Proinflammatory Cytokine Signaling Research Articles

Biologics-Induced Immunophenotypic Cross-Switching in Patients with Psoriasis and Atopic Dermatitis.

Antibody-based therapies that inhibit pro-inflammatory cytokine signalling are commonly used in dermatology. Paradoxically, these biological agents may induce or exacerbate paradoxical reactions. Recently, it has been reported that the treatment of eczema with dupilumab can lead to the development of psoriasiform eruptions, which we called psoriasiform paradoxical reactions (P-PRs). Conversely, cases of eczematous paradoxical reactions (E-PRs) have also been described in patients with psoriasis treated with biologics. To summarise the case characteristics and disease features of phenotypic transition between psoriasis and eczematoid dermatitis, and to explore the mechanism or connection related to biological agents or patients' genetic characteristics, a systematic review was conducted for P-PRs in atopic dermatitis and E-PRs in patients with psoriasis treated with corresponding biological agents, respectively. We identified a series of P-PRs in 42 atopic dermatitis cases treated with dupilumab. The time to onset of P-PRs typically ranged from weeks to months, with a mean latency period of 22.65 weeks. Almost all patients presented with new-onset P-PRs. Simultaneously, we reviewed 22 articles reporting 51 patients with psoriasis with biological agent-induced E-PRs, which occurred on average at 24.47 weeks, 72.55% of them induced by IL-17A inhibitors. 48.98% (24/49) of cases reported a positive personal history of atopy, which may suggest an increased risk of biological agent-induced paradoxical eruptions. Overall, the improvement or resolution upon discontinuation of the inciting biologics was relatively common, and further studies are needed to estimate the real prevalence and unveil the pathophysiological mechanisms of these paradoxical events.

Bystander effect of SARS-CoV-2 spike protein on human monocytic THP-1 cell activation and initiation of prothrombogenic stimulus representing severe COVID-19

BackgroundHypercoagulable state and thromboembolic complications are potential life-threatening events in COVID-19 patients. Our previous studies demonstrated that SARS-CoV-2 infection as well as viral spike protein expressed epithelial cells exhibit senescence with the release of inflammatory molecules, including alarmins.FindingsWe observed extracellular alarmins present in the culture media of SARS-CoV-2 spike expressing cells activate human THP-1 monocytes to secrete pro-inflammatory cytokines to a significant level. The release of THP-1 derived pro-inflammatory cytokine signature correlated with the serum of acute COVID-19 patient, but not in post-COVID-19 state. Our study suggested that the alarmins secreted by spike expressing cells, initiated phagocytosis property of THP-1 cells. The phagocytic monocytes secreted complement component C5a and generated an autocrine signal via C5aR1 receptor. The C5a-C5aR1 signal induced formation of monocyte mediated extracellular trap resulted in the generation of a prothrombogenic stimulus with activating platelets and increased tissue factor activity. We also observed an enhanced C5a level, platelet activating factor, and high tissue factor activity in the serum of acute COVID-19 patients, but not in recovered patients.ConclusionOur present study demonstrated that SARS-CoV-2 spike protein modulates monocyte responses in a paracrine manner for prothrombogenic stimulus by the generation of C5a complement component.

Decreased Innate Migration of Pro-Inflammatory M1 Macrophages through the Mesothelial Membrane Is Affected by Ceramide Kinase and Ceramide 1-P.

The retrograde flow of endometrial tissues deposited into the peritoneal cavity occurs in women during menstruation. Classically (M1) or alternatively (M2) activated macrophages partake in the removal of regurgitated menstrual tissue. The failure of macrophage egress from the peritoneal cavity through the mesothelium leads to chronic inflammation in endometriosis. To study the migration differences of macrophage phenotypes across mesothelial cells, an in vitro model of macrophage egress across a peritoneal mesothelial cell monolayer membrane was developed. M1 macrophages were more sessile, emigrating 2.9-fold less than M2 macrophages. The M1 macrophages displayed a pro-inflammatory cytokine signature, including IL-1α, IL-1β, TNF-α, TNF-β, and IL-12p70. Mass spectrometry sphingolipidomics revealed decreased levels of ceramide-1-phosphate (C1P), an inducer of migration in M1 macrophages, which correlated with its poor migration behavior. C1P is generated by ceramide kinase (CERK) from ceramide, and blocking C1P synthesis via the action of NVP231, a specific CERK chemical inhibitor, prohibited the emigration of M1 and M2 macrophages up to 6.7-fold. Incubation with exogenously added C1P rescued this effect. These results suggest that M1 macrophages are less mobile and have higher retention in the peritoneum due to lower C1P levels, which contributes to an altered peritoneal environment in endometriosis by generating a predominant pro-inflammatory cytokine environment.

