Semen searching when sperm is absent.

Sexual assault cases have varying factors that may mask semen findings when analysing evidence at the forensic laboratory. Semenogelin (Sg) is a potential marker for the identification of semen even at azoospermy or when few sperm cells are found. The current study examined Sg in normospermic and azoospermic donors as an internal evaluation of sensitivity, specificity and interference. The impact of a historical review of 53 judicial sexual assault cases over a five-year period was also analysed. The use of varying tests was of importance to prioritize certain samples within cases. Semen findings by Sg were then compared to prostate-specific antigen (PSA), phosphatase enzyme (AP) and Y-chromosome presence, the latter being used in an attempt to link semen fluid identification with obtaining a male DNA profile. Test findings were the highest ever registered for Sg (1:400,000), PSA (1:800,000), AP (1:25,000) and sperm cytology (SC) (1:50,000). Our results demonstrated the usefulness of using the Sg marker to avoid a false semen-negative result (6% cases), particularly in cases where sperm was absent or scarce (11% spermatozoa positive cases). Results were expressed in categories according to the set: Sg-PSA-AP. Thus, categories I (full positive, 46%), VI (full negative, 27%) and III (Sg/PSA positive; 11%) were the most frequent and Y-chromosome was obtained in 59%, 12% and 12% ratios, respectively. In conclusion, Sg was recommended for the workflow procedure of semen investigation when sperm absence is expected either from azoospermic/oligospermic or normospermic semen, especially before/after ejaculation.

Non-tuberculous mycobacteria in children: muddying the waters of tuberculosis diagnosis.

Non-tuberculous mycobacteria (NTM) are a large family of acid-fast bacteria, widespread in the environment. In children, NTM cause lymphadenitis, skin and soft tissue infections, and occasionally also lung disease and disseminated infections. These manifestations can be indistinguishable from tuberculosis on the basis of clinical and radiological findings and tuberculin skin testing. A diagnostic and therapeutic problem for respiratory physicians and other clinicians is therefore evident, particularly in settings where childhood tuberculosis is common, and bacteriological confirmation of any mycobacterial disease is difficult because of low availability of laboratory services in low-resource settings and the inherent paucibacillary nature of mycobacterial disease in childhood. The epidemiology of NTM varies by world region, and attempts to understand the burden of NTM disease and to identify risk factors in the paediatric population are hampered by inadequate mandatory NTM reporting and the overlap of clinical presentation with tuberculosis. The immune response to both NTM and Mycobacterium tuberculosis is based on cellular immunity and relies on the type-1 cytokine pathway. The disruption of this immune response by genetic or acquired mechanisms, such as mendelian susceptibility to mycobacterial disease or HIV, might result in predisposition to mycobacterial infections. Published diagnostic and management guidelines do not provide specific advice for diagnosis of NTM in children, from whom the quantity and quality of diagnostic samples are often suboptimum. Treatment of NTM infections is very different from the treatment of tuberculosis, depends on the strain and anatomical site of infection, and often involves antibiotic combinations, surgery, or both. In this Review, we summarise the epidemiological and clinical features of NTM infection in children, with a specific focus on the implications for public health in settings with a high endemic burden of childhood tuberculosis.

Clinicopathologic correlations of renal microthrombosis and inflammatory markers in proliferative lupus nephritis.

INTRODUCTION: Microthrombosis is often observed in lupus nephritis (LN) lesions, but its clinical significance is unknown. We evaluated the clinicopathologic correlations of renal microthrombosis and inflammatory markers in LN. METHODS: Kidney biopsies from 58 patients with systemic lupus erythematosus (SLE) proliferative nephritis were analyzed with immunohistochemistry (IHC) for intravascular platelet aggregates (CD61), macrophagic infiltration (CD68), and activated complement deposition (C4d). Clinical data at the time of kidney biopsy and follow-up were analyzed with regard to pathologic IHC data. RESULTS: Microthrombosis was present in 52% of the tissues. It was significantly more prevalent in patients with antiphospholipid antibodies (aPLs) (62% versus 42%). The presence of microthrombosis significantly correlated with higher macrophagic infiltration. Macrophagic infiltration but not microthrombosis was significantly correlated with C4d deposition. Only macrophagic infiltration showed a correlation with SLE and renal activity (proteinuria and active sediment), whereas neither the presence of CD61+ microthrombi nor the extent of C4d deposition correlated with LN severity or outcome. CONCLUSIONS: Microthrombosis is associated with higher macrophagic infiltration in LN but does not seem to increase independently the severity of renal damage. Macrophagic infiltration was the best marker of SLE and renal activity in this LN series.

