Dental pulp is a dynamic tissue able to resist external irritation during tooth decay by using immunocompetent cells involved in innate and adaptive responses. To better understand the immune response of pulp toward gram-negative bacteria, we analyzed biological mediators and immunocompetent cells in rat incisor pulp experimentally inflamed by either lipopolysaccharide (LPS) or saline solution (phosphate-buffered saline [PBS]). Untreated teeth were used as control. Expression of pro- and anti-inflammatory cytokines, chemokine ligands, growth factors, and enzymes were evaluated at the transcript level, and the recruitment of the different leukocytes in pulp was measured by fluorescence-activated cell-sorting analysis after 3 h, 9 h, and 3 d post-PBS or post-LPS treatment. After 3 d, injured rat incisors showed pulp wound healing and production of reparative dentin in both LPS and PBS conditions, testifying to the reversible pulpitis status of this model. IL6, <em>IL1</em>-<em>β</em>, TNF-α, CCL2, CXCL1, CXCL2, MMP9, and iNOS gene expression were significantly upregulated after 3 h of LPS stimulation as compared with PBS. The immunoregulatory cytokine <em>IL1</em>0 was also upregulated after 3 h, suggesting that LPS stimulates not only inflammation but also immunoregulation. Fluorescence-activated cell-sorting analysis revealed a significant, rapid, and transient increase in leukocyte levels 9 h after PBS and LPS stimulation. The quantity of dendritic cells was significantly upregulated with LPS versus PBS. Interestingly, we identified a myeloid-derived suppressor cell-enriched cell population in noninjured rodent incisor dental pulp. The percentage of this population, known to regulate immune response, was higher 9 h after inflammation triggered with PBS and LPS as compared with the control. Taken together, these data offer a better understanding of the mechanisms involved in the regulation of dental pulp immunity that may be elicited by gram-negative bacteria.

OBJECTIVE

The goal of this study was to determine the presence early of markers of endoplasmic reticulum stress (ERS) and insulin resistance in the offspring from dams fed HFD (HFD-O) or standard chow diet (SC-O) during pregnancy and lactation.

METHODS

To address this question, we evaluated the hypothalamic and hepatic tissues in recently weaned mice (d28) and the hypothalamus of newborn mice (d0) from dams fed HFD or SC during pregnancy and lactation.

RESULTS

Body weight, adipose tissue mass, and food intake were more accentuated in HFD-O mice than in SC-O mice. In addition, intolerance to glucose and insulin was higher in HFD-O mice than in SC-O mice. Compared with SC-O mice, levels of hypothalamic IL1-β mRNA, NFκB protein, and p-JNK were increased in HFD-O mice. Furthermore, compared with SC-O mice, hypothalamic AKT phosphorylation after insulin challenge was reduced, while markers of ERS (p-PERK, p-eIF2α, XBP1s, GRP78, and GRP94) and p-AMPK were increased in the hypothalamic tissue of HFD-O at d28 but not at d0. These damages to hypothalamic signaling were accompanied by increased triglyceride deposits, activation of NFκB, p-JNK, p-PERK and p-eIF2α.

CONCLUSIONS

These point out lactation period as maternal trigger for metabolic changes in the offspring. These changes may occur early and quietly contribute to obesity and associated pathologies in adulthood. Although in rodents the establishment of ARC neuronal projections occurs during the lactation period, in humans it occurs during the third trimester. Gestational diabetes and obesity in this period may contribute to impairment of energy homeostasis.

