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.

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.

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.

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.

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.

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.

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.

Klotho sensitive regulation of dendritic cell functions by vitamin E.

BACKGROUND: Dendritic cells (DCs) are the most potent professional antigen-presenting cells for naive T cells to link innate and acquired immunity. Klotho, an anti-aging protein, participates in the regulation of Ca2+ dependent migration in DCs. Vitamin E (VitE) is an essential antioxidant to protect cells from damage and elicits its inhibitory effects on NF-κB-mediated inflammatory response. However, the roles of VitE on mouse DC functions and the contribution of klotho to those effects both are unknown. The present study explored the effects of VitE on klotho expression, maturation, ROS production and migration in DCs. METHODS: The mouse bone marrow cells were isolated and cultured with GM-CSF to attain bone marrow-derived DCs (BMDCs). Cells were stimulated with LPS (100 ng/ml) in the presence or absence of VitE (500 µM). RT-PCR and immunoprecipitation methods were employed to determine klotho expression, ELISA to determine cytokine release, flow cytometry to analyze number of CD86+CD11c+ cells, the intracellular expression of cytokines and reactive oxygen species (ROS) production and a transwell migration assay to trace migration. RESULTS: Klotho transcript level and this hormone secretion in DC supernatant were enhanced by VitE treatment and further increased in the presence of NF-κB inhibitor Bay 11-7082 (10 µM). Moreover, VitE treatment inhibited IL-12p70 protein expression of, ROS accumulation in and CCL21-dependent migration of LPS-triggered mature DCs, these effects were reversed following klotho silencing. CONCLUSION: The up-regulation of klotho by VitE could contribute to the inhibitory effects of VitE on NF-κB-mediated DC functional maturation. The events might contribute to immunotherapeutic effect of VitE on the pathophysiology of klotho-related disease.

Human milk composition differs in healthy mothers and mothers with celiac disease.

PURPOSE: To investigate whether breast-milk composition and microbiota differ in healthy mothers and mothers with celiac disease (CD) to ultimately contribute to identify additional factors determining CD risk. METHODS: Breast-milk samples from healthy mothers (n = 12) and mothers with CD (n = 12) were collected. Cytokines and secretory immunoglobulin A (sIgA) were analyzed by bead-arrays and flow cytometry and human milk oligosaccharides (HMOs) were assessed by capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. Breast-milk microbiota composition was analyzed by conventional and quantitative real-time PCR. RESULT: Breast milk from CD mothers showed significantly lower levels of interleukin (IL) 12p70 (P < 0.042), transforming growth factor (TGF)-ß1 (P < 0.018) and sIgA (P < 0.003) and almost significantly lower levels of interferon (IFN)-γ (P < 0.058). Six mothers in each group belonged to the secretor Le(a-b+) type, one to the secretor Le(a-b-) type and five to the non-secretor Le(a+b-) type. CD mothers of non-secretor Le(a+b-) type showed increased Lacto-N-tetraose content (P < 0.042) compared with healthy mothers. CD mothers' milk showed reduced gene copy numbers of Bifidobacterium spp. (P < 0.026) and B. fragilis group (P < 0.044). CONCLUSION: CD mothers' breast milk is characterized by a reduced abundance of immunoprotective compounds (TGF-ß1 and sIgA) and bifidobacteria. The reduction in these components could theoretically diminish the protective effects of breast-feeding on the child's future risk of developing CD.

The TLR7 agonist Imiquimod promote the immunogenicity of mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSCs) are considered the best candidate in stem cells therapy due to their multipotent differentiation ability, low expression of co-stimulatory molecules (CD80, CD86, CD34 and HLA-II) and immunosuppression effects on in vivo immune responses. MSCs were now widely used in clinical trials but received no encourage results. The major problem was the fate of engrafted MSCs in vivo could not be defined. Some studies indicated that MSCs could induce immune response and result in the damage and rejection of MSCs. As toll like receptors (TLRs) are important in inducing of immune responses, in this study we study the role of TLR7 in mediating the immune status of MSCs isolated from umbilical cord. RESULTS: Our results indicated that TLR7 agonist Imiquimod could increase the proliferation of PBMC isolated from healthy human volunteers and release of lactate dehydrogenase (LDH) in supernatant from PBMC-UCMSCs co-culture system. Flow cytometry and quantitative PCR also confirmed the regulated expression of surface co-stimulatory molecules and pro-inflammatory genes (IL-6, IL-8, IL-12, TGF-ß and TNF-α). And the down-regulation expression of stem cell markers also confirmed the loss of stemness of UCMSCs. We also found that the osteo-differentiation ability of UCMSCs was enhanced in the presence of Imiquimod. CONCLUSION: To our knowledge, this is the first report that activation of TLR7 pathway increases the immunogenicity of UCMSCs. Extensive researches have now been conducted to study whether the change of immune status will be help in tumor rejection based on the tumor-tropism of MSCs.

