A role of proinflammation has been implicated in the pathogenesis of diabetes, but the up-stream regulatory signals and molecular signatures are poorly understood. While histone modifications such as changes in histone deacetylase (HDAC) are emerging as novel epigenetic biomarkers, there is lack of studies to demonstrate their clinical relevance in diabetes. Therefore, we investigated the extent of HDAC machinery and inflammatory signals in peripheral blood mononuclear cells (PBMCs) from patients with type 2 diabetes mellitus (T2DM) compared to control subjects.

HDAC3 activity was significantly (p < 0.05) increased in patients with T2DM compared to control subjects. While subtypes of HDACs were differentially expressed at their transcriptional levels in patients with type 2 diabetes, the most prominent observation is the significantly (p < 0.05) elevated messenger RNA (mRNA) levels of HDAC3. Expression levels of Sirt1 which represents the class III HDAC were decreased significantly in T2DM (p < 0.05). Plasma levels of both TNF-α and IL-6 were significantly higher (p < 0.05) in patients with type 2 diabetes compared to control subjects. Among the proinflammatory mediators, the mRNA expression of MCP-1, IL1-β, NFκB, TLR2, and TLR4 were also significantly (p < 0.05) increased in T2DM. Transcriptional levels of DBC1 (deleted in breast cancer 1, which is a negative regulator of HDAC3) were seen significantly reduced in PBMCs from T2DM. Interestingly, HDAC3 activity/HDAC3 mRNA levels positively correlated to proinflammation, poor glycemic control, and insulin resistance.

Striking message from this study is that while looking for anti-inflammatory strategies and drugs with novel mode of action for T2DM, discovering and designing specific inhibitors targeted to HDAC3 appears promising.

BACKGROUND

Tumor microenvironment conferred by stromal (mesenchymal) stem cells (MSCs) plays a key role in tumor development, progression, and response to therapy. Defining the role of MSCs in tumorigenesis is crucial for their safe utilization in regenerative medicine. Herein, we conducted comprehensive investigation of the cross-talk between human MSCs (hMSCs) and 12 cancer cell lines derived from breast, prostate, colon, head/neck and skin.

METHODS

Human bone marrow-derived MSC line expressing green fluorescence protein (GFP) (hMSC-GFP) were co-cultured with the following cancer cell lines: (MCF7, BT-20, BT-474, MDA-MB-468, T-47D, SK-BR-3, MDA-MB-231, PC-3, HT-29, MDA-MB-435s, and FaDu) and changes in their morphology were assessed using fluorescent microscopy. For cellular tracking, cells were labeled with Vybrant DiO, DiL, and DiD lipophilic dyes. Time-lapse microscopy was conducted using Nikon BioStation IM-Q. Stable expression of mCherry, and luciferase genes was achieved using lentiviral technology. IL1-Beta neutralizing experiments were conducted using soluble recombinant IL-1R (srIL-1R). Changes in gene expression in sorted hMSCs were assessed using Agilent microarray platform while data normalization and bioinformatics were conducted using GeneSpring software.

RESULTS

We observed a dynamic interaction between cancer cells and hMSCs. High CDH1 (E-cadherin) and low IL1-Beta expression by cancer cells promoted reorganization of hMSCs into a niche-like formation, which was dependent on direct cell-cell contact. Our data also revealed transfer of cellular components between cancer cells and hMSCs as one possible mechanism for intercellular communication. Global gene expression analysis of sorted hMSCs following co-culturing with MCF7 and BT-20 cells revealed enrichment in signaling pathways related to bone formation, FAK and MAPKK signaling. Co-culturing hMSCs with MCF7 cells increased their growth evidenced by increase in Ki67 and PCNA staining in tumor cells in direct contact with hMSCs niche. On the other hand, co-culturing hMSCs with FaDu, HT-29 or MDA-MB-231 cells led remarkable decline in their cell growth.

CONCLUSIONS

Dynamic interaction exists between hMSCs and cancer cells. CDH1 and IL1-Beta expression by cancer cells mediates the crosstalk between hMSCs and cancer cells. We propose a model where hMSCs act as the first line of defense against cancer cell growth and spread.

Sublethal oxidative stresses increase the proportions of human fibroblasts positive for senescence associated beta-galactosidase activity and accelerate the transition in the fibroblast morphotypes characterising fibroblast ageing. Stimulation of fibroblasts with TNF-alpha or IL-1alpha transiently increases the production of reactive oxygen species (ROS) in human fibroblasts. Here we propose that repeated stimulation of WI-38 fibroblasts with TNF-alpha or IL-1alpha can generate enough ROS to accelerate the transition in the fibroblast morphotypes and increase the proportion of cells positive for senescence associated beta-galactosidase activity. The involvement of ROS is suggested by experiments where the stimulation of fibroblasts with TNF-alpha or IL-1alpha are performed in the presence of N-acetylcysteine which increases the intracellular antioxidant potential. It is proposed that the decrease in the proportions of morphotypes I and II, and the increase in the proportions of morphotypes III to VI observed after successive stimulation with TNF-alpha or IL1-alpha is attributed to an increased ROS production occurring during the stimulation.

