Human milk oligosaccharides differentially support gut barrier integrity and enhance Th1 and Th17 cell effector responses in vitro.

Human milk oligosaccharides (HMOs) can modulate the intestinal barrier and regulate immune cells to favor the maturation of the infant intestinal tract and immune system, but the precise functions of individual HMOs are unclear. To determine the structure-dependent effects of individual HMOs (representing different structural classes) on the intestinal epithelium as well as innate and adaptive immune cells, we assessed fucosylated (2'FL and 3FL), sialylated (3'SL and 6'SL) and neutral non-fucosylated (LNT and LNT2) HMOs for their ability to support intestinal barrier integrity, to stimulate the secretion of chemokines from intestinal epithelial cells, and to modulate cytokine release from LPS-activated dendritic cells (DCs), M1 macrophages (MØs), and co-cultures with naïve CD4+ T cells. The fucosylated and neutral non-fucosylated HMOs increased barrier integrity and protected the barrier following an inflammatory insult but exerted minimal immunomodulatory activity. The sialylated HMOs enhanced the secretion of CXCL10, CCL20 and CXCL8 from intestinal epithelial cells, promoted the secretion of several cytokines (including IL-10, IL-12p70 and IL-23) from LPS-activated DCs and M1 MØs, and increased the secretion of IFN-γ and IL-17A from CD4+ T cells primed by LPS-activated DCs and MØs while reducing the secretion of IL-13. Thus, 3'SL and 6'SL supported Th1 and Th17 responses while reducing Th2 responses. Collectively, our data show that HMOs exert structure-dependent effects on the intestinal epithelium and possess immunomodulatory properties that confer benefits to infants and possibly also later in life.

Triple-Color FluoroSpot Analysis of Polyfunctional Antigen-Specific T Cells by Quantification of Spot-Forming Units and Relative Spot Volumes.

Switching from ELISpot to FluoroSpot enables the analysis of spot-forming units representing cells producing different cytokines as well as the frequencies of spots derived from cells co-secreting multiple cytokines. Due to the fluorescent read-out signal, sophisticated reader instruments can also measure the relative spot volume, making it possible to differentiate between spots generated by cells secreting different levels of one or more cytokines. Here we describe how triple FluoroSpot assays can be used to define polyfunctional T cells secreting multiple cytokines and how different T-cell populations can differ in the levels of cytokines they secrete.

Systemic and local responses of cytokines and tissue histology following intramammary lipopolysaccharide challenge in dairy cows.

During bovine mastitis, immune responses include the release of cytokines and the recruitment of leukocytes, resulting in profound structural and functional changes in the mammary gland. Our aims were to delineate systemic and local cytokine responses and to quantify histological changes in the mammary tissue of lactating cows after acute intramammary lipopolysaccharide (LPS) challenge. Ten multiparous dairy cows were paired to either treatment (TRT) or control (CON) groups. For TRT cows, one side of the udder was randomly assigned to receive treatment with LPS (50 µg in 10 mL of saline, TL) into both the front and rear quarters; the contralateral quarters received saline (10 mL). Udder-halves of CON cows were similarly assigned randomly to receive either saline (10 mL, CS) or no infusion (untreated). Temporal changes in the concentrations of 15 cytokines in the blood (0, 3, 6, 12, and 24 h relative to the LPS infusion) and in mammary tissue (0, 3, and 12 h) were determined, as were concomitant changes in mammary histology. The cytokines IL-6, IL-10, MCP-1, and MIP-1ß showed a systemic response as their concentrations were significantly different in the plasma of TRT cows as compared with CON cows after LPS challenge. The cytokines IL-1α, IL-1ß, IL-6, IL-8, IL-17A, IL-36RA, IP-10, MCP-1, MIP-1α, MIP-1ß, TNF-α, and VEGF-A showed a local response in TL glands, and 8 cytokines, IL-1ß, IL-6, IL-10, IL-17A, IL-36RA, IP-10, MIP-1ß, and VEGF-A showed systemic changes in the nonchallenged mammary glands adjacent to LPS-infused glands. Endotoxin challenge evoked changes in the histology of mammary tissue that included a 5.2- and 7.2-fold increases in the number of neutrophils in alveolar lumens at 3 h and 12 h, respectively. In summary, LPS challenge induced specific local and systemic responses in cytokine induction and elicited neutrophil infiltration in bovine mammary tissue.

