DNA circuit-based immunoassay for ultrasensitive protein pattern classification.

Cytokines are important immune modulators, and pivotal biomarkers for the diagnostic of various diseases. In standard analytical procedure, each protein is detected individually, using for instance gold standard ELISA protocols or nucleic acid amplification-based immunoassays. In recent years, DNA nanotechnology has been employed for creating sophisticated biomolecular systems that perform neuromorphic computing on molecular inputs, opening the door to concentration pattern recognition for biomedical applications. This work introduces immuno-PUMA (i-PUMA), an isothermal amplification-based immunoassay for ultrasensitive protein detection. The assay couples the convenience of supported format of an ELISA protocol with the computing capabilities of a DNA/enzyme circuit. We demonstrate a limit of detection of 2.1 fM, 8.7 fM and 450 aM for IL12, IL4 and IFNγ cytokines, respectively, outperforming the traditional ELISA format. i-PUMA's versatility extends to molecular computation, allowing the creation of 2-input perceptron-like classifiers for IL12 and IL4, with tunable weight sign and amplitude. Overall, i-PUMA represents a sensitive, low-cost, and versatile immunoassay with potential applications in multimarker-based sample classification, complementing existing molecular profiling techniques.

Air pollution exposure is associated with rhinitis in older US adults via specific immune mechanisms.

BACKGROUND: Pathophysiology of rhinitis in older adults is largely unknown. We tested whether air pollution is associated with this condition and how immune mechanisms may play a role in this relationship. METHODS: We analyzed cross-sectional data from the National Social Life, Health, and Aging Project, a nationally representative study of older adults born between 1920 and 1947. Particulate matter ≤2.5 µm (PM2.5 ) air pollution exposure estimates were generated using validated spatiotemporal models. Presence of rhinitis was defined based on medication use (≥1: intranasal medications: steroids, antihistamines, lubricants, and/or decongestants, and/or oral medications: antihistamines and/or decongestants). K-means cluster analysis (Jaccard method) was used to group 13 peripheral blood cytokines into 3 clusters to facilitate functional determination. We fitted multivariate logistic regressions to correlate PM2.5 exposure with presence of rhinitis, controlling for confounders, and then determined the role of cytokines in this relationship. RESULTS: Long- (but not short-) term exposure to PM2.5 was associated with presence of rhinitis: 3-year exposure window, odds ratio (OR) = 1.32, 95% confidence interval (CI): 0.98, 1.80, per 1 standard deviation (SD) PM2.5 increase. Inclusion of cytokine cluster in the model led to a modestly stronger effect of PM2.5 exposure on rhinitis (OR = 1.37; 95% CI: 1.00, 1.87; 3-year exposure window). The particular immune profile responsible for this result was composed of elevated IL-3, IL-12, and IFN-γ (OR = 4.86, 95% CI: 1.10, 21.58, immune profile-PM2.5 exposure interaction term). CONCLUSION: We show for the first time that IL-3, IL-12, and IFN-γ explain in part the relationship between PM2.5 exposure and rhinitis in older US adults. If confirmed, these immune pathways may be used as therapeutic targets.

Increased circulating interleukin-23 level in patients with sarcoidosis.