Toll-like receptors (TLRs) that sense the microbial pathogens are important components of host immune system. TLRs play key roles in the innate defence mechanism against pathogens, in the development of adaptive immunity, and are possibly the major determinants of the susceptibility to infections. To study the resistance pattern in different breeds of chicken, a comprehensive understanding of TLR4 signalling pathways is required. We investigated the TLR-4 pathway regulated gene expressions in PBMCs of chicken breeds of Broiler (Cobb), Aseel, Dahlem Red and Ghagus upon LPS treatment using Quantitative RT-PCR approach. Several genes were found to be up regulated in both TLR-induced MyD88-dependent and MyD88-independent pathways. These genes include TLR4 (Toll-like receptor 4), MyD88 (Myeloid differentiation primary response gene 88), TRAF6 (TNF receptor associated factor 6), TRIF (TIR domain containing adapter inducing interferon beta), the transcription factors NFkB (Nuclear factor kappa B), IRF7 (Interferon regulatory factor 7) and IFN β (Interferon beta). We have also studied inflammatory cytokines such as IL2, IL6, IL8, IL1 β and TNF α to further understand the downstream signalling of TLR4 pathway. These results showed that higher expression of TLR signalling activation via both MyD88-dependent and TRIF-dependent pathways are more beneficial to chicken mononuclear cells mediated innate immunity. We observed TRIF dependent pathway in Aseel and Ghagus breeds. Our results are in concurrent with general observation that Aseel breed is comparatively more resistant, Ghagus and broilers are moderately resistant and Dahlem Red is comparatively more susceptible to bacterial infections.

OBJECTIVE

Proinflammatory cytokines released during inflammation can cause hyperexcitability in pain transmission neurons, leading to hyperalgesia and allodynia. Polymorphisms in interleukin 1 (IL-1) family of genes (IL1A, IL1B) and in IL-1 receptor antagonist (IL-1Ra, coded by IL1RN) may therefore induce alterations in cytokine levels/effects and pain related response. Our purpose was to investigate the influence of polymorphisms in IL1A/B/RN on cytokine serum levels and its correlation with pain intensity, performance status, adverse effects, metastases and breakthrough pain in Caucasian cancer patients.

METHODS

Serum IL-1α/β levels of 74 cancer patients were measured by competitive enzyme immunosorbent assay. All patients were also genotyped for the polymorphisms in IL1A (rs17561), IL1B (rs1143634) and IL1RN (rs419598) with Real-Time PCR. Results were then correlated to the appearance of bone or CNS metastases and several pain-related parameters.

RESULTS

IL-1β rs1143634 homozygous for T allele were associated with lower levels of IL1-β (p=0.032, Mann-Whitney test) and presented a trend for lower levels of pain (p=0.06, Fisher's Exact Test). Also, IL1-β levels were related with cancer onset status, since a four-fold increase probability of metastatic disease was observed in high IL-1β individuals (OR=4.074, p=0.010, Pearson χ(2) test). Among the female patients presenting metastatic disease and carriers of the TT genotype we observed a trend to lower levels of IL1-β (p=0.053, Pearson χ(2) test).

CONCLUSIONS

Our results indicate that genetic variation at IL1-β gene may influence serum levels of IL1-β, with proportional consequences in cancer-related pain.

OBJECTIVE

Proinflammatory cytokines are known to provoke degradative signaling cascades that promote extracellular matrix disintegration in articular cartilage. Because integration of the repair tissue into the surrounding native cartilage to produce a mechanically stable interface has a profound impact on the viability and functionality of the restored joint surface, this study examined the effects of proinflammatory cytokines on the properties of tissue-engineered cartilage in the context of integration.

METHODS

Using an established in vitro cartilage defect model, we examined the integration of chondrocyte-laden agarose constructs into native articular cartilage and the biochemical and biomechanical alterations of these implants upon treatment with interleukin 1-beta (IL1-beta) and tumor necrosis factor-alpha (TNF-alpha). Additionally, we probed extracellular regulated kinase (ERK) signaling involvement in response to proinflammatory cytokines.

