Both Humoral and Cellular Immunity Limit the Ability of Live Attenuated Influenza Vaccines to Promote T Cell Responses.

One potential advantage of live attenuated influenza vaccines (LAIVs) is their ability to establish both virus-specific Ab and tissue-resident memory T cells (TRM) in the respiratory mucosa. However, it is hypothesized that pre-existing immunity from past infections and/or immunizations prevents LAIV from boosting or generating de novo CD8+ T cell responses. To determine whether we can overcome this limitation, we generated a series of drifted influenza A/PR8 LAIVs with successive mutations in the hemagglutinin protein, allowing for increasing levels of escape from pre-existing Ab. We also inserted a CD8+ T cell epitope from the Sendai virus nucleoprotein (NP) to assess both generation of a de novo T cell response and boosting of pre-existing influenza-specific CD8+ T cells following LAIV immunization. Increasing the level of escape from Ab enabled boosting of pre-existing TRM, but we were unable to generate de novo Sendai virus NP+ CD8+ TRM following LAIV immunization in PR8 influenza-immune mice, even with LAIV strains that can fully escape pre-existing Ab. As these data suggested a role for cell-mediated immunity in limiting LAIV efficacy, we investigated several scenarios to assess the impact of pre-existing LAIV-specific TRM in the upper and lower respiratory tract. Ultimately, we found that deletion of the immunodominant influenza NP366-374 epitope allowed for sufficient escape from cellular immunity to establish de novo CD8+ TRM. When combined, these studies demonstrate that both pre-existing humoral and cellular immunity can limit the effectiveness of LAIV, which is an important consideration for future design of vaccine vectors against respiratory pathogens.

Influenza A virus coinfection dynamics are shaped by distinct virus-virus interactions within and between cells.

When multiple viral populations propagate within the same host environment, they often shape each other's dynamics. These interactions can be positive or negative and can occur at multiple scales, from coinfection of a cell to co-circulation at a global population level. For influenza A viruses (IAVs), the delivery of multiple viral genomes to a cell substantially increases burst size. However, despite its relevance for IAV evolution through reassortment, the implications of this positive density dependence for coinfection between distinct IAVs has not been explored. Furthermore, the extent to which these interactions within the cell shape viral dynamics at the level of the host remains unclear. Here we show that, within cells, diverse coinfecting IAVs strongly augment the replication of a focal strain, irrespective of their homology to the focal strain. Coinfecting viruses with a low intrinsic reliance on multiple infection offer the greatest benefit. Nevertheless, virus-virus interactions at the level of the whole host are antagonistic. This antagonism is recapitulated in cell culture when the coinfecting virus is introduced several hours prior to the focal strain or under conditions conducive to multiple rounds of viral replication. Together, these data suggest that beneficial virus-virus interactions within cells are counterbalanced by competition for susceptible cells during viral propagation through a tissue. The integration of virus-virus interactions across scales is critical in defining the outcomes of viral coinfection.

Utility of Human In Vitro Data in Risk Assessments of Influenza A Virus Using the Ferret Model.

As influenza A viruses (IAV) continue to cross species barriers and cause human infection, the establishment of risk assessment rubrics has improved pandemic preparedness efforts. In vivo pathogenicity and transmissibility evaluations in the ferret model represent a critical component of this work. As the relative contribution of in vitro experimentation to these rubrics has not been closely examined, we sought to evaluate to what extent viral titer measurements over the course of in vitro infections are predictive or correlates of nasal wash and tissue measurements for IAV infections in vivo. We compiled data from ferrets inoculated with an extensive panel of over 50 human and zoonotic IAV (inclusive of swine-origin and high- and low-pathogenicity avian influenza viruses associated with human infection) under a consistent protocol, with all viruses concurrently tested in a human bronchial epithelial cell line (Calu-3). Viral titers in ferret nasal wash specimens and nasal turbinate tissue correlated positively with peak titer in Calu-3 cells, whereas additional phenotypic and molecular determinants of influenza virus virulence and transmissibility in ferrets varied in their association with in vitro viral titer measurements. Mathematical modeling was used to estimate more generalizable key replication kinetic parameters from raw in vitro viral titers, revealing commonalities between viral infection progression in vivo and in vitro. Meta-analyses inclusive of IAV that display a diverse range of phenotypes in ferrets, interpreted with mathematical modeling of viral kinetic parameters, can provide critical information supporting a more rigorous and appropriate contextualization of in vitro experiments toward pandemic preparedness. IMPORTANCE Both in vitro and in vivo models are employed for assessing the pandemic potential of novel and emerging influenza A viruses in laboratory settings, but systematic examinations of how well viral titer measurements obtained in vitro align with results from in vivo experimentation are not frequently performed. We show that certain viral titer measurements following infection of a human bronchial epithelial cell line are positively correlated with viral titers in specimens collected from virus-inoculated ferrets and employ mathematical modeling to identify commonalities between viral infection progression between both models. These analyses provide a necessary first step in enhanced interpretation and incorporation of in vitro-derived data in risk assessment activities and highlight the utility of employing mathematical modeling approaches to more closely examine features of virus replication not identifiable by experimental studies alone.

