Identification and multimodal characterization of a specialized epithelial cell type associated with Crohn's disease.

Crohn's disease (CD) is a complex chronic inflammatory disorder with both gastrointestinal and extra-intestinal manifestations associated immune dysregulation. Analyzing 202,359 cells from 170 specimens across 83 patients, we identify a distinct epithelial cell type in both terminal ileum and ascending colon (hereon as 'LND') with high expression of LCN2, NOS2, and DUOX2 and genes related to antimicrobial response and immunoregulation. LND cells, confirmed by in-situ RNA and protein imaging, are rare in non-IBD controls but expand in active CD, and actively interact with immune cells and specifically express IBD/CD susceptibility genes, suggesting a possible function in CD immunopathogenesis. Furthermore, we discover early and late LND subpopulations with different origins and developmental potential. A higher ratio of late-to-early LND cells correlates with better response to anti-TNF treatment. Our findings thus suggest a potential pathogenic role for LND cells in both Crohn's ileitis and colitis.

A Case Report of a Filipino Boy with Childhood Cataract and Clinically Diagnosed Roberts Syndrome.

Childhood cataract is a common cause of visual impairment. Familial types are uncommon among Filipinos. Furthermore, it is not common to have one that follows an autosomal dominant pattern of inheritance but with associated syndromic presentation like Roberts syndrome which is an autosomal recessive disorder. This is a case of a 9-year-old Filipino boy with cataract in the left eye associated with low-set ears, facial asymmetry, underdeveloped nasal ala, cleft lip and palate, macroglossia, micrognathia, short right shin, and absent feet. Patient was clinically diagnosed with Roberts syndrome. We present a clinically diagnosed Roberts syndrome (RS), the first reported RS in a Filipino in local and international literature to our knowledge with an autosomal dominant childhood cataract. Genetic testing can assist in the confirmation of this case.

A Case Report of Inferior Oblique Reattachment after Inferomedial Orbital Wall Reconstruction from Total Maxillectomy.

The detachment and reattachment of inferior oblique (IO) have been done by ophthalmologists in inferomedial orbital wall fracture repair to avoid inducing a new diplopia in patients post-operatively. However, doing them in orbital wall reconstruction in patient who underwent maxillectomy for a malignancy has not been described yet. We describe a case where a disinserted inferior oblique was reattached after titanium mesh implantation to prevent diplopia after the surgery. This is the case of a 40-year-old male diagnosed with recurrent bilateral nasomaxillary ameloblastoma who underwent total maxillectomy of the right with removal of the inferomedial orbital wall, and detachment and reattachment of inferior oblique with no resulting inferior oblique palsy and diplopia from hypotropia or incyclotorsion. This is the first report, to our knowledge, to describe reattachment of IO in its approximate insertion during reconstruction of the inferomedial orbital wall after total maxillectomy from a malignancy with no resulting diplopia.

Alleviating tiling effect by random walk sliding window in high-resolution histological whole slide image synthesis.

Multiplex immunofluorescence (MxIF) is an advanced molecular imaging technique that can simultaneously provide biologists with multiple (i.e., more than 20) molecular markers on a single histological tissue section. Unfortunately, due to imaging restrictions, the more routinely used hematoxylin and eosin (H&E) stain is typically unavailable with MxIF on the same tissue section. As biological H&E staining is not feasible, previous efforts have been made to obtain H&E whole slide image (WSI) from MxIF via deep learning empowered virtual staining. However, the tiling effect is a long-lasting problem in high-resolution WSI-wise synthesis. The MxIF to H&E synthesis is no exception. Limited by computational resources, the cross-stain image synthesis is typically performed at the patch-level. Thus, discontinuous intensities might be visually identified along with the patch boundaries assembling all individual patches back to a WSI. In this work, we propose a deep learning based unpaired high-resolution image synthesis method to obtain virtual H&E WSIs from MxIF WSIs (each with 27 markers/stains) with reduced tiling effects. Briefly, we first extend the CycleGAN framework by adding simultaneous nuclei and mucin segmentation supervision as spatial constraints. Then, we introduce a random walk sliding window shifting strategy during the optimized inference stage, to alleviate the tiling effects. The validation results show that our spatially constrained synthesis method achieves a 56% performance gain for the downstream cell segmentation task. The proposed inference method reduces the tiling effects by using 50% fewer computation resources without compromising performance. The proposed random sliding window inference method is a plug-and-play module, which can be generalized for other high-resolution WSI image synthesis applications. The source code with our proposed model are available at https://github.com/MASILab/RandomWalkSlidingWindow.git.

