Chondrocytes constantly receive external stimuli, which regulates remodeling. An optimal level of mechanical stress is essential for maintaining chondrocyte homeostasis, however, excessive mechanical stress induces inflammatory cytokines and protease, such as MMPs. Therefore, excessive mechanical stress is considered to be one of the main causes to cartilage destruction leading to osteoarthritis (OA). Integrins are well known as cell adhesion molecules and act as receptors for extracellular matrix (ECM), and are believed to control intracellular signaling pathways both physically and chemically as a mechanoreceptor. However, few studies have focused on the roles and functions of integrins in inflammation caused by excessive mechanical stress. In this study, we examined the relationship between integrins (αVβββ, TNF-α, MMP-3 and MMP-13 induced by excessive mechanical stress. In addition, protein expression of IL1-β and MMP-13 were also inhibited by the addition of cilengitide. Next, we investigated the involvement of intracellular signaling pathways in stress-induced integrin signaling in chondrocytes by using western blotting. The levels of p-FAK, p-ERK, p-JNK and p-p38 were enhanced by excessive mechanical stress and the enhancement was suppressed by treatment with cilengitide. In conclusion, this study revealed that excessive mechanical stress may activate integrin αVβββ, TNF-α, and MMP-3 and MMP-13 through the phosphorylation of FAK and MAPKs. This article is protected by copyright. All rights reserved.

Injury of the periodontium followed by inflammatory response often leads to root resorption. Resorption is accomplished by osteoclasts and their generation may depend on an interaction with the cells in direct contact with the root, the cementoblasts. Our study aimed to investigate the role of human cementoblasts in the formation of osteoclasts and the effect of interleukin (IL)-1β hereupon. Extracted teeth from healthy volunteers were subjected to sequential digestion by type I collagenase and trypsin. The effect of enzymatic digestion on the presence of cells on the root surface was analyzed by histology. Gene expression of primary human cementoblasts (pHCB) was compared with a human cementoblast cell line (HCEM). The pHCBs were analyzed for their expression of IL-1 receptors as well as of receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG). In a co-culture system consisting of osteoclast precursors (blood monocytes) and pHCBs, the formation of osteoclasts and their resorptive activity was assessed by osteo-assay and ivory slices. The cells obtained after a 120 min enzyme digestion expressed the highest level of bone sialoprotein, similar to that of HCEM. This fraction of isolated cells also shared a similar expression pattern of IL-1 receptors (IL1-R1 and IL1-R2). Treatment with IL-1β potently upregulated RANKL expression but not of OPG. pHCBs were shown to induce the formation of functional osteoclasts. This capacity was significantly stimulated by pretreating the pHCBs with IL-1β prior to their co-culture with human blood monocytes. Our study demonstrated that cementoblasts have the capacity to induce osteoclastogenesis, a capacity strongly promoted by IL-1β. These results may explain why osteoclasts can be formed next to the root of teeth.

Stress is associated with pathophysiology of both irritable bowel syndrome (IBS) and hypertension. Angiotensin receptor blockers (ARB) have anti-inflammatory properties via inhibition of angiotensin II (Ang II)/Ang II type I receptor axis (AT1). Inhibition of the classical RAS pathway is also involved in upregulation of angiotensin converting enzyme-2 (ACE2), which activates the Ang-(1-7)/Mas pathway to counteract inflammatory signaling and acts as a partner of the amino acid transporter, B0AT-1, to absorb tryptophan for regulation of microbiota-gut-brain axis. In this study, we determined the effects of ARB irbesartan on stress-induced intestinal inflammation. C57BL/6J mice were subjected to 2-week intermittent restraint stress. They were orally treated during the stress with either vehicle, 3 or 10 mg/kg/day irbesartan. Restraint stress resulted in colon inflammation with higher histological damage scores, increased expression of Nox4, TLR-4 and IL1-β, accumulation of reactive oxygen species (ROS), and activation of the ACE-angiotensin II-AT1 receptor axis. Stress also downregulated intestinal amino acid transporter, ACE2/B0AT-1, and activity of intestinal mammalian target of rapamycin (mTOR) and p70 S6 kinase (p70S6K), resulting in decrease in α-defensins, changes in intestinal microbial contents, and perturbation of tryptophan metabolism with activation of the kynurenine pathway. Administration of irbesartan inhibited activation of stress-induced AT1 pathway to reduce intestinal ROS accumulation and inflammation, restored expression of ACE2/B0AT-1, activity of mTOR and p70S6K, dysbiosis and tryptophan metabolism. Our results suggest that AT1 is a potentially suitable therapeutic target in stress-induced intestinal inflammation, and that irbesartan could be beneficially suitable for the treatment of stressed patients with IBS.

