Blood monocyte activation in rheumatoid arthritis: increased monocyte adhesiveness, integrin expression, and cytokine release

Infiltration of the synovium by mononuclear cells, namely lymphocytes and monocytes, is one of the main features of rheumatoid arthritis (RA) and is considered to be responsible for the development of the disease. In this study in 31 consecutive patients with RA, we investigated whether peripheral blood monocytes exhibited markers of cellular activation related to cell migration. Using flow cytometry with the respective specific antibodies, we studied the expression of integrins CD11a, CD11b, CD11c, CD49d (VLA-4), and CD49e (VLA-5) on monocytes from patients with RA and from normal (N) subjects. IL-1β, IL-6, and tumour necrosis factor-alpha (TNF-α) production by cultured monocytes was measured by immunoassay. Adhesiveness of monocytes was studied on various surfaces (plastic, human fibronectin, gelatin-coated plasma, subendothelial matrix) and on cultured endothelial cells under basal conditions or after stimulation by IL-1β. An increased number of CD14⁺ monocytes (Mo) from RA patients expressed the CD11b molecule (RA Mo = 90·3%, N Mo = 83·4%, P < 0·005). The expression of CD11b on CD14⁺ monocytes was significantly increased in RA patients (median fluorescence intensity (FI): RA Mo = 145 (range 80–466) units; normal Mo = 95 (range 24–164) units; P < 0·003). Production of extracellular IL-1β and IL-6 by RA monocytes was significantly enhanced compared with monocytes from normal subjects (IL-1β: RA = 2·65 ± 0·91 ng/ml versusN = 1·35 ± 0·85 pg/ml, P < 0·05; IL-6: RA = 4·83 ± 0·90 ng/ml versusN = 2·40 ± 0·95 ng/ml, P < 0·05). Compared with normal monocytes, RA monocytes exhibited increased adhesion to the various surfaces studied (plastic, P < 0·01; fibronectin, P < 0·01; and gelatin-coated normal or RA plasma, P < 0·01) as well as to unstimulated (P < 0·01) and IL-1β-stimulated endothelial cells (IL-1β for 4 h, P < 0·05; IL-1β for 24 h, P < 0·05). In our study, blood monocytes from RA patients exhibited features of activation related to cell adhesion.     

Corticosteroids restore the balance between locally produced Th1 and Th2 cytokines and immunoglobulin isotypes to normal in sarcoid lung

In this study, we have investigated the balance between Th1- and Th2-like activity in the lungs in sarcoidosis and have determined the effect of corticosteroid treatment on this. Twenty-one patients with acute untreated sarcoidosis were investigated by bronchoalveolar lavage (BAL) and compared with 11 normal volunteers. Sixteen of the sarcoid patients required corticosteroid therapy and seven of these were reinvestigated after 2–3 months'' treatment. In order to assess Th1- and Th2-like activity in the lungs, IgG subclasses and IgE were measured in BAL fluid and serum, and IL-2, IL-4 and interferon-gamma (IFN-γ) in BAL. In patients with untreated sarcoidosis, albumin-corrected BAL/serum ratios for IgG4 and IgE were significantly reduced (IgG4, 1.04±0.18 (mean±s.e.m.); IgE 9.58±3.11) compared with those in normal controls (IgG4 5.3±0.72, P<0.001; IgE 67.7±28.9, P<0.01). Estimates of actual levels of immunoglobulins produced in the lungs were also made and showed extremely high levels of total IgG in sarcoid patients (39.56±8.2 mg/l ) compared with controls (1.17±0.5 mg/l, P<0.001). Although there was no difference between the groups in amount of IgG4 locally produced, the proportion of total IgG which was IgG4 was greatly reduced in those with sarcoidosis (1.6±0.4% compared with 38.5±3.2%; P<0.001). Lavage levels of IL-4 were also reduced in sarcoid patients (IL-4 2.103±0.21 pg/ml) compared with those in normals (IL-4 6.8±1.05; P<0.001). Levels of IL-2 were lower (7.63±0.51 pg/ml compared with 9.4±0.95 pg/ml), but this difference was not significant. IFN-γ, however, could not be detected above 0.4 pg/ml in any of the normal lavage fluid, but was detectable in 12/21 patients with sarcoidosis (χ²=7.74; P<0.001). These changes reverted towards normal on treatment with oral corticosteroids. The mean albumin-corrected BAL/serum ratio for IgG4 before treatment was 0.88±0.33 compared with 5.5±2.1 (P<0.05) on treatment, and for IgE before treatment 9.52±2.15 compared with 50.8±17.9 (P<0.05) on treatment. Total IgG produced in the lung fell from 26.16±7.9 to 6.12±2.4 mg/l (P<0.001) on treatment, and the proportion of IgG4 locally produced rose from 2.3±0.8% to 23.9±6.1% (P<0.01). The mean level of IL-4 in lavage before treatment was 2.53±0.34 pg/ml compared with 4.7±0.34 (P<0.001) on treatment. Levels of IL-2 also rose significantly on treatment from 8.74±0.95 pg/ml before to 14.44±1.38 pg/ml (P<0.001) on treatment. Levels of IFN-γ fell from 1.65±0.43 pg/ml before treatment to undetectable levels in all patients (P<0.001) on treatment. These results demonstrate an imbalance between Th1- and Th2-like activity in the lungs in sarcoidosis, with suppression of Th2 and increase in Th1. Corticosteroid therapy restores the normal balance between Th1 and Th2 cytokines and immunoglobulins in the lungs, suggesting an effect on local immune regulation.     