In granulosa cells labeled to isotopic steady-state with [3H]serine, addition of interleukin-1 beta (IL1 beta) or bacterial sphingomyelinase (SMase) induced a rapid decrease (approximately 60% by 10 min) in cellular [3H]Sphingomyelin content and a prolonged generation (up to 60 min) of [3H]ceramide, the immediate lipid-moiety generated in response to sphingomyelin hydrolysis. In FSH-treated cells, IL1 beta (0.3-30 ng/ml) inhibited progesterone biosynthesis in a dose-dependent manner, an effect that was also observed in cells exposed to increasing concentrations of bacterial SMase (0.003-0.3 U/ml) or the membrane-permeable ceramide analogue N-hexanoylsphingosine (C6-cer:0.1-10 microM). Abrogation of progesterone biosynthesis was not a sole consequence of inadequate cAMP biosynthesis because cyclic nucleotide levels remained elevated (3- to 4-fold over untreated cultures) after addition of IL1 beta, SMase, or two different cell permeable ceramide analogues (C2-cer and C6-cer) to gonadotropin-stimulated granulosa cells. Moreover, taken into account that exogenous SMase or C6-cer partially abolished progesterone biosynthesis induced by But2cAMP (0.5 mM) or cholera toxin (CTX: 1 microgram/ml), the above mentioned results support the notion that activation of the sphingomyelin pathway exerts its inhibitory effects on granulosa cell steroidogenic activity at site(s) of action both proximal and distal to cAMP generation. As determined by RT-PCR analysis, the inhibitory effect of IL1 beta, SMase, or C6-cer on gonadotropin-stimulated steroidogenesis was accompanied by arrested transcription of the mitochondrial cholesterol side chain cleavage enzyme (P450scc) and 3 beta-hydroxysteroid dehydrogenase/delta 5-4isomerase, the two FSH-inducible steps involved in progesterone biosynthesis. Although bacterial SMase or the ceramide analogue C6-cer alone did not exactly reproduce the effect of IL1 beta on granulosa cell prostaglandin E2 (PGE2) biosynthesis, both agents augmented net PGE2 production and messenger RNA levels of the inducible prostaglandin endoperoxide synthase/cyclooxygenase (PGHS-2) in cytokine-treated cells. Although the effect on PGHS-2 messenger RNA may account for the facilitatory role of ceramide on IL1 beta-induced PGE2 biosynthesis, neither SMase nor the membrane-permeant ceramide analogue were able to augment prostaglandin accumulation in the presence of exogenously added arachidonate precursor. Collectively, whereas these results show that ceramide triggers a negative-effector pathway that is both necessary and sufficient to reproduce the inhibitory effect of IL1 beta on FSH-stimulated granulosa cell steroidogenesis, they also support the notion that sphingomyelin hydrolysis may be important for cytokine-induced PGHS-2 expression but not sufficient to reproduce IL1 beta-stimulated PGE2 biosynthesis.

To investigate the mechanisms of proteolysis within the glioma, and tissue reactions against glioblastoma, immunohistochemical detection both outside and inside of the tumor was performed using seven brains with glioblastoma that were obtained from autopsies. Immunohistochemistry was performed to detect vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMP)-1,-2,-9, membrane-type matrix metalloproteinase (MT-MMP), interleukin (IL)1-beta, and IL-6. The data were translated into color graphics and the localization of these proteins was analyzed. In glial cells around the tumor, GFAP, VEGF, MMP-2, and MT-MMP were strongly expressed. Moreover, IL1-beta was also expressed strongly in the glial cells at the periphery of the tumor. IL-6 was recognized outside of the tumor, but was expressed only in the swollen astrocytes and normal pyramidal cells. These data suggest that in the periphery of the tumor, tissue reconstruction processes take place with concomitant degradation of the matrix by MMP-2 and MT-MMP, as well as vascular remodeling promoted by VEGF. The fact that IL1-beta, but not IL-6, was expressed strongly in the glial cells around the tumor, may indicate that these proteins expressed outside of the tumor are not utilized for tumor growth, but may be used to guard the tumor against invasions, such as immune response.

Recent findings have revealed the complexity of the transcriptional landscape in mammalian cells. One recently described class of novel transcripts are the Cytoplasmic Intron-sequence Retaining Transcripts (CIRTs), hypothesized to confer post-transcriptional regulatory function. For instance, the neuronal CIRT KCNMA1i16 contributes to the firing properties of hippocampal neurons. Intronic sub-sequence retention within IL1-β mRNA in anucleate platelets has been implicated in activity-dependent splicing and translation. In a recent study, we showed CIRTs harbor functional SINE ID elements which are hypothesized to mediate dendritic localization in neurons. Based on these studies and others, we hypothesized that CIRTs may be present in a broad set of transcripts and comprise novel signals for post-transcriptional regulation. We carried out a transcriptome-wide survey of CIRTs by sequencing micro-dissected subcellular RNA fractions. We sequenced two batches of 150-300 individually dissected dendrites from primary cultures of hippocampal neurons in rat and three batches from mouse hippocampal neurons. After statistical processing to minimize artifacts, we found a broad prevalence of CIRTs in the neurons in both species (44-60% of the expressed transcripts). The sequence patterns, including stereotypical length, biased inclusion of specific introns, and intron-intron junctions, suggested CIRT-specific nuclear processing. Our analysis also suggested that these cytoplasmic intron-sequence retaining transcripts may serve as a primary transcript for ncRNAs. Our results show that retaining intronic sequences is not isolated to a few loci but may be a genome-wide phenomenon for embedding functional signals within certain mRNA. The results hypothesize a novel source of cis-sequences for post-transcriptional regulation. Our results hypothesize two potentially novel splicing pathways: one, within the nucleus for CIRT biogenesis; and another, within the cytoplasm for removing CIRT sequences before translation. We also speculate that release of CIRT sequences prior to translation may form RNA-based signals within the cell potentially comprising a novel class of signaling pathways.