Immune function in the vagina of hen oviduct is essential to prevent infection by microorganisms colonizing in the cloaca. The aim of this study was to determine whether CpG oligodeoxynucleotides (CpG-ODN) stimulate the expression of avian β-defensins (AvBDs) in hen vaginal cells. Specific questions were whether CpG-ODN affects the expression of AvBDs and proinflammatory cytokines and whether the cytokines affect AvBDs expression in vaginal cells. The dispersed vaginal cells of White Leghorn laying hens were cultured and stimulated by different doses of lipopolysaccharide (LPS), CpG-ODN, interleukin 1β (IL1B), or IL6. The cultured cell population contained epithelial cells, fibroblast-like cells, and CD45-positive leukocytes. The immunoreactive AvBD3, -10, and -12 were localized in the mucosal epithelium in the section of the vagina. The expression of AvBDs, IL1B, and IL6 was analyzed by quantitative RT-PCR. RT-PCR analysis showed the expression of AvBD1, -3, -4, -5, -10, and -12 in the cultured vaginal cells without stimulation. Toll-like receptors (TLRs) 4 and 21, which recognize LPS and CpG-ODN respectively and IL1 and IL6 receptors (IL1R1 and IL6R) were also expressed in them. The expression of IL1B, IL6, and AvBD10 and -12 was upregulated by LPS, whereas only IL1B and IL6 were upregulated by CpG-ODN. IL1B stimulation upregulated AvBD1 and -3 expression, whereas IL6 stimulation did not cause changes in AvBDs expression. These results suggest that CpG-ODN derived from microbes upregulates the expression of IL1B and IL6 by interaction with TLR21 and then IL1B induces AvBD1 and -3 to prevent infection in the vagina.

Earlier studies showed that both tumor necrosis factor (TNF) and interleukin-1 (IL1) can inhibit virus replication in cultured cells. However, in human FS-4 fibroblasts, in which recombinant human TNF protected cells from encephalomyocarditis (EMC) virus infection, recombinant human IL1 alpha and IL1 beta failed to induce antiviral protection. Moreover, both forms of IL1 inhibited the development of the TNF-induced antiviral state. To elucidate the mechanism of this inhibition, we examined the effect of IL1 on the synthesis of interferon-beta (IFN-beta), stimulated with polyinosinate.polycytidylate [poly(I).poly(C)]. When added 2 h or more before poly(I).poly(C), both forms of IL1 had a strong inhibitory effect on IFN-beta synthesis, as determined by antiviral assay of the IFN-beta protein or by quantitation of IFN-beta mRNA levels in Northern blot analysis. However, when IL1 was added simultaneously with poly(I).poly(C), or 2 h after poly(I).poly(C), IFN-beta synthesis was increased. The inhibitory action of IL1 on poly(I).poly(C)-induced IFN-beta synthesis was abolished in the presence of cycloheximide, suggesting that it is mediated indirectly by an IL1-induced product in the FS-4 cells. In addition to its ability to inhibit IFN-beta synthesis, IL1 also caused a partial reversal of the antiviral action of IFN-beta. In contrast to IL1, TNF did not inhibit IFN-beta synthesis, nor did it interfere with the antiviral action of IFN-beta. Simultaneous addition of TNF and poly(I).poly(C) to FS-4 cells enhanced IFN-beta synthesis. Under proper conditions TNF and IFN-beta showed an additive antiviral effect.

Accumulating evidence suggests that the functional properties of alloactivated T cells may depend upon the microenvironment in which the T cells reside. For instance, we showed previously that heparan sulfate, a biologically active polysaccharide present on cell surfaces and extracellular matrices, modulates the proliferative responses of splenocytes through enhancement of cytokine and prostaglandin production by macrophages. Here we report that under conditions of suboptimal stimulation, heparan sulfate causes discrete alterations in the functional responses of murine cytolytic T cells. When present in a 5-day mixed leukocyte culture (MLC), heparan sulfate mediates an increase, from 3- to 10-fold, in T cell-mediated cytotoxicity. This increase is dose dependent and most pronounced when heparan sulfate is present in the highest concentration during the first 24 hr of the culture period. On the other hand, when added during the last 48-72 hr of an MLC, heparan sulfate decreases cytotoxicity by 3- to 30-fold. Neutralizing antibodies against IL-1 alpha, but not antibodies against IL1 beta, IL-6, or TNF alpha/beta, abrogate the heparan sulfate-mediated increase in cytotoxicity, suggesting that the increase depended in part upon the production of IL-1 alpha. However, studies in which exogenous IL-1 was added to MLC showed that increased cytotoxicity was not due only to increased cytokine production. Augmentation of cytotoxicity was in part independent of T cell help, as depletion of CD4+ cells from the responder population before MLC, or addition of neutralizing anti-murine IL-2 antibodies plus human IL-2 to the MLC, did not abrogate the stimulatory effect of heparan sulfate. Heparan sulfate-treated CD8+ lymphoblasts isolated after 7 days in MLC demonstrated an increased cytotoxicity, elevated intracellular serine esterase, and perforin levels compared with lymphoblasts from control MLC. The decrease in cytotoxicity observed when heparan sulfate was present during the last several days of an MLC was likely mediated by PGE2, as elevated levels of PGE2 were detected in MLC supernatants of heparan sulfate-treated cultures, and because the decrease was not observed in the presence of indomethacin. Our results are consistent with the idea that the metabolism of heparan sulfate, an endogenous component of parenchymal tissues, may regulate the tempo and magnitude of alloreactive cytotoxic T cell responses.