Yolkin, a Polypeptide Complex from Egg Yolk, Affects Cytokine Levels and Leukocyte Populations in Broiler Chicken Blood and Lymphoid Organs after In Ovo Administration.

Yolkin is a polypeptide complex isolated from hen egg yolk that exhibits immunomodulating properties. The aim of the present study was to determine whether in-ovo-delivered yolkin affects leukocyte populations and cytokine levels in broiler chickens. The experiment was carried out on eggs from Ross 308 broiler breeder birds. Yolkin was administered in ovo on the 18th day of incubation, once, at the following three doses: 1, 10, or 100 µg/egg. The immunological parameters were assessed in 1-, 7-, 14-, 21-, 28-, 35-, and 42-day-old birds kept under farming conditions and routinely vaccinated. The leukocyte populations were determined in the thymus, spleen, and blood. The cytokine (IL-1ß, IL-2, IL-6, and IL-10) levels were determined in the plasma of the broiler chickens. Each experimental group included eight birds. The most pronounced effect of yolkin was an increase in the population of T cells, both CD4+ and CD8+, mainly in the blood. This effect on the lymphocyte subsets may be valuable regarding chicken immune responses, mainly against T-dependent antigens, during infection or after vaccination.

Clinical, oral immunological and microbiological shifts during and after pregnancy.

OBJECTIVES: Physiological changes and shifts in the oral microbiota composition during pregnancy may affect the maternal immune system. Uncomplicated pregnancy is associated with a T-helper (Th) 2 predominant cytokine regulation (anti-inflammatory), while oral health deterioration during pregnancy is reflected by severe gingival inflammation, a primarily Th1 cytokine phenotype (pro-inflammatory), and oral microbiome alterations. This prospective observational study aimed to evaluate Th cytokine shifts and changes in the oral microbiota composition in saliva of women before and after birth. MATERIAL AND METHODS: Saliva (n = 96) was collected before and 6 months after birth, and medical, oral health, and periodontal status were assessed. In a multiplex immunoassay, 10 cytokines were simultaneously analyzed and cumulative Th1 and Th2 cytokine levels and Th1/Th2 ratio were calculated for all groups. Putative periodontal pathogens (n = 6) were evaluated by quantitative real-time polymerase chain reaction. RESULTS: Th2 cytokine levels were significantly lower (p = 0.014) while pro-inflammatory cytokine levels were significantly higher (p < 0.01) during pregnancy than postpartum. Similar Th1 levels were found between the groups (p = 0.143). Th1 and Th2 cytokines positively correlated with periodontal parameters (p < 0.001) and levels of studied bacteria during pregnancy (p < 0.05). CONCLUSIONS: This study identified a significantly increased Th1/Th2 cytokine ratio during pregnancy and a positive association with putative periodontal pathogens. This immunological and microbiological deregulation in the oral milieu during pregnancy is suggestive of a destructive inflammatory periodontal profile. STUDY REGISTRATION: Clinical Trials.gov (Record BAP-2015). CLINICAL RELEVANCE: Understanding altered oral immunological and microbiological regulation patterns during pregnancy may help improve the inflammatory periodontal profile in pregnant women.

Association of Periodontitis and Atopic Dermatitis with the Levels of IL-13, IL-31, and TSLP in the Gingival Crevicular Fluid.

