Repeatability and reproducibility of the Forskolin-induced swelling (FIS) assay on intestinal organoids from people with Cystic Fibrosis.

BACKGROUND: The forskolin-induced swelling (FIS) assay measures CFTR function on patient-derived intestinal organoids (PDIOs) and may guide treatment selection for individuals with Cystic Fibrosis (CF). The aim of this study is to demonstrate the repeatability and reproducibility of the FIS assay following a detailed Standard Operating Procedure (SOP), thus advancing the validation of the assay for precision medicine (theranostic) applications. METHODS: Over a 2-year period, FIS responses to CFTR modulators were measured in four European labs. PDIOs from six subjects with CF carrying different CFTR genotypes were used to assess the repeatability and reproducibility across the dynamic range of the assay. RESULTS: Technical, intra-assay repeatability was high (Lin's concordance correlation coefficient (CCC) 0.95-0.98). Experimental, within-subject repeatability was also high within each lab (CCCs all >0.9). Longer-term repeatability (>1 year) showed more variability (CCCs from 0.67 to 0.95). The reproducibility between labs was also high (CCC ranging from 0.92 to 0.97). Exploratory analysis also found that between-lab percentage of agreement of dichotomized CFTR modulator outcomes for predefined FIS thresholds ranged between 78 and 100 %. CONCLUSIONS: The observed repeatability and reproducibility of the FIS assay within and across different labs is high and support the use of FIS as biomarker of CFTR function in the presence or absence of CFTR modulators.

Centralized intestinal organoid generation is a feasible and safe approach for personalized medicine as demonstrated in the HIT-CF Europe Organoid Study.

BACKGROUND: Patient-derived intestinal organoids (PDIOs) show great potential as in vitro drug testing platform for personalised medicine in Cystic Fibrosis and oncology. PDIOs can be generated by culturing adult stem cells obtained through rectal forceps biopsy or suction biopsy, but the safety of these procedures and the success rates of generating organoids after shipment to a centralized lab using these procedures has not been studied in this context. We here report the safety and success rates of both biopsy procedures and the subsequent generation of PDIOs in the international multicentre HIT-CF Organoid Study. METHODS: 502 biopsy procedures were conducted, on 489 adult people with Cystic Fibrosis from 33 different hospitals across 12 countries. Depending on the preference of the hospital, either rectal forceps biopsies or suction biopsies were obtained and internationally shipped to a central laboratory for organoid generation. RESULTS: No adverse events were reported for 280 forceps biopsy procedures, while 222 rectal suction biopsy procedures resulted in 2 adverse events, namely continued bleeding and a probably nonrelated gastroenteritis. The success rate of organoid generation from all biopsies was 95%, and the main reason for failure was insufficient sample viability (3.2%). CONCLUSION: Our results indicate that both rectal suction biopsy and forceps biopsy procedures are safe procedures. The high success rates of PDIO generation from the obtained tissue samples demonstrate the feasibility of the organoid technology for personalised in vitro testing in an international setting.

Paneth cell ontogeny in term and preterm ovine models.

Introduction: Paneth cells are critically important to intestinal health, including protecting intestinal stem cells, shaping the intestinal microbiome, and regulating host immunity. Understanding Paneth cell biology in the immature intestine is often modeled in rodents with little information in larger mammals such as sheep. Previous studies have only established the distribution pattern of Paneth cells in healthy adult sheep. Our study aimed to examine the ontogeny, quantification, and localization of Paneth cells in fetal and newborn lambs at different gestational ages and with perinatal transient asphyxia. We hypothesized that ovine Paneth cell distribution at birth resembles the pattern seen in humans (highest concentrations in the ileum) and that ovine Paneth cell density is gestation-dependent. Methods: Intestinal samples were obtained from 126-127 (preterm, with and without perinatal transient asphyxia) and 140-141 (term) days gestation sheep. Samples were quantified per crypt in at least 100 crypts per animal and confirmed as Paneth cells through in immunohistochemistry. Results: Paneth cells had significantly higher density in the ileum compared to the jejunum and were absent in the colon. Discussion: Exposure to perinatal transient asphyxia acutely decreased Paneth cell numbers. These novel data support the possibility of utilizing ovine models for understanding Paneth cell biology in the fetus and neonate.

