Higher Levels Of TNF-alpha Research Articles

Association between TNF-α and Entamoeba histolytica Diarrhea

An association between tumor necrosis factor alpha (TNF-alpha) and Entamoeba histolytica diarrhea was assessed in a cohort of 138 non-related Bangladeshi children who have been prospectively followed since 2001. Peripheral blood mononuclear cells (PBMCs) obtained at study entry were purified, cultured, and stimulated with soluble amebic antigen before cytokine measurement from supernatant. Higher levels of TNF-alpha were associated with increased risk of first (P = 0.01) and recurrent E. histolytica-related diarrheal episodes (P = 0.005). Children who developed E. histolytica diarrhea had significantly higher TNF-alpha protein levels than those who experienced asymptomatic E. histolytica infection (P value = 0.027) or no infection (P value = 0.017). Microarray studies performed using RNA isolated from acute and convalescent whole blood and colon biopsy samples revealed higher but non-significant TNF-alpha messenger RNA (mRNA) levels in subjects with acute E. histolytica diarrhea compared with convalescence. We conclude that there is an association between higher TNF-alpha production and E. histolytica diarrhea.

Immunocompetence effects of polysaccharide of snakegourd root on human peripheral blood mononuclear cells in vitro

To establish the method of promoting human peripheral blood mononuclear cell proliferation by polysaccharide of snakegourd root and identify the effects of polysaccharide of snakegourd root on lymphocyte proliferation, T lymphocyte subsets and the different levels of TNF-alpha and IL-6. The polysaccharide of snakegourd root preparations were purified with dialysis and ethanol precipitation. The healthy human PBMC were used as the target cells for screening potency of the drugs. MTT colorimetry was established to examine the levels of lymphocyte proliferation on human PBMC by polysaccharide of snakegourd root in vitro. The percents of lymphocyte subsets (CD3+, CD4+, CD8+ T lymphocyte) and the different levels of TNF-a and IL-6 in PBMC were analysed by FCM and ELISA, respectively. 1.0-50.0 mmol x L(-1) of polysaccharides of snakegourd root showed the significant effects of promoting proliferation of human PBMC (P < 0.05). The percents of CD3+, CD4+, CD8+ T lymphocytes in PBMC treated with 5.0 and 10.0 mmol x L(-1) of polysaccharides of snakegourd root were significantly higher than those of the control group (P < 0.05). The levels of TNF-alpha and IL-6 were significantly higher than those of the control group after 1.0, 5.0, 10.0 mmol x L(-1) of polysaccharides of snakegourd root stimulation on the human PBMC at 8 hours (P < 0.05). The significant effects on promoting lymphocyte proliferation and activation of the polysaccharide of snakegourd root are confirmed in this study. The percents of lymphocyte subsets are increased in different degrees by the polysaccharide of snakegourd root. The high levels of TNF-alpha and IL-6 are secreted after the polysaccharides of snakegourd root stimulation on the human PBMC, which lays a foundation for further elucidating the immunocompetence effects and mechanism of the polysaccharide of snakegourd root.

Synergistic effects of lipopolysaccharides from periodontopathic bacteria on pro-inflammatory cytokine production in anex vivowhole blood model

Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia have been strongly associated with chronic periodontitis. This disease is characterized by an accumulation of inflammatory cells in periodontal tissue and subgingival sites. The secretion of high levels of inflammatory cytokines by those cells is believed to contribute to periodontal tissue destruction. The aim of this study was to investigate the inflammatory response of whole blood from periodontitis patients following challenges with whole cells of P. gingivalis, T. denticola, and T. forsythia or their lipopolysaccharides (LPS), individually and in combination. Whole blood collected from seven periodontitis patients was stimulated with whole cells or LPS and the production of interleukin (IL)-1beta, IL-6, IL-8, and tumor necrosis factor alpha (TNF-alpha) were quantified by enzyme-linked immunosorbent assays. The mono and mixed challenges with whole bacterial cells or LPS induced the secretion of high amounts of IL-1beta, IL-6, IL-8, and TNF-alpha by the mixed leukocyte population from periodontitis patients. In addition, P. gingivalis LPS, T. denticola LPS, and T. forsythia LPS acted in synergy to induce high levels of IL-1beta and TNF-alpha. This study suggests that P. gingivalis, T. denticola, and T. forsythia may contribute to the immunodestructive host response characteristic of periodontitis through synergistic effects of their LPS on the inflammatory response induced by a mixed population of leukocytes.