Sepsis is a complex biphasic syndrome characterized by both pro- and anti-inflammatory immune states. Whereas early sepsis mortality is caused by an acute, deleterious pro-inflammatory response, the second sepsis phase is governed by acute immunosuppression, which predisposes patients to long-term risk for life-threatening secondary infections. Despite extensive basic research and clinical trials, there is to date no specific therapy for sepsis, and mortality rates are on the rise. Although IFN-β is one of the most-studied cytokines, its diverse effects are not fully understood. Depending on the disease or type of infection, it can have beneficial or detrimental effects. As IFN-β has been used successfully to treat diverse diseases, emphasis has been placed on understanding the role of IFN-β in sepsis. Analyses of mouse models of septic shock attribute a pro-inflammatory role to IFN-β in sepsis development. As anti-inflammatory treatments in humans with antibodies to TNF-α or IL1-β resulted disappointing, cytokine modulation approaches were discouraged and neutralization of IFN-β has not been pursued for sepsis treatment. In the case of patients with delayed sepsis and immunosuppression, there is a debate as to whether the use of specific cytokines would restore the deactivated immune response. Recent reports show an association of low IFN-β levels with the hyporesponsive state of monocytes from sepsis patients and after endotoxin tolerance induction. These data, discussed here, project a role for IFN-β in restoring monocyte function and reversing immunosuppression, and suggest IFN-β-based additive immunomodulatory therapy. The dichotomy in putative therapeutic approaches, involving reduction or an increase in IFN-β levels, mirrors the contrasting nature of the early hyperinflammatory state and the delayed immunosuppression phase.

Human cytomegalovirus (HCMV) is secreted apically from villous trophoblasts, thus congenital infection is not likely to occur by basal release across the basement membrane. As an alternative route, we hypothesize that an HCMV-infected villous syncytiotrophoblast (ST) upregulates intercellular adhesion molecule (ICAM)-1, causing blood monocytes to bind to the ST and induce apoptosis. Purified (>99.99%) populations of human villous trophoblasts were differentiated into an ST-like culture, infected with HCMV strain AD169, and assessed for ICAM-1 expression by immunofluorescence. Infection strongly upregulated ICAM-1 24 h after challenge. ICAM-1 was also stimulated by transfection with viral genes IE2-55, IE1-72, and IE2-86, but not by UV-inactivated virus. Infection with a green fluorescent protein recombinant virus allowed infection and ICAM-1 expression to be topographically located. We found that ICAM-1 was expressed on both infected and noninfected cells. Furthermore, antibody to tumor necrosis factor (TNF)alpha and, to a lesser extent, interleukin (IL)1 beta inhibited ICAM-1 upregulation on noninfected cells but not on infected cells. We conclude that HCMV IE proteins stimulate ICAM-1 expression on villous trophoblasts by paracrine release of TNF alpha and IL1 beta, as well as by a direct effect on infected cells.

Periodontitis is very prevalent worldwide and is one of the major causes of tooth loss in adults. About 80% of the worldwide population use medicinal plants for their health care. Stemodia maritima L. (S. maritima) antioxidant and antimicrobial effects in vitro as well as anti-inflammatory properties. Herein, the potential therapeutic effect of S. maritima was assessed in rats subjected to experimental periodontitis (EP). EP was induced in female Wistar rats by nylon thread ligature around 2nd upper left molars for 11 days. Animals received (per os) S. maritima (0.2; 1 or 5 mg/kg) or vehicle (saline + DMSO) 1 h before ligature and then once daily for 11 days. The naive group had no manipulation. After this time-point, the animals were terminally anesthetized, and the maxillae were removed for morphometric and histological analyzes (HE). Gingival tissues were dissected to cytokine levels detection (TNF-α, IL1-β, CINC-1, and IL-10), enzymes superoxide dismutase (SOD), and catalase (CAT) analysis, as well as gene expression (TNF-α, IL-1β, RANK, and iNOS) by qRT-PCR. Systemic parameters (weight variation, plasma levels of hepatic enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT), creatinine, total alkaline phosphatase (TALP), and bone alkaline phosphatase (BALP) were performed. Histological analysis of the stomach, liver, kidney, and heart was also performed. S. maritima (5 mg/kg) decreased alveolar bone loss, TNF-α and CINC-1 gingival levels, oxidative stress, and transcription of TNF-α, IL1-β, RANK, and iNOS genes. It elevated both BALP activity and IL-10 gingival levels. The animals showed no any signs of toxicity. In conclusion, S. maritima reduced pro-inflammatory cytokine production, oxidative stress, and alveolar bone loss in a pre-clinical trial of periodontitis. S. maritima is a potential tool for controlling the development of periodontitis.

