P40 Levels Research Articles

Identification of novel cytokine to judge the diagnosis and clinical phenotype of adult-onset Still’s disease

This study aimed to identify biomarkers to distinguish adult-onset Still’s disease (AOSD) and to predict disease phenotypes. In total, 49 patients diagnosed with AOSD and 200 patients with common diseases (controls) were included in the analysis. The levels of 69 cytokines were analyzed using a multi-suspension cytokine array. Cytokine cluster analysis was performed to identify specific molecular networks. Furthermore, random forest analysis and logistic regression analysis were used to rank cytokines based on their importance and to determine specific biomarkers for identification of AOSD patients and phenotypes. Patients with AOSD demonstrated significantly higher macrophage migration inhibitory factor (MIF) and interleukin (IL)-12(p40) serum levels than controls and patients with rheumatoid arthritis. Serum levels of chemokine (C-C motif) ligand (CCL) 8 and CCL22 were significantly lower in AOSD patients with a polycyclic systemic disease phenotype and could be differentiated with high accuracy from the other phenotypes (cutoff value for CCL8 = 122.7 pg/mL, CCL22 = 593.3 pg/mL, sensitivity 66.7%, specificity 87.1%, area under the curve 0.843). Combined MIF and IL-12(p40) levels may represent a biomarker for differentiating patients with AOSD from those with other diseases. The chemokine profiles of AOSD with a polycyclic systemic disease phenotype may differ from other phenotypes.

Extracellular Domain of IL-10 Receptor Chain-2 (IL-10R2) and Its Arginine-Containing Peptides Are Susceptible Substrates for Human Prostate Kallikrein-2 (KLK2).

The kallikrein-related peptidase KLK2 has restricted expression in the prostate luminal epithelium, and its protein target is unknown. The present work reports the hydrolytic activities of KLK2 on libraries of fluorescence resonance energy-transfer peptides from which the sequence SYRIF was the most susceptible substrate for KLK2. The sequence SYRIF is present at the extracellular N-terminal segment (58SYRIF63Q) of IL-10R2. KLK2 was fully active at pH 8.0-8.2, found only in prostate inflammatory conditions, and strongly activated by sodium citrate and glycosaminoglycans, the quantities and structures controlled by prostate cells. Bone-marrow-derived macrophages (BMDM) have IL-10R2 expressed on the cell surface, which is significantly reduced after KLK2 treatment, as determined by flow cytometry (FACS analysis). The IL-10 inhibition of the inflammatory response to LPS/IFN-γ in BMDM cells due to decreased nitric oxide, TNF-α, and IL-12 p40 levels is significantly reduced upon treatment of these cells with KLK2. Similar experiments with KLK3 did not show these effects. These observations indicate that KLK2 proteolytic activity plays a role in prostate inflammation and makes KLK2 a promising target for prostatitis treatment.

Circulating cytokines levels and osteoarthritis: A Mendelian randomization study

BackgroundPrevious traditional observational studies have suggested the contribution of several cytokines and growth factors to the development of osteoarthritis (OA). This study aimed to determine the association of circulating cytokine and growth factor levels with OA. MethodsWe used two-sample Mendelian randomization (MR) to explore the causality between circulating cytokine and growth factor levels and OA [including knee or hip OA (K/HOA), knee OA (KOA), and hip OA (HOA)]. Summary level data for circulating cytokine and growth factor levels were sourced from a genome-wide association study (GWAS) involving 8,293 participants of Finnish ancestry. Single-nucleotide polymorphisms related to K/HOA (39,427 cases and 378,169 controls), KOA (24,955 cases and 378,169 controls), and HOA (15,704 cases and 378,169 controls) were obtained from a previous GWAS. The inverse variance weighted (IVW) method was primarily used for our MR analysis. For exposures to only one relevant SNP as IV, we used the Wald ratio as the major method to assess causal effects. We also conducted a series of sensitivity analyses to improve the robustness of the results. ResultsCirculating vascular endothelial growth factor levels were suggestively associated with an increased risk of K/HOA (odds ratio (OR) = 1.034; 95 % confidence interval (CI) = 1.013–1.055; P = 0.001), KOA (OR = 1.034; 95 % CI = 1.014–1.065; P = 0.002), and HOA (OR = 1.039; 95 % CI = 1.003–1.067; P = 0.034). Circulating interleukin (IL)-12p70 levels was suggestively associated with K/HOA (OR = 1.047; 95 % CI = 1.018–1.077; P = 0.001), KOA (OR = 1.058; 95 % CI = 1.022–1.095; P = 0.001), and HOA (OR = 1.044; 95 % CI = 1.000–1.091; P = 0.048). Circulating IL-18 levels were suggestively associated with HOA (OR = 1.068; 95 % CI = 1.014–1.125; P = 0.012). However, limited evidence exists to support causal genetic relationships between other circulating cytokines, growth factor levels and K/HOA, KOA, and HOA. ConclusionsOur MR analysis provides suggestive evidence of causal relationships between circulating cytokines and growth factors levels and OA, providing new insights into the etiology of OA.

