Cytokine competent gut-joint migratory T Cells contribute to inflammation in the joint.

Although studies have identified the presence of gut-associated cells in the enthesis of joints affected by spondylarthritis (SpA), a direct link through cellular transit between the gut and joint has yet to be formally demonstrated. Using KikGR transgenic mice to label in situ and track cellular trafficking from the distal colon to the joint under inflammatory conditions of both the gut and joint, we demonstrate bona-fide gut-joint trafficking of T cells from the colon epithelium, also called intraepithelial lymphocytes (IELs), to distal sites including joint enthesis, the pathogenic site of SpA. Similar to patients with SpA, colon IELs from the TNFΔARE/+ mouse model of inflammatory bowel disease and SpA display heightened TNF production upon stimulation. Using ex vivo stimulation of photo-labeled gut-joint trafficked T cells from the popliteal lymph nodes of KikGR and KikGR TNFΔARE/+ we saw that the CD4+ photo-labeled population was highly enriched for IL-17 competence in healthy as well as arthritic mice, however in the TNFΔARE/+ mice these cells were additionally enriched for TNF. Using transfer of magnetically isolated IELs from TNF+/+ and TNFΔARE/+ donors into Rag1 -/- hosts, we confirmed that IELs can exacerbate inflammatory processes in the joint. Finally, we blocked IEL recruitment to the colon epithelium using broad spectrum antibiotics in TNFΔARE/+ mice. Antibiotic-treated mice had reduced gut-joint IEL migration, contained fewer Il-17A and TNF competent CD4+ T cells, and lessened joint pathology compared to untreated littermate controls. Together these results demonstrate that pro-inflammatory colon-derived IELs can exacerbate inflammatory responses in the joint through systemic trafficking, and that interference with this process through gut-targeted approaches has therapeutic potential in SpA.

Cytokines Produced in Response to Varicella-Zoster Virus Infection of ARPE-19 Cells Stimulate Lymphocyte Chemotaxis.

Posterior uveitis is an ocular complication that can occur with reactivation of varicella-zoster virus (VZV). It may lead to loss of vision due to retinal detachment and chronic inflammation, which often causes more severe disease than the virus infection itself. To increase our understanding of the immune response, we infected the retinal pigment epithelial (RPE) cell line, ARPE-19, with cell-associated VZV and compared its response to that of the MeWo cell line using multiplex assays. We observed (1) a difference in the magnitude and kinetics of cytokine responses between the 2 cell types and (2) differential migration of CD4+ and CD8+ T cells towards these cytokines. Thus, our data provide information about the cytokine and lymphocytic responses to VZV infection of RPE cells, thereby providing a useful platform for future studies to address mechanisms underlying the immunopathology of VZV-associated posterior uveitis.

Antibody responses to influenza a H1N1 vaccine compared to the circulating strain in influenza vaccine recipients during the 2013/2014 season in North America.

BACKGROUND: Influenza strain A/California/07/2009 H1N1 (H1N1-09) reemerged in 2013/2014 as the predominant cause of illness. We sought to determine if antigenic drift may have contributed to the decreased responses to influenza vaccine. METHODS: Fifty adults who received trivalent inactivated influenza vaccine (IIV3) and 56 children who received live attenuated quadrivalent influenza vaccine (LAIV4) had hemagglutination inhibition (HAI) and microneutralizing (MN) antibodies measured in plasma against H1N1-09 and H1N1 2013/2014 (H1N1-14) influenza. Partial sequencing of the hemagglutinin gene (nt 280-780) was performed on 38 clinical isolates and the vaccine prototype. RESULTS: In IIV3 recipients, HAI and MN titers against H1N1-14 were significantly lower than against H1N1-09 (p<0.0001 and 0.04, respectively). In LAIV4 recipients, only MN titers were significantly lower (p=0.02) for H1N1-09 compared with H1N1-14. A combined analysis showed significantly lower HAI and MN titers for H1N1-14 compared with H1N1-09 (p=0. 016 and 0.008, respectively). All 38 clinical isolates encoded the HA gene K166Q non-synonymous substitution; other non-synonymous substitutions were observed in <10% of the clinical isolates. CONCLUSIONS: 2013/2014 IIV3 and LAIV4 recipients had consistently lower MN antibody titers against H1N1-14 compared with H1N1-09. The HA K166Q mutation, located in a neutralizing epitope, probably contributed to these findings.

