A targeted amplicon next-generation sequencing assay for tryptase genotyping to support personalized therapy in mast cell-related disorders.

Tryptase, the most abundant mast cell granule protein, is elevated in severe asthma patients independent of type 2 inflammation status. Higher active ß tryptase allele counts are associated with higher levels of peripheral tryptase and lower clinical benefit from anti-IgE therapies. Tryptase is a therapeutic target of interest in severe asthma and chronic spontaneous urticaria. Active and inactive allele counts may enable stratification to assess response to therapies in asthmatic patient subpopulations. Tryptase gene loci TPSAB1 and TPSB2 have high levels of sequence identity, which makes genotyping a challenging task. Here, we report a targeted next-generation sequencing (NGS) assay and downstream bioinformatics analysis for determining polymorphisms at tryptase TPSAB1 and TPSB2 loci. Machine learning modeling using multiple polymorphisms in the tryptase loci was used to improve the accuracy of genotyping calls. The assay was tested and qualified on DNA extracted from whole blood of healthy donors and asthma patients, achieving accuracy of 96%, 96% and 94% for estimation of inactive α and ßΙΙΙFS tryptase alleles and α duplication on TPSAB1, respectively. The reported NGS assay is a cost-effective method that is more efficient than Sanger sequencing and provides coverage to evaluate known as well as unreported tryptase polymorphisms.

Gestational Trophoblastic Disease with Coexisting Progressing Pregnancy: Personalised Treatment Modalities.

Purpose: Gestational trophoblastic disease (GTD) coexisting with a steadily progressing pregnancy is an extremely rare condition presented in the literature as a single case or case series of successful delivery. The purpose of this study was to describe five cases of GTD and present possible management strategies for such patients. Methods: Clinical data of five pregnancies with coexisting GTD were identified within the Almazov National Medical Research Centre from 2018 to 2021. Results: Three cases of multiple pregnancies with complete hydatidiform moles and two cases of singleton pregnancies with intraplacental choriocarcinoma and invasive hydatidiform moles were identified. Three pregnancies were prolonged and ended with preterm deliveries. Malignant transformation of the GTD accounted for 60% of the cases. The condition of newborns was based on the level of prematurity and functional immaturity, and in all cases, it was aggravated by anemia. Conclusion: GTD coexisting with progressing pregnancy is threatened by the risks of preterm delivery, miscarriage, hemorrhage, and disease progression and requires monitoring in a multidisciplinary clinic experienced in the management of patients with malignant tumors during pregnancy. In cases of prolonged pregnancy against the background of GTD, we suggest the following monitoring during pregnancy: pelvic, abdominal ultrasound/MRI (without contrast), prenatal invasive fetal karyotype testing in cases of singleton pregnancy, lung X-ray/CT with uterine shielding, weekly assessment of ß-hCG levels, and dynamic monitoring of the fetus. The following postnatal monitoring should be performed: morphological examination of the placenta, weekly assessment of ß-hCG levels up to normalization, then monthly assessment up to six months, and control of ß-hCG level of the newborn.

Evaluation of the reproducibility of responses to nasal allergen challenge and effects of inhaled nasal corticosteroids.