BACKGROUND: Sarcoidosis is a Th1-mediated chronic inflammatory disease characterized by non-caseating granulomas. Its pathogenesis is not yet clear, but the possible role of various proinflammatory cytokines is being discussed. AIM: This study aims to determine serum cytokine (IL-6, IL-12, IL-17, and IL-23) levels in patients with sarcoidosis, and to determine a possible correlation with clinical and laboratory findings of the disease. MATERIAL AND METHOD: Forty-four biopsy-proven sarcoidosis patients followed up at a single centre and 41 healthy volunteers were included in the study. Demographic, clinical, laboratory, and radiological data of all patients were recorded. Serum samples from the patients and the control group were taken and IL-6, IL-12, IL-17, IL-23 were measured by ELISA method. RESULTS: Of the 44 sarcoidosis patients, 13(29.5%) were male and 31(70.5%) were female. Average patient age was 47.4 years, mean disease duration was 3.2 years. Twenty-one (47.7%) patients had erythema nodosum, three (6.8%) had uveitis, 40(90.9%) had arthralgia, 23(52.3%) had ankle arthritis, 15(34.1%) had enthesitis. Laboratory evaluation showed increased serum ACE levels in 24(54.5%) patients, increased serum calcium levels in 11 (25%) patients, increased serum D3 levels in 5(11.4%) patients, increased ESR and CRP levels in 22(50%) and 23(52.3%) patients, respectively. Compared with the control group higher serum IL-23 levels were found in the patients with sarcoidosis (p=.01). Serum IL-23 was associated with ankle arthritis (p=.02). Serum IL-6, IL-12, and IL-17 levels were similar in the sarcoidosis patients and the control group (p=.128, p=.212, p=.521 respectively). CONCLUSION: In our study, we found increased serum IL-23 in patients with sarcoidosis, while serum IL-6, IL-12, and IL-17 were detected as normal. Although our results are somewhat contradictory to other studies in the literature, the question should still be whether sarcoidosis is a Th1/Th17 disease. Multicentre studies are needed in this regard.

IL-12 Contributes to the Development of Asthma by Targeting HIF-1α/NLRP3 Pathway through Runx3.

INTRODUCTION: The aim of the study was to determine the role and mechanism of runt-related transcription factor 3 (Runx3) in the development of asthma. METHODS: An asthma mouse model was constructed and validated by hematoxylin-eosin analysis of lung tissue and noninvasive enhanced pause (Penh) evaluation of airway hyperresponsiveness. Then, the levels of Runx3 and interleukin (IL)-12 in peripheral blood and lung tissue were detected by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. By use Runx3+/- mice, the effect of Runx3 downregulation on ovalbumin (OVA)-induced asthma was investigated. After stimulated by different doses of IL-12, the expressions of Runx3, hypoxia inducible factor-1α (HIF-1α), and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) in BEAS-2B cells were tested through Western blot and immunofluorescence. Subsequently, BEAS-2B cells treated with 20 ng/mL IL-12 were divided into control, Runx3 overexpression negative control, Runx3 overexpression, HIF-1α inhibitor, and Runx3 overexpression + HIF-1α agonist groups. The Western blot, immunofluorescence, and ELISA indicators were tested repeatedly. RESULTS: The increased number of inflammatory cells and Penh value confirmed the success of the asthma mouse model. IL-12 expression was significantly increased, and Runx3 was reduced in asthma mice compared with wild-type mice. Meanwhile, the level of immunoglobulin E (IgE) in serum, cytokines in bronchoalveolar lavage fluid, and IL-12, HIF-1α, NLRP3 in the lung were significantly elevated in Runx3+/- mice. With the increase of IL-12 concentration, Runx3 protein expression decreased, while HIF-1α and NLRP3 expression increased. Further mechanistic studies suggest that Runx3 ameliorates IL-12-induced BEAS-2B injury by inhibiting HIF-1α/NLRP3 pathway. CONCLUSION: These results suggested that IL-12 contributes to the development of asthma by targeting HIF-1α/NLRP3 pathway through Runx3, thus providing a novel strategy for asthma therapy.

Evaluation of IL-23p19/Ebi3 (IL-39) gingival crevicular fluid levels in periodontal health, gingivitis, and periodontitis.

