rheumatoid arthritis (RA) targets specific molecules that mediate and sustain the clinical manifestations of this complex illness. Compared with the general population, patients with RA die prematurely, in part due to associated cardiovascular disease. Even though the mechanisms by which premature atherosclerosis develops in RA is unknown, chronic inflammation may play a major role. This review connects current knowledge of the pathophysiology of RA with data available in the literature related to thrombospondin-1 (TSP1), transforming growth factor beta (TGFbeta and connective tissue growth factor (CTGF) and their relationship with cardiovascular disease in RA. The TSP1/TGFbeta/CTGF axis may contribute in the pro-inflammatory and pro-atherogenic state in patients affected with RA. In fact, increased TSP1 plasma levels are found in patients of RA. TGFbeta is activated by TSP1 through a non-enzymatic mechanism and is constitutively overexpressed by synovial fibroblasts from RA patients. Activation of TGFbeta pathway in synovial fibroblasts and other cells including neutrophils leads to downstream upregulation of CTGF. Overexpression of CTGF is associated with angiogenesis, fibrosis, atherosclerotic blood vessels and erosive arthritis lesions. Recent RA therapies emphasize the need for aggressive control of the activity of the disease to prevent premature atherosclerosis in RA patients. The complexity and heterogeneity of RA as judged by response to a wide spectrum of treatments mandates the elucidation of unknown pro-inflammatory pathways playing a major role in this disease. The TSP1/TGFbeta/CTFG axis represents one of these pro-inflammatory pathways that may result in the development of promising therapeutic strategies to prevent chronic inflammation and thus premature atherosclerosis in RA.

Thrombospondin-1 (TSP1/THBS1) plays a major role in the pathophysiology of rheumatoid arthritis (RA); however, its interface with the cytokine network involved in RA has not been delineated. Correlations were performed between plasma levels of TSP1 and selected cytokines from blood samples collected from 20 patients affected by RA and 13 healthy donors (control). Plasma levels of TSP1 and tissue growth factor beta (TGFbeta) were determined by standard enzyme-linked immunosorbent assay, and cytokines were measured by protein profiling rolling-circle amplification (RCA). TSP1 circulating levels in plasma were found significantly increased in the RA patients when compared with control individuals (P = 0.039). The plasma levels of TGFbeta were also increased in the RA patients, which indicates a statistical trend. Cytokine levels of interleukin (IL)-4, IL-5, IL-12, chemokine CXC 10 (CXCL10/IP10), and chemokine CC 4 (CCL4)/MIP1beta were significantly increased in the RA patients when compared with the control group. In summary, this study demonstrates increased plasma levels of TSP1, which correlated with increased levels of proinflammatory cytokines in plasma of RA patients. More detailed research is required to explore the cytokine imprint yielded by this study and its interface with TSP1 and TGFbeta.

Objective To compare inflammatory peripheral arthritis in wild-type and high molecular weight kininogen (HK)–deficient rats, both on the genetically susceptible Lewis background. Methods By backcrossing Brown-Norway HK-deficient rats with Lewis rats for 6 generations, 2 new strains were produced, wild-type F6 and HK-deficient F6, each with a 98.5% Lewis genome. Inflammatory arthritis was induced by intraperitoneal injection of peptidoglycan–polysaccharide (PG-PS), and the clinical, histopathologic, and biochemical responses were compared in both strains. Results Eighteen days after PG-PS injection, rats with normal concentrations of HK showed weight loss and marked increase in hind ankle diameter with severe synovial inflammation and cartilage abnormalities. In contrast, HK-deficient rats showed no weight loss (P < 0.05), no increase in hind ankle diameter (P < 0.05), and an absence of inflammatory changes (P < 0.05), as measured by the histologic and morphometric Mankin grading system for synovial and cartilage injury. Conclusion Plasma HK is a key mediator of acute and chronic inflammatory arthritis in genetically susceptible Lewis rats.