Tuberculosis in pediatric solid organ and hematopoietic stem cell transplant recipients.

The burden of tuberculosis after pediatric solid organ transplant or hematopoietic stem cell transplantation has not been well characterized. We report 7 pediatric cases with disseminated (4/7) or pulmonary (3/7) tuberculosis after solid organ transplant (n=6) or hematopoietic stem cell transplantation (n=1) during 26 years. The outcome was favorable in 6 patients. Isoniazid-induced hepatitis and rifampin interactions were common.

CXCL12 gene expression is upregulated by hypoxia and growth arrest but not by inflammatory cytokines in rheumatoid synovial fibroblasts.

OBJECTIVES: CXCL12 is a constitutively expressed chemokine with important homeostatic functions. Increased CXCL12 expression has been observed in several inflammatory conditions, including rheumatoid arthritis (RA). This study was undertaken to identify potential mechanisms of regulation of CXCL12 gene expression by human fibroblasts under normal or inflammatory conditions. METHODS: Synovial fibroblasts (SF) were cultured from RA and osteoarthritis (OA) synovial tissues. CXCL12 mRNA expression was analysed by real time quantitative RT-PCR in RA-SF under different growth conditions, and exposed to hypoxia or to different pro-inflammatory factors. A 5'CXCL12 -1.4 kb promoter region fragment was cloned in a luciferase reporter plasmid and its activity analysed in human fibroblasts. RESULTS: CXCL12 mRNA expression was not constitutively increased in RA- compared to OA-SF. LPS, pro-inflammatory cytokines or growth factors did not induce CXCL12 mRNA expression in SF. Hypoxia and growth arrest by either serum starvation or confluent growth induced CXCL12 mRNA and protein expression in SF. Constitutive and induced expression of CXCL12 in fibroblasts was regulated at the transcriptional level by specific regions of the -1.4 kb promoter. CONCLUSIONS: Pro-inflammatory factors and cytokines do not up-regulate CXCL12 gene expression in SF. Growth arrest and hypoxia are potentially important inducers of CXCL12 expression in human fibroblasts and operate by regulating transcriptional activity of the promoter.

Desafio diagnóstico

Paciente varón de 11 meses que ingresa con febrícula, rinorrea y tos progresiva de una semana de evolución. Se acompaña de lesiones papulo-eritematosas de 1-3mm de diámetro, pruriginosas, algunas confluentes, que aparecen en brazo izquierdo y se extienden progresivamente a brazos, piernas, cara y cuero cabelludo. No tiene afectación de tronco, palmas ni plantas. Tiene escasas excoriaciones por rascado en pierna izquierda a la altura de la rodilla

Hepcidin treatment in Hfe-/- mice diminishes plasma iron without affecting erythropoiesis.

BACKGROUND: Iron is essential for mammalian metabolism and its cellular concentration is controlled by regulating its acquisition and storage. Haemochromatosis is a condition involving iron overload that is characterised by increased duodenal iron absorption and a progressive accumulation of iron in vital organs. Hepcidin is the main hormone that regulates iron homoestasis and it is secreted by the liver. MATERIALS AND METHODS: We have studied how extended hepcidin administration affects the iron load status, plasma and tissue iron concentration, erythropoiesis and the expression of proteins involved on iron homeostasis in haemochromatotic (Hfe(-/-)) and wild-type mice. RESULTS: Hepcidin reverted the high plasma iron concentrations in Hfe(-/-) mice to normal values. The high concentration of hepatic iron was not altered in the liver of these Hfe(-/-) mice. Hepcidin administration did not disturb erythropoiesis in either Hfe(-/-) or wild-type mice and likewise, hepcidin did not modify the expression of any protein analysed in the liver, duodenum or spleen of Hfe(-/-) and wild-type mice. These data confirm that hepcidin administration diminishes plasma iron concentrations. CONCLUSION: Treatment with sustained doses of hepcidin diminishes plasma iron concentrations in Hfe(-/-) mice.

