Translational Utility of Organoid Models for Biomedical Research on Gastrointestinal Diseases.

Organoid models have recently been utilized to study 3D human-derived tissue systems to uncover tissue architecture and adult stem cell biology. Patient-derived organoids unambiguously provide the most suitable in vitro system to study disease biology with the actual genetic background. With the advent of much improved and innovative approaches, patient-derived organoids can potentially be used in regenerative medicine. Various human tissues were explored to develop organoids due to their multifold advantage over the conventional in vitro cell line culture approach and in vivo models. Gastrointestinal (GI) tissues have been widely studied to establish organoids and organ-on-chip for screening drugs, nutraceuticals, and other small molecules having therapeutic potential. The function of channel proteins, transporters, and transmembrane proteins was also explained. The successful application of genome editing in organoids using the CRISPR-Cas approach has been reported recently. GI diseases such as Celiac disease (CeD), Inflammatory bowel disease (IBD), and common GI cancers have been investigated using several patient-derived organoid models. Recent advancements on organoid bio-banking and 3D bio-printing contributed significantly in personalized disease management and therapeutics. This article reviews the available literature on investigations and translational applications of patient-derived GI organoid models, notably on elucidating gut-microbial interaction and epigenetic modifications.

A duodenal mucosa transcriptome study identified reduced expression of a novel gene CDH18 in celiac disease.

BACKGROUND: Celiac disease (CD) a complex immune disease that affects duodenal mucosa. Identification of tissue specific biomarkers is expected to improve the existing biopsy based CD diagnosis. AIMS: To investigate the differentially expressed genes (DEGs) in duodenal mucosa tissue to identify clinically relevant gene expression pattern in CD. METHODS: Whole RNA extracted from the duodenal biopsies of three CD patients and four non-CD controls were sequenced. Significant DEGs were identified. Prioritized DEGs were validated using qRT-PCR in an independent group (CD=23; Control=26). Enriched pathways were analyzed, protein-protein interaction networks were evaluated. RESULTS: 923 DEGs comprising of 135 up-regulated, and 788 down-regulated genes, with p-value≤0.05; log2FC>2 or <-2 were identified. A novel down-regulated gene CDH18 (p = 0.03; log2FC=-0.74) was identified. Previously known CXCL9 was replicated. CDH18, a trans-membrane protein was found to interact with other CDH proteins, α/ß catenins, and other membrane transporters such as SLC and ABCB. Pathways and protein networks contributing in channel activity (p = 2.15E-12), membrane transporters (p = 2.15E-12), and cellular adhesion (p = 8.05E-6) were identified. CONCLUSIONS: CDH18, a novel DEG identified in the present study is a pivotal gene involved in maintaining epithelial membrane organization and integrity. The functional significance of lower expression of CDH18 in pathogenesis of CD warranted to be investigated. CDH18 expression could be tested for its effectiveness in diagnostic, prognostic and therapeutic purposes.

Implications of vitamin D deficiency in systemic inflammation and cardiovascular health.

Clinical, epidemiological, and molecular studies have sufficiently highlighted the vitality of vitamin D [25(OH)D and 1,25(OH)2D] in human health and wellbeing. Globally, vitamin D deficiency (VDD) has become a public health concern among all age groups. There is a very high prevalence of VDD per the estimates from several epidemiological studies on different ethnic populations. But, population-specific scales do not support these estimates to define VDD clinically and consistent genetic associations. However, clinical studies have shown the relevance of serum vitamin D screening and oral supplementation in improving health conditions, pointing toward a more prominent role of vitamin D in health and wellness. Routinely, the serum concentration of vitamin D is measured to determine the deficiency and is correlated with physiological conditions and clinical symptoms. Recent research points toward a more inclusive role of vitamin D in different disease pathologies and is not just limited to otherwise bone health and overall growth. VDD contributes to the natural history of systemic ailments, including cardiovascular and systemic immune diseases. Considering its significant impact on premature morbidity and mortality, there is a compelling need to comprehensively review and document the direct and indirect implications of VDD in immune system deregulation, systemic inflammatory conditions, and cardio-metabolism. The recommendations from this review call for furthering our research concerning vitamin D and its direct and indirect implications.