Comparison of Congenital Rubella Syndrome Cases at a Philippine Tertiary Hospital from 2009-2012 to 2019-2022.

Background and Objective: The Philippines does not have a national congenital rubella syndrome (CRS) surveillance or registry. Regular monitoring of CRS cases in hospitals, including in a Philippine tertiary hospital, helped in the past to provide clinico-epidemiologic data on CRS. This study aimed to continue providing clinico-epidemiologic data on CRS cases seen in the Philippine tertiary hospital from 2009-2012 and 2019-2022 and compare the cases seen from said timelines. Methods: A cross-sectional study was used, employing chart review of patients newly diagnosed with CRS from 2009-2012 and 2019-2022 in the Department of Ophthalmology and Visual Sciences at the Philippine tertiary hospital. Results: Forty-two patients newly diagnosed with CRS from 2009-2012 and 2019-2022 were included. Only 14 (33%) were serologically-confirmed cases (albeit qualitatively). Median age (first and third interquartile ranges) at consult was 1 year (0.4, 2.5). Twenty-four (57%) patients had maternal history of rashes and/or fever. Trimester of pregnancy when mother became symptomatic was not significantly correlated with chief complaint (p=0.20) and numbers of ophthalmic (p=0.68) and systemic manifestations (p=0.32). Cataract was the most common ophthalmic manifestation present in 40 (95%) patients. Twenty-six (62%) patients had other associated systemic findings of which hearing loss was the most common. Only 29 of 40 patients with cataract underwent lensectomy, with 23 patients having poor visual prognosis prior to surgery (5 with nystagmus alone, 10 with nystagmus and strabismus, and 8 with strabismus alone). Discussion: Using ophthalmic manifestations as primary indicator, this study provided an update on the CRS cases in the country. Laboratory confirmation remains a challenge in diagnosing CRS as the tests are costly and not widely available. There was increase from 2009-2012 compared to 2019-2022 in number of patients who underwent surgical treatment for cataract but visual outcomes were suboptimal due to delay in consultation. Although there was a decrease in number of CRS cases seen in the Philippine tertiary hospital, this cannot be attributed to increased rubella-containing vaccine (RCV) coverage alone. Conclusion: Provision of data from individual hospital-based studies similar to this highlights the need for a national CRS surveillance system or registry. This can better gauge the burden of CRS and identify the gap in RCV coverage.

Retinoblastoma in the Southern Philippines: Clinical Outcomes of Retinoblastoma Patients in a Davao Tertiary Hospital.

Background: Retinoblastoma is the most common intraocular cancer in childhood in the Philippines. Most data though on demographics, clinical profile, treatment options, and outcomes in the country are from the National Capital Region. Objectives: This study aimed to describe the demographics, clinical profile, treatment done, and outcomes of retinoblastoma patients seen in a public tertiary referral center in Davao from 2011-2020 to make available literature more representative of the status of retinoblastoma in the Philippines. Methods: An analytical cross-sectional study was conducted using the records of retinoblastoma patients seen in a tertiary government hospital located in Davao Region from January 2011 to December 2020. Results: There were 157 patients included in the analysis. Seventy-three (46%) were female with 44% coming from the Davao Region. One hundred seven (69%) patients had unilateral disease. Median age at initial consultation for patients with unilateral disease was significantly older than those with bilateral disease (p<0.003). Tumors were extraocular in 82 (40%) eyes. In the intraocular group, 36% of the eyes belonged to International Classification of Retinoblastoma (ICRB) Groups D and E. Enucleation was the most commonly performed treatment. Survival rate was 28%.This is the first report to provide epidemiologic and clinical data on retinoblastoma in the literature, including survival data, from Mindanao. Advanced stages and extraocular cases of retinoblastoma remain high. Delay of consultation contributed to the prognosis and clinical outcome of the disease. Conclusion: Advanced stages and extraocular cases of retinoblastoma remain significantly high in the country, even in Mindanao.

