Structure Reveals Mechanisms of Viral Suppressors that Intercept a CRISPR RNA-Guided Surveillance Complex.

Genetic conflict between viruses and their hosts drives evolution and genetic innovation. Prokaryotes evolved CRISPR-mediated adaptive immune systems for protection from viral infection, and viruses have evolved diverse anti-CRISPR (Acr) proteins that subvert these immune systems. The adaptive immune system in Pseudomonas aeruginosa (type I-F) relies on a 350 kDa CRISPR RNA (crRNA)-guided surveillance complex (Csy complex) to bind foreign DNA and recruit a trans-acting nuclease for target degradation. Here, we report the cryo-electron microscopy (cryo-EM) structure of the Csy complex bound to two different Acr proteins, AcrF1 and AcrF2, at an average resolution of 3.4 Å. The structure explains the molecular mechanism for immune system suppression, and structure-guided mutations show that the Acr proteins bind to residues essential for crRNA-mediated detection of DNA. Collectively, these data provide a snapshot of an ongoing molecular arms race between viral suppressors and the immune system they target.

Structure Reveals a Mechanism of CRISPR-RNA-Guided Nuclease Recruitment and Anti-CRISPR Viral Mimicry.

Bacteria and archaea have evolved sophisticated adaptive immune systems that rely on CRISPR RNA (crRNA)-guided detection and nuclease-mediated elimination of invading nucleic acids. Here, we present the cryo-electron microscopy (cryo-EM) structure of the type I-F crRNA-guided surveillance complex (Csy complex) from Pseudomonas aeruginosa bound to a double-stranded DNA target. Comparison of this structure to previously determined structures of this complex reveals a ∼180-degree rotation of the C-terminal helical bundle on the "large" Cas8f subunit. We show that the double-stranded DNA (dsDNA)-induced conformational change in Cas8f exposes a Cas2/3 "nuclease recruitment helix" that is structurally homologous to a virally encoded anti-CRISPR protein (AcrIF3). Structural homology between Cas8f and AcrIF3 suggests that AcrIF3 is a mimic of the Cas8f nuclease recruitment helix.

Anti-CRISPR proteins function through thermodynamic tuning and allosteric regulation of CRISPR RNA-guided surveillance complex.

CRISPR RNA-guided detection and degradation of foreign DNA is a dynamic process. Viruses can interfere with this cellular defense by expressing small proteins called anti-CRISPRs. While structural models of anti-CRISPRs bound to their target complex provide static snapshots that inform mechanism, the dynamics and thermodynamics of these interactions are often overlooked. Here, we use hydrogen deuterium exchange-mass spectrometry (HDX-MS) and differential scanning fluorimetry (DSF) experiments to determine how anti-CRISPR binding impacts the conformational landscape of the type IF CRISPR RNA guided surveillance complex (Csy) upon binding of two different anti-CRISPR proteins (AcrIF9 and AcrIF2). The results demonstrate that AcrIF2 binding relies on enthalpic stabilization, whereas AcrIF9 uses an entropy driven reaction to bind the CRISPR RNA-guided surveillance complex. Collectively, this work reveals the thermodynamic basis and mechanistic versatility of anti-CRISPR-mediated immune suppression. More broadly, this work presents a striking example of how allosteric effectors are employed to regulate nucleoprotein complexes.

From a child who IS a problem to a child who HAS a problem: fixed period school exclusions and mental health outcomes from routine outcome monitoring among children and young people attending school counselling.

