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Summary: Secretory granule (SG) fusion is an intermediate step in SG biogenesis. However, the precise mechanism of this process is not completely understood. We show that Golgi-derived mast cell (MC) SGs enlarge through a mechanism that is dependent on phosphoinositide (PI) remodeling and fusion with LC3+ late endosomes (amphisomes), which serve as hubs for the fusion of multiple individual SGs. Amphisome formation is regulated by the tyrosine phosphatase PTPN9, while the subsequent SG fusion event is additionally regulated by the tetraspanin protein CD63 and by PI4K. We also demonstrate that fusion with amphisomes imparts to SGs their capacity of regulated release of exosomes. Finally, we show that conversion of PI(3,4,5)P3 to PI(4,5)P2 and the subsequent recruitment of dynamin stimulate SG fission. Our data unveil a key role for lipid-regulated interactions with the endocytic and autophagic systems in controlling the size and number of SGs and their capacity to release exosomes.

Background: While statin therapy is the preferred treatment for hyperlipidemia, literature supports the low-density lipoprotein (LDL) lowering effects associated with red yeast rice, berberine, and Silybum marianum. Dietary supplements may be perceived as a more affordable alternative to prescription medication. Objective: We determined cost-effectiveness of generic pravastatin versus single-ingredient dietary supplements in relation to LDL lowering effect. Methods: Data from meta-analyses and systematic reviews was extracted to calculate pooled weighted mean LDL differences amongst generic pravastatin and single ingredient dietary supplements. The effect was then divided by average 30-day costs and compared amongst agents. Results: The greatest difference was seen in pravastatin 40 mg [MD -57.88 mg/dL (95%CI: - 64.80 to −50.96)], followed by pravastatin 10 mg [MD -41.30 mg/dL (95%CI: 63.30 to - 19.40)], red yeast rice [MD -25.39 (95%CI: −32.98 to −17.81)], berberine [MD -15.13 (95%CI: −21.78 to −8.48)], and Silybum marianum [MD -9.51 mg/dL (95%CI: −22.13 to - 0.10)]. were divided by mean difference to calculate cost per mg/dL reduction in LDL. Cost-effectiveness was greatest for pravastatin 10 mg [$0.66/mg/dL LDL reduction (range: $0.39 to $1.13)], followed by pravastatin 40 mg [$0.74/mg/dL LDL reduction (range: $0.66 to $0.84)], berberine [$0.81/mg/dL LDL reduction (range: $0.56 to $1.44)], red yeast rice [$0.84/mg/dL reduction (range: $0.67 to $1.13)], and Silybum marianum [$0.88/mg/dL LDL reduction (range: $0.38 to $82.02)]. Conclusion: Pravastatin is most cost-effective in each scenario whether or not prescription insurance is utilized.

Oral immunotherapy (OIT) clinical trials have helped a subset of participants achieve sustained unresponsiveness (SU) to the cognate allergen. Here the authors analyse immune cells from participants from one peanut OIT trial and show that CD8+ T cell differentiation status at baseline may help to predict the likelihood of achieving SU.

Abstract Background Environmental fluctuation during embryonic and fetal development can permanently alter an organism’s morphology, physiology, and behaviour. This phenomenon, known as developmental plasticity, is particularly relevant to reptiles that develop in subterranean nests with variable oxygen tensions. Previous work has shown hypoxia permanently alters the cardiovascular system of snapping turtles and may improve cardiac anoxia tolerance later in life. The mechanisms driving this process are unknown but may involve epigenetic regulation of gene expression via DNA methylation. To test this hypothesis, we assessed in situ cardiac performance during 2 h of acute anoxia in juvenile turtles previously exposed to normoxia (21% oxygen) or hypoxia (10% oxygen) during embryogenesis. Next, we analysed DNA methylation and gene expression patterns in turtles from the same cohorts using whole genome bisulfite sequencing, which represents the first high-resolution investigation of DNA methylation patterns in any reptilian species. Results Genome-wide correlations between CpG and CpG island methylation and gene expression patterns in the snapping turtle were consistent with patterns observed in mammals. As hypothesized, developmental hypoxia increased juvenile turtle cardiac anoxia tolerance and programmed DNA methylation and gene expression patterns. Programmed differences in expression of genes such as SCN5A may account for differences in heart rate, while genes such as TNNT2 and TPM3 may underlie differences in calcium sensitivity and contractility of cardiomyocytes and cardiac inotropy. Finally, we identified putative transcription factor-binding sites in promoters and in differentially methylated CpG islands that suggest a model linking programming of DNA methylation during embryogenesis to differential gene expression and cardiovascular physiology later in life. Binding sites for hypoxia inducible factors (HIF1A, ARNT, and EPAS1) and key transcription factors activated by MAPK and BMP signaling (RREB1 and SMAD4) are implicated. Conclusions Our data strongly suggests that DNA methylation plays a conserved role in the regulation of gene expression in reptiles. We also show that embryonic hypoxia programs DNA methylation and gene expression patterns and that these changes are associated with enhanced cardiac anoxia tolerance later in life. Programming of cardiac anoxia tolerance has major ecological implications for snapping turtles, because these animals regularly exploit anoxic environments throughout their lifespan.

