[The fabrication and related properties study of chitosan-poly (lactide-co-glycolide) double-walled microspheres loaded with nerve growth factor].

OBJECTIVE: To evaluate the feasibility of the chitosan-poly (lactide-co-glycolide) (PLGA) double-walled microspheres for sustained release of bioactive nerve growth factor (NGF) in vitro. METHODS: NGF loaded chitosan-PLGA double-walled microspheres were prepared by emulsion-ionic method with sodium tripolyphosphate (TPP) as an ionic cross-linker. The double-walled microspheres were cross-linked by different concentrations of TPP [1%, 3%, 10% ( W/ V)]. NGF loaded PLGA microspheres were also prepared. The outer and inner structures of double-walled microspheres were observed by light microscopy, scanning electron microscopy, confocal laser scanning microscopy, respectively. The size and distribution of microspheres and fourier transform infra red spectroscopy (FT-IR) were analyzed. PLGA microspheres with NGF or chitosan-PLGA double-walled microspheres cross-linked by 1%, 3%, and 10%TPP concentration (set as groups A, B, C, and D respectively) were used to determine the degradation ratio of microspheres in vitro and the sustained release ratio of NGF in microspheres at different time points. The bioactivity of NGF (expressed as the percentage of PC12 cells with positive axonal elongation reaction) in the sustained release solution of chitosan-PLGA double-walled microspheres without NGF (set as group A1) was compared in groups B, C, and D. RESULTS: The chitosan-PLGA double-walled microspheres showed relative rough and spherical surfaces without aggregation. Confocal laser scanning microscopy showed PLGA microspheres were evenly uniformly distributed in the chitosan-PLGA double-walled microspheres. The particle size of microspheres ranged from 18.5 to 42.7 µm. The results of FT-IR analysis showed ionic interaction between amino groups and phosphoric groups of chitosan in double-walled microspheres and TPP. In vitro degradation ratio analysis showed that the degradation ratio of double-walled microspheres in groups B, C, and D appeared faster in contrast to that in group A. In addition, the degradation ratio of double-walled microsphere in groups B, C, and D decreased when the TPP concentration increased. There were significant differences in the degradation ratio of each group ( P<0.05). In vitro sustained release ratio of NGF showed that when compared with PLGA microspheres in group A, double-walled microspheres in groups B, C, and D released NGF at a relatively slow rate, and the sustained release ratio decreased with the increase of TPP concentration. Except for 84 days, there was significant difference in the sustained release ratio of NGF between groups B, C, and D ( P<0.05). The bioactivity of NGF results showed that the percentage of PC12 cells with positive axonal elongation reaction in groups B, C, and D was significantly higher than that in group A1 ( P<0.05). At 7 and 28 days of culture, there was no significant difference between groups B, C, and D ( P>0.05); at 56 and 84 days of culture, the percentage of PC12 cells with positive axonal elongation reaction in groups C and D was significantly higher than that in group B ( P<0.05), and there was no significant difference between groups C and D ( P>0.05). CONCLUSION: NGF loaded chitosan-PLGA double-walled microspheres have a potential clinical application in peripheral nerve regeneration after injury.

Combined utilization of untimed single urine of MCP-1 and TWEAK as a potential indicator for proteinuria in lupus nephritis: A case-control study.

The aim of this study was to determine whether combined utilization of untimed single urine monocyte chemoattractant protein 1 (uMCP-1) and tumor necrosis factor (TNF)-like weak inducer of apoptosis (uTWEAK) could serve as a screening test for proteinuria in patients with lupus nephritis (LN).A case-control study that contained 39 biopsy-proven LN patients, 20 non-LN systemic lupus erythematosus (SLE) patients, and 10 healthy controls (HCs) were carried out. Correlations between uMCP-1, uTWEAK, and traditional clinical markers were analyzed by Spearman correlation test. Diagnostic values of uMCP-1, uTWEAK, and urine albumin/creatinine ratio (uACR) in the assessment of proteinuria were investigated by receiver operating characteristic (ROC) curves.Biopsy-proven LN patients showed higher levels of uMCP-1 and uTWEAK than non-LN patients. uMCP-1 and uTWEAK were elevated in renal active patients (rSLEDAI ≥4). Both uMCP-1 and uTWEAK showed significant correlation with patients' rSLEDAI, 24-hour urine proteinuria (24hr UP), and anti-double-stranded DNA (anti-dsDNA) antibodies. No correlations of these 2 biomarkers between cystatin C (Cys-C), creatinine (Cr), and blood urea nitrogen (BUN) were observed. An algorithm combining the moderate sensitivity of uMCP-1 and high specificity of uTWEAK displayed great specificity and sensitivity for proteinuria screening.Both uMCP-1 and uTWEAK were positively correlated with the impairments of LN, and the combined utility of untimed single uMCP-1 and uTWEAK might be used as potential predictors for proteinuria in LN.

