Long-term gut colonization with ESBL-producing Escherichia coli in participants without known risk factors from the southeastern United States.

We evaluated gut carriage of extended spectrum beta lactamase producing Enterobacteriaceae (ESBL-E) in southeastern U.S. residents without recent in-patient healthcare exposure. Study enrollment was January 2021-February 2022 in Athens, Georgia, U.S. and included a diverse population of 505 adults plus 50 child participants (age 0-5). Based on culture-based screening of stool samples, 4.5% of 555 participants carried ESBL-Es. This is slightly higher than reported in studies conducted 2012-2015, which found carriage rates of 2.5-3.9% in healthy U.S. residents. All ESBL-E confirmed isolates (n=25) were identified as Escherichia coli. Isolates belonged to 11 sequence types, with 48% classified as ST131. Ninety six percent of ESBL-E isolates carried a blaCTX-M gene. Isolated ESBL-Es frequently carried virulence genes as well as multiple classes of antibiotic resistance genes. Long-term colonization was common, with 64% of ESBL-E positive participants testing positive when rescreened three months later. One participant yielded isolates belonging to two different E. coli sequence types that carried blaCTX-M-1 genes on near-identical plasmids, suggesting intra-gut plasmid transfer. Isolation of E. coli on media without antibiotics revealed that ESBL-E. coli typically made up a minor fraction of the overall gut E. coli population, although in some cases they were the dominant strain. ESBL-E carriage was not associated with a significantly different stool microbiome composition. However, some microbial taxa were differentially abundant in ESBL-E carriers. Together, these results suggest that a small subpopulation of US residents are long-term, asymptomatic carriers of ESBL-Es, and may serve as an important reservoir for community spread of these ESBL genes.

"A peer support worker can really be there supporting the youth throughout the whole process": a qualitative study exploring the role of peer support in providing substance use services to youth.

BACKGROUND: Youth (ages 12-24) rarely access services and supports to address substance use concerns. Peer support can facilitate service engagement and has been associated with positive substance use recovery outcomes in adults, yet few studies have examined this role among youth specifically. As such, this qualitative study explored the role of peer support in providing substance use services to youth in British Columbia and how best to support them in their role. METHODS: Participatory action research methods were used by partnering with youth who had lived/living experience of substance use, including peer support workers, to co-design the research protocol and materials. An initial focus group and subsequent interviews were held with 18 peer support workers who provide services to youth (ages 12-24) based on their own lived experience with mental health and/or substance use. The discussions were audio-recorded, transcribed verbatim, and analysed thematically using an inductive approach. RESULTS: Peer support workers' core experiences providing substance use services to youth centred around supporting youth throughout the whole process. This was accomplished by meeting youth where they are at, providing individualized care, and bridging the gap between other services and supports. However, participants experienced multiple organizational barriers hindering their ability to support youth and stressed the importance of having an employer who understands the work you are doing. This involved having someone advocating for the peer support role to promote collaboration, empowering peers to set boundaries and define their own role, and providing adequate training and mentorship. Finally, peer support workers described how their lived experience bridges connection and de-stigmatization at the individual, organizational, and community level, which was unique to their role. CONCLUSIONS: Peer support plays a unique role in youths' substance use journeys, given their own lived experience and flexibility within their role. However, their position is often misunderstood by employers and other service providers, leaving peers with inadequate support, training, and mentorship to do their job. The findings from this study call for improved integration of peer support into service environments, as well as standardized training that is in-depth and continuous.

The Relationship between Sense of Belonging and Well-Being Outcomes in Emerging Adults with Care Experience.

Robust social resources that lead to a healthy sense of belonging are imperative during emerging adulthood. However, young adults with alternative care experience, such as residential or foster care, often lack the social resources necessary to transition to adulthood successfully. Though some research has examined the importance of social resources in this population, less is known about a sense of belonging, which is associated with these social resources. The current study examined the association between care experience, belonging, and well-being outcomes among emerging adults (n = 703) who were separated from their biological parents during care and received alternative care in 16 nations. The presence of belonging was associated with type of alternative care, age at separation, and reason for separation. There was also an association between belonging and well-being outcomes, including homelessness and suicidal ideation. Adults lacking a sense of belonging reported higher rates of homelessness and suicidal ideation, lower life satisfaction, and lower resilience. The study has global implications for enhancing social support for emerging adults with care experience in order to facilitate a sense of belonging as a social resource.