RESULTS

The time-dependent accumulation of extracellular matrix and concomitant increase in Young's modulus observed in the absence of cytokines was significantly decreased upon IL1-beta and TNF-alpha treatment. Push-out test showed the highest interface strength in hybrid cultures maintained without cytokines, which was significantly lowered with IL1-beta and TNF-alpha treatment. Histological characteristics of the interface region are consistent with the biochemical findings. Treatment with an inhibitor of ERK pathway antagonized the deleterious effects caused by both cytokines.

CONCLUSIONS

This study is the first to show the functional catastrophic effects of IL1-beta and TNF-alpha on the biochemical, structural, and integrative properties of tissue-engineered cartilage and their significant counteraction by the blockade of ERK signaling pathway. With the discovery of new potential chemical entities, ERK inhibitor may emerge as a new therapeutic approach for functional integration and mechanical integrity of an engineered cartilage to the host tissue and, therefore, enhance long-term viability and functionality of the restored joint surface.

BACKGROUND

A crucial step in the metastatic spread of ovarian cancer (OC) is the adhesion and implantation of tumor cells to the peritoneal mesothelium. In order to study this step in the cascade, we derived a pro-metastatic human ovarian carcinoma cell line (MFOC3) from the non-metastatic FOC3 line.

METHODS

Molecular profiling of the isogeneic lines identified differentially expressed genes, and investigation for a role in dissemination for specific factors was achieved by development of a co-culture adhesion assay utilizing monolayers of human mesothelial cells.

RESULTS

After murine intraperitoneal inoculation, the FOC3 cell line formed no metastases, but the MFOC3 subline formed metastases in>> 80% of SCID mice. MFOC3 cells also adhered 2-3 times more avidly to mesothelial monolayers. This adhesion was inhibited by neutralizing antibodies to IL-1β and enhanced by recombinant IL-1β (p < 0.01). IL-1β induced mesothelial cell β1-integrin, and an antibody to this subunit also inhibited the adhesion of MFOC3 to mesothelial cells in vitro and significantly reduced metastases in vivo. Immunohistochemical analysis of a cohort of 96 ovarian cancer cases showed that negative IL-1β expression was significantly associated with an improved overall survival rate.

CONCLUSIONS

These results suggest that a IL-1β/β1-integrin axis plays a role in ovarian tumor cell adhesion to mesothelia, a crucial step in ovarian cancer dissemination.

OBJECTIVE

Nanoemulsions (LDE) with a lipid composition resembling that of LDL can concentrate in aortic lesions and when associated with anti-blastic agents, such as paclitaxel or etoposide, decrease atherosclerotic lesions induced in rabbits. Our aim was to test the association of a lipophilic derivative of methotrexate, didodecyl-methotrexate (ddMTX) to LDE on the lesions and on the expression of pro-inflammatory and anti-inflammatory genes.

METHODS

Twenty male New Zealand rabbits were fed 1 % cholesterol diet for 60 days. Starting from day 30, 10 animals were treated with 4 weekly LDE-ddMTX injections (4 mg/kg, I.V.) and 10 with LDE injections (20 mg LDE total lipid mass/kg).

RESULTS

LDE-ddMTX reduced the size of the lesion areas by 65 % and the intima-media ratio by 2-fold. Reduction of intimal macrophage was 67 % and of apoptotic cells was 88 %. Smooth muscle cells migration into the intima was unaffected. LDE-ddMTX treatment diminished metalloproteinase-9 in the intima. In aortas of atherosclerotic rabbits, downregulation of 6 pro-inflammatory genes, TNF-α, MCP-1, IL-1β, IL-18, MMP-9, MMP-12 and upregulation of the anti-inflammatory IL-10 gene were observed. Incubation of LDE-ddMTX with HUVEC cells led to downregulation of TNF-α IL1-β VAP-1, TLR2 and CXCL2.

CONCLUSIONS

LDE-ddMTX is potentially useful to threat atherosclerosis by acting on inflammatory processes which are instrumental in the development of the disease.

Chronic inflammation is associated with up-regulation of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and excessive inflammatory cytokine secretion by M1 macrophages. The anti-inflammatory cytokine interleukin (IL)-4 converts pro-inflammatory M1 macrophages into an anti-inflammatory and tissue-regenerative M2 phenotype, thus reducing inflammation and enhancing tissue regeneration. We have generated NF-κB responsive, or constitutively active IL-4 expression lentiviral vectors transduced into murine bone marrow-derived mesenchymal stromal cells (MSCs). MSCs with a constitutively active IL-4 expression vector produced large quantities of IL-4 continuously, whereas IL-4 secretion was significantly induced by lipopolysaccharide (LPS) in the NF-κB sensing MSCs. In contrast, LPS had no effect on MSCs with IL-4 secretion driven by a constitutively active promoter. We also found that intermittent and continuous LPS treatment displayed distinct NF-κB activation profiles, and this regulation was independent of IL-4 signaling. The supernatant containing IL-4 from the LPS-treated MSCs suppressed M1 marker (inducible nitric oxide synthase [iNOS] and tumor necrosis factor alpha [TNFα]) expression and enhanced M2 marker (Arginase 1, CD206 and IL1 receptor antagonist [IL1Ra]) expression in primary murine macrophages. The IL-4 secretion at the basal, non-LPS induced level was sufficient to suppress TNFα and enhance Arginase 1 at a lower level, but had no significant effects on iNOS, CD206 and IL1Ra expression. Finally, IL-4 secretion at basal or LPS-induced levels significantly suppressed osteogenic differentiation of MSCs. Our findings suggest that the IL-4 secreting MSCs driven by NF-κB sensing or constitutive active promoter have great potential for mitigating the effects of chronic inflammation and promoting earlier tissue regeneration.