Organoids as a biomarker for personalized treatment in metastatic colorectal cancer: drug screen optimization and correlation with patient response.

BACKGROUND: The inability to predict treatment response of colorectal cancer patients results in unnecessary toxicity, decreased efficacy and survival. Response testing on patient-derived organoids (PDOs) is a promising biomarker for treatment efficacy. The aim of this study is to optimize PDO drug screening methods for correlation with patient response and explore the potential to predict responses to standard chemotherapies. METHODS: We optimized drug screen methods on 5-11 PDOs per condition of the complete set of 23 PDOs from patients treated for metastatic colorectal cancer (mCRC). PDOs were exposed to 5-fluorouracil (5-FU), irinotecan- and oxaliplatin-based chemotherapy. We compared medium with and without N-acetylcysteine (NAC), different readouts and different combination treatment set-ups to capture the strongest association with patient response. We expanded the screens using the optimized methods for all PDOs. Organoid sensitivity was correlated to the patient's response, determined by % change in the size of target lesions. We assessed organoid sensitivity in relation to prior exposure to chemotherapy, mutational status and sidedness. RESULTS: Drug screen optimization involved excluding N-acetylcysteine from the medium and biphasic curve fitting for 5-FU & oxaliplatin combination screens. CellTiter-Glo measurements were comparable with CyQUANT and did not affect the correlation with patient response. Furthermore, the correlation improved with application of growth rate metrics, when 5-FU & oxaliplatin was screened in a ratio, and 5-FU & SN-38 using a fixed dose of SN-38. Area under the curve was the most robust drug response curve metric. After optimization, organoid and patient response showed a correlation coefficient of 0.58 for 5-FU (n = 6, 95% CI -0.44,0.95), 0.61 for irinotecan- (n = 10, 95% CI -0.03,0.90) and 0.60 for oxaliplatin-based chemotherapy (n = 11, 95% CI -0.01,0.88). Median progression-free survival of patients with resistant PDOs to oxaliplatin-based chemotherapy was significantly shorter than sensitive PDOs (3.3 vs 10.9 months, p = 0.007). Increased resistance to 5-FU in patients with prior exposure to 5-FU/capecitabine was adequately reflected in PDOs (p = 0.003). CONCLUSIONS: Our study emphasizes the critical impact of the screening methods for determining correlation between PDO drug screens and mCRC patient outcomes. Our 5-step optimization strategy provides a basis for future research on the clinical utility of PDO screens.

Estimating infection prevalence using the positive predictive value of self-administered rapid antigen diagnostic tests: An exploration of SARS-CoV-2 surveillance data in the Netherlands from May 2021 to April 2022.

Measuring the severity of the disease of SARS-CoV-2 is complicated by the lack of valid estimations for the prevalence of infection. Self-administered rapid antigen diagnostic tests (Ag-RDTs) were available in the Netherlands since March 2021, requiring confirmation by reverse-transcription polymerase chain reaction (RT-PCR) for positive results. We explored the possibility of utilizing the positive predictive value (PPV) of Ag-RDTs to estimate SARS-CoV-2 prevalence. We used data from all Public Health service testing facilities between 3 May 2021 and 10 April 2022. The PPV was calculated by dividing the number of positive RT-PCR results by the total number of confirmation tests performed, and used to estimate the prevalence and compared with the number of COVID-19 hospital admissions. In total 3,599,894 cases were included. The overall PPV was 91.8% and 88.8% were symptomatic. During our study period, the estimated prevalence ranged between 2-22% in symptomatic individuals and 2-14% in asymptomatic individuals, with a correlation between the estimated prevalence and hospital admissions two weeks later (r = 0.68 (p<0.01) and r = 0.60 (p<0.01) for symptomatic/asymptomatic individuals). The PPV of Ag-RDTs can help estimate changes in SARS-CoV-2 prevalence, especially when used in conjunction with other surveillance systems. However, the used method probably overestimated the true prevalence because of unmonitored differences in test propensity between individuals.