Enhanced binding of lymphocytes from patients with multiple sclerosis to tumour necrosis factor-alpha (TNF-α)-treated endothelial monolayers: associations with clinical relapse and adhesion molecule expression

This study investigated the adherent properties and adhesion molecule expression of blood mononuclear cells (MNC) from a total of 84 patients with multiple sclerosis (MS). The MNC from MS patients were significantly more adherent than cells from normal healthy subjects to endothelial monolayers pretreated with 0.01 U/ml TNF-α (103% increase; P = 0.002), 0.1 U/ml TNF-α (80% increase; P< 0.01) and 1.0 U/ml TNF-α (41% increase; P< 0.02), and to endothelium pretreated with 10 U/ml IL-1β (44% increase; P< 0.05) and 100 U/ml interferon-gamma (IFN-γ) (100% increase; P< 0.05). This augmented adhesion was a property of the lymphocytes, in particular CD4⁺ cells, and was inversely related to the time of onset of clinical relapse. The percentage of lymphocytes bearing the adhesion molecules CD49d, CD29 and CD62L was increased in MS blood, but the level of CD29 and CD62L expression was reduced. We infer that circulating lymphocytes in MS are predisposed to cross endothelial barriers at sites where inflammation has already commenced.     

MicroRNA-29 mediates TGFβ1-induced extracellular matrix synthesis by targeting wnt/β-catenin pathway in human orbital fibroblasts

Purpose: Transforming growth factor β1 (TGFβ1) is very important in the synthesis and degradation of extracellular matrix (ECM) and also in the mediation of human orbital fibroblasts (OFs) proliferation. MicroRNA-29 (MiR-29) plays an important role in this process. In the present study, the effects of TGFβ1 on the expression of miR-29 and whether miR-29 is involved in pro-survival signaling pathways mediated by TGFβ1 were examined in human OFs. Methods: Detecting the influence of TGFβ1 on the expression of miR-29a/b/c by real-time PCR analysis. Using 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) to detecting the influence of miR-29 on the increased proliferation caused by TGF-β1 on the human orbital fibroblasts. Using soft agar assay to detecting the influence of miR-29 on the increased colony formation caused by TGF-β1 on the human orbital fibroblasts. Western blot was used to detect the specific mechanisin. Results: TGFβ1 treatment decreases the expression of miR-29 in OFs. In the cultured OFs, the value of optical density (OD) in the group treated with miR-29 is lower than that in the group treated without miR-29 (P < 0.05). In the cultured OFs, the ratio of colony formation in the group treated with miR-29 is lower than that in the group treated without miR-29 (P < 0.05). In OFs, miR-29 decreases the secretion of Wnt3a and activation of β-catenin whether the treatment of TGFβ1 was used or not. MiR-29 decreases expression of Collagen, type I, alpha 1 (COL1A1) through down-regulation of wnt/β-catenin pathway. Conclusions: In OFs TGFβ1 treatment decreases expression of miR-29 which can cause the inhibition of normal ability of TGFβ1. MiR-29 inhibits TGFβ1-induced proliferation of OFS cell and decreases colony formation of OFS cell after TGFβ1 treatment. MiR-29 Mediates TGFβ1-induced Extracellular matrix synthesis through activation of Wnt/β-catenin pathway in human OFs.     