Distinct Th1, Th2 and Treg cytokines balance in chronic periapical granulomas and radicular cysts

Periapical lesions are a host response that involves immune reaction to prevent dissemination of bacteria from an infected root canal. The purpose of this study was to evaluate the levels of nitric oxide (NO), IL-4, TGF-beta, tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) in chronic periapical lesions and to determine their possible association with clinical and radiographic parameters. Seventeen human radicular cysts and 30 periapical granulomas were used in this study. Cytokines and NO were assessed by enzyme-linked immunosorbent assay and by the Griess reaction respectively confirmed by immunohistochemical. TNF-alpha and IFN-gamma were detected in 10% of granulomas and in 41.2% and 70% of radicular cysts. IL-4 was reactive in 24% of cysts, and TGF-beta was positive in all samples. Patients with tenderness showed significantly higher levels of IFN-gamma and IL-4 (P < 0.05). Swelling was associated with high levels of TNF-alpha, IFN-gamma, and IL-4 (P < 0.05). Lesions presenting bone resorption were associated with high levels of NO (P < 0.05). Periapical granulomas display a regulatory environment characterized by high TGF-beta and low inflammatory cytokine levels, while radicular cysts has mist Th1 and Th2 inflammatory reaction with the presence of IFN-gamma, TNF-alpha, and IL-4.

Failure of Interferon γ to Induce the Anti-Inflammatory Interleukin 18 Binding Protein in Familial Hemophagocytosis

BackgroundFamilial hemophagocytosis (FHL) is a rare disease associated with defects in proteins involved in CD8+ T-cell cytotoxicity. Hyperactivation of immune cells results in a perilous, Th1-driven cytokine storm. We set out to explore the regulation of cytokines in an FHL patient who was clinically stable on low-dose immunosuppressive therapy after bone marrow transplantation over a six-month period. During this period, chimerism analyses showed that the fraction of host cells was between 1 and 10%. Both parents of the patient as well as healthy volunteers were studied for comparison.Methods/Principal FindingsUsing ELISA, quantitative real-time PCR, and clinical laboratory methods, we investigated constitutive and inducible cytokines, polymorphisms, and clinical parameters in whole blood and whole blood cultures. Although routine laboratory tests were within the normal range, the chemokines IP-10 and IL-8 as well as the cytokine IL-27p28 were increased up to 10-fold under constitutive and stimulated conditions compared to healthy controls. Moreover, high levels of IFNγ and TNFα were produced upon stimulation. Unexpectedly, IFNγ induction of IL-18 binding protein (IL-18BP) was markedly reduced (1.6-fold vs 5-fold in controls). The patient's mother featured intermediately increased cytokine levels, whereas levels in the father were similar to those in the controls.Conclusions/SignificanceSince IL-18 plays a major role in perpetuating hemophagocytosis, the failure of IFNγ to induce IL-18BP may constitute a fundamental pathogenetic mechanism. Furthermore, increased production of IL-8 and IL-27 appears to be associated with this disease. Such dysregulation of cytokines was also found in the heterozygous parents, providing a novel insight into genotype-phenotype correlation of FHL which may encourage future research of this rare disease.