Systemic administration of dorsomorphin relieves inflammatory nociception in the mouse formalin test.

Inflammatory pain is the most important clinical symptom of inflammatory diseases. Despite intensive research into inflammatory pain mechanisms, the majority of analgesics available are based on mechanistic classes of compounds that have been known for many years, as a result, inflammatory pain control remains a challenge for drug design in the context of clinically unmet needs in terms of safety and efficacy. A growing literature supports that pro-inflammatory cytokine signaling plays a crucial role in the development of inflammatory pain. Modulation of the pro-inflammatory cytokine may hold the key to improved pain management. Previous studies have reported that dorsomorphin played key roles in inflammation. But the role of dorsomorphin in the formalin-induced inflammatory nociception in mice has never been reported. Here, we report a new function of dorsomorphin which can inhibit formalin-induced inflammatory nociception in mice. The antinociceptive effect of dorsomorphin mainly depended on inhibiting the p38 MAPK/c-fos signaling and regulating inflammatory mediators.

Repeated Use of Morphine Induces Anxiety by Affecting a Proinflammatory Cytokine Signaling Pathway in the Prefrontal Cortex in Rats.

We examined the role of toll-like receptors (TLRs) and proinflammatory cytokine signaling pathways in the prefrontal cortex (PFC) in anxiety-like behaviors after repeated use of morphine. Morphine (10mg/kg) was used twice daily for 8days to induce morphine dependence in male Wistar rats. On day 8, opioid dependence was confirmed by measuring naloxone-precipitated withdrawal signs. On days 1 and 8, anxiety-like behaviors were evaluated using a light/dark box test. Expression of TLR1 and 4, proinflammatory cytokines, and some of the downstream signaling molecules was also evaluated in the bilateral PFC at mRNA and protein levels following morphine dependence. The results revealed that morphine caused anxiolytic-like effects on day 1 while induced anxiety following 8days of repeated injection. On day 8, a significant decrease in TLR1 expression was detected in the PFC in morphine-dependent rats, but TLR4 remained unaffected. Repeated morphine injection significantly increased IL1-β, TNFα, and IL6 expression, but decreased IL1R and TNFR at mRNA and protein levels except for IL6R at the protein level in the PFC. The p38α mitogen-activated protein (MAP) kinase expression significantly increased but the JNK3 expression decreased in the PFC in morphine-dependent rats. Repeated injection of morphine also significantly increased the NF-κB expression in the PFC. Further, significant increases in Let-7c, mir-133b, and mir-365 were detected in the PFC in morphine-dependent rats. We conclude that TLR1 and proinflammatory cytokines signaling pathways in the PFC are associated with the anxiogenic-like effects of morphine following its chronic use in rats via a MAP kinase/NF-κB pathway.

Von Willebrand factor links primary hemostasis to innate immunity

The plasma multimeric glycoprotein von Willebrand factor (VWF) plays a critical role in primary hemostasis by tethering platelets to exposed collagen at sites of vascular injury. Recent studies have identified additional biological roles for VWF, and in particular suggest that VWF may play an important role in regulating inflammatory responses. However, the molecular mechanisms through which VWF exerts its immuno-modulatory effects remain poorly understood. In this study, we report that VWF binding to macrophages triggers downstream MAP kinase signaling, NF-κB activation and production of pro-inflammatory cytokines and chemokines. In addition, VWF binding also drives macrophage M1 polarization and shifts macrophage metabolism towards glycolysis in a p38-dependent manner. Cumulatively, our findings define an important biological role for VWF in modulating macrophage function, and thereby establish a novel link between primary hemostasis and innate immunity.

CD169-mediated restrictive SARS-CoV-2 infection of macrophages induces pro-inflammatory responses.