Specific Genetic Polymorphisms Contributing in Differential Binding of Gliadin Peptides to HLA-DQ and TCR to Elicit Immunogenicity in Celiac Disease.

Immunogenicity of gliadin peptides in celiac disease (CD) is majorly determined by the pattern of molecular interactions with HLA-DQ and T-cell receptors (TCR). Investigation of the interactions between immune-dominant gliadin peptides, DQ protein, and TCR are warranted to unravel the basis of immunogenicity and variability contributed by the genetic polymorphisms. Homology modeling of HLA and TCR done using Swiss Model and iTASSER, respectively. Molecular interactions of eight common deamidated immune-dominant gliadin with HLA-DQ allotypes and specific TCR gene pairs were evaluated. Docking of the three structures was performed with ClusPro2.0 and ProDiGY was used to predict binding energies. Effects of known allelic polymorphisms and reported susceptibility SNPs were predicted on protein-protein interactions. CD susceptible allele, HLA-DQ2.5 was shown to have considerable binding affinity to 33-mer gliadin (ΔG = - 13.9; Kd = 1.5E - 10) in the presence of TRAV26/TRBV7. Higher binding affinity was predicted (ΔG = - 14.3, Kd = 8.9E - 11) when TRBV28 was replaced with TRBV20 paired with TRAV4 suggesting its role in CD predisposition. SNP rs12722069 at HLA-DQ8 that codes Arg76α forms three H-bonds with Glu12 and two H-bonds with Asn13 of DQ2 restricted gliadin in the presence of TRAV8-3/TRBV6. None of the HLA-DQ polymorphisms was found to be in linkage disequilibrium with reported CD susceptibility markers. Haplotypic presentations of rs12722069-G, rs1130392-C, rs3188043-C and rs4193-A with CD reported SNPs were observed in sub-ethnic groups. Highly polymorphic sites of HLA alleles and TCR variable regions could be utilized for better risk prediction models in CD. Therapeutic strategies by identifying inhibitors or blockers targeting specific gliadin:HLA-DQ:TCR binding sites could be investigated.

Celiac disease-associated loci show considerable genetic overlap with neuropsychiatric diseases but with limited transethnic applicability.

Clinical and public health research has revealed the co-occurrence of several neuropsychiatric diseases among patients with celiac disease (CD). The significant presence of CD-specific autoantibodies in patients with neuropsychiatric diseases and vice versa are often reported. To explain the genetic basis of such frequent disease co-occurrence and investigate the underlying common pathways/processes, we performed an extensive cross-disease association study followed by supporting in silico functional validation of the leads. Genomewide association study (GWAS) data for CD and eight commonly co-occurring neuropsychiatric diseases from Caucasian populations were analysed, and the shared loci were determined.We performed Immunochip-based fine mapping of these overlapping association signals in an independent European CD data and tested their cross-ethnic transferability using CD association data from the genetically distinct north Indian population. This study identified 12 shared loci between the two diseases with genomewide significance (P = 5e-8). Of these five loci, namely NFIA, KIA1109, NOTCH4-TSBP1-PBX2, HLA-DQA1 and CSK replicated in an independent Dutch cohort representing European ancestry. Three of these loci, namely NFIA, NOTCH4-TSBP1-PBX2 and HLA-DQA1 that are common between CD, anxiety, migraine and schizophrenia respectively withstood locus transferability test in north Indians. Tissue-specific eQTL analysis of SNPs from transferable loci revealed expression QTL effects in brain tissue besides the small intestine and whole blood. Pathway analysis and evidence of epigenetic regulation highlighted the potential contribution of these SNPs to disease pathology. The replicable and transferable association of genetic variants from MHC locus and their functional implications suggest the process of antigen presentation and adaptive/innate immune response regulated by non-HLA genes in the locus may dominate the shared pathogenesis of CD and neuropsychiatric diseases. Functional validation of the shared candidate genes is warranted to unravel the molecular mechanism for the co-occurrence of CD and specific neuropsychiatric diseases.