Immature blood vessels in rheumatoid synovium are selectively depleted in response to anti-TNF therapy.

BACKGROUND: Angiogenesis is considered an important factor in the pathogenesis of Rheumatoid Arthritis (RA) where it has been proposed as a therapeutic target. In other settings, active angiogenesis is characterized by pathologic, immature vessels that lack periendothelial cells. We searched for the presence of immature vessels in RA synovium and analyzed the dynamics of synovial vasculature along the course of the disease, particularly after therapeutic response to TNF antagonists. METHODOLOGY/PRINCIPAL FINDINGS: Synovial arthroscopic biopsies from RA, osteoarthritis (OA) and normal controls were analyzed by double labeling of endothelium and pericytes/smooth muscle mural cells to identify and quantify mature/immature blood vessels. To analyze clinicopathological correlations, a cross-sectional study on 82 synovial biopsies from RA patients with variable disease duration and severity was performed. A longitudinal analysis was performed in 25 patients with active disease rebiopsied after anti-TNF-alpha therapy. We found that most RA synovial tissues contained a significant fraction of immature blood vessels lacking periendothelial coverage, whereas they were rare in OA, and inexistent in normal synovial tissues. Immature vessels were observed from the earliest phases of the disease but their presence or density was significantly increased in patients with longer disease duration, higher activity and severity, and stronger inflammatory cell infiltration. In patients that responded to anti-TNF-alpha therapy, immature vessels were selectively depleted. The mature vasculature was similarly expanded in early or late disease and unchanged by therapy. CONCLUSION/SIGNIFICANCE: RA synovium contains a significant fraction of neoangiogenic, immature blood vessels. Progression of the disease increases the presence and density of immature but not mature vessels and only immature vessels are depleted in response to anti-TNFalpha therapy. The different dynamics of the mature and immature vascular fractions has important implications for the development of anti-angiogenic interventions in RA.

Human inflammatory synovial fibroblasts induce enhanced myeloid cell recruitment and angiogenesis through a hypoxia-inducible transcription factor 1alpha/vascular endothelial growth factor-mediated pathway in immunodeficient mice.

OBJECTIVE: Hyperplasia and phenotypic changes in fibroblasts are often observed in chronic inflammatory lesions, and yet the autonomous pathogenic contribution of these changes is uncertain. The purpose of this study was to analyze the intrinsic ability of fibroblasts from chronically inflamed synovial tissue to drive cell recruitment and angiogenesis. METHODS: Fibroblasts from patients with rheumatoid arthritis (RA) or osteoarthritis (OA), as well as fibroblasts from healthy synovial tissue and healthy skin, were cultured and subcutaneously engrafted into immunodeficient mice. Cell infiltration and angiogenesis were analyzed in the grafts by immunohistochemical studies. The role of vascular endothelial growth factor (VEGF), CXCL12, and hypoxia-inducible transcription factor 1alpha (HIF-1alpha) in these processes was investigated using specific antagonists or small interfering RNA (siRNA)-mediated down-regulation of HIF-1alpha in fibroblasts. RESULTS: Inflammatory (OA and RA) synovial fibroblasts, compared with healthy dermal or synovial tissue fibroblasts, induced a significant enhancement in myeloid cell infiltration and angiogenesis in immunodeficient mice. These activities were associated with increased constitutive and hypoxia-induced expression of VEGF, but not CXCL12, in inflammatory fibroblasts compared with healthy fibroblasts. VEGF and CXCL12 antagonists significantly reduced myeloid cell infiltration and angiogenesis. Furthermore, targeting of HIF-1alpha expression by siRNA or of HIF-1alpha transcriptional activity by the small molecule chetomin in RA fibroblasts significantly reduced both responses. CONCLUSION: These results demonstrate that chronic synovial inflammation is associated with stable fibroblast changes that, under hypoxic conditions, are sufficient to induce inflammatory cell recruitment and angiogenesis, both of which are processes relevant to the perpetuation of chronic inflammation.