The Relationship Between Spirituality and Resilience and Well-being: a Study of 529 Care Leavers from 11 Nations.

Research has found that individuals who were separated from parental care and experienced alternative care settings during childhood are more likely to have poor outcomes as adults. This highlights the importance of understanding factors that are related to resilience and well-being for care leavers. A growing body of research has supported the importance of spirituality in our understanding of resilience and well-being. However, little work to date has examined the relationship of spirituality to outcomes in care leavers. The current study investigated the relationships between spirituality, resilience, well-being, and health in a sample of 529 care leavers from 11 nations. It also examined how different themes of spirituality were related to specific outcome variables. Data revealed that spirituality was significantly associated with higher life satisfaction, better mental and physical health, and more resilience even when accounting for current age, gender, age at separation, Human Development Index scores, and childhood adversity. Furthermore, findings indicate that different themes of spirituality are related to specific outcome variables, even when accounting for demographic information. Findings indicate that spirituality may play an important role in resilience and well-being for care leavers. Implications and limitations are discussed.

A qualitative study exploring how young people perceive and experience substance use services in British Columbia, Canada.

BACKGROUND: Substance use among youth (ages 12-24) is troublesome given the increasing risk of harms associated. Even more so, substance use services are largely underutilized among youth, most only accessing support when in crisis. Few studies have explored young people's help-seeking behaviours to address substance use concerns. To address this gap, this study explored how youth perceive and experience substance use services in British Columbia (BC), Canada. METHODS: Participatory action research methods were used by partnering with BC youth (under the age of 30) from across the province who have lived and/or living experience of substance use to co-design the research protocol and materials. An initial focus group and interviews were held with 30 youth (ages 12-24) with lived and/or living experience of substance use, including alcohol, cannabis, and illicit substances. The discussions were audio-recorded, transcribed verbatim, and analyzed thematically using a data-driven approach. RESULTS: Three main themes were identified and separated by phase of service interaction, starting with: Prevention/Early intervention, where youth described feeling unworthy of support; Service accessibility, where youth encountered many barriers finding relevant substance use services and information; and Service delivery, where youth highlighted the importance of meeting them where they are at, including supporting those who have milder treatment needs and/or do not meet the diagnosis criteria of a substance use disorder. CONCLUSIONS: Our results suggest a clear need to prioritize substance use prevention and early interventions specifically targeting youth and young adults. Youth and peers with lived and/or living experience should be involved in co-designing and co-delivering such programs to ensure their relevance and credibility among youth. The current disease model of care leaves many of the needs of this population unmet, calling for a more integrated youth-centred approach to address the multifarious concerns linked to young people's substance use and service outcomes and experiences.

Real-World Comparison of Bezlotoxumab to Standard of Care Therapy for Prevention of Recurrent Clostridioides difficile Infection in Patients at High Risk for Recurrence.

BACKGROUND: Bezlotoxumab (BEZ) is a monoclonal antibody used to prevent recurrent Clostridioides difficile infection (rCDI). This study investigates BEZ effectiveness in relation to rCDI and patient-specific risk factors in a real-world setting. METHODS: A matched, retrospective cohort study was conducted from 2015 to 2019 to compare BEZ to historical standard of care (SoC) therapy with vancomycin or fidaxomicin. The primary outcome was incidence of 90-day rCDI. Secondary outcomes were incidence of all-cause hospital readmission and all-cause mortality at 90 days, infusion-related reactions, and incidence of heart failure exacerbation. Baseline confounding was addressed using inverse probability of treatment weighting (IPTW). RESULTS: Overall, 107 participants were included (54 BEZ and 53 SoC). Mean number of prior CDI episodes was 2, median number of risk factors for rCDI was 4, and 28% of participants had severe CDI. Incidence of 90-day rCDI was 11% BEZ vs 43% SoC (P = < .001) and 90-day all-cause readmission was 40% BEZ vs 64% SoC (P = .011). In IPTW-adjusted analyses, BEZ was associated with significantly reduced odds of rCDI (odds ratio [OR], 0.14 [95% confidence interval {CI}: .05-.41]) and all-cause readmission (OR, 0.36 [95% CI: .16-.81]). No safety signals were detected with BEZ use. CONCLUSIONS: BEZ is effective for the prevention of rCDI and reduction in all-cause hospital readmission for patients at high risk for recurrence, supporting current guideline recommendations.