GammaGateR: semi-automated marker gating for single-cell multiplexed imaging.

MOTIVATION: Multiplexed immunofluorescence (mIF) is an emerging assay for multichannel protein imaging that can decipher cell-level spatial features in tissues. However, existing automated cell phenotyping methods, such as clustering, face challenges in achieving consistency across experiments and often require subjective evaluation. As a result, mIF analyses often revert to marker gating based on manual thresholding of raw imaging data. RESULTS: To address the need for an evaluable semi-automated algorithm, we developed GammaGateR, an R package for interactive marker gating designed specifically for segmented cell-level data from mIF images. Based on a novel closed-form gamma mixture model, GammaGateR provides estimates of marker-positive cell proportions and soft clustering of marker-positive cells. The model incorporates user-specified constraints that provide a consistent but slide-specific model fit. We compared GammaGateR against the newest unsupervised approach for annotating mIF data, employing two colon datasets and one ovarian cancer dataset for the evaluation. We showed that GammaGateR produces highly similar results to a silver standard established through manual annotation. Furthermore, we demonstrated its effectiveness in identifying biological signals, achieved by mapping known spatial interactions between CD68 and MUC5AC cells in the colon and by accurately predicting survival in ovarian cancer patients using the phenotype probabilities as input for machine learning methods. GammaGateR is a highly efficient tool that can improve the replicability of marker gating results, while reducing the time of manual segmentation. AVAILABILITY AND IMPLEMENTATION: The R package is available at https://github.com/JiangmeiRubyXiong/GammaGateR.

Cross-scale multi-instance learning for pathological image diagnosis.

Analyzing high resolution whole slide images (WSIs) with regard to information across multiple scales poses a significant challenge in digital pathology. Multi-instance learning (MIL) is a common solution for working with high resolution images by classifying bags of objects (i.e. sets of smaller image patches). However, such processing is typically performed at a single scale (e.g., 20× magnification) of WSIs, disregarding the vital inter-scale information that is key to diagnoses by human pathologists. In this study, we propose a novel cross-scale MIL algorithm to explicitly aggregate inter-scale relationships into a single MIL network for pathological image diagnosis. The contribution of this paper is three-fold: (1) A novel cross-scale MIL (CS-MIL) algorithm that integrates the multi-scale information and the inter-scale relationships is proposed; (2) A toy dataset with scale-specific morphological features is created and released to examine and visualize differential cross-scale attention; (3) Superior performance on both in-house and public datasets is demonstrated by our simple cross-scale MIL strategy. The official implementation is publicly available at https://github.com/hrlblab/CS-MIL.

Coronavirus M Protein Trafficking in Epithelial Cells Utilizes a Myosin Vb Splice Variant and Rab10.

The membrane (M) glycoprotein of coronaviruses (CoVs) serves as the nidus for virion assembly. Using a yeast two-hybrid screen, we identified the interaction of the cytosolic tail of Murine Hepatitis Virus (MHV-CoV) M protein with Myosin Vb (MYO5B), specifically with the alternative splice variant of cellular MYO5B including exon D (MYO5B+D), which mediates interaction with Rab10. When co-expressed in human lung epithelial A549 and canine kidney epithelial MDCK cells, MYO5B+D co-localized with the MHV-CoV M protein, as well as with the M proteins from Porcine Epidemic Diarrhea Virus (PEDV-CoV), Middle East Respiratory Syndrome (MERS-CoV) and Severe Acute Respiratory Syndrome 2 (SARS-CoV-2). Co-expressed M proteins and MYO5B+D co-localized with endogenous Rab10 and Rab11a. We identified point mutations in MHV-CoV M that blocked the interaction with MYO5B+D in yeast 2-hybrid assays. One of these point mutations (E121K) was previously shown to block MHV-CoV virion assembly and its interaction with MYO5B+D. The E to K mutation at homologous positions in PEDV-CoV, MERS-CoV and SARS-CoV-2 M proteins also blocked colocalization with MYO5B+D. The knockdown of Rab10 blocked the co-localization of M proteins with MYO5B+D and was rescued by re-expression of CFP-Rab10. Our results suggest that CoV M proteins traffic through Rab10-containing systems, in association with MYO5B+D.