Emerging epidemiological evidence links atopic dermatitis (AD) and periodontitis, although the mechanisms remain unclear. Th2-derived cytokines are key in the development of both diseases, and different gingival crevicular fluid (GCF) profiles among healthy and diseased subjects have been previously reported. This case-control study examined the GCF levels of interleukins (IL)-13, IL-31, and thymic stromal lymphopoietin (TSLP) in 29 subjects with moderate-to-severe AD and 33 controls. All subjects underwent comprehensive clinical and oral evaluations, followed by GCF collection. GCF levels of IL-13, IL-31, and TSLP were assessed using a multiplex-bead immunoassay. Demographic and periodontal parameters were similar among groups (p > 0.05). The GCF levels of IL-31 and TSLP were higher in AD subjects compared to controls (p < 0.05), whereas no significant differences in the GCF levels of IL-13 were noticed (p = 0.377). Moderate-to-severe AD was positively associated with the GCF levels of IL-31 and TSLP, whereas severe periodontitis was negatively associated with IL-31 (p < 0.05). The GCF levels of IL-13 showed no significant associations with either condition (p = 0.689). There was no significant interaction between AD and periodontitis for IL-31 (p < 0.869). These results suggest that AD and periodontitis independently influence the GCF levels of IL-31 in opposing ways, whereas AD alone influences the levels of TSLP.

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.

Detection of pro-inflammatory cytokines in healthy canine tears using Canine Cytokine SpikeMix™ mass spectrometry via multiple reaction monitoring.

PURPOSE: To evaluate the efficacy of the Canine Cytokine SpikeMix™ and MRM-MS for detecting pro-inflammatory cytokines in canine tears from healthy research Beagles. METHODS: A complete ophthalmic examination was performed on 15 healthy research Beagles to verify no ophthalmic diseases. Tears were collected OU by placing a Weck-Cel® cellulose spear in the ventral conjunctival fornix for 1 min. The Weck-Cel® spear was placed in a 2.0 mL tube with a centrifuge filter forcing tears to flow through the filter into the bottom of the tube. The tears were analyzed using the Canine Cytokine SpikeMix™ and MRM-MS. Descriptive data from this study was reported as the normalized total peak area (nTPA) and median (range) using data imported from the online MRM-MS Skyline program. RESULTS: The level of 16 pro-inflammatory cytokines was successfully detected in all 15 dogs. The four cytokines with the highest median amounts in the samples were IL-2 = 0.1243 (0.019-6.7289), IL-6 = 0.964 (0.0036-16.9365), TNFα = 0.1644 (0.0096-0.7138), and CSF-2 = 0.4022 (0.1475-2.6208). CONCLUSIONS: This study revealed that 16 pro-inflammatory cytokines in canine tears from healthy dogs can be detected with Canine Cytokine SpikeMix™ and MRM-MS.

Calligraphy of Nanoplasmonic Bioink-Based Multiplex Immunosensor for Precision Immune Monitoring and Modulation.

Immunomodulation therapies have attracted immense interest recently for the treatment of immune-related diseases, such as cancer and viral infections. This new wave of enthusiasm for immunomodulators, predominantly revolving around cytokines, has spurred emerging needs and opportunities for novel immune monitoring and diagnostic tools. Considering the highly dynamic immune status and limited window for therapeutic intervention, precise real-time detection of cytokines is critical to effectively monitor and manage the immune system and optimize the therapeutic outcome. The clinical success of such a rapid, sensitive, multiplex immunoanalytical platform further requires the system to have ease of integration and fabrication for sample sparing and large-scale production toward massive parallel analysis. In this article, we developed a nanoplasmonic bioink-based, label-free, multiplex immunosensor that can be readily "written" onto a glass substrate via one-step calligraphy patterning. This facile nanolithography technique allows programmable patterning of a minimum of 3 µL of nanoplasmonic bioink in 1 min and thus enables fabrication of a nanoplasmonic microarray immunosensor with 2 h simple incubation. The developed immunosensor was successfully applied for real-time, parallel detection of multiple cytokines (e.g., interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and transforming growth factor-beta (TGF-ß)) in immunomodulated macrophage samples. This integrated platform synergistically incorporates the concepts of nanosynthesis, nanofabrication, and nanobiosensing, showing great potential in the scalable production of label-free multiplex immunosensing devices with superior analytical performance for clinical applications in immunodiagnostics and immunotherapy.