Effectiveness of Bezlotoxumab for Prevention of Recurrent Clostridioides difficile Infection Among Transplant Recipients.

BACKGROUND: Bezlotoxumab significantly reduces the incidence of recurrent Clostridioides difficile infection (CDI); however, limited data are available in solid organ transplant (SOT) and hematopoietic cell transplant (HCT) recipients. METHODS: We conducted a single-center retrospective analysis comparing recurrent CDI in SOT and HCT recipients receiving standard of care alone (oral vancomycin, fidaxomicin, or metronidazole) or bezlotoxumab plus standard of care. The primary outcome was 90-day incidence of recurrent CDI, and secondary outcomes included 90-day hospital readmission, mortality, and incidence of heart failure exacerbation. RESULTS: Overall, 94 patients received bezlotoxumab plus standard of care (n = 38) or standard of care alone (n = 56). The mean age was 53 years; patients had a median of 3 prior Clostridioides difficile episodes and 4 risk factors for recurrent infection. Most patients were SOT recipients (76%), with median time to index CDI occurring 2.7 years after transplantation. Ninety-day recurrent CDI occurred in 16% (6/38) in the bezlotoxumab cohort compared to 29% (16/56) in the standard of care cohort (P = .13). Multivariable regression revealed that bezlotoxumab was associated with significantly lower odds of 90-day recurrent CDI (odds ratio, 0.28 [95% confidence interval, .08-.91]). There were no differences in secondary outcomes, and no heart failure exacerbations were observed. CONCLUSIONS: In a cohort of primarily SOT recipients, bezlotoxumab was well tolerated and associated with lower odds of recurrent CDI at 90 days. Larger, prospective trials are needed to confirm these findings among SOT and HCT populations.

Adverse Childhood Experiences and Problematic Media Use: Perceptions of Caregivers of High-Risk Youth.

Youth with a history of adverse childhood experiences (ACEs) are more likely to develop risky health behaviors. With the increase of media use in the general population, it is likely that these high-risk youth are developing maladaptive behaviors associated with media use (i.e., problematic media use). The goals of this article are (1) to describe symptoms of problematic media use in high-risk youth and (2) to determine whether ACEs are related to problematic media use in this population. Data were collected through online questionnaires from 348 parents or legal guardians of children ages 5 to 18 years, the majority of whom had been adopted. Parents and guardians reported on the child's history of ACEs and completed the Problematic Media Use Measure-Short Form (PMUM-SF). Almost half of the participants reported that their child had a history of four or more ACEs (48.9%). Caregivers of foster or adopted children reported more symptoms of problematic media use than those reporting on their biological children. After adjusting for covariates, the number of ACEs predicted problematic media use above and beyond variance explained by demographic factors or screen time amount. Children with a history of ACEs had higher problematic media use compared to children without ACEs.

"What We Want Is More Access ": Experiences of Supportive Cancer Care and Strategies for Advancement in a Canadian Provincial Cancer Care Organization.

OBJECTIVES: Despite calls for better supportive care, patients and families still commonly bear significant responsibility for managing the physical and mental health and social challenges of being diagnosed with and treated for cancer. As such, there is increased advocacy for integrated supportive care to ease the burden of this responsibility. The purpose of this study was to understand patient and caregiver experiences with supportive care to advance its delivery at a large provincial cancer care organization in Canada. METHOD: We used a qualitative descriptive approach to analyze focus groups with patients and caregivers from seven sites across the large provincial cancer care organization. RESULTS: Focus group participants (n = 69) included cancer patients (n = 57) and caregivers (n = 12). Participants highlighted positive and negative aspects of their experience and strategies for improvement. These are depicted in three themes: (1) improving patient and provider awareness of services; (2) increasing access; (3) enhancing coordination and integration. Participants' specific suggestions included centralizing relevant information about services, implementing a coach or navigator to help advocate for access, and delivering care virtually. CONCLUSIONS: Participants highlighted barriers to access and made suggestions for improving supportive care that they believed would reduce the burden associated with trying to manage their cancer journey.

