A pilot study of multifamily therapy group for young adults with anorexia nervosa: Reconnecting for recovery.

OBJECTIVE: We tested the feasibility, acceptability, and preliminary effect sizes on outcome measures of Reconnecting for Recovery (R4R) Multifamily Therapy Group for young adults with anorexia nervosa (AN). METHOD: Ten participants (mean age = 23 years, SD = 3.6) meeting Diagnostic and Statistical Manual of Mental Disorders 5th Edition criteria for a restrictive eating disorder (AN or other specified feeding and eating disorder) and 14 family members received 16 R4R outpatient sessions over 26 weeks. Feasibility and acceptability were evaluated by recruitment and retention rates and patient/family member suitability scores. Outcomes were determined utilizing the Eating Disorder Examination (EDE), weight (body mass index), and Difficulties in Emotion Regulation Scale. RESULTS: All participants and 12 family members were retained, and the majority found R4R acceptable. EDE global score and lack of emotional awareness improved significantly from baseline (BL) to end-of-treatment (EOT) and BL to 6-month follow-up (6MFU) with moderate to large effect sizes (0.47-1.41). Limited access to emotion regulation strategies (LAERS) improved significantly from BL to 6MFU (moderate effect size; 0.57). Improvements in LAERS from BL to EOT (0.32) and weight from BL to EOT and BL to 6MFU were not significant (effect sizes 0.16-0.22). DISCUSSION: Findings provide preliminary evidence that R4R is feasible, acceptable, and produces clinically significant changes in targeted outcomes.

A Universal Next-Generation Sequencing Protocol To Generate Noninfectious Barcoded cDNA Libraries from High-Containment RNA Viruses.

Several biosafety level 3 and/or 4 (BSL-3/4) pathogens are high-consequence, single-stranded RNA viruses, and their genomes, when introduced into permissive cells, are infectious. Moreover, many of these viruses are select agents (SAs), and their genomes are also considered SAs. For this reason, cDNAs and/or their derivatives must be tested to ensure the absence of infectious virus and/or viral RNA before transfer out of the BSL-3/4 and/or SA laboratory. This tremendously limits the capacity to conduct viral genomic research, particularly the application of next-generation sequencing (NGS). Here, we present a sequence-independent method to rapidly amplify viral genomic RNA while simultaneously abolishing both viral and genomic RNA infectivity across multiple single-stranded positive-sense RNA (ssRNA+) virus families. The process generates barcoded DNA amplicons that range in length from 300 to 1,000 bp, which cannot be used to rescue a virus and are stable to transport at room temperature. Our barcoding approach allows for up to 288 barcoded samples to be pooled into a single library and run across various NGS platforms without potential reconstitution of the viral genome. Our data demonstrate that this approach provides full-length genomic sequence information not only from high-titer virion preparations but it can also recover specific viral sequence from samples with limited starting material in the background of cellular RNA, and it can be used to identify pathogens from unknown samples. In summary, we describe a rapid, universal standard operating procedure that generates high-quality NGS libraries free of infectious virus and infectious viral RNA. IMPORTANCE This report establishes and validates a standard operating procedure (SOP) for select agents (SAs) and other biosafety level 3 and/or 4 (BSL-3/4) RNA viruses to rapidly generate noninfectious, barcoded cDNA amenable for next-generation sequencing (NGS). This eliminates the burden of testing all processed samples derived from high-consequence pathogens prior to transfer from high-containment laboratories to lower-containment facilities for sequencing. Our established protocol can be scaled up for high-throughput sequencing of hundreds of samples simultaneously, which can dramatically reduce the cost and effort required for NGS library construction. NGS data from this SOP can provide complete genome coverage from viral stocks and can also detect virus-specific reads from limited starting material. Our data suggest that the procedure can be implemented and easily validated by institutional biosafety committees across research laboratories.

Distinct differences in the responses of the human pancreatic ß-cell line EndoC-ßH1 and human islets to proinflammatory cytokines.

While insulinoma cells have been developed and proven to be extremely useful in studies focused on mechanisms controlling ß-cell function and viability, translating findings to human ß-cells has proven difficult because of the limited access to human islets and the absence of suitable insulinoma cell lines of human origin. Recently, a human ß-cell line, EndoC-ßH1, has been derived from human fetal pancreatic buds. The purpose of this study was to determine whether human EndoC-ßH1 cells respond to cytokines in a fashion comparable to human islets. Unlike most rodent-derived insulinoma cell lines that respond to cytokines in a manner consistent with rodent islets, EndoC-ßH1 cells fail to respond to a combination of cytokines (IL-1, IFN-γ, and TNF) in a manner consistent with human islets. Nitric oxide, produced following inducible nitric oxide synthase (iNOS) expression, is a major mediator of cytokine-induced human islet cell damage. We show that EndoC-ßH1 cells fail to express iNOS or produce nitric oxide in response to this combination of cytokines. Inhibitors of iNOS prevent cytokine-induced loss of human islet cell viability; however, they do not prevent cytokine-induced EndoC-ßH1 cell death. Stressed human islets or human islets expressing heat shock protein 70 (HSP70) are resistant to cytokines, and, much like stressed human islets, EndoC-ßH1 cells express HSP70 under basal conditions. Elevated basal expression of HSP70 in EndoC-ßH1 cells is consistent with the lack of iNOS expression in response to cytokine treatment. While expressing HSP70, EndoC-ßH1 cells fail to respond to endoplasmic reticulum stress activators, such as thapsigargin. These findings indicate that EndoC-ßH1 cells do not faithfully recapitulate the response of human islets to cytokines. Therefore, caution should be exercised when making conclusions regarding the actions of cytokines on human islets when using this human-derived insulinoma cell line.

