Bone Marrow Aspiration Concentrate and Platelet Rich Plasma for Osteochondral Repair in a Porcine Osteochondral Defect Model

Background

Bone marrow aspiration concentrate (BMAC) may possess a high potency for cartilage and osseous defect healing because it contains stem cells and multiple growth factors. Alternatively, platelet rich plasma (PRP), which contains a cocktail of multiple growth factors released from enriched activated thrombocytes may potentially stimulate the mesenchymal stem cells (MSCs) in bone marrow to proliferate and differentiate.

Methods

A critical size osteochondral defect (10×6 mm) in both medial femoral condyles was created in 14 Goettinger mini-pigs. All animals were randomized into the following four groups: biphasic scaffold alone (TRUFIT BGS, Smith & Nephew, USA), scaffold with PRP, scaffold with BMAC and scaffold in combination with BMAC and PRP. After 26 weeks all animals were euthanized and histological slides were cut, stained and evaluated using a histological score and immunohistochemistry.

Results

The thrombocyte number was significantly increased (p = 0.049) in PRP compared to whole blood. In addition the concentration of the measured growth factors in PRP such as BMP-2, BMP-7, VEGF, TGF-β1 and PDGF were significantly increased when compared to whole blood (p<0.05). In the defects of the therapy groups areas of chondrogenic tissue were present, which stained blue with toluidine blue and positively for collagen type II. Adding BMAC or PRP in a biphasic scaffold led to a significant improvement of the histological score compared to the control group, but the combination of BMAC and PRP did not further enhance the histological score.

Conclusions

The clinical application of BMAC or PRP in osteochondral defect healing is attractive because of their autologous origin and cost-effectiveness. Adding either PRP or BMAC to a biphasic scaffold led to a significantly better healing of osteochondral defects compared with the control group. However, the combination of both therapies did not further enhance healing.     

Integration of Stem Cell to Chondrocyte-Derived Cartilage Matrix in Healthy and Osteoarthritic States in the Presence of Hydroxyapatite Nanoparticles

We investigated the effectiveness of integrating tissue engineered cartilage derived from human bone marrow derived stem cells (HBMSCs) to healthy as well as osteoarthritic cartilage mimics using hydroxyapatite (HA) nanoparticles immersed within a hydrogel substrate. Healthy and diseased engineered cartilage from human chondrocytes (cultured in agar gels) were integrated with human bone marrow stem cell (HBMSC)-derived cartilaginous engineered matrix with and without HA, and evaluated after 28 days of growth. HBMSCs were seeded within photopolymerizable poly (ethylene glycol) diacrylate (PEGDA) hydrogels. In addition, we also conducted a preliminary in vivo evaluation of cartilage repair in rabbit knee chondral defects treated with subchondral bone microfracture and cell-free PEGDA with and without HA. Under in vitro conditions, the interfacial shear strength between tissue engineered cartilage derived from HBMSCs and osteoarthritic chondrocytes was significantly higher (p < 0.05) when HA nanoparticles were incorporated within the HBMSC culture system. Histological evidence confirmed a distinct spatial transition zone, rich in calcium phosphate deposits. Assessment of explanted rabbit knees by histology demonstrated that cellularity within the repair tissues that had filled the defects were of significantly higher number (p < 0.05) when HA was used. HA nanoparticles play an important role in treating chondral defects when osteoarthritis is a co-morbidity. We speculate that the calcified layer formation at the interface in the osteoarthritic environment in the presence of HA is likely to have attributed to higher interfacial strength found in vitro. From an in vivo standpoint, the presence of HA promoted cellularity in the tissues that subsequently filled the chondral defects. This higher presence of cells can be considered important in the context of accelerating long-term cartilage remodeling. We conclude that HA nanoparticles play an important role in engineered to native cartilage integration and cellular processes.     

Carnosol Inhibits Pro-Inflammatory and Catabolic Mediators of Cartilage Breakdown in Human Osteoarthritic Chondrocytes and Mediates Cross-Talk between Subchondral Bone Osteoblasts and Chondrocytes

Aim

The aim of this work was to evaluate the effects of carnosol, a rosemary polyphenol, on pro-inflammatory and catabolic mediators of cartilage breakdown in chondrocytes and via bone-cartilage crosstalk.

Materials and Methods

Osteoarthritic (OA) human chondrocytes were cultured in alginate beads for 4 days in presence or absence of carnosol (6 nM to 9 μM). The production of aggrecan, matrix metalloproteinase (MMP)-3, tissue inhibitor of metalloproteinase (TIMP)-1, interleukin (IL)-6 and nitric oxide (NO) and the expression of type II collagen and ADAMTS-4 and -5 were analyzed. Human osteoblasts from sclerotic (SC) or non-sclerotic (NSC) subchondral bone were cultured for 3 days in presence or absence of carnosol before co-culture with chondrocytes. Chondrocyte gene expression was analyzed after 4 days of co-culture.