Meta-analysis confirmed genetic susceptibility conferred by multiple risk variants from CTLA4 and SERPINA1 in granulomatosis with polyangiitis.

BACKGROUND: Granulomatosis with polyangiitis (GPA) is a rare systemic autoimmune disease. Smaller sample size and complex nature of the disease pathogenesis has made it challenging to perform well-powered genetic investigations. We performed a systematic review based meta-analysis in GPA to investigate the genetic susceptibility conferred by non-human leukocyte antigen (non-HLA) candidate genes. METHODS: A systematic review was performed using web-based literature search and eligible studies were included following inclusion-exclusion criteria. Studies were evaluated for their quality of evidence and study outcome was assessed using the Newcastle-Ottawa Scale and Grades of Research, Assessment, Development and Evaluation tools. Reviewer's agreement was accessed through Cohen's κ value. Meta-analyses were performed using RevMan 5 tool. Meta-odds ratio (meta-OR) and Z test P value were evaluated to estimate the genetic susceptibility for each of the variants. RESULTS: Eighteen studies were found eligible and 7 genetic variants from only 4 genes, namely CTLA4, PRTN3, SERPINA1 and PTPN22 could be studied for meta-analysis. rs231775-G (49-G) (Meta-OR = 1.42 [1.14-1.76]; P = .001) of CTLA4 and rs7151526-A (Meta-OR = 2.70 [1.51-4.85]; P = .0008) of SERPINA1 were confirmed to be predisposing alleles, and rs5742909-C (318-C) (Meta-OR = 0.65 [0.44-0.97]; P =.03) of CTLA4 was found to be protective for GPA. In concordance with the genetic association of rs7151526-A, serological marker for the same variant "Z" allele of SERPINA1 was found to be predisposing (Meta-OR = 12.60 [5.01-31.68]; P < .00001) for GPA. CONCLUSION: Genetic variants confirmed in this study play critical roles in T-cell mediated immune function and could be significantly implicated in GPA. Molecular pathology studies are warranted to confirm their role. These markers could be used for efficient patient classification and disease management.

A meta-analysis suggests the association of reduced serum level of vitamin D and T-allele of Fok1 (rs2228570) polymorphism in the vitamin D receptor gene with celiac disease.

Purpose: As an immune-modulator, vitamin D is known to regulate immune response and is implicated in disease pathogenesis. Celiac disease (CD) is a systemic autoimmune disease and susceptibility conferred by vitamin D metabolism is under investigation. Studies on the association of vitamin D metabolism and genetic polymorphisms are expected to explain CD pathogenesis. We performed a systematic review-based meta-analysis to investigate the 25(OH)D serum levels and susceptibility conferred by the genetic variants of VDR in CD. Methods: Systematic review was conducted through a web-based literature search following stringent study inclusion-exclusion criteria. The Newcastle-Ottawa Scale and GRADE tools were used to assess the quality of evidence in studies and the study outcome. Cohen's κ value was estimated to access the reviewer's agreement. RevMan 5.4.1 was used to perform the meta-analyses. Weighted mean difference and Meta p-value was assessed for 25(OH)D serum levels. Meta-odds ratio and Z-test p-value were evaluated to estimate the allelic susceptibility of VDR variants. Results: A total of 8 out of 12 studies were evaluated for "25(OH)D" serum level, while four studies were found eligible for SNPs (Bsm1, Apa1, Fok1, and Taq1) of VDR. Significantly higher levels [WMD = 5.49, p < 0.00001] of 25(OH)D were observed in healthy controls than in patients with CD. rs2228570-T (Fok1) [Meta-OR = 1.52, p = 0.02] was confirmed to be predisposing allele for CD. Conclusion: Reduced serum level of 25(OH)D and association of Fok1 T-allele of VDR confirmed in this study plays a critical role in immunomodulation and maintaining barrier integrity, which is majorly implicated in CD.

