An Allosteric Anti-tryptase Antibody for the Treatment of Mast Cell-Mediated Severe Asthma.

Severe asthma patients with low type 2 inflammation derive less clinical benefit from therapies targeting type 2 cytokines and represent an unmet need. We show that mast cell tryptase is elevated in severe asthma patients independent of type 2 biomarker status. Active ß-tryptase allele count correlates with blood tryptase levels, and asthma patients carrying more active alleles benefit less from anti-IgE treatment. We generated a noncompetitive inhibitory antibody against human ß-tryptase, which dissociates active tetramers into inactive monomers. A 2.15 Å crystal structure of a ß-tryptase/antibody complex coupled with biochemical studies reveal the molecular basis for allosteric destabilization of small and large interfaces required for tetramerization. This anti-tryptase antibody potently blocks tryptase enzymatic activity in a humanized mouse model, reducing IgE-mediated systemic anaphylaxis, and inhibits airway tryptase in Ascaris-sensitized cynomolgus monkeys with favorable pharmacokinetics. These data provide a foundation for developing anti-tryptase as a clinical therapy for severe asthma.

ARF6, a component of intercellular bridges, is essential for spermatogenesis in mice.

Male germ cells are connected by intercellular bridges (ICBs) in a syncytium due to incomplete cytokinesis. Syncytium is thought to be important for synchronized germ cell development by interchange of cytoplasmic factors via ICBs. Mammalian ADP-ribosylation factor 6 (ARF6) is a small GTPase that is involved in many cellular mechanisms including but not limited to regulating cellular structure, motility, vesicle trafficking and cytokinesis. ARF6 localizes to ICBs in spermatogonia and spermatocytes in mice. Here we report that mice with global depletion of ARF6 in adulthood using Ubc-CreERT2 display no observable phenotypes but are male sterile. ARF6-deficient males display a progressive loss of germ cells, including LIN28A-expressing spermatogonia, and ultimately develop Sertoli-cell-only syndrome. Specifically, intercellular bridges are lost in ARF6-deficient testis. Furthermore, germ cell-specific inactivation using the Ddx4-CreERT2 results in the same testicular morphological phenotype, showing the germ cell-intrinsic requirement of ARF6. Therefore, ARF6 is essential for spermatogenesis in mice and this function is conserved from Drosophila to mammals.

A FLOWERING LOCUS T ortholog is associated with photoperiod-insensitive flowering in hemp (Cannabis sativa L.).

Hemp (Cannabis sativa L.) is an extraordinarily versatile crop, with applications ranging from medicinal compounds to seed oil and fibre products. Cannabis sativa is a short-day plant, and its flowering is highly controlled by photoperiod. However, substantial genetic variation exists for photoperiod sensitivity in C. sativa, and photoperiod-insensitive ("autoflower") cultivars are available. Using a bi-parental mapping population and bulked segregant analysis, we identified Autoflower2, a 0.5 Mbp locus significantly associated with photoperiod-insensitive flowering in hemp. Autoflower2 contains an ortholog of the central flowering time regulator FLOWERING LOCUS T (FT) from Arabidopsis thaliana which we termed CsFT1. We identified extensive sequence divergence between alleles of CsFT1 from photoperiod-sensitive and insensitive cultivars of C. sativa, including a duplication of CsFT1 and sequence differences, especially in introns. Furthermore, we observed higher expression of one of the CsFT1 copies found in the photoperiod-insensitive cultivar. Genotyping of several mapping populations and a diversity panel confirmed a correlation between CsFT1 alleles and photoperiod response, affirming that at least two independent loci involved in the photoperiodic control of flowering, Autoflower1 and Autoflower2, exist in the C. sativa gene pool. This study reveals the multiple independent origins of photoperiod insensitivity in C. sativa, supporting the likelihood of a complex domestication history in this species. By integrating the genetic relaxation of photoperiod sensitivity into novel C. sativa cultivars, expansion to higher latitudes will be permitted, thus allowing the full potential of this versatile crop to be reached.

2024 ACC/AHA/AACVPR/APMA/ABC/SCAI/SVM/SVN/SVS/SIR/VESS Guideline for the Management of Lower Extremity Peripheral Artery Disease: A Report of the American College of Cardiology/American Heart Association Joint Committee on Clinical Practice Guidelines.