Rapid return of children in residential care to family as a result of COVID-19: Scope, challenges, and recommendations.

BACKGROUND: As a result of the COVID-19 pandemic, some governments have mandated that residential care providers rapidly return children and youth to family. OBJECTIVE: The goal of the present study was to better understand the scope and characteristics of rapid return, and to provide data-informed recommendations for service providers working with this population. PARTICIPANTS AND SETTING: Representatives from 67 non-government organizations (NGOs) providing residential care that were government-mandated to rapidly return children and youth to family completed a brief online survey. They collectively serve 12,494 children in 14 nations. METHODS: Using a mixed methods design, results examined 1) characteristics of the rapid return mandate, 2) preparation received by children and families, 3) support services provided since the return, and 4) primary concerns for children and families. RESULTS: Data revealed that rapid return was characterized by compressed timelines that did not allow for adequate child and family assessment and preparation. However, all respondents indicated they believed at least some families would be able to remain intact safely with appropriate support. Primary concerns for children and families related to unresolved antecedents to separation, lack of economic capacity, limited monitoring, and lack of access to education. CONCLUSIONS: Based on the findings, 9 recommendations were made for service providers working with children and families that have been rapidly reunified as a result of the COVID-19 pandemic.

Data-informed recommendations for services providers working with vulnerable children and families during the COVID-19 pandemic.

BACKGROUND: The COVID-19 pandemic and associated response measures have led to unprecedented challenges for service providers working with vulnerable children and families around the world. OBJECTIVE: The goal of the present study was to better understand the impact of the pandemic and associated response measures on vulnerable children and families and provide data-informed recommendations for public and private service providers working with this population. PARTICIPANTS AND SETTING: Representatives from 87 non-government organizations (NGOs) providing a variety of direct services (i.e. residential care, family preservation, foster care, etc.) to 454,637 vulnerable children and families in 43 countries completed a brief online survey. METHODS: Using a mixed methods design, results examined 1) ways in which children and families have been directly impacted by COVID-19, 2) the impact of the pandemic on services provided by NGOs, 3) government responses and gaps in services for this population during the pandemic, and 4) strategies that have been effective in filling these gaps. RESULTS: Data revealed that the pandemic and restrictive measures were associated with increased risk factors for vulnerable children and families, including not having access to vital services. The NGOs experienced government restrictions, decreased financial support, and inability to adequately provide services. Increased communication and supportive activities had a positive impact on both NGO staff and the families they serve. CONCLUSIONS: Based on the findings, ten recommendations were made for service providers working with vulnerable children and families during the COVID-19 pandemic.

Evidências de Validade da Champion's Health Belief Model Scale para o Brasil / Evidencias de Validez de la Champion's Health Belief Model Scale para Brasil / Champion's Health Belief Model Scale Validity Evidence for Brazil

Resumo Objetivo Realizar tradução e adaptação transcultural da Champion's Health Belief Model Scale (CHBMS) para uso no Brasil, no rastreamento mamográfico, e verificar as evidências de validade da versão brasileira desta escala. Métodos Estudo metodológico, realizado com 206 mulheres frequentadoras de unidade básica de saúde, na cidade de Fortaleza-CE, Brasil, de agosto de 2015 a dezembro de 2017. A escala passou por processo de tradução e adaptação transcultural, incluindo validação de face e conteúdo. Posteriormente, foram verificadas as evidências de validade (1.Validade baseada na estrutura interna, avaliada mediante análise exploratória, com rotação ortogonal varimax e retenção de fatores por análises paralelas; 2. Confiabilidade, a partir da homogeneidade por alfa de Cronbach e estabilidade por teste-reteste). Resultados Na validação de face e conteúdo, o instrumento apresentou boa aceitação entre os juízes e o público-alvo. O modelo final da análise fatorial exploratória resultou em escala com sete itens, divididos em três domínios, com variância explicada de 71,4%, com alfa de Cronbach variando de 0,50 a 0,88. Para confiabilidade da escala, o r de Pearson e o ρ de Spearman mostraram alta confiabilidade (0,997 e 0,986). Conclusão A versão brasileira da Champion's Health Belief Model Scale apresenta boas evidências de validade baseada na estrutura interna e é confiável, podendo ser empregada no Brasil para avaliação do monitoramento da adesão à mamografia.