Epidemiology and genetics of granulomatosis with polyangiitis.

Granulomatosis with polyangiitis (GPA) previously known as Wegener's granulomatosis (WG) is a rare rheumatic disease affecting subjects of all ages. Prevalence and incidence of this systemic disease greatly varies across different ethnic groups. GPA is the commonest form of ANCA-associated vasculitis (AAV) with PR3 positivity among 85-95% of the cases. Scientific investigations of GPA is warranted because its severity, clinical heterogeneity, fast disease manifestation and end-organ damage. The etiology of GPA is still unknown. Major role of HLA and non-HLA genes with immune functions were identified, however, very limited replication was observed in different ethnic populations. In the present review, we have discussed the updates on the global epidemiology and contribution of HLA and major non-HLA genes/loci in GPA. We have also highlighted the cross disease association of GPA associated genes that may help in better disease management and predictive medicine. We proposed that high-resolution HLA typing and development of genetic risk model would help in early disease diagnosis and understanding the prognosis.

FTIR based approach to study EnaC mechanosensory functions.

The pulmonary epithelial sodium ion channel (ENaC) is gaining importance for its sodium gating and mechanosensitive roles. The mechano functional studies on ENaC suggest direct molecular interactions between the ENaC protein with cytoskeleton microtubules and other extracellular matrix components. Also, in few mechanotransduction studies, ENaC was shown to respond both to membrane stretch as well as cell volume changes. However, the conformational characteristic of ENaC during sodium and mechano gating are yet to be fully elucidated. Thus obtaining ENaC protein conformational spectrum based on Fourier Transform Infrared Radiation (FTIR) spectroscopy in solution will be useful in predicting the nature of conformational changes occurring during any cell volume changes in an epithelial cell. The conformational spectrum looks promising in studying the disease biology of cystic fibrosis (CF) and CF like conditions that arise due to abnormal ion conductance membrane proteins and subsequent frequent fluid retentions. This review article presents the basics of epithelial ENaC protein as a gated mechanosensor and FTIR for developing fluid dynamics of ENaC protein. This can be applied to develop an ENaC based quantum mechanosensor for the prognosis as well as diagnosis of cystic fibrosis (CF) and allied lung diseases.

Multiple allelic associations from genes involved in energy metabolism were identified in celiac disease.

Energy metabolism is a critical factor that influences disease pathogenesis. Recent high-throughput genomic studies have enabled us to look into disease biology with greater details. Celiac disease (CD) is an inflammatory autoimmune disease where ~60 non-HLA genes were identified which in conjunction with HLA genes explain ~55% of the disease heritability. In this study we aimed to identify susceptibility energy metabolism genes and investigate their role in CD. We re-analysed published Immunochip genotyping data, which were originally analysed for CD association studies in north Indian and Dutch population. 269 energy metabolism genes were tested. Meta-analysis was done for the identified SNPs. To validate the functional implications of identified markers and/or genes, in silico functional annotation was performed. Six SNPs were identified in north Indians, of which three markers from two loci were replicated in Dutch. rs2071592 (PMeta=5.01e-75) and rs2251824 (PMeta=1.87e-14) from ATP6V1G2-NFKBIL1-DDX39B locus and rs4947331 (PMeta= 9.85e-13) from NEU1 locus were found significantly associated. Identified genes are key regulators of cellular energy metabolism and associated with several immune mediated diseases. In silico functional annotation showed significant biological relevance of these novel markers and genes. FDI approved therapeutics against ATP6V1G2 and NEU1 are currently in use to treat chronic and inflammatory diseases. This study identified two pathogenic loci, originally involved in energy metabolism. Extensive investigation showed their synergistic role in CD pathogenesis by promoting immune mediated enteric inflammation. Proposed CD pathogenesis model in this study needs to be tested through tissue-on-chip and in vivo methods to ensure its translational application.