Robert Kaplan, Hematology Fellow, Department ofMedicine and Sol Sherry Thrombosis Research Center,Temple University School of Medicine: The patient is a64-year-old Caucasian female with a history of hyper-tension and diabetes mellitus who presented with a four-month history of weight loss and fatigue. Laboratoryevaluation revealed a pancytopenia initially suspected tobe related to her multiple medications. Despite changesin her therapeutic regimen, there was no significant im-provement in her blood cell counts. A subsequent bonemarrow aspirate revealed atypical promyelocytes withabundant Auer rods accounting for 35% of the marrowcells. Cytogenetic studies detected the t(15,17)(q

Administration of the thienopyridine P2Y12 receptor antagonist, clopidogrel, increased the erosive arthritis induced by peptidoglycan polysaccharide (PG-PS) in rats or by injection of the arthritogenic K/BxN serum in mice. To determine if the detrimental effects are caused exclusively by clopidogrel, we evaluated prasugrel, a third-generation thienopyridine pro-drug, that contrary to clopidogrel is mostly metabolized into its active metabolite in the intestine. Prasugrel effects were examined on the PG-PS-induced arthritis rat model. Erosive arthritis was induced in Lewis rats followed by treatment with prasugrel for 21 days. Prasugrel treated arthritic animals showed a significant increase in the inflammatory response, compared with untreated arthritic rats, in terms of augmented macroscopic joint diameter associated with significant signs of inflammation, histomorphometric measurements of the hind joints and elevated platelet number. Moreover, fibrosis at the pannus, assessed by immunofluorescence of connective tissue growth factor, was increased in arthritic rats treated with prasugrel. In addition to the arthritic manifestations, hepatomegaly, liver granulomas and giant cell formation were observed after PG-PS induction and even more after prasugrel exposure. Cytokine plasma levels of IL-1 beta, IL-6, MIP1 alpha, MCP1, IL-17 and RANTES were increased in arthritis-induced animals. IL-10 plasma levels were significantly decreased in animals treated with prasugrel. Overall, prasugrel enhances inflammation in joints and liver of this animal model. Since prasugrel metabolites inhibit neutrophil function ex-vivo and the effects of both clopidogrel and prasugrel metabolites on platelets are identical, we conclude that the thienopyridines metabolites might exert non-platelet effects on other immune cells to aggravate inflammation.

Increased life expectancy and the need for long-term antiretroviral therapy have brought new challenges to the clinical management of HIV-infected individuals. The prevalence of osteoporosis and fractures is increased in HIV-infected patients; thus optimal strategies for risk management and treatment in this group of patients need to be defined. Prevention of bone loss is an important component of HIV care as the HIV population grows older. Understanding the mechanisms by which HIV infection affects bone biology leading to osteoporosis is crucial to delineate potential adjuvant treatments. This review focuses on HIV-induced osteoporosis within the context of microRNAs (miRNAs) by reviewing first basic concepts of bone biology as well as current knowledge of the role of miRNAs in bone development. Evidence that HIV-associated osteoporosis is in part independent of therapies employed to treat HIV (HAART) is supported by cross-sectional and longitudinal studies and is the focus of this review.

Severe progressive failure of multiple organ systems has emerged during the past three decades as a common cause of death among patients in intensive care units. Sepsis is now better defined as systemic inflammatory response syndrome (SIRS), but its mortality rate has not changed and it continues to be a major health problem. Endotoxin interacts with plasma proteins and contributes to the pathophysiology of SIRS. Information is limited on the effect of endotoxin on human coagulation and fibrinolytic proteins in vivo, as well as on the cell response involved in the cytokine cascade. For this reason we performed quantitative assays to establish the sequence of events that occurs in vivo in the regulation of the contact and fibrinolytic pathways as well as in the cytokine cascade as a response to a single dose administration of endotoxin to normal non-smoking human volunteers.

This Report from the Field documents a series of interventions developed by Temple University Health System and School of Medicine through participation in the RWJF initiative entitled Hablamos Juntos. The report delineates outcomes to date demonstrating that these interventions have met the challenge of improving patient provider communication for Latinos.