BACKGROUND

The zitter (zi/zi) rat, a loss-of-function mutant of the glycosylated transmembrane protein attractin (atrn), exhibits widespread age-dependent spongiform degeneration, hypomyelination, and abnormal metabolism of reactive oxygen species (ROS) in the brain. To date, the mechanisms underlying these phenotypes have remained unclear.

RESULTS

Here, we show differentiation defects in zi/zi oligodendrocytes, accompanied by aberrant extension of cell-processes and hypomyelination. Axonal bundles were relatively preserved during postnatal development. With increasing in age, the injured oligodendrocytes in zi/zi rats become pathological, as evidenced by the accumulation of iron in their cell bodies. Immunohistochemical analysis revealed that atrn expression was absent from an oligodendrocyte lineage, including A2B5-positive progenitors and CNPase-positive differentiated cells. The number and distribution of Olig2-positive oligodendrocyte progenitors was unchanged in the zi/zi brain. Furthermore, an in vitro differentiation assay of cultured oligodendrocyte progenitors prepared from zi/zi brains revealed their normal competence for proliferation and differentiation into mature oligodendrocytes. Interestingly, we demonstrated the accelerated recruitment of ED1-positive macrophages/microglia to the developing zi/zi brain parenchyma prior to the onset of hypomyelination. Semiquantitative RT-PCR analysis revealed a significant up-regulation of CD26 and IL1-beta in the zi/zi brain during this early postnatal stage.

CONCLUSIONS

We demonstrated that the onset of the impairment of oligodendrocyte differentiation occurs in a non-cell autonomous manner in zi/zi rats. Hypomyelination of oligodendrocytes was not due to a failure of the intrinsic program of oligodendrocytes, but rather, was caused by extrinsic factors that interrupt oligodendrocyte development. It is likely that macrophage/microglial activation in the zi/zi CNS leads to disturbances in oligodendrocyte differentiation via deleterious extrinsic factors, such as the cytokine IL1-beta or ROS. Atrn might be involved in the activation of brain macrophages/microglia by suppressing excessive migration of monocytes into the CNS, or by accelerating the transformation of brain monocytes into resting microglia. Understanding the pathogenesis of the zi/zi rat may provide novel insights into the developmental interaction betweens macrophages/microglia and cells of an oligodendrocyte lineage.

Intervertebral disc degeneration is implicated as a leading cause of low back pain. Persistent, local inflammation within the disc nucleus pulposus (NP) and annulus fibrosus (AF) is an important mediator of disc degeneration and negatively impacts the performance of therapeutic stem cells. There is a lack of validated large animal models of disc degeneration that recapitulate clinically-relevant local inflammation. We recently described a goat model of disc degeneration in which increasing doses of chondroitinase ABC (ChABC) were used to reproducibly induce a spectrum of degenerative changes. The objective of this study was to extend the clinical relevance of this model by establishing whether these degenerative changes are associated with local expression of inflammatory cytokines and catabolic enzymes. Degeneration was induced in goat lumbar discs using ChABC at different doses. After 12 weeks, degeneration severity was determined histologically and using quantitative MRI. Expression levels of inflammatory cytokines (TNF-α, IL-1β and IL-6) and catabolic enzymes (MMPs-1 and 13, and ADAMTS-4) were assessed as the percentage of immunopositive cells in the NP and AF. With the exception of MMP-1, cytokine and enzyme expression levels were significantly elevated in ChABC-treated discs in the NP and AF. Expression levels of TNF-α, IL1-β, and ADAMTS-4 were positively correlated with histological grade, while all cytokines and ADAMTS-4 were negatively correlated with MRI T2 and T1ρ scores. These results demonstrate that degenerate goat discs exhibit elevated expression of clinically-relevant inflammatory mediators, and further validate this animal model as a platform for evaluating new therapeutic approaches for disc degeneration. This article is protected by copyright. All rights reserved.

BACKGROUND

The aim of the present study was to evaluate the effects of hyperbaric oxygenation on lipid peroxidation, on the release of circulating cytokines (TNFa, IL6, IL1b) and endothelin-1 (ET1).

METHODS

METHODS

single arm, prospective study.

METHODS

ICU hyperbaric division of a University Hospital.

METHODS

fifteen healthy volunteers (10 male and 5 female, mean age 32+/-7 years) studied during hyperbaric oxygenation divided at random into two groups: group A (7 subjects) and group B (8 subjects).

METHODS

Both groups were consecutively pressurized at 2 atmospheres (2 atm abs) and 2.8 atm abs, with a constant descending rate of 1 m/min; in accordance with the experimental design, group A breathed pure oxygen continuously through facial masks and group B breathed chamber air during pressurization.

METHODS

Twenty millilitres of blood were drawn from all individuals at the following times: 1) basal, before HBO; 2) after 10 min at 2 atm abs; 3) after 10 min at 2.8 atm abs; 4) 30 min after the end of HBO. In all collected samples thiobarbituric reacting substances were evaluated, using the spectrophotometric technique, IL1 TNF and IL6 serum levels by ELISA and endothelin 1 plasma levels by radioimmunoassay.