BACKGROUND: Exclusion from school is a disciplinary tool with an increasingly recognised relationship to poor mental health among children and young people. We explored the relationship between mental health and school exclusion for a cohort of children and young people receiving one to one counselling. METHOD: We analysed routinely collected data from a diverse UK sample of children and young people aged between four and 16 years old and receiving school-based counselling (n = 6712 students from 308 primary and 61 secondary schools). Fixed period school exclusion rates (number of sessions) were compared between the academic year before and the academic year in which the child attended counselling. Mental health (Strengths and Difficulties Questionnaire) was compared at baseline and at the end of the intervention (after between 16-22 counselling sessions depending on the phase of education). RESULTS: Despite more complex and severe initial difficulties, and facing greater adversity, children and young people who experienced school exclusion prior to counselling demonstrated a significant reduction in subsequent sessions of school exclusion in the academic year that the counselling took place (from two full school weeks to half a school week). Moreover, over 74% of the students had fewer reported exclusions and more than half (56.14%) did not have any further subsequent exclusions. They also had better mental health measured by the teacher reported SDQ (pre-intervention M = 18.94, SD = 6.83 vs. postintervention M = 15.67, SD = 7.56, t(310) = 8.23, p < .001) or by the parents (pre-intervention M = 18.09, SD = 6.42 vs. postintervention M = 14.0, SD = 6.99, t(171) = 7.71, p < .001). CONCLUSIONS: School-based mental health interventions may positively influence educational engagement as well as mental health. Providers should, therefore, monitor both to explore the impact of their interventions. The identification of poor mental health may alter staff perceptions and management of challenging pupils, which future studies should explore.

Impact of counselling provision in primary schools on child and adolescent mental health service referral rates: a longitudinal observational cohort study.

BACKGROUND: In the United Kingdom, schools play an increasingly important role in supporting young peoples' mental health. While there is a growing evidence base to support the effectiveness of school-based interventions, less is known about how these provisions impact on local Child and Adolescent Mental Health Service (CAMHS) referral rates. There is a concern that an increase in school-based provision might lead to an increase in CAMHS referrals and overwhelm services. We aimed to examine the longitudinal association between Place2Be counselling provision in primary schools on CAMHS referral rates in South London. METHOD: This was a retrospective cohort study using linked data from the National Pupil Database (NPD) and CAMHS referrals to the South London and Maudsley's NHS Foundation Trust (SLaM) identified through the Clinical Record Interactive Search (CRIS) tool. The cohort included a total of 285 state-maintained primary schools in four London boroughs for the academic years of 2007-2012. During the study period, 23 of these schools received school-based mental health provision from Place2Be. The primary outcome was the incident rate ratio (IRR) of school-level accepted CAMHS referrals in 2012/13 in schools with, or without, Place2Be provision. RESULTS: There was no significant association between elevated rates of CAMHS referral and Place2Be provision, even after comprehensive adjustment for school-level and pupil characteristics (IRR 0.91 (0.67-1.23)). School-level characteristics, including higher proportion of white-British pupils (IRR 1.009 (1.002-1.02)), medical staff ratio (IRR 6.49 (2.05-20.6)) and poorer Ofsted school inspection ratings (e.g. IRR 1.58 (1.06-2.34) for 'Requires Improvement' vs. 'Outstanding') were associated with increased CAMHS referral rates. CONCLUSIONS: Place2Be provision did not result in increased specialist mental health referrals; however, other school-level characteristics did. Future research should investigate pupils' Place2Be clinical outcomes, as well the outcomes of individuals referred to CAMHS to better understand which needs are being met by which services.

Longer-term effects of school-based counselling in UK primary schools.