Resumen Objetivo Realizar la traducción y adaptación transcultural de la Champion's Health Belief Model Scale (CHBMS) para su aplicación en Brasil en el rastreo mamográfico y para verificar las evidencias de validez de la versión brasileña de esta escala. Métodos Estudio metodológico, realizado con 206 mujeres que asistían con frecuencia a una unidad básica de salud, en la ciudad de Fortaleza, estado de Ceará, Brasil, de agosto de 2015 a diciembre de 2017. La escala pasó por un proceso de traducción y adaptación transcultural, que incluyó validación aparente y de contenido. Posteriormente, se verificaron las evidencias de validez (1. Validez basada en la estructura interna, evaluada mediante análisis exploratorio, con rotación ortogonal varimax y retención de factores por análisis paralelos; 2. Confiabilidad a partir de la homogeneidad por alfa de Cronbach y estabilidad por test-retest). Resultados En la validación aparente y de contenido, el instrumento presentó buena aceptación entre los jueces y el público destinatario. El modelo final del análisis factorial exploratorio tuvo como resultado una escala con siete ítems, divididos en tres dominios, con varianza explicada de 71,4%, con alfa de Cronbach que varía de 0,50 a 0,88. En la confiabilidad de la escala, el r de Pearson y el ρ de Spearman demostraron alta confiabilidad (0,997 y 0,986). Conclusión La versión brasileña de la Champion's Health Belief Model Scale presenta buenas evidencias de validez basada en la estructura interna y es confiable, por lo que puede emplearse en Brasil para evaluar el monitoreo de la adherencia a la mamografía.

Abstract Objective Performing translation and cross-cultural adaptation of the Champion's Health Belief Model Scale (CHBMS) for use in Brazil for mammographic screening, and verify the validity evidence of the Brazilian version of this scale. Methods Methodological study, conducted with 206 women attending a Basic Health Unit, in the city of Fortaleza, state of Ceará, Brazil, from August 2015 to December 2017. The scale went through a process of translation and cross-cultural adaptation, including face and content validation. Afterwards, validity evidence was verified (1.Vality based on internal structure, assessed by exploratory analysis, with varimax orthogonal rotation and retention of factors by parallel analyzes; 2. Reliability from Cronbach's alpha homogeneity and test-retest stability). Results In face and content validation, the tool showed good acceptance among the judges and the target audience. The final exploratory factor analysis model resulted in a seven-item scale, divided into three domains, with an explained variance of 71.4%, with Cronbach's alpha ranging from 0.50 to 0.88. For scale reliability, Pearson r and Spearman ρ showed high reliability (0.997 and 0.986). Conclusion The Brazilian version of Champion's Health Belief Model Scale provides good evidence of validity based on internal structure and is reliable. It may be used in Brazil to assess mammography compliance monitoring.

Attention focus and self-touch in toddlers: The moderating effect of attachment security.

The superior self-regulation and attention-regulation abilities of securely attached children have been repeatedly demonstrated. However, the mechanisms that allow securely attached children to exhibit higher levels of attention focus than insecurely attached (anxious-ambivalent and anxious-avoidant) children need to be explored. One possible mechanism that has been hypothesized to play a role in focusing attention is self-touch. Previous research has shown that 10-year-old children exhibit more bilateral self-touch (i.e., both hands are simultaneously moving onto each other or on the body, and both hands are in contact with each other or with the body), but not lateral self-touch (i.e., one hand is moving on the other hand or on the body, and the hand is in contact with the other hand or with the body), when they focus attention on a task. Because bilateral coordination is still developing during childhood, we expected that lateral self-touch, instead of bilateral self-touch, may be associated with attention focus for toddlers. The objectives of the present study were to examine whether securely attached toddlers exhibit more self-touch, particularly lateral self-touch, while they focus on a task than while they do not focus on a task. We expected to find that the association between lateral self-touch and attention focus is not as strong for insecurely attached toddlers. Data from forty-nine mother-child dyads were employed for analyses. The attachment classification of the children was determined using the Strange Situation. The duration of attention focus and self-touch behavior during a reading task were coded. An association between lateral self-touch and attention focus was found for children of all attachment classifications. This association was particularly strong for securely attached children. We discuss the possibility that securely attached toddlers may use lateral self-touch to regulate attention.