OBJECTIVES: IL-23p19/Ebi3 (IL-39) was described as a new IL-12 family member. The aim of this study is to evaluate the gingival crevicular fluid (GCF) IL-39 levels in periodontal diseases and health and to correlate them to GCF levels of IL-1ß and periostin. MATERIALS AND METHODS: Sixty-six adult patients were included in the study. The study design was comprised of three groups, each containing 22 individuals: the periodontally healthy (PH), gingivitis (G), and periodontitis (P) groups. The clinical periodontal parameters were recorded and GCF samples were collected from the participants. GCF interleukin (IL)-39, IL-1ß, and periostin levels were examined using the enzyme-linked immunosorbent assay. RESULTS: GCF IL­1ß, periostin, and IL-39 levels were higher in the P and G groups than in the PH group (p < 0.001). Positive correlations were detected between all GCF biochemical parameters and clinical periodontal parameters (p < 0.05). In the multivariate generalized linear regression analysis, the P (ß = 37.6, 95% CI = 22.9-52.4) and G (ß = 28.4, 95% CI = 15.8-41) groups were associated with GCF IL-39 levels (p < 0.001). CONCLUSION: IL-39 levels were elevated in the presence of periodontal disease paralleling the increase in IL­1ß and periostin levels. IL-39 may have a role in the periodontal inflammation process. STATEMENT OF CLINICAL RELEVANCE: IL-39, a new cytokine from the IL-12 family, can be a possible predictor marker of periodontal diseases.

Hair cortisol concentration in anxiety disorders: exploration of relationships with symptom severity and inflammatory markers.

BACKGROUND: Hair cortisol concentration (HCC) can be used to periodically assess hypothalamic-pituitary-adrenal (HPA) axis function, and appears correlated with prolonged exposure to stress. METHODS: Serial assessment (at Baseline, Week 6 and Week 12) of participants (n = 35) with anxiety disorders by psychopathological rating scales, with assays of HCC and levels of peripheral anti- and pro-inflammatory cytokines. Patients underwent antidepressant treatment for an initial 6 weeks, followed by cyclo-oxygenase inhibitor-2 (COX-2) inhibitor (celecoxib) augmentation or 'treatment as usual' for a further 6 weeks. RESULTS: At Baseline (n = 35), HCC was elevated in patients with single-episode but not recurrent-episode anxiety disorders, mean IL-12p70 levels were low, and mean TNF-α levels were elevated. Following 6 weeks of antidepressant treatment (n = 33), mean HCC was within the normal range but mean IL-2 level was low. Celecoxib augmentation (n = 18) was associated with a reduction in anxiety symptoms and normalisation of mean IL-2 levels. LIMITATIONS: Small sample size. Not all participants were assessed at all time points. CONCLUSION: Serial assessment of HCC is practicable in patients with anxiety disorders. These preliminary findings warrant further investigation in larger samples.

Tumor Suppressors-HTRA Proteases and Interleukin-12-in Pediatric Asthma and Allergic Rhinitis Patients.

Background and objective: Allergy belongs to a group of mast cell-related disorders and is one of the most common diseases of childhood. It was shown that asthma and allergic rhinitis diminish the risk of various cancers, including colon cancer and acute lymphoblastic leukemia. On the other hand, asthma augments the risk of lung cancer and an increased risk of breast cancer in patients with allergy has been observed. Thus, the relation between allergy and cancer is not straightforward and furthermore, its biological mechanism is unknown. The HTRA (high temperature requirement A) proteases promote apoptosis, may function as tumor suppressors and HTRA1 is known to be released by mast cells. Interleukin-12 (Il-12) is an important cytokine that induces antitumor immune responses and is produced mainly by dendritic cells that co-localize with mast cells in superficial organs. Material and methods: In the present study we have assessed with ELISA plasma levels of the HTRA proteins, Il-12, and of the anti-HTRA autoantibodies in children with allergy (40) and in age matched controls (39). Children are a special population, since they usually do not have comorbidities and take not many drugs the processes we want to observe are not influenced by many other factors. Results: We have found a significant increase of HTRA1, 2 and 3, and of the Il-12 levels in the children with atopy (asthma and allergic rhinitis) compared to controls. Conclusion: Our results suggest that the HTRA1-3 and Il-12 levels might be useful in analyzing the pro- and antioncogenic potential in young atopic patients.

Targeted double domain nanoplex based on galactosylated polyethylenimine enhanced the delivery of IL-12 plasmid.