Immunohistochemical detection of intravascular platelet microthrombi in patients with lupus nephritis and anti-phospholipid antibodies.

OBJECTIVES: To evaluate whether the use of platelet immunohistochemistry (IHC) markers improves the sensitivity of histological methods to detect microthrombosis in SLE nephritis and aPLs and to analyse the clinicopathological correlations of microthrombosis in this setting. METHODS: Kidney biopsy specimens from 65 patients with SLE, including 36 with positive aPLs, were studied by IHC using antibodies against platelet glycoproteins CD41 and CD61. Clinical data at the time of kidney biopsy and during a mean follow-up of 7.5 years after biopsy were recorded and analysed with regard to histological or IHC data. RESULTS: Histological lesions previously defined as APS nephropathy were found in 33% of the SLE kidney biopsies and were not associated with positive aPLs. Microthrombi detected as intravascular CD61(+) platelet deposits were present in 43% of the tissues and were significantly associated with positive aPLs, but not with histological APS nephropathy, nephritis manifestations nor with renal outcome. Histological APS lesions but not CD61(+) microthrombi correlated with an older age at nephritis presentation, previous cardiovascular risk factors and worse renal outcome. CONCLUSIONS: Immunodetection of intravascular CD61(+) platelet aggregates is more sensitive than histological evaluation to detect acute microthrombosis and provides a better correlation with aPLs in SLE patients. In contrast, histological lesions consistent with APS nephropathy were not associated with aPLs but with cardiovascular risk factors and worse renal outcome.

Differential expression of vasoactive intestinal peptide and its functional receptors in human osteoarthritic and rheumatoid synovial fibroblasts.

OBJECTIVE: Vasoactive intestinal peptide (VIP) has shown potent antiinflammatory effects in murine arthritis and ex vivo in human rheumatoid arthritis (RA) synovial cells. To investigate the potential endogenous participation of this system in the pathogenesis of RA, we analyzed the expression and regulation of VIP and its functional receptors in human fibroblast-like synoviocytes (FLS) from patients with osteoarthritis (OA) and patients with RA. METHODS: The expression of VIP was studied by reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay, and immunofluorescence in cultured FLS, and by immunohistochemical analysis in synovial tissue. The expression and function of the potential VIP receptors in FLS were studied by RT-PCR, determination of intracellular cAMP production, cell membrane adenylate cyclase (AC) activity, and interleukin-6, CCL2, and CXCL8 production in response to VIP or specific agonists and antagonists. RESULTS: VIP expression was detected in human FLS at the messenger RNA and protein levels, and it was significantly decreased in RA FLS compared with OA FLS. VIP receptor type 1 (VPAC1) was the dominant AC-coupled receptor in OA FLS, in contrast with RA FLS, in which VPAC2 was dominant. Tumor necrosis factor alpha-treated OA FLS reproduced the VIP and VPAC receptor expression pattern of RA FLS. The antagonistic effects of VIP on FLS proinflammatory factor production were reproduced by VPAC1- and VPAC2-specific agonists in OA FLS and RA FLS, respectively. CONCLUSION: VIP expression is down-regulated in RA and in tumor necrosis factor alpha-treated FLS, suggesting that down-regulation of this endogenous antiinflammatory factor may contribute to the pathogenesis of RA. In RA FLS, VPAC2 mediates the antiinflammatory effects of VIP, suggesting that VPAC2 agonists may be an alternative to VIP as antiinflammatory agents.

