BackgroundDamage-associated molecular patterns (DAMPs) are proposed to drive aberrant stimulation of Toll-like receptors (TLRs) in the rheumatoid arthritis (RA) joints resulting in increased ...expression of proinflammatory cytokines and chemokines. In the recent study we demonstrated that the neutrophil-derived lactoferrin (LTF) induces inflammatory response in RA synovial fibroblasts (RASF) via TLR-4 (Ref.). However, the molecular mechanisms of TLR-4 signaling in activated RASF are still unclear.ObjectivesTo clarify the molecular mechanisms of TLR-4 signaling pathways in activated RASF.MethodsRecombinant human neutrophil-derived LTF was used as one of the TLR-4 ligands. RASF were treated with LTF and/or TNF-α, and the expression of proinflammatory cytokines and chemokines, such as IL-6, IL-8 and CCL20 in RASF was measured by RT-qPCR and ELISA. To repress the TLR-4 signaling pathways, a small molecular inhibitor of TLR-4 (TAK-242), TAK1 inhibitor (5Z-7-Oxozeaenol), nuclear factor kappa B (NF-kB) inhibitor (BMS345541), and p38 mitogen activated protein kinase (MAPK) inhibitor (SB202190) were used. The role of nuclear factor of activated T cells 5 (NFAT5) in the TLR-4 signaling in RASF was investigated using a small interfering RNA targeting NFAT5.ResultsStimulation of RASF with LTF significantly increased the expression of IL-6, IL-8 and CCL20 mRNA and proteins (p=0.01). LTF enhanced the expression of these cytokine and chemokine mRNA in RASF stimulated by TNFα. TAK-242 completely repressed the expression of these cytokines and chemokines in RASF stimulated by LTF, while the TAK-1 inhibitor did not suppress the expression of these cytokines and chemokines in RASF. The NF-kB inhibitor, but not the p38MAPK inhibitor, partially repressed the expression of IL-6 and IL-8 mRNA induced by LTF. However, neither the NF-kB inhibitor nor p38MAPK inhibitor repressed the expression of CCL20 mRNA. Interestingly, silencing of NFAT5 significantly decreased the basal expression of IL-6, IL-8 and CCL20 mRNA in RASF. Additionally, silencing of NFAT5 significantly repressed the expression of not only IL-6, IL-8, but also of CCL20 mRNA in RASF treated by LTF.ConclusionsThese findings suggest that NFAT5 plays an important role as a critical regulator in the proinflammatory response of RASF mediated by the TLR-4 signaling pathway.ReferencesUmekita K, et al. Neutrophil-Derived Lactoferrin Regulates the Activity of NFAT5 in Rheumatoid Arthritis Synovial Fibroblasts Via Toll-like Receptor 4. 2015 ACR/ARHP Annual Meeting (San Francisco)AcknowledgementWe thank Ms. Yuki Kaseda, Ms. Ayaka Miyamoto and Dr. Yatsuki Aratake for their excellent technical assistance.Disclosure of InterestNone declared
To substantiate the finding of interstitial myocardial fibrosis in the transplanted heart and to characterize the collagen profile of the transplanted heart, we studied endomyocardial biopsy ...specimens from 30 heart transplants and four heart-lung transplants at 1 to 82 months after transplantation. Indirect immunofluorescent techniques with affinity-purified antibodies for collagen types I, III, IV, and V were used. The degree of interstitial collagen present was scored. The amount of type I collagen was increased in transplants from distant donors (mean ischemia time, 154 minutes) compared with those from on-site donors (mean ischemic time, 59 minutes): collagen scores 1.1 and 1.7, respectively (P less than .01). There was a trend toward a positive correlation, not statistically significant, between cyclosporine dose and collagen III deposition (r = .35), collagen IV (r = .38), and collagen V (r = .56). There was a negative correlation between number of rejection episodes and mean cyclosporine dose (r = -.41) and amount of collagen III (r = -.42) or collagen IV (r = -.42). No correlations were found between collagen deposition and any other variables studied. These results confirm the mixed nature of the collagen deposited and suggest that some fibrosis is related to cyclosporine administration rather than to the number of prior healed rejection episodes.