Children's mental health is deteriorating while access to child and adolescent mental health services is decreasing. Recent UK policy has focused on schools as a setting for the provision of mental health services, and counselling is the most common type of school-based mental health provision. This study examined the longer-term effectiveness of one-to-one school-based counselling delivered to children in UK primary schools. Data were drawn from a sample of children who received school-based counselling in the UK in the 2015/16 academic year, delivered by a national charitable organisation. Mental health was assessed at baseline, immediately post-intervention, and approximately 1 year post-intervention, using the Strengths and Difficulties Questionnaire (SDQ) completed by teachers and parents. Paired t tests compared post-intervention and follow-up SDQ total difficulties scores with baseline values. Propensity score matching was then used to identify a comparator group of children from a national population survey, and linear mixed effects models compared trajectories of SDQ scores in the two groups. In the intervention group, teacher and parent SDQ total difficulties scores were lower at post-intervention and longer-term follow-up compared to baseline (teacher: baseline 14.42 (SD 7.18); post-intervention 11.09 (6.93), t(739) = 13.78, p < 0.001; follow-up 11.27 (7.27), t(739) = 11.92, p < 0.001; parent: baseline 15.64 (6.49); post-intervention 11.90 (6.78), t(361 = 11.29, p < 0.001); follow-up 11.32 (7.19), t(361) = 11.29, p < 0.001). The reduction in SDQ scores was greater in the intervention compared to the comparator group (likelihood ratio test comparing models with time only versus time plus group-by-time interaction: χ2 (3) = 24.09, p < 0.001), and model-predicted SDQ scores were lower in the intervention than comparator group for 2 years post-baseline. A one-to-one counselling intervention delivered to children in UK primary schools predicted improvements in mental health that were maintained over a 2 year follow-up period.

Cas1 and the Csy complex are opposing regulators of Cas2/3 nuclease activity.

The type I-F CRISPR adaptive immune system in Pseudomonas aeruginosa (PA14) consists of two CRISPR loci and six CRISPR-associated (cas) genes. Type I-F systems rely on a CRISPR RNA (crRNA)-guided surveillance complex (Csy complex) to bind foreign DNA and recruit a trans-acting nuclease (i.e., Cas2/3) for target degradation. In most type I systems, Cas2 and Cas3 are separate proteins involved in adaptation and interference, respectively. However, in I-F systems, these proteins are fused into a single polypeptide. Here we use biochemical and structural methods to show that two molecules of Cas2/3 assemble with four molecules of Cas1 (Cas2/32:Cas14) into a four-lobed propeller-shaped structure, where the two Cas2 domains form a central hub (twofold axis of symmetry) flanked by two Cas1 lobes and two Cas3 lobes. We show that the Cas1 subunits repress Cas2/3 nuclease activity and that foreign DNA recognition by the Csy complex activates Cas2/3, resulting in bidirectional degradation of DNA targets. Collectively, this work provides a structure of the Cas1-2/3 complex and explains how Cas1 and the target-bound Csy complex play opposing roles in the regulation of Cas2/3 nuclease activity.

Mechanism of CRISPR-RNA guided recognition of DNA targets in Escherichia coli.

In bacteria and archaea, short fragments of foreign DNA are integrated into Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) loci, providing a molecular memory of previous encounters with foreign genetic elements. In Escherichia coli, short CRISPR-derived RNAs are incorporated into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Recent structures of Cascade capture snapshots of this seahorse-shaped RNA-guided surveillance complex before and after binding to a DNA target. Here we determine a 3.2 Å x-ray crystal structure of Cascade in a new crystal form that provides insight into the mechanism of double-stranded DNA binding. Molecular dynamic simulations performed using available structures reveal functional roles for residues in the tail, backbone and belly subunits of Cascade that are critical for binding double-stranded DNA. Structural comparisons are used to make functional predictions and these predictions are tested in vivo and in vitro. Collectively, the results in this study reveal underlying mechanisms involved in target-induced conformational changes and highlight residues important in DNA binding and protospacer adjacent motif recognition.

IL-35 stimulation of CD39+ regulatory T cells confers protection against collagen II-induced arthritis via the production of IL-10.

IL-35 is produced by regulatory T cells, and this novel cytokine can downregulate Th17 cell development and inhibit autoimmune inflammation. In this work, an rIL-35, as a single-chain fusion between murine IL-12p35 and EBV-induced gene 3, was expressed in yeast. This rIL-35 inhibited OVA-specific cellular and Ab responses in OVA-challenged recipients of DO11.10 CD4+ T cells. Likewise, IL-35 inhibited clinical manifestation of collagen-induced arthritis or could cease further disease exacerbation upon initiation of IL-35 treatment. Exogenous IL-35 treatments suppressed Th1 and Th17 cells and promoted CD39 expression by CD4+ T cells. Sorted CD25-CD39+CD4+ T cells from IL-35-treated mice produced IL-10 and, upon adoptive transfer, were sufficiently potent to inhibit subsequent development of inflammation in mice with collagen-induced arthritis, whereas sorted CD25+CD39+CD4+ T cells showed reduced potency. IL-35 treatments of IL-10-/- mice failed to induce protective CD39+CD4+ T cells, demonstrating the effector role of IL-10 by IL-35 immunosuppression.