Exacerbated and persistent innate immune response marked by pro-inflammatory cytokine expression is thought to be a major driver of chronic COVID-19 pathology. Although macrophages are not the primary target cells of SARS-CoV-2 infection in humans, viral RNA and antigens in activated monocytes and macrophages have been detected in post-mortem samples, and dysfunctional monocytes and macrophages have been hypothesized to contribute to a protracted hyper-inflammatory state in COVID-19 patients. In this study, we demonstrate that CD169, a myeloid cell specific I-type lectin, facilitated ACE2-independent SARS-CoV-2 fusion and entry in macrophages. CD169-mediated SARS-CoV-2 entry in macrophages resulted in expression of viral genomic and subgenomic RNAs with minimal viral protein expression and no infectious viral particle release, suggesting a post-entry restriction of the SARS-CoV-2 replication cycle. Intriguingly this post-entry replication block was alleviated by exogenous ACE2 expression in macrophages. Restricted expression of viral genomic and subgenomic RNA in CD169+ macrophages elicited a pro-inflammatory cytokine expression (TNFα, IL-6 and IL-1β) in a RIG-I, MDA-5 and MAVS-dependent manner, which was suppressed by remdesivir treatment. These findings suggest that de novo expression of SARS-CoV-2 RNA in macrophages contributes to the pro-inflammatory cytokine signature and that blocking CD169-mediated ACE2 independent infection and subsequent activation of macrophages by viral RNA might alleviate COVID-19-associated hyperinflammatory response.

The cellular landscape of the normal kidney allograft: Main players balancing the alloimmune response.

Despite recent advances made in short-term outcomes; minimal improvements have been observed in long-term kidney transplantation outcomes. Due to an imbalance between organ transplant availability and patient waiting list, expanding kidney allograft longevity is a critical need in the field. Prior studies have either focused on early ischemic and immunological conditions affecting kidney allografts (e.g., delayed graft function, acute rejection) or late stage chronic injury when interventions are no longer feasible. However, studies characterizing kidney allografts with normal function by its cellular distribution, cell-cell interactions, and associated molecular pathways are lacking. Herein, we used single nuclei RNA-sequencing to uncover the cellular landscape and transcriptome of the normal kidney allograft. We profiled 40,950 nuclei from seven human kidney biopsies (normal native, N = 3; normal allograft, N = 4); normal allograft protocol biopsies were collected ≥15-months post-transplant. A total of 17 distinct cell clusters were identified with proximal tubules (25.70 and 21.01%), distal tubules (15.22 and 18.20%), and endothelial cells (EC) (4.26 and 9.94%) constituting the major cell populations of normal native and normal allograft kidneys, respectively. A large proportion of cycling cells from normal native kidneys were in G1-phase (43.96%) whereas cells from normal allograft were predominantly in S-phase (32.69%). This result suggests that transcriptional differences between normal native and normal allograft biopsies are dependent on the new host environment, immunosuppression, and injury-affliction. In the normal allograft, EC-specific genes upregulated metabolism, the immune response, and cellular growth, emphasizing their role in maintaining homeostasis during the ongoing alloreactive stress response. Immune cells, including B (2.81%), macrophages (24.96%), monocytes (15.29%), natural killer (NK) (12.83%), neutrophils (8.44%), and T cells (14.41%, were increased in normal allografts despite lack of histological or clinical evidence of acute rejection. Phenotypic characterization of immune cell markers supported lymphocyte activation and proinflammatory cytokines signaling pathways (i.e., IL-15, IL-32). The activation of B, NK, and T cells reveals potential immune cells underlying subclinical inflammation and repair. These single nuclei analyses provide novel insights into kidney and immune cell associated signaling pathways that portray kidney grafts with normal allograft function beyond 2-years post-transplant, revealing a novel perspective in understanding long-term allograft graft survival.

Malnutrition leads to increased inflammation and expression of tuberculosis risk signatures in recently exposed household contacts of pulmonary tuberculosis.