Effect of acute exercise on the levels of salivary cortisol, tumor necrosis factor-α and nitric oxide

The aim of the present study was to assess the levels of salivary cortisol, tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) before, during and after acute exercise. Acute exercise was induced using a standard treadmill test with Bruce protocol in ten physically active male participants. Unstimulated saliva was collected before, during and after exercise. The levels of salivary cortisol and TNF-alpha were assessed by enzyme immunoassays. Salivary NO was determined by the Griess reagent. The results showed that both salivary cortisol and TNF-alpha increased and peaked at 14 min during exercise and then decreased. The levels of NO were increased up to 1 h after exercise and subsequently lowered after 24 h. The results of the present study suggest that acute exercise may induce high levels of salivary cortisol, TNF-alpha and NO.

Cytokine patterns and survival in haemodialysis patients

Increased pro-inflammatory cytokine levels are associated with decreased survival. We performed factor analyses to determine if pro-inflammatory and anti-inflammatory cytokines in haemodialysis (HD) patients load onto one or two discrete factors and assessed if patients with a specific pattern of high pro-inflammatory cytokines have decreased survival compared to patients with a high anti-inflammatory cytokine pattern. We evaluated 231 HD patients and analyzed them based on the three most common cytokine distribution patterns seen: a high pro-inflammatory group, a high anti-inflammatory group and all others. Survival and Cox regression analyses were performed. Factor analyses of individual cytokines showed that they loaded onto a single factor. Sixty-five patients had a pro-inflammatory pattern of high IL-1, IL-6 and TNF-alpha levels and low anti-inflammatory parameters, including IL-2, IL-4, IL-5, IL-12, CH50 and T-cell number. The next most frequent cytokine pattern was found in 20 patients with high levels of anti-inflammatory parameters. The patients with high pro-inflammatory cytokines had decreased survival compared to patients without a characteristic cytokine pattern. Further research is needed to better define the underlying causes of increased inflammation among end-stage renal disease patients and to apply anti-inflammatory therapies that may mitigate adverse effects on patient outcomes.

Selection for Genetic Variation Inducing Pro-Inflammatory Responses under Adverse Environmental Conditions in a Ghanaian Population

BackgroundChronic inflammation is involved in the pathogenesis of chronic age-associated, degenerative diseases. Pro-inflammatory host responses that are deleterious later in life may originate from evolutionary selection for genetic variation mediating resistance to infectious diseases under adverse environmental conditions.Methodology/Principal FindingsIn the Upper-East region of Ghana where infection has remained the leading cause of death, we studied the effect on survival of genetic variations at the IL10 gene locus that have been associated with chronic diseases. Here we show that an IL10 haplotype that associated with a pro-inflammatory innate immune response, characterised by low IL-10 (p = 0.028) and high TNF-α levels (p = 1.39×10−3), was enriched among Ghanaian elders (p = 2.46×10−6). Furthermore, in an environment where the source of drinking water (wells/rivers vs. boreholes) influences mortality risks (HR 1.28, 95% CI [1.09–1.50]), we observed that carriers of the pro-inflammatory haplotype have a survival advantage when drinking from wells/rivers but a disadvantage when drinking from boreholes (pinteraction = 0.013). Resequencing the IL10 gene region did not uncover any additional common variants in the pro-inflammatory haplotype to those SNPs that were initially genotyped.Conclusions/SignificanceAltogether, these data lend strong arguments for the selection of pro-inflammatory host responses to overcome fatal infection and promote survival in adverse environments.

Paracoccin from Paracoccidioides brasiliensis; purification through affinity with chitin and identification of N‐acetyl‐β‐D‐glucosaminidase activity