Performance of ultrasound to monitor Achilles enthesitis in patients with ankylosing spondylitis during TNF-a antagonist therapy.

Enthesitis is considered as the primary anatomical lesion in ankylosing spondylitis (AS). We aimed to investigate the potential of ultrasound to detect early changes after TNF-a antagonist therapy of Achilles enthesitis of AS patients. One hundred AS patients with active disease, requiring TNF-a antagonist therapy, were included (etanercept n = 25, infliximab n = 25, adalimumab n = 25, non-biologic disease-modifying antirheumatic drugs (DMARDs) n = 25). Physical examination was performed to evaluate disease activity and detect Achilles enthesitis and/or retrocalcaneal bursitis. Ultrasound of the Achilles enthesitis was performed bilaterally. Follow-up examinations were performed 3 months after the initiation of therapy. Gray scale (GS) scores, Power Doppler (PD) scores, and total additive scores (TS) decreased significantly during TNF-a antagonist therapy but not in traditional non-biologic traditional DMARDs group. The bath ankylosing spondylitis disease activity index (BASDAI), bath ankylosing spondylitis metrology index (BASMI), bath ankylosing spondylitis functional index (BASFI), and Maastricht ankylosing spondylitis enthesitis score (MASES) all showed significant improvements. When three different TNF-a antagonists were analyzed separately, no significant difference was observed in GS, PD, and total scores. Subclinical Achilles enthesitis, detected only with GS ultrasound, is present in a subset of AS patients and a significant improvement can be demonstrated after 3 months of TNF-a antagonist therapy. Doppler ultrasound provides a reliable estimation to monitor the therapeutic response to TNF antagonists in AS patients with Achilles enthesitis. TNF-a antagonists have been shown to be effective in decreasing ultrasound signs of enthesitis after 3 months of therapy in AS patients.

Platelets induce a proinflammatory phenotype in monocytes via the CD147 pathway in rheumatoid arthritis.

INTRODUCTION: Activated platelets exert a proinflammatory action that can be largely ascribed to their ability to interact with monocytes. However, the mechanisms that promote dynamic changes in monocyte subsets in rheumatoid arthritis (RA) have not been clearly identified. The aim of this study was to determine whether platelet activation and the consequent formation of monocyte-platelet aggregates (MPA) might induce a proinflammatory phenotype in circulating monocytes in RA. METHODS: The surface phenotype of platelets and the frequencies of monocyte subpopulations in the peripheral blood of RA patients were determined using flow cytometry. Platelets were sorted and co-cultured with monocytes. In addition, monocyte activation was assessed by measuring the nuclear factor κB (NF-κB) pathway. The disease activity was evaluated using the 28-joint disease activity score. RESULTS: Platelet activation, circulating intermediate monocytes (Mon2) and MPA formation were significantly elevated in RA, especially in those with active disease status. Furthermore, Mon2 monocytes showed higher CD147 expression and responded to direct cell contact with activated platelets with higher cytokine production and matrix metallopeptidase 9 (MMP-9) secretion, which increased the expression of CD147. After the addition of specific antibodies for CD147, those effects were abolished. Furthermore, the NF-κB-driven inflammatory pathway may be involved in this process. CONCLUSION: These findings indicate an important role of platelet activation and the consequent formation of MPA in the generation of the proinflammatory cytokine milieu and for the promotion and maintenance of the pathogenically relevant Mon2 monocyte compartment in RA, which is likely to play an important role in the pathogenesis of autoimmunity.