Clotrimazole ameliorates chronic mild stress-induced depressive-like behavior in rats; crosstalk between the HPA, NLRP3 inflammasome, and Wnt/ß-catenin pathways.

Depression is a major emotional disorder that has a detrimental effect on quality of life. The chronic mild stress (CMS)-depression model was adopted in rats to evaluate the neurotherapeutic effect of Clotrimazole (CLO) and investigate the possible mechanisms of its antidepressant action via its impact on the hypothalamic pituitary adrenal (HPA) axis and the stress hormone, cortisol. It was found that azole antifungals affect steroidogenesis and the HPA axis. Behavioral, histopathological, inflammatory, and apoptotic pathways were assessed. Serum cortisol, inflammasome biomarkers, hippocampal NLRP3, caspase-1, and IL-18, and the canonical Wnt/ß-catenin neurogenesis biomarkers, Wnt3a, and non-phosphorylated ß-catenin levels were also determined. Different stressors were applied for 28 days to produce depressive-like symptoms, and CLO was administered at a daily dose of 30 mg/kg body weight. Subsequently, behavioral and biochemical tests were carried out to assess the depressive-like phenotype in rats. Stressed rats showed increased immobility time in the forced swimming test (FST), decreased grooming time in the splash test (ST), increased serum cortisol levels, increased inflammasome biomarkers, and decreased neurogenesis. However, administration of CLO produced significant antidepressant-like effects in rats, which were accompanied by a significant decrease in immobility time in FST, an increase in grooming time in ST, a decrease in serum cortisol level, a decrease in inflammasome biomarkers, and an increase in neurogenesis biomarkers. The antidepressant mechanism of CLO involves the HPA axis and the anti-inflammatory effect, followed by neurogenesis pathway activation. Therefore, CLO may have the potential to be a novel antidepressant candidate.

Depressive symptoms among adolescents in six sub-Saharan African countries: A pooled analysis of associated factors.

There is a need to identify risk factors, including nutrition-related factors, for depressive disorders among sub-Saharan African (SSA) adolescents. We examined the association of multiple measures with depressive symptoms among adolescents living across six SSA countries. Building on previous analyses, we used data from a cross-sectional study conducted from 2015 to 2017 among adolescents aged 10-19 years in six SSA countries (N = 7512). Depressive symptoms were defined as highest tertile of the 6-item Kutcher Adolescent Depression Scale score. Using mixed-effects Poisson regression models, we pooled data across sites and examined the association of sociodemographic, nutrition, and other indices with depressive symptoms. We additionally assessed effect modification by sex, age, and school-going status. We observed higher risk of depressive symptoms among girls (adjusted risk ratio [RR]: 1.29, 95 % confidence interval [95 % CI]: 1.05-1.58, P = 0.016), older adolescents (RR for 18-19 years: 1.59, 95 % CI: 1.44-1.76, P < 0.001), and adolescents experiencing bullying (RR: 1.43, 95 % CI: 1.26-1.62, P < 0.001) or violence (RR: 1.34, 95 % CI: 1.24-1.45, P < 0.001). Adolescents experiencing food insecurity also had a higher risk of depressive symptoms (RR: 1.90, 95 % CI: 1.64-2.19, P < 0.001) along with those consuming ≥ 5 servings of fruit and vegetables per day (RR: 1.18, 95 % CI: 1.03-1.34, P = 0.015); conversely, those who consumed grains, roots and tubers in the past day were at decreased risk (RR: 0.73, 95 % CI: 0.69-0.77, P < 0.001). There was no strong evidence of effect modification of associations. This study reinforces the potential role of multiple sociodemographic and nutrition-related measures on risk of depressive symptoms in these populations.