The dimorphic fungus Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, the most frequent systemic mycosis in Latin America. Our group has been working with paracoccin, a P. brasiliensis lectin with MM 70 kDa, which is purified by affinity with immobilized N-acetylglucosamine (GlcNAc). Paracoccin has been described to play a role in fungal adhesion to extracellular matrix components and to induce high and persistent levels of TNFalpha and nitric oxide production by macrophages. In the cell wall, paracoccin colocalizes with the beta-1,4-homopolymer of GlcNAc into the budding sites of the P. brasiliensis yeast cell. In this paper we present a protocol for the chitin-affinity purification of paracoccin. This procedure provided higher yields than those achieved by means of the technique based on the affinity of this lectin with GlcNAc and had an impact on downstream assays. SDS-PAGE and Western blot analysis revealed similarities between the N-acetylglucosamine- and chitin-bound fractions, confirmed by MALDI-TOF-MS of trypsinic peptides. Western blot of two-dimensional gel electrophoresis of the yeast extract showed a major spot with M(r) 70,000 and pI approximately 5.63. Moreover, an N-acetyl-beta-D-glucosaminidase activity was reported for paracoccin, thereby providing new insights into the mechanisms that lead to cell wall remodelling and opening new perspectives for its structural characterization.

Correlations between adipocytokines and insulin resistance in women with polycystic ovary syndrome

To investigate the correlations between adipocytokines and insulin resistance in women with polycystic ovary syndrome. Sixty women with polycystic ovary syndrome aged 19-34 years old were divided into 2 groups: group A [n = 36, BMI > or = 25 kg/m(-2) or WHR (waist height ratio) > 0.85] and group B (n = 24, BMI < 25 kg/m(-2) and WHR < or = 0.85). Twenty-six healthy infertile women with a mean age of 26 + or - 8 years old served as controls and they were named as group C (n = 26, BMI < 25 kg/m(-2) and WHR < or = 0.85). Anthropometric measurements, hormonal profiles and metabolic profiles were compared between three groups. Plasma leptin, CRP and free fatty acid were measured by enzyme-linked immunosorbent assay (ELISA). Adiponectin, interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were measured by radioimmunoassay. Insulin resistance was estimated by homeostasis model assessment (HOMA). (1) Adiponectin and TNF-alpha levels were significantly higher in group B (17 + or - 9) mg/L and (1.0 + or - 0.3) mg/L respectively than in group A (10 + or - 7) mg/L and (0.8 + or - 0.2) mg/L respectively. Leptin was lower in group B (24 + or - 13) microg/L and group C than in group A (42 + or - 21) microg/L (all P < 0.01). IL-6 was higher in group A (173 + or - 184) ng/L and group B (184 + or - 44) ng/L than in group C (P < 0.05 and P < 0.01 respectively). No significant difference of circulating level of CRP was found between these 3 groups. (2) Leptin was positively correlated with BMI, WHR and HOMA-IR (all P < 0.01) whereas adiponectin was negatively correlated with BMI, WHR and HOMA-IR (P < 0.01; P < 0.01, P < 0.05 respectively). Multivariate regression showed that adiponectin was the most significant predictor of HOMA-IR and it explained 16.5% of variance. The higher levels of leptin and TNF-alpha, the lower level of adiponectin in obese PCOS and the higher level of TNF-alpha and IL-6 in non-obese PCOS suggested different adipocytokines play different roles of insulin resistance in PCOS.

Normalization of A2A and A3 adenosine receptor up‐regulation in rheumatoid arthritis patients by treatment with anti–tumor necrosis factor α but not methotrexate

To investigate A(1), A(2A), A(2B), and A(3) adenosine receptors in lymphocytes and neutrophils from patients with early rheumatoid arthritis (ERA) as well as from RA patients treated with methotrexate (MTX) or anti-tumor necrosis factor alpha (anti-TNFalpha), as compared with those in age-matched healthy controls, and to examine correlations between the status and functionality of adenosine receptors and TNFalpha release and NF-kappaB activation. Adenosine receptors were analyzed by saturation binding assays and Western blot analyses. We investigated the potency of typical A(2A) and A(3) agonists in the production of cAMP in control subjects, ERA patients, and RA patients treated with MTX or anti-TNFalpha. In a separate cohort of RA patients, TNFalpha release and NF-kappaB activation were evaluated in plasma and nuclear extracts, respectively. In ERA patients, we found a high density and altered functionality of A(2A) and A(3) receptors. The binding and functional parameters of A(2A) and A(3) receptors normalized after anti-TNFalpha, but not MTX, treatment. TNFalpha release was increased in ERA patients and in MTX-treated RA patients, whereas in anti-TNFalpha-treated RA patients, release was comparable to that in the controls. NF-kappaB activation was elevated in ERA patients and in MTX-treated RA patients. Anti-TNFalpha treatment mediated decreased levels of NF-kappaB activation. A(2A) and A(3) receptor up-regulation in ERA patients and in MTX-treated RA patients was associated with high levels of TNFalpha and NF-kappaB activation. Treatment with anti-TNFalpha normalized A(2A) and A(3) receptor expression and functionality. This new evidence of A(2A) and A(3) receptor involvement opens the possibility of exploiting their potential role in human diseases characterized by a marked inflammatory component.