RESULTS

In both groups, TBARS levels showed a twofold increase (p<0.05) in relation to the baseline, during and after hyperbaric oxygenation. Serum IL6 and IL1b values did not significantly change over the study in any of the volunteers. TNFa amounts significantly increased (p<0.05) during HBO, at 2 atm abs and 2.8 atm abs in both groups, with almost twofold increments. ET1 plasma values increased (p<0.05) in all volunteers during and after HBO: at 2 atm abs (range 7 to 24 pg/ml), 2.8 atm abs (range 7 to 19 pg/ml) and 30 min after (range 8 to 17 pg/ml) in relation to baseline (range 4 to 12 pg/ml). All the studied compounds had a similar trend in the two groups.

CONCLUSIONS

Hyperbaric oxygenation in healthy volunteers can induce not only lipid peroxidation, but also liberation of compounds such as TNFa and endothelins, no matter whether pure oxygen is breathed or not. These results suggest that the phenomenon behind this release might be leukocyte activation as induced by HBO. The possible role of ET1 in determining vasoconstriction occurring during HBO is also suggested.

BACKGROUND

Dengue viral infection is an acute infection that has the potential to have severe complications as its major sequela. Currently, there is no routine laboratory biomarker with which to predict the severity of dengue infection or monitor the effectiveness of standard management. Hence, this meta-analysis compared biomarker levels between dengue fever (DF) and severe dengue infections (SDI) to identify potential biomarkers for SDI.

METHODS

Data concerning levels of cytokines, chemokines, and other potential biomarkers of DF, dengue hemorrhagic fever, dengue shock syndrome, and severe dengue were obtained for patients of all ages and populations using the Scopus, PubMed, and Ovid search engines. The keywords "(IL1* or IL-1*) AND (dengue*)" were used and the same process was repeated for other potential biomarkers, according to Medical Subject Headings terms suggested by PubMed and Ovid. Meta-analysis of the mean difference in plasma or serum level of biomarkers between DF and SDI patients was performed, separated by different periods of time (days) since fever onset. Subgroup analyses comparing biomarker levels of healthy plasma and sera controls, biomarker levels of primary and secondary infection samples were also performed, as well as analyses of different levels of severity and biomarker levels upon infection by different dengue serotypes.

RESULTS

Fifty-six studies of 53 biomarkers from 3,739 dengue cases (2,021 DF and 1,728 SDI) were included in this meta-analysis. Results showed that RANTES, IL-7, IL-8, IL-10, IL-18, TGF-b, and VEGFR2 levels were significantly different between DF and SDI. IL-8, IL-10, and IL-18 levels increased during SDI (95% CI, 18.1-253.2 pg/mL, 3-13 studies, n = 177-1,909, I2 = 98.86%-99.75%). In contrast, RANTES, IL-7, TGF-b, and VEGFR2 showed a decrease in levels during SDI (95% CI, -3238.7 to -3.2 pg/mL, 1-3 studies, n = 95-418, I2 = 97.59%-99.99%). Levels of these biomarkers were also found to correlate with the severity of the dengue infection, in comparison to healthy controls. Furthermore, the results showed that IL-7, IL-8, IL-10, TGF-b, and VEGFR2 display peak differences between DF and SDI during or before the critical phase (day 4-5) of SDI.

CONCLUSIONS

This meta-analysis suggests that IL-7, IL-8, IL-10, TGF-b, and VEGFR2 may be used as potential early laboratory biomarkers in the diagnosis of SDI. This can be used to predict the severity of dengue infection and to monitor the effectiveness of treatment. Nevertheless, methodological and reporting limitations must be overcome in future research to minimize variables that affect the results and to confirm the findings.

The estrogenic character of tamoxifen and raloxifene was studied on three different genes, an ERE-reporter construct and two endogenous genes, sex hormone binding globulin (SHBG) and pS2, in two variants of the human liver carcinoma cell line HepG2. On the ERE-reporter construct and the pS2 gene both tamoxifen and raloxifene acted as pure estrogen antagonists, whereas on the SHBG gene they functioned as partial estrogens/antiestrogens at concentrations below 1 microM and as full "agonists" at concentrations higher than 1 microM. The fold stimulatory effect of tamoxifen and raloxifene on SHBG protein expression was similar in the estrogen receptor (ER) expressing HepG2 cells (HepER3) and the parental non-ER expressing HepG2 cells at concentrations above 1 microM. In contrast, the 17beta-estradiol analogue moxestrol stimulated SHBG expression only in the HepER3 cells. Both tamoxifen and raloxifene had an additive effect to estrogen receptor-dependent SHBG gene expression in the HepER3 cells in the presence of saturating concentrations of moxestrol. However, a significant difference was observed in that a much higher concentration of moxestrol was required to see an additive effect of raloxifene compared to tamoxifen. The cytokine IL1-beta completely blocked the tamoxifen-dependent induction of SHBG gene expression in HepER3 cells, but only partly blocked the effect of moxestrol mediated by the ER. In conclusion, our results suggest that the mechanism for the liver-selective "estrogenic" character of tamoxifen and raloxifene is mediated by a non-ER dependent pathway.

