Increased SLAMF7+CD8+ T cells are associated with the pathogenesis of experimental autoimmune pancreatitis in mice.

BACKGROUND: IgG4-related autoimmune pancreatitis (AIP) is considered to be a T cell-mediated autoimmune disease. However, CD8+ T cells have only received brief mention, and have yet to be completely studied. The study aimed to investigate the expression of signaling lymphocytic activation molecule family 7 (SLAMF7) on CD8+ T cells and the features of SLAMF7+CD8+ T cells in MRL/Mp mice with AIP. METHODS: A murine model of AIP was established by intraperitoneal injection with polyinosinic:polycytidylic acid (poly I:C) for 8 weeks. Dexamethasone treatment was daily administrated for the last 2 weeks during a 6-week course of poly I:C. SLAMF7 expression on CD8+ T cells in the spleen and pancreas was detected by flow cytometry. Granzyme B (GZMB) and cytokines including IFN-γ, TNF-α, and IL-2, were monitored in an in vitro T cell activation assay. Dexamethasone suppression assays were performed to downregulate SLAMF7 expression on T cells upon T cell receptor stimulation. RESULTS: AIP in MRL/Mp mice was induced by repeated intraperitoneal administration of poly I:C and CD8+ T cells were increased in the inflamed pancreas. SLAMF7+CD8+ T cells were elevated in the spleen and pancreas of AIP mice. SLAMF7+CD8+ T subsets produced more GZMB, IFN-γ, TNF-α and IL-2 than SLAMF7-CD8+ T subsets. Dexamethasone treatment ameliorated pancreatic inflammatory and fibrosis of AIP. Dexamethasone could downregulate SLAMF7+CD8+ T cells and reduce GZMB, IFN-γ and TNF-α levels both in vitro and in vivo. CONCLUSIONS: Increased SLAMF7+CD8+ T cells exhibit enhanced cytotoxicity and cytokines secretion capacity, which may be involved in the pathogenesis of AIP.

Natural killer cell-mediated contact dermatitis-like reaction induced by treatment with TLR3 ligand poly(I:C).

INTRODUCTION: Poly(I:C) is recognised by endosomal Toll-like receptor 3 (TLR3) and activates cytotoxic CD8(+) lymphocytes and natural killer (NK) cells. It has been shown that the viral TLR3 agonist induces robust and long-lasting T-cell-mediated responses. In addition, TLR3 modulates the contact hypersensitivity reaction. OBJECTIVE: This study aimed to determine whether poly(I:C) injection can induce NK-mediated hapten reactivity in mice. METHODS: Mice were treated with poly(I:C), and their response to dinitrofluorobenzene hapten was measured by assessing ear swelling and serum interferon gamma (IFN-γ) production. Adoptive cell transfer and cell sorting were used to investigate the mechanism of the reaction, and the phenotype of poly(I:C)-activated liver NK cells was determined by flow cytometry analysis. RESULTS: The results showed that poly(I:C) administration increased ear swelling, serum IFN-γ levels and the response to hapten in both immunocompetent and T- and B-cell-deficient mice. Only liver poly(I:C)-activated DX5(+) NK cells were able to transfer reactivity to hapten into a naive recipient. Induction of liver NK cells after poly(I:C) administration was TLR3/TRIF- and IFN-γ-dependent, interleukin 12-independent, and not modulated by MyD88. CONCLUSION: This study provides new insights into how poly(I:C) stimulates NK-mediated reactivity to hapten and suggests that liver NK cells may modulate the immune response to non-pathogenic factors during viral infection.

Maternal behaviours and adult offspring behavioural deficits are predicted by maternal TNFα concentration in a rat model of neurodevelopmental disorders.