Philippine retinoblastoma initiative multi-eye center study 2010-2020.

AIM: To provide a comprehensive and more representative national data on the disease, especially on treatment options and outcomes, and to determine access of retinoblastoma patients from Luzon, Visayas and Mindanao to eye care, and determine if access is associated with delay in consultation, staging and outcomes. METHODS: Cohort study of retinoblastoma patients seen in eleven institutions located in the three major areas of the Philippines namely Luzon, Vizayas and Mindanao from 2010-2020. RESULTS: Totally 636 patients, involving 821 eyes, were included. Majority (57%) were from Luzon and were seen in institutions in Luzon (72%). Annually, 58±10 new cases were seen with 71% having unilateral disease. Median delay of consultation remained long at 9 (3, 17)mo, longest in patients with unilateral disease (P<0.02) and those from the Visayas (P<0.003). Based on the International Retinoblastoma Staging System, only 35% of patients had Stage 1 while 47% already had extraocular disease. Enucleation was the most common treatment received by 484 patients while intravenous chemotherapy was received by 469. There were 250 (39%) patients alive, 195 (31%) dead, 85 (13%) abandoned, 17 (3%) refused and 89 (14%) with no data. CONCLUSION: This study presents the largest cohort of retinoblastoma patients in the Philippines in terms of patients' and participating institutions' number and geographical location and type of institution (private and public). It also presents more comprehensive data on the treatments used and outcomes (survival, globe salvage, and vision retention rates). Delay in consultation was still long among patients leading to advanced disease stage and lower survival rate. Despite increasing capacity to diagnose and manage retinoblastoma in the country, the delay of consultation remains long primarily due to accessibility issues to eye care institutions especially in the Visayas and financial concerns. The delay was still significant that overall survival rate remain low.

Inhibition of autocrine HGF maturation overcomes cetuximab resistance in colorectal cancer.

Although amplifications and mutations in receptor tyrosine kinases (RTKs) act as bona fide oncogenes, in most cancers, RTKs maintain moderate expression and remain wild-type. Consequently, cognate ligands control many facets of tumorigenesis, including resistance to anti-RTK therapies. Herein, we show that the ligands for the RTKs MET and RON, HGF and HGFL, respectively, are synthesized as inactive precursors that are activated by cellular proteases. Our newly generated HGF/HGFL protease inhibitors could overcome both de novo and acquired cetuximab resistance in colorectal cancer (CRC). Conversely, HGF overexpression was necessary and sufficient to induce cetuximab resistance and loss of polarity. Moreover, HGF-induced cetuximab resistance could be overcome by the downstream MET inhibitor, crizotinib, and upstream protease inhibitors. Additionally, HAI-1, an endogenous inhibitor of HGF proteases, (i) was downregulated in CRC, (ii) exhibited increased genomic methylation that correlated with poor prognosis, (iii) HAI-1 expression correlated with cetuximab response in a panel of cancer cell lines, and (iv) exogenous addition of recombinant HAI-1 overcame cetuximab resistance in CC-HGF cells. Thus, we describe a targetable, autocrine HAI-1/Protease/HGF/MET axis in cetuximab resistance in CRC.

A case report of a Filipino boy with childhood cataract and clinically diagnosed Roberts Syndrome

@#Childhood cataract is a common cause of visual impairment. Familial types are uncommon among Filipinos. Furthermore, it is not common to have one that follows an autosomal dominant pattern of inheritance but with associated syndromic presentation like Roberts syndrome which is an autosomal recessive disorder. This is a case of a 9-year-old Filipino boy with cataract in the left eye associated with low-set ears, facial asymmetry, underdeveloped nasal ala, cleft lip and palate, macroglossia, micrognathia, short right shin, and absent feet. Patient was clinically diagnosed with Roberts syndrome. We present a clinically diagnosed Roberts syndrome (RS), the first reported RS in a Filipino in local and international literature to our knowledge with an autosomal dominant childhood cataract. Genetic testing can assist in the confirmation of this case.

