A human autoimmune organoid model reveals IL-7 function in coeliac disease.

In vitro models of autoimmunity are constrained by an inability to culture affected epithelium alongside the complex tissue-resident immune microenvironment. Coeliac disease (CeD) is an autoimmune disease in which dietary gluten-derived peptides bind to the major histocompatibility complex (MHC) class II human leukocyte antigen molecules (HLA)-DQ2 or HLA-DQ8 to initiate immune-mediated duodenal mucosal injury1-4. Here, we generated air-liquid interface (ALI) duodenal organoids from intact fragments of endoscopic biopsies that preserve epithelium alongside native mesenchyme and tissue-resident immune cells as a unit without requiring reconstitution. The immune diversity of ALI organoids spanned T cells, B and plasma cells, natural killer (NK) cells and myeloid cells, with extensive T-cell and B-cell receptor repertoires. HLA-DQ2.5-restricted gluten peptides selectively instigated epithelial destruction in HLA-DQ2.5-expressing organoids derived from CeD patients, and this was antagonized by blocking MHC-II or NKG2C/D. Gluten epitopes stimulated a CeD organoid immune network response in lymphoid and myeloid subsets alongside anti-transglutaminase 2 (TG2) autoantibody production. Functional studies in CeD organoids revealed that interleukin-7 (IL-7) is a gluten-inducible pathogenic modulator that regulates CD8+ T-cell NKG2C/D expression and is necessary and sufficient for epithelial destruction. Furthermore, endogenous IL-7 was markedly upregulated in patient biopsies from active CeD compared with remission disease from gluten-free diets, predominantly in lamina propria mesenchyme. By preserving the epithelium alongside diverse immune populations, this human in vitro CeD model recapitulates gluten-dependent pathology, enables mechanistic investigation and establishes a proof of principle for the organoid modelling of autoimmunity.

DRA involvement in linaclotide-stimulated bicarbonate secretion during loss of CFTR function.

Duodenal bicarbonate secretion is critical to epithelial protection, as well as nutrient digestion and absorption, and is impaired in cystic fibrosis (CF). We examined if linaclotide, typically used to treat constipation, may also stimulate duodenal bicarbonate secretion. Bicarbonate secretion was measured in vivo and in vitro using mouse and human duodenum (biopsies and enteroids). Ion transporter localization was identified with confocal microscopy, and de novo analysis of human duodenal single-cell RNA sequencing (scRNA-Seq) data sets was performed. Linaclotide increased bicarbonate secretion in mouse and human duodenum in the absence of cystic fibrosis transmembrane conductance regulator (CFTR) expression (Cftr-knockout mice) or function (CFTRinh-172). Na+/H+ exchanger 3 inhibition contributed to a portion of this response. Linaclotide-stimulated bicarbonate secretion was eliminated by down-regulated in adenoma (DRA, SLC26A3) inhibition during loss of CFTR activity. ScRNA-Seq identified that 70% of villus cells expressed SLC26A3, but not CFTR, mRNA. Loss of CFTR activity and linaclotide increased apical brush border expression of DRA in non-CF and CF differentiated enteroids. These data provide further insights into the action of linaclotide and how DRA may compensate for loss of CFTR in regulating luminal pH. Linaclotide may be a useful therapy for CF individuals with impaired bicarbonate secretion.

Simpson-Golabi-Behmel syndrome type 1 with normal birth parameters.

A newborn baby born at 34 weeks and 5 days gestation was admitted for prematurity, dysmorphic features and congenital heart defects. Antenatal scan at 21 weeks showed a large-for-gestational-age foetus with a large abdominal circumference and liver, ventricular septal defect, right prominent renal pelvis and echogenic bowel. Antenatal genetic tests for overgrowth syndromes were negative. The mother had early onset pre-eclampsia. After birth, an overgrowth syndrome was still suspected despite the baby having normal birth parameters. Raw data of the trio whole exome sequencing from the amniocentesis sample were manually inspected. Hemizygous exon 7 deletion in the GPC3 gene was found, and a postnatal diagnosis of Simpson-Golabi-Behmel syndrome, a rare overgrowth syndrome, was made. This case report discusses the significance of antenatal findings, an atypical presentation of a rare syndrome and the obstacles of diagnostic genetic testing.