Exposure to inflammatory stressors during fetal development is a major risk factor for neurodevelopmental disorders (NDDs) in adult offspring. Maternal immune activation (MIA), induced by infection, causes an acute increase in pro-inflammatory cytokines which can increase the risk for NDDs directly by inducing placental and fetal brain inflammation, or indirectly through affecting maternal care behaviours thereby affecting postnatal brain development. Which of these two potential mechanisms dominates in increasing offspring risk for NDDs remains unclear. Here, we show that acute systemic maternal inflammation induced by the viral mimetic polyinosinic:polycytidylic acid (poly I:C) on gestational day 15 of rat pregnancy affects offspring and maternal behaviour, offspring cognition, and expression of NDD-relevant genes in the offspring brain. Dams exposed to poly I:C elicited an acute increase in the pro-inflammatory cytokine tumour necrosis factor (TNF; referred to here as TNFα), which predicted disruption of key maternal care behaviours. Offspring of poly I:C-treated dams showed early behavioural and adult cognitive deficits correlated to the maternal TNFα response, but, importantly, not with altered maternal care. We also found interacting effects of sex and treatment on GABAergic gene expression and DNA methylation in these offspring in a brain region-specific manner, including increased parvalbumin expression in the female adolescent frontal cortex. We conclude that the MIA-induced elevation of TNFα in the maternal compartment affects fetal neurodevelopment leading to altered offspring behaviour and cognition. Our results suggest that a focus on prenatal pathways affecting fetal neurodevelopment would provide greater insights into the mechanisms underpinning the TNFα-mediated genesis of altered offspring behaviour and cognition following maternal inflammation.

Maternal immune activation leads to defective brain-blood vessels and intracerebral hemorrhages in male offspring.

Intracerebral hemorrhages are recognized risk factors for neurodevelopmental disorders and represent early biomarkers for cognitive dysfunction and mental disability, but the pathways leading to their occurrence are not well defined. We report that a single intrauterine exposure of the immunostimulant Poly I:C to pregnant mice at gestational day 9, which models a prenatal viral infection and the consequent maternal immune activation, induces the defective formation of brain vessels and causes intracerebral hemorrhagic events, specifically in male offspring. We demonstrate that maternal immune activation promotes the production of the TGF-ß1 active form and the consequent enhancement of pSMAD1-5 in males' brain endothelial cells. TGF-ß1, in combination with IL-1ß, reduces the endothelial expression of CD146 and claudin-5, alters the endothelium-pericyte interplay resulting in low pericyte coverage, and increases hemorrhagic events in the adult offspring. By showing that exposure to Poly I:C at the beginning of fetal cerebral angiogenesis results in sex-specific alterations of brain vessels, we provide a mechanistic framework for the association between intragravidic infections and anomalies of the neural vasculature, which may contribute to neuropsychiatric disorders.

Maternal immune activation accelerates puberty initiation and alters mechanical allodynia in male and female C57BL6/J mice.

The mechanisms that link maternal immune activation (MIA) with the onset of neurodevelopmental disorders remain largely unclear. Accelerated puberty is also associated with a heightened risk for psychopathology in later life, but there is a dearth of evidence on the impacts of maternal infection on pubertal timing. We examined the effects of MIA on reproductive development, mechanical allodynia, and sensorimotor gating in juvenile, adolescent, and adult male and female mice. Moreover, we investigated hypothalamic neural markers associated with the reproductive and stress axes. Finally, we tested the mitigating effects of environmental enrichment (EE), which has clinical relevancy in human rehabilitation settings. Our results show that administration of polyinosinic-polycytidylic acid (poly(I:C)) on gestational day 12.5 led to early preputial separation, vaginal openings, and age of first estrus in offspring. MIA exposure altered pain sensitivity across development and modestly altered prepulse inhibition. The downregulation of Nr3c1 and Oprk mRNA in the hypothalamus of juvenile mice suggests that MIA's effects may be mediated through disruption of hypothalamic-pituitary-adrenal axis activity. In contrast, life-long housing with EE rescued many of these MIA-induced consequences. Overall, our findings suggest that accelerated puberty may be associated with the deleterious effects of infection during pregnancy and the onset of psychopathology.

Cysteine Peptidase Cathepsin X as a Therapeutic Target for Simultaneous TLR3/4-mediated Microglia Activation.