OBJECTIVE

Dyslipidemia is one of the main risk factors for atherosclerosis, usually the underlying cause of cardiovascular diseases which are the major cause of morbidity and mortality in developed countries. The aim of this study was to assess the effects and the advantages of a combined dietary supplementation with PUFA n-3, vitamin E, niacin and gamma-oryzanol on lipid profile, inflammatory status and oxidative balance.

RESULTS

Fifty-seven dyslipidemic volunteers were randomly assigned to receive: placebo (group A, 19 subjects); PUFA n-3 and vitamin E (group <em>B</em>, 18 subjects); the same as <em>B</em> plus gamma-oryzanol and niacin (group C, 20 subjects). Lipid profile, reactive oxygen species (ROS), total antioxidant capacity (TAC), vitamin E, interleukin 1-<em>beta</em> (<em>IL1</em>-<em>beta</em>), tumor necrosis factor (TNF-alpha) and thromboxane <em>B</em>2 (TX<em>B</em>2) were determined at baseline (T0) and after four months (T1). All dyslipidemic subjects showed, at baseline, oxidative stress and, after four months, all biochemical markers improved significantly in groups treated with dietary supplementation. Particularly in group C all lipid patterns improved significantly.

CONCLUSIONS

Our findings demonstrate that the strategy of combining different compounds, which protect each other and act together at different levels of the lipid chain production, improves lipid profile, inflammatory and oxidative status, allowing us to reduce the dose of each compound under the threshold of its side effects.

Reduced expression of autophagy regulators has been observed in pathological cartilage in humans and mice. The present study aimed to investigate the synergistic therapeutic effect of promotion of chondrocyte autophagy via exposure to sinomenium (SIN) encapsulated by chitosan microspheres (CM-SIN) and photo-crosslinked gelatin methacrylate (GelMA) hydrogel, with the goal of evaluating CM-SIN as a treatment for patients with osteoarthritis. First, we fabricated and characterized GelMA hydrogels and chitosan microspheres. Next, we measured the effect of SIN on cartilage matrix degradation induced by IL1-β in chondrocytes and an ex vivo model. SIN ameliorated the pathological changes induced by IL1-β at least partially through activation of autophagy. Moreover, we surgically induced osteoarthritis in mice, which were injected intra-articularly with CM-SIN and GelMA. Cartilage matrix degradation and chondrocyte autophagy were evaluated 4 and 8 weeks after surgery. Treatment with the combination of CM-SIN and GelMA retarded the progression of surgically induced OA. SIN ameliorated cartilage matrix degradation at least partially by inducing autophagy in vivo. Our results demonstrate that injection of the combination of GelMA hydrogel and CM-SIN could be a promising strategy for treating patients with osteoarthritis.

Defective regulation of type I interferon response is associated with severe inflammatory phenotypes and autoimmunity. Type I interferonopathies are a clinically heterogenic group of Mendelian diseases with a constitutive activation of this pathway that might present as atypical, severe, early onset rheumatic diseases. Skin vasculopathy with chilblains and livedo reticularis, interstitial lung disease, and panniculitis are common. Recent studies have implicated abnormal responses to nucleic acid stimuli or defective regulation of downstream effector molecules in disease pathogenesis. As observed for IL1-β and autoinflammatory diseases, knowledge of the defects responsible for type I interferonopathies will likely promote the development of targeted therapy.

BACKGROUND

Adults with major depressive disorder (MDD) show elevated levels of IL-6 and TNF-alpha. Studies of adolescents with MDD or anxiety disorders (AD) are few and present conflicting results.

METHODS

We studied plasma cytokines in a clinical sample of adolescent females with MDD and/or clinical AD (n=60, mean age 16.8 years), compared to healthy controls (n=44; mean age 16.5 years).

RESULTS

The clinical sample showed significantly higher values of IL-2 (Z=-4.09, p>0.0001), IL1-beta (Z=-2.40, p<0.05) and IL-10 (Z=-2.38, p<0.05) as compared to controls. The subgroup of the clinical sample not treated with SSRIs had a significant difference of IL-6 (Z=-2.26, p<0.05) in addition to the difference of IL-2 and IL1-beta, but showed no difference of IL-10 as compared to the controls. SSRI treatment was related to IL-6, explaining 26% of the variance in the clinical sample after controlling for BMI and symptom severity. In the clinical sample, levels of IL-6 and IFN-gamma were positively correlated with self-assessed symptoms of anxiety and/or depression (corr.coeff 0.35 resp 0.40 at p<0.05).

CONCLUSIONS

The cross-sectional design does not allow for conclusions on causality. The sample sizes were relatively small and a large drop-out in the clinical sample may have influenced the representativity.

CONCLUSIONS

The study suggests that pro-inflammatory cytokines are part of the pathophysiology of emotional disorders in adolescent females and that SSRIs have anti-inflammatory properties. The findings prompt further studies on the specific mechanisms involved and may contribute to the development of more effective treatment and prevention.