Neutralization of p40 Homodimer and p40 Monomer Leads to Tumor Regression in Patient-Derived Xenograft Mice with Pancreatic Cancer.

Pancreatic cancer is a highly aggressive cancer with a high mortality rate and limited treatment options. It is the fourth leading cause of cancer in the US, and mortality is rising rapidly, with a 12% relative 5-year survival rate. Early diagnosis remains a challenge due to vague symptoms, lack of specific biomarkers, and rapid tumor progression. Interleukin-12 (IL-12) is a central cytokine that regulates innate (natural killer cells) and adaptive (cytokine T-lymphocytes) immunity in cancer. We demonstrated that serum levels of IL-12p40 homodimer (p402) and p40 monomer (p40) were elevated and that of IL-12 and IL-23 were lowered in pancreatic cancer patients compared to healthy controls. Comparably, human PDAC cells produced greater levels of p402 and p40 and lower levels of IL-12 and IL-23 compared to normal pancreatic cells. Notably, neutralization of p402 by mAb a3-1d and p40 by mAb a3-3a induced the death of human PDAC cells, but not normal human pancreatic cells. Furthermore, we demonstrated that treatment of PDX mice with p402 mAb and p40 mAb resulted in apoptosis and tumor shrinkage. This study illustrates a new role of p402 and p40 monomer in pancreatic cancer, highlighting possible approaches against this deadly form of cancer with p402 and p40 monomer immunotherapies.

Unveiling COVID-19 Secrets: Harnessing Cytokines as Powerful Biomarkers for Diagnosis and Predicting Severity.

In coronavirus disease (COVID-19), inflammation takes center stage, with a cascade of cytokines released, contributing to both inflammation and lung damage. The objective of this study is to identify biomarkers for diagnosing and predicting the severity of COVID-19. Cytokine levels were determined in the serum from venous blood samples collected from 100 patients with COVID-19 and 50 healthy controls. COVID-19 patients classified based on the Modified Early Warning (MEWS) score. Cytokine concentrations were determined with a multiplex ELISA kit (Bio-Plex Pro™ Human Cytokine Screening Panel). The concentrations of all analyzed cytokines were elevated in the serum of COVID-19 patients relative to the control group, but no significant differences were observed in interleukin-9 (IL-9) and IL-12 p70 levels. In addition, the concentrations of IL-1α, IL-1β, IL-1ra, IL-2Rα, IL-6, IL-12 p40, IL-18, and tumor necrosis factor alpha (TNFα) were significantly higher in symptomatic patients with accompanying pneumonia without respiratory failure (stage 2) than in asymptomatic/mildly symptomatic patients (stage 1). The study revealed that IL-1ra, IL-2Rα, IL-6, IL-8, IL-12 p40, IL-16, and IL-18 levels serve as potential diagnostic biomarkers in COVID-19 patients. Furthermore, elevated IL-1α levels proved to be valuable in assessing the severity of COVID-19.

IL-12p40 is essential but not sufficient for Francisella tularensis LVS clearance in chronically infected mice.

