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Macrophages are critical in the pathogenesis of rheumatoid arthritis (RA). We recently demonstrated that FLIP is necessary for the differentiation and/or survival of macrophages. We also showed that FLIP is highly expressed in RA synovial macrophages. This study was undertaken to determine if a reduction in FLIP in mouse macrophages reduces synovial tissue macrophages and ameliorates serum-transfer arthritis.Mice with Flip deleted in myeloid cells (FlipIn contrast to expectations, FlipOur observations suggest that reducing FLIP in macrophages by increasing the number of antiinflammatory macrophages may be an effective therapeutic approach to suppress inflammation, depending on the disease stage.

Background The risk for developing cardiovascular disease is greater in patients with rheumatoid arthritis (RA) than in the general population. While patients with RA also have dyslipidemia, the impact of dyslipidemia on the severity of inflammatory arthritis and associated cardiovascular disease is unclear. Currently, there are conflicting results regarding arthritis incidence in apolipoprotein E (ApoE) deficient mice, which spontaneously exhibit both hyperlipidemia and atherosclerosis. Here, we utilize a distinct approach to investigate the contribution of a hyperlipidemic environment on the development of arthritis and atherosclerosis in mice lacking ApoE. Methods K/BxN serum transfer-induced arthritis (STIA) was assessed in C57BL/6 (control) and ApoE−/− mice using clinical indices and immunohistochemical staining. Ankle synoviums were processed for flow cytometry. Aortic atherosclerosis was quantitated using Sudan IV staining. Serum cholesterol and cytokine levels were determined via enzymatic and luminex bead-based assays, respectively. Results ApoE−/− mice developed a sustained and enhanced semi-chronic inflammatory arthritis as compared to control mice. ApoE−/− mice had increased numbers of foamy macrophages, enhanced joint inflammation and amplified collagen deposition versus controls. The presence of arthritis did not exacerbate serum cholesterol levels or significantly augment the level of atherosclerosis in ApoE−/− mice. However, arthritic ApoE−/− mice exhibited a marked elevation of IL-6 as compared to non-arthritic ApoE−/− mice and arthritic C57BL/6 mice. Conclusions Loss of ApoE potentiates a semi-chronic inflammatory arthritis. This heightened inflammatory response was associated with an increase in circulating IL-6 and in the number of foamy macrophages within the joint. Moreover, the foamy macrophages within the arthritic joint are reminiscent of those within unstable atherosclerotic lesions and suggest a pathologic role for foamy macrophages in propagating arthritis. Electronic supplementary material The online version of this article (doi:10.1186/s12967-016-0912-y) contains supplementary material, which is available to authorized users.

Objective To examine the expression of ADAM-10 in rheumatoid arthritis (RA) synovial tissue (ST) and the role it plays in angiogenesis. Methods ADAM-10 expression was determined using immunohistology, Western blotting, and quantitative polymerase chain reaction. In order to examine the role of ADAM-10 in angiogenesis, we performed in vitro Matrigel tube formation and chemotaxis assays using human microvascular endothelial cells (HMVECs) transfected with control or ADAM-10 small interfering RNA (siRNA). To determine whether ADAM-10 plays a role in angiogenesis in the context of RA, we performed Matrigel assays using a coculture system of HMVECs and RA synovial fibroblasts. Results Endothelial cells and lining cells within RA ST expressed high levels of ADAM-10 compared with cells within osteoarthritis ST and normal ST. ADAM-10 expression was significantly elevated at the protein and messenger RNA levels in HMVECs and RA synovial fibroblasts stimulated with proinflammatory mediators compared with unstimulated cells. ADAM-10 siRNA–treated HMVECs had decreased endothelial cell tube formation and migration compared with control siRNA–treated HMVECs. In addition, ADAM-10 siRNA–treated HMVECs from the RA synovial fibroblast coculture system had decreased endothelial cell tube formation compared with control siRNA–treated HMVECs. Conclusion These data show that ADAM-10 is overexpressed in RA and suggest that ADAM-10 may play a role in RA angiogenesis. ADAM-10 may be a potential therapeutic target in inflammatory angiogenic diseases such as RA.

Summary A variety of salivary gland–type lesions occur in the breast. Three cases of a novel mammary carcinoma arising in a background of salivary gland metaplasia and morphologically similar to basal cell adenocarcinoma of the salivary gland are presented. The clinical presentation, morphologic features, treatment, and follow-up of these cases are discussed.

Autoimmunity occurs through a break in tolerance, which is considered to be mediated by a failure to delete autoreactive immune cells. The central mechanism for deleting cells is controlled by the apoptotic machinery. Apoptosis occurs via two distinct pathways, an extrinsic pathway involving transduction of an apoptotic signal following aggregation of a death receptor, such as Fas, to its ligand, Fas ligand (FasL), and an intrinsic pathway that signals through the mitochondria and is regulated by the bcl-2 family. In the extrinsic pathway, binding of homotrimeric FasL to Fas facilitates recruitment of both Fas-associated death domain protein (FADD) and pro-caspase-8 leading to the activation of caspase-8 and subsequent degradative phase of apoptosis (1). This process may be inhibited by cellular FADD-like IL-1β-converting enzyme (FLICE)-inhibitory protein (cFLIP), which is also recruited by FADD and acts as an endogenous suppressor of the Fas pathway (1). Mice mutant for Fas (lpr) mice show normal postnatal development, but over time exhibit splenomegaly, lymphadenopathy, hypergammaglobulinemia, autoantibodies, glomerulonephritis, and the presence of double negative T-cells (CD3+CD4-CD8-B220+), with disease severity highly dependent upon strain background (2). The advent of conditional deletion of genes has yielded novel, unexpected, and controversial results regarding specific effects of loss of Fas within the various hematopoietic cell populations. While previous studies have extensively examined the role that Fas plays in lymphocytes and dendritic cells (3-5), few or no studies have directly examined the cell-intrinsic role of Fas in myeloid cells and its contribution to autoimmunity. Here, mice were generated that conditionally lack Fas only in the myeloid cell compartment (CreLysMFasflox/flox). These mice displayed a disruption in monocyte/macrophage and granulocyte homeostasis that led to altered activation of dendritic cells and lymphocytes in a paracrine fashion. Moreover, CreLysMFasflox/flox mice presented with splenomegaly, as well as autoantibodies and proinflammatory cytokines/chemokines. Furthermore, deposited immune complexes in kidneys and glomerulonephritis were observed in mice with myeloid cell-specific loss of Fas. Taken together, these data indicate that loss of Fas in myeloid cells is sufficient to induce an inflammatory phenotype in mice reminiscent of SLE-like disease, suggesting a role of specifically targeting Fas in its treatment.

Objectives/Hypothesis: Diagnosis and treatment of gastroesophageal and laryngopharyngeal reflux disease has significantly increased over recent years. The larynx is highly sensitive to the effects of LPRD and is similarly responsive to proton pump inhibitor pharmacotherapy. The hypothesis of the study was that proton pump activity exists in the human larynx and plays a functional role in normal and/or pathological laryngeal tissue. Study Design: Pathological investigation. Methods: Two fresh human cadaveric larynges (one male and one female larynx) were obtained as part of an exempt protocol from the Human Subjects Committee and were formalin fixed and paraffin embedded. Banked human stomach tissue was also obtained for use as comparative positive and negative control specimens. Sections were immuno-stained with monoclonal antibodies reactive with both alpha and beta subunits of the H + /K + -ATPase (proton) pump. Specimens were reviewed for staining pattern and intensity. Results: Stomach parietal cells (known to produce gastric acid) exhibited strongly positive staining for both the alpha and beta subunits of the proton pump. There was no staining in stomach cells that were not morphologically consistent with the parietal celL In the human larynx there were strong focal and identical staining patterns in the serous cells and ducts of the minor seromucinous glands by both alpha and beta monoclonals to the proton pump. There was variable staining in the laryngeal epithelium that was thought to be consistent with artifact staining resulting from tissue processing. Conclusion: The H + /K + -ATPase (proton) pump is present in serous cells and ducts of submucosal glands in the human larynx. Proton pump inhibitor pharmacotherapy may have a site of action in seromucinous glands of the human larynx, with possible relevance for patients treated for chronic laryngitis with or without laryngopharyngeal reflux disease.