The emerging role of heparanase in tumor initiation, growth, metastasis, and chemoresistance is well recognized and is encouraging the development of heparanase inhibitors as anticancer drugs. Unlike the function of heparanase in cancer cells, very little attention has been given to heparanase contributed by cells composing the tumor microenvironment. Here we used a genetic approach and examined the behavior and function of macrophages isolated from wild-type (WT) and heparanase-knockout (Hpa-KO) mice. Hpa-KO macrophages express lower levels of cytokines (e.g., TNFα, IL1-β) and exhibit lower motility and phagocytic capacities. Intriguingly, inoculation of control monocytes together with Lewis lung carcinoma (LLC) cells into Hpa-KO mice resulted in nearly complete inhibition of tumor growth. In striking contrast, inoculating LLC cells together with monocytes isolated from Hpa-KO mice did not affect tumor growth, indicating that heparanase is critically required for activation and function of macrophages. Mechanistically, we describe a linear cascade by which heparanase activates Erk, p38, and JNK signaling in macrophages, leading to increased c-Fos levels and induction of cytokine expression in a manner that apparently does not require heparanase enzymatic activity. These results identify heparanase as a key mediator of macrophage activation and function in tumorigenesis and cross-talk with the tumor microenvironment.

BACKGROUND

Ex vivo expansion of umbilical cord blood (UCB) is attempted to increase cell numbers to overcome the limitation of cell dose. Presently, suspension cultures or feeder mediated co-cultures are performed for expansion of hematopoietic stem cells (HSCs). Mesenchymal stem cells (MSCs) have proved to be efficient feeders for the maintenance of HSCs. Here, we have established MSCs-HSCs co-culture system with MSCs isolated from less invasive and ethically acceptable sources like umbilical cord tissue (C-MSCs) and placenta (P-MSCs). MSCs derived from these tissues are often compared with bone marrow derived MSCs (BM-MSCs) which are considered as a gold standard. However, so far none of the studies have directly compared C-MSCs with P-MSCs as feeders for ex vivo expansion of HSCs. Thus, we for the first time performed a systematic comparison of hematopoietic supportive capability of C and P-MSCs using paired samples.

METHODS

UCB-derived CD34(+) cells were isolated and co-cultured on irradiated C and P-MSCs for 10 days. C-MSCs and P-MSCs were isolated from the same donor. The cultures comprised of serum-free medium supplemented with 25 ng/ml each of SCF, TPO, Flt-3 L and IL-6. After 10 days cells were collected and analyzed for phenotype and functionality.

RESULTS

C-MSCs and P-MSCs were found to be morphologically and phenotypically similar but exhibited differential ability to support ex vivo hematopoiesis. Cells expanded on P-MSCs showed higher percentage of primitive cells (CD34(+)CD38(-)), CFU (Colony forming unit) content and LTC-IC (Long term culture initiating cells) ability. CD34(+) cells expanded on P-MSCs also exhibited better in vitro adhesion to fibronectin and migration towards SDF-1α and enhanced NOD/SCID repopulation ability, as compared to those grown on C-MSCs. P-MSCs were found to be closer to BM-MSCs in their ability to expand HSCs. P-MSCs supported expansion of functionally superior HSCs by virtue of reduction in apoptosis of primitive HSCs, higher Wnt and Notch activity, HGF secretion and cell-cell contact. On the other hand, C-MSCs facilitated expansion of progenitors (CD34(+)CD38(+)) and differentiated (CD34(-)CD38(+)) cells by secretion of IL1-α, β, MCP-2, 3 and MIP-3α.

CONCLUSIONS

P-MSCs were found to be better feeders for ex vivo maintenance of primitive HSCs with higher engraftment potential than the cells expanded with C-MSCs as feeders.

Doxorubicin (Dox) is an effective antineoplastic agent used to treat cancers, but its use is limited as Dox induces adverse cardiotoxic effects. Dox-induced cardiotoxicity (DIC) can lead to heart failure and death. There is no study that investigates whether embryonic stem cell-derived exosomes (ES-Exos) in DIC can attenuate inflammation-induced pyroptosis, pro-inflammatory M1 macrophages, inflammatory cell signaling, and adverse cardiac remodeling. For this purpose, we transplanted ES-Exos and compared with ES-cells (ESCs) to examine pyroptosis, inflammation, cell signaling, adverse cardiac remodeling, and their influence on DIC induced cardiac dysfunction. Therefore, we used C57BL/6J mice ages 10 ± 2 weeks and divided them into four groups (n = 6-8/group): Control, Dox, Dox + ESCs, and Dox + ES-Exos. Our data shows that the Dox treatment significantly increased expression of inflammasome markers (TLR4 and NLRP3), pyroptotic markers (caspase-1, IL1-β, and IL-18), cell signaling proteins (MyD88, p-P38, and p-JNK), pro-inflammatory M1 macrophages, and TNF-α cytokine. This increased pyroptosis, inflammation, and cell signaling proteins were inhibited with ES-Exos or ESCs. Moreover, ES-Exos or ESCs increased M2 macrophages and anti-inflammatory cytokine, IL-10. Additionally, ES-Exos or ESCs treatment inhibited significantly cytoplasmic vacuolization, myofibril loss, hypertrophy, and improved heart function. In conclusion, for the first time we demonstrated that Dox-induced pyroptosis and cardiac remodeling are ameliorated by ES-Exos or ESCs.