MENTAL STATE LANGUAGE DEVELOPMENT: THE LONGITUDINAL ROLES OF ATTACHMENT AND MATERNAL LANGUAGE.

Maternal mental state language is thought to influence children's mental state language and sociocognitive understanding (e.g., theory of mind), but the mechanism is unclear. The current study examined the longitudinal development of mental state language in mother-child interactions. The methodology included assessments of the child and/or mother-child dyad at six time points between 12 to 52 months of the child's age. Measures determined child's attachment style and language abilities, and mental state language used by mother and child during a block-building task. Results showed that (a) mental state talk, including belief and desire language, increased over time; (b) there were differences between the type of mental state words used by the mother in insecure versus secure dyads; (c) there were differences in patterns of mental state words used in both mothers and children in insecure versus secure dyads; and (d) attachment appeared to exert a consistent influence over time.

Decrease in Behavioral Problems and Trauma Symptoms Among At-Risk Adopted Children Following Web-Based Trauma-Informed Parent Training Intervention.

Children who have experienced early adversities are at risk for behavioral problems and trauma symptoms. Using a two-group, pre-post intervention design, the authors evaluated the effectiveness of an online parent training for Trust-Based Relational Intervention, a trauma-informed, attachment-based intervention, in reducing behavioral problems and trauma symptoms in at-risk adopted children. Children of parents in the treatment group (n = 48) demonstrated significant decreases in behavioral problems and trauma symptoms after intervention. Scores for children in a matched-sample control group did not change. Findings suggest this intervention can effectively reduce behavioral problems and trauma symptoms in children with histories of adversities.

Decrease in Behavioral Problems and Trauma Symptoms Among At-Risk Adopted Children Following Trauma-Informed Parent Training Intervention.

Children who have experienced early adversities are at risk for behavioral problems and trauma symptoms. Using a two-group, pre-post intervention design, the current study evaluated the effectiveness of a parent training utilizing Trust-Based Relational Intervention, a trauma-informed, attachment-based intervention, in reducing behavioral problems and trauma symptoms in at-risk adopted children. Children of parents in the treatment group (n = 48) demonstrated significant decreases in behavioral problems on the Strengths and Difficulties Questionnaire and significant decreases in trauma symptoms on the Trauma Symptoms Checklist for Young Children after intervention. Scores for children in a matched-sample control group did not change. Findings suggest that Trust-Based Relational Intervention is effective at addressing many behavioral problems and trauma symptoms in children with histories of adversities.

Formation of orthopoxvirus cytoplasmic A-type inclusion bodies and embedding of virions are dynamic processes requiring microtubules.

In cells infected with some orthopoxviruses, numerous mature virions (MVs) become embedded within large, cytoplasmic A-type inclusions (ATIs) that can protect infectivity after cell lysis. ATIs are composed of an abundant viral protein called ATIp, which is truncated in orthopoxviruses such as vaccinia virus (VACV) that do not form ATIs. To study ATI formation and occlusion of MVs within ATIs, we used recombinant VACVs that express the cowpox full-length ATIp or we transfected plasmids encoding ATIp into cells infected with VACV, enabling ATI formation. ATI enlargement and MV embedment required continued protein synthesis and an intact microtubular network. For live imaging of ATIs and MVs, plasmids expressing mCherry fluorescent protein fused to ATIp were transfected into cells infected with VACV expressing the viral core protein A4 fused to yellow fluorescent protein. ATIs appeared as dynamic, mobile bodies that enlarged by multiple coalescence events, which could be prevented by disrupting microtubules. Coalescence of ATIs was confirmed in cells infected with cowpox virus. MVs were predominantly at the periphery of ATIs early in infection. We determined that coalescence contributed to the distribution of MVs within ATIs and that microtubule-disrupting drugs abrogated coalescence-mediated MV embedment. In addition, MVs were shown to move from viral factories at speeds consistent with microtubular transport to the peripheries of ATIs, whereas disruption of microtubules prevented such trafficking. The data indicate an important role for microtubules in the coalescence of ATIs into larger structures, transport of MVs to ATIs, and embedment of MVs within the ATI matrix.