Immunoregulatory properties of vasoactive intestinal peptide in human T cell subsets: implications for rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease whose pathogenesis is not completely understood. Unbalanced Th1/Th2 T-cell polarization has been suggested to play a pathogenetic role and therefore, modulation of T-cell polarization is a potential therapeutic target. Vasoactive intestinal peptide (VIP) is a broadly distributed peptide that exerts anti-inflammatory and immunomodulatory effects, in the collagen-induced arthritis (CIA) murine model of RA, and ex vivo, in synovial cells from RA patients. In the present study, we have found that polyclonal stimulation of peripheral blood lymphocytes (PBL) from RA patients produces higher levels of inflammatory mediators and lower levels of Th1 cytokines than PBL from healthy controls; moreover, VIP has negligible effects on inflammatory mediators and Th1 cytokines produced by PBL from healthy controls but favours Th2 profile and enhanced IL-10 production after stimulation. VIP increases the levels of IL-10 and IL-4 in the supernatant of human CD4(+)CD45RA(+) cells cultured in a non-conditioned or a Th2-conditioned situation. In contrast, VIP does not modify the production of these cytokines in a Th1-conditioned medium. In summary, VIP can differentially modify the functional capacity of human lymphocytes by inducing Th2/Treg differentiation depending on their previous phenotype.

Ectopic lymphoid neogenesis in psoriatic arthritis.

BACKGROUND: Ectopic lymphoid neogenesis (LN) occurs in rheumatoid synovium, where it is thought to drive local antigen-dependent B cell development and autoantibody production. This process involves the expression of specific homing chemokines and the development of high endothelial venules (HEV). OBJECTIVE: To investigate whether these mechanisms occur in psoriatic arthritis (PsA) synovium, where autoantibodies have not been described and the organisation and function of B cells is not clear, and to analyse their clinical correlates. METHODS: Arthroscopic synovial biopsy specimens from patients with PsA before and after tumour necrosis factor alpha blockade were characterised by immunohistochemical analysis for T/B cell segregation, peripheral lymph node addressin (PNAd)-positive HEV, and the expression of CXCL13, CCL21 and CXCL12 chemokines in relation to the size of lymphoid aggregates. RESULTS: Lymphoid aggregates of variable sizes were observed in 25 of 27 PsA synovial tissues. T/B cell segregation was often observed, and was correlated with the size of lymphoid aggregates. A close relationship between the presence of large and highly organised aggregates, the development of PNAd+ HEV, and the expression of CXCL13 and CCL21 was found. Large organised aggregates with all LN features were found in 13 of 27 tissues. LN in PsA synovitis was not related to the duration, pattern or severity of the disease. The synovial LN pattern remained stable over time in persistent synovitis, but a complete response to treatment was associated with a regression of the LN features. CONCLUSIONS: LN occurs frequently in inflamed PsA synovial tissues. Highly organised follicles display the characteristic features of PNAd+ HEV and CXCL13 and CCL21 expression, demonstrating that the microanatomical bases for germinal centre formation are present in PsA. The regression of LN on effective treatment indicates that the pathogenic and clinical relevance of these structures in PsA merits further investigation.

Optimising stable retroviral transduction of primary human synovial fibroblasts.

Fibroblast like synoviocytes are the main resident cells in normal joints and are known to play a major role in the pathogenesis of rheumatoid arthritis. Efficient gene targeting of fibroblast like synoviocytes (FLS) is a major goal of current ex vivo gene therapy approaches for the treatment of rheumatoid arthritis. However, there is a need to improve viral systems capable of delivering genes to human rheumatoid fibroblasts and attempts have been made to develop a protocol for high efficiency, reproducible gene transfer using a replication-defective retrovirus vector. The effects of different experimental conditions were examined as well as those related to cellular and viral features on the efficiency of transducing the retroviral-driven expression of enhanced green fluorescent protein (EGFP) to FLS harvested from patients with rheumatoid arthritis. The optimal method established involved a double round of infection by centrifugation with a resting period of 4h between rounds. This approach led to the transduction of 30-70% of FLS obtained from nine patients with rheumatoid arthritis. Consistent transduction efficiencies were achieved in repeat assays such that it could be inferred that the variations observed were attributable to the specific characteristics of each cell line. This simple protocol renders a consistent and reproducible efficiency of rheumatoid fibroblast transduction and makes stable gene targeting using non-replicating retrovirus derived vectors an affordable option for the treatment of rheumatoid arthritis.