The free radical nitric oxide (NO(*)) is known to play a dual role in human physiology and pathophysiology. At low levels, NO(*) can protect cells; however, at higher levels, NO(*) is a known cytotoxin, having been implicated in tumor angiogenesis and progression. While the majority of research devoted to understanding the role of NO(*) in cancer has to date been tissue-specific, we herein review underlying commonalities of NO(*) which may well exist among tumors arising from a variety of different sites. We also discuss the role of NO(*) in human physiology and pathophysiology, including the very important relationship between NO(*) and the glutathione-transferases, a class of protective enzymes involved in cellular protection. The emerging role of NO(*) in three main areas of epigenetics-DNA methylation, microRNAs, and histone modifications-is then discussed. Finally, we describe the recent development of a model cell line system in which human tumor cell lines were adapted to high NO(*) (HNO) levels. We anticipate that these HNO cell lines will serve as a useful tool in the ongoing efforts to better understand the role of NO(*) in cancer.

Objective Rheumatoid arthritis (RA) is a destructive autoimmune disease characterized by an increased inflammation in the joint. Therapies which activate the apoptotic cascade may have potential as a future therapy for RA, however few therapeutics fit this category. Recently, therapies that mimic the action of Bcl-2 homology 3 (BH3) domain-only proteins such as Bim have shown success in preclinical studies of cancer but their potential in autoimmune disease is unknown.

The free radical nitric oxide (NO) is known to play an important role in the biology of human cancers, including lung cancer. However, it is still not clear how elevated amounts of nitric oxide affect tumor development and propagation. Herein we develop an in vitro model system to study these effects in lung tumor cells. Two cell lines—one human lung adenocarcinoma (A549) and one mouse adenocarcinoma (LP07) cell line—were adaptively grown in increasing concentrations of the NO donor DETA-NONOate over several months. Both cell lines were successfully adapted to high levels of NO (HNO). Experiments validated the adaptation occurred as a result of the exogenous NO produced by the DETA-NONOate, and was not merely a response to the chemical composition of DETA-NONOate. No morphological differences were observed between cells that were adapted to the HNO and cells which did not undergo the adaptation process (i.e., "parent cells"). Parent cells were unable to survive when placed directly in media containing high levels of DETA-NONOate, suggesting that the adapted cells underwent a biological change enabling them to survive and grow in a HNO environment. The adapted cells were found to grow faster than the parent cells under both normal growth conditions and stressful growth conditions (serum-less media, growth on soft agar) even when the DETA-NONOate was removed from the HNO culture media. These adapted cell lines can serve as a novel tool for use in future experiments designed to better understand the role nitric oxide plays in lung cancer.

Much of the work aimed at elucidating the pathogenesis of osteonecrosis (ON) of the femoral head has focused on bone blood supply, with little attention to the surrounding synovial tissue (ST). We hypothesized that patients with ON exhibit synovial inflammation. Using immunohistological techniques, we found that a large population of patients with ON had synovial inflammation. Moreover, a population of ON patients had inflamed ST without having an inflammatory disease co-morbidity. The inflammatory infiltrate in these patients comprised primarily CD4(+) T cells and CD68(+) macrophages, the latter of which expressed increased levels of cellular adhesion molecules. Our results suggest the presence of a previously unrecognized population of ON patients without a diagnosed inflammatory co-morbidity and a highly inflammed synovium consisting primarily of a macrophage and CD4(+) T-cell infiltrate.

Objective. SSc is characterized by microvascular abnormalities and leucocyte infiltration. Previous studies have suggested a proadhesive phenotype in SSc skin, but the functional consequences of this phenotype are not fully understood. Molecules known to mediate leucocyte adhesion include those present at intracellular junctions, such as junctional adhesion molecule-B (JAM-B), JAM-C and CD99, as well as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). The aim of this study was to examine adhesive interactions in SSc skin. Methods. The expression of JAM-B, JAM-C, CD99, ICAM-1 and VCAM-1 in SSc skin was determined by immunohistology and cell surface ELISA. Myeloid U937 cell–SSc dermal fibroblast adhesion assays or in situ adhesion assays to SSc skin were performed. Results. JAM-C and CD99 expression on endothelial cells (ECs) in SSc skin was decreased compared with expression on normal ECs. CD99 was overexpressed on mononuclear cells in SSc skin and on SSc dermal fibroblasts. Neutralizing ICAM-1 inhibited the binding of U937 cells to SSc dermal fibroblasts. In addition, blocking both ICAM-1 and VCAM-1 inhibited U937 cell adhesion to either proximal (less involved) or distal (more involved) SSc skin. Conclusions. These studies show that JAM-C and CD99 are aberrantly expressed in SSc skin. However, these adhesion molecules do not mediate myeloid cell–SSc skin adhesion. In contrast, we demonstrate an important role for ICAM-1 and VCAM-1 in the retention of myeloid cells in SSc skin, suggesting that targeting these molecules may be useful SSc therapies.

Chemokines, including RANTES/CCL5 and MCP-1/CCL2, are highly expressed in the joints of patients with rheumatoid arthritis, and they promote leukocyte migration into the synovial tissue. This study was conducted to determine whether the inhibition of RANTES and MCP-1 therapeutically was capable of ameliorating rat of adjuvant-induced arthritis (AIA). Postonset treatment of AIA using a novel inhibitor for endogenous MCP-1 (P8A-MCP-1) improved clinical signs of arthritis and histological scores measuring joint destruction, synovial lining, macrophage infiltration, and bone erosion. Using immunohistochemistry, ELISA, real-time RT-PCR, and Western blot analysis, we defined joint inflammation, bony erosion, monocyte migration, proinflammatory cytokines, and bone markers, and p-p38 levels were reduced in rat AIA treated with P8A-MCP-1. In contrast, neither the dominant-negative inhibitor for endogenous RANTES (44AANA47-RANTES) nor the CCR1/CCR5 receptor antagonist, methionylated-RANTES, had an effect on clinical signs of arthritis when administered after disease onset. Additionally, therapy with the combination of 44AANA47-RANTES plus P8A-MCP-1 did not ameliorate AIA beyond the effect observed using P8A-MCP-1 alone. Treatment with P8A-MCP-1 reduced joint TNF-α, IL-1β, and vascular endothelial growth factor levels. P8A-MCP-1 also decreased p38 MAPK activation in the joint. Our results indicate that inhibition of MCP-1 with P8A-MCP-1 after the onset of clinically detectable disease ameliorates AIA and decreases macrophage accumulation, cytokine expression, and p38 MAPK activation within the joint.

The seventh cholera pandemic that started in 1961 was caused by Vibrio cholerae O1 strains of the El Tor biotype. These strains produce the pore-forming toxin hemolysin, a characteristic used clinically to distinguish classical and El Tor biotypes. Even though extensive in vitro data on the cytolytic activities of hemolysin exist, the connection of hemolysin to virulence in vivo is not well characterized. To study the contribution of hemolysin and other accessory toxins to pathogenesis, we utilized the model of intestinal infection in adult mice sensitive to the actions of accessory toxins. In this study, we showed that 4- to 6-week-old streptomycin-fed C57BL/6 mice were susceptible to intestinal infection with El Tor strains, which caused rapid death at high doses. Hemolysin had the predominant role in lethality, with a secondary contribution by the multifunctional autoprocessing RTX (MARTX) toxin. Cholera toxin and hemagglutinin/protease did not contribute to lethality in this model. Rapid death was not caused by increased dissemination due to a damaged epithelium since the numbers of CFU recovered from spleens and livers 6 h after infection did not differ between mice inoculated with hemolysin-expressing strains and those infected with non-hemolysin-expressing strains. Although accessory toxins were linked to virulence, a strain defective in the production of accessory toxins was still immunogenic since mice immunized with a multitoxin-deficient strain were protected from a subsequent lethal challenge with the wild type. These data suggest that hemolysin and MARTX toxin contribute to vaccine reactogenicity but that the genes for these toxins can be deleted from vaccine strains without affecting vaccine efficacy.