BackgroundMost individuals exposed to Mycobacterium tuberculosis (Mtb) develop latent tuberculosis infection (LTBI) and remain at risk for progressing to active tuberculosis disease (TB). Malnutrition is an important risk factor driving progression from LTBI to TB. However, the performance of blood-based TB risk signatures in malnourished individuals with LTBI remains unexplored. The aim of this study was to determine if malnourished and control individuals had differences in gene expression, immune pathways and TB risk signatures.MethodsWe utilized data from 50 tuberculin skin test positive household contacts of persons with TB - 18 malnourished participants (body mass index [BMI] < 18.5 kg/m2) and 32 controls (individuals with BMI ≥ 18.5 kg/m2). Whole blood RNA-sequencing was conducted to identify differentially expressed genes (DEGs). Ingenuity Pathway Analysis was applied to the DEGs to identify top canonical pathways and gene regulators. Gene enrichment methods were then employed to score the performance of published gene signatures associated with progression from LTBI to TB.ResultsMalnourished individuals had increased activation of inflammatory pathways, including pathways involved in neutrophil activation, T-cell activation and proinflammatory IL-1 and IL-6 cytokine signaling. Consistent with known association of inflammatory pathway activation with progression to TB disease, we found significantly increased expression of the RISK4 (area under the curve [AUC] = 0.734) and PREDICT29 (AUC = 0.736) progression signatures in malnourished individuals.ConclusionMalnourished individuals display a peripheral immune response profile reflective of increased inflammation and a concomitant increased expression of risk signatures predicting progression to TB. With validation in prospective clinical cohorts, TB risk biomarkers have the potential to identify malnourished LTBI for targeted therapy.

521MO Efficacy of pembrolizumab monotherapy (PM) for advanced clear cell gynaecological cancer (CCGC): Phase II PEACOCC trial

Advanced CCGC has poor prognosis with 2nd line objective response rates (ORR) to standard chemotherapy ranging from 0-8%. As PD-L1 expression, PD-1+ tumour infiltrating lymphocytes, pro-inflammatory cytokine signalling, high tumour mutational burden and preliminary clinical activity with PD-1 inhibitors are described in CCGC, we investigated PM for advanced CCGC. PEACOCC is a Phase II, multicentre, single arm, academic, UK trial in patients (pts) with advanced CCGC who had ≥1 prior line of chemotherapy with progression (PD) at study entry. Pembrolizumab 200mg iv q21 days was given until PD (RECIST v1.1), unacceptable adverse event (AE), 2 years (y) PM reached, patient or clinician decision. The primary endpoint was progression-free survival (PFS) rate at 12 weeks (w) (H0≤15%; H1≥33%; 5% 1-sided a; 90% power). Secondary endpoints included ORR, duration of response (DOR), PFS, overall survival (OS) and safety. From 4/3/19–20/10/21 49pts were enrolled, 48 evaluable. Median age 58.5y (32–77y), ECOG 0/1 54.2%/45.8%, 85.4% ovarian CCGC. Median prior lines systemic therapy 2 (1–6); 19pts (39.6%) had received anti-angiogenic therapy. 42pts completed median 4 cycles PM (1–25), 6pts (12.5%) continue PM. 16.7% pts had Grade (G)3 treatment-related AE (TRAE) of hyperthyroidism (n=2), acute kidney injury (AKI), raised alanine aminotransferase, raised alkaline phosphatase, anaemia, encephalitis and diabetic ketoacidosis (DKA). There were no G4 or G5 TRAE. 3pts (6.3%) discontinued PM due to TRAEs. The PFS rate at 12w was 43.8% (90%CI:31.5-56.6) exceeding the pre-stated lower bound of 15%. Best ORR was 25.0% (90%CI:15.1-37.3) [1 complete, 11 partial], with 1y DOR rate 47.7% (95%CI:14.1-75.6). After a median follow-up of 2.1y, median PFS was 12.2w (95%CI:5.9-32.9) and median OS 71.0w (95%CI:29.1-137.6). The PEACOCC trial indicates that PM is a highly effective therapy in heavily pre-treated pts with advanced CCGC: 43.8% pts were alive and progression-free at 12w. Furthermore, clinical outcomes were durable with limited toxicity. These promising results justify consideration of pembrolizumab monotherapy as a new standard-of-care for advanced CCGC.

Neuroinflammation in Alzheimer's Disease: Current Progress in Molecular Signaling and Therapeutics.