Screening of immunomodulatory and adhesive Lactobacillus with antagonistic activities against Salmonella from fermented vegetables.

The purpose of this study was to select strains of lactic acid bacteria (LAB) by their in vitro adhesive and immunomodulatory properties for potential use as probiotics. In this study, 16 randomly selected LAB strains from fermented vegetables (sauerkraut, bean and cabbage) were first screened for their tolerance to acid, bile salts, pepsin and pancreatin, bacterial inhibitory activities and abilities to adherence to Caco-2 cells. Then, 4 strains with the highest adhesion abilities were selected for further studies of their immunomodulatory properties and inhibitory effects against Salmonella adhesion and invasion to Caco-2 cells in vitro. The results showed that these 16 LAB strains effectively survived in simulated gastrointestinal condition and inhibited growth of six tested pathogens. Lactobacillus rhamnosus P1, Lactobacillus plantarum P2, Lactobacillus rhamnosus P3 and Lactobacillus casei P4 had the highest abilities to adhere to Caco-2 cells. Furthermore, L. plantarum P2 strain showed higher abilities to induce expression of tumor necrosis factor-α and interleukin-12 by splenic monocytes and strongly inhibited the adhesion and invasion of S. enteritidis ATCC13076 to Caco-2 cells. These results suggest that Lactobacillus strains P2 could be used as a probiotic candidate in food against Salmonella infection.

[Role of nerve growth factor in differentiation of dendritic cells].

OBJECTIVE: To explore the effect of nerve growth factor (NGF) on the  differentiation of murine bone marrow-derived dendritic cells (DCs) in vitro.
 METHODS: The bone marrow cells of femur and tibia from healthy C57B -L/6 mice were isolated and divided into 4 groups: a phosphate buffered saline (PBS) group (PBS group), a NGF group, a granulocyte monocyte colony stimulating factor (GM-CSF) plus interleukin 4 (IL-4) group (GM-CSF+IL-4 group), and a GM-CSF plus IL-4 and NGF group (n=6 in each group). The positive rate of CD11c+ and the proportion of CD8a- were compared at the 7th day among the different groups by flow cytometry. The immature DCs were acquired by classic methods with GM-CSF and IL-4. The purified DCs were obtained by magnetic bead positive selection for CD11c+ cells. The immature DCs were divided into 4 groups: a PBS group, a NGF group, a LPS group, and a NGF+LPS group (n=6 in each group), which were incubated with PBS, NGF, LPS and NGF+LPS, respectively. Cytokine levels of IL-6, IL-10 and IL-12 were detected by ELISA after 24 hours..
 RESULTS: 1) the percentage of CD11c+ DCs in the NGF group were more than that in the PBS group, and lower than that in the the GM- CSF+IL-4 group (both P<0.05). There was no difference between the GM-CSF + IL-4 group and the NGF+GM-CSF+IL-4 group (P>0.05). CD8a- DCs were dominant in these four groups; 2) NGF could further up-regulate the LPS-induced cytokine secretion from DCs, such as IL-6, IL-10, and IL-12 (all P<0.05), but NGF alone had no such effect (all P<0.05).
 CONCLUSION: NGF can promote the murine bone-marrow cells differentiation into CD11c+ DCs, with CD8a-subset; NGF could enhance LPS-induced cytokine secretion from DCs (IL-6, IL-10 and IL-12).

Effect of the N-butanoyl glutathione (GSH) derivative and acyclovir on HSV-1 replication and Th1 cytokine expression in human macrophages.

Macrophages are an important defense against in vivo herpes simplex virus (HSV) infection by early cytokine secretion; however, they can be infected by HSV-1 and they may be compromised in their ability to produce cytokines. In this paper, we studied the expression of two Th1 cytokines, interleukin (IL)-12 and IL-27, upon HSV-1 infection of human macrophages, and how it is regulated by treatment with two antiviral drugs exerting their anti-HSV-1 activity through different mechanisms of action. We found that infection does not alter intra-macrophage thiol content, while it induces mRNA expression of IL-12 p35 and IL-12 p40 as well as of IL-27 p28 and IL-27 EBI3, as revealed by RT-PCR. The increased expression of mRNA is accompanied by increased production of IL-12 p40 and IL-27 p28 protein, as detected in the culture supernatants by ELISA. The two antiviral drugs tested were acyclovir (ACV), commonly used to treat herpes virus infections, and an N-butanoyl glutathione (GSH) derivative, GSH-C4. While ACV inhibits viral DNA polymerase, GSH-C4 inhibits virus replication by interfering with protein folding and maturation of viral particles. Indeed, GSH-C4, altering the intracellular redox state, may modulate the Th1/Th2 balance favoring Th1-type response. Our data show that both drugs inhibit HSV-1 replication in macrophages, without significantly affecting cytokine mRNA levels. Nonetheless, lower levels of IL-12 p40 and IL-27 p28 proteins were found in the supernatants of macrophages treated with either GSH-C4 or ACV, likely as an indirect consequence of inhibited HSV-1 replication.