Microglia are resident macrophages in the central nervous system that are involved in immune responses driven by Toll-like receptors (TLRs). Microglia-mediated inflammation can lead to central nervous system disorders, and more than one TLR might be involved in these pathological processes. The cysteine peptidase cathepsin X has been recognized as a pathogenic factor for inflammation-induced neurodegeneration. Here, we hypothesized that simultaneous TLR3 and TLR4 activation induces synergized microglia responses and that these phenotype changes affect cathepsin X expression and activity. Murine microglia BV2 cells and primary murine microglia were exposed to the TLR3 ligand polyinosinic-polycytidylic acid (poly(I:C)) and the TLR4 ligand lipopolysaccharide (LPS), individually and simultaneously. TLR3 and TLR4 co-activation resulted in increased inflammatory responses compared to individual TLR activation, where poly(I:C) and LPS induced distinct patterns of proinflammatory factors together with different patterns of cathepsin X expression and activity. TLR co-activation decreased intracellular cathepsin X activity and increased cathepsin X localization at the plasma membrane with concomitant increased extracellular cathepsin X protein levels and activity. Inhibition of cathepsin X in BV2 cells by AMS36, cathepsin X inhibitor, significantly reduced the poly(I:C)- and LPS-induced production of proinflammatory cytokines as well as apoptosis. Additionally, inhibiting the TLR3 and TLR4 common signaling pathway, PI3K, with LY294002 reduced the inflammatory responses of the poly(I:C)- and LPS-activated microglia and recovered cathepsin X activity. We here provide evidence that microglial cathepsin X strengthens microglia activation and leads to subsequent inflammation-induced neurodegeneration. As such, cathepsin X represents a therapeutic target for treating neurodegenerative diseases related to excess inflammation.

Gender-specific development of experimental autoimmune cholangitis induced by double-stranded RNA.

Here we investigated the gender difference in murine cholangitis resembling human primary biliary cholangitis (PBC) caused by synthetic double-stranded RNA, and underlying hepatic innate immune responses. Female C57Bl/6 mice given repeated injections of polyinosinic-polycytidylic acid (poly I:C) for 24 weeks developed overt cholangitis with positive serum anti-mitochondria-M2 antibody, whereas male mice showed minimal pathological changes without induction in autoantibody. Poly I:C induced hepatic inflammatory cytokines and type-I interferons predominantly in females. Hepatic expression levels of toll-like receptor (TLR) 3 and melanoma differentiation-associated protein (MDA) 5 were equivalent in both genders; however, both mRNA and protein levels of retinoic acid-inducible gene (RIG)-I were nearly doubled in female livers. Following 4-week injections of poly I:C, not only hepatic RIG-I, but also TLR3 and MDA5 showed female-predominance. Moreover, hepatic RIG-I levels were 25% lower in ovariectomized mice, whereas supplementation of 17 ß-estradiol enhanced hepatic RIG-I expression, as well as cytokine induction. These results clearly indicate that hepatic RIG-I expression is potentiated by estrogen, and triggers gender-dependent hepatic innate immune response against double-stranded RNA, which most likely play a pivotal role in the pathogenesis of autoimmune cholangiopathies including PBC.

Co-Silencing of Tissue Transglutaminase-2 and Interleukin-15 Genes in a Celiac Disease Mimetic Mouse Model Using a Nanoparticle-in-Microsphere Oral System.