Immune Responses to Circulating and Vaccine Viral Strains in HIV-Infected and Uninfected Children and Youth Who Received the 2013/2014 Quadrivalent Live-Attenuated Influenza Vaccine.

The live-attenuated influenza vaccine (LAIV) has generally been more efficacious than the inactivated vaccine in children. However, LAIV is not recommended for HIV-infected children because of insufficient data. We compared cellular, humoral, and mucosal immune responses to the 2013-2014 LAIV quadrivalent (LAIV4) in HIV-infected and uninfected children 2-25 years of age (yoa). We analyzed the responses to the vaccine H1N1 (H1N1-09), to the circulating H1N1 (H1N1-14), which had significant mutations compared to H1N1-09 and to B Yamagata (BY), which had the highest effectiveness in 2013-2014. Forty-six HIV-infected and 56 uninfected participants with prior influenza immunization had blood and nasal swabs collected before and after LAIV4 for IFNγ T and IgG/IgA memory B-cell responses (ELISPOT), plasma antibodies [hemagglutination inhibition (HAI) and microneutralization (MN)], and mucosal IgA (ELISA). The HIV-infected participants had median CD4+ T cells = 645 cells/µL and plasma HIV RNA = 20 copies/mL. Eighty-four percent were on combination anti-retroviral therapy. Regardless of HIV status, significant increases in T-cell responses were observed against BY, but not against H1N1-09. H1N1-09 T-cell immunity was higher than H1N1-14 both before and after vaccination. LAIV4 significantly increased memory IgG B-cell immunity against H1N1-14 and BY in uninfected, but not in HIV-infected participants. Regardless of HIV status, H1N1-09 memory IgG B-cell immunity was higher than H1N1-14 and lower than BY. There were significant HAI titer increases after vaccination in all groups and against all viruses. However, H1N1-14 MN titers were significantly lower than H1N1-09 before and after vaccination overall and in HIV-uninfected vaccinees. Regardless of HIV status, LAIV4 increased nasal IgA concentrations against all viruses. The fold-increase in H1N1-09 IgA was lower than BY. Overall, participants <9 yoa had decreased BY-specific HAI and nasal IgA responses to LAIV4. In conclusion, HIV-infected and uninfected children and youth had comparable responses to LAIV4. H1N1-09 immune responses were lower than BY and higher than H1N1-14, suggesting that both antigenic mismatches between circulating and vaccine H1N1 and lower immunogenicity of the H1N1 vaccine strain may have contributed to the decreased H1N1 effectiveness of 2013-2014 LAIV4.

Gammaherpesvirus small noncoding RNAs are bifunctional elements that regulate infection and contribute to virulence in vivo.