Method for fabrication and verification of conjugated nanoparticle-antibody tuning elements for multiplexed electrochemical biosensors.

There is a critical need for more accurate, highly sensitive and specific assay for disease diagnosis and management. A novel, multiplexed, single sensor using rapid and label free electrochemical impedance spectroscopy tuning method has been developed. The key challenges while monitoring multiple targets is frequency overlap. Here we describe the methods to circumvent the overlap, tune by use of nanoparticle (NP) and discuss the various fabrication and characterization methods to develop this technique. First sensors were fabricated using printed circuit board (PCB) technology and nickel and gold layers were electrodeposited onto the PCB sensors. An off-chip conjugation of gold NP's to molecular recognition elements (with verification technique) is described as well. A standard covalent immobilization of the molecular recognition elements is also discussed with quality control techniques. Finally use and verification of sensitivity and specificity is also presented. By use of gold NP's of various sizes, we have demonstrated the possibility and shown little loss of sensitivity and specificity in the molecular recognition of inflammatory markers as "model" targets for our tuning system. By selection of other sized NP's or NP's of various materials, the tuning effect can be further exploited. The novel platform technology developed could be utilized in critical care, clinical management and at home health and disease management.

Local levels of inflammatory mediators in uncontrolled type 2 diabetic subjects with chronic periodontitis.

AIM: The aim of this study was to evaluate the levels of a wide panel of cyto/chemokines in the gingival crevicular fluid (GCF) of uncontrolled type 2 diabetic subjects as compared with non-diabetic subjects with periodontitis. METHODS: Twenty-six uncontrolled type 2 diabetic subjects (glycated haemoglobin levels >7.5%) and 20 non-diabetic subjects with chronic periodontitis were enrolled in this study. The levels of 14 cyto/chemokines were measured in the GCF of healthy and diseased sites of the diabetic and non-diabetic subjects using multiplex bead immunoassays. RESULTS: The concentrations of eotaxin, macrophage inflammatory protein-1α, granulocyte-macrophage colony-stimulating factor, interleukin (IL)-6, tumour necrosis factor-α and IL-12 were higher in healthy and diseased sites of diabetic than non-diabetic subjects, after adjustment for multiple comparisons (p < 0.0035). CONCLUSION: Uncontrolled type 2 diabetes mellitus modulated the local levels of several cyto/chemokines at both healthy and diseased periodontal sites in favour of a proinflammatory profile, which may partially explain the greater susceptibility of diabetic subjects to periodontal breakdown.

Serotype-dependent response of human dendritic cells stimulated with Aggregatibacter actinomycetemcomitans.

AIM: Different serotypes of Aggregatibacter actinomycetemcomitans have been described based on the lipopolysaccharide (LPS)-O-polysaccharide antigenicity. In turn, a distinct effect of A. actinomycetemcomitans serotypes has been described on cell proliferation and pro-inflammatory cytokine production in different human cells. This study was aimed to investigate the differential dendritic cell (DC) response when stimulated with different bacterial strains belonging to the most prevalent serotypes of A. actinomycetemcomitans (a-c). MATERIALS AND METHODS: Dendritic cells were obtained from healthy subjects and stimulated with increasing multiplicity of infection (MOI = 10(-1) -10(2)) of A. actinomycetemcomitans, serotypes a-c, or their lipopolysaccharide (10-50 ng/ml). The levels for interferon (IFN)-γ, tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-5, IL-6, IL-10, IL-12 and IL-23 were quantified by real-time RT-PCR and ELISA. RESULTS: Variable DC responses were detected when stimulated with the different strains of A. actinomycetemcomitans. DCs stimulated with A. actinomycetemcomitans strains belonging to the serotype b or their purified LPS expressed higher levels of IL-1ß, IL-6, IL-12, IL-23, IFN-γ and TNF-α than DCs stimulated with the other serotypes. CONCLUSIONS: Aggregatibacter actinomycetemcomitans strains belonging to the serotype b demonstrated a higher capacity to trigger Th1 and Th17-type cytokine production on DCs. These increased potential is likely explained by a higher immunogenicity of their LPS.

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.