Results

In chondrocytes, type II collagen expression was significantly enhanced in the presence of 3 μM carnosol (p = 0.008). MMP-3, IL-6, NO production and ADAMTS-4 expression were down-regulated in a concentration-dependent manner by carnosol (p<0.01). TIMP-1 production was slightly increased at 3 μM (p = 0.02) and ADAMTS-5 expression was decreased from 0.2 to 9 μM carnosol (p<0.05). IL-6 and PGE₂ production was reduced in the presence of carnosol in both SC and NSC osteoblasts while alkaline phosphatase activity was not changed. In co-culture experiments preincubation of NSC and SC osteoblasts wih carnosol resulted in similar effects to incubation with anti-IL-6 antibody, namely a significant increase in aggrecan and decrease in MMP-3, ADAMTS-4 and -5 gene expression by chondrocytes.

Conclusions

Carnosol showed potent inhibition of pro-inflammatory and catabolic mediators of cartilage breakdown in chondrocytes. Inhibition of matrix degradation and enhancement of formation was observed in chondrocytes cocultured with subchondral osteoblasts preincubated with carnosol indicating a cross-talk between these two cellular compartments, potentially mediated via inhibition of IL-6 in osteoblasts as similar results were obtained with anti-IL-6 antibody.     

Sputum IgE and Cytokines in Asthma: Relationship with Sputum Cellular Profile

Background

Local IgE production may play a role in asthma pathogenesis. The aim of the study was to assess sputum total IgE and cytokines in asthmatics according to sputum cellular phenotype.

Methods

We studied 122 subjects including 22 non atopic healthy subjects, 41 eosinophilic (sputum eosinophils ≥3%), 16 neutrophilic (sputum neutrophils >76%) and 43 pauci-granulocytic asthmatics (sputum eosinophils <3% and sputum neutrophils ≤76%) recruited from the asthma clinic at CHU Liege.Sputum supernatant total IgE (tIgE) was measured by ImmunoCAP and sputum supernatant cytokines (IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IFN-γ and TNF-α) were measured with the Luminex xMAP Technology by using commercially available Fluorokine MAP kits.

Results

After concentrating sputum samples, total IgE was detectable in the majority of subjects. Sputum IgE was raised in asthmatics when compared to healthy subjects. Overall, asthmatics did not significantly differ from healthy subjects with respect to cytokine levels. The eosinophilic asthma phenotype, however, was characterised by raised sputum tIgE, IL-5 and IL-13 compared to healthy subjects (p<0.001, p<0.001 and p<0.05 respectively) and pauci-granulocytic asthma (p<0.01, p<0.001 and p<0.05 respectively) and raised IL-5 compared to neutrophilic asthma (p<0.01). When patients were classified according to sputum IgE levels, it appeared that IL-5, IL-6, IL-17 and TNF-α sputum supernatant levels were raised in the “IgE high” asthmatics (IgE ≥0.1 kU/l) when compared to “IgE low” asthmatics (IgE<0.1 kU/l).

Conclusion

The eosinophilic asthma phenotype was associated with raised sputum IgE and a Th2 cytokine profile. Raised sputum IgE was associated with a heterogeneous cytokine overproduction.     

Prospective evaluation of serum biomarker levels and cartilage repair by autologous chondrocyte transplantation and subchondral drilling in a canine model

Introduction

The purpose of this study was to evaluate serum chondroitin sulfate (CS) and hyaluronic acid (HA) levels and the capability of cartilage repair of full-thickness cartilage defects after treatment with two different fundamental surgical techniques: autologous chondrocyte transplantation (AC) and subchondral drilling (SD).

Methods

A 4-mm-diameter full-thickness cartilage defect was created in each of 10 skeletally mature male outbred dogs. The dogs were randomly separated into two groups. Groups A and B were treated with AC and SD, respectively. An evaluation was made at the 24th week of the experiment. Serum was analyzed prospectively – preoperatively and at 6-week intervals – for CS and HA levels by enzyme-linked immunosorbent assay (ELISA) and ELISA-based assays, respectively.

Results

The cartilage repair assessment score (median ± standard deviation) of group A (9.5 ± 2.5) was significantly higher than that of group B (2.5 ± 1.3) (P < 0.05). Group A also demonstrated a better quality of hyaline-like cartilage repair. Prospective analysis of serum WF6 and HA levels between the two groups did not show any significant difference. Serum WF6 levels at the 24th week of the experiment had a negative correlation (r = -0.69, P < 0.05) with the cartilage repair assessment score, whereas serum HA levels tended to correlate positively (r = 0.46, 0.1 <P < 0.05).

Conclusions

AC treatment provides superior results to SD treatment, according to morphology, histology, and cartilage marker levels. AC treatment demonstrated a smoother surface, less fissure, better border integration, and a more reliable outcome of repairing cartilage. Moreover, a decreasing level of serum WF6, which correlated with good quality of the repairing tissue at the end of the follow-up period, was found predominantly in the AC group. Serum WF6 therefore should be further explored as a sensitive marker for the noninvasive therapeutic evaluation of cartilage repair procedures.     