The objective of the present investigation was to design a targeted polyethylenimine (PEI)-based polyplex by conjugating lactose bearing galactose groups on low molecular weight PEI (LMW PEI) grafted to a high molecular weight PEI (HMW PEI) via a succinic acid linker in order to restore the amine content of the whole conjugate used for ligand conjugation. The PEI conjugate was synthesized and characterized in terms of buffering capacity, particle size, zeta potential, plasmid condensation ability, and protection of DNA against degrading enzymes. Also, the transfection efficiency and cytotoxicity were evaluated in the cell line over-expressing asialoglycoprotein receptors (ASGPRs) and compared with the cells lacking the receptors. The results demonstrated the ability of PEI conjugate in condensation of plasmid DNA and protection against enzyme degradation. The PEI conjugate formed nanoparticles of around 75 nm with higher buffering capacity compared with unmodified PEI. The polyplexes prepared by the modified PEI could increase the level of transgene up to four folds in the cells over-expressing the receptor. The results demonstrated the separation of targeting and delivery domains could be considered as a strategy to restore the amine content of the PEI molecule utilized for targeting ligand conjugation.

Effects of SSRIs on peripheral inflammatory cytokines in patients with Generalized Anxiety Disorder.

BACKGROUND: Extensive research into psychoneuroimmunology has led to substantial advances in our understanding of the reciprocal interactions between the central nervous system and the immune system in neuropsychiatric disorders. To date, inflammation has been implicated in the pathogenesis of depression and anxiety. The immunomodulating effects of antidepressants on depression have been reported, however, there is no evidence of the similar effects of antidepressants on anxiety. The aim of the study was to investigate the effects of selective serotonin reuptake inhibitors (SSRIs) on peripheral inflammatory cytokines in patients with first episode generalized anxiety disorder (GAD). METHODS: A prospective cohort design was employed: 42 patients with first episode GAD were treated with either escitalopram or sertraline for 12 weeks. Anxiety was measured by the Generalized Anxiety Disorder Scale and the State Trait Anxiety Inventory, serum pro-inflammatory cytokine levels were measured by the enzyme-linked immunosorbent assay (ELISA), and CRP determined by an immunoturbidimetric method before and after SSRIs treatment RESULTS: Baseline levels of anxiety and pro-inflammatory cytokines including IL-1α, IL-6, IL-8, IL-12, IFN-γ, and CRP were significantly reduced after treatment of SSRIs (p < 0.05 in all cases). In addition, the change of anxiety measures co-vary with the change of peripheral cytokine levels (p < 0.05 in all cases). The regression model revealed that log transformed baseline levels of CRP and IL-6 predicted treatment response (p < 0.05 in both cases). CONCLUSIONS: This study is the first to investigate the effects of SSRIs on pro-inflammatory cytokines in patients with first episode GAD. The findings indicate moderate acute anti-inflammatory effects of SSRIs in GAD, and suggest that these anti-inflammatory effects may underlie anxiolytic effects of SSRIs. The study also indicates that serum levels of CRP and IL-6 may predict treatment response. However, data from randomized controlled trials is warranted to confirm these findings.

Low-Level Primary Blast Induces Neuroinflammation and Neurodegeneration in Rats.