Hyperoxia exposure in premature infants increases the risk of subsequent lung diseases such as asthma and bronchopulmonary dysplasia. Fibroblasts help maintain bronchial and alveolar integrity. Thus understanding mechanisms by which hyperoxia influences fibroblasts is critical. Cellular senescence is increasingly recognized as important to pathophysiology of multiple diseases. We hypothesized that clinically-relevant moderate hyperoxia (<50% O₂) induces senescence in developing fibroblasts. Using primary human fetal lung fibroblasts, we investigated effects of 40% O₂ on senescence, ER stress and autophagy pathways. Fibroblasts were exposed to 21% or 40% O₂ for 7 days with etoposide as positive control to induce senescence, evaluated by morphological changes, β-galactosidase activity, and DNA damage markers. Senescence-associated secretory phenotype (SASP) profile of inflammatory and pro-fibrotic markers was further assessed. Hyperoxia decreased proliferation but increased cell size. SA-β-gal activity and DNA damage response, cell cycle arrest in G2/M phase and marked upregulation of phosphorylated p53 and p21 were noted. Reduced autophagy was noted with hyperoxia. mRNA expression of pro-inflammatory and pro-fibrotic factors (TNF-α, IL-1, IL-8, MMP3) was elevated by hyperoxia or etoposide. Hyperoxia increased several SASP factors (PAI-1, IL1-α, IL1-β, IL-6, LAP, TNF-α). The secretome of senescent fibroblasts promoted extracellular matrix formation by naïve fibroblasts. Overall, we demonstrate that moderate hyperoxia enhances senescence in primary human fetal lung fibroblasts with reduced autophagy but not enhanced ER stress. The resulting SASP is pro-fibrotic and may contribute to abnormal repair in the lung following hyperoxia.

In an attempt to identify cell surface molecules involved in recognition phenomena between cells such as keratinocytes and melanocytes and putatively target biological responses modifiers to keratinocytes, we undertook the detection of cell surface sugar specific receptors: membrane lectins. Keratinocyte membrane lectins were found to bind synthetic glycoproteins (neoglycoproteins) carrying either alpha-L-fucosyl or alpha-L-rhamnosyl residues. Fluorescence microscopy observations indicate that cultured keratinocytes are able to bind these two neoglycoproteins while frozen sections of human skin labelled with neoglycoprotein-coated covaspheres show that the selectivity of the binding to keratinocytes is restricted to alpha-L-rhamnosyl-BSA. Keratinocytes were adapted to grow on collagen; harvesting conditions allowing the analysis of keratinocytes by flow cytometry are described. This technique allows the quantification of the binding at 4 degrees C, and the estimation of the endocytosis of F-, neoglycoproteins: F-, alpha-L-Rha-BSA and F-, alpha-L-Fuc-BSA were efficiently internalized. Thereafter, alpha-L-rhamnose-substituted liposomes containing 5-(6)carboxyfluorescein were prepared in order to follow the delivery of the fluorescent dye into cells. This was measured both by flow cytometry and by spectrofluorimetry. The expression of surface lectins was checked upon action of cytokines (IL1 alpha, IL1 beta, IL2 and TNF) which are known as biological response modifiers of keratinocytes.

Neonatal sepsis is a significant health issue associated with high mortality. Immune responses associated with neonatal sepsis, such as proinflammatory cytokine production, are believed to play a central role in the pathogenesis of this disease. In the present study, serum levels of the proinflammatory cytokines TNF-α, IL1-β, and IL-6 and the anti-inflammatory cytokines IL-4 and IL-10 were evaluated for 25 subjects with neonatal sepsis. We observed that subjects with late onset of sepsis (LOS), as well as those with early onset of sepsis (EOS), had a substantial increase in serum TNF-α. In contrast to EOS, subjects with LOS demonstrated a significant increase in serum levels IL-6 and IL-10. Additionally, we observed a significant difference in cytokine profiles between acute and postacute cases of neonatal sepsis. For instance, the level of proinflammatory cytokines, such as TNF-α and IL-6, was elevated in the acute phase, whereas the production of anti-inflammatory cytokines, such as IL-10, became substantially upregulated during the postacute phase. Additionally, no correlation was observed between cytokine levels and CRP levels or lymphocyte counts. Thus, in contrast to CRP levels and lymphocyte counts, examination of the cytokine profile can provide valuable information when determining the most effective therapy for treating neonatal sepsis. This information may be useful to physicians when determining if anti-inflammatory or immune stimulatory therapy is warranted.