IL-12p40 plays an important role in F. tularensis Live Vaccine Strain (LVS) clearance that is independent of its functions as a part of the heterodimeric cytokines IL-12p70 or IL-23. In contrast to WT, p35, or p19 knockout (KO) mice, p40 KO mice infected with LVS develop a chronic infection that does not resolve. Here, we further evaluated the role of IL-12p40 in F. tularensis clearance. Despite reduced IFN-γ production, primed splenocytes from p40 KO and p35 KO mice appeared functionally similar to those from WT mice during in vitro co-culture assays of intramacrophage bacterial growth control. Gene expression analysis revealed a subset of genes that were upregulated in re-stimulated WT and p35 KO splenocytes, but not p40 KO splenocytes, and thus are candidates for involvement in F. tularensis clearance. To directly evaluate a potential mechanism for p40 in F. tularensis clearance, we reconstituted protein levels in LVS-infected p40 KO mice using either intermittent injections of p40 homodimer (p80) or treatment with a p40-producing lentivirus construct. Although both delivery strategies yielded readily detectable levels of p40 in sera and spleens, neither treatment had a measurable impact on LVS clearance by p40 KO mice. Taken together, these studies demonstrate that clearance of F. tularensis infection depends on p40, but p40 monomers and/or dimers alone are not sufficient.

Abstract 6025: Baseline cytokine profiling in naïve and tumor-bearing Balb/c and C57BL/6 Charles River mice

Abstract Background: Syngeneic studies are still the backbone of oncology research. These rely on immunocompetent mice to model anti-cancer immune responses elicited by experimental treatments. Cytokines mediate these responses through cell signaling, which contributes to an immunosuppressive milieu in the tumor microenvironment. Determining the secretion patterns of these protein modulators can help understand pathogenesis, disease progression and resistance to targeted therapies. To evaluate biological effects of potential interventions, we compared systemic cytokine activities in naïve and untreated tumor-bearing Balb/c and C57BL/6 mice. Study Details: Charles River wild type Balb/c (n=9) and C57BL/6 (n=10) mice were used for all experiments. CT26 and B16.F10 tumor cell lines for inoculations were purchased from ATCC (Manassas, VA). Serum samples were collected from colon carcinoma (Balb/c) and melanoma (C57BL/6) bearing mice as well as tumor-free animals. For rapid multiplex measurements on the Meso Scale Discovery platform the V-PLEX Proinflammatory Panel 1 Mouse Kit was used (cat no. K15048D-1). IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, KC/GRO, IL-10, IL-12p70, and TNF-α were detected simultaneously by electrochemiluminescence within the manufacturer’s specification for each analyte. Results: Sera from CT26 and B16.F10 tumor-bearing mice exhibited higher average concentrations of IFN-γ, IL1-β, IL-4, IL-10, KC/GRO and TNF-α, when compared to naïve mice of the Balb/c or C57BL/6 background, respectively; however, IL-2 production was found increased in tumor-bearing Balb/c and decreased in tumor-bearing C57BL/6 mice. Though known to be promoting tumor growth, IL-6 was only measurable in three out of ten B16.F10-bearing mice and below the limit of detection in CT26-bearing mice. IL-5 and IL-12 p70 levels showed no difference between CT26-bearing and naïve Balb/c mice, whereas both cytokines were elevated in the B16.F10 model over naïve C57BL/6 mice. Conclusions: Circulating serum cytokines have been quantified in commonly used syngeneic mouse strains with and without tumor burden. These reference data provide baseline levels to compare soluble proinflammatory biomarkers suitable for assessing potential immunomodulatory effects of experimental cancer treatments in combination with other pharmacodynamic readouts. Citation Format: Kenneth Munroe, Christoph S. Eberle, Robert Mihalek, Stephen Festin. Baseline cytokine profiling in naïve and tumor-bearing Balb/c and C57BL/6 Charles River mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6025.

Regression of Triple-Negative Breast Cancer in a Patient-Derived Xenograft Mouse Model by Monoclonal Antibodies against IL-12 p40 Monomer.