Celiac disease is a chronic inflammatory condition characterized by activation of the immune system in response to deamidation of gluten peptides brought about by tissue transglutaminase-2 (TG2). Overexpression of interleukin-15 (IL-15) in the intestinal epithelium and the lamina propria leads to the dysregulation of the immune system, leading to epithelial damage. The goal of this study was to develop an RNA interference therapeutic strategy for celiac disease using a combination of TG2 and IL-15 gene silencing in the inflamed intestine. TG2 and IL-15 silencing siRNA sequences, along with scrambled control, were encapsulated in a nanoparticle-in-microsphere oral system (NiMOS) and administered in a poly(I:C) mouse model of celiac disease. Single TG2 and IL-15 siRNA therapy and the combination showed effective gene silencing in vivo. Additionally, it was found that IL-15 gene silencing alone and combination in the NiMOS significantly reduced other proinflammatory cytokines. The tissue histopathology data also confirmed a reduction in immune cell infiltration and restoration of the mucosal architecture and barrier function in the intestine upon treatment. Overall, the results of this study show evidence that celiac disease can be potentially treated with an oral microsphere formulation using a combination of TG2 and IL-15 RNA interference therapeutic strategies.

Viral mimetic triggers cerebral arteriopathy in juvenile brain via neutrophil elastase and NETosis.

Stroke is among the top ten causes of death in children but has received disproportionally little attention. Cerebral arteriopathies account for up to 80% of childhood arterial ischemic stroke (CAIS) cases and are strongly predictive of CAIS recurrence and poorer outcomes. The underlying mechanisms of sensitization of neurovasculature by viral infection are undefined. In the first age-appropriate model for childhood arteriopathy-by administration of viral mimetic TLR3-agonist Polyinosinic:polycytidylic acid (Poly-IC) in juvenile mice-we identified a key role of the TLR3-neutrophil axis in disrupting the structural-functional integrity of the blood-brain barrier (BBB) and distorting the developing neurovascular architecture and vascular networks. First, using an array of in-vivo/post-vivo vascular imaging, genetic, enzymatic and pharmacological approaches, we report marked Poly-IC-mediated extravascular leakage of albumin (66kDa) and of a small molecule DiI (∼934Da) and disrupted tight junctions. Poly-IC also enhanced the neuroinflammatory milieu, promoted neutrophil recruitment, profoundly upregulated neutrophil elastase (NE), and induced neutrophil extracellular trap formation (NETosis). Finally, we show that functional BBB disturbances, NETosis and neuroinflammation are markedly attenuated by pharmacological inhibition of NE (Sivelestat). Altogether, these data reveal NE/NETosis as a novel therapeutic target for viral-induced cerebral arteriopathies in children.

Trial to evaluate the immunogenicity and safety of a melanoma helper peptide vaccine plus incomplete Freund's adjuvant, cyclophosphamide, and polyICLC (Mel63).

BACKGROUND: Peptide vaccines designed to stimulate melanoma-reactive CD4+ T cells can induce T cell and antibody (Ab) responses, associated with enhanced overall survival. We hypothesized that adding toll-like receptor 3 agonist polyICLC to an incomplete Freund's adjuvant (IFA) would be safe and would support strong, durable CD4+ T cell and Ab responses. We also hypothesized that oral low-dose metronomic cyclophosphamide (mCy) would be safe, would reduce circulating regulatory T cells (T-regs) and would further enhance immunogenicity. PARTICIPANTS AND METHODS: An adaptive design based on toxicity and durable CD4+ T cell immune response (dRsp) was used to assign participants with resected stage IIA-IV melanoma to one of four study regimens. The regimens included a vaccine comprising six melanoma peptides restricted by Class II MHC (6MHP) in an emulsion with IFA alone (Arm A), with IFA plus systemic mCy (Arm B), with IFA+ local polyICLC (Arm C), or with IFA+ polyICLC+ mCy (Arm D). Toxicities were recorded (CTCAE V.4.03). T cell responses were measured by interferon γ ELIspot assay ex vivo. Serum Ab responses to 6MHP were measured by ELISA. Circulating T-regs were assessed by flow cytometry. RESULTS: Forty-eight eligible participants were enrolled and treated. Early data on safety and dRsp favored enrollment on arm D. Total enrollment on Arms A-D were 3, 7, 6, and 32, respectively. Treatment-related dose-limiting toxicities (DLTs) were observed in 1/7 (14%) participants on arm B and 2/32 (6%) on arm D. None exceeded the 25% DLT threshold for early closure to enrollment for any arm. Strong durable T cell responses to 6MHP were detected ex vivo in 0%, 29%, 67%, and 47% of participants on arms A-D, respectively. IgG Ab responses were greatest for arms C and D. Circulating T-regs frequencies were not altered by mCy. CONCLUSIONS: 6MHP vaccines administered with IFA, polyICLC, and mCy were well tolerated. The dRsp rate for arm D of 47% (90% CI 32 to 63) exceeded the 18% (90% CI 11 to 26) rate previously observed with 6MHP in IFA alone. Vaccination with IFA+ polyICLC (arm C) also showed promise for enhancing T cell and Ab responses.

