Transcription factor networks directing the development, function, and evolution of innate lymphoid effectors.

Mammalian lymphoid immunity is mediated by fast and slow responders to pathogens. Fast innate lymphocytes are active within hours after infections in mucosal tissues. Slow adaptive lymphocytes are conventional T and B cells with clonal antigen receptors that function days after pathogen exposure. A transcription factor (TF) regulatory network guiding early T cell development is at the core of effector function diversification in all innate lymphocytes, and the kinetics of immune responses is set by developmental programming. Operational units within the innate lymphoid system are not classified by the types of pathogen-sensing machineries but rather by discrete effector functions programmed by regulatory TF networks. Based on the evolutionary history of TFs of the regulatory networks, fast effectors likely arose earlier in the evolution of animals to fortify body barriers, and in mammals they often develop in fetal ontogeny prior to the establishment of fully competent adaptive immunity.

Identification of transcriptional regulators in the mouse immune system.

The differentiation of hematopoietic stem cells into cells of the immune system has been studied extensively in mammals, but the transcriptional circuitry that controls it is still only partially understood. Here, the Immunological Genome Project gene-expression profiles across mouse immune lineages allowed us to systematically analyze these circuits. To analyze this data set we developed Ontogenet, an algorithm for reconstructing lineage-specific regulation from gene-expression profiles across lineages. Using Ontogenet, we found differentiation stage-specific regulators of mouse hematopoiesis and identified many known hematopoietic regulators and 175 previously unknown candidate regulators, as well as their target genes and the cell types in which they act. Among the previously unknown regulators, we emphasize the role of ETV5 in the differentiation of γδ T cells. As the transcriptional programs of human and mouse cells are highly conserved, it is likely that many lessons learned from the mouse model apply to humans.

Intrathymic programming of effector fates in three molecularly distinct γδ T cell subtypes.

Innate γδ T cells function in the early phase of immune responses. Although innate γδ T cells have often been studied as one homogenous population, they can be functionally classified into effector subsets on the basis of the production of signature cytokines, analogous to adaptive helper T cell subsets. However, unlike the function of adaptive T cells, γδ effector T cell function correlates with genomically encoded T cell antigen receptor (TCR) chains, which suggests that clonal TCR selection is not the main determinant of the differentiation of γδ effector cells. A high-resolution transcriptome analysis of all emergent γδ thymocyte subsets segregated on the basis of use of the TCR γ-chain or δ-chain indicated the existence of three separate subtypes of γδ effector cells in the thymus. The immature γδ subsets were distinguished by unique transcription-factor modules that program effector function.

The necroptosis adaptor RIPK3 promotes injury-induced cytokine expression and tissue repair.

Programmed necrosis or necroptosis is an inflammatory form of cell death that critically requires the receptor-interacting protein kinase 3 (RIPK3). Here we showed that RIPK3 controls a separate, necrosis-independent pathway of inflammation by regulating cytokine expression in dendritic cells (DCs). Ripk3(-/-) bone-marrow-derived dendritic cells (BMDCs) were highly defective in lipopolysaccharide (LPS)-induced expression of inflammatory cytokines. These effects were caused by impaired NF-κB subunit RelB and p50 activation and by impaired caspase 1-mediated processing of interleukin-1ß (IL-1ß). This DC-specific function of RIPK3 was critical for injury-induced inflammation and tissue repair in response to dextran sodium sulfate (DSS). Ripk3(-/-) mice exhibited an impaired axis of injury-induced IL-1ß, IL-23, and IL-22 cytokine cascade, which was partially corrected by adoptive transfer of wild-type DCs, but not Ripk3(-/-) DCs. These results reveal an unexpected function of RIPK3 in NF-κB activation, DC biology, innate inflammatory-cytokine expression, and injury-induced tissue repair.

A network of high-mobility group box transcription factors programs innate interleukin-17 production.

How innate lymphoid cells (ILCs) in the thymus and gut become specialized effectors is unclear. The prototypic innate-like γδ T cells (Tγδ17) are a major source of interleukin-17 (IL-17). We demonstrate that Tγδ17 cells are programmed by a gene regulatory network consisting of a quartet of high-mobility group (HMG) box transcription factors, SOX4, SOX13, TCF1, and LEF1, and not by conventional TCR signaling. SOX4 and SOX13 directly regulated the two requisite Tγδ17 cell-specific genes, Rorc and Blk, whereas TCF1 and LEF1 countered the SOX proteins and induced genes of alternate effector subsets. The T cell lineage specification factor TCF1 was also indispensable for the generation of IL-22 producing gut NKp46(+) ILCs and restrained cytokine production by lymphoid tissue inducer-like effectors. These results indicate that similar gene network architecture programs innate sources of IL-17, independent of anatomical origins.