Although some therapies are available for regular breast cancers, there are very few options for triple-negative breast cancer (TNBC). Here, we demonstrated that serum level of IL-12p40 monomer (p40) was much higher in breast cancer patients than healthy controls. On the other hand, levels of IL-12, IL-23 and p40 homodimer (p402) were lower in serum of breast cancer patients as compared to healthy controls. Similarly, human TNBC cells produced greater level of p40 than p402. The level of p40 was also larger than p402 in serum of a patient-derived xenograft (PDX) mouse model. Accordingly, neutralization of p40 by p40 mAb induced death of human TNBC cells and tumor shrinkage in PDX mice. While investigating the mechanism, we found that neutralization of p40 led to upregulation of human CD4+IFNγ+ and CD8+IFNγ+ T cell populations, thereby increasing the level of human IFNγ and decreasing the level of human IL-10 in PDX mice. Finally, we demonstrated the infiltration of human cytotoxic T cells, switching of tumor-associated macrophage M2 (TAM2) to TAM1 and suppression of transforming growth factor β (TGFβ) in tumor tissues of p40 mAb-treated PDX mice. Our studies identify a possible new immunotherapy for TNBC in which p40 mAb inhibits tumor growth in PDX mice.

Immunomodulatory Effects of Asiaticoside Against Shigella flexneri-Infected Macrophages

Macrophages provide the first line of defense against Shigella flexneri infection in the gastrointestinal tract by inducing a variety of inflammatory and antimicrobial responses. Secondary metabolites of plants are used as drugs against infections that are resistant to common antibiotics. In this study, the innate effects of asiaticoside on the proinflammatory activity of mouse macrophages infected with S. flexneri were investigated. The viability of the infected mouse macrophages were examined using viability assay, while the pro-inflammatory cytokines productions were determined using the enzyme-linked immunosorbent assay (ELISA) for determination of IL-1β, IL-12 p40 and TNF-α levels. The production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) protein were determined using the Griess assay and western blot, respectively. Statistical analyses were performed using the Statistical Package of Social Sciences (SPSS) software, version 20. The data obtained from independent experiments (n = 3) were presented as the mean ± standard error of mean (SEM). The results showed that, asiaticoside stimulated the infected macrophages by stimulating increased production of TNF-α, IL-12 p40 and NO as well as increased expression of iNOS in a dose-dependent manner. In contrast the viability of the cells and the production of IL-1β and were reduced also in a dose-dependent manner when compared to untreated cells. These results indicate that asiaticoside has immunomodulatory effects on the innate immune function of infected macrophages, showing the potential use of this compound to reduce the clinical symptoms of the infections.

SiRNA-based identification of IBD-related targets in human monocyte-derived dendritic cells

Inflammatory bowel disease (IBD) is thought to be caused by an aberrant host response to the commensal enteric flora in genetically susceptible individuals. Dendritic cells (DCs) play a key role in the regulation of this response as they sample gut commensals. In healthy individuals DCs actively contribute to tolerance upon recognition of these resident bacteria, whereas in individuals with IBD, DCs will initiate an inflammatory response. To mimic the disease response in vitro, human monocyte-derived DCs were matured with E. coli causing the cells to produce high levels of the pro-inflammatory cytokine IL-12/IL-23p40 (p40) and low levels of the anti-inflammatory cytokine IL-10. A siRNA-based screening assay was developed and screened to identify potential therapeutic targets that shift this balance towards an immunosuppressive state with lower levels of p40 and higher levels of IL-10. The screening assay was optimized and quality controlled using non-targeting controls and positive control siRNAs targeting IL12B and TLR4 transcripts. In the primary screen, smartpool siRNAs were screened for reduction in p40 expression, induction of IL-10 levels, or increase in IL-10:p40 ratios without affecting cell viability. All potential targets were taken forward into a confirmation screen in a different DC donor in which four individual siRNAs per target were screened. At least two siRNAs per target should have an effect to be considered a valid target. This screen resulted in a concise list of ten genes, of which their role in DC maturation is currently being investigated.

