Functional Effects of Probiotic Lactiplantibacillus plantarum in Alleviation Multidrug-Resistant Escherichia coli-Associated Colitis in BALB/c Mice Model.

Multidrug-resistant Escherichia coli (MDR-E. coli) is a global health concern. Lactic acid bacteria (LAB) are important probiotics that have beneficial effects on health, and in recent years, their influences in preventing foodborne pathogens-induced colitis have attracted much attention. Therefore, this study aimed to investigate the oral administration of Lactiplantibacillus plantarum NWAFU-BIO-BS29 as an emerging approach to alleviate MDR-E. coli-induced colitis in BALB/c mice model. To illustrate the mode of action of NWAFU-BIO-BS29 interventions with the gut microbiota and immune responses, the changes on the colonic mucosal barrier, regulatory of the gene expressions of inflammatory cytokines, re-modulating the intestinal microflora, and changes in physiological parameters were studied. The results indicated that daily supplementation of 200 µL fresh bacteria for 7 days had ameliorated the associated colitis and partially prevented the infection. The modes of action by ameliorating the inflammatory response, which destructed villous and then affected the intestinal barrier integrity, reducing the secretion of interleukins (6 and ß) and tumor necrosis factor (TNF-α) in serum by 87.88-89.93%, 30.73-35.98%, and 19.14-22.32%, respectively, enhancing the expressions of some epithelial integrity-related proteins in the mouse mucous layer of mucins 2 and 3, Claudin-1, and Occludin by 130.00-661.85%, 27.64-57.35%, 75.52-162.51%, and 139.36-177.73%, respectively, and 56.09-73.58% for toll-like receptor (TLR4) in colon tissues. Notably, the mouse gut microbiota analysis showed an increase in the relative abundance of beneficial bacteria, including Lactobacillus, Bacteriodales bacterium, Candidatus Saccharimonas, Enterorhabdus, and Bacilli. Furthermore, the probiotic promoted the proliferation of epithelia and goblet cells by increasing short-chain fatty acids (SCFAs) levels by 19.23-31.39%. In conclusion, L. plantarum NWAFU-BIO-BS29 has potential applications and can be considered a safe dietary supplement to ameliorate the colitis inflammation symptoms of MDR-E. coli infection.

Influence of Diclofenac Potassium versus Prednisolone on Postendodontic Pain and Pulpal Interleukin-8 Expression in Symptomatic Irreversible Pulpitis Cases: A Randomized Placebo-controlled Trial.

AIM: This prospective, randomized, double-blind clinical trial investigated the impact of diclofenac potassium, prednisolone, and placebo as oral premedication on postendodontic pain and pulpal interleukin (IL)-8 expression in patients with irreversible pulpitis. METHODS: Thirty-six patients undergoing conventional endodontic treatment were assigned into one of 3 groups (n = 12). Pulpal blood samples were taken after access cavity preparation and stored until they were analyzed using enzyme-linked immunosorbent asssay for quantification of IL-8. Postendodontic pain was scored using the visual analogue scale. Outcome data were statistically analyzed using one-way analysis of variance, Kruskal-Wallis, Friedman's, Dunn's, Chi-square, and Fisher's exact tests and Spearman's correlation coefficient. The significance level (α) was set at 0.05. RESULTS: Apart from preoperative pain scores, all groups had similar baseline characteristics (P > .05). Immediate postendodontic pain scores had a significant difference between all groups (P < .05) where placebo group showed the highest score. There was no significant difference between all groups at 6 and 12 hours postoperatively (P > .05). Furthermore, there was no significant difference in the incidence of postendodontic pain and in mean IL-8 levels between the 3 groups (P > .05). CONCLUSIONS: The only impact the premedications had was on the immediate postendodontic pain intensity, and they had no influence on the later time points, incidence of postendodontic pain or pulpal IL-8 levels.

Harvesting methods of umbilical cord-derived mesenchymal stem cells from culture modulate cell properties and functions.