OBJECTIVE

Although the influence of age on clinical and laboratory features has been widely demonstrated in many arthropathies, studies on elderly onset >> 60 years) psoriatic arthritis (EOPsA) are rare. This study compares manifestations at onset and two year outcome of EOPsA with those of younger onset PsA (YOPsA).

METHODS

Sixty-six consecutive PsA patients with disease duration < 1 year, 16 EOPsA >> 60 years) and 50 YOPsA (< or = 60 years) were admitted to a prospective study. Clinical, laboratory, and radiographic assessment were carried out at admission and after two years. HLA class I and bone scintigraphy were also recorded. In 10 patients with EOPsA and 24 with YOPsA it was possible to obtain synovial fluid, which was subsequently analysed for local inflammatory indices, including interleukin (IL) 1 beta, IL6, and IL8.

RESULTS

Presenting manifestations of EOPsA differed from YOPsA in number of active joints (mean (SD)) (12.2 (6.3) v 6.7 (4.6), p < 0.001), foot bone erosions (2.7 (1.2) v 1.1 (1.1), p < 0.001), erythrocyte sedimentation rate (64.2 (35.3) v 30.5 (30.0) mm 1st h, p < 0.001), C reactive protein (3.9 (2.0) v 1.3 (1.3) mg/dl, p < 0.001) and synovial fluid IL1 beta (8.0 (4.7) v 3.0 (3.0) pg/ml, p < 0.001) and IL6 (828.2 (492.6) v 469.3 (201.4) pg/ml, p < 0.005). No differences were found in the number of subjects with dactylitis, pitting oedema, HLA-B27, or signs of sacroiliac and sternoclavicular joint involvement at bone scintigraphy. After two years, progression was more evident in EOPsA than in YOPsA, as the number of new erosions in the hands and also the C reactive protein were higher in EOPsA patients.

CONCLUSIONS

PsA has a more severe onset and a more destructive outcome in elderly people (onset>> 60 years) than in younger subjects. This behaviour may be influenced by immune changes associated with aging, as suggested by the higher concentrations of IL1 beta and IL6 found in the synovial fluid of EOPsA than in YOPsA.

The case of a 59-year-old female with a longstanding hemiplegia who developed unilateral psoriatic arthritis and unilateral psoriasis on the non-hemiplegic side only is reported. Investigations showed an erythrocyte sedimentation rate of 58 mm/h, with all classes of rheumatoid factor negative in serum and synovial fluid (SF). An inflammatory infiltrate was demonstrated in both knee joints despite the absence of clinical involvement on the hemiplegic side. In addition, while neurofilament and substance P (SP) immunoreactivity was demonstrated in both synovial specimens, conspicuously less SP was present in the clinically involved synovial membrane. Finally, SF levels of SP and IL1 beta were raised in the clinically involved knee only.

Sertoli cells are necessary to provide adequate levels of lactate for germ cell development. Lactate production is hormonally regulated by follicle-stimulating hormone (FSH) and by a large set of intratesticular regulators such as interleukin-1 beta (IL1 beta) and basic fibroblast growth factor (bFGF). Little is known regarding the critical step in the production of this metabolite, viz., the entrance of glucose into the cell as mediated by GLUTs. The aim of the present study was to investigate the expression of the glucose transporters GLUT1 and GLUT3 and its possible regulation by FSH, IL1 beta, and bFGF in Sertoli cells at two different time-points in sexual development. Sertoli cells retaining the ability to undergo mitosis (obtained from 8-day-old rats) and in the process of terminal differentiation (obtained from 20-day-old rats) were examined. Testicular tissue sections and Sertoli cell monolayers obtained from 8- and 20-day-old rats showed positive immunostaining for GLUT1 and GLUT3 proteins. GLUT1 and GLUT3 mRNA levels were detected at the two ages analyzed. Treatment of Sertoli cells obtained from 8- and 20-day-old rats with FSH, IL1 beta, and bFGF for various periods of time (12, 24, and 48 h) increased GLUT1 without changing GLUT3 mRNA levels. Our results thus show that Sertoli cells express GLUT1 and GLUT3 throughout pubertal development, and that, in Sertoli cells, only GLUT1 is regulated by hormones during pubertal development. Hormonal regulation of GLUT1 expression and consequently glucose uptake and lactate production may be a key molecular event in the regulation of spermatogenesis by hormones.