How the location of superoxide generation influences the ß-cell response to nitric oxide.

Cytokines impair the function and decrease the viability of insulin-producing ß-cells by a pathway that requires the expression of inducible nitric oxide synthase (iNOS) and generation of high levels of nitric oxide. In addition to nitric oxide, excessive formation of reactive oxygen species, such as superoxide and hydrogen peroxide, has been shown to cause ß-cell damage. Although the reaction of nitric oxide with superoxide results in the formation of peroxynitrite, we have shown that ß-cells do not have the capacity to produce this powerful oxidant in response to cytokines. When ß-cells are forced to generate peroxynitrite using nitric oxide donors and superoxide-generating redox cycling agents, superoxide scavenges nitric oxide and prevents the inhibitory and destructive actions of nitric oxide on mitochondrial oxidative metabolism and ß-cell viability. In this study, we show that the ß-cell response to nitric oxide is regulated by the location of superoxide generation. Nitric oxide freely diffuses through cell membranes, and it reacts with superoxide produced within cells and in the extracellular space, generating peroxynitrite. However, only when it is produced within cells does superoxide attenuate nitric oxide-induced mitochondrial dysfunction, gene expression, and toxicity. These findings suggest that the location of radical generation and the site of radical reactions are key determinants in the functional response of ß-cells to reactive oxygen species and reactive nitrogen species. Although nitric oxide is freely diffusible, its biological function can be controlled by the local generation of superoxide, such that when this reaction occurs within ß-cells, superoxide protects ß-cells by scavenging nitric oxide.

A gammaherpesvirus Bcl-2 ortholog blocks B cell receptor-mediated apoptosis and promotes the survival of developing B cells in vivo.

Gammaherpesviruses such as Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8) establish lifelong latency in their hosts and are associated with the development of several types of malignancies, including a subset of B cell lymphomas. These viruses are thought to co-opt the process of B cell differentiation to latently infect a fraction of circulating memory B cells, resulting in the establishment of a stable latency setpoint. However, little is known about how this infected memory B cell compartment is maintained throughout the life of the host. We have previously demonstrated that immature and transitional B cells are long-term latency reservoirs for murine gammaherpesvirus 68 (MHV68), suggesting that infection of developing B cells contributes to the maintenance of lifelong latency. During hematopoiesis, immature and transitional B cells are subject to B cell receptor (BCR)-mediated negative selection, which results in the clonal deletion of autoreactive B cells. Interestingly, numerous gammaherpesviruses encode homologs of the anti-apoptotic protein Bcl-2, suggesting that virus inhibition of apoptosis could subvert clonal deletion. To test this, we quantified latency establishment in mice inoculated with MHV68 vBcl-2 mutants. vBcl-2 mutant viruses displayed a marked decrease in the frequency of immature and transitional B cells harboring viral genome, but this attenuation could be rescued by increased host Bcl-2 expression. Conversely, vBcl-2 mutant virus latency in early B cells and mature B cells, which are not targets of negative selection, was remarkably similar to wild-type virus. Finally, in vivo depletion of developing B cells during chronic infection resulted in decreased mature B cell latency, demonstrating a key role for developing B cells in the maintenance of lifelong latency. Collectively, these findings support a model in which gammaherpesvirus latency in circulating mature B cells is sustained in part through the recurrent infection and vBcl-2-mediated survival of developing B cells.

Modulation of B-cell tolerance by murine gammaherpesvirus 68 infection: requirement for Orf73 viral gene expression and follicular helper T cells.

Viruses such as Epstein-Barr virus (EBV) have been linked to mechanisms that support autoantibody production in diseases such as systemic lupus erythematosus. However, the mechanisms by which viruses contribute to autoantibody production remain poorly defined. This stems in part, from the high level of seropositivity for EBV (> 95%) and the exquisite species specificity of EBV. In this study we overcame these problems by using murine gammaherpesvirus 68 (MHV68), a virus genetically and biologically related to EBV. We first showed that MHV68 drives autoantibody production by promoting a loss of B-cell anergy. We next showed that MHV68 infection resulted in the expansion of follicular helper T (Tfh) cells in vivo, and that these Tfh cells supported autoantibody production and a loss of B-cell anergy. Finally, we showed that the expansion of Tfh cells and autoantibody production was dependent on the establishment of viral latency and expression of a functional viral gene called Orf73. Collectively, our studies highlighted an unexpected role for viral latency in the development of autoantibodies following MHV68 infection and suggest that virus-induced expansion of Tfh cells probably plays a key role in the loss of B-cell anergy.

Ccr5 regulates inflammatory gene expression in response to encephalomyocarditis virus infection.

Encephalomyocarditis virus (EMCV) is capable of stimulating inflammatory gene expression by macrophages as a result of interactions between EMCV capsid proteins and cell surface receptors. In this study, biochemical and genetic approaches identified a role for Ccr5, a chemokine receptor, in transducing the signals of EMCV infection that result in the expression of inflammatory genes in macrophages. Antibody neutralization and gene knockout strategies were used to show that the presence of Ccr5 is required for EMCV-stimulated mitogen-activated protein (MAP) kinase and nuclear factor-kappa B (NF-κB) activation, and the subsequent expression of the inflammatory gene-inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2). Ccr5 appears to participate in the early control of virus replication: EMCV mRNA accumulates to sevenfold higher levels in Ccr5-deficient mice when compared to wild-type controls. These findings support a regulatory role for Ccr5 in the antiviral response to EMCV in which this chemokine receptor participates in regulation of inflammatory gene expression in response to virus infection.