Introduction: Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) are promising candidates for stem cell therapy. Various methods such as enzymatic treatment, cell scraping, and temperature reduction using temperature-responsive cell culture dishes have been employed to culture and harvest UC-MSCs. However, the effects of different harvesting methods on cell properties and functions in vitro remain unclear. In this study, we investigated the properties and functions of UC-MSC using various cell-harvesting methods. Methods: UC-MSC suspensions were prepared using treatments with various enzymes, cell scraping, and temperature reduction in temperature-responsive cell culture dishes. UC-MSC sheets were prepared in a temperature-responsive cell culture dish. The properties and functions of the UC-MSC suspensions and sheets were assessed according to Annexin V staining, lactate dehydrogenase (LDH) assay, re-adhesion behavior, and cytokine secretion analysis via enzyme-linked immunosorbent assay. Results: Annexin V staining revealed that accutase induced elevated UC-MSC apoptosis. Physical scraping using a cell scraper induced a relatively high LDH release due to damaged cell membranes. Dispase exhibited relatively low adhesion from initial incubation until 3 h. UC-MSC sheets exhibited rapid re-adhesion at 15 min and cell migration at 6 h. UC-MSC sheets expressed higher levels of cytokines such as HGF, TGF-ß1, IL-10, and IL-6 than did UC-MSCs in suspension. Conclusions: The choice of enzyme and physical scraping methods for harvesting UC-MSCs significantly influenced their activity and function. Thus, selecting appropriate cell-harvesting methods is important for successful stem cell therapy.

In vitro immunoregulatory role of recombinant Ancylostoma ceylanicum calreticulin.

Ancylostoma ceylanicum is a zoonotic soil-derived nematode that parasitizes the intestines of humans and animals (dogs and cats), leading to malnutrition and iron-deficiency anemia. Helminth parasites secrete calreticulin (CRT), which regulates or blocks the host's immune response. However, no data on A. ceylanicum calreticulin (Ace-CRT) are available. We investigated the biological function of recombinant Ace-CRT (rAce-CRT). rAce-CRT showed reliable antigenicity and stimulated the proliferation of mouse splenocytes and canine peripheral blood mononuclear cells. Quantitative reverse-transcription PCR assays revealed that rAce-CRT primarily promoted the expression of T helper 2 cytokines, particularly IL-13, in canine peripheral blood lymphocytes. rAce-CRT inhibited complement-mediated sheep erythrocyte hemolysis in vitro. Our findings indicate that Ace-CRT plays an immunomodulatory role and may be a promising candidate molecule for a hookworm vaccine.

Expression of Inflammatory Mediators in Biofilm Samples and Clinical Association in Multiple Sclerosis Patients in Remission-A Pilot Study.

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of unknown etiology that affects the central nervous system and can lead to neurological impairment. Our aim was to determine whether MS patients also show inflammatory changes in the oral cavity more frequently than healthy individuals. For this purpose, we examined plaque samples for various mediators and their correlation with clinical findings. A study group (MS) and a control group were examined and compared. The plaque samples were analyzed for the expression of interleukins (IL-2, -6, -10), matrix metalloproteinases (MMP-7, MMP-9), and a surface antigen CD90 by quantitative real-time PCR. The clinical parameters examined were the Mombelli plaque index; bleeding on probing (BOP) index; periodontal pocket depth; and decayed, missing, and filled tooth (DMFT) index. The expression of MMP9 was significantly (p = 0.035) higher in the control group. The expression of IL-2 was increased four-fold in the MS group; however, this difference was not statistically significant. The mean PD (p < 0.001) and BOP index (p = 0.029) values were increased in the study group. The clinical parameters of the BOP index and PD were significantly amplified in the MS patients. However, no causal relationship between the investigated inflammatory mediators and the clinical findings could be established in this case series.

Comparative immunohistochemical analysis of inflammatory cytokines in distinct subtypes of Sweet syndrome.