Purpose To test the hypothesis that transcatheter arterial embolization (TAE) of VX2 rabbit liver tumors increases the expression of hypoxia-inducible factor-1α (HIF-1α), a transcription factor that regulates the expression of pro-angiogenic genes. Materials and Methods VX2 tumors were implanted in the livers of eight New Zealand white rabbits. Once tumor growth was seen at T2-weighted turbo spin-echo magnetic resonance (MR) imaging, four of the eight rabbits underwent TAE with 45–150-μm polyvinyl alcohol particles. The remaining four rabbits served as non-TAE controls. The TAE end point was stasis of antegrade blood flow. All rabbits were sacrificed for tumor harvest 2 hours after TAE. Tumor tissue and corresponding normal liver tissue in each rabbit liver were stained with anti-human HIF-1α monoclonal antibody and reviewed with light microscopy. Percentages of stained viable tumor and normal liver cells were compared by using the Mann-Whitney U test (α = 0.05). Results In eight rabbits with 24 discrete liver tumors, the mean percentage (±standard deviation) of positive HIF-1α–stained cells in the TAE group was greater than that in the control group (19% ± 7.0 vs 12% ± 8.0, respectively) ( P = .05). Normal liver tissue in both the TAE and control groups showed no HIF-1α staining. Conclusion Although HIF-1α is not expressed in normal rabbit liver parenchyma—even after TAE—HIF-1α expression is present in implanted VX2 rabbit liver tumors and significantly increased in lesions that have undergone embolization.

To better define important cell subsets expressing activation markers in rheumatoid arthritis (RA), we compared selective lymphocyte and monocyte B7H1, B7H2, B7RP.1, B7RP.2, and inducible costimulatory molecule (ICOS) expression from normal peripheral blood (NL PB), RA PB, and RA synovial fluid (SF) by multicolor flow cytometry and immunohistochemistry. RA SF memory lymphocytes expressed B7RP.1 and B7RP.2, suggesting that T-cells may function as antigen presenting cells (APCs) in RA joints. We found similar results for ICOS expression. RA SF CD141 monocytes also expressed B7RP.1 (an ICOS ligand) and the homologous ligand B7RP.2, identifying monocytes as potential mediators of antigen processing and lymphocyte activation in RA. Furthermore, we found an increased population of RA SF CD141 monocytes expressing B7H1 and B7H2. [The FACS analysis was supported by immunohistochemistry, showing intense lymphocyte and APC (macrophages with dendritic morphology) ICOS staining in RA synovial tissue (ST). Overall, these results define elevated populations of memory T-lymphocytes expressing proinflammatory B7 molecules in RA SF that either stimulate T cells through ICOS (via ICOS ligands B7RP.1 and B7RP.2), or down-regulate RA ST T-lymphocytes through B7H1 and B7H2.] Therefore, in the same joint, there may exist positive and negative influences on the inflammatory response, and perhaps, the negative signals dominate as joint inflammation resolves. q 2007 International Society for Analytical Cytology

Objective Rheumatoid arthritis (RA) is a chronic inflammatory disease that is characterized by hypertrophy of the synovial tissue, leukocyte infiltration, angiogenesis, and ultimately joint destruction. Mucins (MUCs) are a family of heavily glycosylated proteins that protect epithelial membranes and are used as ligands for cell adhesion. MUC gene expression has been found to be altered in many cancers and inflammatory states. This study was undertaken to examine its expression in synovial tissue (ST) and role in arthritis. Methods We performed immunohistochemistry, Western blotting, and reverse transcriptase–polymerase chain reaction to determine expression patterns of MUC1, MUC2, MUC3, and MUC5AC in RA, osteoarthritic (OA), and normal human ST. Results MUC3 was expressed in synovial lining cells, macrophages, and fibroblasts. Significantly more RA (n = 12) and OA (n = 13) synovial lining cells expressed MUC3 than did normal synovial lining cells (n = 7) (22% and 24% versus 0.4%, respectively; P < 0.05). Additionally, macrophages in RA and OA ST expressed significantly more MUC3 than did macrophages in normal ST (50% and 51% versus 10%, respectively; P < 0.05). MUC5AC was expressed at low levels in synovial lining cells, macrophages, and endothelial cells in RA and OA ST, and was barely expressed in normal ST. MUC1 and MUC2 proteins were not detected in ST. Messenger RNA (mRNA) for MUC3 and MUC5AC was detected in ST, and mRNA for MUC3 was detected in cultured ST fibroblasts. Conclusion These data demonstrate up-regulated MUC expression by ST cells and suggest a novel role of MUC3 and MUC5AC in the pathogenesis of arthritis.

Few studies have explored the mechanistic basis for the apparent paradoxical effects of nitric oxide and its interrelated redox species (NO(X)) in cancer biology. Our aim was to determine the differential effects of the redox state and kinetics of nitrosative species on the key cancer processes of apoptosis. Therefore, a murine lung adenocarcinoma cell line was exposed to various NO(X) donor compounds differing in redox state and delivery kinetics. DNA strand breaks (DSBs) were measured by the alkaline single-cell gel electrophoresis assay (the COMET assay) and correlated with cell viability by the MTT and soft agar colony assays, while caspase enzymatic activity was measured using an in vitro fluorogenic caspase assay. Finally, cDNA microarrays defined apoptosis-related gene expression alterations resultant from these NO(X) donors. Exogenous NO(X) differentially influences DSBs, and apoptosis-related cell death and expression based on the redox state and kinetics of NO(X) delivery. In our murine lung adenocarcinoma model we have demonstrated differential effects of NO(X) based on the mode of delivery and redox state. These data suggest that the development of NO(X)-based cancer chemotherapy must consider the redox state and kinetics of delivery into their logical design.

Introduction Although caspase-8 is a well-established initiator of apoptosis and suppressor of necroptosis, recent evidence suggests that this enzyme maintains functions beyond its role in cell death. As cells of the innate immune system, and in particular macrophages, are now at the forefront of autoimmune disease pathogenesis, we examined the potential involvement of caspase-8 within this population. Methods CreLysMCasp8fl/fl mice were bred via a cross between Casp8fl/fl mice and CreLysM mice, and RIPK3−/−CreLysMCasp8fl/fl mice were generated to assess the contribution of receptor-interacting serine-threonine kinase (RIPK)3. Immunohistochemical and immunofluorescence analyses were used to examine renal damage. Flow cytometric analysis was employed to characterize splenocyte distribution and activation. CreLysMCasp8fl/fl mice were treated with either Toll-like receptor (TLR) agonists or oral antibiotics to assess their response to TLR activation or TLR agonist removal. Luminex-based assays and enzyme-linked immunosorbent assays were used to measure cytokine/chemokine and immunoglobulin levels in serum and cytokine levels in cell culture studies. In vitro cell culture was used to assess macrophage response to cell death stimuli, TLR activation, and M1/M2 polarization. Data were compared using the Mann–Whitney U test. Results Loss of caspase-8 expression in macrophages promotes onset of a mild systemic inflammatory disease, which is preventable by the deletion of RIPK3. In vitro cell culture studies reveal that caspase-8–deficient macrophages are prone to a caspase-independent death in response to death receptor ligation; yet, caspase-8–deficient macrophages are not predisposed to unchecked survival, as analysis of mixed bone marrow chimeric mice demonstrates that caspase-8 deficiency does not confer preferential expansion of myeloid populations. Loss of caspase-8 in macrophages dictates the response to TLR activation, as injection of TLR ligands upregulates expression of costimulatory CD86 on the Ly6ChighCD11b+F4/80+ splenic cells, and oral antibiotic treatment to remove microbiota prevents splenomegaly and lymphadenopathy in CreLysMCasp8fl/fl mice. Further, caspase-8–deficient macrophages are hyperresponsive to TLR activation and exhibit aberrant M1 macrophage polarization due to RIPK activity. Conclusions These data demonstrate that caspase-8 functions uniquely in macrophages by controlling the response to TLR activation and macrophage polarization in an RIPK-dependent manner. Electronic supplementary material The online version of this article (doi:10.1186/s13075-015-0794-z) contains supplementary material, which is available to authorized users.