Injury, dysbiosis, and filaggrin deficiency drive skin inflammation through keratinocyte IL-1α release.

BACKGROUND: Atopic dermatitis (AD) is associated with epidermal barrier defects, dysbiosis, and skin injury caused by scratching. In particular, the barrier-defective epidermis in patients with AD with loss-of-function filaggrin mutations has increased IL-1α and IL-1ß levels, but the mechanisms by which IL-1α, IL-1ß, or both are induced and whether they contribute to the aberrant skin inflammation in patients with AD is unknown. OBJECTIVE: We sought to determine the mechanisms through which skin injury, dysbiosis, and increased epidermal IL-1α and IL-1ß levels contribute to development of skin inflammation in a mouse model of injury-induced skin inflammation in filaggrin-deficient mice without the matted mutation (ft/ft mice). METHODS: Skin injury of wild-type, ft/ft, and myeloid differentiation primary response gene-88-deficient ft/ft mice was performed, and ensuing skin inflammation was evaluated by using digital photography, histologic analysis, and flow cytometry. IL-1α and IL-1ß protein expression was measured by means of ELISA and visualized by using immunofluorescence and immunoelectron microscopy. Composition of the skin microbiome was determined by using 16S rDNA sequencing. RESULTS: Skin injury of ft/ft mice induced chronic skin inflammation involving dysbiosis-driven intracellular IL-1α release from keratinocytes. IL-1α was necessary and sufficient for skin inflammation in vivo and secreted from keratinocytes by various stimuli in vitro. Topical antibiotics or cohousing of ft/ft mice with unaffected wild-type mice to alter or intermix skin microbiota, respectively, resolved the skin inflammation and restored keratinocyte intracellular IL-1α localization. CONCLUSIONS: Taken together, skin injury, dysbiosis, and filaggrin deficiency triggered keratinocyte intracellular IL-1α release that was sufficient to drive chronic skin inflammation, which has implications for AD pathogenesis and potential therapeutic targets.

IL-22 derived from γδ T cells restricts Staphylococcus aureus infection of mechanically injured skin.

BACKGROUND: Staphylococcus aureus is an opportunistic pathogen that colonizes the skin of patients with atopic dermatitis (AD) and aggravates their disease. Neutrophils and the cytokines IL-17A and IL-17F, which drive the expression of the neutrophil-attracting chemokines, are important for the clearance of S aureus infection. The cytokine IL-22 is often coproduced by IL-17-secreting cells. The levels of IL-22 are elevated in AD skin lesions. OBJECTIVE: We sought to determine the role of IL-22 in the clearance of S aureus infection of mouse skin subjected to tape stripping, a surrogate for scratching, a cardinal feature of AD. METHODS: S aureus was applied to the tape-stripped skin of wild-type and Il22-/- mice. Bacterial burden was evaluated by enumerating colony-forming units. Quantitative PCR and ELISA were performed to quantify Il22 mRNA and IL-22 protein in mouse and human skin. Flow cytometry was used to enumerate neutrophils in the skin. RESULTS: Scratching the skin of healthy adults and tape stripping of mouse skin induced local expression of Il22 mRNA and IL-22 protein. Induction of Il22 expression by tape stripping was dependent on IL-23 and γδ T cells. Clearance of S aureus from tape-stripped skin was significantly impaired in Il22-/- mice. Neutrophil infiltration and upregulation of expression of genes encoding the antimicrobial peptides antigen-6/urokinase-type plasminogen activator receptor related protein-1 and ß-DEFENSIN 14 and the chemokine (C-X-C motif) ligand following tape stripping were significantly impaired in Il22-/- mice. CONCLUSIONS: These findings show that IL-22 is important for limiting the growth of S aureus on mechanically injured skin and caution that IL-23 and IL-22 blockade in patients with AD may enhance susceptibility to staphylococcal skin infection.

Management of Patients With Pancreatic Cysts: Analysis of Possible False-Negative Cases of Malignancy.