Local IL-17 Production Exerts a Protective Role in Murine Experimental Glomerulonephritis

IL-17 is a pro-inflammatory cytokine implicated in the pathogenesis of glomerulonephritis and IL-17 deficient mice are protected from nephrotoxic nephritis. However, a regulatory role for IL-17 has recently emerged. We describe a novel protective function for IL-17 in the kidney. Bone marrow chimeras were created using wild-type and IL-17 deficient mice and nephrotoxic nephritis was induced. IL-17 deficient hosts transplanted with wild-type bone marrow had worse disease by all indices compared to wild-type to wild-type bone marrow transplants (serum urea p<0.05; glomerular thrombosis p<0.05; tubular damage p<0.01), suggesting that in wild-type mice, IL-17 production by renal cells resistant to radiation is protective. IL-17 deficient mice transplanted with wild-type bone marrow also had a comparatively altered renal phenotype, with significant differences in renal cytokines (IL-10 p<0.01; IL-1β p<0.001; IL-23 p<0.01), and macrophage phenotype (expression of mannose receptor p<0.05; inducible nitric oxide synthase p<0.001). Finally we show that renal mast cells are resistant to radiation and produce IL-17, suggesting they are potential local mediators of disease protection. This is a novel role for intrinsic cells in the kidney that are radio-resistant and produce IL-17 to mediate protection in nephrotoxic nephritis. This has clinical significance as IL-17 blockade is being trialled as a therapeutic strategy in some autoimmune diseases.     

Enhanced activity of NLRP3 inflammasome in peripheral blood cells of patients with active rheumatoid arthritis

IntroductionInterleukin-1β (IL-1β) is a major inflammatory cytokine, produced predominantly by innate immune cells through NLRP3-inflammasome activation. Both intrinsic and extrinsic danger signals may activate NLRP3. Genetic variations in NLRP3-inflammasome components have been reported to influence rheumatoid arthritis (RA) susceptibility and severity. We sought to assess the activity of NLRP3-inflammasome in patients with active RA compared to healthy individuals.MethodIntracellular protein expression of NLRP3, ASC, pro- and active caspase-1, pro- and active IL-1β was assessed by immunoblotting both at baseline and upon inflammasome activation. NLRP3 function (IL-1β secretion) was assessed upon priming of TLR2 (Pam(3)CysSK(4), TLR3 (poly(I:C)) or TLR4 (LPS) and ATP sequential treatment. We used caspase inhibitors (casp-1, 3/7 and 8) to assess their contribution to IL-1β maturation. All experiments were performed in whole blood cells.ResultsActive RA patients (n = 11) expressed higher basal intracellular levels of NLRP3 (p < 0.008), ASC (p < 0.003), active caspase-1 (p < 0.02) and pro-IL-1β (p < 0.001). Upon priming with TLR4 (LPS) and ATP, RA-derived cell extracts (n = 7) displayed increased expression of NLRP3 (p < 0.01) and active caspase-1 (p < 0.001). Secreted IL-1β in culture supernatants from whole blood cells activated with TLR4 (LPS) or TLR3 agonist (poly(I:C)) plus ATP was higher in RA patients (n = 20) versus controls (n = 18) (p < 0.02 for both). Caspase-1 inhibition significantly reduced IL-1β secretion induced by all stimuli, whereas caspase-8 inhibition affected only TLR4 and TLR3 cell priming.ConclusionPatients with active RA have increased expression of NLRP3 and NLRP3-mediated IL-1β secretion in whole blood cells upon stimulation via TLR3 and TLR4 but not TLR2. In these patients, IL-1β secretion seems to be predominately driven by caspase-1 and caspase-8. Targeting NLRP3 or downstream caspases may be of benefit in suppressing IL-1β production in RA.     

Omega 3 Fatty Acids Promote Macrophage Reverse Cholesterol Transport in Hamster Fed High Fat Diet

The aim of this study was to investigate macrophage reverse cholesterol transport (RCT) in hamster, a CETP-expressing species, fed omega 3 fatty acids (ω3PUFA) supplemented high fat diet (HFD). Three groups of hamsters (n = 6/group) were studied for 20 weeks: 1) control diet: Control, 2) HFD group: HF and 3) HFD group supplemented with ω3PUFA (EPA and DHA): HFω3. In vivo macrophage-to-feces RCT was assessed after an intraperitoneal injection of ³H-cholesterol-labelled hamster primary macrophages.Compared to Control, HF presented significant (p<0.05) increase in body weight, plasma TG (p<0.01) and cholesterol (p<0.001) with an increase in VLDL TG and in VLDL and LDL cholesterol (p<0.001).Compared to HF, HFω3 presented significant decrease in body weight. HFω3 showed less plasma TG (p<0.001) and cholesterol (p<0.001) related to a decrease in VLDL TG and HDL cholesterol respectively and higher LCAT activity (p<0.05) compared to HF. HFω3 showed a higher fecal bile acid excretion (p<0.05) compared to Control and HF groups and higher fecal cholesterol excretion (p<0.05) compared to HF. This increase was related to higher gene expression of ABCG5, ABCA1 and SR-B1 in HFω3 compared to Control and HF groups (<0.05) and in ABCG1 and CYP7A1 compared to HF group (p<0.05). A higher plasma efflux capacity was also measured in HFω3 using ³H- cholesterol labeled Fu5AH cells.In conclusion, EPA and DHA supplementation improved macrophage to feces reverse cholesterol transport in hamster fed HFD. This change was related to the higher cholesterol and fecal bile acids excretion and to the activation of major genes involved in RCT.     