Dendritic cells (DCs) are critical for immune homeostasis. To target DCs, we generated a mouse line with Flip deficiency in cells that express cre under the CD11c promoter (CD11c-Flip-KO). CD11c-Flip-KO mice spontaneously develop erosive, inflammatory arthritis, resembling rheumatoid arthritis, which is dramatically reduced when these mice are crossed with Rag−/− mice. The CD8α+ DC subset is significantly reduced, along with alterations in NK cells and macrophages. Autoreactive CD4+ T cells and autoantibodies specific for joint tissue are present, and arthritis severity correlates with the number of autoreactive CD4+ T cells and plasmablasts in the joint-draining lymph nodes. Reduced T regulatory cells (Tregs) inversely correlate with arthritis severity, and the transfer of Tregs ameliorates arthritis. This KO line identifies a model that will permit in depth interrogation of the pathogenesis of rheumatoid arthritis, including the role of CD8α+ DCs and other cells of the immune system., Dendritic cells are critical for initiation of immune responses and for induction of tolerance. Here the authors show that deletion of survival factor c-flip in CD11c-expressing cells subset perturbs CD8a+ dendritic cell, NK and macrophage pools, and leads to development of autoimmune arthritis.

Objective: Rheumatoid arthritis (RA) is characterized by hyperplasia of the synovial lining, inflammation, and destruction of cartilage and bone. Since there are only a few detectable cells undergoing apoptosis in the joint, it is possible that a defect in apoptosis may contribute to synovial hyperplasia. This study sought to identify and characterize the direct role of apoptotic regulators in a mouse model of inflammatory arthritis. Methods Using a serum transfer model, experimental arthritis was induced in mice lacking the proapoptotic Bcl-2 family genes Bak (Bak−/−), Bax (Bax−/−), or Bim (Bim−/−), as compared with wild-type (WT) control mice. Physical examination for edema of the ankles and histopathologic analysis of ankle sections were used to determine the severity of arthritis. The serum and ankles were examined for production of chemokines and cytokines using enzyme-linked immunosorbent or Luminex-based assays. Results Bim−/− mice displayed increased severity and prolongation of arthritis. In contrast, Bak−/− and Bax−/− mice showed no difference in the severity of arthritis as compared with WT mice. In addition, Bim−/− mice had elevated levels of proinflammatory chemokines and cytokines, decreased joint and serum production of antiinflammatory cytokines, fewer TUNEL-positive cells, and reduced levels of active caspase 3 as compared with WT mice. Conclusion These studies are the first to demonstrate a role for the proapoptotic Bcl-2 protein Bim in the effector phase of RA. The findings indicate that Bim potentially functions to repress the effector phase of arthritis by regulating the milieu of the joint and serum, and by inducing apoptosis.

One of the central functions of cyclin-dependent kinase inhibitors, such as p21, p27, or p16, is to prevent entry into the cell cycle. However, the question remains as to whether they have other functions in the cell. We previously demonstrated that overexpression of p21 in fibroblasts isolated from patients with rheumatoid arthritis decreases the production of pro-inflammatory molecules. Overexpression of p21 has been also shown to reduce the development of experimental arthritis in mice and rats. To explore the role of endogenous p21 in the development of arthritis, we induced arthritis in p21(-/-) mice using the K/BxN serum transfer model of arthritis. Mice deficient in p21 were more resistant to serum transfer-induced arthritis (K/BxN) than wild-type (wt) control mice. Fewer macrophages were detected in p21(-/-) as compared to wt joints following transfer of K/BxN serum. Chemotaxis assays of bone marrow-derived macrophages from p21(-/-) and wt mice revealed no difference in migration. However, there was a substantial decrease in inflammatory monocytes circulating in peripheral blood and in monocyte precursors in bone marrow of p21(-/-) mice as compared to wt mice. Adoptive transfer of wt bone marrow-derived macrophages into p21(-/-) mice restored the sensitivity to serum transfer-induced arthritis. These data suggest a novel role for p21 in regulating the development and/or differentiation of monocytic populations that are crucial for the induction of inflammatory arthritis.

Rheumatoid arthritis (RA) is characterized by profound mononuclear cell (MNC) recruitment into synovial tissue (ST), thought to be due in part to tumor necrosis factor alpha (TNFalpha), a therapeutic target for RA. Although chemokines may also be involved, the mechanisms remain unclear. We undertook this study to examine the participation of CXCL16, a novel chemokine, in recruitment of MNCs to RA ST in vivo and to determine the signal transduction pathways mediating this process.Using a human RA ST-SCID mouse chimera, immunohistochemistry, enzyme-linked immunosorbent assay, real-time reverse transcription-polymerase chain reaction, flow cytometry, and in vitro chemotaxis assays, we defined the expression and function of CXCL16 and its receptor, CXCR6, as well as the signal transduction pathways utilized by them for MNC homing in vitro and in vivo.CXCL16 was markedly elevated in RA synovial fluid (SF) samples, being as high as 145 ng/ml. Intense macrophage and lining cell staining for CXCL16 in RA ST correlated with increased CXCL16 messenger RNA levels in RA ST compared with those in osteoarthritis and normal ST. By fluorescence-activated cell sorting analysis, one-half of RA SF monocytes and one-third of memory lymphocytes expressed CXCR6. In vivo recruitment of human MNCs to RA ST implanted in SCID mice occurred in response to intragraft injection of human CXCL16, a response similar to that induced by TNFalpha. Lipofection of MNCs with antisense oligodeoxynucleotides for ERK-1/2 resulted in a 50% decline in recruitment to engrafted RA ST and a 5-fold decline in recruitment to regional lymph nodes. Interestingly, RA ST fibroblasts did not produce CXCL16 in response to TNFalpha in vitro, suggesting that CXCL16 protein may function in large part independently of TNFalpha.Taken together, these results point to a unique role for CXCL16 as a premier MNC recruiter in RA and suggest additional therapeutic possibilities, targeting CXCL16, its receptor, or its signaling pathways.

Objective. To identify disease-specific gene expression profiles in patients with rheumatoid arthritis (RA), using complementary DNA (cDNA) microarray analyses on lymphoblastoid B cell lines (LCLs) derived from RA-discordant monozygotic (MZ) twins. Methods. The cDNA was prepared from LCLs derived from the peripheral blood of 11 pairs of RAdiscordant MZ twins. The RA twin cDNA was labeled with cy5 fluorescent dye, and the cDNA of the healthy co-twin was labeled with cy3. To determine relative expression profiles, cDNA from each twin pair was combined and hybridized on 20,000-element microarray chips. Immunohistochemistry and real-time polymerase chain reaction were used to detect the expression of selected gene products in synovial tissue from patients with RA compared with patients with osteoarthritis and normal healthy controls. Results. In RA twin LCLs compared with healthy co-twin LCLs, 1,163 transcripts were significantly differentially expressed. Of these, 747 were overexpressed and 416 were underexpressed. Gene ontology analysis revealed many genes known to play a role in apoptosis, angiogenesis, proteolysis, and signaling. The 3 most significantly overexpressed genes were laeverin (a novel enzyme with sequence homology to CD13), 11hydroxysteroid dehydrogenase type 2 (a steroid pathway enzyme), and cysteine-rich, angiogenic inducer 61 (a known angiogenic factor). The products of these genes, heretofore uncharacterized in RA, were all abundantly expressed in RA synovial tissues. Conclusion. Microarray cDNA analysis of peripheral blood–derived LCLs from well-controlled patient populations is a useful tool to detect RA-relevant genes and could help in identifying novel therapeutic targets.