AIM: The "2024 ACC/AHA/AACVPR/APMA/ABC/SCAI/SVM/SVN/SVS/SIR/VESS Guideline for the Management of Lower Extremity Peripheral Artery Disease" provides recommendations to guide clinicians in the treatment of patients with lower extremity peripheral artery disease across its multiple clinical presentation subsets (ie, asymptomatic, chronic symptomatic, chronic limb-threatening ischemia, and acute limb ischemia). METHODS: A comprehensive literature search was conducted from October 2020 to June 2022, encompassing studies, reviews, and other evidence conducted on human subjects that was published in English from PubMed, EMBASE, the Cochrane Library, CINHL Complete, and other selected databases relevant to this guideline. Additional relevant studies, published through May 2023 during the peer review process, were also considered by the writing committee and added to the evidence tables where appropriate. STRUCTURE: Recommendations from the "2016 AHA/ACC Guideline on the Management of Patients With Lower Extremity Peripheral Artery Disease" have been updated with new evidence to guide clinicians. In addition, new recommendations addressing comprehensive care for patients with peripheral artery disease have been developed.

Inhibiting Isoprenylation Suppresses FcεRI-Mediated Mast Cell Function and Allergic Inflammation.

IgE-mediated mast cell activation is a driving force in allergic disease in need of novel interventions. Statins, long used to lower serum cholesterol, have been shown in multiple large-cohort studies to reduce asthma severity. We previously found that statins inhibit IgE-induced mast cell function, but these effects varied widely among mouse strains and human donors, likely due to the upregulation of the statin target, 3-hydroxy-3-methylgutaryl-CoA reductase. Statin inhibition of mast cell function appeared to be mediated not by cholesterol reduction but by suppressing protein isoprenylation events that use cholesterol pathway intermediates. Therefore, we sought to circumvent statin resistance by targeting isoprenylation. Using genetic depletion of the isoprenylation enzymes farnesyltransferase and geranylgeranyl transferase 1 or their substrate K-Ras, we show a significant reduction in FcεRI-mediated degranulation and cytokine production. Furthermore, similar effects were observed with pharmacological inhibition with the dual farnesyltransferase and geranylgeranyl transferase 1 inhibitor FGTI-2734. Our data indicate that both transferases must be inhibited to reduce mast cell function and that K-Ras is a critical isoprenylation target. Importantly, FGTI-2734 was effective in vivo, suppressing mast cell-dependent anaphylaxis, allergic pulmonary inflammation, and airway hyperresponsiveness. Collectively, these findings suggest that K-Ras is among the isoprenylation substrates critical for FcεRI-induced mast cell function and reveal isoprenylation as a new means of targeting allergic disease.

Review of the role of the endogenous opioid and melanocortin systems in the restless legs syndrome.

Restless legs syndrome (RLS) is responsive to opioid, dopaminergic and iron-based treatments. Receptor blocker studies in RLS patients suggest that the therapeutic efficacy of opioids is specific to the opioid receptor and mediated indirectly through the dopaminergic system. An RLS autopsy study reveals decreases in endogenous opioids, ß-endorphin and perhaps Met-enkephalin in the thalamus of RLS patients. A total opioid receptor knock-out (mu, delta and kappa) and a mu-opioid receptor knock-out mouse model of RLS show circadian motor changes akin to RLS and, although both models show sensory changes, the mu-opioid receptor knock mouse shows circadian sensory changes closest to those seen in idiopathic RLS. Both models show changes in striatal dopamine, anaemia and low serum iron. However, only in the total receptor knock-out mouse do we see the decreases in serum ferritin that are normally found in RLS. There are also decreases in serum iron when wild-type mice are administered a mu-opioid receptor blocker. In addition, the mu-opioid receptor knock-out mouse also shows increases in striatal zinc paralleling similar changes in RLS. Adrenocorticotropic hormone and α-melanocyte stimulating hormone are derived from pro-opiomelanocortin as is ß-endorphin. However, they cause RLS-like symptoms and periodic limb movements when injected intraventricularly into rats. These results collectively suggest that an endogenous opioid deficiency is pathogenetic to RLS and that an altered melanocortin system may be causal to RLS as well.

A randomized double-blinded trial to assess recurrence of systemic allergic reactions following COVID-19 mRNA vaccination.