Microglia Specific Drug Targeting Using Natural Products for the Regulation of Redox Imbalance in Neurodegeneration.

Microglia, a type of innate immune cell of the brain, regulates neurogenesis, immunological surveillance, redox imbalance, cognitive and behavioral changes under normal and pathological conditions like Alzheimer's, Parkinson's, Multiple sclerosis and traumatic brain injury. Microglia produces a wide variety of cytokines to maintain homeostasis. It also participates in synaptic pruning and regulation of neurons overproduction by phagocytosis of neural precursor cells. The phenotypes of microglia are regulated by the local microenvironment of neurons and astrocytes via interaction with both soluble and membrane-bound mediators. In case of neuron degeneration as observed in acute or chronic neurodegenerative diseases, microglia gets released from the inhibitory effect of neurons and astrocytes, showing activated phenotype either of its dual function. Microglia shows neuroprotective effect by secreting growths factors to heal neurons and clears cell debris through phagocytosis in case of a moderate stimulus. But the same microglia starts releasing pro-inflammatory cytokines like TNF-α, IFN-γ, reactive oxygen species (ROS), and nitric oxide (NO), increasing neuroinflammation and redox imbalance in the brain under chronic signals. Therefore, pharmacological targeting of microglia would be a promising strategy in the regulation of neuroinflammation, redox imbalance and oxidative stress in neurodegenerative diseases. Some studies present potentials of natural products like curcumin, resveratrol, cannabidiol, ginsenosides, flavonoids and sulforaphane to suppress activation of microglia. These natural products have also been proposed as effective therapeutics to regulate the progression of neurodegenerative diseases. The present review article intends to explain the molecular mechanisms and functions of microglia and molecular dynamics of microglia specific genes and proteins like Iba1 and Tmem119 in neurodegeneration. The possible interventions by curcumin, resveratrol, cannabidiol, ginsenosides, flavonoids and sulforaphane on microglia specific protein Iba1 suggest possibility of natural products mediated regulation of microglia phenotypes and its functions to control redox imbalance and neuroinflammation in management of Alzheimer's, Parkinson's and Multiple Sclerosis for microglia-mediated therapeutics.

Contributions of human ACE2 and TMPRSS2 in determining host-pathogen interaction of COVID-19.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is at present an emerging global public health crisis. Angiotensin converting enzyme 2 (ACE2) and trans-membrane protease serine 2 (TMPRSS2) are the two major host factors that contribute to the virulence of SARS-CoV-2 and pathogenesis of coronavirus disease-19 (COVID-19). Transmission of SARS-CoV-2 from animal to human is considered a rare event that necessarily requires strong evolutionary adaptations. Till date no other human cellular receptors are identified beside ACE2 for SARS-CoV-2 entry inside the human cell. Proteolytic cleavage of viral spike (S)-protein and ACE2 by TMPRSS2 began the entire host-pathogen interaction initiated with the physical binding of ACE2 to S-protein. SARS-CoV-2 S-protein binds to ACE2 with much higher affinity and stability than that of SARS-CoVs. Molecular interactions between ACE2-S and TMPRSS2-S are crucial and preciously mediated by specific residues. Structural stability, binding affinity and level of expression of these three interacting proteins are key susceptibility factors for COVID-19. Specific protein-protein interactions (PPI) are being identified that explains uniqueness of SARS-CoV-2 infection. Amino acid substitutions due to naturally occurring genetic polymorphisms potentially alter these PPIs and poses further clinical heterogeneity of COVID-19. Repurposing of several phytochemicals and approved drugs against ACE2, TMPRSS2 and S-protein have been proposed that could inhibit PPI between them. We have also identified some novel lead phytochemicals present in Azadirachta indica and Aloe barbadensis which could be utilized for further in vitro and in vivo anti-COVID-19 drug discovery. Uncovering details of ACE2-S and TMPRSS2-S interactions would further contribute to future research on COVID-19.