Role of selected salivary inflammatory cytokines in the diagnosis and prognosis of oral squamous cell carcinoma. A Systematic Review and Meta-analysis

Background: Oral squamous cell carcinoma (OSCC) is gradually increasing its incidence in our society. Unfortunately, this entity is diagnosed at an advanced stage in most patients, a fact that implies greater difficulty in its treatment and a worse prognosis. This systematic review aims to assess whether the cytokines IL-6, IL-8 and TNF-α are potential salivary biomarkers that allow early diagnosis of cancer. Material and methods: An electronic search was performed in three databases (Pubmed, Scopus and Web of Science). We used the following keywords: "salivary cytokines", "saliva cytokines", "salivary interleukins", "biomarkers", "oral squamous cell carcinoma" and "diagnosis", combined with the Boolean operators "AND" and "OR". Results: 128 publications were found and finally 23 articles were included in the review and 15 in the meta-analysis. It has been observed that the majority of OSCC patients express higher salivary concentrations of IL-6, IL-8 and TNF-α compared to the control (CL) and premalignant lesion (OPML) groups. It has also been observed that the different premalignant lesions do not have statistically significant differences in the salivary concentration of the cytokines, and on the other hand, differences have been observed between the different TNM stages. The meta-analysis has shown that the difference in concentration of IL-6, IL-8 and TNF-α is statistically significant between the CL group and the OSCC, and also between the CL group and OPML. Conclusions: There is sufficient evidence to affirm that IL-6, IL-8 and TNF-α are useful salivary cytokines in the early diagnosis and prognosis of OSCC. Although future studies are necessary to establish greater reliability of these biomarkers and thus be able to develop a valid diagnostic test. (AU)

Bone marrow-derived mononuclear cells ameliorate neurological function in chronic cerebral infarction model mice via improvement of cerebral blood flow.

BACKGROUND AIMS: Stroke is a frequently observed neurological disorder that might lead to permanent and severe disability. Recently, various regenerative therapies have been developed, some of which have already been applied clinically. However, their outcomes have not been fully satisfactory. In particular, the development of regenerative therapies for chronic ischemic stroke is greatly needed. Herein intracerebral administration of bone marrow-derived mononuclear cells (BM-MNCs) was assessed as a potential treatment for chronic ischemic stroke using a severe combined immunodeficiency mouse model characterized by minimal vascular variation unrelated to immunodeficiency. METHODS: A reproducible model of permanent middle cerebral artery occlusion was prepared, and intracerebral BM-MNC transplantation was performed 14 days after stroke induction in the infarcted brain. RESULTS: Sensorimotor behavioral function and cerebral blood flow were significantly improved upon treatment with BM-MNCs compared to control medium injection. The transplanted cells exhibited characteristics of the vascular endothelium and microglia/macrophages. Significant angiogenesis and suppression of astrogliosis and microgliosis were observed in the affected brain. Messenger RNA expression analysis showed significant increases in anti-inflammatory cytokines, A2 astrocyte/anti-inflammatory microglia markers and vascular endothelial markers such as vascular endothelial growth factor and significant decreases in pro-inflammatory cytokines and A1 astrocyte/pro-inflammatory microglia markers following BM-MNC transplantation. CONCLUSIONS: These results suggest that intracerebral administration of BM-MNCs should be considered an effective cell therapy for chronic stroke.

Compositional variations in metal nanoparticle components of welding fumes impact lung epithelial cell toxicity.

Welding fumes contain harmful metals and gas by-products associated with development of lung dysfunction, asthma, bronchitis, and lung cancer. Two prominent welding fume particulate metal components are nanosized iron (Fe) and manganese (Mn) which might induce oxidative stress and inflammation resulting in pulmonary injury. Welding fume toxicity may be dependent upon metal nanoparticle (NP) components. To examine toxicity of welding fume NP components, a system was constructed for controlled and continuous NP generation from commercial welding and customized electrodes with varying proportions of Fe and Mn. Aerosols generated consisted of nanosized particles and were compositionally consistent with each electrode. Human alveolar lung A459 epithelial cells were exposed to freshly generated metal NP mixtures at a target concentration of 100 µg/m3 for 6 hr and then harvested for assessment of cytotoxicity, generation of reactive oxygen species (ROS), and alterations in the expression of genes and proteins involved in metal regulation, inflammatory responses, and oxidative stress. Aerosol exposures decreased cell viability and induced increased ROS production. Assessment of gene expression demonstrated variable up-regulation in cellular mechanisms related to metal transport and storage, inflammation, and oxidative stress based upon aerosol composition. Specifically, interleukin-8 (IL-8) demonstrated the most robust changes in both transcriptional and protein levels after exposure. Interleukin-8 has been determined to serve as a primary cytokine mediating inflammatory responses induced by welding fume exposures in alveolar epithelial cells. Overall, this study demonstrated variations in cellular responses to metal NP mixtures suggesting compositional variations in NP content within welding fumes may influence inhalation toxicity.