Per a 10 protease activity modulates CD40 expression on dendritic cell surface by nuclear factor-kappaB pathway

Serine protease activity of Per a 10 from Periplaneta americana modulates dendritic cell (DC) functions by a mechanism(s) that remains unclear. In the present study, Per a 10 protease activity on CD40 expression and downstream signalling was evaluated in DCs. Monocyte-derived DCs from cockroach-allergic patients were treated with proteolytically active/heat-inactivated Per a 10. Stimulation with active Per a 10 demonstrated low CD40 expression on DCs surface (P < 0·05), while enhanced soluble CD40 level in the culture supernatant (P < 0·05) compared to the heat-inactivated Per a 10, suggesting cleavage of CD40. Per a 10 activity reduced the interleukin (IL)-12 and interferon (IFN)-γ secretion by DCs (P < 0·05) compared to heat-inactivated Per a 10, indicating that low CD40 expression is associated with low levels of IL-12 secretion. Active Per a 10 stimulation caused low nuclear factor-kappa B (NF-κB) activation in DCs compared to heat-inactivated Per a 10. Inhibition of the NF-κB pathway suppressed the CD40 expression and IL-12 secretion by DCs, further indicating that NF-κB is required for CD40 up-regulation. CD40 expression activated the tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6), thereby suggesting its involvement in NF-κB activation. Protease activity of Per a 10 induced p38 mitogen-activated protein kinase (MAPK) activation that showed no significant effect on CD40 expression by DCs. However, inhibiting p38 MAPK or NF-κB suppressed the secretion of IL-12, IFN-γ, IL-6 and TNF-α by DCs. Such DCs further reduced the secretion of IL-4, IL-6, IL-12 and TNF-α by CD4⁺ T cells. In conclusion, protease activity of Per a 10 reduces CD40 expression on DCs. CD40 down-regulation leads to low NF-κB levels, thereby modulating DC-mediated immune responses.     

Inflammatory Responses in Blood Samples of Human Immunodeficiency Virus-Infected Patients with Pulmonary Infections

We analyzed the characteristics of the inflammatory response occurring in blood during pulmonary infections in human immunodeficiency virus (HIV)-infected patients. A prospective study of consecutive hospital admissions of HIV-infected patients with new-onset radiologic pulmonary infiltrates was carried out in a tertiary university hospital from April 1998 to May 2001. Plasma cyclic AMP receptor protein (CRP), interleukin 1β (IL-1β), IL-6, IL-8, IL-10, and tumor necrosis factor alpha (TNF-α) levels were determined at the time of admission and 4, 5, and 6 days later. Patients were included in a protocol addressed to study etiology and outcome of disease. A total of 249 episodes of infection were included, with the main diagnoses being bacterial pneumonia (BP) (118 episodes), Pneumocystis carinii pneumonia (PCP) (41 episodes), and mycobacteriosis (36 episodes). For these three patient groups, at the time of admission the median CRP and cytokine levels were as follows: CRP, 10.2, 3.8 and 5 mg/dl, respectively (P = 0.0001); IL-8, 19, 3, and 2.9 pg/ml (P = 0.045); and TNF-α, 46.4, 44, and 75 pg/ml, respectively (P = 0.029). There were no significant differences in levels of IL-1β, IL-6, or IL-10 among the patient groups. A total of 23 patients died. At the time of admission, HIV-infected patients with BP had higher plasma CRP and IL-8 levels than did PCP and mycobacteriosis patients. TNF-α levels were higher in patients with mycobacteriosis. An elevated IL-8 level (>61 pg/ml) at the time of admission was an independent factor associated with higher mortality (odds ratio, 12; 95% confidence interval, 1.2 to 235.5).     

Effect of rTsP53 on the M1/M2 activation of bone-marrow derived macrophage in vitro

We investigated that if rTsP53 could be used to activate bone-marrow derived macrophage (BMDM) into M2 macrophage and stop M1 macrophage activation. After 72 h incubation in blank culture medium, cells with PE-CCR7 (-) and FITC-CD206 (-) was extracted and its mean proportion was 92.30 ± 0.22%. With the stimulation of 20 μg/ml IFN-γ for 72 h, cells with PE-CCR7 (+) was extracted and its mean proportion was 16.24 ± 0.82%. With the stimulation of IL-3/IL-14 (both 10 μg/ml) for 72 h, cells with FICT-CD206 (+) was extracted and its mean proportion was 87.32 ± 4.29%. Co-incubation with different dose of rTsP53 (0.001 μg/ml, 0.01 μg/ml, 0.1 μg/ml, 1 μg/ml, 2 μg/ml, 5 μg/ml, 10 μg/ml, respectively) for 72 h, FITC-CD206 (+) macrophage was extracted. The mean proportion in each group was 1.09 ± 0.22%, 2.13 ± 0.13%, 4.91 ± 0.07%, 5.48 ± 0.29%, 9.81 ± 0.06%, 12.83 ± 0.55%, 17.87 ± 0.02%, respectively. The dose of rTsP53 was significantly positive correlated to the proportion of FITC-CD206 (+) macrophage. Co-incubation with 20 μg/ml IFN-γ and 5 μg/ml rTsP53 for 72 h, cells with PE-CCR7 (+) was extracted and its mean proportion was 10.60 ± 0.19%. Compared to that of mere co-incubation with IFN-γ, there was significant difference between the two groups. ELISA showed that Th1 cytokines’ (IFN-γ, IL-6 and TNF-α) level decreased in the culture medium supernatant of BMDM co-incubated with rTsP53. There was negative correlation between the Th1 cytokines’ level and the dose of rTsP53. Both Th2 cytokines (IL-4 and IL-13) and regulatory cytokines in the culture medium increased. There was positive correlation between the Th2 cytokines’ level and the dose of rTsP53. There was also positive correlation between the regulatory cytokines’ level and the dose of rTsP53. Compared to that of BMDM co-incubated with IFN-γ, levels of TNF-α and IL-6 were significant lower than that of BMDM co-incubated with both IFN-γ and rTsP53 (both P < 0.05), while the levels of IL-4 and TGF-β were significant higher (both P < 0.05). There was no significant difference in the levels of IL-13 and IL-10 between the two groups.     