We performed a combined approach to identify suspected allergy to knee arthroplasty (TKR): patch test (PT), lymphocyte transformation test (LTT), histopathology (overall grading; T- and B-lymphocytes, macrophages, and neutrophils), and semiquantitative Real-time-PCR-based periprosthetic inflammatory mediator analysis (IFNγ, TNFα, IL1-β, IL-2, IL-6, IL-8, IL-10, IL1β). We analyzed 25 TKR patients with yet unexplained complications like pain, effusion, and reduced range of motion. They consisted of 20 patients with proven metal sensitization (11 with PT reactions; 9 with only LTT reactivity). Control specimens were from 5 complicated TKR patients without metal sensitization, 12 OA patients before arthroplasty, and 8 PT patients without arthroplasty. Lymphocytic infiltrates were seen and fibrotic (Type IV membrane) tissue response was most frequent in the metal sensitive patients, for example, in 81% of the PT positive patients. The latter also had marked periprosthetic IFNγ expression. 8/9 patients with revision surgery using Ti-coated/oxinium based implants reported symptom relief. Our findings demonstrate that combining allergy diagnostics with histopathology and periprosthetic cytokine assessment could allow us to design better diagnostic strategies.

CPT-11 is a drug used as chemotherapy for colorectal cancer. CPT-11 causes toxic side-effects in patients. CPT-11 toxicity has been attributed to the activity of intestinal microbiota, however, intestinal microbiota may also have protective effects in CP!-11 chemotherapy. This study aimed to elucidate mechanisms through which microbiota and dietary fibres could modify host health. Rats bearing a Ward colon carcinoma were treated with a two-cycle CPT-11/5-fluorouracil therapy recapitulating clinical therapy of colorectal cancer. Animals were fed with a semi-purified diet or a semi-purified diet was supplemented with non-digestible carbohydrates (isomalto-oligosaccharides, resistant starch, fructo-oligosaccharides, or inulin) in 3 independent experiments. Changes in intestinal microbiota, bacteria translocating to mesenteric lymphnodes, cecal GUD activity, and cecal SCFA production, and the intestinal concentration of CPT-11 and its metabolites were analysed. Non-digestible carbohydrates significantly influenced feed intake, body weight and other indicators of animal health. The identification of translocating bacteria and their quantification in cecal microbiota indicated that overgrowth of the intestine by opportunistic pathogens was not a major contributor to CPT-11 toxicity. Remarkably, fecal GUD activity positively correlated to body weight and feed intake but negatively correlated to cecal SN-38 concentrations and IL1-β. The reduction in CPT-11 toxicity by non-digestible carbohydrates did not correlate to stimulation of specific bacterial taxa. However, cecal butyrate concentrations and feed intake were highly correlated. The protective role of intestinal butyrate production was substantiated by a positive correlation of the host expression of MCT1 (monocarboxylate transporter 1) with body weight as well as a positive correlation of the abundance of bacterial butyryl-CoA gene with cecal butyrate concentrations. These correlations support the interpretation that the influence of dietary fibre on CPT-11 toxicity is partially mediated by an increased cecal production of butyrate.

State of the art. Osteoarthritis (OA) is a chronic articular disease characterized by cartilage degradation and osteophyte formation. OA physiopathology is multifactorial and involves mechanical and hereditary factors. So far, there is neither preventive medicine to delay cartilage breakdown nor curative treatment. Objectives. To investigate pro-inflammatory paracrine interactions between human primary chondrocytes and macrophages following interleukin-1-β (IL-1β) treatment; to evaluate the molecular mechanism responsible for the inhibitory effect of resveratrol. Results. The activation of NF-κB in chondrocytes by IL-1β induced IL-6 secretion. The latter will then activate STAT3 protein in macrophages. Moreover, STAT3 was able to positively regulate IL-6 secretion, as confirmed by the doubling level of IL-6 in the coculture compared to macrophage monoculture. These experiments confirm the usefulness of the coculture model in the inflammatory arthritis-linked process as a closer biological situation to the synovial joint than separated chondrocytes and macrophages. Il also demonstrated the presence of an inflammatory amplification loop induced by IL-1β. Resveratrol showed a strong inhibitory effect on the pro-inflammatory marker secretion. The decrease of IL-6 secretion is dependent on the NFκB inhibition in the chondrocytes. Such reduction of the IL-6 level can limit STAT3 activation in the macrophages, leading to the interruption of the inflammatory amplification loop. Conclusion. These results increase our understanding of the anti-inflammatory actions of resveratrol and open new potential approaches to prevent and treat osteoarthritis.