BMP7 Gene Transfer via Gold Nanoparticles into Stroma Inhibits Corneal Fibrosis In Vivo

This study examined the effects of BMP7 gene transfer on corneal wound healing and fibrosis inhibition in vivo using a rabbit model. Corneal haze in rabbits was produced with the excimer laser performing -9 diopters photorefractive keratectomy. BMP7 gene was introduced into rabbit keratocytes by polyethylimine-conjugated gold nanoparticles (PEI2-GNPs) transfection solution single 5-minute topical application on the eye. Corneal haze and ocular health in live animals was gauged with stereo- and slit-lamp biomicroscopy. The levels of fibrosis [α-smooth muscle actin (αSMA), F-actin and fibronectin], immune reaction (CD11b and F4/80), keratocyte apoptosis (TUNEL), calcification (alizarin red, vonKossa and osteocalcin), and delivered-BMP7 gene expression in corneal tissues were quantified with immunofluorescence, western blotting and/or real-time PCR. Human corneal fibroblasts (HCF) and in vitro experiments were used to characterize the molecular mechanism mediating BMP7’s anti-fibrosis effects. PEI2-GNPs showed substantial BMP7 gene delivery into rabbit keratocytes in vivo (2×10⁴ gene copies/ug DNA). Localized BMP7 gene therapy showed a significant corneal haze decrease (1.68±0.31 compared to 3.2±0.43 in control corneas; p<0.05) in Fantes grading scale. Immunostaining and immunoblot analyses detected significantly reduced levels of αSMA (46±5% p<0.001) and fibronectin proteins (48±5% p<0.01). TUNEL, CD11b, and F4/80 assays revealed that BMP7 gene therapy is nonimmunogenic and nontoxic for the cornea. Furthermore, alizarin red, vonKossa and osteocalcin analyses revealed that localized PEI2-GNP-mediated BMP7 gene transfer in rabbit cornea does not cause calcification or osteoblast recruitment. Immunofluorescence of BMP7-transefected HCFs showed significantly increased pSmad-1/5/8 nuclear localization (>88%; p<0.0001), and immunoblotting of BMP7-transefected HCFs grown in the presence of TGFβ demonstrated significantly enhanced pSmad-1/5/8 (95%; p<0.001) and Smad6 (53%, p<0.001), and decreased αSMA (78%; p<0.001) protein levels. These results suggest that localized BMP7 gene delivery in rabbit cornea modulates wound healing and inhibits fibrosis in vivo by counter balancing TGFβ1-mediated profibrotic Smad signaling.     

Insulin Resistance in PCOS Patients Enhances Oxidative Stress and Leukocyte Adhesion: Role of Myeloperoxidase

Cardiovascular diseases and oxidative stress are related to polycystic ovary syndrome (PCOS) and insulin resistance (IR). We have evaluated the relationship between myeloperoxidase (MPO) and leukocyte activation in PCOS patients according to homeostatic model assessment of IR (HOMA-IR), and have explored a possible correlation between these factors and endocrine and inflammatory parameters. This was a prospective controlled study conducted in an academic medical center. The study population consisted of 101 PCOS subjects and 105 control subjects. We divided PCOS subjects into PCOS non-IR (HOMA-IR<2.5) and PCOS IR (HOMA-IR>2.5). Metabolic and anthropometric parameters, total and mitochondrial reactive oxygen species (ROS) production, MPO levels, interactions between human umbilical vein endothelial cells and leukocytes, adhesion molecules (E-selectin, ICAM-1 and VCAM-1) and proinflammatory cytokines (IL-6 and TNF-α) were evaluated. Oxidative stress was observed in PCOS patients, in whom there was an increase in total and mitochondrial ROS production and MPO levels. Enhanced rolling flux and adhesion, and a decrease in polymorphonuclear cell rolling velocity were also detected in PCOS subjects. Increases in IL-6 and TNF-α and adhesion molecules (E-selectin, ICAM-1 and VCAM-1) were also observed, particularly in the PCOS IR group, providing evidence that inflammation and oxidative stress are related in PCOS patients. HOMA-IR was positively correlated with hsCRP (p<0.001, r = 0.304), ROS production (p<0.01, r = 0.593), leukocyte rolling flux (p<0.05, r = 0.446), E-selectin (p<0.01, r = 0.436) and IL-6 (p<0.001, r = 0.443). The results show an increase in the rate of ROS and MPO levels in PCOS patients in general, and particularly in those with IR. Inflammation in PCOS induces leukocyte-endothelium interactions and a simultaneous increase in IL-6, TNF-α, E-selectin, ICAM-1 and VCAM-1. These conditions are aggravated by the presence of IR.     