Expression of COX-2, IL-1β, TNF-α and IL-4 in epithelium of serrated adenoma,adenoma and hyperplastic polyp

Introduction

Colon polyps and inflammatory process play the key role in neoplasia of colorectal cancer. In recent years there have been many publications on the malignancy of hyperplastic polyp (HP) which according to the WHO classification is a non-neoplastic polyp. The aim of this study is to determine the expression of inflammatory proteins COX-2, IL-1β, TNF-α and IL-4 in the epithelium of colorectal polyps.

Material and methods

In the study, 144 colorectal polyps were analyzed. The groups of HP, classical (A) and serrated adenomas (SA) and normal mucosa (control) according to histopathological studies were selected. Immunohistochemical examinations Rusing antibodies against COX-2, IL-1β, TNF-α and IL-4 were performed. The expression of analyzed protein was evaluated using modified Remmele-Stegner scale (0-16).

Results

Statistical analysis revealed higher expression of TNF-α (16 ±3.87 vs. 1 ±5.06), IL-1β (12 ±4 vs 8 ±2.72), COX-2 (9 ±2.54 vs. 8 ±3.14) and IL-4 (12 ±3.45 vs. 4 ±3.35) in SA polyps compared to the control (p < 0.001). The HP had an increased level of expression of TNF-α (12 ±3.72 vs. 1 ±5.06, p < 0.005), COX-2 (8.5 ±1.97 vs. 8 ±3.14, p < 0.012) and IL-4 (12 ±3.46 vs. 4 ±3.35, p < 0.001). Significantly higher expression of IL-4 (12 ±2.32 vs. 4 ±3.35, p < 0.001) and IL-1β (16 ±4.32 vs. 8 ±2.72, p < 0.044) in A compared to the control were observed.

Conclusions

Expression of inflammatory factors differed between polyps. Inflammation accompanied the serrated structures which occur in polyps. The inflammatory process affects the development of colorectal polyps. The HP may predispose to malignancy.     

Anti-inflammatory effect of peroxisome proliferator-activated receptor-γ (PPAR-γ) on non-obese diabetic mice with Sjogren’s syndrome

Objective: To investigate the anti-inflammatory effect of peroxisome proliferator-activated receptor-γ (PPAR-γ) on non-obese diabetic mice (NOD mice) with Sjogren’s syndrome. Methods: 22 eight-week-old female NOD mice were randomly divided into 2 groups. Rosiglitazone and normal saline were administered in the PPAR-γ group and the control group respectively. At the age of 9, 12 and 15 weeks, one mouse in each group was sacrificed respectively, and the remaining mice were sacrificed at the age of 18 weeks. Blood were obtained by cardiac puncture, and salivary glands were resected. The degree of salivary gland damage and infiltration of lymphocytes were examined by H&E staining. The level of IL-1β, IL-4, IL-6 and TNF-α in serum were measured by ELISA. The mRNA expression level of IL-1β, IL-4, IL-6 and TNF-α in MSG were detected by Real-time PCR. Expression of PPAR-γ in the salivary glands was detected by Immunohistochemistry. Results: Compared with the control group, mice in the PPAR-γ group showed that (1) histopathologic changes in the salivary glands were significantly ameliorated; (2) at the age of 18 weeks, IL-6 [(25.86 ± 7.32) vs (37.41 ± 11.34)] and TNF-α [(56.88 ± 22.19) vs (78.61 ± 20.76)] were expressed significantly lower and IL-4 [(25.76 ± 12.65) vs (12.11 ± 3.70)] was expressed significantly higher in serum (P < 0.05); (3) the expression of TNF-α was significantly decreased and the expression of IL-4 was significantly increased in MSG (P < 0.05). Conclusion: PPAR-γ ameliorates Sjogren’s syndrome on NOD mice effectively. The mechanism may be related to the reduction of Th1 cytokines and change of T helper cell balance from Th1 to Th2.     