Fas activation of a proinflammatory program in rheumatoid synoviocytes and its regulation by FLIP and caspase 8 signaling.

OBJECTIVE: The expansion of an aggressive population of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) synovium occurs despite their expression of functional death receptors and exposure to death receptor ligands. FLS can survive Fas challenge because of the constitutive expression of FLIP apoptosis inhibitor. We investigated whether Fas signaling plays a pathogenetic role by activating a nonapoptotic proinflammatory program in RA FLS. METHODS: Cultured RA FLS were stimulated with an agonistic anti-Fas antibody in the presence or absence of the caspase inhibitor Z-VAD-FMK or after RNA interference with a short hairpin RNA expression plasmid directed against FLIP. NF-kappaB and activator protein 1 (AP-1) activation was studied by electrophoretic mobility shift assays and p65 immunofluorescence analysis, and expression of messenger RNA (mRNA) for monocyte chemoattractant protein 1, interleukin-8, IkappaB alpha, and matrix metalloproteinases (MMPs) 1, 9, and 13 was examined by reverse transcription-polymerase chain reaction. Chemotactic activity of Fas-activated FLS-conditioned media was studied in Transwell migration assays. RESULTS: Fas stimulation activated NF-kappaB and AP-1, and this response required caspase activity, since Z-VAD-FMK inhibitor precluded it. FLIP was processed to p43 protein after Fas stimulation in a caspase-dependent manner, and inhibition of FLIP expression resulted in reduced Fas-triggered NF-kappaB activation. Fas stimulation increased expression of mRNA for IkappaB alpha, MMPs, and chemokines, and Fas-activated RA FLS displayed increased chemotactic activity for monocytic cells. CONCLUSION: Fas triggering may contribute to the proinflammatory features of RA FLS by activating NF-kappaB and AP-1 and by expression of relevant target genes, such as MMPs and chemokines. Fas proinflammatory signaling is dependent upon caspase activity and FLIP expression. These data implicate FLIP as a potentially important molecular switch that turns the Fas signaling away from apoptosis and toward induction of a proinflammatory phenotype in RA FLS.

CXCL12 is displayed by rheumatoid endothelial cells through its basic amino-terminal motif on heparan sulfate proteoglycans.

The chemokine CXCL12 (also known as stromal cell-derived factor, SDF-1) is constitutively expressed by stromal resident cells and is involved in the homeostatic and inflammatory traffic of leukocytes. Binding of CXCL12 to glycosaminoglycans on endothelial cells (ECs) is supposed to be relevant to the regulation of leukocyte diapedesis and neoangiogenesis during inflammatory responses. To improve our understanding of the relevance of this process to rheumatoid arthritis (RA), we have studied the mechanisms of presentation of exogenous CXCL12 by cultured RA ECs. RA synovial tissues had higher levels of CXCL12 on the endothelium than osteoarthritis (OA) tissues; in both, CXCL12 colocalized to heparan sulfate proteoglycans (HSPGs) and high endothelial venules. In cultured RA ECs, exogenous CXCL12alpha was able to bind in a CXCR4-independent manner to surface HSPGs. Desulfation of RA EC HSPGs by pretreatment with sodium chlorate, or by replacing in a synthetic CXCL12alpha the residues Lys24 and Lys27 by Ser (CXCL12alpha-K2427S), decreased or abrogated the ability of the chemokine to bind to RA ECs. Ex vivo, synovial ECs from patients with either OA or RA displayed a higher CXCL12-binding capacity than human umbilical vein ECs (HUVECs), and in HUVECs the binding of CXCL12 was increased on exposure to tumor necrosis factor-alpha or lymphotoxin-alpha1beta2. Our findings indicate that CXCL12 binds to HSPGs on ECs of RA synovium. The phenomenon relates to the interaction of HSPGs with a CXCL12 domain with net positive surface charge located in the first beta strand, which encompasses a canonical BXBB HSPG-binding motif. Furthermore, we show that the attachment of CXCL12 to HSPGs is upregulated by inflammatory cytokines. Both the upregulation of a constitutive chemokine during chronic inflammation and the HSPG-dependent immobilization of CXCL12 in EC surfaces are potential sites for therapeutic intervention.