A Multi-center Genome-wide Association Study of Cervical Dystonia.

BACKGROUND: Several monogenic causes for isolated dystonia have been identified, but they collectively account for only a small proportion of cases. Two genome-wide association studies have reported a few potential dystonia risk loci; but conclusions have been limited by small sample sizes, partial coverage of genetic variants, or poor reproducibility. OBJECTIVE: To identify robust genetic variants and loci in a large multicenter cervical dystonia cohort using a genome-wide approach. METHODS: We performed a genome-wide association study using cervical dystonia samples from the Dystonia Coalition. Logistic and linear regressions, including age, sex, and population structure as covariates, were employed to assess variant- and gene-based genetic associations with disease status and age at onset. We also performed a replication study for an identified genome-wide significant signal. RESULTS: After quality control, 919 cervical dystonia patients compared with 1491 controls of European ancestry were included in the analyses. We identified one genome-wide significant variant (rs2219975, chromosome 3, upstream of COL8A1, P-value 3.04 × 10-8 ). The association was not replicated in a newly genotyped sample of 473 cervical dystonia cases and 481 controls. Gene-based analysis identified DENND1A to be significantly associated with cervical dystonia (P-value 1.23 × 10-6 ). One low-frequency variant was associated with lower age-at-onset (16.4 ± 2.9 years, P-value = 3.07 × 10-8 , minor allele frequency = 0.01), located within the GABBR2 gene on chromosome 9 (rs147331823). CONCLUSION: The genetic underpinnings of cervical dystonia are complex and likely consist of multiple distinct variants of small effect sizes. Larger sample sizes may be needed to provide sufficient statistical power to address the presumably multi-genic etiology of cervical dystonia. © 2021 International Parkinson and Movement Disorder Society.

Genome-wide analysis of the H3K4 histone demethylase RBP2 reveals a transcriptional program controlling differentiation.

Retinoblastoma protein (pRB) mediates cell-cycle withdrawal and differentiation by interacting with a variety of proteins. RB-Binding Protein 2 (RBP2) has been shown to be a key effector. We sought to determine transcriptional regulation by RBP2 genome-wide by using location analysis and gene expression profiling experiments. We describe that RBP2 shows high correlation with the presence of H3K4me3 and its target genes are separated into two functionally distinct classes: differentiation-independent and differentiation-dependent genes. The former class is enriched by genes that encode mitochondrial proteins, while the latter is represented by cell-cycle genes. We demonstrate the role of RBP2 in mitochondrial biogenesis, which involves regulation of H3K4me3-modified nucleosomes. Analysis of expression changes upon RBP2 depletion depicted genes with a signature of differentiation control, analogous to the changes seen upon reintroduction of pRB. We conclude that, during differentiation, RBP2 exerts inhibitory effects on multiple genes through direct interaction with their promoters.

Viral expansion after transfer is a primary driver of influenza A virus transmission bottlenecks.