Integral inequalities for closed linear Weingarten submanifolds in the product spaces.

An integral inequality for closed linear Weingarten 𝑚-submanifolds with parallel normalized mean curvature vector field (pnmc lw-submanifolds) in the product spaces 𝑀𝑛(𝑐) × â„, 𝑛 > 𝑚 ≥ 4, where 𝑀𝑛(𝑐) is a space form of constant sectional curvature 𝑐 ∈ {-1, 1}, is proved. As an application is shown that the sharpness in this inequality is attained in the totally umbilical hypersurfaces, and in a certain family of standard product of the form 𝕊1(√1 - 𝑟2) × ð•Šð‘š-1(𝑟) with 0 < 𝑟 < 1 when 𝑐 = 1. In the case where 𝑐 = -1, is obtained an integral inequality whose sharpness is attained only in the totally umbilical hypersurfaces. When 𝑚 = 2 and 𝑚 = 3, an integral inequality is also obtained with equality happening in the totally umbilical hypersurfaces.

Femoral Occlusion during Neonatal Cardiopulmonary Resuscitation Improves Outcomes in an Ovine Model of Perinatal Cardiac Arrest.

BACKGROUND: The goal of chest compressions during neonatal resuscitation is to increase cerebral and coronary blood flow leading to the return of spontaneous circulation (ROSC). During chest compressions, bilateral femoral occlusion may increase afterload and promote carotid and coronary flow, an effect similar to epinephrine. Our objectives were to determine the impact of bilateral femoral occlusion during chest compressions on the incidence and timing of ROSC and hemodynamics. METHODOLOGY: In this randomized study, 19 term fetal lambs in cardiac arrest were resuscitated based on the Neonatal Resuscitation Program guidelines and randomized into two groups: femoral occlusion or controls. Bilateral femoral arteries were occluded by applying pressure using two fingers during chest compressions. RESULTS: Seventy percent (7/10) of the lambs in the femoral occlusion group achieved ROSC in 5 ± 2 min and three lambs (30%) did not receive epinephrine. ROSC was achieved in 44% (4/9) of the controls in 13 ± 6 min and all lambs received epinephrine. The femoral occlusion group had higher diastolic blood pressures, carotid and coronary blood flow. CONCLUSION: Femoral occlusion resulted in faster and higher incidence of ROSC, most likely due to attaining increased diastolic pressures, coronary and carotid flow. This is a low-tech intervention that can be easily adapted in resource limited settings, with the potential to improve survival and neurodevelopmental outcomes.

Coupling of coastal activity with tidal cycles is stronger in tool-using capuchins (Cebus capucinus imitator).

Terrestrial mammals exploiting coastal resources must cope with the challenge that resource availability and accessibility fluctuate with tidal cycles. Tool use can improve foraging efficiency and provide access to structurally protected resources that are otherwise unavailable (e.g. molluscs and fruits). To understand how variable accessibility of valuable resources shapes behavioural patterns, and whether tool use aids in the efficient exploitation of intertidal resources, we compared the relationship between tidal cycles and activity patterns of tool-using versus non-tool-using groups of white-faced capuchin monkeys on Jicarón Island in Coiba National Park, Panama. Although tool use on Jicarón is localized to a small stretch of coast (approx. 1 km), all coastal groups forage on intertidal resources. Using more than 5 years of camera trap data at varying distances from the coast, we found that capuchins on Jicarón showed increased coastal activity during specific parts of the tidal cycle, and that this relationship differed between tool-using and non-tool-using groups, as well as between seasons. Activity patterns of tool-using capuchins were more strongly and consistently tied to tidal cycles compared with non-tool-users, indicating that tool use might allow for more efficient exploitation of tidal resources. Our findings highlight the potential of tool use to aid niche expansion.