BACKGROUND: Similar immune responses in the nasal and bronchial mucosa implies that nasal allergen challenge (NAC) is a suitable early phase experimental model for drug development targeting allergic rhinitis (AR) and asthma. We assessed NAC reproducibility and the effects of intranasal corticosteroids (INCS) on symptoms, physiology, and inflammatory mediators. METHODS: 20 participants with mild atopic asthma and AR underwent three single blinded nasal challenges each separated by three weeks (NCT03431961). Cohort A (n = 10) underwent a control saline challenge, followed by two allergen challenges. Cohort B (n = 10) underwent a NAC with no treatment intervention, followed by NAC with 14 days pre-treatment with saline nasal spray (placebo), then NAC with 14 days pre-treatment with INCS (220 µg triamcinolone acetonide twice daily). Nasosorption, nasal lavage, blood samples, forced expiratory volume 1 (FEV1), total nasal symptom score (TNSS), peak nasal inspiratory flow (PNIF) were collected up to 24 h after NAC. Total and active tryptase were measured as early-phase allergy biomarkers (≤30 min) and IL-13 and eosinophil cell counts as late-phase allergy biomarkers (3-7 h) in serum and nasal samples. Period-period reproducibility was assessed by intraclass correlation coefficients (ICC), and sample size estimates were performed using effect sizes measured after INCS. RESULTS: NAC significantly induced acute increases in nasosorption tryptase and TNSS and reduced PNIF, and induced late increases in nasosorption IL-13 with sustained reductions in PNIF. Reproducibility across NACs varied for symptoms and biomarkers, with total tryptase 5 min post NAC having the highest reproducibility (ICC = 0.91). Treatment with INCS inhibited NAC-induced IL-13 while blunting changes in TNSS and PNIF. For a similar crossover study, 7 participants per treatment arm are needed to detect treatment effects comparable to INCS for TNSS. CONCLUSION: NAC-induced biomarkers and symptoms are reproducible and responsive to INCS. NAC is suitable for assessing pharmacodynamic activity and proof of mechanism for drugs targeting allergic inflammation.

Trial protocol for the study of recommendation system DiaCompanion with personalized dietary recommendations for women with gestational diabetes mellitus (DiaCompanion I).

Background: Gestational diabetes mellitus (GDM) is a common complication of pregnancy associated with serious adverse outcomes for mothers and their offspring. Achieving glycaemic targets is the mainstream in the treatment of GDM in order to improve pregnancy outcomes. As GDM is usually diagnosed in the third trimester of pregnancy, the time frame for the intervention is very narrow. Women need to get new knowledge and change their diet very quickly. Usually, these patients require additional frequent visits to healthcare professionals. Recommender systems based on artificial intelligence could partially substitute healthcare professionals in the process of educating and controlling women with GDM, thus reducing the burden on the women and healthcare systems. We have developed a mobile-based personalized recommendation system DiaCompanion I with data-driven real time personal recommendations focused primarily on postprandial glycaemic response prediction. The study aims to clarify the effect of using DiaCompanion I on glycaemic levels and pregnancy outcomes in women with GDM. Methods: Women with GDM are randomized to 2 treatment groups: utilizing and not utilizing DiaCompanion I. The app provides women in the intervention group the resulting data-driven prognosis of 1-hour postprandial glucose level every time they input their meal data. Based on the predicted glucose level, they can adjust their current meal so that the predicted glucose level falls within the recommended range below 7 mmol/L. The app also provides reminders and recommendations on diet and lifestyle to the participants of the intervention group. All the participants are required to perform 6 blood glucose measurements a day. Capillary glucose values are retrieved from the glucose meter and if not available, from the woman's diary. Additionally, data on glycaemic levels during the study and consumption of major macro- and micronutrients will be collected using the mobile app with electronic report forms in the intervention group. Women from the control group receive standard care without the mobile app. All participants are prescribed with insulin therapy if needed and modifications in their lifestyle. A total of 216 women will be recruited. The primary outcome is the percentage of postprandial capillary glucose values above target (>7.0 mmol/L). Secondary outcomes include the percentage of patients requiring insulin therapy during pregnancy, maternal and neonatal outcomes, glycaemic control using glycated hemoglobin (HbA1c), continuous glucose monitoring data and other blood glucose metrics, the number of patient visits to endocrinologists and acceptance/satisfaction of the two strategies assessed using a questionnaire. Discussion: We believe that the approach including DiaCompanion I will be more effective in patients with GDM for improving glycaemic levels and pregnancy outcomes. We also expect that the use of the app will help reduce the number of clinic visits. Trial registration number: ClinicalTrials.gov, Identifier NCT05179798.