Molecular profiling and functional delineation of peroxiredoxin 3 (HaPrx3) from the big-belly seahorses (Hippocampus abdominalis) and understanding their immunological responses.

Peroxiredoxins (Prxs) are ubiquitously expressed antioxidant proteins that can protect aerobic organisms from oxidative stress. Here, we characterized the HaPrx3 homolog at the molecular level from big-belly seahorse (Hippocampus abdominalis) and analyzed its functional activities. The coding sequence of HaPrx3 consists of 726 bp, which encodes 241 amino acids. The predicted molecular weight and theoretical isoelectric point (pI) of HaPrx3 was 26.20 kDa and 7.04, respectively. Multiple sequence alignments revealed that the arrangements of domains, catalytic triads, dimers, and decamer interfaces of HaPrx3 were conserved among Prx sequences of other organisms. According to the phylogenetic analysis, HaPrx3 is clustered with the teleost Prx3 subclade. The highest transcript level of HaPrx3 was detected in the ovary tissue among fourteen healthy fish tissues. The mRNA levels of HaPrx3 in blood and liver tissues were significantly (P < 0.05) upregulated in response to lipopolysaccharide (LPS), polyinosinic-polycytidylic (poly I:C), Edwardsiella tarda, and Streptococcus iniae, suggesting its involvement in immune responses. Under functional properties, insulin disulfide reduction assay confirmed the oxidoreductase activity of recombinant HaPrx3. A cell viability assay and Hoechst staining indicated cell survival ability and reduction of apoptotic activity, respectively. Moreover, a peroxidase activity assay verified peroxidase activity, while a metal-catalyzed oxidation (MCO) assay indicated the DNA protection ability of HaPrx3. Collectively, it is concluded that HaPrx3 may play a significant role in oxidative stress and immune responses against pathogenic infections in big-belly seahorses.

Sulforaphane suppresses polyinosinic­polycytidylic acid­stimulated release of cytokines, chemokines and MMPs by human corneal fibroblasts.

Viral corneal infection is a common cause of visual impairment and blindness. Polyinosinic­polycytidylic acid, or poly(I:C), is similar to viral double­stranded RNA in structure and has been implicated in the release of a variety of cytokines, chemokines and matrix metalloproteinases (MMPs) by corneal fibroblasts. Sulforaphane (SFN) is an isothiocyanate compound found in cruciferous vegetables. The present study investigated the potential effect of SFN on the poly(I:C)­stimulated release of cytokines, chemokines and MMPs in human corneal fibroblasts (HCFs). ELISA showed that SFN was associated with a time­ and dose­dependent reduction in poly(I:C)­stimulated production of interleukin (IL)­8, chemoattractant protein­1, IL­6, MMP­1 and MMP­3 by HCFs. Western blot analysis indicated that SFN suppressed the function of poly(I:C) by modulating mitogen­activated protein kinases (MAPKs), including p38 and extracellular signal­regulated kinase (ERK), activator protein­1 (AP­1) component c­Jun and the kinase, Akt, and the phosphorylation and degradation of the nuclear factor (NF)­κB inhibitor IκB­α. Immunofluorescence analysis revealed that SFN attenuated the production of poly(I:C)­induced nuclear translocation of the NF­κB p65 subunit. Reverse transcription­quantitative PCR analysis revealed that SFN prevented the poly(I:C)­induced upregulation of Toll­like receptor 3 (TLR3) mRNA expression in HCFs. No significant cytotoxic effect of SFN on HCFs was observed. In summary, SFN attenuated the poly(I:C)­induced production of proinflammatory chemokines, cytokines and MMPs by HCFs, by inhibiting TLR3, MAPK (p38 and ERK), AP­1, Akt and NF­κB signaling. SFN may therefore be a potential novel treatment for viral corneal infection by limiting immune cell infiltration.