This study investigates the ways in which two proinflammatory cytokines, tumor necrosis factor alpha (TNF) and interleukin-1 beta (IL1), cause increased production of prostaglandin E2 (PGE2) in rabbit articular chondrocytes (RAC). Rabbit articular chondrocytes in primary culture were incubated with IL1, TNF, or both. Arachidonic acid (AA) release, PGE2 production, and the activities of cytosolic phospholipase A2 (cPLA2), secreted phospholipase A2 (sPLA2), and cyclooxygenase (COX) were measured. The mRNA levels of cPLA2, sPLA2, and COX-2 were also measured by Northern blotting, using specific complementary DNA probes. Incubation of IL1-stimulated RAC with TNF further increased PGE2 production. This synergy did not involve PLA2 stimulation, as there were no increases in AA release, cPLA2 and sPLA2 activities, or mRNA. In contrast, TNF increased the effect of IL1 on COX-2 activity and mRNA level. These results show that TNF and IL1 act in synergy in PGE2 production in articular chondrocytes. As sPLA2 and cPLA2 do not seem to be involved, COX-2 appears to be the best target for a specific anti-inflammatory strategy against cartilage degradation.

Palmitoleic acid (PMA) has anti-inflammatory and antidiabetic activities. Here we tested whether these effects of PMA on glucose homeostasis and liver inflammation, in mice fed with high-fat diet (HFD), are PPAR-α dependent. C57BL6 wild-type (WT) and PPAR-α-knockout (KO) mice fed with a standard diet (SD) or HFD for 12 weeks were treated after the 10th week with oleic acid (OLA, 300 mg/kg of b.w.) or PMA 300 mg/kg of b.w. Steatosis induced by HFD was associated with liver inflammation only in the KO mice, as shown by the increased hepatic levels of IL1-beta, IL-12, and TNF-α; however, the HFD increased the expression of TLR4 and decreased the expression of IL1-Ra in both genotypes. Treatment with palmitoleate markedly attenuated the insulin resistance induced by the HFD, increased glucose uptake and incorporation into muscle in vitro, reduced the serum levels of AST in WT mice, decreased the hepatic levels of IL1-beta and IL-12 in KO mice, reduced the expression of TLR-4 and increased the expression of IL-1Ra in WT mice, and reduced the phosphorylation of NF ����B (p65) in the livers of KO mice. We conclude that palmitoleate attenuates diet-induced insulin resistance, liver inflammation, and damage through mechanisms that do not depend on PPAR-α.

OBJECTIVE

To investigate the effects of Clostridium butyricum (C. butyricum) on experimental gastric ulcers (GUs) induced by alcohol, restraint cold stress, or pyloric ligation in mice, respectively.

METHODS

One hundred and twenty mice were randomly allocated into three types of gastric ulcer models (n = 40 each), induced by alcohol, restraint cold stress, or pyloric ligation. In each GU model, 40 mice were allocated into four groups (n = 10 each): the sham control group; model group (GU induction without pretreatment); C. butyricum group (GU induction with C. butyricum pretreatment); and Omeprazole group (GU induction with Omeprazole pretreatment). The effects of C. butyricum were evaluated by examining the histological changes in the gastric mucosal erosion area, the activities of superoxide dismutase (SOD) and catalase (CAT), the level of malondialdehyde (MDA), and the contents of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, leukotriene B4 (LTB4) and 6-keto-PGF-1α (degradation product of PGI2) in the gastric tissue.

RESULTS

Our data showed that C. butyricum significantly reduced the gastric mucosal injury area and ameliorated the pathological conditions of the gastric mucosa. C. butyricum not only minimized the decreases in activity of SOD and CAT, but also reduced the level of MDA in all three GU models used in this study. The accumulation of IL1-β, TNF-α and LBT4 decreased, while 6-keto-PGF-1α increased with pretreatment by C. butyricum in all three GU models.

CONCLUSIONS

Our data demonstrated the protective effects of pretreatment with C. butyricum on anti-oxidation and anti-inflammation in different types of GU models in mice. Further studies are needed to explore its potential clinical benefits.