SDRQuerier: A Visual Querying Framework for Cross-National Survey Data Recycling.

Public opinion surveys constitute a widespread, powerful tool to study peoples' attitudes and behaviors from comparative perspectives. However, even global surveys can have limited geographic and temporal coverage, which can hinder the production of comprehensive knowledge. To expand the scope of comparison, social scientists turn to ex-post harmonization of variables from datasets that cover similar topics but in different populations and/or at different times. These harmonized datasets can be analyzed as a single source and accessed through various data portals. However, the Survey Data Recycling (SDR) research project has identified three challenges faced by social scientists when using data portals: the lack of capability to explore data in-depth or query data based on customized needs, the difficulty in efficiently identifying related data for studies, and the incapability to evaluate theoretical models using sliced data. To address these issues, the SDR research project has developed the SDRQuerier, which is applied to the harmonized SDR database. The SDRQuerier includes a BERT-based model that allows for customized data queries through research questions or keywords (Query-by-Question), a visual design that helps users determine the availability of harmonized data for a given research question (Query-by-Condition), and the ability to reveal the underlying relational patterns among substantive and methodological variables in the database (Query-by-Relation), aiding in the rigorous evaluation or improvement of regression models. Case studies with multiple social scientists have demonstrated the usefulness and effectiveness of the SDRQuerier in addressing daily challenges.

High serum granulocyte-colony stimulating factor characterises neutrophilic COPD exacerbations associated with dysbiosis.

INTRODUCTION: COPD exacerbations are heterogeneous and can be triggered by bacterial, viral, or noninfectious insults. Exacerbations are also heterogeneous in neutrophilic or eosinophilic inflammatory responses. A noninvasive peripheral biomarker of COPD exacerbations characterised by bacterial/neutrophilic inflammation is lacking. Granulocyte-colony stimulating factor (G-CSF) is a key cytokine elevated during bacterial infection and mediates survival, proliferation, differentiation and function of neutrophils. OBJECTIVE: We hypothesised that high peripheral G-CSF would be indicative of COPD exacerbations with a neutrophilic and bacterial phenotype associated with microbial dysbiosis. METHODS: Serum G-CSF was measured during hospitalised exacerbation (day 0 or D0) and after 30 days of recovery (Day30 or D30) in 37 subjects. In a second cohort, serum and sputum cytokines were measured in 59 COPD patients during stable disease, at exacerbation, and at 2-weeks and 6-weeks following exacerbation. RESULTS: Serum G-CSF was increased during exacerbation in a subset of patients. These exacerbations were enriched for bacterial but not viral or type-2 biologies. The median serum G-CSF level was 1.6-fold higher in bacterial exacerbation compared to nonbacterial exacerbation (22 pg·mL-1 versus 13 pg·mL-1, p=0.0007). Serum G-CSF classified bacterial exacerbations with an area under the curve (AUC) for the receiver operating characteristic (ROC) curve equal to 0.76. Exacerbations with a two-fold or greater increase in serum G-CSF were characterised by neutrophilic inflammation, with increased sputum and blood neutrophils, and high sputum interleukin (IL)-1ß, IL-6 and serum amyloid A1 (SAA1) levels. These exacerbations were preceded by dysbiosis, with decreased microbiome diversity and enrichment of respiratory pathogens such as Haemophilus and Moraxella. Furthermore, serum G-CSF at exacerbation classified neutrophilic-dysbiotic exacerbations (AUC for the ROC curve equal to 0.75). CONCLUSIONS: High serum G-CSF enriches for COPD exacerbations characterised by neutrophilic inflammation with underlying bacterial dysbiosis.

Effect of intranasal corticosteroid treatment on allergen-induced changes in group 2 innate lymphoid cells in allergic rhinitis with mild asthma.