Phytomedicine based natural flavonoids have potent antioxidant, anti-inflammatory, and neuroprotective activities against neurodegenerative diseases. The aim of the present study is to investigate the potent neuroprotective and antioxidant potential effects of fisetin (natural flavonoid) against central nervous system (CNS)-insult, lipopolysaccharide (LPS)-induced reactive oxygen species (ROS), neuroinflammation, neurodegeneration, and synaptic/memory deficits in adult mice. The mice were injected intraperitoneally (i.p.) with LPS (250 μg/kg/day for 1 week) and a fisetin dosage regimen (20 mg/kg/day i.p. for 2 weeks, 1 week pre-treated to LPS and 1 week co-treated with LPS). Behavioral tests, and biochemical and immunofluorescence assays were applied. Our results revealed that fisetin markedly abrogated the LPS-induced elevated ROS/oxidative stress and activated phosphorylated c-JUN N-terminal Kinase (p-JNK) in the adult mouse hippocampus. Fisetin significantly alleviated LPS-induced activated gliosis. Moreover, fisetin treatment inhibited LPS-induced activation of the inflammatory Toll-like Receptors (TLR4)/cluster of differentiation 14 (CD14)/phospho-nuclear factor kappa (NF-κB) signaling and attenuated other inflammatory mediators (tumor necrosis factor-α (TNF-α), interleukin-1 β (IL1-β), and cyclooxygenase (COX-2). Furthermore, immunoblotting and immunohistochemical results revealed that fisetin significantly reversed LPS-induced apoptotic neurodegeneration. Fisetin improved the hippocampal-dependent synaptic and memory functions in LPS-treated adult mice. In summary, our results strongly recommend that fisetin, a natural potent antioxidant, and neuroprotective phytomedicine, represents a promising, valuable, and therapeutic candidate for the prevention and treatment of neurodegenerative diseases.

Reparative macrophages play an important role in cardiac repair post-myocardial infarction (MI). Bone marrow mononuclear cells (BM-MNCs) have been investigated as a donor for cell therapy but with limited clinical success. These cells, however, may be utilized as a source for reparative macrophages. This translational study aimed to establish a robust in vitro protocol to produce functional reparative macrophages from BM-MNCs and to establish pre-clinical evidence of the efficacy of reparative macrophage transplantation for the treatment of MI. Mouse BM-MNCs were treated with M-CSF plus IL-4, IL-10, TGF-β+F4/80⁺CD206⁺ reparative macrophages. Expression and secretion of tissue repair-related factors including IGF-1, TGF-βIL1-ra were remarkably enhanced in reparative macrophages compared to BM-MNCs. These cells were transplanted in a mouse MI model, resulting in evident improvement in cardiac function recovery, compared to BM-MNC transplantation. Histological studies showed that reparative macrophage transplantation enhanced myocardial tissue repair including augmented microvascular formation, reduced cardiomyocyte hypertrophy and attenuated interstitial fibrosis. Moreover, survival of reparative macrophages in the heart post-transplantation was increased compared to BM-MNCs. Reparative macrophage transplantation also increased host-derived reparative macrophages in part through TGF-β secretion. In conclusion, concomitant M-CSF + IL-4 treatment effectively produced reparative macrophages from BM-MNCs in vitro. Transplantation of produced reparative macrophage achieved a superior therapeutic efficacy, compared to BM-MNC transplantation, through the enhanced quantity and quality of donor cell engraftment. Further development of this advanced cell-based therapy is warranted.

Secreted frizzled-related protein 4 (SFRP4) is a member of secreted protein family with sequence similarity to frizzled receptors of wingless-related integration site (Wnt) signaling pathways. These proteins control diverse functions from embryonic development to adults in many organisms including humans. Initially, SFRPs were recognized as antagonists of Wnt signaling and supposed to interact with Wnts. Further research demonstrated their interactions to frizzled receptors and a functional diversity was related to these proteins, Wnt signaling potentiation in addition to modulation. SFRP4 is the largest member of SFRP family and is implicated in many diseases including obesity, type 2 diabetes (T2D), and cancer. SFRP4 acts as a biomarker for T2D and was expressed several years before clinical diagnosis of disease. This review mainly focusses on the role of SFRP4 in obesity and how it can lead to β-cell failure and ultimately to T2D. The role of SFRP4 in adipose tissues causing increased production of adipokines lead to the oxidative stress in pancreas that particularly have low amount of antioxidant enzymes in pancreatic β-cells leading to failure in exocytosis of insulin containing granules causing T2D. Obesity-induced inflammation is a principal factor in pathogenesis of insulin resistance as well as metabolic syndrome. Pro-inflammatory cytokines have potential to cause insulin resistance in skeletal muscles, adipose tissue, and liver via inhibition of insulin signal transduction. Secretion of SFRP4 is mediated by interleukin 1-β (IL1-β). This review highlights the molecular mechanisms by which SFRP4 leads to T2D. Understanding of molecular mechanism and targeting SFRP4 could help to eradicate or reduce chances of developing T2D.

