Tumor-associated macrophages trigger MAIT cell dysfunction at the HCC invasive margin.

Mucosal-associated invariant T (MAIT) cells represent an abundant innate-like T cell subtype in the human liver. MAIT cells are assigned crucial roles in regulating immunity and inflammation, yet their role in liver cancer remains elusive. Here, we present a MAIT cell-centered profiling of hepatocellular carcinoma (HCC) using scRNA-seq, flow cytometry, and co-detection by indexing (CODEX) imaging of paired patient samples. These analyses highlight the heterogeneity and dysfunctionality of MAIT cells in HCC and their defective capacity to infiltrate liver tumors. Machine-learning tools were used to dissect the spatial cellular interaction network within the MAIT cell neighborhood. Co-localization in the adjacent liver and interaction between niche-occupying CSF1R+PD-L1+ tumor-associated macrophages (TAMs) and MAIT cells was identified as a key regulatory element of MAIT cell dysfunction. Perturbation of this cell-cell interaction in ex vivo co-culture studies using patient samples and murine models reinvigorated MAIT cell cytotoxicity. These studies suggest that aPD-1/aPD-L1 therapies target MAIT cells in HCC patients.

Immunosuppressive CD29+ Treg accumulation in the liver in mice on checkpoint inhibitor therapy.

OBJECTIVE: Liver metastases are often resistant to immune checkpoint inhibitor therapy (ICI) and portend a worse prognosis compared with metastases to other locations. Regulatory T cells (Tregs) are one of several immunosuppressive cells implicated in ICI resistance of liver tumours, but the role played by Tregs residing within the liver surrounding a tumour is unknown. DESIGN: Flow cytometry and single-cell RNA sequencing were used to characterise hepatic Tregs before and after ICI therapy. RESULTS: We found that the murine liver houses a Treg population that, unlike those found in other organs, is both highly proliferative and apoptotic at baseline. On administration of αPD-1, αPD-L1 or αCTLA4, the liver Treg population doubled regardless of the presence of an intrahepatic tumour. Remarkably, this change was not due to the preferential expansion of the subpopulation of Tregs that express PD-1. Instead, a subpopulation of CD29+ (Itgb1, integrin ß1) Tregs, that were highly proliferative at baseline, doubled its size in response to αPD-1. Partial and full depletion of Tregs identified CD29+ Tregs as the prominent niche-filling subpopulation in the liver, and CD29+ Tregs demonstrated enhanced suppression in vitro when derived from the liver but not the spleen. We identified IL2 as a critical modulator of both CD29+ and CD29- hepatic Tregs, but expansion of the liver Treg population with αPD-1 driven by CD29+ Tregs was in part IL2-independent. CONCLUSION: We propose that CD29+ Tregs constitute a unique subpopulation of hepatic Tregs that are primed to respond to ICI agents and mediate resistance.

Efficacy of Wolbachia-Infected Mosquito Deployments for the Control of Dengue.

BACKGROUND: Aedes aegypti mosquitoes infected with the wMel strain of Wolbachia pipientis are less susceptible than wild-type A. aegypti to dengue virus infection. METHODS: We conducted a cluster-randomized trial involving releases of wMel-infected A. aegypti mosquitoes for the control of dengue in Yogyakarta, Indonesia. We randomly assigned 12 geographic clusters to receive deployments of wMel-infected A. aegypti (intervention clusters) and 12 clusters to receive no deployments (control clusters). All clusters practiced local mosquito-control measures as usual. A test-negative design was used to assess the efficacy of the intervention. Patients with acute undifferentiated fever who presented to local primary care clinics and were 3 to 45 years of age were recruited. Laboratory testing was used to identify participants who had virologically confirmed dengue (VCD) and those who were test-negative controls. The primary end point was symptomatic VCD of any severity caused by any dengue virus serotype. RESULTS: After successful introgression of wMel into the intervention clusters, 8144 participants were enrolled; 3721 lived in intervention clusters, and 4423 lived in control clusters. In the intention-to-treat analysis, VCD occurred in 67 of 2905 participants (2.3%) in the intervention clusters and in 318 of 3401 (9.4%) in the control clusters (aggregate odds ratio for VCD, 0.23; 95% confidence interval [CI], 0.15 to 0.35; P = 0.004). The protective efficacy of the intervention was 77.1% (95% CI, 65.3 to 84.9) and was similar against the four dengue virus serotypes. The incidence of hospitalization for VCD was lower among participants who lived in intervention clusters (13 of 2905 participants [0.4%]) than among those who lived in control clusters (102 of 3401 [3.0%]) (protective efficacy, 86.2%; 95% CI, 66.2 to 94.3). CONCLUSIONS: Introgression of wMel into A. aegypti populations was effective in reducing the incidence of symptomatic dengue and resulted in fewer hospitalizations for dengue among the participants. (Funded by the Tahija Foundation and others; AWED ClinicalTrials.gov number, NCT03055585; Indonesia Registry number, INA-A7OB6TW.).