An assessment of peripheral immunity in patients with sarcoidosis using measurements of serum vitamin D3, cytokines and soluble CD23

The aetiology of the peripheral anergy in sarcoidosis is unclear. To investigate this further we measured the serum levels of several factors important in different aspects of immune regulation to obtain a profile of those factors which promote and inhibit immune activation in sarcoidosis. Thirty-seven patients with sarcoidosis and 20 healthy controls of similar sex and age comprised the study group. Serum IL-10, interferon-gamma (IFN-γ), soluble CD23 (sCD23), IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1β and tumour necrosis factor-alpha (TNF-α) were measured using in-house ELISAs. Vitamin D3 was measured using a radioreceptor assay. Serum levels of sCD23 and IL-10 were significantly elevated in patients with sarcoidosis relative to controls (median 13.9 versus 9.5 arbitrary units/ml, P < 0.01 for sCD23, and 9.6 versus 5.0 pg/ml, P < 0.04 for IL-10). Regardless of steroid therapy or disease activity, serum levels of IFN-γ, TNF-α, IL-1β, GM-CSF and IL-8 were no different in patients with sarcoidosis and controls. Vitamin D3 levels were significantly higher in patients with sarcoidosis versus normal controls (medians 78.0 versus 56.0, P < 0.001), active sarcoidosis (n = 20) versus inactive disease (n = 17) (medians 81.5 versus 66.0, P < 0.03) and active sarcoidosis versus controls (medians 81.5 versus 56.0, P < 0.0002). The levels were no different between patients with inactive sarcoidosis and controls. We suggest that IL-10 and vitamin D3 may contribute to the peripheral anergy in sarcoidosis. The elevated serum sCD23 suggests an increase in peripheral humoral immunity. Consistent with a quiescent peripheral immune system, factors capable of monocyte/macrophage activation (TNF-α, IFN-γ, GM-CSF and IL-8) were not elevated in the peripheral circulation.     

Chronic sinusitis refractory to standard management in patients with humoral immunodeficiencies

Chronic refractory sinusitis is a common feature in patients with primary immunodeficiencies. The efficacy of standard therapeutic strategies is questionable. In an open trial we evaluated the efficacy of azithromycin, N-acetylcysteine and topical intranasal beclomethasone (100 μg twice daily for 6 weeks) in 16 patients with primary immunodeficiencies (median age 13.5 years, range 5–32 years). All patients suffered from chronic sinusitis despite regular immunoglobulin replacement therapy every 3 weeks. Magnetic resonance imaging (MRI) scans were performed before and after 6 weeks of treatment to evaluate morphological changes in the paranasal sinuses. Nasal swabs and washings were taken for microbial analysis and measurement of inflammatory mediators (IL-8, tumour necrosis factor-alpha (TNF-α), eosinophilic cationic protein (ECP)) before and post therapy. Inflammatory mediators in nasal secretions were significantly elevated in patients: IL-8 median 2436 pg/ml (range 441–5435 pg/ml), TNF-α 37.3 pg/ml (3.75–524 pg/ml) and ECP 33 ng/ml (1.5–250 ng/ml) versus age-matched healthy controls: IL-8 median 212 pg/ml (99–825 pg/ml), TNF-α 3.77 pg/ml (2.8–10.2 pg/ml) and ECP 1.5 ng/ml (1.5–14.8 ng/ml) (P < 0.0001). Inflammation of the maxillary sinuses was confirmed by MRI scans in all patients, additionally infection of the ethmoidal and frontal sinuses was recorded in five patients. Bacterial growth appeared in 11 out of 16 cultures. In spite of therapy, no improvement in sinal inflammation visualized by MRI was achieved. Moreover, no significant decrease in pathogens and levels of inflammatory mediators could be detected (IL-8 1141 pg/ml, 426–4556 pg/ml; TNF-α 13.9 pg/ml, 4.1–291.6 pg/ml; ECP 32.3 ng/ml, 3.7–58.4 ng/ml). Our results demonstrate that conventional management of sinusitis is of little benefit in patients with chronic refractory sinusitis with an underlying immunodeficiency. More studies are needed to test antibiotic regimens, probably combined with surgical drainage and anti-inflammatory agents.     