BACKGROUND: Systemic allergic reactions (sARs) following coronavirus disease 2019 (COVID-19) mRNA vaccines were initially reported at a higher rate than after traditional vaccines. OBJECTIVE: We aimed to evaluate the safety of revaccination in these individuals and to interrogate mechanisms underlying these reactions. METHODS: In this randomized, double-blinded, phase 2 trial, participants aged 16 to 69 years who previously reported a convincing sAR to their first dose of COVID-19 mRNA vaccine were randomly assigned to receive a second dose of BNT162b2 (Comirnaty) vaccine and placebo on consecutive days in a blinded, 1:1 crossover fashion at the National Institutes of Health. An open-label BNT162b2 booster was offered 5 months later if the second dose did not result in severe sAR. None of the participants received the mRNA-1273 (Spikevax) vaccine during the study. The primary end point was recurrence of sAR following second dose and booster vaccination; exploratory end points included biomarker measurements. RESULTS: Of 111 screened participants, 18 were randomly assigned to receive study interventions. Eight received BNT162b2 second dose followed by placebo; 8 received placebo followed by BNT162b2 second dose; 2 withdrew before receiving any study intervention. All 16 participants received the booster dose. Following second dose and booster vaccination, sARs recurred in 2 participants (12.5%; 95% CI, 1.6 to 38.3). No sAR occurred after placebo. An anaphylaxis mimic, immunization stress-related response (ISRR), occurred more commonly than sARs following both vaccine and placebo and was associated with higher predose anxiety scores, paresthesias, and distinct vital sign and biomarker changes. CONCLUSIONS: Our findings support revaccination of individuals who report sARs to COVID-19 mRNA vaccines. Distinct clinical and laboratory features may distinguish sARs from ISRRs.

Genetic Mapping, Identification, and Characterization of a Candidate Susceptibility Gene for Powdery Mildew in Cannabis sativa.

Powdery mildew (PM) in Cannabis sativa is most frequently caused by the biotrophic fungus Golovinomyces ambrosiae. Based on previously characterized variation in susceptibility to PM, biparental populations were developed by crossing the most resistant cultivar evaluated, 'FL 58', with a susceptible cultivar, 'TJ's CBD'. F1 progeny were evaluated and displayed a range of susceptibility, and two were self-pollinated to generate two F2 populations. In 2021, the F2 populations (n = 706) were inoculated with PM and surveyed for disease severity. In both F2 populations, 25% of the progeny were resistant, while the remaining 75% showed a range of susceptibility. The F2 populations, as well as selected F1 progeny and the parents, were genotyped with a single-nucleotide polymorphism array, and a consensus genetic map was produced. A major effect quantitative trait locus on C. sativa chromosome 1 (Chr01) and other smaller-effect quantitative trait loci (QTL) on four other chromosomes were identified. The most associated marker on Chr01 was located near CsMLO1, a candidate susceptibility gene. Genomic DNA and cDNA sequencing of CsMLO1 revealed a 6.8-kb insertion in FL 58, relative to TJ's CBD, of which 846 bp are typically spliced into the mRNA transcript encoding a premature stop codon. Molecular marker assays were developed using CsMLO1 sequences to distinguish PM-resistant and PM-susceptible genotypes. These data support the hypothesis that a mutated MLO susceptibility gene confers resistance to PM in C. sativa and provides new genetic resources to develop resistant cultivars. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Aryl hydrocarbon receptor activation alters immune cell populations in the lung and bone marrow during coronavirus infection.

Respiratory viral infections are one of the major causes of illness and death worldwide. Symptoms associated with respiratory infections can range from mild to severe, and there is limited understanding of why there is large variation in severity. Environmental exposures are a potential causative factor. The aryl hydrocarbon receptor (AHR) is an environment-sensing molecule expressed in all immune cells. Although there is considerable evidence that AHR signaling influences immune responses to other immune challenges, including respiratory pathogens, less is known about the impact of AHR signaling on immune responses during coronavirus (CoV) infection. In this study, we report that AHR activation significantly altered immune cells in the lungs and bone marrow of mice infected with a mouse CoV. AHR activation transiently reduced the frequency of multiple cells in the mononuclear phagocyte system, including monocytes, interstitial macrophages, and dendritic cells in the lung. In the bone marrow, AHR activation altered myelopoiesis, as evidenced by a reduction in granulocyte-monocyte progenitor cells and an increased frequency of myeloid-biased progenitor cells. Moreover, AHR activation significantly affected multiple stages of the megakaryocyte lineage. Overall, these findings indicate that AHR activation modulates multiple aspects of the immune response to a CoV infection. Given the significant burden of respiratory viruses on human health, understanding how environmental exposures shape immune responses to infection advances our knowledge of factors that contribute to variability in disease severity and provides insight into novel approaches to prevent or treat disease.NEW & NOTEWORTHY Our study reveals a multifaceted role for aryl hydrocarbon receptor (AHR) signaling in the immune response to coronavirus (CoV) infection. Sustained AHR activation during in vivo mouse CoV infection altered the frequency of mature immune cells in the lung and modulated emergency hematopoiesis, specifically myelopoiesis and megakaryopoiesis, in bone marrow. This provides new insight into immunoregulation by the AHR and extends our understanding of how environmental exposures can impact host responses to respiratory viral infections.