Insulin Modulates Inflammatory Cytokine Release in Acute Stages and Augments Expression of Adhesion Molecules and Leukocytes in Lungs on Chronic Stages of Paracoccidioidomycosis.

Type 1 diabetes mellitus (T1D) is caused by partial destruction of the insulin-producing beta cells in the pancreas and is a major issue for public health care worldwide. Reduced or impaired immunological responses, which render patients more susceptible to infections, have been observed in T1D, and this dysfunction is often related to a lack of insulin in the blood. Paracoccidioidomycosis is an important systemic mycosis endemic in Latin America. To evaluate the effects of T1D on this fungal infection and the modulatory effects of insulin, we induced diabetes in C57Bl/6 male mice (alloxan, 60 mg/kg), infected the mice (Pb18, 1 x 106 cells), and treated the mice with neutral protamine Hagedorn (NPH) insulin (2 IU/600 mg/dL blood glucose). Twenty-four hours after infection, infected diabetic mice showed reduced secretion of interferon (IFN)-γ and interleukine (IL)-12 p70 compared to infected nondiabetic controls. On the 45th day of infection, infected diabetic mice presented higher IFN-γ levels, a higher tumor necrosis factor (TNF)-α:IL-10 ratio, and lower adhesion molecule expression levels than nondiabetic mice. In the in vitro experiments, alveolar macrophages from diabetic animals showed reduced phagocytic activity compared to those from control animals at 4, 12, and 24 h. In infected diabetic mice, treatment with insulin restored IL-12 p70 levels at 24 h of infection, reduced IFN-γ levels and the TNF-α:IL-10 ratio at 45 days, and restored vascular cell adhesion molecule (VCAM)-1 expression in pulmonary blood vessels, and this treatment reduced the diminished phosphorylation of extracellular signal-regulated kinases (ERK) and increased nuclear factor-kappa-B(iκb)-α and jun amino-terminal kinases (JNK) p46 levels in infected nondiabetic mice. In addition, insulin promoted increased phagocytic activity in the alveolar macrophages of diabetic mice. These data suggest that T1D mice are more susceptible to Pb18 infection and that insulin modulates this inflammation in diabetic mice by augmenting the expression of adhesion molecules and leukocytes in the lungs and by reducing chronic inflammation.

Inhibitory effect of particulate matter on toll-like receptor 9 stimulated dendritic cells by downregulating mitogen-activated protein kinase and NF-κB pathway

ABSTRACT Ambient particulate matter (PM) is associated with adverse health consequences. However, the influence of PM on the innate immune system is poorly understood. The aim of the present study was to examine the effect of diesel particulate matter 2.5 μm (PM2.5, SRM1650b) on dendritic cells. PM2.5 significantly reduced cytokine levels of interleukin (IL)-12 p40, IL-6 and TNF-α levels in CpG-DNA (TLR9 ligand)-stimulated dendritic cells. To determine the mechanisms underlying this observed inhibition induced by PM2.5, western blot analysis was conducted. PM2.5 was found to downregulate ERK1/2, JNK1/2, p38 MAPKs, and NF-κB pathways. PM2.5 exposure decreased TLR9-dependent NF-κB and activator protein (AP-1) reporter luciferase activities. Our findings demonstrate that PM2.5 reduced the production of cytokines which may be associated with inhibition of MAPK and NF-κB signaling pathway. Further, data suggest the immunosuppressive effect of PM2.5 on the innate immune cells may lead to serious damage to the host immune system.

Evaluation of Tear Protein Markers in Dry Eye Disease with Different Lymphotoxin-Alpha Expression Levels