Intestinal Secretory Factor Released by Macrophages Stimulated with Clostridium difficile Toxin A: Role of Interleukin 1β

Clostridium difficile toxin A is associated with enterocolitis in animals and humans. However, the mechanisms of its secretory and damaging effects are not totally understood. In this work, we examined the intestinal secretion of electrolytes and water caused by supernatants from macrophages stimulated with toxin A in rabbit ileal mucosa mounted in Üssing chambers. We also investigated the mechanism by which the intestinal secretory factor (ISF) is released from stimulated macrophages. Supernatants from macrophages stimulated with toxin A caused potent intestinal secretion (change in short-circuit current [ΔIsc], 76 μA · cm⁻²; P < 0.01). The release of the ISF was pertussis toxin sensitive (reduction, 61%; P < 0.01) and was also reduced (P < 0.05) by a protein synthesis inhibitor (67%), protease inhibitors (57%), a phospholipase A₂ inhibitor (54%), a cyclo-oxygenase inhibitor (62%), a dual cyclo- and lipoxygenase inhibitor (48%), a platelet-activating factor (PAF) receptor antagonist (55%), and tumor necrosis factor alpha (TNF-α) synthesis inhibitors (48%). However, this release was not inhibited by a lipo-oxygenase inhibitor. Monoclonal anti-interleukin 1β (IL-1β) but not anti-IL-1α antibody blocked (72%; P < 0.01) the secretory action of the ISF, as did recombinant human IL-1 receptor antagonist (80%; P < 0.01). High levels of IL-1β (3,476 pg/ml) were detected by an enzyme-linked immunosorbent assay in the above supernatants. Furthermore, the addition of IL-1β to the serosal side caused a potent secretory effect (ΔIsc, 80 μA · cm⁻²; P < 0.01). These results show that macrophages stimulated with toxin A release an ISF capable of provoking intestinal secretion. The regulation of this factor is dependent upon the activation of the G protein. In addition, prostaglandins, PAF, and TNF-α are involved in the release of the ISF. We conclude that IL-1β is probably the ISF released by macrophages in response to toxin A.     

Calreticulin promotes angiogenesis via activating nitric oxide signalling pathway in rheumatoid arthritis

Calreticulin (CRT) is a multi-functional endoplasmic reticulum protein implicated in the pathogenesis of rheumatoid arthritis (RA). The present study was undertaken to determine whether CRT was involved in angiogenesis via the activating nitric oxide (NO) signalling pathway. We explored the profile of CRT expression in RA (including serum, synovial fluid and synovial tissue). In order to investigate the role of CRT on angiogenesis, human umbilical vein endothelial cells (HUVECs) were isolated and cultured in this study for in-vitro experiments. Our results showed a significantly higher concentration of CRT in serum (5·4 ± 2·2 ng/ml) of RA patients compared to that of osteoarthritis (OA, 3·6 ± 0·9 ng/ml, P < 0·05) and healthy controls (HC, 3·7 ± 0·6 ng/ml, P < 0·05); and significantly higher CRT in synovial fluid (5·8 ± 1·2 ng/ml) of RA versus OA (3·7 ± 0·3 ng/ml, P < 0·05). High levels of CRT are expressed in synovial membrane localized predominantly to inflammatory cells and synovial perivascular areas in both the lining and sublining layers of RA synovial tissue (RAST). Increased nitric oxide (NO) production and phosphorylation level of endothelial nitric oxide synthase (eNOS) were measured in HUVECs following CRT stimulation, while the total eNOS expression was not significantly changed. Furthermore, CRT promoted the proliferation, migration and tube formation of HUVECs, which were significantly inhibited by a specific eNOS inhibitor. These findings suggested that CRT may be involved in angiogenesis events in RA through NO signalling pathways, which may provide a potential therapeutic target in the treatment of RA.     

Usefulness of Serum Leucine-Rich Alpha-2 Glycoprotein as a Disease Activity Biomarker in Patients with Rheumatoid Arthritis

Our study aimed to investigate whether serum leucine-rich alpha-2-glycoprotein (LRG) levels are elevated in patients with rheumatoid arthritis (RA). In addition, we assessed their correlation with disease activity parameters and pro-inflammatory cytokine, tumor necrosis factor-α (TNF-α). Our study included 69 patients with RA and 48 age- and sex-matched healthy controls. Serum concentrations of TNF-α and LRG were determined by enzyme-linked immunosorbent assay. Serum LRG concentrations were significantly elevated in patients with RA compared with those in healthy controls (30.8±14.4 vs. 22.2±6.1 ng/mL; P<0.001). In patients with RA, serum LRG levels were found to be correlated with disease activity score 28 (DAS28), erythrocyte sedimentation rate, and C-reactive protein levels (γ=0.671; γ=0.612; and γ=0.601, P<0.001, respectively), but not with serum TNF-α levels. Serum LRG levels in patients with an active disease status (DAS28≥2.6) were significantly higher than those in remission (DAS28<2.6) (36.45±14.36 vs. 24.63±8.81 ng/mL; P<0.001). Our findings suggest that serum LRG could contribute to the inflammatory process independent of TNF-α and it may be a novel biomarker for assessing inflammatory activity in patients with RA.