Background: A dysregulated immune response has been implicated in Sweet syndrome (SS) pathogenesis; however, cytokine profiles across different conditions associated with SS - including adult-onset immunodeficiency (AOID) due to anti-interferon (IFN)-γ autoantibodies - remain unknown. Objective: To investigate alterations in inflammatory cytokines in skin lesions of distinct subtypes of SS. Methods: Skin biopsies were collected from 42 AOID- and 52 non-AOID-associated SS patients and 18 healthy controls. The comparative immunohistochemical study was conducted using monoclonal antibodies against interleukin (IL)-1ß, IL-6, IL-17, IFN-γ, and tumor necrosis factor-α on paraffin-embedded sections. The quantitative percentage positivity and intensity were calculated using computer-based image analysis. Results: The results showed stronger and more diffuse dermal immunoreactivity for IFN-γ and IL-17 in the AOID-associated (p < 0.001 and p < 0.001, respectively) and non-AOID-associated SS (p < 0.001 and p < 0.001, respectively) groups. However, no significant differences in the levels of these two cytokines were observed between the AOID- and non-AOID-associated SS groups. Increased expression of IFN-γ together with IL-17 was also noted in almost all subtypes among non-AOID-associated SS. Conclusions: These results demonstrate that IFN-γ and IL-17 are implicated in immunopathology of all SS subtypes, including AOID-associated SS, despite the presence of anti-IFN-γ autoantibodies.

Cytokine mRNA expression in the bronchoalveolar lavage cells from horses affected by different equine asthma subtypes.

Equine asthma (EA) is a respiratory syndrome associated with the increase of different leukocyte populations in the bronchoalveolar lavage fluid (BALF). Its pathogenetic mechanisms remain unclear. This study aimed to evaluate the associations between the mRNA expression of different cytokines in the BALF, different EA subtypes and lung function. Fifteen horses underwent physical examination, airway endoscopy, BALF cytology and lung function testing (8/15). One horse did not have evidence of EA and was used as healthy reference, while the others were classified as affected by neutrophilic or mixed granulocytic EA. Cells isolated from the residual BALF were used for IL-1ß, IL-2, IFN-γ, IL-4, IL-17A genes expression by quantitative RT-PCR., Cytokine expression was compared between groups, and their correlations with BALF leukocyte and lung function were evaluated. IL-1ß expression was positively correlated with BALF neutrophils count (p=0.038, r=0.56) and with increased expiratory resistance (p=0.047, r=0.76). IFN-γ was correlated with BALF mast cells (p=0.029, r=0.58). IL-4 was higher in horses with mixed granulocytic EA than neutrophilic (p=0.008), positively correlated with BALF mast cells (p=0.028, r=0.59) and inversely with whole-breath (p=0.046, r=-0.76) and expiratory reactance (p=0.003, r=-0.93). Finally, IL-17A was inversely correlated with expiratory reactance (p=0.009, r=-0.92). These results support that multiple immune responses are involved in EA pathogenesis; innate, Th2, and Th17 responses. Innate immunity appeared associated with neutrophilic inflammation, and Th2 response with increased mast cells. The role of Th1 response in EA remains questionable.

sTREM2 Differentially Affects Cytokine Expression in Myeloid-Derived Cell Models via MAPK-JNK Signaling Pathway.

TREM2 is a critical innate immune receptor primarily expressed on myeloid-derived cells, such as microglia and macrophages. Mutations in TREM2 are linked to several neurodegenerative diseases including Alzheimer's disease (AD). TREM2 can be cleaved from the cell membrane and released as soluble TREM2 (sTREM2). sTREM2 levels are shown to peak prior to AD, with its levels fluctuating throughout disease progression. However, the mechanism by which sTREM2 may affect innate immune responses is largely uncharacterized. In this study, we investigated whether sTREM2 can induce inflammatory response in myeloid-derived THP-1 monocytes and macrophages and characterized the signaling mechanisms involved. Our results show that sTREM2 was capable of stimulating the expression of several inflammatory cytokines in THP-1 cells throughout the time course of 2 h to 8 h but inducing anti-inflammatory cytokine expression at later time points. A TREM2 antibody was capable of inhibiting the expression of some cytokines induced by sTREM2 but enhancing others. The complex of sTREM2/TREM2 antibody was shown to enhance IL-1ß expression, which was partially blocked by an NLRP3 specific inhibitor, indicating that the complex activated the NRLP3 inflammasome pathway. sTREM2 was also shown to have differential effects on cytokine expression in M0, M1, and M2 macrophages differentiated from THP-1 cells. sTREM2 has a more stimulating effect on cytokine expression in M0 macrophages, less of an effect on M2 macrophages, and some inhibitory effects on cytokine expression in M1 macrophages at early time points. Analyses of several signaling pathways revealed that sTREM2-induced expression of cytokines occurs mainly through MAPK-JNK signaling. Our work reveals differential effects of sTREM2 on cytokine expression profiles of THP-1 cells and macrophages and demonstrates that the MAPK-JNK signaling pathway is mainly responsible for sTREM2-induced cytokine expression.