The objectives of this research were to confirm the effect of compounds derived from milk fermented by Lactobacillus helveticus (LH-2) on the nonspecific host defence system, and isolate and characterize the active peptides that mediate the immune response. The cell-free supernatant obtained from the fermented milk and its fractions were tested in vitro for immuno-modulating activity using murine macrophages (RAW 264.7 cell line). Cytokine production (Interleukin-6 (IL-6), Tumor Necrosis Factor-alpha (TNF-alpha), and Interleukin-1beta (IL1-beta)), nitric oxide (NO) production and phagocytosis were used as biomarkers. Macrophages stimulated with cell-free supernatant of fermented milk showed higher production of cytokines and NO compared with macrophages stimulated with LPS (Lipopolysaccharide) and a commercial immunomodulator derived from beta-casein (f54-59). Phagocytosis was observed by macrophages stimulated with the supernatant. Two of nine fractions collected from the supernatant using size exclusion chromatography produced the highest response when used to stimulate macrophages. The results of the dose-response study of the effect of the fraction with the highest stimulation effect on the production of TNF-alpha showed a direct correlation between protein concentration and TNF-alpha release. The fraction contained four novel peptides, three derived from the hydrolysis of beta-casein and one from the hydrolysis of alpha-lactalbumin. These results confirm that fermentation of milk by Lactobacillus helveticus (LH-2) results in the production of specific peptides capable of modulating macrophage activity.

Growth hormone (GH) has been used as anabolic therapy to treat catabolic patients. In a recent study, however, administration of high doses of GH to critically ill adults was associated with an increase in morbidity and mortality. Preponderance of septic shock and uncontrolled infections as causes of death in these patients suggests an immuno-modulatory effect of GH. Our hypothesis was that GH treatment may modulate the production of proinflammatory cytokines, which are implicated in sepsis. In our study, human monocytes in whole blood were activated with lipopolysaccaharide (LPS) (1-100 ng/ml) purified from a clinical isolate of group B Neisseria meningitidis in the presence of a high dose of GH (100 ng/ml). The subsequent proinflammatory cytokine response was analysed by intracellular cytokine staining and flow cytometry. Our results show that GH enhances IL1-alpha, IL-6 and TNF-alpha production by LPS activated monocytes in whole blood. The modulation of cytokines by GH may be responsible for the adverse consequences of GH in critically ill patients.

BACKGROUND

The clinical efficacy of camptothecin (CPT), a drug specifically targeting topoisomerase I (TopoI), is under evaluation for the treatment of malignant gliomas. Due to the high unresponsiveness of these tumours to chemotherapy, it would be very important to study the signalling network that drives camptothecin outcome in this type of cancer cells. To address this issue, we had previously compared the expression profile of human U87-MG glioblastoma cells with that of a CPT-resistant counterpart, giving evidence that the development of a robust inflammatory response was the main transcriptional effect associated with CPT resistance. Here we report time-related changes and cell line specific patterns of gene expression after CPT treatment by using two p53 wild-type glioblastoma cell lines, U87-MG and DBTRG-05, with different sensitivities to TopoI inhibition.

RESULTS

First, we demonstrated that CPT treatment brings the two cell lines to completely different outcomes: accelerated senescence in U87-MG and apoptosis in DBTRG-05 cells. Then, to understand the different susceptibility to CPT, we used oligo-microarray to identify the genes whose expression was regulated during a time-course treatment, ranging from 2 h to 72 h. The statistical analysis of microarray data by MAANOVA (MicroArray ANalysis Of VAriance) showed much less modulated genes in apoptotic DBTRG-05 cells (155) with respect to the senescent U87-MG cells (3168), where the number of down-regulated genes largely exceeded that of the up-regulated ones (80% vs. 20%). Despite this great difference, the two data-sets showed a large overlapping (60% circa) mainly due to the expression of early stress responsive genes. The use of High-Throughput GoMINER and EASE tools, for functional analysis of significantly enriched GO terms, highlighted common cellular processes and showed that U87-MG and DBTRG-05 cells shared many GO terms, which are related to the down-regulation of cell cycle and mitosis and to the up-regulation of cell growth inhibition and DNA damage.Furthermore, the down-regulation of MYC and DP1 genes, which act as key transcription factors in cell growth control, together with the inhibition of BUB1, BUB3 and MAD2 mRNAs, which are known to be involved in the spindle checkpoint pathway, were specifically associated with the execution of senescence in U87-MG cells and addressed as critical factors that could drive the choice between different CPT-inducible effectors programs. In U87-MG cells we also found inflammation response and IL1-beta induction, as late transcriptional effects of Topo I treatment but these changes were only partially involved in the senescence development, as shown by IL1-beta gene silencing.