The 14th International Podocyte Conference 2023: from podocyte biology to glomerular medicine.

The 14th International Podocyte Conference took place in Philadelphia, Pennsylvania, USA from May 23 to 26, 2023. It commenced with an early-career researchers' meeting on May 23, providing young scientists with a platform to present and discuss their research findings. Throughout the main conference, 29 speakers across 9 sessions shared their insights on podocyte biology, glomerular medicine, novel technologic advancements, and translational approaches. Additionally, the event featured 3 keynote lectures addressing engineered chimeric antigen receptor T cell- and mRNA-based therapies and the use of biobanks for enhanced disease comprehension. Furthermore, 4 brief oral abstract sessions allowed scientists to present their findings to a broad audience. The program also included a panel discussion addressing the challenges of conducting human research within the American Black community. Remarkably, after a 5-year hiatus from in-person conferences, the 14th International Podocyte Conference successfully convened scientists from around the globe, fostering the presentation and discussion of crucial research findings, as summarized in this review. Furthermore, to ensure continuous and sustainable education, research, translation, and trial medicine related to podocyte and glomerular diseases for the benefit of patients, the International Society of Glomerular Disease was officially launched during the conference.

Rationale and design of the Nephrotic Syndrome Study Network (NEPTUNE) Match in glomerular diseases: designing the right trial for the right patient, today.

Glomerular diseases are classified using a descriptive taxonomy that is not reflective of the heterogeneous underlying molecular drivers. This limits not only diagnostic and therapeutic patient management, but also impacts clinical trials evaluating targeted interventions. The Nephrotic Syndrome Study Network (NEPTUNE) is poised to address these challenges. The study has enrolled >850 pediatric and adult patients with proteinuric glomerular diseases who have contributed to deep clinical, histologic, genetic, and molecular profiles linked to long-term outcomes. The NEPTUNE Knowledge Network, comprising combined, multiscalar data sets, captures each participant's molecular disease processes at the time of kidney biopsy. In this editorial, we describe the design and implementation of NEPTUNE Match, which bridges a basic science discovery pipeline with targeted clinical trials. Noninvasive biomarkers have been developed for real-time pathway analyses. A Molecular Nephrology Board reviews the pathway maps together with clinical, laboratory, and histopathologic data assembled for each patient to compile a Match report that estimates the fit between the specific molecular disease pathway(s) identified in an individual patient and proposed clinical trials. The NEPTUNE Match report is communicated using established protocols to the patient and the attending nephrologist for use in their selection of available clinical trials. NEPTUNE Match represents the first application of precision medicine in nephrology with the aim of developing targeted therapies and providing the right medication for each patient with primary glomerular disease.

Association of COVID-19 Versus COVID-19 Vaccination With Kidney Function and Disease Activity in Primary Glomerular Disease: A Report of the Cure Glomerulonephropathy Study.