Polyethylene Terephthalate Hydrolases in Human Gut Microbiota and Their Implications for Human Health.

Polyethylene terephthalate (PET), primarily utilized for food and beverage packaging, consistently finds its way into the human gut, thereby exerting adverse effects on human health. PET hydrolases, critical for the degradation of PET, have been predominantly sourced from environmental microbial communities. Given the fact that the human gut harbors a vast and intricate consortium of microorganisms, inquiry into the presence of potential PET hydrolases within the human gut microbiota becomes imperative. In this investigation, we meticulously screened 22,156 homologous sequences that could potentially encode PET hydrolases using the hidden Markov model (HMM) paradigm, drawing from 4984 cultivated genomes of healthy human gut bacteria. Subsequently, we methodically validated the hydrolytic efficacy of five selected candidate PET hydrolases on both PET films and powders composed of micro-plastics (MPs). Notably, our study also unveiled the influence of both diverse PET MP powders and their resultant hydrolysates on the modulation of cytokine expression in macrophages. In summary, our research underscores the ubiquitous prevalence and considerable potential of the human gut microbiota in PET hydrolysis. Furthermore, our study significantly contributes to the holistic evaluation of the potential health hazards posed by PET MPs to human well-being.

A CONSORT-guided, randomized controlled clinical trial of nebulized administration of dexamethasone and saline on lower airway cytokine mRNA expression in horses with moderate asthma.

BACKGROUND: Nebulized administration of dexamethasone on cytokine regulation in horses with moderate asthma has not been investigated. OBJECTIVE: To investigate the changes in expression of inflammatory cytokine mRNA after nebulized administration of dexamethasone treatment of horses with moderate asthma. ANIMALS: Horses with naturally occurring moderate asthma (n = 16) and healthy control horses (n = 4). All horses were kept in a dusty environment during the study. METHODS: Prospective, parallel, randomized, controlled, blinded clinical trial. Blood endogenous cortisol, tracheal mucus, and bronchoalveolar lavage (BAL) were sampled before and after 13 days treatment with either nebulized administration of dexamethasone (15 mg once daily) or 0.9% saline (3 mL). Treatment groups were randomly allocated via randomization function (Microsoft Excel). Amplification of target mRNA in BAL fluid (IL-1ß, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-17, IL-23, IFN-γ, Eotaxin-2, and TNF-α) was achieved by qPCR, and the relative expression software tool was used to analyze BAL inflammatory cytokine mRNA. RESULTS: Horses treated with nebulized administration of dexamethasone had increased relative expression of IL-5 (1.70-fold), IL-6 (1.71-fold), IL-17 (3.25-fold), IL-12 (1.66-fold), and TNF-α (1.94-fold), and decreased relative expression of IL-23 (1.76-fold; P = .04) in samples collected on Day 14, in comparison to samples collected on Day 0 (all P < .05). Horses treated with nebulized administration of saline had no significant difference in the relative expression of any gene (all P > .05). CONCLUSIONS AND CLINICAL IMPORTANCE: Nebulized administration of dexamethasone was associated with increased expression of inflammatory cytokine mRNA. There was no improvement in inflammatory airway cytology associated with either dexamethasone or saline treatment.

Effect of vibration of the vortex mixer on the red blood cells.