CONCLUSIONS

By comparing the transcription profile of two glioblastoma cell lines treated with camptothecin, we were able to identify the common cellular pathways activated upon Topo I inhibition. Moreover, our results helped in identifying some key genes whose expression seemed to be associated with the execution of senescence or apoptosis in U87-MG and DBTRG-05 cells, respectively.

OBJECTIVE

In this study, we sought to investigate the dynamic changes in the levels of TNF-α, IL-1β and LPS in the gingival crevicular fluid (GCF) in a rat model of diabetes mellitus (DM) and periodontitis (PD). Additionally, we evaluated alveolar bone loss and the histopathological response associated with experimental diabetes mellitus and experimental periodontitis.

METHODS

DM and PD were induced together in 15 rats (group 1) by streptozotocin injection and ligature induction. Periodontitis alone was produced by ligature induction in 15 rats (group 2), diabetes alone was produced by streptozotocin injection in 15 rats (group 3), and fifteen systemically and periodontally healthy rats were used as controls (group 4). The gingival TNF-α, IL-1β and LPS levels were measured by using ELISA method. Periodontal destruction was assessed by measuring the alveolar bone loss. Periodontal inflammation was quantified by histopathological grading in H&E stained samples.

RESULTS

Higher levels of TNF-α, IL1-β and LPS, increased alveolar bone loss and more serve histopathology were found in group 1 compared with group 2, group 3 and group 4 (p< 0.05). The quantities of TNF-α, IL1-β and LPS, the amount of alveolar bone loss and the severity of the histopathological finding were greater in group 2 than group 3 and group 4 (p< 0.05). Group 3 demonstrated higher levels of TNF-α, IL1-β and LPS, increased alveolar bone loss and more serve histopathology than group 4 (p< 0.05). Statistically significant differences were noted between all of the groups.

CONCLUSIONS

These data indicate that DM may lead to enhanced TNF-α, IL1-β and LPS production in the periodontal tissues. The resorption values of alveolar bone and the histological inflammation were more severe in rats with periodontitis and diabetes mellitus than in those with periodontitis alone, diabetes mellitus alone and control rats. Our findings are consistent with the hypothesis that hyperglycemia contributes to the heightened inflammatory response associated with periodontitis.

BACKGROUND

Certain inflammatory biomarkers increase with age, provide information about general burden of illness and could cause or reflect any collateral damage to healthy cells and organs. However, comparative studies to predict adverse outcomes are missing. Therefore, our study validated and identified the principal prognostic marker to predict important adverse outcomes in the oldest old from an extensive battery of serum inflammatory markers.

METHODS

A large battery of potential 'inflammaging' markers (IL-1α, IL1-β, IL-4, IL-6, IL-8, IL-10, TNF-α, IFN-γ, EGF, VEGF, MCP-1, usCRP, prealbumin) was assessed in a representative sample of 415 heterogenic individuals 80years of age or older in the BELFRAIL study. Kaplan-Meier, Cox proportional hazards and CART analyses determined the overall prognostic value of these markers for predicting all-cause, cardiovascular and non-cardiovascular mortality as well as hospitalization.

RESULTS

Serum IL-6 and usCRP levels were strongly associated with time of survival, independent of cause of death. Serum IL-6 levels had the most robust dose-response relationship with mortality. To a lesser extent, IL-10 and IL-1β were associated with all-cause mortality but were restricted to non-cardiovascular or cardiovascular mortality, respectively. Having a low IL-6 at baseline (<1.77pg/ml) could predict 90% of those who were not at risk for all-cause mortality after 3years, even after adjusting for confounders. Similarly, we observed an 83.6% chance of identifying those cases with 0 or 1 hospitalization using low IL-6 serum levels.

CONCLUSIONS

The results suggest that a single measurement of low IL-6 serum levels is the first choice to guide clinical practice in the oldest old and could summarize the short-term risk of death and hospitalization.

OBJECTIVE

Higher systemic levels of pro-inflammatory biomarkers and low adiponectin are associated with increased risk of type 2 diabetes, but their associations with changes in glycaemic deterioration before onset of diabetes are poorly understood. We aimed to study whether inflammation-related biomarkers are associated with 5-year changes in glucose and insulin, HbA1c, insulin sensitivity and beta-cell function before the diagnosis of type 2 diabetes and whether these associations may be bidirectional.

METHODS

We used multiple repeat measures (17 891 person-examinations from 7683 non-diabetic participants) from the Whitehall II study to assess whether circulating high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL6), IL1 receptor antagonist (IL1Ra) and adiponectin are associated with subsequent changes in glycaemia, insulin, insulin resistance and beta-cell function (based on oral glucose tolerance tests). We examined bidirectionality by testing if parameters of glucose metabolism at baseline are associated with changes in inflammation-related biomarkers.