Effects of Moringa oleifera Seed Oil on Cultured Human Sebocytes In Vitro and Comparison with Other Oil Types.

The seeds of Moringa oleifera (horseradish tree) contain about 40% of one of the most stable vegetable oils (Moringa seed oil). Therefore, the effects of Moringa seed oil on human SZ95 sebocytes were investigated and were compared with other vegetable oils. Immortalized human SZ95 sebocytes were treated with Moringa seed oil, olive oil, sunflower oil, linoleic acid and oleic acid. Lipid droplets were visualized by Nile Red fluorescence, cytokine secretion via cytokine antibody array, cell viability with calcein-AM fluorescence, cell proliferation by real-time cell analysis, and fatty acids were determined by gas chromatography. Statistical analysis was performed by the Wilcoxon matched-pairs signed-rank test, the Kruskal-Wallis test and Dunn's multiple comparison test. The vegetable oils tested stimulated sebaceous lipogenesis in a concentration-dependent manner. The pattern of lipogenesis induced by Moringa seed oil and olive oil was comparable to lipogenesis stimulated by oleic acid with also similar fatty acid secretion and cell proliferation patterns. Sunflower oil induced the strongest lipogenesis among the tested oils and fatty acids. There were also differences in cytokine secretion, induced by treatment with different oils. Moringa seed oil and olive oil, but not sunflower oil, reduced the secretion of pro-inflammatory cytokines, in comparison to untreated cells, and exhibited a low n-6/n-3 index. The anti-inflammatory oleic acid detected in Moringa seed oil probably contributed to its low levels of pro-inflammatory cytokine secretion and induction of cell death. In conclusion, Moringa seed oil seems to concentrate several desired oil properties on sebocytes, such as high content level of the anti-inflammatory fatty acid oleic acid, induction of similar cell proliferation and lipogenesis patterns compared with oleic acid, lipogenesis with a low n-6/n-3 index and inhibition of secretion of pro-inflammatory cytokines. These properties characterize Moringa seed oil as an interesting nutrient and a promising ingredient in skin care products.

Exposure of Tissue-Engineered Cartilage Analogs to Synovial Fluid Hematoma After Ankle Fracture Is Associated With Chondrocyte Death and Altered Cartilage Maintenance Gene Expression.