Alzheimer's disease, a neurodegenerative disease with amyloid beta accumulation as a major hallmark, has become a dire global health concern as there is a lack of clear understanding of the causative agent. It is a major cause of dementia which is increasing exponentially with age. Alzheimer's disease is marked by tau hyperphosphorylation and amyloid beta accumulation that robs people of their memories. Amyloid beta deposition initiated a spectrum of microglia-activated neuroinflammation, and microglia and astrocyte activation elicited expressions of various inflammatory and anti-inflammatory cytokines. Neuroinflammation is one of the cardinal features of Alzheimer's disease. Pro-inflammatory cytokine signaling plays multifarious roles in neurodegeneration and neuroprotection. Induction of proinflammatory signaling leads to discharge of immune mediators which affect functions of neurons and cause cell death. Sluggish anti-inflammatory system also contributes to neuroinflammation. Numerous pathways like NFκB, p38 MAPK, Akt/mTOR, caspase, nitric oxide, and COX are involved in triggering brain immune cells like astrocytes and microglia to secrete inflammatory cytokines such as tumor necrosis factor, interleukins, and chemokines and participate in Alzheimer's disease pathology. PPAR-γ agonists tend to boost the phagocytosis of amyloid beta and decrease the inflammatory cytokine IL-1β. Recent findings suggest the cross-link between gut microbiota and neuroinflammation contributing in AD which has been explained in this study. The role of cellular, molecular pathways and involvement of inflammatory mediators in neuroinflammation has also been described; targeting them could be a potential therapeutic strategy for treatment of AD.

Porphyromonas gingivalis and dental stem cells crosstalk amplify inflammation and bone loss in the periodontitis niche.

Periodontitis is the sixth most prevalent disease, and almost 3.5 billion people are affected globally by dental caries and periodontal diseases. The microbial shift from a symbiotic microbiota to a dysbiotic microbiota in the oral cavity generally initiates periodontal disease. Pathogens in the periodontal microenvironment interact with stem cells to modulate their regenerative potential. Therefore, this review focuses on the interaction between microbes and stem cells in periodontitis conditions. Microbes direct dental stem cells to secrete a variety of pro-inflammatory cytokines and chemokines, which increase the inflammatory burden in the damaged periodontal tissue, which further aggravates periodontitis. Microbial interaction also decreases the osteogenic differentiation potential of dental stem cells by downregulating alkaline phosphatase, runt-related transcription factor 2, type 1 collagen, osteocalcin, osteopontin, and so on. Microbe and stem cell interaction amplifies pro-inflammatory cytokine signaling in the periodontitis niche, decreasing the osteogenic commitment of dental stem cells. A clear understanding of microbial stem cell interactions is crucial in designing regenerative therapies using stem cells in the management of periodontitis.

Melatonin: Regulation of Viral Phase Separation and Epitranscriptomics in Post-Acute Sequelae of COVID-19.

The relentless, protracted evolution of the SARS-CoV-2 virus imposes tremendous pressure on herd immunity and demands versatile adaptations by the human host genome to counter transcriptomic and epitranscriptomic alterations associated with a wide range of short- and long-term manifestations during acute infection and post-acute recovery, respectively. To promote viral replication during active infection and viral persistence, the SARS-CoV-2 envelope protein regulates host cell microenvironment including pH and ion concentrations to maintain a high oxidative environment that supports template switching, causing extensive mitochondrial damage and activation of pro-inflammatory cytokine signaling cascades. Oxidative stress and mitochondrial distress induce dynamic changes to both the host and viral RNA m6A methylome, and can trigger the derepression of long interspersed nuclear element 1 (LINE1), resulting in global hypomethylation, epigenetic changes, and genomic instability. The timely application of melatonin during early infection enhances host innate antiviral immune responses by preventing the formation of “viral factories” by nucleocapsid liquid-liquid phase separation that effectively blockades viral genome transcription and packaging, the disassembly of stress granules, and the sequestration of DEAD-box RNA helicases, including DDX3X, vital to immune signaling. Melatonin prevents membrane depolarization and protects cristae morphology to suppress glycolysis via antioxidant-dependent and -independent mechanisms. By restraining the derepression of LINE1 via multifaceted strategies, and maintaining the balance in m6A RNA modifications, melatonin could be the quintessential ancient molecule that significantly influences the outcome of the constant struggle between virus and host to gain transcriptomic and epitranscriptomic dominance over the host genome during acute infection and PASC.