Tolerogen-induced interferon-producing killer dendritic cells (IKDCs) protect against EAE.

Natural killer (NK) cells and dendritic cells (DCs) have been shown to link the innate and adaptive immune systems. Likewise, a new innate cell subset, interferon-producing killer DCs (IKDCs), shares phenotypic and functional characteristics with both DCs and NK cells. Here, we show IKDCs play an essential role in the resolution of experimental autoimmune encephalomyelitis (EAE) upon treatment with the tolerizing agent, myelin oligodendrocyte glycoprotein (MOG), genetically fused to reovirus protein σ1 (termed MOG-pσ1). Activated IKDCs were recruited subsequent MOG-pσ1 treatment of EAE, and disease resolution was abated upon NK1.1 cell depletion. These IKDCs were able to kill activated CD4(+) T cells and mature dendritic DCs, thus, contributing to EAE remission. In addition, IKDCs were responsible for MOG-pσ1-mediated MOG-specific regulatory T cell recruitment to the CNS. The IKDCs induced by MOG-pσ1 expressed elevated levels of HVEM for interactions with cognate ligand-positive cells: LIGHT(+) NK and T(eff) cells and BTLA(+) B cells. Further characterization revealed these activated IKDCs being MHC class II(high), and upon their adoptive transfer (CD11c(+)NK1.1(+)MHC class II(high)), IKDCs, but not CD11c(+)NK1.1(+)MHC class II(intermediate/low) (unactivated) cells, conferred protection against EAE. These activated IKDCs showed enhanced CD107a, PD-L1, and granzyme B expression and could present OVA, unlike unactivated IKDCs. Thus, these results demonstrate the interventional potency induced HVEM(+) IKDCs to resolve autoimmune disease.

Simulated-Scenario and Peer-Mentorship Curriculum to Train Prehospital Providers in the Practice of Mass Gathering Medicine.

OBJECTIVE: A mass gathering medicine training program was established for a 7,200-seat arena. The objectives of this study were to describe the program schema and determine its impact in preparing novice emergency medical technicians (EMTs) to manage the difficulties of large-venue emergency medical services (EMS). METHODS: Optional, anonymous surveys were administered to EMTs. Novice EMTs were assessed pre-/post-program implementation, and both novice and experienced EMTs completed self-reported Likert scales. Data were analyzed with nonparametric methods. RESULTS: A total of 43/56 responses (response rate = 76.8%) were received. Only 37.2% of providers felt prepared to work mass gatherings before the training, and 60.5% stated that their previous education did not prepare them for large-venue challenges. After the training program, novice EMTs were significantly associated with increased knowledge of large-venue EMS procedures (P = 0.0170), higher proficiency using extrication equipment (P = 0.0248), increased patient care skills (P = 0.0438), and both increased confidence working events (P = 0.0002) and better teamwork during patient encounters (P = 0.0001). The majority of EMTs reported the program as beneficial. CONCLUSION: Upon hire, EMS providers felt unprepared to work large-venue EMS. The analyses demonstrated that this training program improved select large-venue emergency skills for prehospital providers and may fill a gap in the education system regarding mass gathering medicine.

Adenovirus F protein as a delivery vehicle for botulinum B.