[The circulating fibrocytes are associated with the lung inflammation and fibrosis of mice with interstitial lung disease].

OBJECTIVE: To study the pathological process and characteristics of bleomycin (BLM)-induced interstitial lung disease (ILD) model and collagen-induced arthritis (CIA) complicated by BLM-induced ILD (CIA-ILD) model in mice and explore the correlations with circulating fibrocytes. METHODS: Ninety mice were randomly divided into normal saline group (S group), BLM group (B group), CIA-BLM group (CB group), with 30 mice in each group. On the 2, 7, 14, 21 and 28 days after BLM challenge, mice of each group were sacrificed. HE staining was used to detect the degrees of acute inflammation and sirius red staining to detect lung fibrosis. Immunohistochemicstry was adopted to detect alpha-smooth muscle actin expression. The peripheral blood was obtained to count the proportion of circulating fibrocytes (CD45⁺COL1⁺) by flow cytometry. The correlations between circulating fibrocytes and pathological changes of ILD were analyzed statistically. RESULTS: Both of the ILD model groups showed the dynamic process from pulmonary alveolitis to fibrosis gradually. Inflammatory lesion in the lungs of B group was the most obvious on the 7-14th day, and then there was a slow rehabilitation process on the 21th and 28th day. CB group began to present alveoli destruction and collagen deposition on the 14th day, and the inflammation was the most severe on the 28th day. Pulmonary hydroxyproline content was obviously ascended in CB group as compared with B group (P<0.05) after 14 days. An increase in the total number of circulating fibrocytes was observed in both B and CB groups. It tended to increase at first, and subsequently declined. The level of circulating fibrocytes was higher in CB group than in B group on day 14, 21 and 28 (P<0.05). The immunohistochemical results showed that there were many myofibroblasts infiltrating in CB group from the 14th day to 28th day. The number of circulating fibrocytes in peripheral blood was positively correlated with pulmonary inflammation, fibrosis degree and hydroxyproline content (r=0.847, 0.826, 0.735, P<0.01). CONCLUSION: The pathological process of CIA-ILD animal model is more close to rheumatoid arthritis-induced ILD development. Circulating fibrocytes may be involved in the inflammation and fibrosis progression of lung.

CD147 up-regulates calcium-induced chemotaxis, adhesion ability and invasiveness of human neutrophils via a TRPM-7-mediated mechanism.

OBJECTIVES: We aimed to investigate whether CD147 can up-regulate the chemotactic, adhesive and invasive properties of human neutrophils and to determine the mechanism underlying this process. METHODS: Human promyelocytic leukaemia cells (HL-60) cells and peripheral blood or synovial fluid neutrophils were isolated from RA patients. Under cyclophilin A (CypA) stimulation, chemotaxis, adhesion potential and invasion ability were assessed using chemotaxis, adhesion and invasiveness assays. Lipid raft isolation and western blot were used to determine the mechanism underlying the effects of CypA stimulation. RESULTS: CD147 up-regulates the calcium-induced chemotaxis, adhesion ability and invasiveness of human neutrophils in RA patients. Transient receptor potential melastatin 7 may be responsible for this phenomenon. CONCLUSION: These findings suggest that in RA patients, abundant CypA up-regulates the calcium-induced chemotactic, adhesive and invasive properties of neutrophils via direct binding to CD147. Cyclophilin-CD147 interactions might contribute to the destruction of cartilage and bone in RA.

Incorporation of chitosan microspheres into collagen-chitosan scaffolds for the controlled release of nerve growth factor.