For many viruses, narrow bottlenecks acting during transmission sharply reduce genetic diversity in a recipient host relative to the donor. Since genetic diversity represents adaptive potential, such losses of diversity are though to limit the opportunity for viral populations to undergo antigenic change and other adaptive processes. Thus, a detailed picture of evolutionary dynamics during transmission is critical to understanding the forces driving viral evolution at an epidemiologic scale. To advance this understanding, we used a novel barcoded virus library and a guinea pig model of transmission to decipher where in the transmission process diversity is lost for influenza A viruses. In inoculated guinea pigs, we show that a high level of viral genetic diversity is maintained across time. Continuity in the barcodes detected furthermore indicates that stochastic effects are not pronounced within inoculated hosts. Importantly, in both aerosol-exposed and direct contact-exposed animals, we observed many barcodes at the earliest time point(s) positive for infectious virus, indicating robust transfer of diversity through the environment. This high viral diversity is short-lived, however, with a sharp decline seen 1-2 days after initiation of infection. Although major losses of diversity at transmission are well described for influenza A virus, our data indicate that events that occur following viral transfer and during the earliest stages of natural infection have a predominant role in this process. This finding suggests that immune selection may have greater opportunity to operate during influenza A transmission than previously recognized.

Observations of Oncologists on Treatment Selection With Interim Positron Emission Tomography-Adapted Approaches in Classic Hodgkin Lymphoma: The Real-World CONNECT Study.

PURPOSE: We surveyed oncologists who treat classic Hodgkin lymphoma (cHL) as part of the CONNECT study to understand the treatment decision-making process, including the impact of positron emission tomography/computed tomography (PET/CT) imaging. METHODS: US physicians self-identifying as oncologists, hematologists, or hematologists/oncologists with ≥2 years of practice experience who treated ≥1 adult with stage III/IV cHL in the frontline setting in the last year were surveyed (October 19-November 16, 2020). Physician demographics, guideline adherence, and PET/CT utilization, interpretation, and access barriers were assessed. RESULTS: In total, 301 physicians participated in the survey. Eighty-eight percent of physicians gave somewhat-to-significant consideration to NCCN guidelines. Most physicians (94%; n = 284) reported obtaining a PET/CT scan at diagnosis; of these physicians, 97% reported obtaining an interim PET/CT scan for stage III/IV cHL, with 65% typically obtaining an interim PET/CT scan after cycle 2. The Deauville 5-point scale (5PS) was the primary scoring system used to review PET/CT results by 62% of physicians, with a positive score defined as ≥3 by 44%, ≥4 by 37%, and ≥2 by 12% of physicians. Fifty-five percent of physicians reported difficulty in obtaining PET/CT scans. CONCLUSION: Although most physicians considered NCCN guidelines when treating patients with stage III/IV cHL, interim PET/CT scans after cycle 2 were not universally obtained. When PET/CT scans were obtained, Deauville 5PS scores were not always provided, and variability existed on what defined a positive score. These findings suggest that opportunities exist for education and improved PET-adapted treatment approaches.

Effect of mutant ß-catenin on liver growth homeostasis and hepatocarcinogenesis in transgenic mice.

BACKGROUND: Mutations in the Wnt signalling pathway molecule ß-catenin are associated with liver cancer. AIMS: Our aim was to confirm the effects of stabilized ß-catenin on liver growth, identify whether those effects were reversible and cell autonomous or non-cell autonomous and to model ß-catenin-induced liver cancer in mice. METHODS: Using a liver-specific inducible promoter, we generated transgenic mice in which the expression of mutant ß-catenin can be induced or repressed within hepatocytes in mice of different ages. RESULTS: Similar to other models, the hepatic expression of mutant ß-catenin in our model beginning in utero or induced in quiescent adult liver resulted in a two-fold liver enlargement and development of disease with a latency of 1-5 months, and mice displayed elevated blood ammonia and altered hepatic gene expression. Our model additionally allowed us to discover that molecular and phenotypic abnormalities were reversible following the inhibition of transgene expression. Hepatocyte transplant studies indicated that mutant ß-catenin could not increase the growth of transgene-expressing foci in either growth-permissive or -restrictive hepatic environments, but still directly altered hepatocyte gene expression. Mice with continuous but focal transgene expression developed hepatic neoplasms after the age of 1 year. CONCLUSIONS: Our findings indicate that hepatocyte gene expression is directly affected by mutant ß-catenin in a cell autonomous manner. However, hepatomegaly associated with diffuse hepatocyte-specific expression of mutant ß-catenin is secondary to liver functional alteration or non-cell autonomous. Both phenotypes are reversible. Nevertheless, some foci of transgene-expressing cells progressed to carcinoma, confirming the association of mutant ß-catenin with liver cancer.

Author Correction: Asthma and/or hay fever as predictors of fertility/impaired fecundity in U.S. women: National Survey of Family Growth.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A method for the unbiased quantification of reassortment in segmented viruses.