GOALS: To examine the utility of integrated molecular pathology (IMP) in managing surveillance of pancreatic cysts based on outcomes and analysis of false negatives (FNs) from a previously published cohort (n=492). BACKGROUND: In endoscopic ultrasound with fine-needle aspiration (EUS-FNA) of cyst fluid lacking malignant cytology, IMP demonstrated better risk stratification for malignancy at approximately 3 years' follow-up than International Consensus Guideline (Fukuoka) 2012 management recommendations in such cases. STUDY: Patient outcomes and clinical features of Fukuoka and IMP FN cases were reviewed. Practical guidance for appropriate surveillance intervals and surgery decisions using IMP were derived from follow-up data, considering EUS-FNA sampling limitations and high-risk clinical circumstances observed. Surveillance intervals for patients based on IMP predictive value were compared with those of Fukuoka. RESULTS: Outcomes at follow-up for IMP low-risk diagnoses supported surveillance every 2 to 3 years, independent of cyst size, when EUS-FNA sampling limitations or high-risk clinical circumstances were absent. In 10 of 11 patients with FN IMP diagnoses (2% of cohort), EUS-FNA sampling limitations existed; Fukuoka identified high risk in 9 of 11 cases. In 4 of 6 FN cases by Fukuoka (1% of cohort), IMP identified high risk. Overall, 55% of cases had possible sampling limitations and 37% had high-risk clinical circumstances. Outcomes support more cautious management in such cases when using IMP. CONCLUSIONS: Adjunct use of IMP can provide evidence for relaxed surveillance of patients with benign cysts that meet Fukuoka criteria for closer observation or surgery. Although infrequent, FN results with IMP can be associated with EUS-FNA sampling limitations or high-risk clinical circumstances.

Lifestyle related factors & impact of metabolic syndrome on quality of life, level of functioning & self-esteem in patients with bipolar disorder & schizophrenia.

BACKGROUND & OBJECTIVES: Though studies have reported high prevalence rates of metabolic syndrome among patients with bipolar disorder (BPAD) and schizophrenia, there is lack of data on the impact of the same on the patients' life. This study was aimed to assess the lifestyle related factors associated with metabolic syndrome (MetS) and to study the impact of MetS on functioning and quality of life (QOL) in patients with BPAD and schizophrenia. METHODS: A total of 102 patients with BPAD and 72 patients with schizophrenia attending the output unit of a tertiary care hospital in north India were evaluated for MetS. These patients were assessed on Health Promoting Lifestyle Profile scale II (HPLP II), World Health Organization QOL -Bref Version (WHOQOL-Bref), Impact of Weight on Quality of Life- Lite version (IWOQOL -Lite), Body weight, Image and Self-esteem Evaluation questionnaire (BWISE), Obesity-related Problem scale (OP scale) and Global Assessment of Functioning (GAF) scale. RESULTS: MetS was associated with lower scores on domains of health responsibility and nutrition habit domain on HPLP-II scale in both groups, and additionally on physical activity and stress management domain in BPAD group. On WHOQOL-Bref, MetS was associated with lower scores on the domains of physical and psychological health in both groups. On IWQOL-Lite, scores on personal distress and self esteem domains were higher in those with obesity in both groups and also on physical activity domain in schizophrenia group. Those with MetS had lower level of functioning as measured by GAF in schizophrenia group. Fulfillment of higher number of criteria of MetS correlated with poorer quality of life and higher problems in both groups. INTERPRETATION & CONCLUSIONS: Many modifiable lifestyle factors increase the risk of MetS. MetS was found to be associated with poorer QOL in patients with BPAD and schizophrenia; in addition, obesity led to poor self-esteem and excessive personal distress.

Integrated molecular pathology accurately determines the malignant potential of pancreatic cysts.