BACKGROUND: The first stage of fracture healing consists of hematoma formation with recruitment of proinflammatory cytokines and matrix metalloproteinases. Unfortunately, when there is an intra-articular fracture, these inflammatory mediators are not retained at the fracture site, but instead, envelop the healthy cartilage of the entire joint via the synovial fluid fracture hematoma (SFFH). These inflammatory cytokines and matrix metalloproteinases are known factors in the progression of osteoarthritis and rheumatoid arthritis. Despite the known inflammatory contents of the SFFH, little research has been done on the effects of the SFFH on healthy cartilage with regard to cell death and alteration in gene expression that could lead to posttraumatic osteoarthritis (PTOA). METHODS: SFFH was collected from 12 patients with intraarticular ankle fracture at the time of surgery. Separately, C20A4 immortalized human chondrocytes were 3-dimensionally cultured to create scaffold-free cartilage tissue analogs (CTAs) to simulate healthy cartilage. Experimental CTAs (n = 12) were exposed to 100% SFFH for 3 days, washed, and transferred to complete media for 3 days. Control CTAs (n = 12) were simultaneously cultured in complete medium without exposure to SFFH. Subsequently, CTAs were harvested and underwent biochemical, histological, and gene expression analysis. RESULTS: Exposure of CTAs to ankle SFFH for 3 days significantly decreased chondrocyte viability by 34% (P = .027). Gene expression of both COL2A1 and SOX9 were significantly decreased after exposure to SFFH (P = .012 and P = .0013 respectively), while there was no difference in COL1A1, RUNX2, and MMP13 gene expression. Quantitative analysis of Picrosirius red staining demonstrated increased collagen I deposition with poor ultrastructural organization in SFFH-exposed CTAs. CONCLUSION: Exposure of an organoid model of healthy cartilage tissue to SFFH after intraarticular ankle fracture resulted in decreased chondrocyte viability, decreased expression of genes regulating normal chondrocyte phenotype, and altered matrix ultrastructure indicating differentiation toward an osteoarthritis phenotype. CLINICAL RELEVANCE: The majority of ankle fracture open reduction and internal fixation does not occur immediately after fracture. In fact, typically these fractures are treated several days to weeks later in order to let the swelling subside. This means that the healthy innocent bystander cartilage not involved in the fracture is exposed to SFFH during this time. In this study, the SFFH caused decreased chondrocyte viability and specific altered gene expression that might have the potential to induce osteoarthritis. These data suggest that early intervention after intraarticular ankle fracture could possibly mitigate progression toward PTOA.

Copper in airborne fine particulate matter (PM2.5) from urban sites causes the upregulation of pro-inflammatory cytokine IL-8 in human lung epithelial A549 cells.

Fine atmospheric particles, such as PM2.5, are strongly related to the onset and exacerbation of inflammatory responses leading to the development of respiratory and cardiovascular diseases. PM2.5 is a complex mixture of tiny particles with different properties (i.e., size, morphology, and chemical components). Moreover, the mechanism by which PM2.5 induces inflammatory responses has not been fully elucidated. Therefore, it is necessary to determine the composition of PM2.5 to identify the main factors causing PM2.5-associated inflammation and diseases. In the present study, we investigated PM2.5 from two sites (Fukue, a remote monitoring site, and Kawasaki, an urban monitoring site) with greatly different environments and PM2.5 compositions. The results of ICP-MS and EDX-SEM indicated that PM2.5 from Kawasaki contained more metals and significantly induced the expression of the pro-inflammatory cytokine gene IL-8 compared to the PM2.5 from Fukue. We also verified the increased secretion of IL-8 protein from exposure to PM2.5 from Kawasaki. We further investigated their effects on inflammatory response and cytotoxicity using metal nanoparticles (Cu, Zn, and Ni) and ions and found that the Cu nanoparticles caused a dose-dependent increase in IL-8 expression together with significant cell death. We also found that Cu nanoparticles enhanced the secretion of IL-8 protein. These results suggest that Cu in PM2.5 is involved in lung inflammation.

Covariates of vaginal microbiota and pro-inflammatory cytokine levels in women of reproductive age.