RATIONALE & OBJECTIVE: Patients with glomerular disease (GN) may be at increased risk of severe COVID-19, yet concerns over vaccines causing disease relapse may lead to vaccine hesitancy. We examined the associations of COVID-19 with longitudinal kidney function and proteinuria and compared these with similar associations with COVID-19 vaccination. STUDY DESIGN: Observational cohort study from July 1, 2021, to January 1, 2023. SETTING & PARTICIPANTS: A prospective observational study network of 71 centers from North America and Europe (CureGN) with children and adults with primary minimal change disease, focal segmental glomerulosclerosis, membranous nephropathy, or IgA nephropathy. EXPOSURE: COVID-19 and COVID-19 vaccination. OUTCOME: Repeated measure of estimated glomerular filtration rate (eGFR); recurrent time-to-event outcome of GN disease worsening as defined by doubling of the urinary protein-creatinine ratio (UPCR) to at least 1.5g/g or increase in dipstick urine protein by 2 ordinal levels to 3+(300mg/dL) or above. ANALYTICAL APPROACH: Interrupted time series analysis for eGFR. Prognostic matched sequential stratification recurrent event analysis for GN disease worsening. RESULTS: Among 2,055 participants, 722 (35%) reported COVID-19 infection; of these, 92 (13%) were hospitalized, and 3 died (<1%). The eGFR slope before COVID-19 infection was-1.40mL/min/1.73m2 (± 0.29 SD); within 6 months after COVID-19 infection, the eGFR slope was-4.26mL/min/1.73m2 (± 3.02 SD), which was not significantly different (P=0.34). COVID-19 was associated with increased risk of worsening GN disease activity (HR, 1.35 [95% CI, 1.01-1.80]). Vaccination was not associated with a change in eGFR (-1.34mL/min/1.73m2±0.15 SD vs-2.16mL/min/1.73m2±1.74 SD; P=0.6) or subsequent GN disease worsening (HR 1.02 [95% CI, 0.79-1.33]) in this cohort. LIMITATIONS: Infrequent or short follow-up. CONCLUSIONS: Among patients with primary GN, COVID-19 infection was severe for 1 in 8 cases and was associated with subsequent worsening of GN disease activity, as defined by proteinuria. By contrast, vaccination against COVID-19 was not associated with change in disease activity or kidney function decline. These results support COVID-19 vaccination for patients with GN. PLAIN-LANGUAGE SUMMARY: In this cohort study of 2,055 patients with minimal change disease, focal segmental glomerulosclerosis, membranous nephropathy, or IgA nephropathy, COVID-19 resulted in hospitalization or death for 1 in 8 cases and was associated with a 35% increase in risk for worsening proteinuria. By contrast, vaccination did not appear to adversely affect kidney function or proteinuria. Our data support vaccination for COVID-19 in patients with glomerular disease.

Large-scale validation of skin prion seeding activity as a biomarker for diagnosis of prion diseases.

Definitive diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) relies on the examination of brain tissues for the pathological prion protein (PrPSc). Our previous study revealed that PrPSc-seeding activity (PrPSc-SA) is detectable in skin of sCJD patients by an ultrasensitive PrPSc seed amplification assay (PrPSc-SAA) known as real-time quaking-induced conversion (RT-QuIC). A total of 875 skin samples were collected from 2 cohorts (1 and 2) at autopsy from 2-3 body areas of 339 cases with neuropathologically confirmed prion diseases and non-sCJD controls. The skin samples were analyzed for PrPSc-SA by RT-QuIC assay. The results were compared with demographic information, clinical manifestations, cerebrospinal fluid (CSF) PrPSc-SA, other laboratory tests, subtypes of prion diseases defined by the methionine (M) or valine (V) polymorphism at residue 129 of PrP, PrPSc types (#1 or #2), and gene mutations in deceased patients. RT-QuIC assays of the cohort #1 by two independent laboratories gave 87.3% or 91.3% sensitivity and 94.7% or 100% specificity, respectively. The cohort #2 showed sensitivity of 89.4% and specificity of 95.5%. RT-QuIC of CSF available from 212 cases gave 89.7% sensitivity and 94.1% specificity. The sensitivity of skin RT-QuIC was subtype dependent, being highest in sCJDVV1-2 subtype, followed by VV2, MV1-2, MV1, MV2, MM1, MM1-2, MM2, and VV1. The skin area next to the ear gave highest sensitivity, followed by lower back and apex of the head. Although no difference in brain PrPSc-SA was detected between the cases with false negative and true positive skin RT-QuIC results, the disease duration was significantly longer with the false negatives [12.0 ± 13.3 (months, SD) vs. 6.5 ± 6.4, p < 0.001]. Our study validates skin PrPSc-SA as a biomarker for the detection of prion diseases, which is influenced by the PrPSc types, PRNP 129 polymorphisms, dermatome sampled, and disease duration.