Purpose To present the clinical data of 35 patients with T3 unresectable hepatocellular carcinoma (HCC) that were treated with 90Y with the specific intent of downstaging to resection, radiofrequency ablation (RFA) candidate, United Network for Organ Sharing (UNOS) stage T2 or liver transplantation. Materials and Methods One hundred fifty patients with unresectable HCC were treated with 90Y microspheres. Of these, 35 patients were UNOS stage T3 at the time of treatment. Patients were followed for clinical toxicities, alterations in model for end-stage-liver disease (MELD) score, tumor response, downstaging to RFA, resection, transplantation, and survival. Results Nineteen of 34 patients (56%) were successfully downstaged from T3 to T2 following treatment. 11 of 34 (32%) patients treated were downstaged to target lesions measuring 3.0 cm or less. Twenty-three of 35 (66%) were downstaged to either T2 status, lesion

Recent pilot research suggested that the H+/K+-ATPase (proton pump), which is the target of pharmacotherapy for laryngopharyngeal reflux disease (LPRD), is associated with human laryngeal submucosal glands. The hypothesis of this study is that proton pump is expressed in the human larynx, and is not solely associated with the parietal cells of the stomach.Fifteen surgical larynx subjects (27 pathologic specimens) containing seromucinous glands from banked tissue were retrospectively obtained after approval from Human Subjects Committee. Banked human stomach tissue was also obtained for comparative positive and negative controls. Sections were immunostained with two monoclonal antibodies selectively reactive with alpha or beta subunits of the H+/K+-ATPase (proton) pump.In the human larynx, positive staining was seen in 14 of 15 subjects. Twenty-six specimens showed consistent staining in the seromucinous cells and ducts for the alpha subunit, and 23 specimens for the beta subunit. Stomach parietal cells exhibited strongly positive staining for both the alpha and beta subunits of the proton pump. There was no staining in stomach cells that were not morphologically consistent with the parietal cell.The H+/K+-ATPase (proton) pump is present in seromucinous cells and ducts in the human larynx, with some variable expression noted. Proton pump involvement in human laryngeal seromucinous glands may explain heightened laryngeal sensitivity in those patients with chronic laryngitis believed to have LPRD. Also, proton pump inhibitor pharmacotherapy may have a site of action in the human larynx, explaining some of the controversies attributable to LPRD.B-3.

Objective/Hypothesis: The role of infectious agents in the etiology of chronic rhinosinusitis with nasal polyposis (CRSwNP) remains unclear. Recent studies have provided indirect evidence of exposure to staphylococcal exotoxins in the blood and polyp tissue of patients with CRSwNP. These exotoxins have the capacity to act as superantigens, bypassing normal antigen processing and directly stimulating a massive inflammatory response. The objective of the study was to analyze mucus and polyp tissue samples from patients with CRSwNP for the presence of staphylococcal exotoxins. Study Design: Prospective study. Methods: Tissue and mucus samples were obtained from 42 patients undergoing endoscopic sinus surgery for chronic rhinosinusitis and 11 normal control patients. Twenty-nine of 42 patients had chronic rhinosinusitis with bilateral nasal polyposis, 2 had antrochoanal polyps, and 11 had chronic rhinosinusitis without nasal polyps. Eleven patients without chronic rhinosinusitis or polyps served as normal control patients. Specimens were analyzed for the presence of five staphylococcal exotoxins (SEA, SEB, SEC, SED, and toxic shock syndrome toxin type 1 [TSST-1]) using enzyme-linked immunosorbent assay (ELISA). Histological analysis of specimens and mean eosinophil counts were correlated with the presence of toxin. Results: At least one toxin was detected in 14 of 29 patients with bilateral nasal polyposis. Nine of the 14 patients also had positive findings for additional toxins. The dominant histological pattern in the CRSwNP patient group was polypoid mucosa with edema, which was found in both ELISA-positive and ELISA-negative patients. Mean eosinophil counts tended to be higher in ELISA-positive patients with polyps compared with patients without toxin detection. No toxin was detected in the 11 specimens taken from normal control patients. Only 1 of the 13 patients with CRS without polyps had positive ELISA results for toxin. Conclusion: The current study demonstrates the presence of superantigen toxins in 14 of 29 patients with CRSwNP, with SEB and TSST-1 being the most common. Further studies are necessary to correlate the presence of toxin with the pathological changes present in polyp tissue.

Following intranasal inoculation, Vibrio cholerae KFV101 (Δ ctxAB Δ hapA Δ hlyA Δ rtxA ) colonizes and stimulates tumor necrosis factor alpha and interleukin 1β (IL-1β) in mice, similar to what occurs with isogenic strain P4 (Δ ctxAB ), but is less virulent and stimulates reduced levels of IL-6, demonstrating a role for accessory toxins in pathogenesis. Morbidity is enhanced in C3H/HeJ mice, indicating that Toll-like receptor 4 is important for infection containment.

Objective CC chemokines and their receptors play a fundamental role in trafficking and activation of leukocytes at sites of inflammation, contributing to joint damage in rheumatoid arthritis. Met-RANTES, an amino-terminal–modified methionylated form of RANTES (CCL5), antagonizes the binding of the chemokines RANTES and macrophage inflammatory protein 1α (MIP-1α; CCL3) to their receptors CCR1 and CCR5, respectively. The aim of this study was to investigate whether Met-RANTES could ameliorate adjuvant-induced arthritis (AIA) in the rat. Methods Using immunohistochemistry, enzyme-linked immunosorbent assay, real-time reverse transcription–polymerase chain reaction, Western blot analysis, adoptive transfer, and chemotaxis, we defined joint inflammation, bony destruction, neutrophil and macrophage migration, Met-RANTES binding affinity to rat receptors, proinflammatory cytokine and bone marker levels, CCR1 and CCR5 expression and activation, and macrophage homing into joints with AIA. Results Administration of Met-RANTES as a preventative reduced the severity of joint inflammation. Administration of Met-RANTES to ankles with AIA showed decreases in inflammation, radiographic soft tissue swelling, and bone erosion. Met-RANTES significantly reduced the number of neutrophils and macrophages at the peak of arthritis compared with saline-injected controls. Competitive chemotaxis in peripheral blood mononuclear cells demonstrated that Met-RANTES inhibited MIP-1α and MIP-1β at 50% inhibition concentrations of 5 nM and 2 nM, respectively. Furthermore, levels of tumor necrosis factor α, interleukin-1β, macrophage colony-stimulating factor, and RANKL were decreased in joints with AIA in the Met-RANTES group compared with the control group. Interestingly, the expression and activation of CCR1 and CCR5 in the joint were down-regulated in the Met-RANTES group compared with the control group. Functionally, Met-RANTES administration decreased adoptively transferred peritoneal macrophage homing into the joint. Conclusion The data suggest that the targeting of Th1-associated chemokine receptors reduce joint inflammation, bone destruction, and cell recruitment into joints with AIA.