PURPOSES: Red blood cells (RBCs) are often subject to vibration during processing, transfusion, and transport. Further research is necessary to understand the effects of vibration on human RBCs and to reduce experimental deviations caused by device vibration. METHODS: Flow cytometry was used in this study to observe the cytokine expression of IgG and IgA and deformation of human red blood cells affected by the vibration of a vortex mixer with varying frequency (750 rpm and 1500 rpm), duration (5 min and 10 min), and container volume (96 well plate and 48 well plate). RESULTS: The size of RBCs in duration of 10 min is obviously smaller than the duration of 5 min. The 10-minute duration led to visibly smaller RBC sizes compared to the 5-minute duration. There was little effect on the size of RBCs in the 10-minute groups from differences in frequency and container volume. However, decreased RBC size can be observed in the 5-minute groups, where frequency is increased or container volume is decreased. Echinocytes were present in photomicrographs of all 10-minute groups, but microstructure of the RBCs was not impacted by vortex mixer vibration. The elevated frequency or reduced container volume results in an increased cytokine expression of IgG within the 5-minute groupings. CONCLUSION: It can be inferred that vibration must not be overlooked due to its potential impact on the shape and cytokine expression of RBCs. Hence, the inclusion of vibration must be taken into consideration in experiments and devices pertaining to RBCs.

Macrophages morphology and cytokine reeducation by ex situ copper thiol complexes.

OBJECTIVE: To study the reeducation effect of copper thiol complexes on macrophage morphology and cytokine expression. METHODS: The effect of copper thiol complexes was assessed on murine macrophages by the cell morphology observed through optical microscopy, while the expression of cytokines by protein abundance after stimulation. A viability experiment was performed on PMBC to confirm that copper complexes do not affect other cells. RESULTS: The M1 shape was reported after treatment with copper thiol complexes at 1-200 µM, while M2 behavior was documented between 50 and 800 µM. Surprisingly, a thin elongate morphology was observed between 400-800 µM like the M2 shape. The expression of M1 cytokines was noted ranging from 1 to 100 µM, with the highest yield at 1 µM (2243 pg/µL) for the copper-penicillamine complex. M2 production behavior was observed at 1-800 µM, with the highest abundance close to 1150 pg/µL (200-400 µM) was quantified from the copper-cysteine complex. Finally, LCCu complexes did not induce a cytotoxic response on PBMC while exhibiting a high IL-4 and IL-10 production, similar to their gold analogs. CONCLUSIONS: The capacity of copper thiol complexes to reeducate M1 to M2 morphoexpression can be promising for cell protection by using copper thiol penicillamine or immuno-regeneration of tissues when using copper thiol cysteine.

Cytokine expression of IL-8 before and after root canal treatment in patients with symptomatic apical periodontitis: A clinical trial.

This study's objective was to compare the cytokine expression of IL-8 in periapical tissues of single-rooted teeth with symptomatic apical periodontitis (SAP) before and after root canal treatments. As well as, comparing IL-8 levels in peri-apical tissues between vital and necrotic teeth with SAP. METHODOLOGY: Thirty-six patients were allocated according to their pulp status into two experimental groups (n = 18) receiving the same treatment protocol; group 1: Vital pulps with SAP, and group 2: non-vital pulps with SAP. Conventional endodontic treatment was done on two visits; isolation and disinfection of the operative field were undertaken, and two-stage access cavity preparation was implemented. The first pre-instrumentation peri-apical sample (S1) was collected prior to cleaning and shaping procedures. A 2.5% NaOCl irrigation was used to thoroughly irrigate the canal after performing root canal preparation utilising the ProTaper Next (PTN) rotary system. After 1 week, the second post-instrumentation peri-apical sample (S2) was collected. Using an ELISA kit, the quantity of IL-8 was evaluated following the collection of all samples. RESULTS: In all pre-instrumentation samples, IL-8 was detected (100%). The level of IL-8 expression was significantly decreased from the S1 to S2 of all samples (p < 0.001). The intra-group comparison showed a statistically significant reduction in the level of IL-8 expression between S1 and S2 in both vital and non-vital groups where p < 0.001* in both groups. The inter-group comparison of levels of IL-8 expression (vital and non-vital) revealed a significant difference between both groups regarding the pretreatment sample with the higher levels of IL-8 shown in the non-vital group (p < 0.001). While in the post-treatment sample, both groups showed a significant reduction in the level of IL-8 expression but the difference between them was not statistically significant (p = 0.226). CONCLUSION: Root canal instrumentation seems to be efficient in decreasing the levels of anti-inflammatory cytokines, namely IL-8. Further research should clarify how intra-canal medicaments affect inflammatory mediator levels.