BACKGROUND AND STUDY AIMS: Current diagnostic testing is inadequate to determine the malignant potential of pancreatic cysts, resulting in overcautious patient management. Integrated molecular pathology (IMP) testing combines molecular analysis with first-line test results (cytology, imaging, and fluid chemistry) to assess the malignant potential of pancreatic cysts. This multicenter study aimed to determine the diagnostic accuracy of IMP for pancreatic adenocarcinoma, and the utility of IMP testing under current guideline recommendations for managing pancreatic cysts. PATIENTS AND METHODS: Patients who had undergone previous IMP testing as prescribed by their physician and for whom clinical outcomes were available from retrospective record review were included (n = 492). Performance was determined by correlation between clinical outcome and previous IMP diagnosis ("benign"/"statistically indolent" vs. "statistically higher risk [SHR]"/ "aggressive") or an International Consensus Guideline (Sendai 2012) criteria model for "surveillance" vs. "surgery." The Cox proportional hazards model determined hazard ratios for malignancy. RESULTS: Benign and statistically indolent IMP diagnoses had a 97 % probability of benign follow-up for up to 7 years and 8 months from initial IMP testing. SHR and aggressive diagnoses had relative hazard ratios for malignancy of 30.8 and 76.3, respectively (both P < 0.0001). Sendai surveillance criteria had a 97 % probability of benign follow-up for up to 7 years and 8 months, but for surgical criteria the hazard ratio was only 9.0 (P < 0.0001). In patients who met Sendai surgical criteria, benign and statistically indolent IMP diagnoses had a > 93 % probability of benign follow-up, with relative hazard ratios for SHR and aggressive IMP diagnoses of 16.1 and 50.2, respectively (both P < 0.0001). CONCLUSION: IMP more accurately determined the malignant potential of pancreatic cysts than a Sendai 2012 guideline management criteria model. IMP may improve patient management by justifying more relaxed observation in patients meeting Sendai surveillance criteria. IMP can more accurately differentiate between the need for surveillance or surgery in patients meeting Sendai surgical criteria.

The added value of using mutational profiling in addition to cytology in diagnosing aggressive pancreaticobiliary disease: review of clinical cases at a single center.

BACKGROUND: This study aimed to better understand the supporting role that mutational profiling (MP) of DNA from microdissected cytology slides and supernatant specimens may play in the diagnosis of malignancy in fine-needle aspirates (FNA) and biliary brushing specimens from patients with pancreaticobiliary masses. METHODS: Cytology results were examined in a total of 30 patients with associated surgical (10) or clinical (20) outcomes. MP of DNA from microdissected cytology slides and from discarded supernatant fluid was analyzed in 26 patients with atypical, negative or indeterminate cytology. RESULTS: Cytology correctly diagnosed aggressive disease in 4 patients. Cytological diagnoses for the remaining 26 were as follows: 16 negative (9 false negative), 9 atypical, 1 indeterminate. MP correctly determined aggressive disease in 1 false negative cytology case and confirmed a negative cytology diagnosis in 7 of 7 cases of non-aggressive disease. Of the 9 atypical cytology cases, MP correctly diagnosed 7 as positive and 1 as negative for aggressive disease. One specimen that was indeterminate by cytology was correctly diagnosed as non-aggressive by MP. When first line malignant (positive) cytology results were combined with positive second line MP results, 12/21 cases of aggressive disease were identified, compared to 4/21 cases identified by positive cytology alone. CONCLUSIONS: When first line cytology results were uncertain (atypical), questionable (negative), or not possible (non-diagnostic/indeterminate), MP provided additional information regarding the presence of aggressive disease. When used in conjunction with first line cytology, MP increased detection of aggressive disease without compromising specificity in patients that were difficult to diagnose by cytology alone.

Assessment of acceptability of black wheat flour products and factors affecting it among Anganwadi beneficiaries and workers: A mixed-method prospective observational study.

Introduction: Malnutrition is very common in India and black wheat might be an acceptable solution to this problem. The aim of the study was to assess acceptability of black wheat flour products and factors affecting it among Anganwadi beneficiaries and workers. Materials and Methods: This was a mixed-method prospective observational study. All the family members enrolled for supplementary nutrition and Anganwadi workers/helpers of three randomly selected Anganwadi centers were taken in the study. For qualitative data, in-depth interview was done, and for quantitative data, 9-point hedonic scale was administered. Braun and Clarke's six-phase data analysis framework was used for qualitative data. Results: A total of 16 pregnant females, 14 lactating females, 16 children, 2 Anganwadi workers, and 3 Anganwadi helpers participated in the study. Thematic analysis of the data revealed five significant themes. It included characteristics of black wheat flour, the process of making the product (experience of making the product), family acceptability, availability, and hygiene. Participants expressed that the black color appearance is one of the negative influencers in the acceptability of black wheat flour. Most of the participants liked the taste as well as the texture. However, kneading, rolling, and puffing were more challenging than traditional wheat flour. On the hedonic scale, the mean rank of acceptability is lowest for color (3.03), followed by puffing (3.49) and highest for texture (4.87) and taste (4.60). Conclusion: Our study results revealed that black wheat is acceptable to the Anganwadi beneficiaries and workers.

Structure and energetics guide dynamic behaviour in a T = 3 icosahedral virus capsid.