The P2Y12 receptor plays a crucial role in the regulation of platelet activation by several agonists, which is irreversibly antagonized by the active metabolite of clopidogrel, a widely used anti-thrombotic drug. In this study, we investigated whether reduction of platelet reactivity leads to reduced inflammatory responses using a rat model of erosive arthritis. We evaluated the effect of clopidogrel on inflammation in Lewis rats in a peptidoglycan polysaccharide (PG-PS)-induced arthritis model with four groups of rats: 1) untreated, 2) clopidogrel-treated, 3) PG-PS-induced, and 4) PG-PS-induced and clopidogrel-treated. There were significant differences between the PG-PS+clopidogrel group when compared to the PG-PS group including: increased joint diameter and clinical manifestations of inflammation, elevated plasma levels of pro-inflammatory cytokines (IL-1 beta, interferon (IFN) gamma, and IL-6), an elevated neutrophil blood count and an increased circulating platelet count. Plasma levels of IL-10 were significantly lower in the PG-PS+clopidogrel group compared to the PG-PS group. Plasma levels of platelet factor 4 (PF4) were elevated in both the PG-PS and the PG-PS+clopidogrel groups, however PF4 levels showed no difference upon clopidogrel treatment, suggesting that the pro- inflammatory effect of clopidogrel may be due to its action on cells other than platelets. Histology indicated an increase in leukocyte infiltration at the inflammatory area of the joint, increased pannus formation, blood vessel proliferation, subsynovial fibrosis and cartilage erosion upon treatment with clopidogrel in PG-PS-induced arthritis animals. In summary, animals treated with clopidogrel showed a pro-inflammatory effect in the PG-PS-induced arthritis animal model, which might not be mediated by platelets. Elucidation of the mechanism of clopidogrel-induced cell responses is important to understand the role of the P2Y12 receptor in inflammation.

Objective: To evaluate the effect of a thrombospondin 1 (TSP1)-derived peptide on inflammation and angiogenesis in an animal model of erosive arthritis and to assess the relationship between TSP1 and connective tissue growth factor (CTGF) in the pathophysiology of rheumatoid arthritis. Methods: Erosive arthritis in Lewis rats was induced by peptidoglycan-polysaccharide (PG-PS). Animals were divided into four groups: (1) negative control and groups receiving, (2) no treatment, (3) treatment with a TSP1-derived peptide, and (4) treatment with a scrambled peptide. Samples obtained from ankle joint, spleen and liver were studied using histology, histomorphometry, immunohistochemistry and RT-PCR. Results: Histological data indicated that the TSP1-derived peptide treatment decreased neovascularization, leukocyte infiltration and thickening of the synovial lining of the joint, and reduced granuloma formation in the spleen and liver when compared to control groups. Higher concentrations of CTGF and TSP1 proteins were observed in the affected areas of animals which did not receive TSP1-derived peptide treatment. Also, immunofluorescence and RT-PCR analyses showed an increase in CTGF protein expression and regulation, respectively, in the tissues of untreated animals when compared to the TSP1-derived peptide treated animals. By immunofluorescence, TSP1 expression was decreased in the TSP1-derived peptide treated animals. Moreover, macrophage/monocyte-specific staining revealed a decrease in cell infiltration in the articular tissue of the TSP1-derived peptide treated animals. Conclusion: Both inflammation and angiogenesis were decreased after TSP1-derived peptide treatment indicating a potential pathway by which TSP1 interaction with neutrophils induces CTGF in RA affected tissues. J. Cell. Physiol. 211: 504–512, 2007. © 2007 Wiley-Liss, Inc.

Objective Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with leukocyte adhesion to and extravasation through vascular endothelium into synovial tissue. Recent evidence indicates that the thrombospondin 1 gene is up-regulated in patients with RA. We have identified a region within the TSP-1 type 3 repeats that inhibits human neutrophil elastase (HNE) and binds to human neutrophils. The present study was undertaken to investigate the therapeutic benefit of this TSP-1-derived peptide sequence and its effect on connective tissue growth factor (CTGF), a protein involved in fibrotic disorders and in neovascularization, which is a hallmark of RA. Methods CTGF gene and protein expression, as well as protein levels of CTGF in the synovium, after treatment with the TSP-1-derived peptide were studied in the peptidoglycan-polysaccharide animal model of erosive arthritis. Results Peptide treatment prevented joint infiltration and inflammation and was associated with reduced circulating antigen levels of HNE and TSP-1. Additionally, CTGF was up-regulated in this experimental model of RA. Treatment with the TSP-1-derived peptide was associated with down-regulation of the message and protein levels of CTGF. Immunofluorescence studies showed that the mean area fraction of CTGF immunoreactivity in the peptide-treated group of animals was significantly less than that in the untreated group. Conclusion These results document a role for TSP-1 in regulating CTGF gene and protein expression in synovial tissue, suggesting a link with the disease course in this model of RA. This TSP-1-derived synthetic peptide may represent an important template for drug development in RA and other inflammatory conditions associated with neutrophil activation.