Graphical Abstract

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Upregulation of microRNA-23a/b promotes tumor progression and confers poor prognosis in patients with gastric cancer

Background and purpose: To investigate the clinical significance of microRNA (miR)-23a and miR-23b expression in human gastric cancer (GC). Methods: Quantitative RT-PCR was performed to detect the expression changes of miR-23a and miR-23b in 160 human GC tissues and paired normal mucosa. The associations between miR-23a and miR-23b expression, and the selected clinicopathological characteristics and patients’ prognosis were also evaluated. Results: MiR-23a (GC vs. Normal: 3.98 ± 1.23 vs. 2.29 ± 1.12, P < 0.001) and miR-23b (GC vs. Normal: 3.70 ± 1.24 vs. 1.58 ± 1.18, P < 0.001) expression were both increased dramatically when compared with paired normal mucosa. Notably, the expression levels of miR-23a in GC tissues were positively correlated with those of miR-23b (Spearman correlation coefficient r = 0.77, P < 0.001). Then, the coexpression of miR-23a and miR-23b (miR-23a-high/miR-23b-high) in GC tissues was significantly associated with the advanced TNM stage (P < 0.001), the presence of lymph node metastasis (P = 0.008) and the great depth of invasion (P = 0.02). Furthermore, both univariate and multivariate analyses showed that miR-23a/miR-23b co-expression was an independent predictor for unfavorable overall survival. Conclusions: These results suggest that the dysregulation of miR-23a and miR-23b may be implicated in the progression of human GC. Combined expression of miR-23a and miR-23b appears to be a valuable marker for prognosis of this disease.     

Effect of miR-200b on retinal endothelial cell function under high glucose environment

As one of the important complications of diabetes, diabetic retinopathy (DR) presented high incidence worldwide. Hyperglycemia is an important promoting factor for DR occurrence and development. It can damage retinal endothelial cell, resulting in retinal structure and function disorder. Studies have shown that miR-200b may involve in regulating DR occurrence and development, but its specific function and mechanism have not been elucidated. This study aimed to investigate miR-200b effect and mechanism on human retinal endothelial cells (hRECs) under high glucose environment. hRECs were cultured under high glucose or normal environment. Real time PCR was applied to detect miR-200b expression. MiR-200b was transfected to hRECs and MTT was used to detect its effect on hRECs proliferation under high glucose environment. Real time PCR and Western blot were performed to determine VEGF and TGFβ1 expression in the retina endothelial cells. MiR-200b expression decreased significantly under high glucose environment, whereas hRECs proliferated obviously. Compared with normal control, VEGF and TGFβ1 mRNA and protein expression increased markedly (P < 0.05). After miR-200b transfection, miR-200b expression increased, while VEGF and TGFβ1 mRNA and protein expression decreased obviously. Compared with high glucose group, hRECs proliferation was inhibited (P < 0.05). MiR-200b can regulate RECs growth and proliferation by changing VEGF and TGFβ1 expression to delay DR.     

Spatial and temporal differences of HMGB1 expression in the pancreas of rats with acute pancreatitis

We aimed to investigate the spatial and temporal differences in expression between HMGB1 and early-stage inflammatory cytokines (IL-1, IL-6 and TNF-α) in pancreas tissue in rats with acute pancreatitis. SD rats (BW 350 ± 30 g, n = 48) were randomly divided into the experimental group (n = 36) which were injected with 5% sodium taurocholate into the bilipancreatic duct retrogradely to produce acute necrotic pancreatitis (ANP) rat models, and the sham-operated (SO) group (n = 12) injected with equal dose of saline. The rats were sacrificed at different time points at 0 h, 3 h, 6 h, 12 h, and 24 h post modeling, respectively. The peripheral blood amylase and different inflammatory factors in ANP rats at different time points were detected by ELISA, and the expression of HMGB1 in the pancreatic tissue was detected by immunohistochemistry, Western blot and Q-PCR methods. Results showed that the serum amylase in the ANP model rats was significantly higher than the sham-operated group (P < 0.05). The early inflammatory factors (IL-1, TNF-α and IL-6) increased quickly at 3 h after the model induction, reached the peak level at 6 h (higher than SO group, P < 0.05), then decreased at 12 h, and at 24 h the levels were lower than those at 12 h (P < 0.05). The HMGB1 level in the pancreatitis tissue did not change significantly at 3 h and 6 h (P > 0.05), however, it increased remarkably at 12 h, and maintained up to 24 h (P > 0.05). As a late inflammatory factor, the expression of HMGB1 in acute pancreatitis was obviously later than the early inflammatory factors IL-1, TNF-α and IL-6. HMGB1 may play a key role in maintaining the development of the acute pancreatitis.     