Olmesartan Decreased Levels of IL-1β and TNF-α, Down-Regulated MMP-2, MMP-9, COX-2, RANK/RANKL and Up-Regulated SOCs-1 in an Intestinal Mucositis Model

Methotrexate (MTX) is a pro-oxidant compound that depletes dihydrofolate pools and is widely used in the treatment of leukaemia and other malignancies. The efficacy of methotrexate is often limited by mucositis and intestinal injury, which are major causes of morbidity in children and adults. The aim of this study was to evaluate the effect of olmesartan (OLM), an angiotensin II receptor antagonist, on an Intestinal Mucositis Model (IMM) induced by MTX in Wistar rats. IMM was induced via intraperitoneal (i.p.) administration of MTX (7 mg/kg) for three consecutive days. The animals were pre-treated with oral OLM at 0.5, 1 or 5 mg/kg or with vehicle 30 min prior to exposure to MTX. Small intestinal homogenates were assayed for levels of the IL-1β, IL-10 and TNF-α cytokines, malondialdehyde and myeloperoxidase activity. Additionally, immunohistochemical analyses of MMP-2, MMP-9, COX-2, RANK/RANKL and SOCS-1 and confocal microscopy analysis of SOCS-1 expression were performed. Treatment with MTX + OLM (5 mg/kg) resulted in a reduction of mucosal inflammatory infiltration, ulcerations, vasodilatation and haemorrhagic areas (p<0.05) as well as reduced concentrations of MPO (p<0.001) and the pro-inflammatory cytokines IL-1β (p<0.001) and TNF-a (p<0.01), and increase anti-inflammatory cytocine IL-10 (p<0.05). Additionally, the combined treatment reduced expression of MMP-2, MMP-9, COX-2, RANK and RANKL(p<0.05) and increased cytoplasmic expression of SOCS-1 (p<0.05). Our findings confirm the involvement of OLM in reducing the inflammatory response through increased immunosuppressive signalling in an IMM. We also suggest that the beneficial effect of olmesartan treatment is specifically exerted during the damage through blocking inflammatory cytocines.     

Sex Bias in Experimental Immune-Mediated,Drug-Induced Liver Injury in BALB/c Mice: Suggested Roles for Tregs,Estrogen, and IL-6

Background and Aims

Immune-mediated, drug-induced liver injury (DILI) triggered by drug haptens is more prevalent in women than in men. However, mechanisms responsible for this sex bias are not clear. Immune regulation by CD4+CD25+FoxP3+ regulatory T-cells (Tregs) and 17β-estradiol is crucial in the pathogenesis of sex bias in cancer and autoimmunity. Therefore, we investigated their role in a mouse model of immune-mediated DILI.

Methods

To model DILI, we immunized BALB/c, BALB/cBy, IL-6–deficient, and castrated BALB/c mice with trifluoroacetyl chloride-haptenated liver proteins. We then measured degree of hepatitis, cytokines, antibodies, and Treg and splenocyte function.

Results

BALB/c females developed more severe hepatitis (p<0.01) and produced more pro-inflammatory hepatic cytokines and antibodies (p<0.05) than did males. Castrated males developed more severe hepatitis than did intact males (p<0.001) and females (p<0.05). Splenocytes cultured from female mice exhibited fewer Tregs (p<0.01) and higher IL-1β (p<0.01) and IL-6 (p<0.05) than did those from males. However, Treg function did not differ by sex, as evidenced by absence of sex bias in programmed death receptor-1 and responses to IL-6, anti-IL-10, anti-CD3, and anti-CD28. Diminished hepatitis in IL-6-deficient, anti-IL-6 receptor α-treated, ovariectomized, or male mice; undetectable IL-6 levels in splenocyte supernatants from ovariectomized and male mice; elevated splenic IL-6 and serum estrogen levels in castrated male mice, and IL-6 induction by 17β-estradiol in splenocytes from naïve female mice (p<0.05) suggested that 17β-estradiol may enhance sex bias through IL-6 induction, which subsequently discourages Treg survival. Treg transfer from naïve female mice to those with DILI reduced hepatitis severity and hepatic IL-6.

Conclusions

17β-estradiol and IL-6 may act synergistically to promote sex bias in experimental DILI by reducing Tregs. Modulating Treg numbers may provide a therapeutic approach to DILI.     