Summary: Granulocytes encompass diverse roles, from fighting off pathogens to regulating inflammatory processes in allergies. These roles are represented by distinct cellular phenotypes that we captured with mass cytometry (CyTOF). Our protocol enables simultaneous evaluation of human basophils, eosinophils, and neutrophils under homeostasis and upon immune activation by anti-Immunoglobulin E (anti-IgE) or interleukin-3 (IL-3). Granulocyte integrity and detection of protein markers were optimized so that rare granulocyte populations could be deeply characterized by single cell mass cytometry.For complete details on the use and execution of this protocol, please refer to Vivanco Gonzalez et al. (2020).

Turtles are iconic reptiles that inhabit a range of ecosystems from oceans to deserts and climates from the tropics to northern temperate regions. Yet, we have little understanding of the genetic adaptations that allow turtles to survive and reproduce in such diverse environments. Common snapping turtles, Chelydra serpentina, are an ideal model species for studying adaptation to climate because they are widely distributed from tropical to northern temperate zones in North America. They are also easy to maintain and breed in captivity and produce large clutch sizes, which makes them amenable to quantitative genetic and molecular genetic studies of traits like temperature-dependent sex determination. We therefore established a captive breeding colony and sequenced DNA from one female using both short and long reads. After trimming and filtering, we had 209.51Gb of Illumina reads, 25.72Gb of PacBio reads, and 21.72 Gb of Nanopore reads. The assembled genome was 2.258 Gb in size and had 13,224 scaffolds with an N50 of 5.59Mb. The longest scaffold was 27.24Mb. BUSCO analysis revealed 97.4% of core vertebrate genes in the genome. We identified 3.27 million SNPs in the reference turtle, which indicates a relatively high level of individual heterozygosity. We assembled the transcriptome using RNA-Seq data and used gene prediction software to produce 22,812 models of protein coding genes. The quality and contiguity of the snapping turtle genome is similar to or better than most published reptile genomes. The genome and genetic variants identified here provide a foundation for future studies of adaptation to climate.

Mast cell neoplasms are one of the most frequently diagnosed malignancies in dogs. The clinical picture, course, and prognosis vary substantially among patients, depending on the anatomic site, grade and stage of the disease. The most frequently involved organ is the skin, followed by hematopoietic organs (lymph nodes, spleen, liver, and bone marrow) and mucosal sites of the oral cavity and the gastrointestinal tract. In cutaneous mast cell tumors, several grading and staging systems have been introduced. However, no comprehensive classification and no widely accepted diagnostic criteria have been proposed to date. To address these open issues and points we organized a Working Conference on canine mast cell neoplasms in Vienna in 2019. The outcomes of this meeting are summarized in this article. The proposed classification includes cutaneous mast cell tumors and their sub-variants defined by grading- and staging results, mucosal mast cell tumors, extracutaneous/extramucosal mast cell tumors without skin involvement, and mast cell leukemia (MCL). For each of these entities, diagnostic criteria are proposed. Moreover, we have refined grading and staging criteria for mast cell neoplasms in dogs based on consensus discussion. The criteria and classification proposed in this article should greatly facilitate diagnostic evaluation and prognostication in dogs with mast cell neoplasms and should thereby support management of these patients in daily practice and the conduct of clinical trials.