Molecular characterization and functional study of a galectin-9 from a teleost fish, Boleophthalmus pectinirostris.

Galectin-9, a tandem-repeat galectin, plays an important role in the regulation of innate immune response against various microbial infections. Here, galectin-9 from mudskipper (Boleophthalmus pectinirostris) was identified and named as BpGal-9. Putative BpGal-9 contains two conserved carbohydrate recognition domains (CRDs), one CRD within N-terminal (N-CRD) and the other one within C-terminal (C-CRD). Multi-alignment analysis indicated that BpGal-9 shared the highest amino acid sequence identity of 64.3 % with that of Southern platyfish (Xiphophorus maculatus). Phylogenetic analysis showed that BpGal-9 grouped tightly with other teleosts galectin-9 and was most closely related to that of Southern platyfish. BpGal-9 transcripts were more abundant in the intestine, and its expression upregulated significantly in the intestine, kidney, spleen, gills, and skin after Edwardsiella tarda infection. Meanwhile, BpGal-9 expression significantly increased in hemocytes and serum of mudskipper infected by E. tarda. The recombinant BpGal-9 (rBpGal-9) and rBpGal-9C-CRD could agglutinate all tested bacteria, whereas rBpGal-9N-CRD could only agglutinate three kinds of bacteria. When targeting the same bacteria, rBpGal-9 showed stronger agglutinating activities than rBpGal-9C-CRD or rBpGal-9N-CRD. In addition, the induction effect of three recombinant proteins on the mRNA expression of anti-inflammatory cytokines (BpIL-10 and BpTGF-ß) was better than that on the pro-inflammatory cytokines (BpIL-1ß and BpTNF-α). Our result suggested that the N-CRD and C-CRD of galectin-9 contribute differently to its multiple functions in innate immunity in teleosts.

Umbilical cord-derived mesenchymal stem cell sheets transplanted subcutaneously enhance cell retention and survival more than dissociated stem cell injections.

BACKGROUND: Human umbilical cord-derived mesenchymal stem cell (hUC-MSC) sheets have recently attracted attention as an alternative approach to injected cell suspensions for stem cell therapy. However, cell engraftment and cytokine expression levels between hUC-MSC sheets and their cell suspensions in vivo have not yet been compared. This study compares hUC-MSC in vivo engraftment efficacy and cytokine expression for both hUC-MSC sheets and cell suspensions. METHODS: hUC-MSC sheets were prepared using temperature-responsive cell culture; two types of hUC-MSC suspensions were prepared, either by enzymatic treatment (trypsin) or by enzyme-free temperature reduction using temperature-responsive cell cultureware. hUC-MSC sheets and suspensions were transplanted subcutaneously into ICR mice through subcutaneous surgical placement and intravenous injection, respectively. hUC-MSC sheet engraftment after subcutaneous surgical transplantation was investigated by in vivo imaging while intravenously injected cell suspensions were analyzing using in vitro organ imaging. Cytokine levels in both transplant site tissues and blood were quantified by enzyme-linked immunosorbent assay. RESULTS: After subcutaneous transplant, hUC-MSC sheets exhibited longer engraftment duration than hUC-MSC suspensions. This was attributed to extracellular matrix (ECM) and cell-cell junctions retained in sheets but enzymatically altered in suspensions. hUC-MSC suspensions harvested using enzyme-free temperature reduction exhibited relatively long engraftment duration after intravenous injection compared to suspensions prepared using trypsin, as enzyme-free harvest preserved cellular ECM. High HGF and TGF-ß1 levels were observed in sheet-transplanted sites compared to hUC-MSC suspension sites. However, no differences in human cytokine levels in murine blood were detected, indicating that hUC-MSC sheets might exert local paracrine rather than endocrine effects. CONCLUSIONS: hUC-MSC sheet transplantation could be a more effective cell therapeutic approach due to enhanced engraftment and secretion of therapeutic cytokines over injected hUC-MSC suspensions.

Immune checkpoint blockade induced shifts in cytokine expression patterns in peripheral blood of head and neck cancer patients are linked to outcome.