Effect of Sfrp5 on Cytokine Release and Insulin Action in Primary Human Adipocytes and Skeletal Muscle Cells

Secreted frizzled-related protein 5 (Sfrp5) is an adipokine with anti-inflammatory and insulin-sensitizing properties in mice. However, the mechanism of Sfrp5 action, especially in humans, is largely unknown. Therefore, cytokine release and insulin signaling were analyzed to investigate the impact of Sfrp5 on inflammation and insulin signaling in primary human adipocytes and skeletal muscle cells (hSkMC). Sfrp5 neither affected interleukin (IL)-6, monocyte chemoattractant protein-1 (MCP-1) and adiponectin release from human adipocytes, nor IL-6 and IL-8 release from hSkMC. In tumor necrosis factor (TNF) α-treated adipocytes, Sfrp5 reduced IL-6 release by 49% (p<0.05), but did not affect MCP-1 and adiponectin release. In MCP-1-treated hSkMC, Sfrp5 did not affect cytokine secretion. In untreated adipocytes, Sfrp5 decreased the insulin-mediated phosphorylation of Akt-Ser473, Akt-Thr308, GSK3α-Ser21 and PRAS40-Thr246 by 34% (p<0.01), 31% (p<0.05), 37% (p<0.05) and 34% (p<0.01), respectively, and the stimulation of glucose uptake by 25% (p<0.05). Incubation with TNFα increased the phosphorylation of JNK and NFκB, and impaired insulin signaling. When Sfrp5 and TNFα were combined, there was no additional effect on insulin signaling and JNK phosphorylation, but phosphorylation of NFκB was reversed to basal levels. Sfrp5 had no effect on insulin signaling in untreated or in MCP-1 treated hSkMC. Thus, Sfrp5 lowered IL-6 release and NFκB phosphorylation in cytokine-treated human adipocytes, but not under normal conditions, and decreased insulin signaling in untreated human adipocytes. Sfrp5 did not act on hSkMC. Therefore, the cellular actions of Sfrp5 seem to depend on the type of tissue as well as its inflammatory and metabolic state.     

Cytokine profiles in the joint depend on pathology,but are different between synovial fluid,cartilage tissue and cultured chondrocytes

Introduction

This study aimed to evaluate whether profiles of several soluble mediators in synovial fluid and cartilage tissue are pathology-dependent and how their production is related to in vitro tissue formation by chondrocytes from diseased and healthy tissue.

Methods

Samples were obtained from donors without joint pathology (n = 39), with focal defects (n = 65) and osteoarthritis (n = 61). A multiplex bead assay (Luminex) was performed measuring up to 21 cytokines: Interleukin (IL)-1α, IL-1β, IL-1RA, IL-4, IL-6, IL-6Rα, IL-7, IL-8, IL-10, IL-13, tumor necrosis factor (TNF)α, Interferon (IFN)γ, oncostatin M (OSM), leukemia inhibitory factor (LIF), adiponectin, leptin, monocyte chemotactic factor (MCP)1, RANTES, basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), vascular growth factor (VEGF).

Results

In synovial fluid of patients with cartilage pathology, IL-6, IL-13, IFNγ and OSM levels were higher than in donors without joint pathology (P ≤0.001). IL-13, IFNγ and OSM were also different between donors with cartilage defects and OA (P <0.05). In cartilage tissue from debrided defects, VEGF was higher than in non-pathological or osteoarthritic joints (P ≤0.001). IL-1α, IL-6, TNFα and OSM concentrations (in ng/ml) were markedly higher in cartilage tissue than in synovial fluid (P <0.01). Culture of chondrocytes generally led to a massive induction of most cytokines (P <0.001). Although the release of inflammatory cytokines was also here dependent on the pathological condition (P <0.001) the actual profiles were different from tissue or synovial fluid and between non-expanded and expanded chondrocytes. Cartilage formation was lower by healthy unexpanded chondrocytes than by osteoarthritic or defect chondrocytes.

Conclusions

Several pro-inflammatory, pro-angiogenic and pro-repair cytokines were elevated in joints with symptomatic cartilage defects and/or osteoarthritis, although different cytokines were elevated in synovial fluid compared to tissue or cells. Hence a clear molecular profile was evident dependent on disease status of the joint, which however changed in composition depending on the biological sample analysed. These alterations did not affect in vitro tissue formation with these chondrocytes, as this was at least as effective or even better compared to healthy chondrocytes.     

Carvedilol Decrease IL-1β and TNF-α, Inhibits MMP-2, MMP-9, COX-2, and RANKL Expression,and Up-Regulates OPG in a Rat Model of Periodontitis