RESULTS

Higher hsCRP and IL6 were associated with increases in fasting insulin, insulin resistance and, for IL6, with beta-cell function after adjustment for confounders. Higher adiponectin was associated with decreases in fasting glucose, HbA1c, fasting insulin, insulin resistance and beta-cell function. The reverse approach showed that 2-h glucose and insulin sensitivity were associated with changes in IL1Ra. Fasting insulin and insulin resistance showed inverse associations with changes in adiponectin.

CONCLUSIONS

Subclinical inflammation is associated with development of increased glycaemia, insulin resistance and beta-cell function in non-diabetic individuals. These findings are consistent with the hypothesis that inflammation-related processes may increase insulin resistance and lead to a compensatory upregulation of beta-cell function.

Cre-mediated excision of loxP sites is widely used in mice to manipulate gene function in a tissue-specific manner. To analyze phenotypic alterations related to Cre-expression, we have used AMH-Cre-transgenic mice as a model system. Different Cre expression levels were obtained by investigation of C57BL/6J wild type as well as heterozygous and homozygous AMH-Cre-mice. Our results indicate that Cre-expression itself in Sertoli cells already has led to oxidative stress and lipid peroxidation (4-HNE lysine adducts), inducing PPARα/γ, peroxisome proliferation and alterations of peroxisome biogenesis (PEX5, PEX13 and PEX14) as well as metabolic proteins (ABCD1, ABCD3, MFP1, thiolase B, catalase). In addition to the strong catalase increase, a NRF2- and FOXO3-mediated antioxidative response (HMOX1 of the endoplasmic reticulum and mitochondrial SOD2) and a NF-κB activation were noted. TGFβ1 and proinflammatory cytokines like IL1, IL6 and TNFα were upregulated and stress-related signaling pathways were induced. Sertoli cell mRNA-microarray analysis revealed an increase of TNFR2-signaling components. 53BP1 recruitment and expression levels for DNA repair genes as well as for p53 were elevated and the ones for related sirtuin deacetylases affected (SIRT 1, 3-7) in Sertoli cells. Under chronic Cre-mediated DNA damage conditions a strong downregulation of Sirt1 was observed, suggesting that the decrease of this important coordinator between DNA repair and metabolic signaling might induce the repression release of major transcription factors regulating metabolic and cytokine-mediated stress pathways. Indeed, caspase-3 was activated and increased germ cell apoptosis was observed, suggesting paracrine effects. In conclusion, the observed wide stress-induced effects and metabolic alterations suggest that it is essential to use the correct control animals (Cre/Wt) with matched Cre expression levels to differentiate between Cre-mediated and specific gene-knock out-mediated effects.

Inflammasome is the cytoplasmic complex responsible for pro-IL1 β cleavage and secretion of IL-1β. Recently our group reported the first association between polymorphisms in the inflammasome receptor NLRP1 and adult-onset systemic lupus erythematosus (SLE) "di per se" and especially in SLE-associated renal disease, suggesting the involvement of NLRP1-inflammasome in the immune dysregulation characteristic of SLE patients. Considering that juvenile-onset SLE (JSLE) is more severe than adult SLE, and that the genetic background plays a major role in the early development of autoimmune diseases, we analysed selected polymorphisms in inflammasome genes (NLRP1, NLRP3, CARD8, IL1B, TNFAIP3) of children and adolescents with JSLE (n = 90) and in healthy controls (n = 144). A single polymorphism in IL1B, and not NLRP1, gene resulted in association with JSLE, suggesting that IL-1 β is involved in the pathogenesis of SLE, but different genes could play specific role in adult- or early-onset disease.

P2X(7) receptor is a ligand-gated ion channel, which can induce the opening of large membrane pores. Here, we provide evidence that the receptor induces pore formation in astrocytes cultured from cortex, but not from the hippocampus. Furthermore, P2X(7) receptor activation promptly induces p38 mitogen-activated protein kinase (MAPK) phosphorylation in cortical but not in hippocampal astrocytes. Given the role of p38 MAPK activation in pore opening, these data suggest that defective coupling of the receptor to the enzyme could occur in hippocampal cultures. The different capabilities of the receptor to open membrane pores cause relevant functional consequences. Upon pore formation, caspase-1 is activated and pro-IL1-beta is cleaved and released extracellularly. The receptor stimulation does not result in interleukin-1beta secretion from hippocampal astrocytes, although the pro-cytokine is present in the cytosol of lipopolysaccharide-primed cultures. These results open the possibility that activation of P2X(7) receptors differently influences the neuroinflammatory processes in distinct brain regions.