Background: Immune-checkpoint blockade (ICB) of programmed-death-1 (PD-1) with pembrolizumab or nivolumab is approved for treating recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC). NadiHN and ADRISK are phase IIB trials investigating in locally advanced (LA) HNSCC having low or high risk of recurrence the potential benefits from adding nivolumab to post-operative radiotherapy or pembrolizumab to cisplatin-based radio-chemotherapy. Methods: Along five randomized controlled ICB trials including NadiHN and ADRISK, blood samples were taken before and after starting ICB in n=25 patients. Concentrations of vascular endothelial growth factor A (VEGF), CCL2 (MCP-1), interleukin-6 (IL-6), IL-8, interferon-gamma (IFN-γ), and CXCL10 (IP-10) pre- and post-ICB in EDTA-anticoagulated plasma and serum were compared. We used receiver operating characteristic (ROC) curves to identify optimal cutoff for defining subgroups before analyzing overall survival (OS) applying Kaplan-Meier plots and multivariate Cox regression. Results: We detected huge heterogeneity between cytokine patterns in pre-and post-ICB plasma and serum. We observed high correlation between concentrations of some cytokines. Despite absent systematic OS differences after ICB with pembrolizumab or nivolumab or between LA-HNSCC versus R/M HNSCC patients, we noticed improved outcome of patients having lower IFN-γ concentrations pre- and post-ICB and following ICB reduced concentrations of VEGF, IL-6, and IL-8 but not MCP-1. Contrarily, increases in IL-6, IL-8, and VEGF levels correlated with impaired outcome. Multivariate Cox regression revealed five independent OS predictors among cytokines; using natural logarithms of their hazard ratios to estimate an individual's risk of dying, three cytokine-expression pattern (CEP)-risk groups with no death within mean (95% confidence interval) follow-up of 29.2 (22.1-36.2) months and median OS of 11.3 (8.8-13.8) and 2.9 (0.4-5.4) months were found. Conclusion: Whereas individual pre- or post-ICB cytokine concentrations in serum or plasma alone failed to predict the survivor group, CEP-risk groups may support the identification of individual patients with long-lasting benefit from ICB.

Effect of rearing systems on immune status, stress parameters, intestinal morphology, and mortality in conventional and local chicken breeds.

The majority of poultry meat used to be sourced from intensively housed birds. However, consumer preference has since demanded poultry producers develop more sustainable farming systems. Although free-range farming is considered beneficial for animal welfare, it is not as easy to standardize as an intensive system, which makes the choice of bird genotype appear crucial for alternative systems. In this study, we aimed to evaluate the effect of conventional and free-range rearing systems on the immune status, stress parameters, intestinal morphology and mortality in commercial hybrids (Ross 308) and local poultry strains, Bionda Piemontese (BP), Robusta Maculata (RM), BP x Sasso (BPxS), and RM x Sasso (RMxS). RNA was extracted from the jejunum and spleen to assess the mRNA expression of IL-2, IL-6, IL-10, IL-18, IL-1ß, inducible nitric oxide synthase (iNOS), toll-like receptor (TLR)-4, and interferon gamma (IFN-γ). The heterophil:lymphocyte (H/L) ratio and intestinal histomorphometric evaluation were also calculated. We found that compared to the conventional system, the rearing system significantly affected the jejunum expression of IL-10, iNOS, IL-2, and IL-6, where these genes were upregulated in free-range system. A significant interaction between the rearing system and the genotype was also shown. More specifically, local breeds showed a significantly higher expression (P < 0.001) of IL-6 in the free-range system compared to the same genotypes in the conventional system. Moreover, IL-6 is constantly upregulated in local breeds within the free-range system compared to Ross hybrids. We also found significantly increased H/L and mortality rates in the latter, compared to the local breeds in the free-range reared system. The jejunum morphology also demonstrated a significantly higher villus height in BP and BPxS compared to the Ross hybrids. Overall, the results of our study confirm that the intense selection for growth in broiler chickens may have reduced their ability to react to the environmental stimuli related to free-range systems, resulting in a lower adaptability to a free-range environment, thus making them inappropriate for any farming system other than the conventional one. On the contrary, local chicken breeds are able to adapt and survive in the free-range system of rearing, and represent a genetic resource especially when adaptability to free-range conditions is required.