UNLABELLED: Many viruses express noncoding RNAs (ncRNAs). The gammaherpesviruses (γHVs), including Epstein-Barr virus, Kaposi's sarcoma-associated herpesvirus, and murine γHV68, each contain multiple ncRNA genes, including microRNAs (miRNAs). While these ncRNAs can regulate multiple host and viral processes in vitro, the genetic contribution of these RNAs to infection and pathogenesis remains largely unknown. To study the functional contribution of these RNAs to γHV infection, we have used γHV68, a small-animal model of γHV pathogenesis. γHV68 encodes eight small hybrid ncRNAs that contain both tRNA-like elements and functional miRNAs. These genes are transcribed by RNA polymerase III and are referred to as the γHV68 TMERs (tRNA-miRNA-encoded RNAs). To determine the total concerted genetic contribution of these ncRNAs to γHV acute infection and pathogenesis, we generated and characterized a recombinant γHV68 strain devoid of all eight TMERs. TMER-deficient γHV68 has wild-type levels of lytic replication in vitro and normal establishment of latency in B cells early following acute infection in vivo. In contrast, during acute infection of immunodeficient mice, TMER-deficient γHV68 has reduced virulence in a model of viral pneumonia, despite having an enhanced frequency of virus-infected cells. Strikingly, expression of a single viral tRNA-like molecule, in the absence of all other virus-encoded TMERs and miRNAs, reverses both attenuation in virulence and enhanced frequency of infected cells. These data show that γHV ncRNAs play critical roles in acute infection and virulence in immunocompromised hosts and identify these RNAs as a new potential target to modulate γHV-induced infection and pathogenesis. IMPORTANCE: The gammaherpesviruses (γHVs) are a subfamily of viruses associated with chronic inflammatory diseases and cancer, particularly in immunocompromised individuals. These viruses uniformly encode multiple types of noncoding RNAs (ncRNAs) that are not translated into proteins. It remains unclear how virus-expressed ncRNAs influence the course and outcome of infection in vivo. Here, we generated a mouse γHV that lacks the expression of multiple ncRNAs. Notably, this mutant virus is critically impaired in the ability to cause disease in immunocompromised hosts yet shows a paradoxical increase in infected cells early during infection in these hosts. While the original mouse virus encodes multiple ncRNAs, the expression of a single domain of one ncRNA can partially reverse the defects of the mutant virus. These studies demonstrate that γHV ncRNAs can directly contribute to virus-induced disease in vivo and that these RNAs may be multifunctional, allowing the opportunity to specifically interfere with different functional domains of these RNAs.

A conserved RNA polymerase III promoter required for gammaherpesvirus TMER transcription and microRNA processing.

Canonical RNA polymerase III (pol III) type 2 promoters contain a single A and B box and are well documented for their role in tRNA and SINE transcription in eukaryotic cells. The genome of Murid herpesvirus 4 (MuHV-4) contains eight polycistronic tRNA-microRNA encoded RNA (TMER) genes that are transcribed from a RNA pol III type 2-like promoter containing triplicated A box elements. Here, we demonstrate that the triplicated A box sequences are required in their entirety to produce functional MuHV-4 miRNAs. We also identify that these RNA pol III type 2-like promoters are conserved in eukaryotic genomes. Human and mouse predicted tRNA genes containing these promoters also show enrichment of alternative RNA pol III transcription termination sequences and are predicted to give rise to longer tRNA primary transcripts.

Genetic manipulation of keratinocyte stem cells with lentiviral vectors.

The epidermis of the skin and its appendages, such as the hair follicles, are formed and maintained by keratinocyte stem cells. Highly efficient and permanent genetic modifications are valuable tools to examine the multipotency and regenerative capacity of keratinocyte stem cells in skin and hair follicle development, homeostasis, and regeneration. Herein, we describe an ex vivo approach by which primary mouse keratinocytes can be permanently manipulated by lentiviral vectors at the genetic level. This protocol can be used to permanently express a gene-of-interest or selectively silence the expression of an endogenous gene, which can be used in preclinical development of gene-based therapies for skin and systemic disorders.

Murine gammaherpesvirus 68 infection protects lupus-prone mice from the development of autoimmunity.