A case report of inferior oblique reattachment after Inferomedial Orbital Wall Reconstruction from Total Maxillectom

@#The detachment and reattachment of inferior oblique (IO) have been done by ophthalmologists in inferomedial orbital wall fracture repair to avoid inducing a new diplopia in patients post-operatively. However, doing them in orbital wall reconstruction in patient who underwent maxillectomy for a malignancy has not been described yet. We describe a case where a disinserted inferior oblique was reattached after titanium mesh implantation to prevent diplopia after the surgery. This is the case of a 40-year-old male diagnosed with recurrent bilateral nasomaxillary ameloblastoma who underwent total maxillectomy of the right with removal of the inferomedial orbital wall, and detachment and reattachment of inferior oblique with no resulting inferior oblique palsy and diplopia from hypotropia or incyclotorsion. This is the first report, to our knowledge, to describe reattachment of IO in its approximate insertion during reconstruction of the inferomedial orbital wall after total maxillectomy from a malignancy with no resulting diplopia.

Retinoblastoma in the Southern Philippines: Clinical outcomes of retinoblastoma patients in a Davao Tertiary Hospital

Background@#Retinoblastoma is the most common intraocular cancer in childhood in the Philippines. Most data though on demographics, clinical profile, treatment options, and outcomes in the country are from the National Capital Region. @*Objectives@#This study aimed to describe the demographics, clinical profile, treatment done, and outcomes of retinoblastoma patients seen in a public tertiary referral center in Davao from 2011-2020 to make available literature more representative of the status of retinoblastoma in the Philippines. @*Methods@#An analytical cross-sectional study was conducted using the records of retinoblastoma patients seen in a tertiary government hospital located in Davao Region from January 2011 to December 2020. @*Results@#There were 157 patients included in the analysis. Seventy-three (46%) were female with 44% coming from the Davao Region. One hundred seven (69%) patients had unilateral disease. Median age at initial consultation for patients with unilateral disease was significantly older than those with bilateral disease (p<0.003). Tumors were extraocular in 82 (40%) eyes. In the intraocular group, 36% of the eyes belonged to International Classification of Retinoblastoma (ICRB) Groups D and E. Enucleation was the most commonly performed treatment. Survival rate was 28%. This is the first report to provide epidemiologic and clinical data on retinoblastoma in the literature, including survival data, from Mindanao. Advanced stages and extraocular cases of retinoblastoma remain high. Delay of consultation contributed to the prognosis and clinical outcome of the disease.@*@#Conclusion. Advanced stages and extraocular cases of retinoblastoma remain significantly high in the country, even in Mindanao.

Comparison of Congenital Rubella Syndrome Cases at a Philippine Tertiary Hospital from 2009-2012 to 2019-2022

Background and Objective@#The Philippines does not have a national congenital rubella syndrome (CRS) surveillance or registry. Regular monitoring of CRS cases in hospitals, including in a Philippine tertiary hospital, helped in the past to provide clinico-epidemiologic data on CRS. This study aimed to continue providing clinico-epidemiologic data on CRS cases seen in the Philippine tertiary hospital from 2009-2012 and 2019-2022 and compare the cases seen from said timelines.@*Methods@#A cross-sectional study was used, employing chart review of patients newly diagnosed with CRS from 2009-2012 and 2019-2022 in the Department of Ophthalmology and Visual Sciences at the Philippine tertiary hospital.@*Results@#Forty-two patients newly diagnosed with CRS from 2009-2012 and 2019-2022 were included. Only 14 (33%) were serologically-confirmed cases (albeit qualitatively). Median age (first and third interquartile ranges) at consult was 1 year (0.4, 2.5). Twenty-four (57%) patients had maternal history of rashes and/or fever. Trimester of pregnancy when mother became symptomatic was not significantly correlated with chief complaint (p=0.20) and numbers of ophthalmic (p=0.68) and systemic manifestations (p=0.32). Cataract was the most common ophthalmic manifestation present in 40 (95%) patients. Twenty-six (62%) patients had other associated systemic findings of which hearing loss was the most common. Only 29 of 40 patients with cataract underwent lensectomy, with 23 patients having poor visual prognosis prior to surgery (5 with nystagmus alone, 10 with nystagmus and strabismus, and 8 with strabismus alone). @*Discussion@#Using ophthalmic manifestations as primary indicator, this study provided an update on the CRS cases in the country. Laboratory confirmation remains a challenge in diagnosing CRS as the tests are costly and not widely available. There was increase from 2009-2012 compared to 2019-2022 in number of patients who underwent surgical treatment for cataract but visual outcomes were suboptimal due to delay in consultation. Although there was a decrease in number of CRS cases seen in the Philippine tertiary hospital, this cannot be attributed to increased rubella-containing vaccine (RCV) coverage alone. @*Conclusion@#Provision of data from individual hospital-based studies similar to this highlights the need for a national CRS surveillance system or registry. This can better gauge the burden of CRS and identify the gap in RCV coverage.