Reassortment of segmented viruses can be an important source of genetic diversity underlying viral evolution and emergence. Methods for the quantification of reassortment have been described but are often cumbersome and best suited for the analysis of reassortment between highly divergent parental strains. While it is useful to understand the potential of divergent parents to reassort, outcomes of such heterologous reassortment are driven by differential selection acting on the progeny and are typically strain specific. To quantify reassortment robustly, a system free of differential selection is needed. We have generated such a system for influenza A virus and for mammalian orthoreovirus by constructing well-matched parental viruses carrying small genetic tags. The method utilizes high-resolution melt technology for the identification of reassortant viruses. Ease of sample preparation and data analysis enables streamlined genotyping of a large number of virus clones. The method described here thereby allows quantification of the efficiency of reassortment and can be applied to diverse segmented viruses.

DAPK3 participates in the mRNA processing of immediate early genes in chronic lymphocytic leukaemia.

Cross-linking of the B-cell receptor (BCR) induces transcriptional activation of immediate early genes (IEGs) including EGR1 and DUSP2 in chronic lymphocytic leukaemia (CLL). Here, we have shown that this transcriptional activation correlated with histone H3 threonine 6 and 11 phosphorylation. Both transcription and histone post-translational modifications are repressed by ibrutinib, a small molecule inhibitor used in CLL treatment. Moreover, we have identified the death-associated protein kinase 3 (DAPK3), as the kinase mediating these histone phosphorylation marks in response to activation of the BCR signalling pathway with this kinase being recruited to RNA polymerase II in an anti-IgM-dependent manner. DAPK inhibition mimics ibrutinib-induced repression of both IEG mRNA and histone H3 phosphorylation and has anti-proliferative effect comparable to ibrutinib in CLL in vitro. DAPK inhibitor does not repress transcription itself but impacts on mRNA processing and has a broader anti-tumour effect than ibrutinib, by repressing both anti-IgM- and CD40L-dependent activation.

Gestational Pesticide Exposure and Child Respiratory Health.

BACKGROUND: Childhood wheeze may be related to pesticide exposure, and diet and genetics (Paroxonase; PON1) may modify the effects of exposure. METHODS: We analyzed data from the HOME Study, a prospective pregnancy and birth cohort, to examine the association of gestational urinary organophosphate (OP) and pyrethroid (3PBA) metabolite concentrations with child wheeze, forced expiratory volume in one second (FEV1) at ages 4 and 5 years, and wheeze trajectory patterns through age 8 years. RESULTS: Among 367 singletons, the frequency of wheeze ranged from 10.6% to 24.1% at each measurement age. OP and 3PBA metabolite concentrations were not associated with wheeze at 8 years or from birth to 8 years, but there were three significant interactions: (1) maternal daily fruit and vegetable consumption (less than daily consumption and increasing 3PBA was associated with wheeze at age 8 years, OR = 1.40), (2) maternal PON1-108 allele (CT/TT genotypes and high DE was associated with wheeze at age 8 years, OR = 2.13, 2.74) and (3) PON1192 alleles (QR/RR genotypes with higher diethylphosphate (DE) and dialkyl phosphate (DAP) were associated with wheeze at age 8 years, OR = 3.84). Pesticide metabolites were not consistently related to FEV1 or wheeze trajectory. CONCLUSIONS: Gestational OP and 3PBA metabolites were associated with child respiratory outcomes in participants with maternal dietary and genetic susceptibility.

Asthma and/or hay fever as predictors of fertility/impaired fecundity in U.S. women: National Survey of Family Growth.

This study addresses whether asthma and/or hay fever predict fertility and impaired fecundity. The lifetime number of pregnancies (fertility) and spontaneous pregnancy losses (impaired fecundity) in 10,847 women representative of the U.S. population 15 to 44 years of age with histories of diagnosed asthma and/or hay fever are analyzed in the 1995 National Survey of Family Growth using multivariable Poisson regression with multiple covariates and adjustments for complex sampling. Smokers have significantly increased fertility compared to nonsmokers. Smokers with asthma only have significantly increased fertility compared to other smokers. Higher fertility is associated with impaired fecundity (ectopic pregnancy, miscarriage, stillbirth). Women with asthma (with and without hay fever) have significantly higher pregnancy losses than women without asthma. With increasing number of pregnancies, smokers have increased pregnancy losses compared to nonsmokers. Smokers, especially those with asthma only, have increased fertility and require special attention as to their family planning needs, reproductive health, and smoking cessation. Women with asthma, regardless of number of pregnancies, and smokers with higher numbers of pregnancies have high risk pregnancies that require optimal asthma/medical management prenatally and throughout pregnancy. Whether a proinflammatory asthma endotype underlies both the increased fertility and impaired fecundity associated with age and smoking is discussed.