This study aimed to assess the correlation between covariates of the vaginal microbiota and local levels of proinflammatory cytokines in women of reproductive age presenting four molecularly defined bacterial community-state types (CSTs). We enrolled 133 non-pregnant women who attended primary care health clinics for routine Pap-testing. Molecular profiling of vaginal microbiota was performed by V3-V4 16S rRNA sequencing. The covariates of vaginal microbiota included were: vaginal pH, total bacterial cell count, diversity (Shannon index), -richness and dominant taxa abundances. Levels of interleukin (IL)-1ß, IL-6, IL-8, and tumour necrosis factor (TNF-α) were measured by enzyme-linked immunosorbent assays in supernatants of cervicovaginal fluids. Nonparametric Kruskal-Wallis test was used to compare microbiota covariates and cytokines among different CSTs. Spearman's tests were performed to assess correlations across the measured parameters. A total of 96 (72.2%) participants had CSTs dominated by Lactobacillus spp. (Lactobacillus crispatus CST I, n=38; Lactobacillus gasseri CST II, n=20; and Lactobacillus iners CST III, n=38). A total of 37 (27.8%) presented the Lactobacillus-depleted CST IV. Total bacterial count was higher in CST II (1.29E+05, 3.40E+04-6.69E+05) compared to other Lactobacillus-dominated CSTs (p=0.0003). The highest values of microbiota diversity (1.85; 0.23-2.68) and richness (27.0; 5.0-37.0) were observed in CST IV (P<0.0001). Lower levels of IL-1ß were observed in CST I (5.4; 0.0-3,256) when compared to CST III (51.7; 0.0-2,616) and to CST IV (56.2; 0.0-3,407) (P=0.008). Levels of IL-6 were higher in CST II (4.13; 0-131.4) than in CST IV (0.0-58.27) (P=0.02). Correlation tests showed an overall distinct profile of CST II when compared to other Lactobacillusdominated CSTs, particularly regarding the correlation between total bacterial load and cytokines (r>0.39). In conclusion, this study provides evidence of a single pro-inflammatory signature of L. gasseri-dominated microbiota in response to bacterial load. Further studies evaluating a broader range of inflammation markers are warranted.

Cytokine expression in gingival crevicular fluid around teeth opposing dental implants and 3-unit fixed partial dentures in a cross-sectional study.

OBJECTIVE: This study aimed to study the cytokines in gingival crevicular fluid (GCF) of the teeth opposing to dental implants and 3-unit fixed partial dentures (FPDs). MATERIALS AND METHODS: A total of 74 participants were recruited for this cross-sectional study. Based on the status of lower first molars, the participants were divided into dental implants group and 3-unit FPDs group. Social index and oral hygiene were recorded. Occlusal loading was evaluated with a T-scan. GCF was sampled from the upper first molar and assessed with a commercial cytokine assay kit. RESULTS: Forty three dental implants patients and 31 3-unit FPDs patients received all of the clinical and laboratory evaluation. The dental implants group had a higher occlusion force distribution on first molars region. IL-10, IL-17, RANK had a higher mean in dental implants group and was associated with occlusion force of first molar. There was a weakly association between IL-10 and dental implants in the binary logistic regression analyses. CONCLUSIONS: In this study, the teeth opposing implants have a higher level of cytokines in the GCF than teeth opposing to 3-unit FPDs in periodontal healthy participants because of the poor osseoperception of dental implants. IL-10 might reflect a higher occlusion force in dental implants region. CLINICAL RELEVANCE: This study provided that different tooth restoration methods could influence the periodontal status of the contact teeth.

Effect of saffron, black seed, and their main constituents on inflammatory cytokine response (mainly TNF-α) and oxidative stress status: an aspect on pharmacological insights.

Tumor necrosis factor-α (TNF-α), an inflammatory cytokine, is produced by monocytes and macrophages. It is known as a 'double-edged sword' because it is responsible for advantageous and disadvantageous events in the body system. The unfavorable incident includes inflammation, which induces some diseases such as rheumatoid arthritis, obesity, cancer, and diabetes. Many medicinal plants have been found to prevent inflammation, such as saffron (Crocus sativus L.) and black seed (Nigella sativa). Therefore, the purpose of this review was to assess the pharmacological effects of saffron and black seed on TNF-α and diseases related to its imbalance. Different databases without time limitations were investigated up to 2022, including PubMed, Scopus, Medline, and Web of Science. All the original articles (in vitro, in vivo, and clinical studies) were collected on the effects of black seed and saffron on TNF-α. Black seed and saffron have therapeutic effects against many disorders, such as hepatotoxicity, cancer, ischemia, and non-alcoholic fatty liver, by decreasing TNF-α levels based on their anti-inflammatory, anticancer, and antioxidant properties. Saffron and black seed can treat a variety of diseases by suppressing TNF-α and exhibiting a variety of activities such as neuroprotective, gastroprotective, immunomodulatory, antimicrobial, analgesic, antitussive, bronchodilator, antidiabetic activity, anticancer, and antioxidant effects. To uncover the beneficial underlying mechanisms of black seed and saffron, more clinical trials and phytochemical research are required. Also, these two plants affect other inflammatory cytokines, hormones, and enzymes, implying that they could be used to treat a variety of diseases.