Introduction: Diabetic retinopathy (DR) is a severe ocular complication of type 2 diabetes (T2DM). Our laboratory has been studying the role of a pro-inflammatory axis comprised by TSP1-TGFb and CTGF in T2DM. The axis is differentially expressed in patients with T2DM and non-proliferative DR (NPDR) when compared with patients with T2DM and proliferative DR (PDR). The differential expression of such axis is accompanied by interesting cytokine profiles as well. The purpose of this study was to evaluate potential microRNAs associated with the above changes in order to understand better the pathophysiology of T2DM. Recent studies indicate that miRNAs transported to exosomes or HDL can be transferred in an active form to recipients cells potentially involving them in cell-to-cell communication. Methods: Institutional approved IRB and informed consent allowed this prospective study to recruit a total of 40 individuals afflicted by T2DM (n=20 NPDR and n=20 PDR) from the Ophthalmology Department Diabetes Clinic at Temple Hospital, normal controls (n=20) were recruited from the Sol Sherry Thrombosis Research Center at Temple. Western-blotting technique was utilized to document the presence of fragments from CTGF in plasma, which have been documented in vitreous fluid to be angiogenic. Multiplexed protein profiling on microarrays by rolling-circle amplification was employed to determine IL-4 and MIP-1b. Commercially available ELISA was the method to determine the plasma concentration of TSP1, TGFb and CTGF. Total RNA was extracted from human plasma using the miRCURY TM RNA isolation kit by Exiqon at their facility in Denmark. Results and Discussion: TSP1, TGFb, CTGF, IL4, MIP-1b were significantly elevated in NPDR patients when compared to PDR patients. Fragments of CTGF were identified more abundant in patients with PDR when compared to NPDR. Only three microRNAs were differentially expressed in the normal group when compared with either NPDR (decreased), namely miR-133a (p Disclosures No relevant conflicts of interest to declare.

Introduction Diabetic retinopathy is the most severe of several ocular complications of diabetes. Diabetes afflicts approximately 6% of the US population, thus retinopathy remains an important problem (PMID: 14702427). Our laboratory has provided evidence of the significant role of a pro-inflammatory axis during chronic inflammation (PMID: 19485899). The axis is comprised of TSP1-TGFβ-CTGF. We now provide evidence of an additional player within the axis, namely IL-4. A series of biochemical measurements were used to assess the role of the above mentioned axis in the progression of disease, from non-proliferative diabetic retinopathy (NPDR) to proliferative diabetic retinopathy (PDR). Methods Institutionally approved IRB and informed consent allowed this prospective study to recruit a total of 40 individuals from a vulnerable population disproportionately afflicted by this disease and representative of the population that we served in North Philadelphia with type-2 diabetes. Western-blotting technique was utilized to document the presence of fragments from CTGF in plasma, which have been shown in vitreous fluid to be angiogenic (PMID: 14988298). Multiplexed protein profiling on microarrays by rolling-circle amplification was employed to determine IL-4, IL-13, and MIP-1β levels. Commercially available ELISA was the method to determine the plasma concentration of TSP1, TGFβ and CTGF. Results and Discussion TSP1 (p Disclosures: No relevant conflicts of interest to declare.

Abstract 3268 Prasugrel is a very potent and effective P2Y12 receptor antagonist, used primarily to prevent thrombotic events in patients managed for percutaneous coronary intervention (PCI). It is not recommended as prophylaxis antithrombotic therapy because prasugrel increases patient's risk of bleeding. Even though indication of prasugrel treatment has been limited to the PCI procedure, there are not reports of the effects of prasugrel in patients with associated inflammatory diseases that undergo for PCI. Thus, we evaluate the effects of prasugrel, for 21 days, in a peptidoglycan polysaccharide (PG-PS)-induced arthritis rat model. Lewis rats were randomly separated into four groups from which only half of the animals were induced with PG-PS. There was a significant increase in the inflammatory changes observed in the PG-PS-induced animals treated with prasugrel compared to the PG-PS-induced control including a) increase joint diameter associated with visible signs of inflammation (during days 4 to 6 [p Disclosures: No relevant conflicts of interest to declare.