BACKGROUND: Immunization with recombinant carboxyl-terminal domain of the heavy chain (Hc domain) of botulinum neurotoxin (BoNT) stimulates protective immunity against native BoNT challenge. Most studies developing a botulism vaccine have focused on the whole Hc; however, since the principal protective epitopes are located within beta-trefoil domain (Hcbetatre), we hypothesize that immunization with the Hcbetatre domain is sufficient to confer protective immunity. In addition, enhancing its uptake subsequent to nasal delivery prompted development of an alternative vaccine strategy, and we hypothesize that the addition of targeting moiety adenovirus 2 fiber protein (Ad2F) may enhance such uptake during vaccination. RESULTS: The Hcbetatre serotype B immunogen was genetically fused to Ad2F (Hcbetatre/B-Ad2F), and its immunogenicity was tested in mice. In combination with the mucosal adjuvant, cholera toxin (CT), enhanced mucosal IgA and serum IgG Ab titers were induced by nasal Hcbetatre-Ad2F relative to Hcbetatre alone; however, similar Ab titers were obtained upon intramuscular immunization. These BoNT/B-specific Abs induced by nasal immunization were generally supported in large part by Th2 cells, as opposed to Hcbetatre-immunized mice that showed more mixed Th1 and Th2 cells. Using a mouse neutralization assay, sera from animals immunized with Hcbetatre and Hcbetatre-Ad2F protected mice against 2.0 LD50. CONCLUSION: These results demonstrate that Hcbetatre-based immunogens are highly immunogenic, especially when genetically fused to Ad2F, and Ad2F can be exploited as a vaccine delivery platform to the mucosa.

Comparing body composition assessment tests in long-term hemodialysis patients.

BACKGROUND: Protein-energy wasting is common in chronic kidney disease and is associated with decreases in body muscle and fat stores and poor outcomes. The accuracy and reliability of field methods to measure body composition is unknown in this population. STUDY DESIGN: Cross-sectional observational study. SETTING & PARTICIPANTS: 118 maintenance hemodialysis patients were seen at the General Clinical Research Center at Harbor-UCLA Medical Center, Torrance, CA. INDEX TESTS: Triceps skinfold, near-infrared interactance, and bioelectrical impedance analysis using the Segal, Kushner, and Lukaski equations. REFERENCE TEST: Dual-energy x-ray absorptiometry (DEXA). RESULTS: Participants (42% women, 52% with diabetes, 40% African Americans, and 38% Hispanics) were aged 49.4 +/- 11.5 (mean +/- SD) years, and had undergone dialysis therapy for 41.1 +/- 32.9 months. Body mass index was 27.0 +/- 6.0 kg/m(2). Using DEXA as the reference test, the bioelectrical impedance analysis-Kushner equation, triceps skinfold, and near-infrared interactance were most accurate of the index tests in estimating total-body fat percentage, whereas bioelectrical impedance analysis-Segal equation and bioelectrical impedance analysis-Lukaski equation overestimated total body fat percentage. Bland-Altman analyses and difference plots showed that bioelectrical impedance analysis-Kushner and near-infrared interactance were most similar to the reference test. Bioelectrical impedance analysis-Kushner, triceps skinfold, and near-infrared interactance had the smallest mean differences from DEXA, especially in women (1.6%, 0.7%, and 1.2%, respectively). Similar results were observed in African American participants (n = 47). LIMITATIONS: Measurements were performed 1 day after a hemodialysis treatment, leading to more fluid retention, which may have affected the reference and index tests differently. CONCLUSIONS: Using DEXA as the reference test, both near-infrared interactance and bioelectrical impedance analysis-Kushner method yield more consistent estimates of total body fat percentage in maintenance hemodialysis patients compared with the other index tests. Near-infrared interactance is not affected by skin color. Field methods with portable devices may provide adequate precision.

AcrIF9 tethers non-sequence specific dsDNA to the CRISPR RNA-guided surveillance complex.

Bacteria have evolved sophisticated adaptive immune systems, called CRISPR-Cas, that provide sequence-specific protection against phage infection. In turn, phages have evolved a broad spectrum of anti-CRISPRs that suppress these immune systems. Here we report structures of anti-CRISPR protein IF9 (AcrIF9) in complex with the type I-F CRISPR RNA-guided surveillance complex (Csy). In addition to sterically blocking the hybridization of complementary dsDNA to the CRISPR RNA, our results show that AcrIF9 binding also promotes non-sequence-specific engagement with dsDNA, potentially sequestering the complex from target DNA. These findings highlight the versatility of anti-CRISPR mechanisms utilized by phages to suppress CRISPR-mediated immune systems.