Role of selected salivary inflammatory cytokines in the diagnosis and prognosis of oral squamous cell carcinoma. A Systematic Review and Meta-analysis.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is gradually increasing its incidence in our society. Unfortunately, this entity is diagnosed at an advanced stage in most patients, a fact that implies greater difficulty in its treatment and a worse prognosis. This systematic review aims to assess whether the cytokines IL-6, IL-8 and TNF-α are potential salivary biomarkers that allow early diagnosis of cancer. MATERIAL AND METHODS: An electronic search was performed in three databases (Pubmed, Scopus and Web of Science). We used the following keywords: "salivary cytokines", "saliva cytokines", "salivary interleukins", "biomarkers", "oral squamous cell carcinoma" and "diagnosis", combined with the Boolean operators "AND" and "OR". RESULTS: 128 publications were found and finally 23 articles were included in the review and 15 in the meta-analysis. It has been observed that the majority of OSCC patients express higher salivary concentrations of IL-6, IL-8 and TNF-α compared to the control (CL) and premalignant lesion (OPML) groups. It has also been observed that the different premalignant lesions do not have statistically significant differences in the salivary concentration of the cytokines, and on the other hand, differences have been observed between the different TNM stages. The meta-analysis has shown that the difference in concentration of IL-6, IL-8 and TNF-α is statistically significant between the CL group and the OSCC, and also between the CL group and OPML. CONCLUSIONS: There is sufficient evidence to affirm that IL-6, IL-8 and TNF-α are useful salivary cytokines in the early diagnosis and prognosis of OSCC. Although future studies are necessary to establish greater reliability of these biomarkers and thus be able to develop a valid diagnostic test.

Effect of a protease-activated receptor-2 antagonist (GB88) on inflammation-related loss of alveolar bone in periodontal disease.

BACKGROUND AND OBJECTIVE: Protease-activated receptor-2 (PAR2 ), a pro-inflammatory G-protein coupled receptor, has been associated with pathogenesis of periodontitis and the resulting bone loss caused by oral pathogens, including the keystone pathogen Porphyromonas gingivalis (P. gingivalis). We hypothesised that administration of a PAR2 antagonist, GB88, might prevent inflammation and subsequent alveolar bone resorption in a mouse model of periodontal disease. METHODS: Periodontitis was induced in mice by oral inoculations with P. gingivalis for a total of eight times over 24 days. The infected mice were treated with either GB88 or vehicle for the duration of the trial. Following euthanasia on day 56, serum was collected and used for the detection of mast cell tryptase. The right maxillae were defleshed and stained with methylene blue to measure the exposed cementum in molar teeth. The left maxillae were prepared for cryosections followed by staining for tartrate-resistant acid phosphatase to identify osteoclasts or with toluidine blue to identify mast cells. Reverse transcription quantitative PCR (RT-qPCR) was used to quantify the expression of inflammatory cytokines in the gingival tissue. Supernatants of T-lymphocyte cultures isolated from the regional lymph nodes were assayed using a cytometric bead array to measure the Th1/Th2/Th17 cytokine levels. RESULTS: Measurement of the exposed cementum showed that GB88 reduced P. gingivalis-induced alveolar bone loss by up to 69%. GB88 also prevented the increase in osteoclast numbers observed in the infected mice. Serum tryptase levels were significantly elevated in both the infected groups, and not altered by treatment. RT-qPCR showed that GB88 prevented the upregulation of Il1b, Il6, Ifng and Cd11b. In T-lymphocyte supernatants, only IFNγ and IL-17A levels were increased in response to infection, but this was prevented by GB88 treatment. CONCLUSIONS: GB88 significantly reduced osteoclastic alveolar bone loss in mice infected with P. gingivalis, seemingly by preventing the upregulation of several inflammatory cytokines. PAR2 antagonism may be an effective treatment strategy for periodontal disease.