Sequencing by avidity enables high accuracy with low reagent consumption.

We present avidity sequencing, a sequencing chemistry that separately optimizes the processes of stepping along a DNA template and that of identifying each nucleotide within the template. Nucleotide identification uses multivalent nucleotide ligands on dye-labeled cores to form polymerase-polymer-nucleotide complexes bound to clonal copies of DNA targets. These polymer-nucleotide substrates, termed avidites, decrease the required concentration of reporting nucleotides from micromolar to nanomolar and yield negligible dissociation rates. Avidity sequencing achieves high accuracy, with 96.2% and 85.4% of base calls having an average of one error per 1,000 and 10,000 base pairs, respectively. We show that the average error rate of avidity sequencing remained stable following a long homopolymer.

Revealing parental mosaicism: the hidden answer to the recurrence of apparent de novo variants.

Mosaicism refers to the presence of two or more populations of genetically distinct cells within an individual, all of which originate from a single zygote. Previous literature estimated the percentage of parental mosaicism ranged from 0.33 to 25.9%. In this study, parents whose children had previously been diagnosed with developmental disorders with an apparent de novo variant were recruited. Peripheral blood, buccal and semen samples were collected from these parents if available for the detection of potential parental mosaicism using droplet digital PCR, complemented with the method of blocker displacement amplification. Among the 20 families being analyzed, we report four families with parental mosaicism (4/20, 20%). Two families have maternal gonosomal mosaicism (EYA1 and EBF3) and one family has paternal gonadal mosaicism (CHD7) with a pathogenic/ likely pathogenic variant. One family has a paternal gonosomal mosaicism with a variant of uncertain significance (FLNC) with high clinical relevance. The detectable variant allele frequency in our cohort ranged from 8.7-35.9%, limit of detection 0.08-0.16% based on our in-house EBF3 assay. Detecting parental mosaicism not only informs family with a more accurate recurrence risk, but also facilitates medical teams to create appropriate plans for pregnancy and delivery, offering the most suitable care.

Early Tracheostomy May Be Performed Safely in Obese COVID-19 Patients Supported on VV-ECMO.

OBJECTIVE: Obese patients with coronavirus disease 2019 (COVID-19)-associated acute respiratory failure (ARDS) often require prolonged intubation. However, data are sparse regarding optimal tracheostomy timing in obese adults with COVID-19 requiring venovenous extracorporeal membrane oxygenation (VV-ECMO). This study retrospectively describes the course of obese patients with COVID-19 who underwent tracheostomy on VV-ECMO between March 2020 and December 2022. METHODS: There were 62 participants with a median age of 43 (interquartile range [IQR] 33 to 53) years and a median body mass index of 42 (IQR 34 to 50) kg/m2 who received VV-ECMO for COVID-19-associated ARDS. Of those, 42 underwent tracheostomy on VV-ECMO, and 50% (n = 21) of the 42 patients underwent early (within 14 days of initiated ventilatory support) tracheostomy. RESULTS: Among patients who received tracheostomies, the combined respiratory tract and lung parenchymal bleeding rate was 29% (n = 12), but only 7% required surgical intervention for bleeding from the tracheostomy site (n = 3). The hospital length of stay (LOS) was 42 (IQR 36 to 57) days, and mortality rate was 38% (n = 16). Tracheostomy timing was not associated with differences in respiratory tract bleeding, mechanical ventilatory support duration, VV-ECMO support duration, intensive care unit LOS, hospital LOS, mortality, or survival probability. CONCLUSIONS: Although an individualized and holistic approach to clinical decision making continues to be necessary, the findings of this study suggest that early tracheostomy may be performed safely in obese patients with COVID-19 on VV-ECMO.

Fibrodysplasia ossificans progressiva in Hong Kong-A case report series.

Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare condition. The diagnosis could be challenging due to its rarity and non-specific presenting symptoms. However, early diagnosis and appropriate management help in preserving patients' function and quality of life. Herein, we report the diagnostic journeys and clinical courses of 8 patients with FOP in Hong Kong and illustrate the challenges involved.

Organoid modeling of lung-resident immune responses to SARS-CoV-2 infection.