BACKGROUND: Allergic rhinitis is characterized by rhinorrhea, nasal congestion, sneezing and nasal pruritus. Group 2 innate lymphoid cells (ILC2s), CD4+ T cells and eosinophils in nasal mucosa are increased significantly after nasal allergen challenge (NAC). Effects of intranasal corticosteroids (INCS) on ILC2s remain to be investigated. METHODS: Subjects (n = 10) with allergic rhinitis and mild asthma were enrolled in a single-blind, placebo-controlled, sequential treatment study and treated twice daily with intranasal triamcinolone acetonide (220 µg) or placebo for 14 days, separated by a 7-day washout period. Following treatment, subjects underwent NAC and upper airway function was assessed. Cells from the nasal mucosa and blood, sampled 24 h post-NAC, underwent flow cytometric enumeration for ILC2s, CD4+ T and eosinophil progenitor (EoPs) levels. Cell differentials and cytokine levels were assessed in nasal lavage. RESULTS: Treatment with INCS significantly attenuated ILC2s, IL-5+ /IL-13+ ILC2s, HLA-DR+ ILC2s and CD4+ T cells in the nasal mucosa, 24 h post-NAC. EoP in nasal mucosa was significantly increased, while mature eosinophils were significantly decreased, 24 h post-NAC in INCS versus placebo treatment arm. Following INCS treatment, IL-2, IL-4, IL-5 and IL-13 were significantly attenuated 24 h post-NAC accompanied by significant improvement in upper airway function. CONCLUSION: Pre-treatment with INCS attenuates allergen-induced increases in ILC2s, CD4+ T cells and terminal differentiation of EoPs in the nasal mucosa of allergic rhinitis patients with mild asthma, with little systemic effect. Attenuation of HLA-DR expression by ILC2s may be an additional mechanism by which steroids modulate adaptive immune responses in the upper airways.

Association of tribbles homologue 1 gene expression in human umbilical vein endothelial cells with duration of intrauterine exposure to hyperglycaemia.

Maternal gestational diabetes mellitus (GDM) is considered to be an important factor that epigenetically predisposes offspring to metabolic and cardiovascular diseases. However, the mechanisms of how intrauterine hyperglycaemia affects offspring have not been thoroughly studied. The mammalian tribbles homologue 1 (TRIB1) gene is associated with plasma lipid concentrations and coronary artery disease (CAD). Our aim was to study the effect of GDM and its treatment terms on the level of TRIB1 gene expression in human umbilical vein endothelial cells (HUVECs) of newborns from women with and without GDM. The study included 50 women with GDM and 25 women without GDM (control group). Women with GDM were divided into three groups according to their gestational age when the treatment of GDM started: 24-28 weeks (GDM1, N = 16), 29-32 weeks (GDM2, N = 25) and >34 weeks (GDM3, N = 9). The levels of TRIB1 gene expression in GDM3, GDM2, GDM1 and control groups were 2.8 ± 1.1, 4.2 ± 2.4, 6.0 ± 3.4 and 8.1 ± 6.1, respectively (p = 0.001). After comparison in pairs the difference was significant for the following pairs: GDM2-control (p = 0.004), GDM3-control (p = 0.002), GDM1-GDM3 (p = 0.012). Notably, if treatment had been started before the 28th week of gestation, the difference in TRIB1 gene expression in HUVECs was not significant (p = 0.320 for comparison between GDM1 and control groups). Our findings support the hypothesis that TRIB1 gene expression in HUVECs depends on the duration of intrauterine exposure to hyperglycaemia.

Two Escape Mechanisms of Influenza A Virus to a Broadly Neutralizing Stalk-Binding Antibody.