Circulating levels of TNF-α and its soluble receptors in the plasma of patients with epithelial ovarian cancer

The significance of circulating levels of TNF-alpha and its soluble receptors (sTNF-Rs) in the plasma of patients with epithelial ovarian cancer (EOC) has not been fully elucidated. The present study was to investigate the relationship of pretreatment plasma levels of TNF-alpha, sTNFR-1 and sTNFR-2 with outcome in 126 patients with EOC. Concentrations of TNF-alpha and sTNF-Rs were determined by enzyme-linked immunosorbent assay (ELISA). Median TNF-alpha and sTNF-Rs levels were significantly higher in EOC patients than in healthy controls. High plasma levels of TNF-alpha and sTNF-Rs were correlated with tumor stage and with reduced mean survival time (MST). The results of the present study suggested that preoperative plasma TNF-alpha and sTNF-Rs levels in EOC patients correlated with the highest risk of cancer progression. Thus, the clinical value of an activated TNF system in EOC needs to be further investigated.

The Proinflammatory Environment in Potential Heart and Lung Donors: Prevalence and Impact of Donor Management and Hormonal Therapy

Brain stem death can elicit a potentially manipulable cardiotoxic proinflammatory cytokine response. We investigated the prevalence of this response, the impact of donor management with tri-iodothyronine (T3) and methylprednisolone (MP) administration, and the relationship of biomarkers to organ function and transplant suitability. In a prospective randomized double-blinded factorially designed study of T3 and MP therapy, we measured serum levels of interleukin-1 and -6 (IL-1 and IL-6), tumor necrosis factor-alpha (TNF-alpha), C-reactive protein, and procalcitonin (PCT) levels in 79 potential heart or lung donors. Measurements were performed before and after 4 hr of algorithm-based donor management to optimize cardiorespiratory function and +/-hormone treatment. Donors were assigned to receive T3, MP, both drugs, or placebo. Initial IL-1 was elevated in 16% donors, IL-6 in 100%, TNF-alpha in 28%, CRP in 98%, and PCT in 87%. Overall biomarker concentrations did not change between initial and later measurements and neither T3 nor MP effected any change. Both PCT (P =0.02) and TNF-alpha (P =0.044) levels were higher in donor hearts with marginal hemodynamics at initial assessment. Higher PCT levels were related to worse cardiac index and right and left ventricular ejection fractions and a PCT level more than 2 ng x mL(-1) may attenuate any improvement in cardiac index gained by donor management. No differences were observed between initially marginal and nonmarginal donor lungs. A PCT level less than or equal to 2 ng x mL(-1) but not other biomarkers predicted transplant suitability following management. There is high prevalence of a proinflammatory environment in the organ donor that is not affected by tri-iodothyronine or MP therapy. High PCT and TNF-alpha levels are associated with donor heart dysfunction.