Molecular cartography uncovers evolutionary and microenvironmental dynamics in sporadic colorectal tumors.

Colorectal cancer exhibits dynamic cellular and genetic heterogeneity during progression from precursor lesions toward malignancy. Analysis of spatial multi-omic data from 31 human colorectal specimens enabled phylogeographic mapping of tumor evolution that revealed individualized progression trajectories and accompanying microenvironmental and clonal alterations. Phylogeographic mapping ordered genetic events, classified tumors by their evolutionary dynamics, and placed clonal regions along global pseudotemporal progression trajectories encompassing the chromosomal instability (CIN+) and hypermutated (HM) pathways. Integrated single-cell and spatial transcriptomic data revealed recurring epithelial programs and infiltrating immune states along progression pseudotime. We discovered an immune exclusion signature (IEX), consisting of extracellular matrix regulators DDR1, TGFBI, PAK4, and DPEP1, that charts with CIN+ tumor progression, is associated with reduced cytotoxic cell infiltration, and shows prognostic value in independent cohorts. This spatial multi-omic atlas provides insights into colorectal tumor-microenvironment co-evolution, serving as a resource for stratification and targeted treatments.

GammaGateR: semi-automated marker gating for single-cell multiplexed imaging.

Motivation: Multiplexed immunofluorescence (mIF) is an emerging assay for multichannel protein imaging that can decipher cell-level spatial features in tissues. However, existing automated cell phenotyping methods, such as clustering, face challenges in achieving consistency across experiments and often require subjective evaluation. As a result, mIF analyses often revert to marker gating based on manual thresholding of raw imaging data. Results: To address the need for an evaluable semi-automated algorithm, we developed GammaGateR, an R package for interactive marker gating designed specifically for segmented cell-level data from mIF images. Based on a novel closed-form gamma mixture model, GammaGateR provides estimates of marker-positive cell proportions and soft clustering of marker-positive cells. The model incorporates user-specified constraints that provide a consistent but slide-specific model fit. We compared GammaGateR against the newest unsupervised approach for annotating mIF data, employing two colon datasets and one ovarian cancer dataset for the evaluation. We showed that GammaGateR produces highly similar results to a silver standard established through manual annotation. Furthermore, we demonstrated its effectiveness in identifying biological signals, achieved by mapping known spatial interactions between CD68 and MUC5AC cells in the colon and by accurately predicting survival in ovarian cancer patients using the phenotype probabilities as input for machine learning methods. GammaGateR is a highly efficient tool that can improve the replicability of marker gating results, while reducing the time of manual segmentation. Availability and Implementation: The R package is available at https://github.com/JiangmeiRubyXiong/GammaGateR.

Topological-Preserving Membrane Skeleton Segmentation in Multiplex Immunofluorescence Imaging.