Tissue-resident immunity underlies essential host defenses against pathogens, but analysis in humans has lacked in vitro model systems where epithelial infection and accompanying resident immune cell responses can be observed en bloc. Indeed, human primary epithelial organoid cultures typically omit immune cells, and human tissue resident-memory lymphocytes are conventionally assayed without an epithelial infection component, for instance from peripheral blood, or after extraction from organs. Further, the study of resident immunity in animals can be complicated by interchange between tissue and peripheral immune compartments. To study human tissue-resident infectious immune responses in isolation from secondary lymphoid organs, we generated adult human lung three-dimensional air-liquid interface (ALI) lung organoids from intact tissue fragments that co-preserve epithelial and stromal architecture alongside endogenous lung-resident immune subsets. These included T, B, NK and myeloid cells, with CD69+CD103+ tissue-resident and CCR7- and/or CD45RA- TRM and conservation of T cell receptor repertoires, all corresponding to matched fresh tissue. SARS-CoV-2 vigorously infected organoid lung epithelium, alongside secondary induction of innate cytokine production that was inhibited by antiviral agents. Notably, SARS-CoV-2-infected organoids manifested adaptive virus-specific T cell activation that was specific for seropositive and/or previously infected donor individuals. This holistic non-reconstitutive organoid system demonstrates the sufficiency of lung to autonomously mount adaptive T cell memory responses without a peripheral lymphoid component, and represents an enabling method for the study of human tissue-resident immunity.

Critical role of down-regulated in adenoma bicarbonate transporter in linaclotide stimulated intestinal bicarbonate secretion.

Duodenal bicarbonate secretion is critical to epithelial protection, nutrient digestion/absorption and is impaired in cystic fibrosis (CF). We examined if linaclotide, typically used to treat constipation, may also alter duodenal bicarbonate secretion. Bicarbonate secretion was measured in vivo and in vitro using mouse and human duodenum. Ion transporter localization was identified with confocal microscopy and de novo analysis of human duodenal single cell RNA sequencing (sc-RNAseq) was performed. Linaclotide increased bicarbonate secretion in mouse and human duodenum in the absence of CFTR expression or function. Linaclotide-stimulated bicarbonate secretion was eliminated by down-regulated in adenoma (DRA) inhibition, regardless of CFTR activity. Sc-RNAseq identified that 70% of villus cells expressed SLC26A3, but not CFTR, mRNA. Linaclotide increased apical membrane expression of DRA in non-CF and CF differentiated enteroids. These data provide insights into the action of linaclotide and suggest linaclotide may be a useful therapy for CF individuals with impaired bicarbonate secretion.

Heart transplantation using donation after circulatory death in the United States.

OBJECTIVES: Heart donation after circulatory death was recently reintroduced in the United States with hopes of increasing donor heart availability. We examined its national use and outcomes. METHODS: The United Network for Organ Sharing database was used to identify validated adult patients undergoing heart transplantation using donation after circulatory death donors (n = 266) and donation after brain death donors (n = 5998) between December 1, 2019, and December 31, 2021, after excluding heart-lung transplants. Propensity score matching was used to create more balanced groups for comparison. RESULTS: The monthly percentage of donation after circulatory death heart transplant increased from 2.5% in December 2019 to 6.8% in December 2021 (P < .001). Twenty-two centers performed donation after circulatory death heart transplants, ranging from 1 to 75 transplants per center. Four centers performed 70% of the national volume. Recipients of donation after circulatory death hearts were more likely to be clinically stable (80.4% vs 41.1% in status 3-6, P < .001), to have type O blood (58.3% vs 39.9%, P < .001), and to wait longer after listing (55, interquartile range, 15-180 days vs 32, interquartile range, 9-160 days, P = .003). Six-month survival was 92.1% (95% confidence interval, 91.3-92.8) after donation after brain death heart transplants and 92.6% (95% confidence interval, 88.1-95.4) after donation after circulatory death heart transplants (hazard ratio, 0.94, 95% confidence interval, 0.57-1.54, P = .79). Outcomes in propensity-matched patients were similar except for higher rates of treated acute rejection in donation after circulatory death transplants before discharge (14.4% vs 8.8%, P = .01). In donation after circulatory death heart recipients, outcomes did not differ based on the procurement technique (normothermic regional perfusion vs direct procurement and perfusion). CONCLUSIONS: Heart transplantation with donation after circulatory death donors has short-term survival comparable to donation after brain death transplants. Broader implementation could substantially increase donor organ availability.