Higher Inflammatory Marker Levels in Older Persons: Associations With 5-Year Change in Muscle Mass and Muscle Strength

There is growing evidence that higher levels of inflammatory markers are associated with physical decline in older persons, possibly through the catabolic effects of inflammatory markers on muscle. The aim of this study was to investigate the association between serum levels of inflammatory markers and loss of muscle mass and strength in older persons. Using data on 2,177 men and women in the Health, Aging, and Body Composition Study, we examined 5-year change in thigh muscle area estimated by computed tomography and grip and knee extensor strength in relation to serum levels of interleukin-6 (IL-6), C-reactive protein, tumor necrosis factor-alpha (TNF-alpha), and soluble receptors (measured in a subsample) at baseline. Higher levels of inflammatory markers were generally associated with greater 5-year decline in thigh muscle area. Most associations, with the exception of soluble receptors, were attenuated by adjustment for 5-year change in weight. Higher TNF-alpha and interleukin-6 soluble receptor levels remained associated with greater decline in grip strength in men. Analyses in a subgroup of weight-stable persons showed that higher levels of TNF-alpha and its soluble receptors were associated with 5-year decline in thigh muscle area and that higher levels of TNF-alpha were associated with decline in grip strength. TNF-alpha and its soluble receptors showed the most consistent associations with decline in muscle mass and strength. The results suggest a weight-associated pathway for inflammation in sarcopenia.

Ethyl pyruvate improves survival in awake hemorrhage

Classical experimental models of hemorrhage are characterized by the use of anesthetics that may interfere with the typical immune responses and pathology of hemorrhage/resuscitation. Thus, therapeutic strategies successful in anesthetized animals might not be beneficial in clinical trials. In this study, we analyzed whether ethyl pyruvate could provide therapeutic benefits during resuscitation in awake (unanesthetized) hemorrhage. Our results indicate that hemorrhage in unanesthetized animals required approximately 25% higher blood withdrawal than anesthetized animals to achieve the same targeted mean arterial blood pressure. Resuscitation with Hextend reestablished circulatory volume and improved survival during resuscitation of awake rodents. Yet, over 75% of the animals resuscitated with Hextend died within the first hours after hemorrhage. Resuscitation with Hextend containing 50 mM ethyl pyruvate protected over 87% of the animals. This survival benefit did not correlate with significant changes in the metabolic markers but with an anti-inflammatory potential during resuscitation. Unlike classical hemorrhage in anesthetized animals, ethyl pyruvate reestablished mean arterial blood pressure significantly earlier than Hextend in unanesthetized rodents. Unanesthetized animals showed twofold higher serum tumor necrosis factor (TNF)-alpha than anesthetized animals subjected to the same blood pressure. This process was not due to the response of a single organ, but affected all the analyzed organs including the lung, heart, spleen, and liver. Although resuscitation with Hextend failed to attenuate systemic TNF-alpha levels, it inhibited TNF-alpha levels in the lung, heart, and liver but not in the spleen. Unlike Hextend, resuscitation with ethyl pyruvate prevented high serum TNF-alpha levels and blunted TNF-alpha responses in all the organs including the spleen. These studies indicate that the inflammatory responses in anesthetized animals differ from that in unanesthetized animals and that awake hemorrhage can provide advantages in the study of anti-inflammatory strategies during resuscitation. Ethyl pyruvate may attenuate systemic inflammatory responses during resuscitation and improve survival in experimental models of awake hemorrhage.

TNF‐α and IL‐4 levels in generalized aggressive periodontitis subjects

The aim of this study was to evaluate tumor necrosis factor (TNF)-alpha and interleukin (IL)-4 levels in healthy sites and sites exhibiting signs of moderate and advanced generalized aggressive periodontitis (GAgP) in the same subject. The following sites were selected for crevicular fluid sampling in the same AgP subject (n = 14): Healthy sites (HS): no marginal bleeding or bleeding on probing (BOP) and probing depth (PD) <or= 3 mm; Moderate sites (MS): BOP and PD between 4 and 6 mm; Advanced sites (AS): BOP and PD >or= 7 mm. One site from periodontally healthy subjects (n = 13) was sampled for use as a control. TNF-alpha and IL-4 levels were measured using ELISA. The total amount of TNF-alpha was lower for control sites, while there were no differences among healthy and diseased sites from GAgP subjects (P < 0.05). The concentration of TNF-alpha was higher in HS, in relation to the other sites (P < 0.05). There were no significant differences among the groups regarding total amounts of IL-4 (P > 0.05), while IL-4 concentration was significantly higher in control sites, when compared with sites from GAgP subjects (P < 0.05). In conclusion, high levels of TNF-alpha and low levels of IL-4 were observed in both healthy and diseased sites within the same generalized AgP individuals.