BackgroundLymph node stromal cells (LNSC) play a crucial role in shaping the immune response and maintaining peripheral tolerance. We developed an experimental model for studying the functional ...capacities of human LNSC during the earliest phases of RA and compared their cellular and molecular characteristics to LNSC from healthy volunteers.MethodsACPA+ RA patients (n=24), ACPA+ RA-risk individuals (n=23) and seronegative healthy controls (n=14;HC) underwent ultrasound-guided inguinal lymph node biopsy. Human LNSCs were isolated and expanded in vitro for cellular (flow cytometry), molecular (methylome, transcriptome and microRNA) and functional (contraction) analyses.ResultsRNA sequencing was performed on LNSC of HC (n=5), ACPA+ RA-risk individuals (n=6) and ACPA+ RA patients (n=4). Of interest, LNSC from ACPA+ RA-risk individuals and ACPA+ RA patients were more similar to each other compared with HC. Pathway analysis of commonly increased genes in RA (-risk) LNSC showed, among others, significant enrichment of pathways affecting actin cytoskeleton, focal adhesion and cell junction.DNA methylation (Illumina HumanMethylation450 array) analyses revealed 459 differentially methylated CpG sites (DMS) in LNSC from ACPA+ RA patients (n=5) versus HC (n=4), 504 DMS between ACPA+ RA-risk individuals (n=3) versus HC and 665 DMS when comparing RA patients with RA-risk individuals (delta β-value >0.1, p<0.05). 34 DMS were different in both RA and RA-risk LNSC compared to healthy LNSC. 80% of these DMS were significantly hypomethylated and associated with antigen processing and presentation (HLA-DRB1), immune response and regulation of actin cytoskeleton.Accordingly, in a gel contraction assay LNSC from ACPA+ RA-risk individuals and RA patients showed impaired collagen contraction compared to healthy LNSC. Healthy LNSC (n=5) covered 26.5% +/-2.5 of the well, while RA-risk (n=4) and RA (n=5) LNSC only covered 33.9% +/-5.9 and 30.6% +/-6.5.ConclusionsThis data point towards alterations in the cytoskeleton and antigen-processing and presentation in LNSC from ACPA+ RA-risk individuals and RA patients. Further studies are required to investigate the influence of this LNSC abnormality on immune responses.Disclosure of InterestC. Ospelt: None declared, E. Karouzakis: None declared, J. Hähnlein: None declared, J. Semmelink: None declared, R. Gay: None declared, P. Tak Employee of: GSK, D. Gerlag Employee of: GSK, S. Gay: None declared, L. van Baarsen: None declared
Les chiffres qui concernent la classe des insectes sont astronomiques. Selon les experts, les estimations globales du nombre d’espèces d’insectes varient entre cinq et dix millions, voire même trente ...millions, de par le Monde. Nous voilà en présence des champions incontestés de la biodiversité ! À ce jour, seules 950 000 espèces sont connues. 7 000 nouvelles espèces sont découvertes chaque année, tandis que 17 500 à 35 000 autres s’éteignent à tout jamais, la plupart avant même d’être découvertes. Cette hécatombe est en partie due au fait que la moitié environ des espèces d’insectes vivent dans les forêts humides tropicales, comme celles de Madagascar. Ces écosystèmes d’une grande richesse voient leurs superficies sans cesse diminuées par un déboisement intensif.
Increasing trend of outpatient management of children with newly diagnosed IDDM. Colorado IDDM Registry, 1978-1988.
J N Kostraba ,
E C Gay ,
M Rewers ,
H P Chase ,
G J Klingensmith and
R F Hamman
...Department of Preventive Medicine, University of Colorado School of Medicine, Denver 80262.
Abstract
OBJECTIVE--To examine the management of newly diagnosed insulin-dependent diabetes mellitus (IDDM) in Colorado over time and
to determine the prevalence of outpatient care at IDDM diagnosis on a statewide basis. RESEARCH DESIGN AND METHODS--The Colorado
IDDM Registry was used to assess medical care at the diagnosis of IDDM in 1182 patients less than 18 yr of age between 1978
and 1988. RESULTS--Twenty-three percent of children with IDDM in Colorado reported never being hospitalized during the diagnosis
period. Treatment of IDDM at diagnosis (outpatient vs. inpatient) did not differ by age, sex, or ethnicity/race. Patients
living in rural counties were less likely to have been treated as outpatients at diagnosis than those living in urban counties.
Physicians at specialized diabetes clinics (e.g., The Barbara Davis Center for Childhood Diabetes and The Childrens Hospital)
were more likely to treat newly diagnosed children in an outpatient setting than physicians not affiliated with these clinics.