Periodontal diseases are initiated primarily by Gram-negative, tooth-associated microbial biofilms that elicit a host response that causes osseous and soft tissue destruction. Carvedilol is a β-blocker used as a multifunctional neurohormonal antagonist that has been shown to act not only as an anti-oxidant but also as an anti-inflammatory drug. This study evaluated whether Carvedilol exerted a protective role against ligature-induced periodontitis in a rat model and defined how Carvedilol affected metalloproteinases and RANKL/RANK/OPG expression in the context of bone remodeling. Rats were randomly divided into 5 groups (n = 10/group): (1) non-ligated (NL), (2) ligature-only (LO), and (3) ligature plus Carvedilol (1, 5 or 10 mg/kg daily for 10 days). Periodontal tissue was analyzed for histopathlogy and using immunohistochemical analysis characterized the expression profiles of MMP-2, MMP-9, COX-2, and RANKL/RANK/OPG and determined the presence of IL-1β, IL-10 and TNF-α, myeloperoxidase (MPO), malonaldehyde (MDA) and, glutathione (GSH). MPO activity in the group with periodontal disease was significantly increased compared to the control group (p<0.05). Rats treated with 10 mg/kg Carvedilol presented with significantly reduced MPO and MDA concentrations (p<0.05) in addition to presenting with reduced levels of the pro-inflammatory cytokines IL-1 β and TNF-α (p<0.05). IL-10 levels in Carvedilol-treated rats remained unaltered. Immunohistochemical analysis demonstrated reduced expression of MMP-2, MMP-9, RANK, RANKL, COX-2, and OPG in rats treated with 10 mg/kg Carvedilol. This study demonstrated that Carvedilol affected bone formation/destruction and anti-inflammatory activity in a rat model of periodontitis.     

Altered Responses to Homeostatic Cytokines in Patients with Idiopathic CD4 Lymphocytopenia

Idiopathic CD4 lymphocytopenia (ICL) is a rare immune deficiency characterized by a protracted CD4⁺ T cell loss of unknown etiology and by the occurrence of opportunistic infections similar to those seen in AIDS. We investigated whether a defect in responses to cytokines that control CD4⁺ T cell homeostasis could play a role in ICL. Immunophenotype and signaling responses to interleukin-7 (IL-7), IL-2, and thymic stromal lymphopoietin (TSLP) were analyzed by flow cytometry in CD4⁺ T cells from 15 ICL patients and 15 healthy blood donors. The induction of phospho-STAT5 after IL-7 stimulation was decreased in memory CD4⁺ T cells of some ICL patients, which correlated with a decreased expression of the IL-7Rα receptor chain (R = 0.74, p<0.005) and with lower CD4⁺ T cell counts (R = 0.69, p<0.005). IL-2 responses were also impaired, both in the Treg and conventional memory subsets. Decreased IL-2 responses correlated with decreased IL-7 responses (R = 0.75, p<0.005), pointing to combined defects that may significantly perturb CD4⁺ T cell homeostasis in a subset of ICL patients. Unexpectedly, responses to the IL-7-related cytokine TSLP were increased in ICL patients, while they remained barely detectable in healthy controls. TSLP responses correlated inversely with IL-7 responses (R = −0.41; p<0.05), suggesting a cross-regulation between the two cytokine systems. In conclusion, IL-7 and IL-2 signaling are impaired in ICL, which may account for the loss of CD4⁺ T cell homeostasis. Increased TSLP responses point to a compensatory homeostatic mechanism that may mitigate defects in γc cytokine responses.     

Retinoic Acid Correlates with Reduced Serum IL-10 And TGF-β in Allergic Rhinitis

Background:Retinoic acid (RA) plays a key role in naïve T cell differentiation into FOXP3+ Treg cell in the respiratory airways. The present study aims to investigate RA and Treg-related cytokine serum levels, salivary IgA levels, FOXP3 and IL-4 gene expression, and the relationships between RA serum levels and Treg-related cytokines in allergic rhinitis (AR) patients and healthy controls.Methods:Salivary IgA and serum IgE, RA, IL-10, and TGF-β concentrations were measured by ELISA in 37 AR patients and 30 age- and sex-matched healthy controls.Results:IL-10 and TGF-β concentrations were significantly less in AR patients than in healthy controls (p< 0.01 and P< 0.0001, respectively). Salivary IgA was significantly greater in patients than in controls (p< 0.05). RA was not significantly different between patients and controls (p> 0.05); however, a significant positive correlation was found between serum RA and both IL-10 and TGF-β in AR patients.Conclusion:Our data suggest that RA may influence AR risk via affecting the TGF-β and IL-10 production.Key Words: Allergic Rhinitis, Interleukin-10, Obesity, Retinoic Acid, Transforming Growth Factor-β     

Association between Inflammation and Cardiac Geometry in Chronic Kidney Disease: Findings from the CRIC Study

Background

Left ventricular hypertrophy (LVH) and myocardial contractile dysfunction are independent predictors of mortality in patients with chronic kidney disease (CKD). The association between inflammatory biomarkers and cardiac geometry has not yet been studied in a large cohort of CKD patients with a wide range of kidney function.

Methods

Plasma levels of interleukin (IL)-1β, IL-1 receptor antagonist (IL-1RA), IL-6, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β, high-sensitivity C-Reactive protein (hs-CRP), fibrinogen and serum albumin were measured in 3,939 Chronic Renal Insufficiency Cohort study participants. Echocardiography was performed according to the recommendations of the American Society of Echocardiography and interpreted at a centralized core laboratory.