Mastocytosis is a hematologic neoplasm characterized by expansion and focal accumulation of neoplastic mast cells (MC) in diverse organs, including the skin, bone marrow (BM), spleen, liver, and gastrointestinal tract. The World Health Organization classification divides the disease into prognostically distinct variants of cutaneous mastocytosis (CM) and systemic mastocytosis (SM). Although this classification remains valid, recent developments in the field and the advent of new diagnostic and prognostic parameters created a need to update and refine definitions and diagnostic criteria in MC neoplasms. In addition, MC activation syndromes (MCAS) and genetic features predisposing to SM and MCAS have been identified. To discuss these developments and refinements in the classification, we organized a Working Conference comprised of experts from Europe and the United States in August 2020. This article reports on outcomes from this conference. Of particular note, we propose adjustments in the classification of CM and SM, refinements in diagnostic criteria of SM variants, including smoldering SM and BM mastocytosis (BMM), and updated criteria for MCAS and other conditions involving MC. CD30 expression in MC now qualifies as a minor SM criterion, and BMM is now defined by SM criteria, absence of skin lesions and absence of B- and C-findings. A basal serum tryptase level exceeding 20 ng/mL remains a minor SM criterion, with recognition that hereditary alpha-tryptasemia and various myeloid neoplasms may also cause elevations in tryptase. Our updated proposal will support diagnostic evaluations and prognostication in daily practice and the conduct of clinical trials in MC disorders.

It is well established that adult vertebrates acclimatizing to hypoxic environments undergo mitochondrial remodeling to enhance oxygen delivery, maintain ATP, and limit oxidative stress. However, many vertebrates also encounter oxygen deprivation during embryonic development. The effects of developmental hypoxia on mitochondrial function are likely to be more profound, because environmental stress during early life can permanently alter cellular physiology and morphology. To this end, we investigated the long-term effects of developmental hypoxia on mitochondrial function in a species that regularly encounters hypoxia during development—the common snapping turtle (Chelydra serpentina). Turtle eggs were incubated in 21% or 10% oxygen from 20% of embryonic development until hatching, and both cohorts were subsequently reared in 21% oxygen for 8 months. Ventricular mitochondria were isolated, and mitochondrial respiration and reactive oxygen species (ROS) production were measured with a microrespirometer. Compared to normoxic controls, juvenile turtles from hypoxic incubations had lower Leak respiration, higher P:O ratios, and reduced rates of ROS production. Interestingly, these same attributes occur in adult vertebrates that acclimatize to hypoxia. We speculate that these adjustments might improve mitochondrial hypoxia tolerance, which would be beneficial for turtles during breath-hold diving and overwintering in anoxic environments.

Mouse IgE and mast cell (MC) functions have been studied primarily using inbred strains. Here, we (a) identified effects of genetic background on mouse IgE and MC phenotypes, (b) defined the suitability of various strains for studying IgE and MC functions, and (c) began to study potentially novel genes involved in such functions. We screened 47 Collaborative Cross (CC) strains, as well as C57BL/6J and BALB/cJ mice, for strength of passive cutaneous anaphylaxis (PCA) and responses to the intestinal parasite Strongyloides venezuelensis (S.v.). CC mice exhibited a diversity in PCA strength and S.v. responses. Among strains tested, C57BL/6J and CC027 mice showed, respectively, moderate and uniquely potent MC activity. Quantitative trait locus analysis and RNA sequencing of BM-derived cultured MCs (BMCMCs) from CC027 mice suggested Sp140 as a candidate gene for MC activation. siRNA-mediated knock-down of Sp140 in BMCMCs decreased IgE-dependent histamine release and cytokine production. Our results demonstrated marked variations in IgE and MC activity in vivo, and in responses to S.v., across CC strains. C57BL/6J and CC027 represent useful models for studying MC functions. Additionally, we identified Sp140 as a gene that contributes to IgE-dependent MC activation.