Plasma Cytokines and Birth Weight as Biomarkers of Vaccine-Induced Humoral Responses in Piglets.

Failure to mount an effective immune response to vaccination leaves individuals at risk for infection and can compromise herd immunity. Vaccine unresponsiveness can range from poor responses “low responders” to a failure to seroconvert “non-responders.” Biomarkers of vaccine unresponsiveness, particularly those measured at the time of vaccination, could facilitate more strategic vaccination programs. We previously reported that pro-inflammatory cytokine signaling within peripheral blood mononuclear cells, elevated plasma interferon-gamma (IFNγ), and low birth weight correlated with vaccine-induced serum IgG titers in piglets that were below the threshold of detectable seroconversion (vaccine non-responders). These observations suggested that plasma IFNγ concentration and birth weight might serve as pre-vaccination biomarkers of vaccine unresponsiveness. To test this hypothesis, piglets (n = 67) from a different production facility were vaccinated with the same commercial Mycoplasma hyopneumoniae bacterin (RespiSure-One) to determine if there was a consistent and significant association between vaccine-induced serum IgG titers and either plasma cytokine concentrations or birth weight. All piglets seroconverted following vaccination with significantly less variability in vaccine-induced serum IgG titers than observed in the previous vaccine trial. Piglets exhibited highly variable birth weights and plasma cytokine concentrations prior to vaccination, but there were no significant associations (p > 0.05) between these variables and vaccine-induced serum IgG titers. There were significant (p < 0.001) differences in plasma IFNγ concentrations among individual litters (n = 6), and plasma IFNγ concentrations decreased in all pigs from birth to 63-days of age. One of the six litters (n = 11 piglets) exhibited significantly elevated plasma IFNγ concentrations during the first 3 weeks of life (p < 0.001) and at the time of vaccination (p < 0.01). This litter, however, had similar vaccine-induced serum IgG titers when compared to the other piglets in this study. Collectively the two studies indicate that while plasma cytokines and birth weight can be associated with vaccine non-responsiveness, their temporal and individual variation, as well as the complexity of the vaccine responsiveness phenotype, make them inconsistent biomarkers for predicting the less extreme phenotype of vaccine low responders.

Upper airway gene expression shows a more robust adaptive immune response to SARS-CoV-2 in children

Unlike other respiratory viruses, SARS-CoV-2 disproportionately causes severe disease in older adults whereas disease burden in children is lower. To investigate whether differences in the upper airway immune response may contribute to this disparity, we compare nasopharyngeal gene expression in 83 children (<19-years-old; 38 with SARS-CoV-2, 11 with other respiratory viruses, 34 with no virus) and 154 older adults (>40-years-old; 45 with SARS-CoV-2, 28 with other respiratory viruses, 81 with no virus). Expression of interferon-stimulated genes is robustly activated in both children and adults with SARS-CoV-2 infection compared to the respective non-viral groups, with only subtle distinctions. Children, however, demonstrate markedly greater upregulation of pathways related to B cell and T cell activation and proinflammatory cytokine signaling, including response to TNF and production of IFNγ, IL-2 and IL-4. Cell type deconvolution confirms greater recruitment of B cells, and to a lesser degree macrophages, to the upper airway of children. Only children exhibit a decrease in proportions of ciliated cells, among the primary targets of SARS-CoV-2, upon infection. These findings demonstrate that children elicit a more robust innate and especially adaptive immune response to SARS-CoV-2 in the upper airway that likely contributes to their protection from severe disease in the lower airway.