Impaired distal colonic pH in adults with cystic fibrosis.

Previous wireless motility capsule (WMC) studies demonstrated decreased small intestinal pH in people with CF (PwCF) however the data is lacking on the colonic pH profile. We re-analyzed previously published WMC data to determine colonic pH/bicarbonate concentration and single cell RNA sequencing (sc-RNAseq) to examine the normal expression of acid-base transporters in the colon/rectum.CF patients showed significantly lower pH and bicarbonate concentration values, particularly in the distal rectosigmoid region. There was no difference in colonic motility parameters between CF and non-CF subjects. SLC26A3 is highly expressed bicarbonate transporter in the colon and rectum, more so than CFTR. While dysmotility can alter intraluminal pH, observed changes likely originate from alterations in intestinal ion transport rather than colonic dysmotility. SLC26A3 is abundantly expressed in the human colon and rectum and may be a therapeutic target for restoration of bicarbonate transport. These findings may help better understand the gastrointestinal symptoms in PwCF.

Evaluating age-based eligibility thresholds for heart re-transplantation - an analysis of the united network for organ sharing database.

BACKGROUND: Risk-adjusted survival after late heart re-transplantation may be comparable to primary transplant, but the efficacy of re-transplantation in older candidates is not established. We evaluated outcomes after heart re-transplantation in recipients > 60 years. METHODS: We identified 1026 adult patients undergoing isolated heart re-transplantation between 2003 and 2020 from the United Network for Organ Sharing database. Older recipients (> 60 years, n=177) were compared to younger recipients (≤ 60 years, n=849). Five and ten-year post-transplant survival was estimated using the Kalpan-Meier method and adjusted with multivariable Cox models. RESULTS: Older recipients were more likely to be male and have diabetes or previous malignancies with higher baseline creatinine. They also more frequently required pre-transplant ECMO (11.9% vs. 6.8%, p=0.02) and received re-transplantation due to primary graft failure (13.6% vs. 8.5%, p=0.03). After the transplant, older recipients had a higher incidence of stroke (6.8% vs. 2.6%, p=0.01) and dialysis requirements (20.3% vs. 13.2%) before discharge (both p<0.05), and more frequently died from malignancy-related causes (16.3% vs. 3.9%, p<0.001). After adjustment, recipient age >60 was associated with an increased risk of both 5-year (HR 1.42, 95% CI 1.02-2.01, p=0.04) and 10-year mortality (HR 1.72, 95% CI 1.20-2.45, p=0.003). Restricted cubic spline showed a non-linear relationship between recipient age and 10-year mortality. CONCLUSIONS: Heart re-transplantation in recipients > 60 years has inferior outcomes compared to younger recipients. Strict patient selection and close follow-up are warranted to ensure the appropriate utilization of donor hearts and to improve long-term outcomes.

Complete Genome Sequences of Genamy16 and NovaSharks, Two Gordonia rubripertincta Bacteriophages Isolated from Soil in Southeastern Florida.

We report on two actinobacteriophages, Genamy16 and NovaSharks, that were isolated from soil in Florida using Gordonia rubripertincta NRRL B-16540. The genomes of both phages are ~65,000 bp, with similar GC contents, and, based on gene content similarity to phages in the Actinobacteriophage Database, were assigned to phage cluster DV.

The Role of Diet Compared to Physical Activity on Women's Cancer Mortality: Results From the Third National Health and Nutrition Examination Survey.