Objective. Chemokine receptors mediate leukocyte migration into inflamed rheumatoid arthritis (RA) synovial tissue (ST). Knowledge of their distribution is crucial for understanding the evolution of the inflammatory process. In this study, we used rat adjuvantinduced arthritis (AIA), a model for RA, to define the temporospatial expression of chemokine receptors. Methods. ST from rats with AIA was immunostained, the percentage of cells expressing each receptor was determined, and findings were correlated with levels of inflammation. Chemokine receptor expression was evaluated on rat macrophages in vitro. Results. CCR1, a receptor for macrophage inflammatory protein 1 (MIP-1)/CCL3 and RANTES/ CCL5, exhibited high constitutive expression on macrophages in AIA. CCR5, binding MIP-1/CCL3 and RANTES/CCL5, was up-regulated on ST macrophages during the course of AIA, correlating with macrophage expression of CCR2, a receptor for monocyte chemoattractant protein 1/CCL2. Endothelial cell (EC) CCR2 was down-regulated as arthritis progressed, inversely correlating with inflammation. CCR3, another RANTES/CCL5 receptor, was constitutively high on macrophages in vivo and in vitro, with down-regulation during AIA. CXCR4, a receptor for stromal cell–derived factor 1/CXCL12), was prominently up-regulated on ECs, preceding the peak of inflammation. Conclusion. These findings show that 1) constitutive expression of CCR1 on macrophages remains high during AIA; 2) CCR2 and CCR3 may play a role in initial recruitment of leukocytes to ST in AIA; 3) macrophage expression of CCR2 and CCR5 may be important for sustaining inflammatory changes; and 4) EC CXCR4 may be a harbinger of inflammatory changes. Our results may help guide chemokine receptor blockade–targeting treatment strategies in inflammatory arthritis.

The Construction Institute's Committee on Social and Environmental Concerns in Construction has examined 24 items of social and environmental concerns in construction that will have to be considered by those who are going to be involved in the reconstruction of the World Trade Center. These topics include the economic aspects of the reconstruction, the planning and design, the environmental issues, the transportation challenges, the contractual problems, the memorial, and the security requirements. During the drafting of this report, the Committee received assistance and guidance from the engineering staff of the Port Authority of New York and New Jersey. Some Committee members visited the World Trade Center site at Ground Zero during the preparation of the report. Also consulted by the Committee were some well-known, highly qualified engineers on various aspects of the report. These contributions from nonmembers of the Committee made the report richer. It is the intent of this report to serve as a starting point for planning and redevelopment.

Glutathione s-transferase pi (GSTpi) is an enzyme that provides cellular protection against redox-mediated damage by free radicals, which have been implicated in carcinogenesis.Forty-three consecutive specimens from 19 patients were reviewed to identify samples of squamous mucosa, Barrett's metaplasia, adenocarcinoma, and peritumoral inflammation. Serial sections were stained with an anti-GSTpi polyclonal antibody, and GSTpi expression was quantified for each histologic group.GSTpi expression was diminished in peritumoral mononuclear inflammatory cells (p.001) compared with squamous epithelium, Barrett's metaplasia, or adenocarcinoma. Barrett's metaplasia exhibited decreased GSTpi expression compared with squamous mucosa (p =.045). GSTpi expression by50% of adenocarcinoma cells was associated with an increased risk (2.25x) of disease at last follow-up.GSTpi is prominently expressed in esophageal squamous mucosa and adenocarcinoma. Mononuclear cells may be susceptible to oxidative damage secondary to weak GSTpi production. GSTpi may protect the tumor cells themselves from the cytotoxic effects of free radicals. The biochemical role of GSTpi expression in malignant transformation deserves further investigation.

The contribution of accessory toxins to the acute inflammatory response to Vibrio cholerae was assessed in a murine pulmonary model. Intranasal administration of an El Tor O1 V. cholerae strain deleted of cholera toxin genes (ctxAB) caused diffuse pneumonia characterized by infiltration of PMNs, tissue damage, and hemorrhage. By contrast, the ctxAB mutant with an additional deletion in the actin-cross-linking repeats-in-toxin (RTX) toxin gene (rtxA) caused a less severe pathology and decreased serum levels of proinflammatory molecules interleukin (IL)-6 and murine macrophage inflammatory protein (MIP)-2. These data suggest that the RTX toxin contributes to the severity of acute inflammatory responses. Deletions within the genes for either hemagglutinin/protease (hapA) or hemolysin (hlyA) did not significantly affect virulence in this model. Compound deletion of ctxAB, hlyA, hapA, and rtxA created strain KFV101, which colonized the lung but induced pulmonary disease with limited inflammation and significantly reduced serum titers of IL-6 and MIP-2. 100% of mice inoculated with KFV101 survive, compared with 20% of mice inoculated with the ctxAB mutant. Thus, the reduced virulence of KFV101 makes it a prototype for multi-toxin deleted vaccine strains that could be used for protection against V. cholerae without the adverse effects of the accessory cholera toxins.

Objectifs La tractographie est une technique d’IRM en tenseur de diffusion (DT) qui permet de representer des structures lineaires in vivo par des traces (tracts). L’objectif etait d’evaluer la validite anatomique de la tractographie des bandelettes neurovasculaires (BNV) peri-prostatiques. Methodes Dix patients eligibles a une prostatectomie totale avaient une sequence DT in vivo au cours de leur IRM-3-Teslas preoperatoire. La piece de prostatectomie etait preparee pour une acquisition ex vivo de 12 h en IRM-7-Teslas (sequences T2, DT). La tractographie etait generee dans le logiciel « TMII-Tracer » sur la plateforme Matlab®. Des coupes prostatiques tous les 5 mm etaient traitees par Hematein-Eosine-Safran (HES) et Proteine S100 (PS100). Le nombre et la position des fibres nerveuses autour de la prostate etaient identifies dans chaque secteur en DT-MRI, puis compares avec l’immunohistochimie. Resultats Les donnees ont pu etre analysees pour 8 patients qui avaient eu une preservation incomplete ou une non-preservation. La tractographie 3 T in vivo generait des tracts non specifiques et ne permettait pas de dissocier les fibres nerveuses. La tractographie 7 T ex vivo generait des tracts des BNV postero-laterales avec un taux de correlation histologique de 58 %. Dans les secteurs lateraux et anterieurs, la correlation entre tractographie et histologie etait inferieure a 30 %. Apres identification histologique prealable des fibres nerveuses, il etait possible de cibler une region d’interet sur la carte 7 T de fraction d’anisotropie. Des tracts specifiques des fibres nerveuses de plus de 200 μm de diametre etaient ainsi obtenus. Conclusion La tractographie des tissus peri-prostatiques est faisable mais sa pertinence anatomique est limitee in vivo. Ex vivo, il est possible d’obtenir des traces specifiques des fibres nerveuses. La duree d’acquisition des images, le haut champ magnetique et le recours a une identification histologique prealable sont des limites pour une application clinique de la tractographie dans l’imagerie des BNV.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease characterized by synovial pannus formation, leukocyte infiltration, and angiogenesis. Adenoviral production of interleukin-13 (IL-13) reduces levels of proinflammatory mediators in an explant model of RA synovial tissue in vitro. To assess this approach in an animal model of arthritis, we compared intra-articular injections of an adenovirus producing rat IL-13 (AxCArIL-13), a control virus, and rat ankles receiving phosphate-buffered saline (PBS) in rat adjuvant-induced arthritis (AIA). We demonstrate that IL-13 levels are normally low in ankles throughout the course of rat AIA. We show that administration of AxCArIL-13 before arthritis onset significantly reduces ankle circumference, paw volume, bony destruction, the number of polymorphonuclear cells (PMNs), the quantity of blood vessels, and levels of monocyte chemoattractant protein (MCP)-1 in ankles. When administered as a treatment to inflamed ankles, AxCArIL-13 decreases articular index scores, paw volumes, bony destruction, vascularization, tumor necrosis factor-alpha (TNF-alpha) levels, and the quantity of monocytes, lymphocytes, and PMNs. Thus, increasing IL-13 levels significantly ameliorates the course of rat AIA, suggesting that similar strategies for the treatment of human RA are worthy of further study.