Uncoupling of Natural Killer cell functional maturation and cytolytic function in NOD mice.

NK cells are innate immune cells that target infected and tumor cells. Mature NK (mNK) cells undergo functional maturation characterized by four distinct stages, during which they acquire their cytotoxic properties. mNK cells from non-obese diabetic (NOD) mice exhibit a defect in functional maturation and have impaired cytotoxic functions. Hence, we tested whether the impaired cytotoxic function observed in mNK cells from NOD mice can be explained by their defect in functional maturation. By comparing the function of mNK cells from B6, B6g7 and NOD mice, we show that the expression of granzyme B is severely impaired in mNK cells from NOD mice, agreeing with their inability to control tumor growth in vivo. The low level of granzyme B expression in mNK cells from NOD mice is found at all stages of functional maturation and is therefore independent of their functional maturation defect. Consequently, this study demonstrates that phenotypic functional maturation of mNK cells can be uncoupled from the acquisition of cytotoxic functions.

Proinflammatory and Immunomodulatory Gene and Protein Expression Patterns in Spinal Cord and Spleen Following Acute and Chronic High Thoracic Injury.

Introduction: In addition to paralysis and loss of sensation, high-level spinal cord injury (SCI) causes sympathetic dysfunction that can lead to autonomic dysreflexia (AD) and chronic immune suppression involving splenic leukopenia. Evidence has shown that treatment with either gabapentin or blockade of TNFα mitigates maladaptive plasticity and the underlying hemodynamic dysfunction, spleen atrophy, and immune dysfunction associated with AD. Because significant improvements long term was noted following treatments only during acute stages of recovery, we sought to systematically examine changes in proinflammatory and immunomodulatory cytokines to ascertain the reason. Methods: Adult female Wistar rats underwent complete T4 spinal transection before euthanasia at systematic intervals from 3 days to 8 weeks after injury. Using qRT-PCR and meso scale discovery (MSD) assays, the gene and protein expression of TNFα and IFNγ in the spleen, upper thoracic (T4-9) and lumbosacral (L5-S6) spinal cords were analyzed. Results: We found that spleen atrophy occurs in a biphasic manner compared to naïve controls, with significant decreases in the spleen mass noted at 3 days and 8 weeks after injury. Splenic TNFα mRNA and protein levels did not change significantly over time, while IFNγ gene expression dipped acutely with trends for increased protein levels at more chronic time points. TNFα protein increased significantly only in thoracic spinal cord segments from 3 to 14 days post-injury. IFNγ mRNA and protein levels remained unelevated in injured spinal cords over time, with trends for increased protein levels at 2 and 8 weeks in the lumbosacral segments. Discussion: Novel temporal-spatial cytokine expression profiles reveal that TNFα protein levels are increased solely in upper thoracic segments after high thoracic SCI, while IFNγ remains unaltered. Splenic leukopenia and latent systemic immunosuppression are not associated with altered TNFα or IFNγ expression in the spleen or spinal cord.

Anoxia Rapidly Induces Changes in Expression of a Large and Diverse Set of Genes in Endothelial Cells.

Sinusoidal endothelial cells are the predominant vascular surface of the bone marrow and constitute the functional hematopoietic niche where hematopoietic stem and progenitor cells receive cues for self-renewal, survival, and differentiation. In the bone marrow hematopoietic niche, the oxygen tension is usually very low, and this condition affects stem and progenitor cell proliferation and differentiation and other important functions of this region. Here, we have investigated in vitro the response of endothelial cells to a marked decrease in O2 partial pressure to understand how the basal gene expression of some relevant biological factors (i.e., chemokines and interleukins) that are fundamental for the intercellular communication could change in anoxic conditions. Interestingly, mRNA levels of CXCL3, CXCL5, and IL-34 genes are upregulated after anoxia exposure but become downmodulated by sirtuin 6 (SIRT6) overexpression. Indeed, the expression levels of some other genes (such as Leukemia Inhibitory Factor (LIF)) that were not significantly affected by 8 h anoxia exposure become upregulated in the presence of SIRT6. Therefore, SIRT6 mediates also the endothelial cellular response through the modulation of selected genes in an extreme hypoxic condition.