OBJECTIVE: Mild blast traumatic brain injury is commonly prevalent in modern combat casualty care and has been associated with the development of neurodegenerative conditions. However, whether primary lower level blast overpressure (LBOP) causes neurodegeneration and neuroinflammation remains largely unknown. The aim of our present study was to determine whether LBOP can cause neuroinflammation and neurodegeneration. METHODS: Anesthetized rats were randomly assigned to LBOP group (70 kPa, n = 5) or sham group (without blast, n = 5). Histopathological and cytokine changes in brain tissue at 5 days post-injury were evaluated by hematoxylin-eosin staining and Bioplex assay, respectively. RESULTS: Histopathological assessment revealed neuronal degeneration and increased density of inflammatory cells in frontal and parietal cortex, hippocampus and thalamus in rats exposed to LBOP. LBOP exposure significantly elevated levels of pro-inflammatory cytokines (EPO, IL-1ß, IL-6, IL-12, IL-18, and TNF-α) and chemokines (GRO and RANTES) as well as of an anti-inflammatory cytokine (IL-13) in the frontal cortex. CONCLUSIONS: This study reveals a role of neuroinflammation in neurodegeneration after mild blast traumatic brain injury. Therapies that target this process might in warfighters might function either by attenuating the development of post-traumatic stress disorder, chronic traumatic encephalopathy and Alzheimer's disease, or by slowing their progression.

Immunotherapeutic effects of recombinant adenovirus encoding interleukin 12 in experimental pulmonary tuberculosis.

High dose of Mycobacterium tuberculosis (Mtb) strain H37Rv administered by intratracheal injection in BALB/c mice induce progressive tuberculosis (TB). In this model, during the first month there is a temporal control of bacillary growth, in coexistence with macrophage activation, granuloma formation and Th-1 response. Then, bacterial proliferation recommences, accompanied by progressive pneumonia and decreasing expression of protective cytokines (IFN-γ and TNF-α). In this model, we studied the IL-12 gene expression kinetics and cellular source. There is a rapid and progressive IL-12 expression peaking at day 14, when granulomas start their formation and numerous macrophages show strong IL-12 immunostaining, while during progressive TB there is a significant decrease of IL-12 expression and occasional macrophages showed IL-12 immunolabeling. In the second part of this study, we determined the immunotherapeutic effect of recombinant adenoviruses that codify IL-12 (AdIL-12). Intratracheal administration of only one dose of AdIL-12 one day before Mtb infection produced significant decrease of bacterial loads, lesser pneumonia and higher expression of TNF-α, IFN-γ and iNOS. When only one dose of AdIL-12 was given in healthy mice cohoused with infected mice with highly virulent and transmissible Mtb, total prevention of infection was conferred. Moreover, when AdIL-12 was administered by intranasal route in animals suffering late active TB after 2 months of infection, a very low pulmonary bacilli burdens was detected. These experimental data confirm that IL-12 is a significant cytokine in the immune protection against Mtb, and gene therapy based in adenoviruses coding this cytokine increased protective immunity and prevent Mtb transmission.

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.

Diosgenin attenuates neuropathic pain in a rat model of chronic constriction injury.

Diosgenin is a steroidal saponin extract from numerous plants, including Solanum and Dioscorea species, and has been reported to possess neuroprotective activity. However, the role of diosgenin in neuropathic pain remains unclear. The present study examined the effects of diosgenin on allodynia and the levels of inflammatory mediators in rats following neuropathic pain evoked by chronic constriction injury (CCI). In addition, the underlying molecular mechanisms involved in diosgenin­induced suppression of neuropathic pain were examined. The results of the present study demonstrated diosgenin reversed CCI­decreased mechanical withdrawal threshold and thermal withdrawal latency. Furthermore, diosgenin inhibited CCI­induced upregulated levels of the pro­inflammatory cytokines tumor necrosis factor­α, interleukin (IL)­1ß and IL­2, and suppressed oxidative stress induced by CCI in the spinal cord. Furthermore, diosgenin significantly inhibited the expression of phosphorylated­p38 mitogen activated protein kinase (MAPK) and nuclear factor (NF)­κB in the spinal cord in CCI rats compared with sham­operated rats. In conclusion, the present study demonstrated that diosgenin attenuates neuropathic pain in CCI rats by inhibiting activation of the p38 MAPK and NF­κB signaling pathways. These results implicate diosgenin in the treatment of neuropathic pain, which merits further clinical investigation.

miR-128 enhances dendritic cell-mediated anti-tumor immunity via targeting of p38.