A HEV-restricted sulfotransferase is expressed in rheumatoid arthritis synovium and is induced by lymphotoxin-alpha/beta and TNF-alpha in cultured endothelial cells.

BACKGROUND: The recruitment of lymphocytes to secondary lymphoid organs relies on interactions of circulating cells with high endothelial venules (HEV). HEV are exclusive to these organs under physiological conditions, but they can develop in chronically-inflamed tissues. The interaction of L-selectin on lymphocytes with sulfated glycoprotein ligands on HEV results in lymphocyte rolling, which represents the initial step in lymphocyte homing. HEV expression of GlcNAc6ST-2 (also known as HEC-GlcNAc6ST, GST-3, LSST or CHST4), an HEV-restricted sulfotransferase, is essential for the elaboration of L-selectin functional ligands as well as a critical epitope recognized by MECA-79 mAb. RESULTS: We examined the expression of GlcNAc6ST-2 in relationship to the MECA-79 epitope in rheumatoid arthritis (RA) synovial vessels. Expression of GlcNAc6ST-2 was specific to RA synovial tissues as compared to osteoarthritis synovial tissues and localized to endothelial cells of HEV-like vessels and small flat-walled vessels. Double MECA-79 and GlcNAc6ST-2 staining showed colocalization of the MECA-79 epitope and GlcNAc6ST-2. We further found that both TNF-alpha and lymphotoxin-alphabeta induced GlcNAc6ST-2 mRNA and protein in cultured human umbilical vein endothelial cells. CONCLUSION: These observations demonstrate that GlcNAc6ST-2 is induced in RA vessels and provide potential cytokine pathways for its induction. GlcNAc6ST-2 is a novel marker of activated vessels within RA ectopic lymphoid aggregates. This enzyme represents a potential therapeutic target for RA.

Down-regulation of FLIP sensitizes rheumatoid synovial fibroblasts to Fas-mediated apoptosis.

OBJECTIVE: Hyperplasia of fibroblast-like synoviocytes (FLS) contributes to chronic inflammation and joint destruction in rheumatoid arthritis (RA). FLICE-inhibitory protein (FLIP) is an antiapoptotic protein that might prevent apoptotic elimination of FLS in response to death ligands such as tumor necrosis factor alpha (TNFalpha) or Fas ligand, which are present in RA synovium. Previous studies on FLIP expression by osteoarthritis (OA) and RA FLS have shown variable results, and the specific role of FLIP as an apoptosis inhibitor in these cells remains unclear. We undertook this study to investigate the expression and antiapoptotic function of FLIP in FLS. METHODS: We studied the expression of FLIP by immunohistochemistry and immunoblotting in synovial tissues or cultured FLS from RA and OA patients. FLS apoptosis was induced by an agonistic anti-Fas monoclonal antibody and FLS were then quantified. We studied the effects of cycloheximide (CHX), TNFalpha, and FLIP antisense oligonucleotide on FLIP expression and FLS apoptotic susceptibility. RESULTS: FLIP(L) was the isoform mainly expressed in lining synoviocytes and cultured FLS. Synovial tissues and cultured FLS from OA and RA tissues displayed similar patterns and levels of expression of FLIP. Fas-induced apoptosis was variable in different FLS lines, but differences between OA and RA groups were not detected. TNFalpha induced increases in FLIP(L) and FLIP(S) expression and protected RA FLS from apoptosis, while CHX induced the opposite effects. Down-regulation of FLIP by antisense oligonucleotide strongly sensitized RA FLS to Fas-mediated apoptosis. CONCLUSION: Apoptosis susceptibility and FLIP expression are similar in OA and RA FLS. Down-regulation of FLIP sensitizes RA FLS to Fas-mediated apoptosis and may be a valuable tool for targeting RA FLS hyperplasia.