Intestinal organoids as an in vitro platform to characterize disposition, metabolism, and safety profile of small molecules.

Intestinal organoids derived from LGR5+ adult stem cells allow for long-term culturing, more closely resemble human physiology than traditional intestinal models, like Caco-2, and have been established for several species. Here we evaluated intestinal organoids for drug disposition, metabolism, and safety applications. Enterocyte-enriched human duodenal organoids were cultured as monolayers to enable bidirectional transport studies. 3D enterocyte-enriched human duodenal and colonic organoids were incubated with probe substrates of major intestinal drug metabolizing enzymes (DMEs). To distinguish human intestinal toxic (high incidence of diarrhea in clinical trials and/or black box warning related to intestinal side effects) from non-intestinal toxic compounds, ATP-based cell viability was used as a readout, and compounds were ranked based on their IC50 values in relation to their 30-times maximal total plasma concentration (Cmax). To assess if rat and dog organoids reproduced the respective in vivo intestinal safety profiles, ATP-based viability was assessed in rat and dog organoids and compared to in vivo intestinal findings when available. Human duodenal monolayers discriminated high and low permeable compounds and demonstrated functional activity for the main efflux transporters Multi drug resistant protein 1 (MDR1, P-glycoprotein P-gp) and Breast cancer resistant protein (BCRP). Human 3D duodenal and colonic organoids also showed metabolic activity for the main intestinal phase I and II DMEs. Organoids derived from specific intestinal segments showed activity differences in line with reported DMEs expression. Undifferentiated human organoids accurately distinguished all but one compound from the test set of non-toxic and toxic drugs. Cytotoxicity in rat and dog organoids correlated with preclinical toxicity findings and observed species sensitivity differences between human, rat, and dog organoids. In conclusion, the data suggest intestinal organoids are suitable in vitro tools for drug disposition, metabolism, and intestinal toxicity endpoints. The possibility to use organoids from different species, and intestinal segment holds great potential for cross-species and regional comparisons.

Patient reported experience measures on HIV viral load testing at public health facilities in Dar es Salaam, Tanzania: A convergent mixed method study.

While viral load (VL) testing is critical to effective treatment of human immunodeficiency virus (HIV), little is known about patients' experiences with, and barriers to VL-testing in the context of HIV infection. We assessed patient reported experience measures (PREMs) on VL-testing in public HIV clinics in Tanzania. In a cross-sectional convergent mixed method study, we collected information on VL test related PREMs, clinical and sociodemographic factors. PREMs were measured using a 5-point Likert scale. Focus Group Discussions (FGDs) explored on experience, access, and barriers to VL-testing. Descriptive statistics summarized patients' factors and PREMs. Logistic regression was used to explore association of patient factors, PREMs and satisfaction with VL-testing services. Thematic analysis was used for qualitative data. A total of 439 (96.48%) respondents completed the survey, 331 (75.40%) were female, median (IQR) age was 41(34, 49) years. A total of 253(57.63%) had a VL test at least once in the past 12 months, of whom 242(96.0%) had VL<1000 copies/ml. Investigating barriers to VL-testing, most participants (>92.0%) reported good or very good health services responsiveness (HSR). A scale of very good was chosen by the majority for being treated with respect 174(39.6%), listened to 173(39.4%), following advice 109(24.8%), being involved in decisions 101(23.0%), and for communication 102(23.3%). Satisfaction on VL-testing services was significantly associated with respondents following care providers' advice, (aOR) = 2.07 [95%CI 1.13-3.78], involvement in decisions aOR = 4.16 [95%CI 2.26-7.66], and communication aOR = 2.27 [95%CI 1.25-4.14]. FGDs findings converged with the survey data, with identified barriers to VL test including lack of autonomy in decision making, little awareness on the benefits of the test, long waiting time, stigma, competing priorities for those with comorbidities and transport costs. Satisfaction on VL-testing was largely a result of involvement in decision making, following care provider's advice and good communication; entities needing universal improvement across the country.