Purpose: Apoptosis index (AI), Bcl-2, and Bax have shown prognostic significance in head and neck squamous cell carcinoma (HNSCCa). Other areas of research have implicated nitric oxide (NO) or its various intermediate species in both proapoptotic and antiapoptotic processes. We have previously shown that NO-generating enzymes are significantly increased during the stepwise progression to HNSCCa. The aim of this study was to explore the interrelationship of NO and a known consequence of NO-related oxidative stress, apoptosis, during this step-wise process. Materials and Methods: Formalin fixed-paraffin embedded tissue samples of 10 normal oral mucosa, 15 reactive/dysplastic lesions, and 17 HNSCCa lesions studied previously were subjected to the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP labeling (TUNEL) assay as well as immunohistochemical staining against Bcl-2, Bax, and p53. Patient charts were reviewed and clinical data were compared. The study pathologist (G.K.H) reviewed these slides blinded to patient identifiers or clinical data. The number of immunopositive cell nuclei or staining intensity was graded, noting the pattern of immunostaining. These staining characteristics were compared with the results of immunostaining previously obtained for endothelial constitutive NO synthase (ecNOS) and nitrotyrosine. Results: Compared with normal oral mucosa, the AI, Bcl-2, Bax, Bcl-2/Bax intensity and frequency ratios, and mutant p53 intensity significantly changed in reactive/dysplastic and HNSCCa lesions (P

Objectives: Amended reports (AmRs) need to follow patients to treating physicians, to avoid erroneous management based on the original diagnosis. This study was undertaken to determine if AmRs followed the patient appropriately. Methods: AmRs with diagnostic changes and discrepancies between ordering and treating physicians were tracked. Chart reviews, electronic medical report (EMR) reviews, and interviews were conducted to establish receipt of the AmR by the correct physician. Results: Seven of 60 AmRs had discrepancies between the ordering and treating physicians, all with malignant diagnoses. The AmR was present in the treating physician’s chart in only one case. Ordering physicians indicated that AmRs were not forwarded to treating physicians when corrected results arrived after patient referral, under the assumption that the new physician was automatically forwarded pathology updates. No harm was documented in any of our cases. In one case with a significant amendment, the correct information was entered in the patient chart based on a tumor board discussion. A review of two electronic health record systems uncovered significant shortcomings in each delivery system. Conclusions: AmRs fail to follow the patient’s chain of referrals to the correct care provider, and EMR systems lack the functionality to address this failure and alert clinical teams of amendments.

Caspase-8, an executioner enzyme in the death receptor pathway, was shown to initiate apoptosis and suppress necroptosis. In this study, we identify a novel, cell death–independent role for caspase-8 in dendritic cells (DCs): DC-specific expression of caspase-8 prevents the onset of systemic autoimmunity. Failure to express caspase-8 has no effect on the lifespan of DCs but instead leads to an enhanced intrinsic activation and, subsequently, more mature and autoreactive lymphocytes. Uncontrolled TLR activation in a RIPK1-dependent manner is responsible for the enhanced functionality of caspase-8–deficient DCs, because deletion of the TLR-signaling mediator, MyD88, ameliorates systemic autoimmunity induced by caspase-8 deficiency. Taken together, these data demonstrate that caspase-8 functions in a cell type–specific manner and acts uniquely in DCs to maintain tolerance.

Polymorphisms in the transcription factor interferon regulatory factor 5 (IRF5) are associated with an increased risk of developing rheumatoid arthritis (RA). This study was undertaken to determine the role of IRF5 in a mouse model of arthritis development.K/BxN serum-transfer arthritis was induced in mice deficient in IRF5, or lacking IRF5 only in myeloid cells, and arthritis severity was evaluated. K/BxN arthritis was also induced in mice deficient in TRIF, Toll-like receptor 2 (TLR2), TLR3, TLR4, and TLR7 to determine the pathways through which IRF5 might promote arthritis. In vitro studies were performed to determine the role of IRF5 in interleukin-1 (IL-1) receptor and TLR signaling.Arthritis severity was reduced in IRF5-deficient, TRIF-deficient, TLR3-deficient, and TLR7-deficient mice. The expression of multiple genes regulating neutrophil recruitment or function and bioactive IL-1β formation was reduced in the joints during active arthritis in IRF5-deficient mice. In vitro studies showed that TLR7 and the TRIF-dependent TLR3 pathway induce proinflammatory cytokine production in disease-relevant cell types in an IRF5-dependent manner.Our findings indicate that IRF5 contributes to disease pathogenesis in inflammatory arthritis. This is likely due at least in part to the role of IRF5 in mediating proinflammatory cytokine production downstream of TLR7 and TLR3. Since TLR7 and TLR3 are both RNA-sensing TLRs, this suggests that endogenous RNA ligands present in the inflamed joint promote arthritis development. These findings may be relevant to human RA, since RNA capable of activating TLR7 and TLR3 is present in synovial fluid and TLR7 and TLR3 are up-regulated in the joints of RA patients.

The expression of endothelial constitutive nitric oxide synthase (NOS3) by squamous dysplasia and carcinomas of the head and neck has previously been described. We sought to compare NOS3 expression in squamous mucosa, glandular metaplasia, and adenocarcinoma of the esophagus.Forty paraffin-embedded specimens from 20 patients with adenocarcinoma were stained with anti-NOS3 monoclonal antibody. The percentage of cells stained and the intensity of staining were determined for squamous epithelium, metaplasia, and adenocarcinoma. Staining characteristics were statistically analyzed according to clinical variables.NOS3 expression was significantly higher in adenocarcinoma and squamous epithelium compared with glandular metaplasia. Among the carcinomas, larger tumor size (T3/4), nodal positivity, and advanced TNM stage (III/IV) significantly correlated with increased NOS3 expression.NOS3 is expressed in reflux-induced lesions of the esophagus. Glandular metaplasia shows basal levels of NOS3 that significantly increase with malignant transformation and tumor progression. The role of free radicals in carcinogenesis is being actively studied.

Objective The chronic inflammation and progressive joint destruction observed in rheumatoid arthritis (RA) are mediated in part by macrophages. A paucity of apoptosis has been observed in RA synovial tissues, yet the mechanism remains unknown. The present study sought to characterize the expression of Fas, Fas ligand (FasL), and Fas-associated death domain–like interleukin-1β–converting enzyme–inhibitory protein (FLIP), and to quantify the apoptosis induced by agonistic anti-Fas antibody, using mononuclear cells (MNC) isolated from the peripheral blood (PB) and synovial fluid (SF) of RA patients. Methods The expression of Fas, FasL, and FLIP and apoptosis induced by agonistic anti-Fas antibody in MNC from the PB and SF of RA patients were determined by flow cytometry. Immunohistochemistry employing a monospecific anti-FLIP antibody was performed on RA and osteoarthritis (OA) synovial tissue. Results CD14-positive monocyte/macrophages from normal and RA PB and from RA SF expressed equivalent levels of Fas and FasL. Furthermore, unlike the CD14-positive PB monocytes, RA SF monocyte/macrophages were resistant to the addition of agonistic anti-Fas antibody. In contrast, both CD14-positive PB and SF monocyte/macrophages were sensitive to apoptosis mediated by a phosphatidylinositol 3-kinase inhibitor. Intracellular staining of the caspase 8 inhibitor, FLIP, in CD14-positive SF monocyte/macrophages revealed a significant up-regulation of FLIP compared with normal and RA PB monocytes. Immunohistochemical analysis of synovial tissue from RA and OA patients revealed increased FLIP expression in the RA synovial lining compared with the OA synovial lining. Furthermore, FLIP expression was observed in the CD68-positive population in the RA synovial lining. Forced reduction of FLIP by a chemical inhibitor resulted in RA SF macrophage apoptosis that was enhanced by agonistic anti-Fas antibody, indicating that FLIP is necessary for SF macrophage survival. Conclusion These data suggest that up-regulation of FLIP in RA macrophages may account for their persistence in the disease. Thus, the targeted suppression of FLIP may be a potential therapeutic strategy for the amelioration of RA.