Abstract 3287 Clopidogrel is a widely used anti-thrombotic drug that prevents platelet activation by irreversibly antagonizing P2Y12 receptor. This drug could be co-administrated in patients afflicted with inflammatory conditions, such as rheumatoid arthritis and lupus. Hence it is of importance to determine if blocking P2Y12 receptors has any therapeutic benefit during the inflammatory response. Therefore, we evaluated the effect of clopidogrel using an animal model of chronic inflammation. Genetically susceptible Lewis rats were injected intraperitoneally with peptidoglycan polysaccharide (PGPS) to develop arthritis and followed for 21 days. Interestingly, we noticed an increased level of inflammation in induced arthritis animals treated with clopidogrel compared with induced arthritis animals alone, including a) increased in joint diameter between day 15 and 21 (p Disclosures: No relevant conflicts of interest to declare.

Abstract 3275 Background: Diabetic retinopathy (DR) is a progressive disease that affects over 4 million people in the United States and is one of the leading causes of blindness. The pathophysiology of the early events (no evidence of DR) leading to diabetic retinopathy is still not fully understood. Recent studies have proposed that levels of TSP1 (Arch Ophthalmol 127:507, 2009) and CTGF (Diabetes Care 27:758, 2004) may play a role in shifting the angiogenic balance and pathogenesis of DR. We postulate that during acute and chronic inflammation, as seen in type-2 diabetes a pro-inflammatory axis comprised by TSP1, transforming growth factor-beta (TGF-b) and CTGF may play a significant role in the progression of NPDR to PDR. Methods: This is a prospective control study comprised to date of: a) six (6) human subjects with NPDR, b) eleven (11) subjects with PDR, c) five (5) subjects without type-2 diabetes but in need of pars plana vitrectomy, and d) seventeen (17) normal human volunteers. Plasma samples were obtained from all groups (a-d) after signing an informed consent approved by our institution's IRB committee. Vitreous fluid (VF) was collected from subjects in b and c. Cytokine profile was determined by Multiplex Assay. TSP1, TGF-b and CTGF were assessed by commercially available ELISA assays and visualized by Western-blotting technique. Results: TNFa in conjunction with other pro-inflammatory cytokines was found elevated in NPDR subjects when compared to subjects in control groups (c-d, P Disclosures: No relevant conflicts of interest to declare.

Abstract 4721 The P2Y12 receptor plays a crucial role in the regulation of platelet activation by several agonists. The active metabolite of clopidogrel, a widely used anti-thrombotic drug, irreversibly antagonizes the P2Y12 receptor. However, treatment with clopidogrel has been associated with joint inflammation. In addition, Boilard E. et al identified micro-particles generated by activated platelets in joint fluid from rheumatoid arthritis (RA) patients. In this study we investigated whether platelet activation leads to changes in inflammatory responses using a rat model of erosive arthritis. Here, we evaluated the effect of clopidogrel on inflammation in Lewis rats in a peptidoglycan polysaccharide (PG-PS)-induced arthritis model with four groups of eight rats each: untreated group, animals receiving clopidogrel (clopidogrel group), animals receiving PG-PS (PG-PS group), and animals receiving PG-PS as well as clopidogrel (PG-PS + clopidogrel group). There were significant differences between the PG-PS + clopidogrel treated group when compared to PG-PS treated group including: increased joint diameter [4.24 ± 0.73 vs. 2.70 ± 1.09; p0.05], and elevated plasma levels of pro-inflammatory cytokines such as IL-1beta, IF gamma, IL-6. Plasma levels of anti-inflammatory cytokine IL-10 were significantly lower in PG-PS + clopidogrel treated group compared to PG-PS group alone. Histological observations indicated an increase in leukocyte infiltration at the inflammatory area of the joint, increased pannus formation and decrease in bone mass upon treatment with clopidogrel in PG-PS-induced arthritis animals. Accordingly to the clinical, histological and cytokine results, we conclude that clopidogrel has a pro-inflammatory effect in PG-PS-induced arthritis animal model and this might implicate that P2Y12 receptor antagonism might enhance inflammatory responses. Disclosures: No relevant conflicts of interest to declare.