Drug-repurposing screen on patient-derived organoids identifies therapy-induced vulnerability in KRAS-mutant colon cancer.

Patient-derived organoids (PDOs) are widely heralded as a drug-screening platform to develop new anti-cancer therapies. Here, we use a drug-repurposing library to screen PDOs of colorectal cancer (CRC) to identify hidden vulnerabilities within therapy-induced phenotypes. Using a microscopy-based screen that accurately scores drug-induced cell killing, we have tested 414 putative anti-cancer drugs for their ability to switch the EGFRi/MEKi-induced cytostatic phenotype toward cytotoxicity. A majority of validated hits (9/37) are microtubule-targeting agents that are commonly used in clinical oncology, such as taxanes and vinca-alkaloids. One of these drugs, vinorelbine, is consistently effective across a panel of >25 different CRC PDOs, independent of RAS mutational status. Unlike vinorelbine alone, its combination with EGFR/MEK inhibition induces apoptosis at all stages of the cell cycle and shows tolerability and effective anti-tumor activity in vivo, setting the basis for a clinical trial to treat patients with metastatic RAS-mutant CRC.

The role of miRNAs in insulin resistance and diabetic macrovascular complications - A review.

Diabetes is the most prevalent metabolic disturbance disease and has been regarded globally as one of the principal causes of mortality. Diabetes is accompanied by several macrovascular complications, including stroke, coronary artery disease (CAD), and cardiomyopathy as a consequence of atherosclerosis. The onset of type 2 diabetes is closely related to insulin resistance (IR). miRNAs have been linked to various metabolic processes, including glucose homeostasis, regulation of lipid metabolism, gluconeogenesis, adipogenesis, glucose transporter type 4 expression, insulin sensitivity, and signaling. Consequently, miRNA dysregulation mediates IR in some target organs, comprising liver, muscle, and adipose tissue. Moreover, miRNAs are crucial in developing diabetes and its associated macrovascular complications through their roles in several signaling pathways implicated in inflammation, apoptosis, cellular survival and migration, the proliferation of vascular smooth muscle cells, neurogenesis, angiogenesis, autophagy, oxidative stress, cardiac remodeling, and fibrosis. Therefore, the purpose of this review is to clarify the role of miRNAs in hepatic, muscle, and adipose tissue IR and explain their roles in the pathogenesis of macrovascular diabetic complications, including stroke, CAD, and cardiomyopathy. Also, explain their roles in gestational diabetes mellitus (GDM). Besides, this review discusses the latest updates on the alteration of miRNA expression in diabetic macrovascular complications.

Molecular and Functional Characterization of Human Intestinal Organoids and Monolayers for Modeling Epithelial Barrier.

BACKGROUND: Patient-derived organoid (PDO) models offer potential to transform drug discovery for inflammatory bowel disease (IBD) but are limited by inconsistencies with differentiation and functional characterization. We profiled molecular and cellular features across a range of intestinal organoid models and examined differentiation and establishment of a functional epithelial barrier. METHODS: Patient-derived organoids or monolayers were generated from control or IBD patient-derived colon or ileum and were molecularly or functionally profiled. Biological or technical replicates were examined for transcriptional responses under conditions of expansion or differentiation. Cell-type composition was determined by deconvolution of cell-associated gene signatures and histological features. Differentiated control or IBD-derived monolayers were examined for establishment of transepithelial electrical resistance (TEER), loss of barrier integrity in response to a cocktail of interferon (IFN)-γ and tumor necrosis factor (TNF)-α, and prevention of cytokine-induced barrier disruption by the JAK inhibitor, tofacitinib. RESULTS: In response to differentiation media, intestinal organoids and monolayers displayed gene expression patterns consistent with maturation of epithelial cell types found in the human gut. Upon differentiation, both colon- and ileum-derived monolayers formed functional barriers, with sustained TEER. Barrier integrity was compromised by inflammatory cytokines IFN-γ and TNF-α, and damage was inhibited in a dose-dependent manner by tofacitinib. CONCLUSIONS: We describe the generation and characterization of human colonic or ileal organoid models capable of functional differentiation to mature epithelial cell types. In monolayer culture, these cells formed a robust epithelial barrier with sustained TEER and responses to pharmacological modulation. Our findings demonstrate that control and IBD patient-derived organoids possess consistent transcriptional and functional profiles that can enable development of epithelial-targeted therapies.