To compare tear protein markers between normal subjects and patients with dry eye (DE) and high and low lymphotoxin-alpha (LT-α) levels. Prospective cross-sectional study. Patients with DE were divided into low(≤700 pg/mL) and high (>700 pg/mL) LT-α groups. Twelve protein markers were measured by microsphere-based immunoassay and ocular surface parameters were determined in right eyes (33 high LT-α DE, 27 low LT-α DE, and 20 control eyes) and left eyes (21 high LT-α DE, 39 low LT-α DE, and 20 control eyes). In both eyes, tumor necrosis factor-α (TNF-α), interleukin (IL)-10, IL-1β, IL-1 receptor antagonist (IL-1Ra), IL-17A, and IL-12/23 p40 levels in high LT-α DE were significantly higher (P < .01) than in low LT-α DE. Significant correlations identified in high LT-α DE were: Standard Patient Evaluation Eye Dryness with IL-10 (R= 0.43, P= .013), IL-1β (R= 0.48, P= .005), and IL-12/23 p40 (R= 0.50, P= .003), IL-12/23 p40 with ocular surface disease index (R= 0.35, P= .049), and epidermal growth factor with corneal fluorescein staining score (R=-0.36, P= .038). Significant correlations in low LT-α DE were: Standard Patient Evaluation Eye Dryness with IL-10 (R=-0.39, P= .046), TNF-α (R=-0.39, P= .047), and IL-17A (R=-0.48, P= .013), ocular surface disease index with TNF-α (R=-0.47, P= .017) and IL-17A (R=-0.46, P= .018), and IL-6 with tear breakup time (R=-0.40, P= .044). Lastly, IL-1Ra levels significantly increased in DE patients, positively correlated with temporal conjunctival hyperemia index, and negatively correlated with Schirmer I test (P < .05). Our study identified tear IL-1Ra level as a potential biomarker to replace the Schirmer I test. Multiple tear protein marker levels increased in high LT-α DE, indicating that high LT-α DE might have a different pathogenesis.

Indirect Regulation and Equilibrium of p35 and p40 Subunits of Interleukin (IL)-12/23 by Ustekinumab in Psoriasis Treatment.

BackgroundUstekinumab, a human-derived monoclonal antibody that targets the p40 subunit of interleukin (IL)-12 and IL-23, has excellent clinical efficacy and safety in treating psoriasis, with a long half-life. However, no reports have described the use of human skin/serum samples to elucidate its molecular mechanisms.Material/MethodsTwenty-four psoriasis patients were enrolled in our double-blind study and randomly divided into placebo and ustekinumab-administered groups. Dynamic changes in psoriasis area-severity index scores, and mRNA and protein levels of p35 and p40 were analyzed at 3 time points (before treatment and during the 12th and 24th weeks of treatment).ResultsUstekinumab initially increased and then decreased p35 mRNA expression, but increased p40 mRNA levels throughout the study. The p35 protein levels were not significantly altered, while p40 protein levels were increased after the first 2 injections but decreased after the third injection.ConclusionsWe concluded that 2 equilibria influence the efficacy of ustekinumab against psoriasis. First, because of the dual roles of p35 in psoriasis pathogenesis, homeostasis occurs between p35 and p40 expression levels. The second balance lies between the upregulation of p40 mRNA levels and the ability of ustekinumab to neutralize the function of the elevated p40 protein.

Stężenie interleukin 6, 8 i 12p40 oraz białka C-reaktywnego u pacjentów z trądzikiem pospolitym o różnym nasileniu zmian skórnych

Stężenie interleukin 6, 8 i 12p40 oraz białka C-reaktywnego u pacjentów z trądzikiem pospolitym o różnym nasileniu zmian skórnych

IL-17A induces osteoblast differentiation by activating JAK2/STAT3 in ankylosing spondylitis