P1526: AUTOIMMUNE HEMOLYTIC ANEMIAS: RELATIONSHIP BETWEEN SINGLE NUCLEOTIDE POLYMORPHISMS OF CYTOKINE GENES AND CLINICAL/HEMATOLOGICAL PARAMETERS

Background: Autoimmune hemolytic anemia (AIHA) is highly heterogeneous and involves several pathogenic mechanisms including autoantibody production, complement activation, and cytokine dysregulation. Hb levels at baseline and the degree of bone marrow compensation are the main predictors of outcome. The contribution of high, intermediate, and low producer genetic profile cytokine genes may give further insights into the pathogenesis of the disease. Aims: To evaluate the frequency of cytokine single nucleotide polymorphisms (SNPs) in warm (wAIHA) and cold agglutinin disease (CAD) and their relationship with clinical and hematological parameters, including the degree of anemia, treatment refractoriness and number of therapies lines. Methods: We investigated SNPs of five cytokine genes [tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma, transforming growth factor (TGF)-beta, interleukin (IL)-6 and IL-10] in 72 Italian warm AIHA patients, using the Cytokine Tray (One Lambda, Canoga Park, CA) according to the manufacturer’s recommendations. Allele and genotype frequency results were compared to those described in the Italian population by Uboldi de Capei et al. (2003) and Poli et al. (2002). Results: One hundred and twenty AIHA patients have been evaluated: median age 63 years (range 17-90), classified as wAIHA (n=75) and CAD (n=45). Demographic and hematological characteristics of patients are described in Figure 1A. Regarding cytokine SNP frequencies (Figure 1B), we observed that the TNF-alpha-308 high producers genotypes (G/G and G/A) were significantly more frequent in AIHA patients (37% vs 16%, p=0.0003 or 37% vs 23%, p=0.01) and in wAIHA (43% vs 16%, p<0.0001 or 43% vs 23%, p=0.0014) compared to controls. No differences were observed in CAD patients versus controls. Likewise, no significant differences between AIHA (including wAIHA and CAD subtypes) and controls were noted regarding other cytokine SNPs. Concerning the clinical severity at presentation, the frequency of TGF-beta (82% vs 58%, p=0.01) and IL-10 (17% vs 5%, p=0.03) high producers was higher in patients with Hb ≤7.5 g/dL (median of all AIHA) versus those with less severe presentation. Similarly, the frequency of high/intermediate IFN-gamma producers was higher within the group with Hb levels ≤7.5 g/dL at onset (75% vs 58%, p=0.03). No characteristic patterns were observed regarding IL-6 and TNF-alpha. Finally, the patterns of TNF-alpha and TGF-beta production were differently distributed considering the number of therapy lines: the first both in all AIHA and in CAD patients (p=0.004 and p=0.01, respectively), the second only in CAD patients (p=0.03). No differences in genotype frequencies of cytokine SNPs were observed regarding the number of relapses. Image:Summary/Conclusion: These preliminary data show that AIHA patients display a more pro-inflammatory cytokine signature at molecular level (TNF-alpha high producer genotype) as compared to the Italian control population. Moreover, cytokine SNPs were also associated with clinical severity at onset, and requirement of multiple treatments.

Signaling differences in peripheral blood mononuclear cells of high and low vaccine responders prior to, and following, vaccination in piglets

Individual variability in responses to vaccination can result in vaccinated subjects failing to develop a protective immune response. Vaccine non-responders can remain susceptible to infection and may compromise efforts to achieve herd immunity. Biomarkers of vaccine unresponsiveness could aid vaccine research and development as well as strategically improve vaccine administration programs. We previously vaccinated piglets (n = 117) against a commercial Mycoplasma hyopneumoniae vaccine (RespiSure-One) and observed in low vaccine responder piglets, as defined by serum IgG antibody titers, differential phosphorylation of peptides involved in pro-inflammatory cytokine signaling within peripheral blood mononuclear cells (PBMCs) prior to vaccination, elevated plasma interferon-gamma concentrations, and lower birth weight compared to high vaccine responder piglets. In the current study, we use kinome analysis to investigate signaling events within PBMCs collected from the same high and low vaccine responders at 2 and 6 days post-vaccination. Furthermore, we evaluate the use of inflammatory plasma cytokines, birthweight, and signaling events as biomarkers of vaccine unresponsiveness in a validation cohort of high and low vaccine responders. Differential phosphorylation events (FDR < 0.05) within PBMCs are established between high and low responders at the time of vaccination and at six days post-vaccination. A subset of these phosphorylation events were determined to be consistently differentially phosphorylated (p < 0.05) in the validation cohort of high and low vaccine responders. In contrast, there were no differences in birth weight (p > 0.5) and plasma IFNγ concentrations at the time of vaccination (p > 0.6) between high and low responders within the validation cohort. The results in this study suggest, at least within this study population, phosphorylation biomarkers are more robust predictors of vaccine responsiveness than other physiological markers.