Timing influences the impact of aryl hydrocarbon receptor activation on the humoral immune response to respiratory viral infection.

Humoral responses to respiratory viruses, such as influenza viruses, develop over time and are central to protection from repeated infection with the same or similar viruses. Epidemiological and experimental studies have linked exposures to environmental contaminants that bind the aryl hydrocarbon receptor (AHR) with modulated antibody responses to pathogenic microorganisms and common vaccinations. Other studies have prompted investigation into the potential therapeutic applications of compounds that activate AHR. Herein, using two different AHR ligands [2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2-(1H-Indol-3-ylcarbonyl)-4-thiazolecarboxylic acid methyl ester (ITE), to modulate the duration of AHR activity, we show that the humoral response to viral infection is dependent upon the duration and timing of AHR signaling, and that different cellular elements of the response have different sensitivities. When AHR activation was initiated prior to infection with influenza A virus, there was suppression of all measured elements of the humoral response (i.e., the frequency of T follicular helper cells, germinal center B cells, plasma cells, and circulating virus-specific antibody). However, when the timing of AHR activation was adjusted to either early (days -1 to +5 relative to infection) or later (days +5 onwards), then AHR activation affected different aspects of the overall humoral response. These findings highlight the importance of considering the timing of AHR activation in relation to triggering an immune response, particularly when targeting the AHR to manipulate disease processes.

Dry-Cleaning Chemicals and a Cluster of Parkinson's Disease and Cancer: A Retrospective Investigation.

BACKGROUND: Environmental exposure to trichloroethylene (TCE), a carcinogenic dry-cleaning chemical, may be linked to Parkinson's disease (PD). OBJECTIVE: The objective of this study was to determine whether PD and cancer were elevated among attorneys who worked near a contaminated site. METHODS: We surveyed and evaluated attorneys with possible exposure and assessed a comparison group. RESULTS: Seventy-nine of 82 attorneys (96.3%; mean [SD] age: 69.5 [11.4] years; 89.9% men) completed at least one phase of the study. For comparison, 75 lawyers (64.9 [10.2] years; 65.3% men) underwent clinical evaluations. Four (5.1%) of them who worked near the polluted site reported PD, more than expected based on age and sex (1.7%; P = 0.01) but not significantly higher than the comparison group (n = 1 [1.3%]; P = 0.37). Fifteen (19.0%), compared to four in the comparison group (5.3%; P = 0.049), had a TCE-related cancer. CONCLUSIONS: In a retrospective study, diagnoses of PD and TCE-related cancers appeared to be elevated among attorneys who worked next to a contaminated dry-cleaning site. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Phylogenomics reveals patterns of ancient hybridization and differential diversification that contribute to phylogenetic conflict in willows, poplars, and close relatives.

Despite the economic, ecological, and scientific importance of the genera Salix L. (willows) and Populus L. (poplars, cottonwoods, and aspens) Salicaceae, we know little about the sources of differences in species diversity between the genera and of the phylogenetic conflict that often confounds estimating phylogenetic trees. Salix subgenera and sections, in particular, have been difficult to classify, with one recent attempt termed a "spectacular failure" due to a speculated radiation of the subgenera Vetrix and Chamaetia. Here, we use targeted sequence capture to understand the evolutionary history of this portion of the Salicaceae plant family. Our phylogenetic hypothesis was based on 787 gene regions and identified extensive phylogenetic conflict among genes. Our analysis supported some previously described subgeneric relationships and confirmed the polyphyly of others. Using an fbranch analysis, we identified several cases of hybridization in deep branches of the phylogeny, which likely contributed to discordance among gene trees. In addition, we identified a rapid increase in diversification rate near the origination of the Vetrix-Chamaetia clade in Salix. This region of the tree coincided with several nodes that lacked strong statistical support, indicating a possible increase in incomplete lineage sorting due to rapid diversification. The extraordinary level of both recent and ancient hybridization in both Salix and Populus have played important roles in the diversification and diversity in these two genera.

Absence of evidence of transfusion transmission risk of Creutzfeldt-Jakob disease in the United States: Results froma 28-year lookback study.