A nasal interleukin-12 DNA vaccine coexpressing Yersinia pestis F1-V fusion protein confers protection against pneumonic plague.

Previous studies have shown that mucosal application of interleukin-12 (IL-12) can stimulate elevated secretory immunoglobulin A (IgA) responses. Since possible exposure to plague is via Yersinia pestis-laden aerosols that results in pneumonic plague, arming both the mucosal and systemic immune systems may offer an added benefit for protective immunity. Two bicistronic plasmids were constructed that encoded the protective plague epitopes, capsular antigen (F1-Ag) and virulence antigen (V-Ag) as a F1-V fusion protein but differed in the amounts of IL-12 produced. When applied nasally, serum IgG and mucosal IgA anti-F1-Ag and anti-V-Ag titers were detectable beginning at week 6 after three weekly doses, and recombinant F1-Ag boosts were required to elevate the F1-Ag-specific antibody (Ab) titers. Following pneumonic challenge, the best efficacy was obtained in mice primed with IL-12(Low)/F1-V vaccine with 80% survival compared to mice immunized with IL-12(Low)/F1, IL-12(Low)/V, or IL-12(Low) vector DNA vaccines. Improved expression of IL-12 resulted in lost efficacy when using the IL-12(High)/F1-V DNA vaccine. Despite differences in the amount of IL-12 produced by the two F1-V DNA vaccines, Ab responses and Th cell responses to F1- and V-Ags were similar. These results show that IL-12 can be used as a molecular adjuvant to enhance protective immunity against pneumonic plague, but in a dose-dependent fashion.

Conformational Dynamics of DNA Binding and Cas3 Recruitment by the CRISPR RNA-Guided Cascade Complex.

Bacteria and archaea rely on CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided adaptive immune systems for sequence specific elimination of foreign nucleic acids. In Escherichia coli, short CRISPR-derived RNAs (crRNAs) assemble with Cas (CRISPR-associated) proteins into a 405-kilodalton multisubunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade binds foreign DNA complementary to the crRNA guide and recruits Cas3, a trans-acting nuclease-helicase required for target degradation. Structural models of Cascade have captured static snapshots of the complex in distinct conformational states, but conformational dynamics of the 11-subunit surveillance complex have not been measured. Here, we use hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) to map conformational dynamics of Cascade onto the three-dimensional structure. New insights from structural dynamics are used to make functional predictions about the mechanisms of the R-loop coordination and Cas3 recruitment. We test these predictions in vivo and in vitro. Collectively, we show how mapping conformational dynamics onto static 3D-structures adds an additional dimension to the functional understanding of this biological machine.

Structural biology. Crystal structure of the CRISPR RNA-guided surveillance complex from Escherichia coli.

Clustered regularly interspaced short palindromic repeats (CRISPRs) are essential components of RNA-guided adaptive immune systems that protect bacteria and archaea from viruses and plasmids. In Escherichia coli, short CRISPR-derived RNAs (crRNAs) assemble into a 405-kilodalton multisubunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Here we present the 3.24 angstrom resolution x-ray crystal structure of Cascade. Eleven proteins and a 61-nucleotide crRNA assemble into a seahorse-shaped architecture that binds double-stranded DNA targets complementary to the crRNA-guide sequence. Conserved sequences on the 3' and 5' ends of the crRNA are anchored by proteins at opposite ends of the complex, whereas the guide sequence is displayed along a helical assembly of six interwoven subunits that present five-nucleotide segments of the crRNA in pseudo-A-form configuration. The structure of Cascade suggests a mechanism for assembly and provides insights into the mechanisms of target recognition.

IFN-γ-deficient mice develop IL-1-dependent cutaneous and musculoskeletal inflammation during experimental brucellosis.