BACKGROUND

Dendritic cells orchestrate pivotal immunological processes mediated by the production of cytokines and chemokines.

OBJECTIVE

To assess whether neutralisation of tumour necrosis factor alpha (TNF alpha) during maturation of dendritic cells affects their phenotype and behaviour, which might explain the beneficial effects of TNF alpha neutralisation in rheumatoid arthritis.

METHODS

Immature and fully matured dendritic cells were cultured from blood monocytes from patients with rheumatoid arthritis and healthy controls following standardised protocols. TNF alpha was neutralised by addition of the p55 soluble TNF alpha receptor, PEGsTNFRI. The effect of TNF alpha neutralisation on the phenotype (CD14, CD16, CD32, CD64, CD80, CD83, CD86, and MHC) of dendritic cells was investigated by flow cytometry. Expression of chemokines (CCL17, CCL18, CCL19, CCL22, CCL3, and CXCL8) and production of IL1 beta and IL6 during dendritic cell differentiation and maturation were examined.

RESULTS

Neutralisation of TNF alpha during the differentiation and maturation of dendritic cells did not result in an altered dendritic cell phenotype in the rheumatoid patients or the healthy controls. In contrast, the expression of CCL17, CCL18, CCL19, CCL22, CCL3, and CXCL8 by dendritic cells was significantly reduced when TNF alpha activity was inhibited during lipopolysaccharide triggered dendritic cell maturation. The production of IL1 beta and IL6 by mature dendritic cells was inhibited by PEGsTNFRI.

CONCLUSIONS

Inhibition of TNF alpha activity during dendritic cell maturation leads to the development of semi-mature cells. These data suggest a novel pathway by which the neutralisation of TNF alpha might exert its therapeutic effects.

Receptor-interacting protein (RIP) is a serine/threonine protein kinase that is critically involved in tumor necrosis factor receptor-1 (TNF-R1)-induced NF-kappa B activation. In a yeast two-hybrid screening for potential RIP-interacting proteins, we identified ZIN (zinc finger protein inhibiting NF-kappa B), a novel protein that specifically interacts with RIP. ZIN contains four RING-like zinc finger domains at the middle and a proline-rich domain at the C terminus. Overexpression of ZIN inhibits RIP-, IKK beta-, TNF-, and IL1-induced NF-kappa B activation in a dose-dependent manner in 293 cells. Domain mapping experiments indicate that the RING-like zinc finger domains of ZIN are required for its interaction with RIP and inhibition of RIP-mediated NF-kappa B activation. Overexpression of ZIN also potentiates RIP- and TNF-induced apoptosis. Moreover, immunofluorescent staining indicates that ZIN is a cytoplasmic protein and that it colocalizes with RIP. Our findings suggest that ZIN is an inhibitor of TNF- and IL1-induced NF-kappa B activation pathways.

OBJECTIVE

To analyze the association of interleukin (IL-1) gene polymorphisms with susceptibility to, and severity of, rheumatoid arthritis (RA) patients, comparing them with the genotype distribution in healthy controls. Also, to assess the influence of IL1-B and IL1RN gene polymorphism on IL-1beta/IL-1Ra plasma levels and response to therapy.

METHODS

We tested the allelic distribution of IL-1B (-511 and +3953) and IL-1RN (variable number of tandem repeats) gene polymorphism in 126 RA patients and 178 healthy blood donors (HBDs). The patients were categorized into two subgroups in relation to the response to methotrexate (MTX) therapy. Group A included 70 RA patients in stable partial remission after 6 months of MTX treatment (MTX-R). Group B included 56 RA patients with active disease despite MTX therapy. This group received antitumor necrosis factor (TNF) biological drugs and were defined MTX-nonresponders (MTX-NR).

RESULTS

None of the two IL-1B (-511 and +3953) gene polymorphisms were significantly different in frequency between RA patients and healthy controls. We observed an increased frequency of the rare allele IL1RN*3 in RA patients with active disease, not responding to MTX therapy (MTX-NR) (4.5%) vs MTX-R (3.6%) and healthy controls (0.8%). Interestingly, RA patients whose genotypes included the IL1RN*long allele (haplotype long-C-T) showed the worse response to MTX. HBDs harboring the IL1RN*2/2 genotype showed significantly lower levels of plasma IL1-Ra, but comparable levels of IL-1beta with regard to subjects with the presence of the IL1RN* long allele. Furthermore, the presence of the TT IL-1B +3953 genotype was associated with lower plasma levels of IL1-Ra, both in HBDs and in RA patients. Carriers of the IL1RN*2 allele responded better to infliximab therapy.