Gammaherpesvirus infections, such as those caused by EBV, have been suggested to promote the development of autoimmunity. To test this idea, we infected healthy WT and lupus-prone B6.Sle123 mice with an EBV-related and rodent-specific gammaherpesvirus, γHV68. Although acute γHV68 infection increased autoantibody levels for 4 to 6 wk, latent infection inhibited these responses for 1 y. The inhibition of autoantibody expression was only observed in B6.Sle123 females and not in males, which already displayed lower autoantibody titers. Contrary to the initial hypothesis, infection of young B6.Sle123 mice, both male and female, resulted in suppression of lymphoid activation and expansion and of glomerular inflammation and sclerosis, preserving kidney function. Moreover, γHV68 infection led to reduced autoantibody titers, lymphoid activation, and glomerular inflammation whether lupus-prone females were infected before or during disease manifestation. Finally, γHV68 infection also inhibited autoantibody production in the genetically distinct MRL/lpr lupus-prone mice. Our findings indicate that γHV68 infection strongly inhibits the development and progression of lupus-like disease in mice that spontaneously develop this condition mediating its beneficial effects at the humoral, cellular, and organ levels. The mechanisms by which the virus exerts this down-modulatory action are not yet clear, but appear to operate via reduced activation of dendritic cells, T cells, and B cells. Gammaherpesviruses coevolved with the vertebrate immune systems, establishing lifelong infections in humans and other mammals. Our findings that γHV68 infection prevents rather than exacerbates autoimmunity in mice suggest that infection with gammaherpesviruses may be protective rather than pathological in most individuals.

Viral cyclins mediate separate phases of infection by integrating functions of distinct mammalian cyclins.

Gammaherpesvirus cyclins have expanded biochemical features relative to mammalian cyclins, and promote infection and pathogenesis including acute lung infection, viral persistence, and reactivation from latency. To define the essential features of the viral cyclin, we generated a panel of knock-in viruses expressing various viral or mammalian cyclins from the murine gammaherpesvirus 68 cyclin locus. Viral cyclins of both gammaherpesvirus 68 and Kaposi's sarcoma-associated herpesvirus supported all cyclin-dependent stages of infection, indicating functional conservation. Although mammalian cyclins could not restore lung replication, they did promote viral persistence and reactivation. Strikingly, distinct and non-overlapping mammalian cyclins complemented persistence (cyclin A, E) or reactivation from latency (cyclin D3). Based on these data, unique biochemical features of viral cyclins (e.g. enhanced kinase activation) are not essential to mediate specific processes during infection. What is essential for, and unique to, the viral cyclins is the integration of the activities of several different mammalian cyclins, which allows viral cyclins to mediate multiple, discrete stages of infection. These studies also demonstrated that closely related stages of infection, that are cyclin-dependent, are in fact genetically distinct, and thus predict that cyclin requirements may be used to tailor potential therapies for virus-associated diseases.

Pediatric emergencies on a US-based commercial airline.

OBJECTIVES: The purpose of this investigation was to determine the incidence and character of pediatric emergencies on a US-based commercial airline and to evaluate current in-flight medical kits. METHODS: In-flight consultations to a major US airline by a member of our staff are recorded in an institutional database. In this observational retrospective review, the database was queried for consultations for all passengers up to 18 years old between January 1, 1995, and December 31, 2002. Consultations were reviewed for type of emergency, use of the medical kit, and unscheduled landings. RESULTS: Two hundred twenty-two pediatric consultations were identified, representing 1 pediatric call per 20,775 flights. The mean age of patients was 6.8 years. Fifty-three emergencies were preflight calls, and 169 were in-flight pediatric consultations. The most common in-flight consultations concerned infectious disease (45 calls, 27%), neurological (25 calls, 15%), and respiratory tract (22 calls, 13%) emergencies. The emergency medical kit was used for 60 emergencies. Nineteen consultations (11%) resulted in flight diversions (1/240,000 flights), most commonly because of in-flight neurological (9) and respiratory tract (5) emergencies. International flights had a higher incidence than domestic flights of consultations and diversions for pediatric emergencies. CONCLUSIONS: The most common in-flight pediatric emergencies involved infectious diseases and neurological and respiratory tract problems. Emergency medical kits should be expanded to include pediatric medications.

Medical director for air medical transport programs.

The NAEMSP recognizes the multifaceted and integral position of a medical director for an air medical transport program and the EMS community at large.