Objective. To examine the relationship between apoptosis and the expression of antiapoptotic proteins in the pathogenesis of experimental inflammatory arthritis. Methods. Clinical and histologic assessment of adjuvant-induced arthritis (AIA) was performed over a 42-day period. The induction of apoptosis was measured by TUNEL analysis, and the antiapoptotic proteins, Bcl-2 and FLIP, were examined by immunohistochemistry with the use of monospecific antibodies. The percentage of Bcl-2- and FLIP-positive cells was correlated with histologic markers of AIA. Results. Arthritis developed by day 14 following adjuvant injection. Few TUNEL-positive cells were observed between days 0 and 21, indicating that apoptosis did not occur at these time points. An increase in the number of TUNEL-positive cells was observed at day 28, particularly outside sites of cartilage or bone erosion, which dramatically declined by day 35. Immunohistochemical analyses of Bcl-2 and FLIP revealed that the synovium was positive for Bcl-2 and FLIP on day 0. On day 14, Bcl-2 was present at the sites of early erosions and correlated with the erosion and inflammation scores. FLIP was also highly expressed at sites of erosion and was localized to the pannus starting on day 21. Although TUNEL positivity peaked at day 28, a time point in which Bcl-2 and FLIP were present, the areas that displayed intense positivity for expression of Bcl-2 and FLIP were TUNEL negative. In addition, the number of neutrophils in the synovial lining and pannus significantly decreased from day 28 to day 35, suggesting that the cells undergoing apoptosis were neutrophils. Furthermore, at day 42 when TUNEL-positive cells were absent, Bcl-2 expression was diminished, while FLIP remained highly expressed in the pannus. Conclusion. The overall percentage of TUNEL-positive cells in the ankle was

Objectives/Hypothesis Oxidative/reductive (redox) DNA damage from radical species such as nitric oxide (NO·) are increasingly being implicated in the development of cancer. Moreover, redox-protective cellular mechanisms, such as glutathione S-transferase, may determine cellular susceptibility to this redox-mediated damage. Methods Formalin-fixed, paraffin-embedded tissue samples of 11 normal oral mucosa, 15 reactive/dysplastic lesions, and 131 head and neck squamous cell carcinomas (HNSCCs) were immunohistochemically stained using a polyclonal antibody against glutathione S-transferase π (GST-π). Slides were reviewed in a blinded fashion by the study pathologist (g.k.h.) and intensity was graded, noting the pattern of immunostaining. These staining characteristics were compared with those obtained using monoclonal antibodies against endothelial constitutive nitric oxide synthase (ecNOS) and nitrotyrosine, a marker of NO·'s pathological nitrosylation of proteins on serial sections of the same tissue. Patient charts were reviewed and clinical data collected. Results The expression of GST-π was significantly increased in reactive/dysplastic and HNSCC samples compared with normal squamous mucosa (P < .001 for both). Furthermore, among the carcinomas the expression of GST-π was significantly increased in higher-grade lesions (P < .02). The expression of GST-π correlated highly with the expression of ecNOS and nitrotyrosine (P < .0001 for both). Conclusions These findings demonstrate that GST-π is upregulated in conjunction with the NO·-generating ecNOS isoform and implicate GST-π in protecting HNSCC from the cytotoxic effects of high concentrations of NO· found in the tumor bed.

OBJECTIVE To examine cytokine and chemokine production during the evolution of rat adjuvant-induced arthritis (AIA), a model of rheumatoid arthritis. METHODS Clinical and laboratory assessment of the course of AIA was performed over a 47-day period. Levels of the cytokines tumor necrosis factor a (TNFalpha), interleukin-1beta (IL-1beta), and IL-6, as well as levels of the chemokines macrophage inflammatory protein 1alpha (MIP-1alpha) and JE, the murine homolog of monocyte chemoattractant protein 1, were determined by enzyme-linked immunosorbent assay in the sera and joints of AIA and control rats. Synovia from AIA rats were (immuno)histochemically analyzed. Results of cytokine and chemokine measurements were correlated with clinical and laboratory markers of inflammation and histology. RESULTS Early (before day 14 post adjuvant injection) and later phases of AIA could be distinguished. Cytokine and chemokine production was increased in AIA versus control rats. The production of TNFalpha, IL-1beta, MIP-1alpha, and, as determined earlier, epithelial neutrophil-activating peptide 78-like protein was abundant prior to and during the course of AIA, while that of IL-6 and JE was elevated in the late phase of AIA. Cytokine and chemokine levels were correlated with the clinical symptoms of arthritis and blood neutrophil counts. Joint levels of IL-1beta showed correlation with synovial lining proliferation and neutrophil ingress into AIA synovium. CONCLUSION Cytokines and chemokines are involved in the clinical, laboratory, and histologic changes underlying AIA. The production of these mediators may be temporally and spatially regulated. These findings may be important for the optimal timing of cytokine and chemokine targeting.

The regulation of proliferation and cell death is vital for homeostasis, but the mechanism that coordinately balances these events in rheumatoid arthritis (RA) remains largely unknown. In RA, the synovial lining thickens in part through increased proliferation and/or decreased synovial fibroblast cell death. Here we demonstrate that the anti-apoptotic protein, Bcl-2, is highly expressed in RA compared with osteoarthritis synovial tissues, particularly in the CD68-negative, fibroblast-like synoviocyte population. To determine the importance of endogenous Bcl-2, an adenoviral vector expressing a hammerhead ribozyme to Bcl-2 (Ad-Rbz-Bcl-2) mRNA was employed. Ad-Rbz-Bcl-2 infection resulted in reduced Bcl-2 expression and cell viability in synovial fibroblasts isolated from RA and osteoarthritis synovial tissues. In addition, Ad-Rbz-Bcl-2-induced mitochondrial permeability transition, cytochrome c release, activation of caspases 9 and 3, and DNA fragmentation. The general caspase inhibitor zVAD.fmk blocked caspase activation, poly(ADP-ribose) polymerase cleavage, and DNA fragmentation, but not loss of transmembrane potential or viability, indicating that cell death was independent of caspase activation. Ectopically expressed Bcl-xL inhibited Ad-Rbz-Bcl-2-induced mitochondrial permeability transition and apoptosis in Ad-Rbz-Bcl-2-transduced cells. Thus, forced down-regulation of Bcl-2 does not induce a compensatory mechanism to prevent loss of mitochondrial integrity and cell death in human fibroblasts.

Background: The prototypic acute phase reactant, C-reactive protein (CRP), is a serum soluble, cyclic pentameric protein, the concentration of which increases markedly within hours of any tissue-damaging, inflammatory event. However, upon dissociation of its pentameric quaternary structure, CRP subunits undergo a spontaneous and irreversible conformational change. The resulting molecule, termed modified CRP or mCRP, has reduced aqueous solubility and a propensity to aggregate into a matrix-like lattice structure. Methods: Using monoclonal antibodies, normal human tissues were immunohistochemically screened for the presence of CRP as well as mCRP antigens. Results: Significant levels of mCRP were detected in the walls of blood vessels associated with normal human tissues. These data indicate that mCRP is a naturally occurring form of CRP and that it is a tissue-based rather than serum-based molecule. Significance: This report describes the localization of a stable form of CRP, mCRP, in blood vessels associated with normal human tissues.

Background Nitric oxide (NO·) and its metabolic byproducts are implicated in carcinogenesis. We examined a marker of NO·-species' pathologic protein nitrosylation, nitrotyrosine, during head and neck squamous cell carcinoma (HNSCCa) development. Materials and Methods Paraffin-embedded tissue samples of normal oral mucosa, squamous hyperplasia/dysplasia, and HNSCCa were immunohistochemically analyzed for staining intensity using an antinitrotyrosine monoclonal antibody, and correlated with retrospective clinical data. Results Significantly higher staining was noted in reactive, dysplastic and HNSCCa samples compared with samples of normal mucosa. Additionally, significant differences in staining were found between various primary sites of the upper aerodigestive tract. Lastly, individual inflammatory cells also stained intensely. Conclusions Increasing amounts of nitrotyrosine staining were found in reactive/dysplastic and HNSCCa lesions compared to normal squamous mucosa. Inflammatory cell staining implicates NO· as a possible mediator of immunosuppression. Given these findings, the role of NO· in mutations leading to and the immunosuppression found in HNSCCa warrants further investigation. © 2000 John Wiley & Sons, Inc. Head Neck 22: 64–70, 2000.