CONCLUSIONS

The results of this study provide evidence of an association between the IL1RN*long allele and RA, the strongest association being observed in RA patients with an aggressive disease resistant to MTX treatment.

The semen of most men with spinal cord injury (SCI) contains sperm with abnormally low motility. Studies suggest that the seminal plasma is the source of this condition. The seminal plasma of men with SCI contains an abnormally high number of white blood cells (WBC), specifically, activated T cells. It is known that activated T cells secrete cytokines and elevated concentrations of cytokines can be harmful to sperm. It is not known if the seminal plasma of men with SCI contains elevated concentrations of cytokines. The purpose of this study was to determine if the seminal plasma of men with SCI contained elevated concentrations of cytokines. Using the method of enzyme-linked immunosorbant assay (ELISA), ten cytokines were measured in the seminal plasma of men with SCI as well as healthy non-SCI control subjects. The cytokines of interest were grouped according to Th1 effector functions: interleukin 1 beta, interleukin 2, interleukin 12, tumor necrosis factor alpha, tumor necrosis factor beta, interferon gamma (IL1 beta, IL2, IL1beta, INF gamma, respectively) and Th2 effector functions: interleukin 4, interleukin 6, interleukin 10, transforming growth factor beta 1 (IL4, IL6, IL1beta 1, respectively). The results showed a predominance of Th1 versus Th2 cytokine production in the seminal plasma of men with SCI compared with that of control subjects. This finding suggests an immunologic basis for infertility as a possible avenue of investigation in these men.

Neural injury is known to trigger inflammatory changes, including the synthesis of proinflammatory cytokines such as interleukin-1-<em>beta</em> (<em>IL1</em><em>beta</em>), tumor necrosis factor-alpha (TNFalpha), and interferon-gamma (IFNgamma) [G. Raivich, L. L. Jones, C. U. A. Kloss, A. Werner, H. Neumann, and G. W. Kreutzberg, 1998, J Neurosci, 18: 5804-5816] that may play a pivotal role in mediating the cellular response in the affected brain tissue. Here we examined the effects of transgenic deletion of receptors for these cytokines on neuronal cell loss in the adult mouse facial motor nucleus after a peripheral, facial nerve cut. Homozygous deletion of <em>IL1</em> receptor 1 (<em>IL1</em>R1), TNF receptor 1 or 2 (TNFR1 or TNFR2), or IFNgamma receptor 1 (IFNgammaR1) alone had no effect but combined deletion of TNFR1 and TNFR2 caused a striking absence of alphaX <em>beta</em>2 integrin/IBA1-double-labeled, phagocytic microglial nodules in the axotomized facial motor nucleus 14 days after nerve cut. Moreover, this combined deletion also led to an almost complete prevention of cell loss by Day 29. Additional neuronal cell counts at Day 60 revealed a second phase of motoneuron cell disappearance, which did not depend on the presence of TNF receptors. However, there was still the same 22% difference in the total number of motoneurons between the wild-type and TNFR1 & 2-deficient mice, underlining the role of TNF ligands and both TNF receptors in mediating the early phase of neuronal cell loss after traumatic injury.

BACKGROUND

The authors previously identified and compared alterations in gene expression in the myocardia of patients with deteriorating heart failure who underwent left ventricular assist device (LVAD) implantation with those of patients with stable end-stage failure (ESF). We hypothesized that matrix metalloproteinases (MMPs) and their endogenous inhibitors, the tissue inhibitors of MMPs (TIMPs), would be implicated in the mechanisms that underlie deteriorating heart failure.

METHODS

Gridded macro-array filters were used to provide a broad overview of MMP and TIMP mRNA expression in heart failure. Precise mRNA levels of TIMP1, MMP1, and beta-spectrin were determined using quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) of myocardial samples from 27 patients with deteriorating heart failure who underwent LVAD implantation, from 17 patients with stable ESF who underwent elective heart transplantation, and from 28 donor organs with good hemodynamic function.

RESULTS

Gridded macro-arrays analysis of pooled failing heart samples determined that TIMP1 mRNA was the most readily detectable TIMP in failing myocardium. Quantitative RT-PCR showed that expression levels in individual patients were similar in patients with stable ESF (1.00 +/- 0.24, n = 17) and in donor organ samples (1.49 +/- 0.22, n = 28) but were significantly increased in the deteriorating heart failure group (5.38 +/- 0.32, n = 26, p < 0.0001 compared with patients with ESF). Similarly, MMP1 levels did not differ between donor and ESF groups but increased in the deteriorating failure group (6.04 +/- 0.50, n = 27, p < 0.001 compared with the ESF group). Levels of beta-II spectrin were the same in all 3 groups. Both TIMP1 and MMP1 showed positive correlation with each other and with previously determined levels of mRNA for both interleukin-1beta (IL-1beta) and IL-6 in this patient series when considering all patients individually, but neither correlated with tumor necrosis factor alpha.