Results

LVH, systolic dysfunction and diastolic dysfunction were present in 52.3%, 11.8% and 76.3% of the study subjects, respectively. In logistic regression analysis adjusted for age, sex, race/ethnicity, diabetic status, current smoking status, systolic blood pressure, urinary albumin- creatinine ratio and estimated glomerular filtration rate, hs-CRP (OR 1.26 [95% CI 1.16, 1.37], p<0.001), IL-1RA (1.23 [1.13, 1.34], p<0.0001), IL-6 (1.25 [1.14, 1.36], p<0.001) and TNF-α (1.14 [1.04, 1.25], p = 0.004) were associated with LVH. The odds for systolic dysfunction were greater for subjects with elevated levels of hs-CRP (1.32 [1.18, 1.48], p<0.001) and IL-6 (1.34 [1.21, 1.49], p<0.001). Only hs-CRP was associated with diastolic dysfunction (1.14 [1.04, 1.26], p = 0.005).

Conclusion

In patients with CKD, elevated plasma levels of hs-CRP and IL-6 are associated with LVH and systolic dysfunction.     

Effects of High-Intensity Swimming on Lung Inflammation and Oxidative Stress in a Murine Model of DEP-Induced Injury

Studies have reported that exposure to diesel exhaust particles (DEPs) induces lung inflammation and increases oxidative stress, and both effects are susceptible to changes via regular aerobic exercise in rehabilitation programs. However, the effects of exercise on lungs exposed to DEP after the cessation of exercise are not clear. Therefore, the aim of this study was to evaluate the effects of high-intensity swimming on lung inflammation and oxidative stress in mice exposed to DEP concomitantly and after exercise cessation. Male Swiss mice were divided into 4 groups: Control (n = 12), Swimming (30 min/day) (n = 8), DEP (3 mg/mL—10 μL/mouse) (n = 9) and DEP+Swimming (n = 8). The high-intensity swimming was characterized by an increase in blood lactate levels greater than 1 mmoL/L between 10th and 30th minutes of exercise. Twenty-four hours after the final exposure to DEP, the anesthetized mice were euthanized, and we counted the number of total and differential inflammatory cells in the bronchoalveolar fluid (BALF), measured the lung homogenate levels of IL-1β, TNF-α, IL-6, INF-ϫ, IL-10, and IL-1ra using ELISA, and measured the levels of glutathione, non-protein thiols (GSH-t and NPSH) and the antioxidant enzymes catalase and glutathione peroxidase (GPx) in the lung. Swimming sessions decreased the number of total cells (p<0.001), neutrophils and lymphocytes (p<0.001; p<0.05) in the BALF, as well as lung levels of IL-1β (p = 0.002), TNF-α (p = 0.003), IL-6 (p = 0.0001) and IFN-ϫ (p = 0.0001). However, the levels of IL-10 (p = 0.01) and IL-1ra (p = 0.0002) increased in the swimming groups compared with the control groups, as did the CAT lung levels (p = 0.0001). Simultaneously, swimming resulted in an increase in the GSH-t and NPSH lung levels in the DEP group (p = 0.0001 and p<0.002). We concluded that in this experimental model, the high-intensity swimming sessions decreased the lung inflammation and oxidative stress status during DEP-induced lung inflammation in mice.     

Secreted Protein Acidic and Rich in Cysteine (SPARC) Exacerbates Colonic Inflammatory Symptoms in Dextran Sodium Sulphate-Induced Murine Colitis

Background

Secreted Protein Acidic and Rich in Cysteine (SPARC) is expressed during tissue repair and regulates cellular proliferation, migration and cytokine expression. The aim was to determine if SPARC modifies intestinal inflammation.

Methods

Wild-type (WT) and SPARC-null (KO) mice received 3% dextran sodium sulphate (DSS) for 7 days. Inflammation was assessed endoscopically, clinically and histologically. IL-1β, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17A, IL-12/IL23p40, TNF-α, IFN-γ, RANTES, MCP-1, MIP-1α, MIP-1β, MIG and TGF-β1 levels were measured by ELISA and cytometric bead array. Inflammatory cells were characterised by CD68, Ly6G, F4/80 and CD11b immunofluorescence staining and regulatory T cells from spleen and mesenteric lymph nodes were assessed by flow cytometry.

Results

KO mice had less weight loss and diarrhoea with less endoscopic and histological inflammation than WT animals. By day 35, all (n = 13) KO animals completely resolved the inflammation compared to 7 of 14 WT mice (p<0.01). Compared to WTs, KO animals at day 7 had less IL1β (p = 0.025) and MIG (p = 0.031) with higher TGFβ1 (p = 0.017) expression and a greater percentage of FoxP3+ regulatory T cells in the spleen and draining lymph nodes of KO animals (p<0.01). KO mice also had fewer CD68+ and F4/80+ macrophages, Ly6G+ neutrophils and CD11b+ cells infiltrating the inflamed colon.

Conclusions

Compared to WT, SPARC KO mice had less inflammation with fewer inflammatory cells and more regulatory T cells. Together, with increased TGF-β1 levels, this could aid in the more rapid resolution of inflammation and restoration of the intestinal mucosa suggesting that the presence of SPARC increases intestinal inflammation.