Human brucellosis exhibits diverse pathological manifestations that can affect almost any organ. In particular, osteoarticular complications are the most common focal manifestation of brucellosis and occur in 40-80% of patients. In immunocompetent mice, Brucella replication is generally restricted to the spleen, liver, and to a lesser extent, LNs, thereby limiting their use for study of focal inflammation often found in brucellosis. Here, we report that nasal, oral, or peritoneal infection of IFN-γ(-/-) mice with WT Brucella melitensis or Brucella abortus results in joint and periarticular tissue inflammation. Histological analysis of the affected joints revealed inflammatory infiltrates and debris within the joint space colocalizing with Brucella antigen. Osteoarthritis, necrosis, periarticular soft tissue inflammation, and substantial brucellae burdens were observed. Oral rifampicin was effective in clearing infection and halting further progression of focal inflammation from infected IFN-γ(-/-) mice, although some symptoms and swelling remained. Elevated IL-1 ß, but not TNF-α, IL-6, or IL-17, was detected in joint homogenates from infected IFN-γ(-/-) mice. Whereas more susceptible to systemic infection, IL-1R(-/-) mice depleted of IFN-γ were more resistant to focal inflammation than WT mice similarly depleted of IFN-γ. Collectively, these results show IFN-γ(-/-) mice represent a potential model for study of focal inflammation attributed to Brucella infection and will allow evaluation of intervention strategies targeting IL-1, IL-1R, or other inflammatory mediators, with the potential to complement antibiotic-based therapies.

Apple polyphenols require T cells to ameliorate dextran sulfate sodium-induced colitis and dampen proinflammatory cytokine expression.

Human IBD, including UC and Crohn's disease, is characterized by a chronic, relapsing, and remitting condition that exhibits various features of immunological inflammation and affects at least one/1000 people in Western countries. Polyphenol extracts from a variety of plants have been shown to have immunomodulatory and anti-inflammatory effects. In this study, treatment with APP was investigated to ameliorate chemically induced colitis. Oral but not peritoneal administration of APP during colitis induction significantly protected C57BL/6 mice against disease, as evidenced by the lack of weight loss, colonic inflammation, and shortening of the colon. APP administration dampened the mRNA expression of IL-1ß, TNF-α, IL-6, IL-17, IL-22, CXCL9, CXCL10, CXCL11, and IFN-γ in the colons of mice with colitis. APP-mediated protection requires T cells, as protection was abated in Rag-1(-/-) or TCRα(-/-) mice but not in IL-10(-/-), IRF-1(-/-), µMT, or TCRδ(-/-) mice. Administration of APP during colitis to TCRα(-/-) mice actually enhanced proinflammatory cytokine expression, further demonstrating a requirement for TCRαß cells in APP-mediated protection. APP treatment also inhibited CXCR3 expression by TCRαß cells, but not B or NK cells, in the colons of mice with colitis; however, depletion of CD4(+) or CD8(+) T cells alone did not abolish APP-mediated protection. Collectively, these results show that oral administration of APP protects against experimental colitis and diminishes proinflammatory cytokine expression via T cells.

A parenteral DNA vaccine protects against pneumonic plague.

The chemokine, lymphotactin (LTN), was tested as a molecular adjuvant using bicistronic DNA vaccines encoding the protective Yersinia capsular (F1) antigen and virulence antigen (V-Ag) as a F1-V fusion protein. The LTN-encoding F1-V or V-Ag vaccines were given by the intranasal (i.n.) or intramuscular (i.m.) routes, and although serum IgG and mucosal IgA antibodies (Abs) were induced, F1-Ag boosts were required for robust anti-F1-Ag Abs. Optimal efficacy against pneumonic plague was obtained in mice i.m.-, not i.n.-immunized with these DNA vaccines. These vaccines stimulated elevated Ag-specific Ab-forming cells and mixed Th cell responses, with Th17 cells markedly enhanced by i.m. immunization. These results show that LTN can be used as a molecular adjuvant to enhance protective immunity against plague.