Although virus capsids appear as rigid, symmetric particles in experimentally determined structures; biochemical studies suggest a significant degree of structural flexibility in the particles. We carried out all-atom simulations on the icosahedral capsid of an insect virus, Flock House Virus, which show intriguing differences in the degree of flexibility of quasi-equivalent capsid subunits consistent with previously described biological behaviour. The flexibility of all the ß and γ subunits of the protein and RNA fragments is analysed and compared. Both γA subunit and RNA fragment exhibit higher flexibility than the γB and γC subunits. The capsid shell is permeable to the bidirectional movement of water molecules, and the movement is heavily influenced by the geometry of the capsid shell along specific symmetry axes. In comparison to the symmetry axes along I5 and I3, the I2 axis exhibits a slightly higher water content. This enriched water environment along I2 could play a pivotal role in facilitating the structural transitions necessary for RNA release, shedding some light on the intricate and dynamic processes underlying the viral life cycle. Our study suggests that the physical characterization of whole virus capsids is the key to identifying biologically relevant transition states in the virus life cycle and understanding the basis of virus infectivity.

Pyridine-2,6-Dicarboxamide Proligands and their Cu(II)/Zn(II) Complexes Targeting Staphylococcus Aureus for the Attenuation of In Vivo Dental Biofilm.

In the pursuit to combat stubborn bacterial infections, particularly those stemming from gram-positive bacteria, this study is an attempt to craft a precision-driven platform characterized by unparalleled selectivity, specificity, and synergistic antimicrobial mechanisms. Leveraging remarkable potential of metalloantibiotics in antimicrobial applications, herein, this work rationally designs, synthesizes, and characterizes a new library of Pyridine-2,6-dicarboxamide ligands and their corresponding transition metal Cu(II)/Zn(II) complexes. The lead compound L11 demonstrates robust antibacterial properties against Staphylococcus aureus (Minimum Inhibitory Concentration (MIC) = 2-16 µg mL-1), methicillin and vancomycin-resistant S. aureus (MIC = 2-4 µg mL-1) and exhibit superior antibacterial activity when compared to FDA-approved vancomycin, the drug of last resort. Additionally, the compound exhibits notable antimicrobial efficacy against resistant enterococcus strains (MIC = 2-8 µg mL-1). To unravel mechanistic profile, advanced imaging techniques including SEM and AFM are harnessed, collectively suggesting a mechanistic pathway involving cell wall disruption. Live/dead fluorescence studies further confirm efficacy of L11 and its complexes against S. aureus membranes. This translational exploration extends to a rat model, indicating promising in vivo therapeutic potential. Thus, this comprehensive research initiative has capabilities to transcends the confines of this laboratory, heralding a pivotal step toward combatting antibiotic-resistant pathogens and advancing the frontiers of metalloantibiotics-based therapy with a profound clinical implication.

SMAD regulatory networks construct a balanced immune system.

A balanced immune response requires combating infectious assaults while striving to maintain quiescence towards the self. One of the central players in this process is the pleiotropic cytokine transforming growth factor-ß (TGF-ß), whose deficiency results in spontaneous systemic autoimmunity in mice. The dominant function of TGF-ß is to regulate the peripheral immune homeostasis, particularly in the microbe-rich and antigen-rich environment of the gut. To maintain intestinal integrity, the epithelial cells, myeloid cells and lymphocytes that inhabit the gut secrete TGF-ß, which acts in both paracrine and autocrine fashions to activate its signal transducers, the SMAD transcription factors. The SMAD pathway regulates the production of IgA by B cells, maintains the protective mucosal barrier and promotes the balanced differentiation of CD4(+) T cells into inflammatory T helper type 17 cells and suppressive FOXP3(+) T regulatory cells. While encounters with pathogenic microbes activate SMAD proteins to evoke a protective inflammatory immune response, SMAD activation and synergism with immunoregulatory factors such as the vitamin A metabolite retinoic acid enforce immunosuppression toward commensal microbes and innocuous food antigens. Such complementary context-dependent functions of TGF-ß are achieved by the co-operation of SMAD proteins with distinct dominant transcription activators and accessory chromatin modifiers. This review highlights recent advances in unravelling the molecular basis for the multi-faceted functions of TGF-ß in the gut that are dictacted by fluid orchestrations of SMADs and their myriad partners.

Expectations of caregivers of persons with severe mental illness from mental health rehabilitation services: A qualitative study.