Abstract 4320 Biologic therapy for rheumatoid arthritis (RA) targets specific molecules that mediate and sustain the clinical manifestations of this complex illness. Compared with the general population, patients with RA are at an increased risk to develop cardiovascular diseases and the precise mechanism(s) of action remain obscure. This laboratory has proposed the existence of a pro-inflammatory axis in RA comprised by thrombospondin-1 (TSP1), transforming growth factor-beta (TGF-b) and CTGF. The present study evaluated plasma levels of TSP1, TGFb and CTGF in patients with RA by ELISA as well as specific cytokines and chemokines. CTGF plasma levels in RA patients (9.2 pg/ml mean, range 3.44–17.08) were found significantly increased (P Disclosures: No relevant conflicts of interest to declare.

Rheumatoid Arthritis (RA) is a chronic, autoimmune disease that affects a vast population worldwide with women being afflicted three times more than men. There is evidence for an increased risk of cardiovascular events in RA patients compared to the general population. These cardiovascular events may be associated with the chronic inflammatory state in which activation of coagulation leads to thrombin generation. Our laboratory has evidence that thrombospondin-1 (TSP1), an adhesive molecule that plays a major role in RA, promotes thrombin generation on the surface of a monocytic cell line (Isordia-Salas et al, Thromb Res.2005;116(6)). We also have documented that disrupting the TSP1 interaction on human neutrophils prevents the development of erosive arthritis in an experimental animal model (Manns et al, Arthritis and Rheumatism54(8), 2006). This observation is mediated by a novel pathway whereby TSP1 induces the up-regulation of Connective Tissue Growth Factor (CTGF). Therefore, to assess whether our in vitro and in vivo observations can be extrapolated to human disease, blood samples were collected from 20 patients afflicted with rheumatoid arthritis and 13 from healthy donors which served as the negative control. Plasma samples were separated and analyzed by Enzyme-Linked Immunosorbent Assay (ELISA) to determine the levels of transforming growth factor beta (TGF-β), protrombin F1+2 fragments (F1+2) and thrombospondin-1, and by multiplexed cytokine protein profiling on microarray by rolling-circle amplification (RCA) to determine cytokine levels. The F1+2 plasma levels showed an elevated trend in the RA group (p=0.06). TSP1 plasma levels were significantly increased in the RA group compared to the normal control (p=0.0004). Pro-inflammatory cytokine levels including IL-1β (p=0.0365), IL-6 (p=0.0029), TNF-α (p=0.0339), interferon-inducible protein 10 (p=0.0003) and macrophage inflammatory protein -1α (p=0.0012) were found elevated in the RA group compared to the normal control group. Some regulatory cytokines such as transforming growth factor-β (0.06), interferon-gamma (p=0.06) and IL-13 (p=0.22) showed no statistically significance between groups, but all of them showed a trend for higher circulation levels in plasma in the RA group. In summary, comparison between normal individuals and RA patients showed an increase in the levels of cytokines in the RA afflicted patients confirming what has been reported in the literature. We were able to correlate an increase in proteolytic factors and TSP1 levels in the RA patients with an increase of pro-inflammatory cytokines. Further studies are needed to elucidate why TSP1 acts as a pro-inflammatory molecule on the neutrophil surface of the RA patients.