The proportion of patients receiving only outpatient care at IDDM diagnosis increased from 6% in 1978 to 35% in 1988. This
increase can be attributed to three factors: 1) an increase in the number of Colorado children diagnosed at The Barbara Davis
Center, where outpatient care is strongly advocated; 2) a change in treatment practices at The Childrens Hospital away from
routine hospitalization at onset; and 3) a steady increase in outpatient care for newly diagnosed diabetic children by physicians
who were not affiliated with the aforementioned specialized diabetes clinics. CONCLUSIONS--The relatively new practice of
outpatient care at diagnosis of IDDM increased between 1978 and 1988 in Colorado, in both specialized diabetes clinics and
physicians' practices not affiliated with specialized clinics.
Chronic afternoon (PM) but not morning injections of melatonin (MEL) induced significant reductions in testicular and seminal
vesicle weights as well as attenuating serum prolactin (PRL) and ...luteinizing hormone (LH) levels. Although there were no treatment-induced
effects on hemipituitary weights, PM-Mel injections led to significant reductions in in vitro PRL secretion and tended to
increase the ability of dopamine to inhibit PRL release. It was also shown that LH-releasing hormone (LHRH) could inhibit
in vitro PRL release from hamster pituitaries. Basal or LHRH-stimulated LH secretion from incubated pituitaries was not affected
by Mel in vivo. From these results we conclude that properly timed Mel injections do not reduce pituitary's ability to secrete
LH but severely attenuate PRL secretion. These findings are similar to those observed in pituitaries from hamsters housed
in short-photoperiod conditions.
Chronic myeloproliferative disorders (MPD) are clonal diseases of the pluripotent hematopoietic stem cell frequently associated with myelofibrosis (MF). There is only indirect evidence indicating ...that the increased deposition of collagen in bone marrow matrix is a secondary phenomenon. A liquid culture system for cloning and growing bone marrow fibroblasts has permitted us to approach more directly the understanding of the pathogenesis of myelofibrosis by comparing the biophysical, growth, and functional characteristics of fibroblasts from normals, MPD patients without MF, and those with MF. In patients with MF, marrow fibroblast colony (CFU-F) formation could not be studied; fibroblasts were grown from marrow expiants. CFU-F from normals and MPD patients exhibited similar cell density distribution and similar cell sedimentation rates. These similarities contrasted sharply with the differences seen when the erythroid and granulocyte-macrophage progenitors were studied by the same methods. There was a marked light density shift and a rapidly sedimenting shift of MPD hematopoietic colony-forming cells. Marrow fibroblasts from MPD patients with and without MF displayed the same in vitro growth characteristics as fibroblasts from normals. Both types of fibroblasts exhibited anchorage and serum dependence, and contact inhibition of growth. Marrow fibroblasts were also characterized for the presence and distribution of fibronectin and collagen types by immunofluorescent staining using monospecific antibodies. Extracellular matrix, membrane-, and cytoplasm-associated fibronectin, type I, type III, and type V collagen showed a similar staining pattern in both normal and myelofibrotic marrow fibroblasts. Plasminogen-depen-dent fibrinolytic activity elicited from normal and myelofibrotic marrow fibroblasts were equivalent. Chromosomal analysis of hematopoietic cells and marrow fibroblasts from Philadelphia chromosome positive chronic myelocytic leukemia patients with and without MF showed that the Philadelphia chromosome was present only in hematopoietic cells. The results of these studies taken together demonstrate that bone marrow collagen-producing cells from MPD patients with and without MF behave in vitro as do those from normals. These findings support the hypothesis that the marrow fibrosis observed in patients with MPD results from a reactive process rather than from a primary disorder affecting the marrow collagen-producing cells.