Mesenchymal Stem Cells (MSC) Promote Aggressive Behavior of Human Breast Cancer Cells (MCF-7) in Vitro- the Role Cytokines (TNF-alpha) and Chemokines

The tumor microenvironment contributes to local tumor growth and may set up a metastatic niche for breast cancer cells. We tested the effects of spontaneously transformed murine mesenchymal stem cells (stMSC; Li et al. Cancer Res 2008) on the migration, proliferation and cell cycle of the MCF-7 human breast cancer cell line.MCF-7 cells were collected from plates held at confluence to establish cell cycle arrest and then grown either in control media (50/50 mix of fresh and conditioned MCF-7 media) or modification of stMSC conditioned media (50/50 mix of fresh and stMSC conditioned media with TNF-alpha, anti-TNF-alpha or isotype control). MCF-7 cells were plated at equal concentrations into 24-well dishes to establish near confluent plates. The MCF-7 monolayer was wounded using a pipette tip, and the area evaluated/photographed at 0, 24 and 48 hours (in triplicate). Media was changed daily. Synchronized cultures were grown in control or MSC media for 0, 24 or 48 hours in triplicates and analyzed by FACS for cell cycle status and standard cell counts. MCF-7 cells grown in MSC cultured media healed 49% (based on wound size) while control wound size was 26% healed at 24 hrs (p=0.00591). At 48 hrs, MCF-7 cells exposed to stMSC media showed 95% healing while MCF-7 cells in control media still retained 50% of the original wound size (50% healing; p=0.00002). The exposure MCF-7 grown in control media and MSC media did not differ in cell cycle progression. MSC media increased proliferation of MCF-7 cells at 24, 48 and 72 hours compared to control media. (p<0.002). In a separate experiments media from stMSC exposed to TNF-alpha led to increased healing of MCF-7 monolayer compared to stMSC conditioned media at 24 hrs (59.5% vs 44.5%; p=0.027). Healing was lower in media from stMSC cultured with anti-TNF blocking antibody compared to stMSC conditioned media at 24 hrs (66.6% vs 95.7%; p=0.002). We have also detected that stMSC conditioned media contains very high levels of TNF-alpha, MCP-1, CCL-5 and also high levels of NF-kB activity in stMSC conditioned media.StMSCs produce factors (such as TNF-alpha) that provide breast cancer cells (MCF-7) with migration and growth advantage. Animal experiments assessing the ability stMSCs to contribute to invasion and migration of breast cancer cells are currently underway.

Plasma from Patients with Sepsis Up-Regulates the Expression of CD49d and CD64 on Blood Neutrophils

An excessive interaction of blood neutrophils with microvascular walls may underlie the organ failure of sepsis. In this study, flow cytometric analysis was used to investigate whether plasma from 22 patients with sepsis altered the expression of the adhesion molecules (CD11a, CD11b, CD49d, and CD62L) on normal blood neutrophils and enhanced their binding to cultured endothelium. Most of the plasma samples from patients with sepsis increased the percentage of neutrophils bearing CD49d (86% samples versus 22% normal plasma samples; P < 0.001) and CD64 (69% samples versus 17% normal plasma samples; P < 0.001). This effect was not seen with plasma from patients with community-acquired infections who did not develop sepsis, nor with plasma from patients with acute or chronic inflammation who had no evidence of infection. A direct association was noted between the percentage of neutrophils expressing CD64 in the blood of patients with sepsis and the ability of plasma from these patients to up-regulate CD64 on normal neutrophils. Although CD62L was present on the majority of neutrophils after incubation with sepsis plasma, it was less apparent when the cells were cultured with normal plasma. The patients' plasma had no effect on neutrophils expressing CD11a and CD11b. High levels of TNF-alpha, IL-6, IL-8, and IL-10 were detected in the patients' blood, but incubation of the recombinant forms of these cytokines with neutrophils, even in the presence of LPS, did not increase CD49d and CD64 expression. The sepsis plasma also enhanced the attachment of neutrophils to untreated and TNF-alpha-treated endothelium, and this binding was impeded by anti-CD49d and anti-CD64 antibodies. We suggest that changes in the phenotype of neutrophils by circulating factors may facilitate their attachment to endothelium, which may be an important factor in the induction of organ dysfunction in severe sepsis.