BACKGROUND: Mental health rehabilitation services (MHRS) play an essential role in addressing the diverse and often complex needs of persons with severe mental illness (SMI). The rehabilitation services are of utmost importance in training these patients to lead an independent and meaningful life. In India, caregivers of persons with SMI play a significant role in the decision-making process concerning treatment and rehabilitation. AIM: The main objective of the present study was to understand and analyze the expectations of caregivers of persons with severe mental illness from mental health rehabilitation services in the Indian context. METHOD: The present study is qualitative in nature. A semi-structured interview schedule was used to capture the caregivers' expectations. Fifteen caregivers who met inclusion and exclusion criteria were interviewed for the study. The data obtained was analyzed using Braun and Clarke's six-phase data analysis framework. RESULTS: Thematic analysis of the data emerged into five themes, namely (1) 'proximity', (2) 'professionalism of staff', (3) 'treatment related expectations', (4) 'welfare benefits', and (5) 'infrastructure'. These themes and their sub-themes were subsequently discussed in the light of existing literature. CONCLUSION: Caregivers of persons with SMI identified the most relevant as well as generic expectations from MHRS. Understanding these expectations can help improvise the existing services delivery system and help institutes, non-government organizations (NGOs) and other concerned entities working in mental health rehabilitation to develop service user friendly rehabilitation models.

Oral versus long-acting injectable antipsychotic in first episode schizophrenia: A 12 weeks interventional study.

Background: There is underutilization of long-acting injectable (LAI) antipsychotics for first-episode schizophrenia (FES) despite having convenient dosing and treatment retention. LAIs are predominantly used for patients with poor compliance, chronic course, and multiple relapses. Materials and Methods: Seventy-two treatment naïve patients with the first episode of Schizophrenia (DSM-5) were assessed for baseline severity of psychopathology using the positive and negative syndrome scale (PANSS) and quality of life (QOL) using the WHOQOL-BREF scale. Patients were randomized to receive either oral haloperidol or LAI haloperidol for a period of 12 weeks. Results: Both the groups had a significant reduction in PANSS scores and improvement in QoL over 12 weeks period (P = 0.0001). The LAI group showed greater adherence and significantly better quality of life than the oral group (P = 0.023). The mean numbers of side effects were less in the LAI group at week 2 as compared to the oral group. Conclusion: LAI haloperidol is similar to oral haloperidol in patients with FES with respect to treatment response and offers benefits in form of a lesser number of side effects during early treatment, overall better adherence rates, and better QOL.

AI-based AlphaFold2 significantly expands the structural space of the autophagy pathway.

ABBREVIATIONS: AF2: AlphaFold2; AF2-Mult: AlphaFold2 multimer; ATG: autophagy-related; CTD: C-terminal domain; ECTD: extreme C-terminal domain; FR: flexible region; MD: molecular dynamics; NTD: N-terminal domain; pLDDT: predicted local distance difference test; UBL: ubiquitin-like.

Gastric emptying scintigraphy: is four hours necessary?

INTRODUCTION: Recommendations for gastric emptying scintigraphy (GES) suggest imaging over 4 hours to better define gastroparesis. AIMS: To determine the value of defining delayed gastric emptying at time points earlier than 4 hours. METHODS: GES was performed with ingestion of a liquid egg white meal with imaging at 0, 0.5, 1, 2, 3, and 4 hours. Patients completed the Patient Assessment of Gastrointestinal Symptoms questionnaire immediately before GES. RESULTS: Of 1499 patients undergoing GES from September 2007 to January 2010 (76.2% were female, mean age of 45.5±0.5 y, 21.3% had diabetes, 9.5% had earlier gastric surgery), 160 (10.7%) had increased gastric retention at 1 hour (>90%), 404 (27%) had increased retention at 2 hours (>60%), 576 (38.4%) had increased retention at 3 hours (>30%), and 629 (42%) had increased retention at 4 hours (>10%). Gastric retention at 4 hours correlated with retention at 3 hours (r=0.890; P<0.001), 2 hours (r=0.738; P<0.001), and 1 hour (r=0.510; P<0.001). Symptoms correlated better with the gastric retention at later time points. The symptoms correlating with gastric retention at 4 hours included early satiety (r=0.170; P<0.01), vomiting (r=0.143; P<0.01), feeling excessively full after meals (r=0.123; P<0.01), and loss of appetite (r=0.122; P<0.01). CONCLUSIONS: Gastric retention at 4 hours correlates well with gastric retention at 3 hours, good at 2 hours, but only fair with gastric retention at 1 hour. Gastric retention at 1 hour may miss 36% of patients found to have delayed gastric emptying at 4 hours. Symptoms (early satiety, vomiting, feeling excessively full after meals, and loss of appetite) correlated better with the gastric retention at later time points.