Background and objectivesMost forms of arthritis occur with a distinctive pattern of joint involvement. To elucidate whether stromal differences between the joints might play a role in the ...susceptibility of joints to develop arthritis, we compared the transcriptome, epigenetic marks and functions of synovial fibroblasts (SF) isolated from different joints.Material and methodsSF were isolated from hand, shoulders, and knees from RA and OA patients. Total RNA was isolated and sequenced on theIlluminaHiSeq 2000 (n = 21). Epigenetic regulation was studied by methyl-DNA -immunoprecipitation and treatment with bromodomain inhibitors. Adhesion to tissue culture plates (n = 20) and proliferation (n = 15) were measured with the xCELLigence-RTCA-DP Instrument. Transwell plates were used to measure migration of leukocytes from healthy donors towards supernatants of cultured SF (n = 14).ResultsBased on their transcriptome profile, SF clustered according to their joint of origin. Hand SF featured increased expression of transcripts in the HOX cluster that are also increased in distal limb buds during embryonic development, eg, HOXD13, HOXA13 and HOTTIP. Accordingly, genes upregulated in hand SF were clustered in biological processes such as ‘limb development’ (enrichment score (ES) 4.3) and ‘tissue morphogenesis’ (ES 3.4). The expression of these developmental transcripts in adult SF was maintained by histone acetylation and lack of DNA methylation. Other biological processes enriched in hand SF were ‘cell-cell signalling’ (ES 2.9), ‘cell migration’ (ES 1.9) and ‘defense response’ (ES 0.9). Genes that were lower expressed in hand SF were mainly enriched in ‘cell adhesion’ (enrichment score 2.3) and ‘embryonic morphogenesis (enrichment score 2.3). Functionally adhesion was significantly lower in handcompared to shoulder SF (cell index 0.6 ± 0.2 vs. 0.9 ± 0.2, p = 0.01). Hand SF proliferated faster than shoulder SF (doubling time: 39 ± 3h vs. 67 ± 15h, p = 0.03) and significantly more leucocytes migrated towards the supernatants from cultured hand SF than shoulder SF.ConclusionsSF from different anatomic locations exhibit significant differences in their transcriptome, particularly in the expression of embryonic transcription factors encoded in the HOX cluster. Hand SF display differences in their chemotactic, proliferative and adhesive properties, which may explain why chronic arthritidis tend to be more destructive in hands compared to other joints.
Objective
To map hypoxic areas in arthritic synovium and to establish the relevance of low oxygen levels to the phenotype of synovial fibroblasts, with special focus on bone degradation.
Methods
To ...analyze the distribution of hypoxia in arthritic joints, the hypoxia marker EF5 was administered to mice with collagen‐induced arthritis (CIA). To evaluate the effect of hypoxia on rheumatoid arthritis synovial fibroblasts (RASFs), reverse suppression subtractive hybridization and complementary DNA array were used. Real‐time polymerase chain reaction, Western blotting, and immunohistochemistry were used to evaluate the expression of inhibitor of DNA binding/differentiation 2 (ID‐2). To investigate the function of ID‐2 in RASFs, cells were transfected either with ID‐2 vector or with ID‐2–specific small interfering RNA.
Results
EF5 staining showed the presence of hypoxia in arthritic joints, particularly at sites of synovial invasion into bone. Differential expression analysis revealed that ID‐2 was strongly induced by hypoxia in RASFs. Immunohistochemical analysis of CIA mouse synovium and human RA synovium showed a strong expression of ID‐2 by RASFs at sites of synovial invasion into bone. Overexpression of ID‐2 in RASFs significantly induced the expression of several factors promoting osteoclastogenesis. The biologic relevance of the potent osteoclastogenesis‐promoting effects was shown by coculture assays of ID‐2–overexpressing RASFs with bone marrow cells, leading to an increased differentiation of osteoclasts from bone marrow precursors.
Conclusion
The data show that hypoxic conditions are present at sites of inflammation and synovial invasion into bone in arthritic synovium. Hypoxia‐induced ID‐2 may contribute to joint destruction in RA patients by promoting synovial fibroblast–dependent osteoclastogenesis.
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Background: The National Quality Forum recently announced accountability measures including use of chemotherapy (ACT) with Stage III colon cancer. This study examines the impact of ...performance reporting using cancer registry data to assess the quality of care for Stage III colon cancer patients using the Commission on Cancer's (CoC) Cancer Program Practice Profile Reports. Cancer registries are the primary source for measurement but adjuvant treatment may be incomplete. Methods: In January 2005 the CoC provided reports to 1,337 hospitals using registry data on over 80,000 Stage III colon cancer patients diagnosed from 1998–2003. Each hospital received a weighted performance rate (PR) and comparison to hospitals of similar types, nationally and regionally. Hospitals could review and correct missing or inaccurate data to provide an updated profile. Results: The initial overall hospital-level PR was 66.2%, hospitals in the top quartile had PRs =80.1%. After 24 months 603 hospitals had corrected data. The mean PR was 74.9%, an increase of 8.7%. The top quartile PR was =89.7%. The PRs for hospitals in the top quartile increased on average by 4.3%, and for hospitals outside this group by 22.7%. The proportion of hospitals with PRs =90% increased from 7.9% to 23.9%. Conclusions: With over two-fifths of hospitals correcting data, the concordance rate with ACT in Stage III colon cancer has significantly increased compared to initial registry data. Reporting quality data and allowing auditing and correction on key quality indicators corrects the data gap in cancer registry out-patient treatment data. Exposure to profile reports can spur local providers to promote better communication with centralized data repositories, significantly contributing to the assessment of care provided to cancer patients.
No significant financial relationships to disclose.
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