Modeling resistance of colorectal peritoneal metastases to immune checkpoint blockade in humanized mice.

BACKGROUND: The immunogenic nature of metastatic colorectal cancer (CRC) with high microsatellite instability (MSI-H) underlies their responsiveness to immune checkpoint blockade (ICB). However, resistance to ICB is commonly observed, and is associated with the presence of peritoneal-metastases and ascites formation. The mechanisms underlying this site-specific benefit of ICB are unknown. METHODS: We created a novel model for spontaneous multiorgan metastasis in MSI-H CRC tumors by transplanting patient-derived organoids (PDO) into the cecum of humanized mice. Anti-programmed cell death protein-1 (PD-1) and anti-cytotoxic T-lymphocytes-associated protein 4 (CTLA-4) ICB treatment effects were analyzed in relation to the immune context of primary tumors, liver metastases, and peritoneal metastases. Immune profiling was performed by immunohistochemistry, flow cytometry and single-cell RNA sequencing. The role of B cells was assessed by antibody-mediated depletion. Immunosuppressive cytokine levels (interleukin (IL)-10, transforming growth factor (TGF)b1, TGFb2, TGFb3) were determined in ascites and serum samples by ELISA. RESULTS: PDO-initiated primary tumors spontaneously metastasized to the liver and the peritoneum. Peritoneal-metastasis formation was accompanied by the accumulation of ascites. ICB completely cleared liver metastases and reduced primary tumor mass but had no effect on peritoneal metastases. This mimics clinical observations. After therapy discontinuation, primary tumor masses progressively decreased, but peritoneal metastases displayed unabated growth. Therapy efficacy correlated with the formation of tertiary lymphoid structures (TLS)-containing B cells and juxtaposed T cells-and with expression of an interferon-γ signature together with the B cell chemoattractant CXCL13. B cell depletion prevented liver-metastasis clearance by anti-CTLA-4 treatment. Peritoneal metastases were devoid of B cells and TLS, while the T cells in these lesions displayed a dysfunctional phenotype. Ascites samples from patients with cancer with peritoneal metastases and from the mouse model contained significantly higher levels of IL-10, TGFb1, TGFb2 and TGFb3 than serum samples. CONCLUSIONS: By combining organoid and humanized mouse technologies, we present a novel model for spontaneous multiorgan metastasis by MSI-H CRC, in which the clinically observed organ site-dependent benefit of ICB is recapitulated. Moreover, we provide empirical evidence for a critical role for B cells in the generation of site-dependent antitumor immunity following anti-CTLA-4 treatment. High levels of immunosuppressive cytokines in ascites may underlie the observed resistance of peritoneal metastases to ICB.

Engineering multicellular living systems-a Keystone Symposia report.

The ability to engineer complex multicellular systems has enormous potential to inform our understanding of biological processes and disease and alter the drug development process. Engineering living systems to emulate natural processes or to incorporate new functions relies on a detailed understanding of the biochemical, mechanical, and other cues between cells and between cells and their environment that result in the coordinated action of multicellular systems. On April 3-6, 2022, experts in the field met at the Keystone symposium "Engineering Multicellular Living Systems" to discuss recent advances in understanding how cells cooperate within a multicellular system, as well as recent efforts to engineer systems like organ-on-a-chip models, biological robots, and organoids. Given the similarities and common themes, this meeting was held in conjunction with the symposium "Organoids as Tools for Fundamental Discovery and Translation".