BackgroundIL-17A has recently emerged as a potential target that regulates the extensive inflammation and abnormal bone formation observed in ankylosing spondylitis (AS). Blocking IL-17A is expected to inhibit bony ankylosis. Here, we investigated the effects of anti IL-17A agents in AS.MethodsTNFα, IL-17A, and IL-12/23 p40 levels in serum and synovial fluid from patients with ankylosing spondylitis (AS), rheumatoid arthritis (RA), osteoarthritis (OA), or healthy controls (HC) were measured by ELISA. Bone tissue samples were obtained at surgery from the facet joints of ten patients with AS and ten control (Ct) patients with noninflammatory spinal disease. The functional relevance of IL-17A, biological blockades, Janus kinase 2 (JAK2), and non-receptor tyrosine kinase was assessed in vitro with primary bone-derived cells (BdCs) and serum from patients with AS.ResultsBasal levels of IL-17A and IL-12/23 p40 in body fluids were elevated in patients with AS. JAK2 was also highly expressed in bone tissue and primary BdCs from patients with AS. Furthermore, addition of exogenous IL-17A to primary Ct-BdCs promoted the osteogenic stimulus-induced increase in ALP activity and mineralization. Intriguingly, blocking IL-17A with serum from patients with AS attenuated ALP activity and mineralization in both Ct and AS-BdCs by inhibiting JAK2 phosphorylation and downregulating osteoblast-involved genes. Moreover, JAK2 inhibitors effectively reduced JAK2-driven ALP activity and JAK2-mediated events.ConclusionsOur findings indicate that IL-17A regulates osteoblast activity and differentiation via JAK2/STAT3 signaling. They shed light on AS pathogenesis and suggest new rational therapies for clinical AS ankylosis.

Correlation between IL-17A/F, IL-23, IL-35 and IL-12/-23 (p40) levels in peripheral blood lymphocyte cultures and disease activity in Behcet's patients.

Behcet's disease is a chronic multisystemic disease with remissions and relapses. Several studies have shown that immune mechanisms play an important role in the development of the disease. In order to assess the association of disease activity with IL-17A/F, IL-23, IL-12/23 (p40) and IL-35 expression, we aimed to investigate production of these cytokines in peripheral blood mononuclear cells (PBMCs) from Behcet's patients and normal controls. Furthermore, we included Systemic Lupus Erythematosus (SLE) as disease control to evaluate the specificity of our data for immunopathogenesis of BD. Totally 15 active, 15 inactive Behcet's patients, 12 active and 12 inactive SLE patients and 12 healthy volunteers were enrolled in the study. Peripheral blood mononuclear cells were separated, lymphocyte cultures were performed and IL-17A/F, IL-12/23 p(40), IL-23, IL-35 cytokine levels were measured by ELISA in culture supernatants in the presence or absence of phytohemagglutinin (PHA) on time-dependent manner. IL-17 A/F levels increased parallel to IL-23 levels in Behcet's and SLE patients. Compared to healthy controls, IL-17 A/F levels were higher in active Behcet's and SLE patients; on the contrary, levels of IL-35 were lower. IL-17A/F, IL-12/23 (p40) and IL-23 levels were detectable most frequently in active Behcet's patients followed by active SLE patients. Our results indicate that IL-17 A/F, IL-23 and IL-12/23 (p40) may play role in the immunopathogenesis of BD so as Th17 and Th1 cell responses. Since IL-35 levels were lower in active Behcet's patients compared to inactive patients and healthy controls, there may be a plasticity between Th17 and Treg cells according to the state of disease activity.

Abstract WMP77: Anti-Inflammatory Signaling by Leukemia Inhibitory Factor is Suppressed in Aged Animals After Stroke