Whole-Slide Imaging, Mutual Information Registration for Multiplex Immunohistochemistry and Immunofluorescence.

Multiplex immunohistochemistry/immunofluorescence (mIHC/mIF) is a developing technology that facilitates the evaluation of multiple, simultaneous protein expressions at single-cell resolution while preserving tissue architecture. These approaches have shown great potential for biomarker discovery, yet many challenges remain. Importantly, streamlined cross-registration of multiplex immunofluorescence images with additional imaging modalities and immunohistochemistry (IHC) can help increase the plex and/or improve the quality of the data generated by potentiating downstream processes such as cell segmentation. To address this problem, a fully automated process was designed to perform a hierarchical, parallelizable, and deformable registration of multiplexed digital whole-slide images (WSIs). We generalized the calculation of mutual information as a registration criterion to an arbitrary number of dimensions, making it well suited for multiplexed imaging. We also used the self-information of a given IF channel as a criterion to select the optimal channels to use for registration. Additionally, as precise labeling of cellular membranes in situ is essential for robust cell segmentation, a pan-membrane immunohistochemical staining method was developed for incorporation into mIF panels or for use as an IHC followed by cross-registration. In this study, we demonstrate this process by registering whole-slide 6-plex/7-color mIF images with whole-slide brightfield mIHC images, including a CD3 and a pan-membrane stain. Our algorithm, WSI, mutual information registration (WSIMIR), performed highly accurate registration allowing the retrospective generation of an 8-plex/9-color, WSI, and outperformed 2 alternative automated methods for cross-registration by Jaccard index and Dice similarity coefficient (WSIMIR vs automated WARPY, P < .01 and P < .01, respectively, vs HALO + transformix, P = .083 and P = .049, respectively). Furthermore, the addition of a pan-membrane IHC stain cross-registered to an mIF panel facilitated improved automated cell segmentation across mIF WSIs, as measured by significantly increased correct detections, Jaccard index (0.78 vs 0.65), and Dice similarity coefficient (0.88 vs 0.79).

Comparison of Collagenase Clostridium histolyticum to Surgery for the Management of Peyronie's Disease: A Randomized Trial.

PURPOSE: Since Food and Drug Administration approval of collagenase Clostridium histolyticum for Peyronie's disease, there has been significant debate regarding its role and comparable efficacy to surgery. MATERIALS AND METHODS: A randomized, controlled trial was performed of Peyronie's disease men treated with either collagenase C histolyticum + RestoreX penile traction therapy + sildenafil or penile surgery + RestoreX penile traction therapy + sildenafil, with 3-month data presented. Primary objectives were overall satisfaction, subjective changes in erectile function, penile sensation, penile length, and changes in the International Index of Erectile Function-Erectile Function Domain score. Secondary outcomes included objective changes in length, curve, adverse events, and other standardized and nonstandardized questionnaires. RESULTS: A total of 40 men were enrolled, with 38 (collagenase C histolyticum group = 19, surgery group = 19) completing treatment and having 3-month data available. All demographic and clinicopathological variables were similar between groups. Following treatment, 50% of men in the collagenase C histolyticum group reported being very satisfied (vs 21% in the surgery group, P = .08) and noted better subjective erectile function (100% vs 68%, P = .03) and penile length (88% vs 16%, P < .0001), lesser impacts on penile sensation (75% vs 11% no change, P < .001), and similar International Index of Erectile Function-Erectile Function Domain changes (+1.5 vs +2.5, P = .91). Objectively, men in the surgery group had greater curve improvements (84% vs 54%, P < .01) and higher rates of adverse events (50 vs 13 events, P < .001) but decreased penile length (-0.5 cm vs +1.0 cm, P < .01). CONCLUSIONS: At 3 months posttreatment, collagenase C histolyticum + RestoreX penile traction therapy + sildenafil results in lesser curve improvements but greater penile length and fewer adverse events, including impacts on subjective erectile function and sensation, than men treated with surgery.

Cytoreduction and HIPEC for Gastric Carcinomatosis: Multi-institutional Analysis of Two Phase II Clinical Trials.