SARS-CoV-2 seroprevalence in the city of Hyderabad, India in early 2021.

Background: COVID-19 emerged as a global pandemic in 2020, spreading rapidly to most parts of the world. The proportion of infected individuals in a population can be reliably estimated via serosurveillance, making it a valuable tool for planning control measures. Our serosurvey study aimed to investigate SARS-CoV-2 seroprevalence in the urban population of Hyderabad at the end of the first wave of infections. Methods: This cross-sectional survey, conducted in January 2021 and including males and females aged 10 years and above, used multi-stage random sampling. 9363 samples were collected from 30 wards distributed over six zones of Hyderabad, and tested for antibodies against SARS-CoV-2 nucleocapsid antigen. Results: Overall seropositivity was 54.2%, ranging from 50% to 60% in most wards. Highest exposure appeared to be among those aged 30-39 and 50-59 years, with women showing greater seropositivity. Seropositivity increased with family size, with only marginal differences among people with varying levels of education. Seroprevalence was significantly lower among smokers. Only 11% of the survey subjects reported any COVID-19 symptoms, while 17% had appeared for COVID-19 testing. Conclusion: Over half the city's population was infected within a year of onset of the pandemic. However, ∼ 46% of people remained susceptible, contributing to subsequent waves of infection.

Functional patient-derived organoid screenings identify MCLA-158 as a therapeutic EGFR × LGR5 bispecific antibody with efficacy in epithelial tumors.

Patient-derived organoids (PDOs) recapitulate tumor architecture, contain cancer stem cells and have predictive value supporting personalized medicine. Here we describe a large-scale functional screen of dual-targeting bispecific antibodies (bAbs) on a heterogeneous colorectal cancer PDO biobank and paired healthy colonic mucosa samples. More than 500 therapeutic bAbs generated against Wingless-related integration site (WNT) and receptor tyrosine kinase (RTK) targets were functionally evaluated by high-content imaging to capture the complexity of PDO responses. Our drug discovery strategy resulted in the generation of MCLA-158, a bAb that specifically triggers epidermal growth factor receptor degradation in leucine-rich repeat-containing G-protein-coupled receptor 5-positive (LGR5+) cancer stem cells but shows minimal toxicity toward healthy LGR5+ colon stem cells. MCLA-158 exhibits therapeutic properties such as growth inhibition of KRAS-mutant colorectal cancers, blockade of metastasis initiation and suppression of tumor outgrowth in preclinical models for several epithelial cancer types.

Burden of HIV-related stigma and associated factors among women living with depression accessing PMTCT services in Dar es Salaam, Tanzania.

HIV-related stigma represents a potent risk factor for a range of poor health outcomes, including mental health symptoms, treatment non-adherence, and substance use. Understanding the role of HIV-related stigma in promoting healthcare outcomes is critical for vulnerable populations, such as pregnant women living with HIV, in contexts with continued high rates of HIV and associated stigma, such as sub-Saharan Africa. The current study examined a range of risk and protective factors for HIV-related stigma with 742 pregnant women (M age = 29.6 years) living with depression and HIV accessing prevention of mother-to-child transmission of HIV (PMTCT) services in Dar es Salaam, Tanzania. Risk factors included depressive symptoms, ART non-adherence, intimate partner violence, food insecurity, and alcohol problems. Protective factors included disclosure of HIV status, social support, an appreciative relationship with their partner, hope, and self-efficacy. Findings highlight key psychosocial and behavioral determinants of HIV-related stigma for pregnant women living with HIV in Tanzania, and can inform perinatal care programming and interventions to optimize mental health and adherence outcomes.