BACKGROUND: Artifical nerve scaffold can be used as a promising alternative to autologous nerve grafts to enhance the repair of peripheral nerve defects. However, current nerve scaffolds lack efficient microstructure and neurotrophic support. METHODS: Microsphere-Scaffold composite was developed by incorporating chitosan microspheres loaded with nerve growth factor (NGF-CMSs) into collagen-chitosan scaffolds (CCH) with longitudinally oriented microchannels (NGF-CMSs/CCH). The morphological characterizations, in vitro release kinetics study, neurite outgrowth assay, and bioactivity assay were evaluated. After that, a 15-mm-long sciatic nerve gap in rats was bridged by the NGF-CMSs/CCH, CCH physically absorbed NGF (NGF/CCH), CCH or nerve autograft. 16 weeks after implantation, electrophysiology, fluoro-gold retrograde tracing, and nerve morphometry were performed. RESULTS: The NGF-CMSs were evenly distributed throughout the longitudinally oriented microchannels of the scaffold. The NGF-CMSs/CCH was capable of sustained release of bioactive NGF within 28 days as compared with others in vitro. In vivo animal study demonstrated that the outcomes of NGF-CMSs/CCH were better than those of NGF/CCH or CCH. CONCLUSION: Our findings suggest that incorporation of NGF-CMSs into the CCH may be a promising tool in the repair of peripheral nerve defects.

Ionically cross-linked chitosan microspheres for controlled release of bioactive nerve growth factor.

Controlled release of neurotrophic factors to target tissue via microsphere-based delivery systems is critical for the treatment strategies of diverse neurodegenerative disorders. The present study aims to investigate the feasibility of the controlled release of bioactive nerve growth factor (NGF) with ionically cross-linked chitosan microspheres (NGF-CMSs). The microspheres were prepared by the emulsion-ionic cross-linking method with sodium tripolyphosphate (STPP) as an ionic cross-linking agent. The size and distribution of the microspheres, SEM images, Fourier transform infra red spectroscopy (FT-IR), encapsulation efficiency, in vitro release tests and bioactivity assay were subsequently evaluated. We found that the microspheres had relatively rough surfaces with mean sizes between 20 and 31µm. FT-IR results provided evidence of ionic interaction between amino groups and phosphoric groups of chitosan and STPP. The NGF encapsulation efficiency ranged from 63% to 88% depending on the concentration of STPP. The in vitro release profiles of NGF from NGF-CMSs were influenced by the concentration of STPP. NGF-CMSs which were cross-linked with higher concentration of STPP showed slower but sustained release of NGF. In addition, the released NGF from NGF-CMSs was capable of maintaining the viability of PC12 cells, as well as promoting their differentiation. Taken together, our findings suggest that NGF-CMSs are capable of releasing bioactive NGF over 7 days, thus having potential application in nerve injury repair.

[Effect of cyclophilin A on monocyte-derived foam cells].

AIM: To observe whether cyclophilin A (CypA)has an effect on macrophage-derived foam cells, and to investigate the involvement of CypA in the development of atherosclerosis. METHODS: The foam cell model was established through incubating the human monocyte line (THP-1 cells) with oxidized low density lipoproteins (ox-LDL). The cells were stained with fresh oil red O to study the morphology of the macrophage-derived foam cells. The cell adhesion, invasion and the production of matrix metalloproteinase (MMPs) of the macrophage-derived foam cells were detected by adhesion assay, invasion assay and gelatin zymography respectively both in the absence or presence of different concentrations of purified CypA (50, 100, 200 µg/L). Then the foam cells were respectively pre-treated with CsA, c7b8f10, HAb18 mAb, and dual treatment of c7b8f10 and HAb18 mAb respectively, to investigate the inhibitory effect on macrophage-derived foam cells. RESULTS: The adhesion, invasion and the production of MMP-9 and MMP-2 were enhanced during the differentiation of monocytes into macrophages (P<0.05). CypA, especially in the concentration of 100 ng/mL, significantly promoted the function of macrophage-derived foam cells (P<0.05). CsA, c7b8f10, HAb18 mAb, and c7b8f10- HAb18 mAb combination dramatically inhibited the function of macrophage-derived foam cells both in the absence or presence of CypA (P<0.05). The c7b8f10- HAb18 mAb combination pretreatment had the most obviously suppressive effect on macrophage-derived foam cells (P<0.05). CONCLUSION: These findings suggest that CypA up regulates the adhesion, the invasion and the expressions of MMP-2 and MMP-9 in macrophage-derived foam cells. The CypA effect is blocked by the pretreatment of the different antagonists. This research might suggest the correlation between atherosclerosis pathogenesis and the vulnerability of atherosclerotic plaques, and thus give us some good ideas for atherosclerosis therapy in future.