Basophil activation tests (BATs) can closely monitor, in vitro, a patient’s propensity to develop type I hypersensitivity reactions. Because of their high specificity and sensitivity, BATs have become promising diagnostic tools, especially in cases with equivocal clinical histories, skin prick test results, and/or levels of specific IgE to allergen extracts. BATs also are useful as tools for monitoring the effects of treatment, since oral immunotherapy (OIT) studies report a diminution in patients’ basophil responsiveness over the course of OIT. This review will discuss the BAT findings obtained before, during, and after OIT for food allergy. We will mainly focus on the association of basophil responsiveness, and alterations in basophil surface markers, with clinical outcomes and other clinical features, such as blood levels of specific IgG and IgE antibodies. The detailed analysis of these correlations will ultimately facilitate the use of BATs, along with other blood biomarkers, to differentiate short-term desensitization versus sustained unresponsiveness and to improve treatment protocols. Given the critical anatomic location of mast cells adjacent to the many IgE+ plasma cells found in the gastrointestinal tissues of allergic individuals, we will also discuss the role of gastrointestinal mast cells in manifestations of food allergies.

Summary: Basophils, the rarest granulocyte, play critical roles in parasite- and allergen-induced inflammation. We applied mass cytometry (CyTOF) to simultaneously asses 44 proteins to phenotype and functionally characterize neutrophils, eosinophils, and basophils from 19 healthy donors. There was minimal heterogeneity seen in eosinophils and neutrophils, but data-driven analyses revealed four unique subpopulations within phenotypically basophilic granulocytes (PBG; CD45+HLA-DR−CD123+). Through CyTOF and fluorescence-activated cell sorting (FACS), we classified these four PBG subpopulations as (I) CD16lowFcεRIhighCD244high (88.5 ± 1.2%), (II) CD16highFcεRIhighCD244high (9.1 ± 0.4%), (III) CD16lowFcεRIlowCD244low (2.3 ± 1.3), and (IV) CD16highFcεRIlowCD244low (0.4 ± 0.1%). Prospective isolation confirmed basophilic-morphology of PBG I–III, but neutrophilic-morphology of PBG IV. Functional interrogation via IgE-crosslinking or IL-3 stimulation demonstrated that PBG I–II had significant increases in CD203c expression, whereas PBG III–IV remained unchanged compared with media-alone conditions. Thus, PBG III–IV could serve roles in non-IgE-mediated immunity. Our findings offer new perspectives in human basophil heterogeneity and the varying functional potential of these new subsets in health and disease.

Background: As there is limited data on the sustainability of desensitization of multifood-oral immunotherapy (multifood-OIT), we conducted a multisite multifood-OIT study to compare the efficacy of successful desensitization with sustained dosing vs discontinued dosing after multifood-OIT. Methods: We enrolled 70 participants, aged 5–22 years with multiple food allergies confirmed by double-blind placebo-controlled food challenges (DBPCFCs). In the open-label phase of the study, all participants received omalizumab (weeks 1–16) and multi-OIT (2–5 allergens; weeks 8–30) and eligible participants (on maintenance dose of each allergen by weeks 28–29) were randomized 1:1:1 to 1 g, 300 mg, or 0 mg arms (blinded, weeks 30–36) and then tested by food challenge at week 36. Success was defined as passing 2 g food challenge to at least 2 foods in week 36. Findings: Most participants were able to reach a dose of 2 g or higher of each of 2, 3, 4, and 5 food allergens (as applicable to the participant's food allergens in OIT) in week 36 food challenges. Using an intent-to-treat analysis, we did not find evidence that a 300 mg dose was effectively different than a 1 g dose in maintaining desensitization, and both together were more effective than OIT discontinuation (0 mg dose) (85% vs 55%, P = 0.03). Fifty-five percent of the intent-to-treat participants and 69% of per protocol participants randomized to the 0 mg arm showed no objective reactivity after 6 weeks of discontinuation. Cross-desensitization was found between cashew/pistachio and walnut/pecan when only one of the foods was part of OIT. No statistically significant safety differences were found between the three arms. Interpretation: These results suggest that sustained desensitization after omalizumab-facilitated multi-OIT best occurs through continued maintenance OIT dosing of either 300 mg or 1 g of each food allergen as opposed to discontinuation of multi-OIT. Funding: Sean N. Parker Center for Allergy and Asthma Research at Stanford University, Jeff and MacKenzie Bezos, NIAID AADCRC U19AI104209. Trial Registration Number: ClinicalTrials.gov number, NCT02626611. Keywords: Food allergy, Oral immunotherapy, Omalizumab, Sustained unresponsiveness, Food allergen