Glycosaminoglycans modulate inflammation and apoptosis in LPS‐treated chondrocytes

Previous studies reported that hyaluronic acid (HA), chondroitin sulphate (CS) and heparan sulphate (HS) were able to reduce the inflammatory process in a variety of cell types after lipopolysaccharide (LPS) stimulation. The aim of this study was to investigate the anti-inflammatory effect of glycosaminoglycans (GAGs) in mouse articular chondrocytes stimulated with LPS. Chondrocyte treatment with LPS (50 microg/ml) generated high levels of TNF-alpha, IL-1beta, IL-6, IFN-gamma, MMP-1, MMP-13, iNOS gene expression and their related proteins, increased NO concentrations (evaluated in terms of nitrites formation), NF-kappaB activation and IkBalpha degradation as well as apoptosis evaluated by the increase in caspase-3 expression and the amount of its related protein. The treatment of chondrocytes using two different doses (0.5 and 1.0 mg/ml) of HA, chondroitin-4-sulphate (C4S), chondroitin-6-sulphate (C6S), HS, keratan sulphate (KS) and dermatan sulphate (DS) produced a number of effects. HA exerted a very small anti-inflammatory and anti-apoptotic effect while it significantly reduced NO levels, although the effect on iNOS expression and activity was extremely slight. C4S and C6S reduced inflammation mediators and the apoptotic process. C6S failed to decrease NO production, although iNOS expression and activity were significantly reduced. HS, like C4S, was able to reduce all the effects stimulated by LPS treatment. KS and DS produced no reduction in any of the parameters considered. These results give further support to the hypothesis that GAGs actively participate in the regulation of inflammatory and apoptotic processes.

Functional Dichotomy of Dendritic Cells following Interaction withLeishmania braziliensis: Infected Cells Produce High Levels of TNF-α, whereas Bystander Dendritic Cells Are Activated to Promote T Cell Responses

Leishmania braziliensis infections are often associated with exaggerated immune responses that can sometimes lead to severe disease associated with high levels of IFN-gamma and TNF-alpha. To explore the role played by dendritic cells (DCs) in these responses, we characterized DCs that were exposed to L. braziliensis. We found that DCs cultured with L. braziliensis parasites up-regulated DC activation markers and produced IL-12 and TNF-alpha. However, not all DCs in the culture became infected, and an analysis of infected and uninfected DCs demonstrated that the up-regulation of activation markers and IL-12 production was primarily confined to the uninfected (bystander) DCs. Further studies with Transwell chambers and parasite fractions indicated that the activation of bystander DCs was mediated by a soluble parasite product, in a type 1 IFN- and MyD88-independent, but TNF-alpha-dependent fashion, and that the activated DCs were more efficient at presenting Ag than control DCs. In contrast, L. braziliensis-infected DCs failed to up-regulate activation markers, but exhibited a dramatic enhancement in their ability to produce TNF-alpha in response to LPS as compared with uninfected DCs. These findings uncover a dual role for DCs in L. braziliensis infection: T cell activation by bystander DCs due to enhanced Ag-presenting capacity following exposure to soluble parasite products, and increased production of TNF-alpha by infected cells that may contribute to the local control of the parasites, but concomitantly induce immunopathology.