Andrographolide sulfate inhibited NF-κB activation and alleviated pneumonia induced by poly I:C in mice.

Pneumonia is a common illness that continues to be the major killer of remaining to be a significant source of morbidity and mortality in the patient population. Many microorganisms cause pneumonia, and now concern is turning to the importance of the cause the new therapies for viral pneumonia. In the current study, we report the effect of andrographolide sulfonate, a water-soluble form of andrographolide (trade name: Xi-Yan-Ping Injection), on poly I: C-induced pneumonia. Andrographolide sulfonate was administrated through intraperitoneal injection to mice with poly I: C-induced pneumonia. Recruitment of airway inflammatory cells, alteration of lung histological induced by Poly I: C were significantly ameliorated by andrographolide sulfonate. The protein levels of pro-inflammatory cytokines in bronchoalveolar fluid (BALF) and serum were reduced by andrographolide sulfonate treatment. The levels of MUC5AC and MUC5B in lung tissue were also suppressed. These results reveal that andrographolide sulfate remarkably alleviated pneumonia induced by poly I:C in mice. Moreover, andrographolide sulfonate markedly inhibited the activation of nuclear factor-κB (NF-κB). Taken together, we demonstrated that andrographolide sulfonate ameliorated poly I: C-induced pneumonia in mice, suggesting the possible use of andrographolide sulfonate for virus-induced pneumonia in clinical.

Increased RNA editing in maternal immune activation model of neurodevelopmental disease.

The etiology of major neurodevelopmental disorders such as schizophrenia and autism is unclear, with evidence supporting a combination of genetic factors and environmental insults, including viral infection during pregnancy. Here we utilized a mouse model of maternal immune activation (MIA) with the viral mimic PolyI:C infection during early gestation. We investigated the transcriptional changes in the brains of mouse fetuses following MIA during the prenatal period, and evaluated the behavioral and biochemical changes in the adult brain. The results reveal an increase in RNA editing levels and dysregulation in brain development-related gene pathways in the fetal brains of MIA mice. These MIA-induced brain editing changes are not observed in adulthood, although MIA-induced behavioral deficits are observed. Taken together, our findings suggest that MIA induces transient dysregulation of RNA editing at a critical time in brain development.

Double-Stranded Structure of the Polyinosinic-Polycytidylic Acid Molecule to Elicit TLR3 Signaling and Adjuvant Activity in Murine Intranasal A(H1N1)pdm09 Influenza Vaccination.

Polyinosinic-polycytidylic acid (PIC) is a potent double-stranded RNA (dsRNA) adjuvant useful in intranasal influenza vaccination. In mice, the intensity and duration of immune responses to PIC correlated with the double-stranded chain length. A rational method to avoid PIC chain extension in PIC production is to use multiple short poly(I) molecules and one long poly(C) molecule for PIC assembly. In this study, we elucidate that a newly developed uPIC100-400 molecule comprising multiple 0.1 kb poly(I) molecules and one 0.4 kb poly(C) molecule effectively enhanced the immune responses in mice, by preventing the challenged viral propagation and inducing hemagglutinin-specific IgA, after intranasal A(H1N1)pdm09 influenza vaccination. Reduced intraperitoneal toxicity of PIC prepared with multiple short poly(I) molecules in mice indicates the widened effective range of uPIC100-400 as an adjuvant. In contrast to uPIC100-400, the PIC molecule comprising multiple 0.05 kb poly(I) molecules failed to elicit mouse mucosal immunity. These results were consistent with TLR3 response but not retinoic acid inducible gene I (RIG-I)-like receptor response in the cell assays, which suggests that the adjuvant effect of PIC in mouse intranasal immunization depends on TLR3 signaling. In conclusion, the double-stranded PIC with reduced toxicity developed in this study would contribute to the development of PIC-adjuvanted vaccines.