BACKGROUND

Hippocampal damages, which are accompanied by inflammation, are among the main causes of epilepsy acquisition. We previously reported that chronic intracerebroventricular (i.c.v.) injection of lipopolysaccharide (LPS) modulates epileptogenesis in rats. There is a network of gap junction channels in the hippocampus that contribute to epileptogenesis. Gap junction channels are formed by oligomeric protein subunits called connexins (Cx). Astrocytic Cx43 and neuronal Cx36 are expressed in the hippocampus. In order to find out the possible role of gap junctions in seizure-modulating effect of LPS and neuroinflammation, we studied the effect of central administration of LPS on expression of Cx36 and Cx43 in rat hippocampus.

METHODS

LPS, 2.5 mug/rat/day, was injected i.c.v. to male Wistar rats for 14 days. mRNA and protein abundance of Cx36, Cx43 and IL1-β were measured in rat hippocampus by real time-PCR, Western blot and ELISA techniques, at the beginning, in the middle, and at the end of the treatment period.

RESULTS

IL1-β protein level was significantly increased 6 h after first injection of LPS. Cx36 and Cx43 mRNA expression did not alter during chronic administration of LPS. A selective decrease in Cx43 protein expression was observed after 7 injections of LPS.

CONCLUSIONS

It is suggested that Cx43 containing gap junctions in the hippocampus is down-regulated in response to chronic injection of LPS. This event can inhibit propagation of toxic and noxious molecules to neighboring cells and modulate hippocampal excitability and epileptogenesis.

Chronic focal encephalitis (CFE) generally presents with seizures that increase in severity and frequency as the disease progresses. Malfunction of synaptic transmission through altered glutamate signaling has been proposed as a likely mechanism triggering CFE. In addition, profuse inflammation is commonly seen in histopathological examination of resected tissue. To further explore the roles of glutamatergic activity and inflammation in this disease, we examined the expression of 52 genes by real time RT-PCR (kinetic RT-PCR or kRT-PCR) in a brain specimen from a CFE patient with active seizures, eight control specimens from patients with several other neurologic disorders, and two from individuals with no recorded history of neurological abnormalities. The CFE specimen displayed a dramatic increase in the expression of several inflammation-related genes (i.e. IL1 beta, IgVH, and IL2R gamma among others) and a striking down-regulation of several GluRs, in particular mGluR4. This type of analysis may prove useful in describing the molecular events underlying intractable epilepsy.

As B-lymphocytes play an important role in innate and adaptive immunity, we aimed to examine the effects of CpG oligodeoxynucleotides (ODNs) on purified tonsil-originating CD19+ B-cells, representing mucosal B-cells. We screened various K-type ODNs, reactive with human B-cells, and tested for the production of immunoglobulins in vitro. Using one CpG-ODN, DSP30, we observed that it could upregulate not only Toll-like receptor 9 (TLR9) mRNA expression in activated B-cells, but also the early expression of CD69 followed by the sequential expression of CD80, CD86 and the nuclear factor (NF)-kappaB pathway. Furthermore, mRNA expression of certain B-cell-derived cytokines was influenced by exposure to DSP30, with a strong upregulation of interleukin 6 (IL-6) and downregulation of IL1-beta. Stimulation of B-cells, co-stimulated with IL-2, IL-10 and soluble CD40 ligand (sCD40L) with different CpG-ODNs, had differing effects on the terminal differentiation in vitro of B-cells into immunoglobulin-secreting cells. TLR9 is involved in innate immunity and the recognition of bound CpG DNA from invading bacterial pathogens. As tonsillar B-cells are mucosal-type B-lymphocytes, this study suggests that CpG-ODNs show promise as mucosal adjuvants in modulating the local production of immunoglobulins of certain classes and subclasses, a crucial issue in vaccine perspectives.

The role of cytokines in the control of beta-2 microglobulin release from various haemopoietic cells was studied in vitro. Cell types investigated were resting cells or blasts of the T cell, B cell or monocyte-macrophage lineage. Mediators used in these experiments were r-IFN-alpha 2c, r-IFN-gamma, r-TNF-alpha, r-TNF-beta, r-IL1, r-IL2 and r-GM-CSF. Nanogram amounts of some of these mediators strongly affected beta-2 microglobulin release in vitro. r-IFN-gamma, r-IFN-alpha 2c and r-IL2 strongly enhanced and r-TNF-alpha and r-TNF-beta occasionally increased shedding of beta-2 microglobulin. r-IL1 and high concentrations of r-IFN-alpha 2c were inhibitory, whereas r-GM-CSF was ineffective. The impact of antigenic stimulation on beta-2 microglobulin release in mixed lymphocyte culture (MLC) was also studied. Stimulation with alloantigens in MLC greatly enhanced beta-2 microglobulin shedding, and this enhancement could be inhibited by a monoclonal antibody neutralizing human IFN-gamma. Among the various cell types studied, macrophages derived from peripheral blood monocytes were most susceptible to cytokine-induced alterations of beta-2 microglobulin shedding. From these data, we conclude that cytokines not only control the static expression of beta-2 microglobulin on the surface of haemopoietic cells but also largely affect their shedding.