CONCLUSIONS

Patients with deteriorating heart failure have increased expression of TIMP1 and MMP1 mRNA. Correlation with pro-inflammatory cytokines suggests common pathways of regulation and potential activation by IL-6 and IL1-beta.

OBJECTIVE

The relationship of plasma cytokine levels to infection, core temperature, and to one another in patients with thermal injury was examined.

BACKGROUND

The response to infection has been associated with cytokines such as interleukin 1 beta (IL1 beta), interleukin 6 (IL6), and tumor necrosis factor alpha (TNF alpha), and these cytokines have been studied in various inflammatory diseases. The authors previously reported that patients with thermal injury have elevated IL1 beta and IL6 plasma levels and that these cytokines may play different roles in the response to thermal injury.

METHODS

IL1 beta, IL6, and TNF alpha were measured by enzyme-linked immunosorbent assay (ELISA) in serial samples of plasma from 27 patients.

RESULTS

IL6 and TNF alpha levels were increased in severely infected patients as compared to patients who remained free of infection, and the IL6 level was higher in infected patients who died than those who survived. There was no apparent relationship between IL1 beta levels and infection. IL6 and IL1 beta were positively correlated with core temperature. The correlations between IL6 and IL1 beta, between IL6 and TNF alpha, and between TNF alpha and IL1 beta were significant.

CONCLUSIONS

These results suggest that IL6 and TNF alpha play a role in the response of burned patients to infection.

The effects of interleukin 7 (IL7) previously known as murine pre-B cell growth factor/lymphopoietin 1 on the growth of murine thymocytes was investigated. In the presence of a suboptimal dose of phytohemagglutinin, IL7 induced a dose-dependent increase in thymocyte proliferation which was comparable to that induced by IL1. Additionally IL7 was shown to synergize with a suboptimal dose of IL1 to enhance thymocyte proliferation. Thymocyte proliferation induced by IL7, like that induced by IL1, was inhibited when either recombinant transforming growth factor (TGF) beta 1 or beta 2 were added at the initiation of culture. Interestingly, IL7-driven thymocyte proliferation was considerably less susceptible to inhibition by TGF-beta 1 or TGF-beta 2 than that induced by IL1. Taken together these results suggest IL7 may activate distinct populations of thymocytes and/or act through a pathway distinct from that utilized by IL1.

The current study was aimed to characterize for the first time the alterations in the characteristic neuro-inflammatory markers triggered by sarin exposure in the rat's brain, and to investigate its dependency on seizure duration. Centrally mediated seizures are a common consequence of exposure to organophosphates (OP) despite conventional treatment with atropine and an oxime. In the present study midazolam, was used to control duration and intensity of seizures. The levels of the pro-inflammatory cytokine peptides IL-1beta, IL-6, TNF-alpha and prostaglandin E2 (PGE2) were monitored at various times after sarin exposure in the hippocampus and cortex of rats treated with midazolam following 5 or 30 min of seizure activity. Biochemical evaluation of brain tissues revealed a significant increase in the level of the pro-inflammatory peptides starting at 2 h and peaking at 2-24 h following sarin. Hippocampal values of IL1-beta increased from 1.2+/-0.1 pg/mg tissue (control), to 2.4+/-0.3 at 2 h (5 min seizure) and to 9.3+/-2.5 at 8h (30 min seizure). PGE2 level in the hippocampus increased up to 24 h following exposure (from 56+/-3 to 175+/-26 and 277+/-28 pg/mg tissue) following 5 and 30 min of seizure activity respectively. Thus, unlike limitation of seizures to 5 min by midazolam, delayed treatment (30 min) resulted in prolonged seizures and pronounced increase in cytokines and PGE2. In addition, a second increase in inflammatory markers was observed 30 days following sarin exposure only in rats treated following 30 min of seizure activity. Histological evaluation of the rat brain, conducted in this study, revealed lack of damage in the hippocampus and piriform cortex with minor lateral ventricles enlargement in few animals following 5 min of sarin-induced seizure activity. In contrast, marked histological damage to the brain was demonstrated following 30 min of seizure activity, consisting severe damage to the hippocampus, piriform cortex and some thalamic nuclei. In summary, a novel characterization of the prolonged central neuro-inflammatory process that accompanies sarin exposure is presented. The timing of the anticonvulsive treatment was shown to be crucial in modulation of the neuro-inflammatory response, and may implicate the consequent long-term brain damage.