Broadly neutralizing antibodies targeting the stalk region of influenza A virus (IAV) hemagglutinin (HA) are effective in blocking virus infection both in vitro and in vivo. The highly conserved epitopes recognized by these antibodies are critical for the membrane fusion function of HA and therefore less likely to be permissive for virus mutational escape. Here we report three resistant viruses of the A/Perth/16/2009 strain that were selected in the presence of a broadly neutralizing stalk-binding antibody. The three resistant viruses harbor three different mutations in the HA stalk: (1) Gln387Lys; (2) Asp391Tyr; (3) Asp391Gly. The Gln387Lys mutation completely abolishes binding of the antibody to the HA stalk epitope. The other two mutations, Asp391Tyr and Asp391Gly, do not affect antibody binding at neutral pH and only slightly reduce binding at low pH. Interestingly, they enhance the fusion ability of the HA, representing a novel mechanism that allows productive membrane fusion even in the presence of antibody and hence virus escape from antibody neutralization. Therefore, these mutations illustrate two different resistance mechanisms used by IAV to escape broadly neutralizing stalk-binding antibodies. Compared to the wild type virus, the resistant viruses release fewer progeny viral particles during replication and are more sensitive to Tamiflu, suggesting reduced viral fitness.

Inhibition of the kinase ITK in a mouse model of asthma reduces cell death and fails to inhibit the inflammatory response.

Interleukin-2 (IL-2)-inducible T cell kinase (ITK) mediates T cell receptor (TCR) signaling primarily to stimulate the production of cytokines, such as IL-4, IL-5, and IL-13, from T helper 2 (TH2) cells. Compared to wild-type mice, ITK knockout mice are resistant to asthma and exhibit reduced lung inflammation and decreased amounts of TH2-type cytokines in the bronchoalveolar lavage fluid. We found that a small-molecule selective inhibitor of ITK blocked TCR-mediated signaling in cultured TH2 cells, including the tyrosine phosphorylation of phospholipase C-γ1 (PLC-γ1) and the secretion of IL-2 and TH2-type cytokines. Unexpectedly, inhibition of the kinase activity of ITK during or after antigen rechallenge in an ovalbumin-induced mouse model of asthma failed to reduce airway hyperresponsiveness and inflammation. Rather, in mice, pharmacological inhibition of ITK resulted in T cell hyperplasia and the increased production of TH2-type cytokines. Thus, our studies predict that inhibition of the kinase activity of ITK may not be therapeutic in patients with asthma.

Dissociation of efficacy and cytokine release mediated by an Fc-modified anti-CD3 mAb in a chronic experimental autoimmune encephalomyelitis model.

Humanization and modification of the Fc region of anti-human CD3 mAbs have greatly expanded their potential use in chronic T cell mediated diseases. However, low levels of cytokine release and immunogenicity may still impact a chronic dosing strategy. We investigated the use of an Fc-modified murine chimeric anti-mouse CD3 (N297A) in the chronic MOG(35-55)-induced EAE mouse model of MS. Two daily doses of 10 microg at the onset of clinical symptoms led to both a reduction in T cell numbers in the blood and a significant, prolonged reduction in the symptoms. Histological examination of the spinal cords at the peak of efficacy confirmed a reduction of infiltrating T cells in the CNS. Analysis of the cerebral spinal fluid from EAE mice showed biologically active levels of N297A. Analysis of the cytokine/chemokine levels in cerebrospinal fluid showed a decrease in GM-CSF, IL-6 and IP-10. The combination of N297A dosing with cyclosporine A (CSA) pretreatment showed a significant decrease of TNFalpha, IL-6 and IP-10 without effect on clinical efficacy. However, pretreatment of CSA significantly reduced the immunogenic response observed following a second course of N297A treatment. Therefore, the side effects of an Fc-modified anti-CD3 mAb may be modulated without affecting efficacy.

Short report: serologic evidence of human ehrlichiosis in Peru.