MiRNA (miR)-128, which is a well­recognized inhibitor of tumor growth, is involved in the anti-tumor function of dendritic cells (DCs). However, the association between miR­128 and the DC­mediated anti­tumor immunity remains to be elucidated. Murine B16 melanoma cells and C57BL/6 male mice were used to obtain marrow­derived DCs. DCs were treated with B16 cell suspension. miR­128 mimic, miR­128 inhibitor, p38 inhibitor or negative control oligonucleotides were transfected into DCs. After transfection, mRNA and protein expression of p38 in DCs was detected via reverse transcription­quantitative polymerase chain reaction and western blotting. The present study demonstrated that the miR­128 abundance in DCs was significantly attenuated by B16 (a melanoma cell line) stimulation and the protein expression level of p38 was increased. Additionally, miR­128 inhibited the protein expression of p38 in DCs in a dose­dependent manner, however no significant effect on the p38 mRNA level was observed. Furthermore, miR­128 mimic or p38 inhibitor decreased the mRNA expression and secretion of interleukin (IL)­6 and IL­10 cytokines and increased the level of IL­12 in DCs, whereas an miR­128 inhibitor exhibited the opposite effects. These findings suggested that miR­128 regulated the immune response of DCs via p38­downstream cytokines. Furthermore, the tumor growth rate, size and weight were markedly decreased and the survival time prolonged, following injection of DCs harboring miR­128 mimic or p38 inhibitor in C57BL/6 mice bearing B16 melanoma. The results therefore suggest that miR­128 enhances the anti­tumor immunity response of DCs via targeting of the p38 mitogen activated protein kinase signaling pathway.

Sinulariolide suppresses LPS­induced phenotypic and functional maturation of dendritic cells.

The dendritic cell (DC) maturation process is essential for the development of T cell responses and immune tolerance. Accordingly, DCs are considered a major target in the development of immunomodulating agents. In the present study, the effect of sinulariolide, an active compound isolated from the cultured soft coral Sinularia flexibilis, on lipopolysaccharide (LPS)­induced murine bone marrow­derived DCs was evaluated. The different phenotypes, cytokine secretion and the mix­lymphocyte reaction of DCs were detected using flow cytometry and ELISA. The experimental results revealed that the phenotypic and functional maturation of DCs stimulated by LPS were markedly reduced by sinulariolide in a concentration­dependent manner, including the expression of co­stimulatory molecules (CD40, CD80 and CD86). In addition, sinulariolide reduced the release of tumor necrosis factor­α, interleukin (IL)­6, IL­12 and nitric oxide from the LPS­activated DCs, decreased their abilities to stimulate allogeneic T cell proliferation, and inhibited LPS­induced nuclear factor­κB pathways. These findings offer novel insight into the immunopharmacological function of sinulariolide and its effects on DCs.

Interleukin-10 Gene-Modified Dendritic Cell-Induced Type 1 Regulatory T Cells Induce Transplant-Tolerance and Impede Graft Versus Host Disease After Allogeneic Stem Cell Transplantation.

BACKGROUND/AIMS: Tr1 cells can induce peripheral tolerance to self- and foreign antigens, and have been developed as a therapeutic tool for the induction of tolerance to transplanted tissue. We explored the feasibility of generating Tr1 cells by using IL-10 gene-modified recipient DCs (DCLV-IL-10) to stimulate donor naive CD4+ T cells. We also investigated some biological properties of Tr1 cells. METHODS: DCLV-IL-10 were generated through DCs transduced with a lentivirus vector carrying the IL-10 gene, and Tr1 cells were produced by using DCLV-IL-10 to stimulate naive CD4+ T cells. The effects of Tr1 cells on T-cell proliferation and the occurrence of graft versus host disease (GVHD) following allogeneic stem-cell transplantation (allo-HSCT) were investigated. RESULTS: The DCLV-IL-10-induced Tr1 cells co-expressed LAG-3 and CD49b. Moreover, they also expressed CD4, CD25, and IL-10, but not Foxp3, and secreted significantly higher levels of IL-10 (1,729.36 ± 185.79 pg/mL; P < 0.001) and INF-γ (1,524.48 ± 168.65 pg/mL; P < 0.01) than the control T cells upon the stimulation by allogeneic DCs. Tr1 cells markedly suppressed T-lymphocyte proliferation and the mixed lymphocytic response (MLR) in vitro. The mice used in the allo-HSCT model had longer survival times and lower clinical and pathological GVHD scores than the control mice. CONCLUSION: IL-10 gene-modified DC-induced Tr1 cells may be used as a potent cellular therapy for the prevention of GVHD after allo-HSCT.