Glutaredoxin 2 from big belly seahorse (Hippocampus abdominalis) and its potential involvement in cellular redox homeostasis and host immune responses.

Glutaredoxins are oxidoreductases present in almost all living organisms. They belong to the thioredoxin superfamily and share the thioredoxin structure and catalytic motif. Glutaredoxin 2 has been identified as a mitochondrial protein in vertebrates. In this study, the sequence of Glutaredoxin 2 from Hippocampus abdominalis (HaGrx2) was analyzed by molecular, transcriptional, and functional assays. In-silico analysis revealed that HaGrx2 shows the highest homology with Hippocampus comes, while distinctly cluster with fish Grx2 orthologs. Tissue distribution analysis showed that HaGrx2 is ubiquitously expressed in all tissues tested, and the highest expression was observed in the brain and skin. Significant HaGrx2 transcript modulation was identified in blood and liver upon injecting bacterial and Pathogen Associated Molecular Patterns. The redox activity of HaGrx2 was revealed by Dehydroascorbic reduction and insulin disulfide reduction activity assays. Further, the deglutathionylation activity of 1 nM HaGrx2 was found to be equivalent to that of 0.84 nM HaGrx1. HaGrx2 exhibited antiapoptotic activity against H2O2-induced oxidative stress in FHM cells. Altogether, the results of this study suggest that HaGrx2 plays a role in redox homeostasis and innate immune responses in fish.

Salivary glands require Aurora Kinase B for regeneration after transient innate immune-mediated injury.

Severe, irreversible salivary gland disease and oral dryness is experienced by sufferers of Sjögren's syndrome and those treated with irradiation for head and neck cancer. Therefore, major efforts have been made in the last decade to unravel key molecular signals that can drive salivary gland (SG) regeneration and functional restoration. However, the earliest molecular determinants that accompany SG regeneration remain incompletely defined. The present study examined the initial mitogenic events marking the regenerative response of the murine submandibular gland (SMG), following innate immune-mediated injury. Local intraductal administration of the synthetic double stranded (ds) RNA polyinosinic-polycytidylic acid (poly (I:C)) widely, but transiently, depleted the acinar and progenitor cells, 24 hours post poly (I:C) introduction. While the progenitor and duct cells started to proliferate and expand at 72 hours, the Mist1-positve acinar cells did not re-appear until 96 hours post poly (I:C) injury. The cellular replenishment during regeneration involved significant upregulation of the cell cycle promoter Aurora kinase B (AURKB). AURKB, which is expressed in healthy proliferating and cancerous cells, is a serine/threonine protein kinase, well known to orchestrate key events in cell division and cytokinesis. However, the expression and role of AURKB in regeneration of post mitotic salivary gland cells has not been previously explored. In vivo inhibition of AURKB using the selective inhibitor Barasertib (AZD1152-HQPA) interfered with SMG recovery from the transient, but severe poly (I:C)-mediated injury and cellular depletion. AURKB deficiency during regeneration of the injured tissues: disrupted cell cycle progression, repressed renewal of Mist1-positive acinar cells and prevented recovery of salivary secretion. The knowledge gained in this study may be utilized in the development of therapeutic targets for irreversible salivary gland disease.

A multipeptide vaccine plus toll-like receptor agonists LPS or polyICLC in combination with incomplete Freund's adjuvant in melanoma patients.