Multiplex immunofluorescence (MxIF) is an emerging imaging technology whose downstream molecular analytics highly rely upon the effectiveness of cell segmentation. In practice, multiple membrane markers (e.g., NaKATPase, PanCK and ß-catenin) are employed to stain membranes for different cell types, so as to achieve a more comprehensive cell segmentation since no single marker fits all cell types. However, prevalent watershed-based image processing might yield inferior capability for modeling complicated relationships between markers. For example, some markers can be misleading due to questionable stain quality. In this paper, we propose a deep learning based membrane segmentation method to aggregate complementary information that is uniquely provided by large scale MxIF markers. We aim to segment tubular membrane structure in MxIF data using global (membrane markers z-stack projection image) and local (separate individual markers) information to maximize topology preservation with deep learning. Specifically, we investigate the feasibility of four SOTA 2D deep networks and four volumetric-based loss functions. We conducted a comprehensive ablation study to assess the sensitivity of the proposed method with various combinations of input channels. Beyond using adjusted rand index (ARI) as the evaluation metric, which was inspired by the clDice, we propose a novel volumetric metric that is specific for skeletal structure, denoted as clDiceSKEL. In total, 80 membrane MxIF images were manually traced for 5-fold cross-validation. Our model outperforms the baseline with a 20.2% and 41.3% increase in clDiceSKEL and ARI performance, which is significant (p<0.05) using the Wilcoxon signed rank test. Our work explores a promising direction for advancing MxIF imaging cell segmentation with deep learning membrane segmentation. Tools are available at https://github.com/MASILab/MxIF_Membrane_Segmentation.

A Specialized Epithelial Cell Type Regulating Mucosal Immunity and Driving Human Crohn's Disease.

Crohn's disease (CD) is a complex chronic inflammatory disorder that may affect any part of gastrointestinal tract with extra-intestinal manifestations and associated immune dysregulation. To characterize heterogeneity in CD, we profiled single-cell transcriptomics of 170 samples from 65 CD patients and 18 non-inflammatory bowel disease (IBD) controls in both the terminal ileum (TI) and ascending colon (AC). Analysis of 202,359 cells identified a novel epithelial cell type in both TI and AC, featuring high expression of LCN2, NOS2, and DUOX2, and thus is named LND. LND cells, confirmed by high-resolution in-situ RNA imaging, were rarely found in non-IBD controls, but expanded significantly in active CD. Compared to other epithelial cells, genes defining LND cells were enriched in antimicrobial response and immunoregulation. Moreover, multiplexed protein imaging demonstrated that LND cell abundance was associated with immune infiltration. Cross-talk between LND and immune cells was explored by ligand-receptor interactions and further evidenced by their spatial colocalization. LND cells showed significant enrichment of expression specificity of IBD/CD susceptibility genes, revealing its role in immunopathogenesis of CD. Investigating lineage relationships of epithelial cells detected two LND cell subpopulations with different origins and developmental potential, early and late LND. The ratio of the late to early LND cells was related to anti-TNF response. These findings emphasize the pathogenic role of the specialized LND cell type in both Crohn's ileitis and Crohn's colitis and identify novel biomarkers associated with disease activity and treatment response.

Patient-derived enteroids provide a platform for the development of therapeutic approaches in microvillus inclusion disease.

Microvillus inclusion disease (MVID), caused by loss-of-function mutations in the motor protein myosin Vb (MYO5B), is a severe infantile disease characterized by diarrhea, malabsorption, and acid/base instability, requiring intensive parenteral support for nutritional and fluid management. Human patient-derived enteroids represent a model for investigation of monogenic epithelial disorders but are a rare resource from MVID patients. We developed human enteroids with different loss-of function MYO5B variants and showed that they recapitulated the structural changes found in native MVID enterocytes. Multiplex immunofluorescence imaging of patient duodenal tissues revealed patient-specific changes in localization of brush border transporters. Functional analysis of electrolyte transport revealed profound loss of Na+/H+ exchange (NHE) activity in MVID patient enteroids with near-normal chloride secretion. The chloride channel-blocking antidiarrheal drug crofelemer dose-dependently inhibited agonist-mediated fluid secretion. MVID enteroids exhibited altered differentiation and maturation versus healthy enteroids. γ-Secretase inhibition with DAPT recovered apical brush border structure and functional Na+/H+ exchange activity in MVID enteroids. Transcriptomic analysis revealed potential pathways involved in the rescue of MVID cells including serum/glucocorticoid-regulated kinase 2 (SGK2) and NHE regulatory factor 3 (NHERF3). These results demonstrate the utility of patient-derived enteroids for developing therapeutic approaches to MVID.