A pilot, open-label, 8-week study evaluating the efficacy, safety and tolerability of adjunctive minocycline for the treatment of bipolar I/II depression.

OBJECTIVES: The objectives of the study were to determine if adjunctive minocycline mitigates depressive symptom severity and improves cognitive function in individuals with bipolar I/II disorder (BD). The study also aimed to determine if changes in depressive and/or cognitive symptoms over the course of treatment were associated with changes in circulating inflammatory cytokine levels. METHODS: A total of 29 (intention-to-treat: n=27) adults meeting DSM-IV-TR criteria for a major depressive episode as part of bipolar I or II disorder (i.e. Hamilton Depression Rating Scale 17-item [HAMD-17] ≥20) were enrolled in an 8-week, open-label study with adjunctive minocycline (100 mg bid). The primary outcome measure was the Montgomery-Åsberg Depression Rating Scale (MADRS). The HAMD-17, Clinical Global Impression-Severity (CGI-S), cognitive test composite scores and plasma cytokines were secondary outcome measures. Plasma cytokines were measured with the 30 V-Plex Immunoassay from Meso Scale Discovery. RESULTS: Adjunctive minocycline was associated with a reduction in depressive symptom severity from baseline to week 8 on the MADRS (P<.001, d=0.835), HAMD-17 (P<.001, d=0.949) and CGI-S (P<.001, d=1.09). Improvement in psychomotor speed, but not verbal memory or executive function, was observed only amongst individuals exhibiting a reduction in depression severity (P=.007, d=0.826). Levels of interleukin (IL)-12/23p40 (P=.002) were increased, while levels of IL-12p70 (P=.001) and C-C motif chemokine ligand 26 (CCL26) (P<.001) were reduced from baseline to week 8. A reduction in CCL26 levels was associated with a less favourable treatment response (P<.001). CONCLUSIONS: Results from the pilot study suggest that adjunctive minocycline may exert antidepressant effects in individuals with bipolar depression, possibly by targeting inflammatory cytokines.

Interleukin-10 and prostaglandin E2 have complementary but distinct suppressive effects on Toll-like receptor-mediated dendritic cell activation in ovarian carcinoma.

Dendritic cells (DC) have the potential to instigate a tumour-specific immune response, but their ability to prime naïve lymphocytes depends on their activation status. Thus, for tumour immunotherapy to be effective, the provision of appropriate DC activation stimuli such as Toll-like receptor (TLR) agonists is crucial in order to overcome immunosuppression associated with the tumour microenvironment. To address this, we investigated how ovarian carcinoma (OC)-associated ascites impedes activation of DC by TLR agonists. Our results show that ascites reduces the TLR-mediated up-regulation of CD86 and partially inhibits the production of the pro-inflammatory cytokines interleukin 6 (IL-6), IL-12 and tumour necrosis factor α (TNFα) in monocyte-derived DC from healthy controls. We further observe an impaired T cell stimulatory capacity of DC upon activation with TLR agonists in the presence of ascites, indicating that their functionality is affected by the immunosuppressive factors. We identify IL-10 and prostaglandin E2 (PGE2) as the pivotal immunosuppressive components in OC-associated ascites compromising TLR-mediated DC activation. Interestingly, IL-10 is present in both ascites from patients with malignant OC and in peritoneal fluid from patients with benign ovarian conditions and both fluids have similar ability to reduce TLR-mediated DC activation. However, depletion of IL-10 from ascites revealed that the presence of paracrine IL-10 is not crucial for ascites-mediated suppression of DC activation in response to TLR activation. Unlike IL-10, PGE2 is absent from peritoneal fluid of patients with benign conditions and selectively reduces TNFα induction in response to TLR-mediated activation in the presence of OC-associated ascites. Our study highlights PGE2 as an immunosuppressive component of the malignant OC microenvironment rendering PGE2 a potentially important target for immunotherapy in OC.