Objective: To determine whether the anti-inflammatory effects of leukemia inhibitory factor (LIF) are altered in aged male and female rats. Methods: Focal ischemia was induced in 3 month old male and 18 month old male/female Sprague-Dawley using the middle cerebral artery occlusion (MCAO) procedure. Animals were treated with PBS or LIF at 6, 24, and 48 h after MCAO (125 μg/kg). Infarct volume in aged animals was quantified with T2-weighted MRI. Functional motor skills were assessed immediately prior to euthanization at 72 h post-MCAO. Protein levels of CD11b and IL-12 p40 in brain and spleen tissue were measured using immunoblotting. Results: At 72 h, there was a trend towards decreased infarct volume in aged male (189 ± 45OD; n=4) and female LIF-treated rats (104 ± 71 OD; n=4) compared to PBS-treated aged male (188 ± 45; n=4) and female rats (179 ± 72; n=5). LIF-treated aged female rats showed significant decreases in circling (p=0.0209) and body swing bias (p=0.05) compared to the PBS-treated aged females. Normalized CD11b levels were significantly in LIF-treated young males (1.1 ± 0.3 OD; n=5) compared to PBS-treated young males (2.5 ± 0.5 OD; n=6; p&lt;0.05). Although aged male (1.2 ± 0.4 OD) and females (1.2 ± 0.3 OD) had lower levels of CD11b in the brain 72 h post-MCAO compared to young males, LIF had no effect on brain CD11b levels in aged rats. LIF increased spleen size (1.6 ± 0.1 g) and splenic CD11b levels (0.11 ± 0.01 OD) compared to PBS-treated females (0.6 ± 0.1 g; 0.08 ± 0.01 OD; p&lt;0.05), and there was a significant positive correlation between these two factors in the aged female rats (r = 0.6775; p&lt;0.05). Furthermore, IL-12 p40 levels in splenic tissue were significantly higher in young male PBS-treated rats (0.38 ± 0.06 OD) compared to aged male (0.12 ± 0.01 OD; p&lt;0.01) and female rats (0.16 ± 0.04 OD; p&lt;0.01). LIF treatment reduced IL-12 p40 levels in the spleen in young rats (0.24 ± 0.04; p&lt;0.05) but not in either of the aged rat groups. Conclusions: LIF is more effective at decreasing splenic IL-12 expression and immune cell infiltration in young rats compared to aged rats. However, LIF counteracts the post-stroke splenic response in aged females but not aged male rats.

Selective neutralization of IL-12 p40 monomer induces death in prostate cancer cells via IL-12–IFN-γ

Cancer cells are adept at evading cell death, but the underlying mechanisms are poorly understood. IL-12 plays a critical role in the early inflammatory response to infection and in the generation of T-helper type 1 cells, favoring cell-mediated immunity. IL-12 is composed of two different subunits, p40 and p35. This study underlines the importance of IL-12 p40 monomer (p40) in helping cancer cells to escape cell death. We found that different mouse and human cancer cells produced greater levels of p40 than p40 homodimer (p402), IL-12, or IL-23. Similarly, the serum level of p40 was much greater in patients with prostate cancer than in healthy control subjects. Selective neutralization of p40, but not p402, by mAb stimulated death in different cancer cells in vitro and in vivo in a tumor model. Interestingly, p40 was involved in the arrest of IL-12 receptor (IL-12R) IL-12Rβ1, but not IL-12Rβ2, in the membrane, and that p40 neutralization induced the internalization of IL-12Rβ1 via caveolin and caused cancer cell death via the IL-12-IFN-γ pathway. These studies identify a role of p40 monomer in helping cancer cells to escape cell death via suppression of IL-12Rβ1 internalization.

Can We Improve Pressure Feedback Methods in Vestibular Evoked Myogenic Potentials by Applying Custom Pressures?

The aim of the present study was to use constant and customized pressure levels to improve the feedback method of the blood pressure cuff technique in order to decrease intra-subject and inter-subject variability. The study was conducted in two stages. In the first stage, the relationship between the pressure level generated in the blood pressure cuff and electromyographic response in the sternocleidomastoid (SCM) muscle was investigated. In the second stage, vestibular evoked myogenic potential (VEMP) measurements were made using a custom-built VEMP chair at a constant pressure level of 40 mmHg (P40) or at 50% of the maximum pressure (Pmax50%) that could be generated by the SCM muscle. VEMP measurements were performed on 100 volunteers consisting of 48 males and 52 females whose ages were between 20 and 68 years. The response rate was 41% on a subject basis and 53% on an ear basis. Response rates were similar in males and females, and they decreased with age. The response rate was significantly lower in 11% of the volunteers who could not generate the stipulated 80 mmHg pressure level. Response rates obtained with P40 and Pmax50% were similar, and p13 and n23 latencies and p13-n23 amplitudes obtained from both sides were also similar. Amplitudes were higher in Pmax50% measurements compared to P40, and amplitudes obtained with P40 levels showed greater variance compared to Pmax50%. The use of Pmax50% provided reduced variation compared to P40; however, it did not have significant clinical implications. Further studies are needed for the control of many factors that are related to amplitude variability.