Congregation of orthopoxvirus virions in cytoplasmic A-type inclusions is mediated by interactions of a bridging protein (A26p) with a matrix protein (ATIp) and a virion membrane-associated protein (A27p).

Some orthopoxviruses, e.g., the cowpox, ectromelia, and raccoonpox viruses, form large, discrete cytoplasmic inclusions within which mature virions (MVs) are embedded by a process called occlusion. These inclusions, which may protect occluded MVs in the environment, are composed of aggregates of the A-type inclusion protein (ATIp), which is truncated in orthopoxviruses such as vaccinia virus (VACV) and variola virus that fail to form inclusions. In addition to an intact ATIp, occlusion requires the A26 protein (A26p). Although VACV contains a functional A26p, determined by complementation of a cowpox virus occlusion-defective mutant, its role in occlusion was unknown. We found that restoration of the full-length ATI gene was sufficient for VACV inclusion formation and the ensuing occlusion of MVs. A26p was present in inclusions even when virion assembly was inhibited, suggesting a direct interaction of A26p with ATIp. Analysis of a panel of ATIp mutants indicated that the C-terminal repeat region was required for inclusion formation and the N-terminal domain for interaction with A26p and occlusion. A26p is tethered to MVs via interaction with the A27 protein (A27p); A27p was not required for association of A26p with ATIp but was necessary for occlusion. In addition, the C-terminal domain of A26p, which mediates A26p-A27p interactions, was necessary but insufficient for occlusion. Taken together, the data suggest a model for occlusion in which A26p has a bridging role between ATIp and A27p, and A27p provides a link to the MV membrane.

Activation of the stress protein response inhibits the STAT1 signalling pathway and iNOS function in alveolar macrophages: role of Hsp90 and Hsp70.

BACKGROUND AND AIM: Alveolar fluid clearance is impaired by inducible nitric oxide synthase (iNOS)/nitric oxide (NO)-dependent mechanisms in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). The activation of the stress protein response (SPR) in alveolar macrophages on iNOS-dependent NO production in response to interferon gamma (IFNgamma), a major cytokine present in the airspace of patients with ALI, was investigated. METHODS: The SPR was activated in murine and primary human alveolar macrophages prior to analysis of signal transducer and activator of transcription factor 1 (STAT1) activation, iNOS mRNA and protein synthesis, and NO production. RESULTS: SPR activation resulted in inhibition of IFNgamma-mediated NO production (p=0.001) with >95% detergent insolubilisation of the STAT1 protein. Its subsequent proteasomal degradation was partially reversed with pretreatment of cells with the chemical chaperone glycerol. This early effect of the SPR was caused by the complete disruption of heat shock protein 90 (Hsp90)-STAT1 binding, as shown by immunoprecipitation. Recovery of STAT1 activation and recovery of iNOS synthesis occurred within 12 h after SPR activation (p=0.02). NO production (as compared with non-SPR controls) did not occur until 48 h later (p=0.02). SPR-induced Hsp70 (Hsp70i) expression caused a late inhibition of NO production (p=0.02). Inhibiting >50% Hsp70i expression recovered NO production to control levels whereas overexpressing Hsp70i in the absence of the SPR inhibited NO production (p=0.02). CONCLUSION: Early inhibition of STAT1 following its dissociation from Hsp90, and later inhibition of iNOS activity by Hsp70i, represent novel mechanisms by which SPR activation modulates the IFNgamma signalling in alveolar macrophages. These results highlight a potential clinical application for Hsp90 inhibitors in modulating NO signalling during the early phase of acute lung injury.