Effects of Lactobacillus plantarum Strain OLL2712 Culture Conditions on the Anti-inflammatory Activities for Murine Immune Cells and Obese and Type 2 Diabetic Mice.

Studies on the health-promoting effects of lactic acid bacteria (LAB) are numerous, but few provide examples of the relationship between LAB function and culture conditions. We verified the effect of differences in culture conditions on Lactobacillus plantarum OLL2712 functionality; this strain exhibits anti-inflammatory activity and preventive effects against metabolic disorders. We measured interleukin-10 (IL-10) and IL-12 production in murine immune cells treated with OLL2712 cells prepared under various culture conditions. The results showed that the IL-10-inducing activities of OLL2712 cells on murine immune cells differed dramatically between OLL2712 groups at different culture phases and using different culture medium components, temperatures, and neutralizing pHs. In particular, exponential-phase cells had much more IL-10-inducing activity than stationary-phase cells. We confirmed that the Toll-like receptor 2 (TLR2) stimulation activity of OLL2712 cells depended on culture conditions in conjunction with IL-10-inducing activity. We also demonstrated functional differences by culture phases in vivo; OLL2712 cells at exponential phase had more anti-inflammatory activity and anti-metabolic-disorder effects on obese and diabetic mice than those by their stationary-phase counterparts. These results suggest that culture conditions affect the functionality of anti-inflammatory LAB.IMPORTANCE While previous studies demonstrated that culture conditions affected the immunomodulatory properties of lactic acid bacteria (LAB), few have comprehensively investigated the relationship between culture conditions and LAB functionality. In this study, we demonstrated several culture conditions of Lactobacillus plantarum OLL2712 for higher anti-inflammatory activity. We also showed that culture conditions concretely influenced the health-promoting functions of OLL2712 in vivo, particularly against metabolic disorders. Further, we characterized a novel mechanism by which changing LAB culture conditions affected immunomodulatory properties. Our results suggest that culture condition optimization is important for the production of LAB with anti-inflammatory activity.

Association between lipoxin A4 and interleukin-12 in gingival crevicular fluid: a preliminary investigation.

BACKGROUND AND OBJECTIVE: The balance between host proinflammatory and anti-inflammatory immune responses is a key determinant for the pathogenesis of periodontal disease. This study aimed to explore the possibility of an association between lipoxin A4 and interleukin-12 in chronic periodontitis. MATERIAL AND METHODS: Forty-five patients with chronic periodontitis and 45 periodontally healthy patients were included in this case-control study. Plaque index, calculus index, gingival index, sulcus bleeding index, full-mouth probing depth and clinical attachment loss were recorded. Gingival crevicular fluid samples were collected and analysed for interleukin-12 and lipoxin A4 using ELISA. RESULTS: The mean concentration of lipoxin A4 was lower in the periodontitis group compared with the periodontally healthy group. There was a negative correlation between interleukin-12 and lipoxin A4 in both groups. There was a negative correlation between clinical attachment loss and lipoxin A4, and a positive correlation between clinical attachment loss and interleukin-12. However, the correlations were statistically insignificant. CONCLUSIONS: The mean interleukin-12 concentration was significantly higher in gingival crevicular fluid from patients with periodontitis than in that from with healthy patients, and the mean lipoxin A4 concentration was lower in patients with periodontitis than in healthy patients. Lipoxin A4 possibly has an inhibitory effect on interleukin-12.