BACKGROUND: For many years, there has been concern about the risk of transmission of classic forms of Creutzfeldt-Jakob disease (CJD) by blood transfusion, particularly after the recognition of such transmission of variant CJD (vCJD). We report on a 28-year lookback study of recipients of blood from donors who subsequently developed CJD. METHODS: Patients with diagnosed CJD and a history of blood donation were identified. Blood centers were asked to provide information about the distribution of the donations and consignees were requested to provide information about the recipients of the donations. Vital status of each available recipient was determined and, if deceased, the reported cause(s) of death were obtained primarily from the National Death Index. All recipients included in the study database contributed person-time up to the last recorded review of vital status. RESULTS: There were 84 eligible donors who gave 3284 transfusable components, and it was possible to evaluate 1245 recipients, totaling 6495 person-years of observation. The mean observation period per recipient was 5.5 years with a maximum of 51 years. No case of CJD or prion disease was reported among the recipient population. DISCUSSION: The study suggests that CJD may not be transfusion-transmissible, a position in agreement with similar findings from two similar European reports amounting to an overall observation period of 15,500 person-years. These studies have supported the conclusion that the risk, if any, of transmission of CJD by blood products is extremely small and remains theoretical.

The Aryl Hydrocarbon Receptor Modulates T Follicular Helper Cell Responses to Influenza Virus Infection in Mice.

T follicular helper (Tfh) cells support Ab responses and are a critical component of adaptive immune responses to respiratory viral infections. Tfh cells are regulated by a network of signaling pathways that are controlled, in part, by transcription factors. The aryl hydrocarbon receptor (AHR) is an environment-sensing transcription factor that modulates many aspects of adaptive immunity by binding a range of small molecules. However, the contribution of AHR signaling to Tfh cell differentiation and function is not known. In this article, we report that AHR activation by three different agonists reduced the frequency of Tfh cells during primary infection of C57BL/6 mice with influenza A virus (IAV). Further, using the high-affinity and AHR-specific agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin, we show that AHR activation reduced Tfh cell differentiation and T cell-dependent B cell responses. Using conditional AHR knockout mice, we demonstrated that alterations of Tfh cells and T cell-dependent B cell responses after AHR activation required the AHR in T cells. AHR activation reduced the number of T follicular regulatory (Tfr) cells; however, the ratio of Tfr to Tfh cells was amplified. These alterations to Tfh and Tfr cells during IAV infection corresponded with differences in expression of BCL6 and FOXP3 in CD4+ T cells and required the AHR to have a functional DNA-binding domain. Overall, these findings support that the AHR modulates Tfh cells during viral infection, which has broad-reaching consequences for understanding how environmental factors contribute to variation in immune defenses against infectious pathogens, such as influenza and severe acute respiratory syndrome coronavirus.

Identifying dose constraints for the parotid ducts to minimize patient-reported xerostomia: Is conventional mean dose sparing of the parotid glands sufficient?

BACKGROUND AND PURPOSE: The aim of this study was to identify dose constraints for the parotid ducts that limit patient-reported xerostomia and estimate whether these constraints are achieved during conventional parotid gland sparing radiation therapy (PGS-RT). METHODS AND MATERIALS: Thirty-eight oropharyngeal squamous cell carcinoma patients were treated prospectively on trial with MRI sialography-guided parotid duct sparing radiation therapy (PDS-RT). PDS-RT explicitly minimizes dose to the parotid ducts in addition to PGS-RT. Parotid duct dose constraints were identified that distinguished patients reporting high and low rates of xerostomia. Atlas-based parotid duct contours were generated on a retrospective cohort of similar patients where the parotid ducts were not contoured nor explicitly spared to estimate the dose received by the parotid ducts during PGS-RT. RESULTS: Patients whose intraglandular parotid ducts or total parotid ducts were planned for a mean dose < 14 Gy and < 12 Gy, respectively, reported significantly (p < 0.01) lower rates of xerostomia at 6 and 12 months post-RT. Patients receiving PDS-RT had average total and intraglandular duct doses of 11.6  and 13.6 Gy, respectively, compared to an estimated 23.8  and 22.1 Gy, for those receiving PGS-RT (p < 0.01). Only 6% (6/108) and 20% (22/108) of patients receiving PGS-RT were estimated to meet the dose constraints for the total ducts and intraglandular ducts, respectively. CONCLUSION: Parotid duct dose thresholds exist that appear to distinguish patients with and without xerostomia. The identified dose thresholds are frequently not met in PGS-RT plans. In addition to reducing the dose to the parotid gland(s), parotid duct sparing may also further reduce xerostomia.