Serum Cytokine Responses during Acute Human Granulocytic Ehrlichiosis

Human granulocytic ehrlichiosis (HGE) is caused by obligate intracellular bacteria in the Ehrlichia phagocytophila group. The disease ranges from subclinical to fatal. We speculated that cell-mediated immunity would be important for recovery from and potentially in the clinical manifestations of HGE; thus, serum tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), gamma interferon (IFN-γ), IL-10, and IL-4 concentrations were studied. IFN-γ (1,035 ± 235 pg/ml [mean ± standard error of the mean]) and IL-10 (118 ± 46 pg/ml) concentrations were elevated in acute-phase sera versus convalescent sera and normal subjects (P≤0.013 and P≤0.018, respectively). TNF-α, IL-1β, and IL-4 levels were not elevated. Cytokine levels in severely and mildly affected patients were not different. HGE leads to induction of IFN-γ-dominated cell-mediated immunity associated with clinical manifestations, recovery from infection, or both.     

Induction of proinflammatory cytokine and antioxidant enzyme gene expression following brief myocardial ischaemia

The purpose of this study was to determine if proinflammatory cytokines are up-regulated during reperfusion following sublethal ischaemia, and whether concurrent up-regulation of antioxidant enzymes occurs. Open-chest rats were subjected to 15 min of ischaemia followed by 1 or 3 h reperfusion (R). Myocardium from the ischaemic zone showed a significantly higher (P < 0.01) generation of thiobarbituric acid-reactive substances at 1 h and 3 h R. Northern blots showed a weak signal in controls for IL-6 mRNA (0.13 ± 0.01); this was elevated to 0.68 ± 0.12 at 1 h and 0.69 ± 0.10 at 3 h R. Neither IL-1β nor tumour necrosis factor-alpha (TNF-α) were detectable in controls. IL-1β rose to 0.78 ± 0.07 at 1 h and 0.51 ± 0.08 at 3 h R, and TNF-α rose to 0.69 ± 0.10 at 1 h and 0.38 ± 0.15 at 3 h R. Western blotting showed no signals in the control, but readily detectable signals at 1 h R; these remained high (IL-6) or decreased (IL-1β and TNF-α) at 3 h R. mRNA analysis for antioxidant enzymes revealed a weak signal in controls for catalase (CAT; 0.16 ± 0.08), glutathione peroxidase (GSH-Px; 0.15 ± 0.06) and superoxide dismutase (SOD; 0.21 ± 0.05). After 1 h R, levels increased significantly for CAT (0.46 ± 0.10; P < 0.025) and GSH-Px (0.51 ± 0.13; P < 0.01), but remained similar to controls for SOD (0.26 ± 0.15). At 3 h R the mRNA levels were significantly elevated for the three enzymes (CAT 0.48 ± 0.13; GSH-Px 0.47 ± 0.10; SOD 0.54 ± 0.08). We conclude that mRNA for proinflammatory cytokines is expressed early in reperfusion, and that the proteins are present in heart tissue. Also, reperfusion is associated with rapid expression of genes for antioxidant enzymes, which may enhance reactive oxygen intermediate (ROI) scavenging.     

Production of interleukins 10 and 12 by peripheral blood mononuclear cells (PBMC) in chronic hepatitis C virus (HCV) infection

We previously reported that interferon-gamma (IFN-γ) production by PBMC in response to HCV core protein was increased in patients with type C chronic liver disease. To understand better the pathophysiology of this disease, we evaluated production of IL-10 and IL-12 by PBMC from 41 patients with chronic HCV infection, including asymptomatic HCV carriers with persistently normal serum ALT values. IL-10 is known to inhibit many effector functions of the immune system, suppressing Th1-type cell development, while IL-12 stimulates differentiation of Th1-type cells, facilitating cell-mediated immunity. IL-10 production was determined by culturing lymphocytes with concanavalin A (Con A), while IL-12 was produced by monocytes in the presence of Staphylococcus aureusCowan 1 (SAC) with or without recombinant HCV core protein, respectively. The cytokine levels in culture supernatants were measured by ELISA. Spontaneous IL-10 production was greater in patients with chronic hepatitis (CH) (229±119 pg/ml, P<0.01) and liver cirrhosis (LC) (185±88 pg/ml, P<0.05) than in controls (119±27 pg/ml), while it was decreased during IFN treatment (70±25 pg/ml). Both HCV core protein and Con A enhanced IL-10 production by cells from HCV-infected patients. IL-12 was not detectable in medium alone cultures, and SAC-induced IL-12 production did not differ between various patient groups and controls. Simultaneous addition of HCV protein resulted in an increase of IL-12 production in chronic liver disease compared with SAC-alone cultures. Addition of IL-10 to the cultures equally suppressed IFN-γ production for both controls and patient groups, but the enhancing effect of IL-12 on IFN-γ production was significantly less in LC than in controls and other patient groups. The findings suggest that secretion of IL-10/IL-12 by cells from control individuals and various patient groups may be different, and that the cytokines might show different effects on IFN-γ production by some cells.