BACKGROUND: Cancer vaccines require adjuvants to induce effective immune responses; however, there is no consensus on optimal adjuvants. We hypothesized that toll-like receptor (TLR)3 agonist polyICLC or TLR4 agonist lipopolysaccharide (LPS), combined with CD4 T cell activation, would support strong and durable CD8+ T cell responses, whereas addition of an incomplete Freund's adjuvant (IFA) would reduce magnitude and persistence of immune responses. PATIENTS AND METHODS: Participants with resected stage IIB-IV melanoma received a vaccine comprised of 12 melanoma peptides restricted by Class I MHC (12MP), plus a tetanus helper peptide (Tet). Participants were randomly assigned 2:1 to cohort 1 (LPS dose-escalation) or cohort 2 (polyICLC). Each cohort included 3 subgroups (a-c), receiving 12MP + Tet + TLR agonist without IFA (0), or with IFA in vaccine one (V1), or all six vaccines (V6). Toxicities were recorded (CTCAE v4). T cell responses were measured with IFNγ ELIspot assay ex vivo or after one in vitro stimulation (IVS). RESULTS: Fifty-three eligible patients were enrolled, of which fifty-one were treated. Treatment-related dose-limiting toxicities (DLTs) were observed in 0/33 patients in cohort 1 and in 2/18 patients in cohort 2 (11%). CD8 T cell responses to 12MP were detected ex vivo in cohort 1 (42%) and in cohort 2 (56%) and in 18, 50, and 72% for subgroups V0, V1, and V6, respectively. T cell responses to melanoma peptides were more durable and of highest magnitude for IFA V6. CONCLUSIONS: LPS and polyICLC are safe and effective vaccine adjuvants when combined with IFA. Contrary to the central hypothesis, IFA enhanced T cell responses to peptide vaccines when added to TLR agonists. Future studies will aim to understand mechanisms underlying the favorable effects with IFA. TRIAL REGISTRATION: The clinical trial Mel58 was performed with IRB (#15781) and FDA approval and is registered with Clinicaltrials.gov on April 25, 2012 (NCT01585350). Patients provided written informed consent to participate. Enrollment started on June 24, 2012.

Prenatal immune activation induces age-related alterations in rat offspring: Effects upon NMDA receptors and behaviors.

Prenatal exposure to polyriboinosinic-polyribocytidylic acid (poly I:C) results in psychotic-like behavior in mature rat offspring as well as enduring modifications of glutamatergic excitatory synaptic transmission. However, little is known about the dynamic behavioral and glutamate N-methyl-D-aspartate (NMDA) receptor changes in rat offspring following poly I:C treatment of pregnant dams. In this study, poly I:C was administered to rats intravenously at a dose of 10 mg/kg on gestational day 9 in order to assess changes in behavior and NMDA receptors in offspring over time. Results demonstrate progressive worsening behaviors in adolescents and adults that manifest as increased anxiety, cognitive impairment, and pre-pulse inhibition deficits. Age-related alteration of NMDA receptors in the prefrontal cortex and hippocampus, either total number or distribution, were observed from weaning to adulthood. These results suggest that abnormalities of NMDA receptors occur prior to obvious schizophrenia-like behavioral manifestations. Hence, NMDA receptors may be potential therapeutic targets to prevent disease development during asymptomatic periods of schizophrenia, and may serve as targets for preventive and/or therapeutic strategies for schizophrenia. Further, PSD95, a scaffolding protein that is a component of the NMDA receptor signaling complex, is increased in the hippocampus of adult offspring, when serious behavioral abnormalities emerge. This result suggests that PSD95 may be involved in behavioral abnormalities of schizophrenia.

Epigenome-wide effects of vitamin D on asthma bronchial epithelial cells.

Vitamin D is a nutrient and a hormone with multiple effects on immune regulation and respiratory viral infections, which can worsen asthma and lead to severe asthma exacerbations. We set up a complete experimental and analytical pipeline for ATAC-Seq and RNA-Seq to study genome-wide epigenetic changes in human bronchial epithelial cells of asthmatic subjects, following treatment of these cells with calcitriol (vitamin D3) and Poly (I:C)(a viral analogue). This approach led to the identification of biologically plausible candidate genes for viral infections and asthma, such as DUSP10 and SLC44A1.