Intracellular regulation of Fas-induced apoptosis in human fibroblasts by extracellular factors and cycloheximide.

Fibroblasts play an important role in reparative and inflammatory processes by synthesizing extracellular matrix components and releasing growth factors and cytokines. Fibroblast apoptosis has been observed at the termination phase of reparative or fibrotic responses, but its regulation in this context is poorly known. We investigated the susceptibility of human dermal fibroblasts (DF) to Fas-induced apoptosis and its regulation by extracellular factors potentially involved in immune-mediated inflammation and repair. DF expressed all components of the Fas apoptotic pathway: surface Fas, Fas-associated protein with death domain, and caspase-8 proteins. However, Fas activation resulted in caspase-8 activation and apoptosis only in the presence of cycloheximide (CHX). DF constitutively expressed Fas-associated death domain-like IL-1-converting enzyme-like inhibitory protein (FLIP) that was drastically down-regulated by CHX. Exogenous growth factors, cytokines, and adherence to the extracellular matrix shifted the balance of FLIP-caspase-8 proteins and modified the susceptibility of DF to Fas- or Fas-CHX-induced apoptosis. Short-term serum deprivation, suspension culture, and pretreatment with IFN-gamma or TNF-alpha increased, whereas long-term serum-free culture and pretreatment with TGF-beta or IL-10 decreased the apoptotic susceptibility of DF. Surface Fas expression was only modified by TNF-alpha and IFN-gamma, whereas all studied factors modified FLIP-caspase-8 protein expression, consistently with their pro- or antiapoptotic effects. Antisense FLIP oligonucleotides prevented resistance to Fas-induced apoptosis in DF. FLIP-caspase-8 balance seems tightly regulated in fibroblasts by extracellular factors that determine their susceptibility to Fas- or Fas-CHX-induced apoptosis. Th1 and Th regulatory cytokines display opposite effects on fibroblast apoptosis that suggest that their pro- or antifibrotic effects involve direct effects on fibroblast survival.

Synoviocyte-derived CXCL12 is displayed on endothelium and induces angiogenesis in rheumatoid arthritis.

CXCL12 (stromal cell-derived factor-1) is a potent CXC chemokine that is constitutively expressed by stromal resident cells. Although it is considered a homeostatic rather than an inflammatory chemokine, CXCL12 has been immunodetected in different inflammatory diseases, but also in normal tissues, ant its potential functions and regulation in inflammation are not well known. In this study, we examined the cellular sources of CXCL12 gene expression and the mechanism and effects of its interactions with endothelial cells in rheumatoid arthritis synovium. We show that CXCL12 mRNA was not overexpressed nor induced in cultured rheumatoid synoviocytes, but it specifically accumulated in the rheumatoid hyperplastic lining layer and endothelium. CXCL12 gene expression was restricted to fibroblast-like synoviocytes, whereas endothelial cells did not express CXCL12 mRNA, but displayed the protein on heparitinase-sensitive factors. CXCL12 colocalized with the angiogenesis marker alpha(v)beta(3) integrin in rheumatoid endothelium and induced angiogenesis in s.c. Matrigel plugs in mice. The angiogenic activity of rheumatoid synovial fluid in vivo was abrogated by specific immunodepletion of CXCL12. Our results indicate that synoviocyte-derived CXCL12 accumulates and it is immobilized on heparan sulfate molecules of endothelial cells, where it can promote angiogenesis and inflammatory cell infiltration, supporting a multifaceted function for this chemokine in the pathogenesis of rheumatoid arthritis.