A serosurvey for human ehrlichiosis caused by Ehrlichia chaffeensis and Anaplasma phagocytophilum was performed in different regions of Peru by using indirect immunofluorescence assays (IFAs). Regions included an urban community in a shantytown in Lima (Pampas) and three rural communities located on the northern coast of Peru (Cura Mori), in the southern Peruvian Andes (Cochapata), and in the Peruvian jungle region (Santo Tomas). An overall E. chaffeensis seroprevalence of 13% (21 of 160) was found by IFA. Seroprevalences in females and males was 15% (16 of 106) and 9% (5 of 53), respectively. Seroprevalences in Cura Mori, Cochapata, Pampas, and Santo Tomas were 25% (10 of 40), 23% (9 of 40), 3% (1 of 40), and 3% (1 of 40), respectively. Seroprevalences in Cura Mori and Cochapata were significantly higher than in Santo Tomas or Pampas (P < 0.01). No sera were reactive to A. phagocytophilum. These findings suggest that human infection with E. chaffeensis occurs in Peru. Further studies are needed to characterize Ehrlichia species in Peru, their vectors and their clinical significance.

Functional characterization of N297A, a murine surrogate for low-Fc binding anti-human CD3 antibodies.

Several low- or non-FcR binding anti-human CD3 monoclonal antibodies have been under investigation for the treatment of autoimmune diseases. To model the mechanism of action of these anti-human CD3 mAbs in the murine system, an Fc-modified anti-mouse CD3 antibody (N297A) was generated. N297A exhibited similar biological effects as Fc-modified anti-human CD3 antibodies including rapid, reversible reduction in peripheral leukocyte numbers, differential modulation of activated versus resting T cells, and reduced levels of induced cytokine release compared to the non-Fc-modified parent antibody. In an in vivo model of colitis induced by adoptive transfer of IL-10-deficient cells, administration of N297A significantly reduced body weight loss. As N297A shared many functional characteristics of non-FcR binding anti-human CD3 mAbs both in vitro and in vivo, it provides a means to model the mechanisms of action of Fc-modified anti-human CD3 antibodies in mouse.

Molecular evidence of a new strain of Ehrlichia canis from South America.

Blood samples from dogs with clinical signs compatible with ehrlichiosis were examined for infection of Ehrlichia canis using PCR, multiplex real-time PCR, and DNA sequencing analysis. Eleven of 25 samples were positive for a new strain of E. canis. This is the first molecular identification of E. canis infection in dogs from Peru.

NF-kappa B regulates BCL3 transcription in T lymphocytes through an intronic enhancer.

Exposure to soluble protein Ags in vivo leads to abortive proliferation of responding T cells. In the absence of a danger signal, artificially provided by adjuvants, most responding cells die, and the remainder typically become anergic. The adjuvant-derived signals provided to T cells are poorly understood, but recent work has identified BCL3 as the gene, of those tested, with the greatest differential transcriptional response to adjuvant administration in vivo. As an initial step in analyzing transcriptional responses of BCL3 in T cells, we have identified candidate regulatory regions within the locus through their evolutionary conservation and by analysis of DNase hypersensitivity. An evolutionarily conserved DNase hypersensitive site (HS3) within intron 2 was found to act as a transcriptional enhancer in response to stimuli that mimic TCR activation, namely, PHA and PMA. In luciferase reporter gene constructs transiently transfected into the Jurkat T cell line, the HS3 enhancer can cooperate not only with the BCL3 promoter, but also with an exogenous promoter from herpes simplex thymidine kinase. Deletional analysis revealed that a minimal sequence of approximately 81 bp is required for full enhancer activity. At the 5' end of this minimal sequence is a kappaB site, as confirmed by EMSAs. Mutation of this site in the context of the full-length HS3 abolished enhancer activity. Cotransfection with NF-kappaB p65 expression constructs dramatically increased luciferase activity, even without stimulation. Conversely, cotransfection with the NF-kappaB inhibitor IkappaBalpha reduced activation. Together, these results demonstrate a critical role for NF-kappaB in BCL3 transcriptional up-regulation by TCR-mimetic signals.