Genetic Knock-out of TNFR1 and TNFR2 in a JAK2-V617F Polycythemia Vera Mouse Model.

Genetic Knock-out of TNFR1 and TNFR2 in a JAK2-V617F Polycythemia Vera Mouse Model.

Pathophysiological Commonality Between Irritable Bowel Syndrome and Metabolic Syndrome: Role of Corticotropin-releasing Factor-Toll-like Receptor 4-Proinflammatory Cytokine Signaling.

Irritable bowel syndrome (IBS) displays chronic abdominal pain with altered defecation. Most of the patients develop visceral hypersensitivity possibly resulting from impaired gut barrier and altered gut microbiota. We previously demonstrated that colonic hyperpermeability with visceral hypersensitivity in animal IBS models, which is mediated via corticotropin-releasing factor (CRF)–Toll-like receptor 4 (TLR4)–proinflammatory cytokine signaling. CRF impairs gut barrier via TLR4. Leaky gut induces bacterial translocation resulting in dysbiosis, and increases lipopolysaccharide (LPS). Activation of TLR4 by LPS increases the production of proinflammatory cytokines, which activate visceral sensory neurons to induce visceral hypersensitivity. LPS also activates CRF receptors to further increase gut permeability. Metabolic syndrome (MS) is a cluster of cardiovascular risk factors, including insulin resistance, obesity, dyslipidemia, and hypertension, and recently several researchers suggested the possibility that impaired gut barrier and dysbiosis with low-grade systemic inflammation are involved in MS. Moreover, TLR4–proinflammatory cytokine contributes to the development of insulin resistance and obesity. Thus, the existence of pathophysiological commonality between IBS and MS is expected. This review discusses the potential mechanisms of IBS and MS with reference to gut barrier and microbiota, and explores the possibility of existence of a pathophysiological link between these diseases with a focus on CRF, TLR4, and proinflammatory cytokine signaling. We also review epidemiological data supporting this possibility, and discuss the potential of therapeutic application of the drugs used for MS to IBS treatment. This notion may pave the way for exploring novel therapeutic approaches for these disorders.

Peripheral apelin mediates visceral hypersensitivity and impaired gut barrier in a rat irritable bowel syndrome model

Growing evidence indicates that visceral hypersensitivity and impaired gut barrier play an important role in the pathophysiology of irritable bowel syndrome (IBS). In animal models, these changes are known to be mediated via corticotropin-releasing factor (CRF)-Toll like receptor 4 (TLR4)-proinflammatory cytokine signaling. Apelin, an endogenous ligand of APJ, was reported to modulate CRF-induced enhanced colonic motility. In this context, we hypothesized that apelin also modulates visceral sensation and gut barrier, and tested this hypothesis. We measured visceral pain threshold in response to colonic balloon distention by abdominal muscle contractions assessed by electromyogram in rats. Colonic permeability was estimated by quantifying the absorbed Evans blue in colonic tissue. Intraperitoneal (ip) administration of [Ala13]-apelin-13, an APJ antagonist, blocked lipopolysaccharide (LPS)- or CRF-induced visceral hypersensitivity and colonic hyperpermeability (IBS model) in a dose-response manner. These inhibitory effects were blocked by compound C, an AMPK inhibitor, NG-nitro-L-arginine methyl ester, a nitric oxide (NO) synthesis inhibitor or naloxone in the LPS model. On the other hand, ip [Pyr1]-apelin-13, an APJ agonist, caused visceral hypersensitivity and colonic hyperpermeability, and these effects were reversed by astressin, a CRF receptor antagonist, TAK-242, a TLR4 antagonist or anakinra, an interleukin-1 receptor antagonist. APJ system modulated CRF-TLR4-proinflammatory cytokine signaling to cause visceral hypersensitivity and colonic hyperpermeability. APJ antagonist blocked these GI changes in IBS models, which were mediated via AMPK, NO and opioid signaling. Apelin may contribute to the IBS pathophysiology, and the inhibition of apelinergic signaling may be a promising therapeutic option for IBS.