There is evidence of shortening of the APTT in patients afflicted with and without cancer, and cancer is a well known underlying condition associated with hypercoagulability. Increased levels of coagulation factors such as VIII, IX, XI, II, and fibrinogen have been demonstrated as independent risk factors for venous thromboembolism (VTE). However, there is evidence that high levels of circulating factor VIII levels are not the only determinant for a shortened APTT (Blood 2004; 104:3631) and likely the same case for the other factors including fibrinogen levels. A shortened APTT in a patient with cancer with no evidence of a thrombotic event could be of clinical value in particular when the cost of measuring individual coagulation factors previously identified as risk factors for VTE would be very high. A lack of understanding of the underlying mechanism leading to a shortened APTT in the absence of VTE limit its clinical use, particularly for the clinical management of the patient presenting at admission with this phenomenon in association with cancer. In this case-control prospective study, we have investigated a potential mechanism associated with a shortened APTT in patients with cancer without evidence of thrombosis. The rationale for this study was a previous observation supporting a functional role of TSP1 in the generation of thrombin on a cell surface (Thrombosis Research 2005; 116: 533). The study was comprised of 69 human subjects subdivided in three separate groups, Group I, was constituted by 23 normal human volunteers, Group II included 23 patients without a shortened APTT and cancer, Group III was comprised of 23 patients with cancer with a shortened APTT (see table). Groups I and II were matched with group III by age and gender. In addition patients in Group II were matched with Group III by the type of cancer. Laboratory measurements included APTT, D-Dimer, soluble E, L, and P-Selectins as well as TSP1. Platelet and neutrophil counts were determined by automated methods. Laboratory measurements demonstrating a significant difference in Group III when compared with Group I and II were P-Selectin and TSP1. These results were independent of the platelet count in Group III. However the significant elevated circulating levels in plasma of P-Selectin in Group III are evidence supporting platelet activation. There was a trend for higher levels of D-Dimer in Group III (P < 0.17) when compared with Group II (P< 0.76), in accordance with previous studies reported in the literature. In summary, this prospective study demonstrates a potential association of a shortened APTT in patients with cancer with elevated circulating levels of soluble P-Selectin and TSP1. Our laboratory is currently investigating in more detail this interesting finding as well as the prospective clinical follow up of patients included in Groups II and III. | Parameter | Group I | Group II | Group III | P Value | |:------------------------------------ | -------- | -------- | --------- | ------- | | Normal Range APTT: 24.2–35.3 seconds | | aPTT | 29.3±2.7 | 28.2±2.3 | 23±1.0 | 0.01 | | sP-Selectin | 23.2±4 | 25.4±9 | 44.0±11 | 0.001 | | TSP1 | 382±39 | 871±496 | 1246±295 | 0.001 | Plasma Determinations

Gene expression of CTGF, a proangiogenic molecule, is up-regulated by serine proteases (FXa) and thrombin (JBC 275, 2000). We have demonstrated that thrombospondin-1 (TSP1) plays a major role in the assembly of the prothrombinase complex on the surface of neutrophils (PMNs) and that PMNs contain the message for CTGF. Rheumatoid arthritis is a chronic inflammatory disease associated with leukocyte adhesion and extravasation with angiogenesis as its hallmark. There is evidence indicating that TSP1 plays a major role in the pathophysiology of RA (JI 171, 2003). In this genetically susceptible rodent model of RA, a single intraperitoneal (i.p.) injection of peptidoglycan-polysaccharide induces an inflammatory immune response characterized by increased joint diameters in a biphasic manner. The acute phase occurs during the first five days with spontaneous reactivation around day 12 resulting in a progressive and irreversible chronic phase. We tested a well-characterized synthetic peptide derived from TSP1-type 3 repeats under three different therapeutic modalities in the experimental model of RA. First, peptide treatment was administered intravenously (i.v.) during the first five days of the experimental protocol representing the acute phase. Second, peptide treatment was administered i.v. during the acute phase but then i.p. every other day during the chronic phase of the experimental protocol. Third, peptide treatment was administered daily i.p. during the acute phase and every other day during the chronic phase. At all times a positive control group was included as well as a negative control group receiving a scrambled peptide. Total RNA was isolated from the ankle joints of the animals and RT-PCR was performed using specific primers for CTGF and G3PDH. Integrated density values were measured and the ratio of CTGF/G3PDH assessed. Immunohistochemical analysis of CTGF was also performed using Bioquant image analysis software to quantitate CTGF immuno expression. In peptide-treated animals CTGF expression in the cells of the synovial lining was significantly decreased when compared to the disease untreated group. Peptide untreated positive control group was associated with upregulation of the CTGF gene expression (2.53±0.33) in the ankle tissue and localized to the synovial membranes as judge by the immunochemical studies (8.7±0.77). In contrast, peptide treatment downregulated CTGF gene expression (0.9±0.41, p