Assessment of risk conferred by coding and regulatory variations of TMPRSS2 and CD26 in susceptibility to SARS-CoV-2 infection in human.

At present, more than 200 countries and territories are directly affected by the coronavirus disease-19 (COVID-19) pandemic. Incidence and case fatality rate are significantly higher among elderly individuals (age>60 years), type 2 diabetes and hypertension patients. Cellular receptor ACE2, serine protease TMPRSS2 and exopeptidase CD26 (also known as DPP4) are the three membrane bound proteins potentially implicated in SARS-CoV-2 infection. We hypothesised that common variants from TMPRSS2 and CD26 may play critical role in infection susceptibility of predisposed population or group of individuals. Coding (missense) and regulatory variants from TMPRSS2 and CD26 were studied across 26 global populations. Two missense and five regulatory SNPs were identified to have differential allelic frequency. Significant linkage disequilibrium (LD) signature was observed in different populations. Modelled protein-protein interaction (PPI) predicted strong molecular interaction between these two receptors and SARS-CoV-2 spike protein (S1 domain). However, two missense SNPs, rs12329760 (TMPRSS2) and rs1129599 (CD26), were not found to be involved physically in the said interaction. Four regulatory variants (rs112657409, rs11910678, rs77675406 and rs713400) from TMPRSS2 were found to influence the expression of TMPRSS2 and pathologically relevant MX1. rs13015258 a 50 UTR variant from CD26 have significant role in regulation of expression of key regulatory genes that could be involved in SARS-CoV-2 internalization. Overexpression of CD26 through epigenetic modification at rs13015258-C allele was found critical and could explain the higher SARS-CoV-2 infected fatality rate among type 2 diabetes.

Association study identified biologically relevant receptor genes with synergistic functions in celiac disease.

Receptors are essential mediators of cellular physiology, which facilitate molecular and cellular cross-talk with the environment. Nearly 20% of the all known celiac disease (CD) genes are receptors by function. We hypothesized that novel biologically relevant susceptibility receptor genes act in synergy in CD pathogenesis. We attempted to identify novel receptor genes in CD by re-analyzing published Illumina Immunochip dense genotype data for a north Indian and  a European (Dutch) cohort. North Indian dataset was screened for 269 known receptor genes. Association statistics for SNPs were considered with minor allele frequency >15% and association P ≤ 0.005 to attend desired study power. Identified markers were tested for cross-ethnic replication in a European CD dataset. Markers were analyzed in-silico to explain their functional significance in CD. Six novel SNPs from MOG (rs29231, p = 1.21e-11), GABBR1 (rs3025643, p = 1.60e-7), OR2H2 (rs1233388, p = 0.0002), ABCF1 (rs9262119, p = 0.0005), ADRA1A (rs10102024, p = 0.003), and ACVR2A (rs7560426, p = 0.004) were identified in north Indians, of which three genes namely, GABBR1 (rs3025643, p = 5.38e-8), OR2H2 (rs1233388, p = 3.29e-5) and ABCF1 (rs9262119, p = 0.0002) were replicated in Dutch. Tissue specific functional annotation, potential epigenetic regulation, co-expression, protein-protein interaction and pathway enrichment analyses indicated differential expression and synergistic function of key genes that could alter cellular homeostasis, ubiquitination mediated phagosome pathway and cellular protein processing to contribute for CD. At present multiple therapeutic compounds/drugs are available targeting GABBR1 and ADRA1A, which could be tested for their effectiveness against CD in controlled drug trials.