Ibrutinib induces chromatin reorganisation of chronic lymphocytic leukaemia cells.

Chronic lymphocytic leukaemia (CLL) is the most common leukaemia in Western countries. It has recently been shown that the homogeneity of the chromatin landscape between CLL cells contrasts with the important observed genetic heterogeneity of the disease. To gain further insight into the consequences of disease evolution on the epigenome's plasticity, we monitored changes in chromatin structure occurring in vivo in CLL cells from patients receiving continuous Ibrutinib treatment. Ibrutinib, an oral inhibitor of the Bruton's tyrosine kinase (BTK) has proved to be remarkably efficient against treatment naïve (TN), heavily pre-treated and high-risk chronic lymphocytic leukaemia (CLL), with limited adverse events. We established that the chromatin landscape is significantly and globally affected in response to Ibrutinib. However, we observed that prior to treatment, CLL cells show qualitative and quantitative variations in chromatin structure correlated with both EZH2 protein level and cellular response to external stimuli. Then, under prolonged exposure to Ibrutinib, a loss of the two marks associated with lysine 27 (acetylation and trimethylation) was observed. Altogether, these data indicate that the epigenome of CLL cells from the peripheral blood change dynamically in response to stimuli and suggest that these cells might adapt to the Ibrutinib "hit" in a process leading toward a possible reduced sensitivity to treatment.

Targeting Aberrant Epigenetic Networks Mediated by PRMT1 and KDM4C in Acute Myeloid Leukemia.

Transcriptional deregulation plays a major role in acute myeloid leukemia, and therefore identification of epigenetic modifying enzymes essential for the maintenance of oncogenic transcription programs holds the key to better understanding of the biology and designing effective therapeutic strategies for the disease. Here we provide experimental evidence for the functional involvement and therapeutic potential of targeting PRMT1, an H4R3 methyltransferase, in various MLL and non-MLL leukemias. PRMT1 is necessary but not sufficient for leukemic transformation, which requires co-recruitment of KDM4C, an H3K9 demethylase, by chimeric transcription factors to mediate epigenetic reprogramming. Pharmacological inhibition of KDM4C/PRMT1 suppresses transcription and transformation ability of MLL fusions and MOZ-TIF2, revealing a tractable aberrant epigenetic circuitry mediated by KDM4C and PRMT1 in acute leukemia.

c-Myc and transforming growth factor α enhance the development of hepatic lesions due to mutant ß-catenin in transgenic mice.

Alterations in the Wnt signaling pathway are associated with diverse cancers, including hepatocellular carcinoma (HCC). The development of HCC is thought to be a multistage process in which multiple genetic alterations are necessary. Few studies have assessed the effect of aberrant Wnt signaling activity in association with other molecular alterations in HCC. Here we sought to determine whether co-overexpression of c-Myc or TGFα, 2 signaling molecules known to contribute to HCC development, enhanced the development of hepatic lesions associated with a stabilized ß-catenin. The coexpression of mutant ß-catenin with either c-Myc or TGFα within hepatocytes increased the severity of hepatic lesions compared with that associated with any of the transgenes expressed individually. The coexpression of mutant ß-catenin with c-Myc or TGFα resulted in severe hepatomegaly necessitating the euthanasia of mice by an average of 156 and 128 d, respectively, after the cessation of doxycycline. The expression of mutant ß-catenin alone resulted in mild to moderate hepatomegaly that prompted the euthanasia of mice by an average of 75 d after the cessation of doxycycline. Collectively, these findings indicate that coexpression of c-Myc or TGFα delays the onset of endstage hepatic disease yet enhances the severity of hepatic lesions due to mutant ß-catenin.

Direct delivery of MIF morpholinos into the zebrafish otocyst by injection and electroporation affects inner ear development.

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.