Background: Among women in the United States, cancer is the second leading cause of death. Prior studies have examined how lifestyle factors, such as diet and physical activity, influence cancer mortality. However, few have evaluated if diet or physical activity has a stronger protective effect for cancer mortality. Therefore, this study aims to evaluate and compare the impacts of diet and physical activity on women's cancer mortality. Methods: Prospective, cross-sectional data were abstracted from the Third US National Health and Nutrition Examination Survey (NHANES III) on female respondents from 1988 to 1994. Physical activity was derived from the CDC's metabolic equivalent (MET) intensity levels. Dietary classifications were derived from the USDA's healthy eating index (HEI). We utilized the National Death Index to obtain mortality follow-up information on our cohort until December 31, 2015. Chi-squared, multivariable Cox regression, and Kaplan-Meier estimates were employed for statistical analyses. Results: Of 3,590 women (median age: 57, range: 40-89), 30% had an obese BMI (BMI≥30 kg/m2). Additionally, 22% of participants self-reported a healthy diet, 69% needed dietary improvement, and 9% had a poor diet. Furthermore, 21% reported physical inactivity, 44% did not meet physical activity guidelines, and 35% met guidelines. On multivariate analysis, healthy diet (HR: 0.70; 95% CI: 0.51-0.98; p = 0.04), but not physical activity (HR: 0.87; 95% CI: 0.55-1.38; p = 0.55), independently predicted for lower cancer mortality. Participants with a healthy diet but low exercise had decreased cancer mortality compared to participants with an unhealthy diet but high exercise (p = 0.01). Conclusions: A healthful diet was associated with lower cancer mortality in women, even after adjusting for obesity, inflammation, and other covariates. In addition, diet may play a stronger role in reducing cancer mortality in women than physical activity.

Efficient derivation of chimeric-antigen receptor-modified TSCM cells.

Chimeric-antigen receptor (CAR) T-cell immunotherapy employs autologous-T cells modified with an antigen-specific CAR. Current CAR-T manufacturing processes tend to yield products dominated by effector T cells and relatively small proportions of long-lived memory T cells. Those few cells are a so-called stem cell memory T (TSCM) subset, which express naïve T-cell markers and are capable of self-renewal and oligopotent differentiation into effector phenotypes. Increasing the proportion of this subset may lead to more effective therapies by improving CAR-T persistence; however, there is currently no standardized protocol for the effective generation of CAR-TSCM cells. Here we present a simplified protocol enabling efficient derivation of gene-modified TSCM cells: Stimulation of naïve CD8+ T cells with only soluble anti-CD3 antibody and culture with IL-7 and IL-15 was sufficient for derivation of CD8+ T cells harboring TSCM phenotypes and oligopotent capabilities. These in-vitro expanded TSCM cells were engineered with CARs targeting the HIV-1 envelope protein as well as the CD19 molecule and demonstrated effector activity both in vitro and in a xenograft mouse model. This simple protocol for the derivation of CAR-TSCM cells may facilitate improved adoptive immunotherapy.

Heart transplantation after total artificial heart bridging-Outcomes over 15 years.

BACKGROUND: Data are limited on outcomes after heart transplantation in patients bridged-to-transplantation (BTT) with a total artificial heart (TAH-t). METHODS: The UNOS database was used to identify 392 adult patients undergoing heart transplantation after TAH-t BTT between 2005 and 2020. They were compared with 11 014 durable left ventricular assist device (LVAD) BTT patients and 22 348 de novo heart transplants (without any durable VAD or TAH-t BTT) during the same period. RESULTS: TAH-t BTT patients had increased dialysis dependence compared to LVAD BTT and de novo transplants (24.7% vs. 2.7% vs. 3.8%) and higher levels of baseline creatinine and total bilirubin (all p < .001). After transplantation, TAH-t BTT patients were more likely to die from multiorgan failure in the first year (25.0% vs. 16.1% vs. 16.1%, p = .04). Ten-year survival was inferior in TAH-t BTT patients (TAH-t BTT 53.1%, LVAD BTT 61.8%, De Novo 62.6%, p < .001), while 10-year survival conditional on 1-year survival was similar (TAH-t BTT 66.8%, LVAD BTT 68.7%, De Novo 69.0%, all p > .20). Among TAH-t BTT patients, predictors of 1-year mortality included higher baseline creatinine and total bilirubin, mechanical ventilation, and cumulative center volume <20 cases of heart transplantation involving TAH-t BTT (all p < .05). CONCLUSION: Survival after TAH-t BTT is acceptable, and patients who survive the early postoperative phase experience similar hazards of mortality over time compared to de novo transplant patients and durable LVAD BTT patients.