INTRODUCTION: There are no approved locoregional therapies for peritoneal carcinomatosis from gastric adenocarcinoma (GA). Cytoreductive surgery with hyperthermic intraperitoneal chemotherapy (CRS-HIPEC) represents a potential treatment for advanced GA with isolated peritoneal metastasis. PATIENTS AND METHODS: Two separate single-institution phase II, single-arm studies evaluating CRS-HIPEC using cisplatin with mitomycin C (NIH: NCT03092518, MDACC: NCT02891447) in patients with GA and confirmed peritoneal metastasis were analyzed. The primary endpoint of each trial was overall survival (OS). Clinical, pathologic, and treatment variables were analyzed for association with outcomes. RESULTS: Over 4 years, 41 patients with peritoneal carcinomatosis from GA underwent CRS-HIPEC. All patients had synchronous peritoneal metastasis and received systemic chemotherapy as front-line therapy. A total of 23 patients also received laparoscopic HIPEC prior to open CRS-HIPEC. The majority (63%, n = 26) were male, and median PCI score at CRS-HIPEC was 2. Median OS was 24.9 months from diagnosis and 14.4 months from CRS-HIPEC. Three-year OS was 25% from diagnosis and 22% from CRS-HIPEC. Median RFS was 7.4 months. The rate of 30-day Clavien-Dindo grade ≥ 3 complications was 32%; specifically, the rate of anastomotic leak was 22%. Multivariable analysis identified the number of pathologically positive lymph nodes as an independent predictor of postoperative OS. CONCLUSIONS: In patients with gastric adenocarcinoma and isolated peritoneal metastasis treated with CRS-HIPEC, 3-year OS was 22% from CRS-HIPEC, and complications were common. The number of pathologic lymph node metastases was inversely correlated with overall survival. Further investigation of CRS-HIPEC for GA should include patient selection based on response to systemic chemotherapy or incorporate novel intraperitoneal treatment strategies.

Efficacy of collagenase Clostridium histolyticum in men with congenital penile curvature.

BACKGROUND: Collagenase Clostridium histolyticum (CCH) has been proven effective in multiple subpopulations of PD men; however, no studies have evaluated its role with congenital penile curvature (CPC). AIM: To evaluate the safety and efficacy of CCH in men with CPC. METHODS: A prospective registry was queried of men undergoing CCH injections at our institution. Beginning in 2016, CCH was administered to CPC men using a similar protocol to PD. A comparative analysis was performed between cohorts to evaluate the safety and efficacy of therapy. OUTCOMES: Objective measures included penile length and curvature, while subjective outcomes included standardized (International Index of Erectile Function and Peyronie's Disease Questionnaire) and non-standardized assessments. Curvature outcomes were categorized as follows: 1 - included all men, with the most recent assessment considered final, and 2 - only men who had completed eight CCH injections or stopped early due to satisfaction. RESULTS: From 2014 to Oct 2021, a total of 453 men (408 PD, 45 CPC) underwent one or more CCH injections. In comparing cohorts, CPC men were younger (33 vs 58 years, p<0.0001), had lesser baseline curvatures (52.5 vs 65°, p<0.01), more ventral curves (25.7 vs 9.2%, p<0.01), and longer penile lengths (12.5 vs 12.0 cm, p=0.04). Following treatment, both cohorts experienced similar curvature improvements. Specifically, PD men experienced 20-25° or 33-35% improvements depending on definition compared to 25-30° or 40-50% in CPC men (all p-values >0.05). CPC and PD men also demonstrated similar changes on standardized questionnaires, with the exception of the Psychological and Physical subdomain, which was more improved in CPC men (-11 vs -4, p<0.01). Baseline curvature was positively correlated with greater absolute (degree) and relative (percent) improvements. Adverse events were similar between groups after controlling for confounders. CLINICAL IMPLICATIONS: CCH may be safely and effectively administered in men with CPC. These data provide the first evidence for the efficacy of a non-surgical therapeutic option in this cohort. STRENGTHS AND LIMITATIONS: Strengths - large, prospective series with standardized assessments; Limitations - non-randomized study, short-term follow-up, and lack of standardized method to differentiate CPC from PD. CONCLUSIONS: CCH may be safely and effectively administered to men with CPC, with similar success rates compared to PD. Increasing curvature was associated with greater absolute (degree) and relative (percent) improvements, supporting the role for CCH in men with mild, moderate, or severe curvatures. External validation is warranted prior to routine implementation.

Management of large kidney stones in the geriatric population.

PURPOSE: The aim of this review is to highlight the unique factors that predispose geriatric patients to nephrolithiasis and to compare the utility and efficacy of surgical techniques in this specific patient population. METHODS: PubMed and EMBASE databases were reviewed, and studies were organized according to surgical treatments. RESULTS: Few prospective studies exist comparing kidney stone removal in the elderly to younger cohorts. In addition, various age cut-offs were used to determine who was considered elderly. Most studies which analyzed Percutaneous Nephrolithotomy (PCNL) found a slightly higher rate of minor complications but comparable stone free rate and operative time. For ureteroscopy (URS) and extracorporeal shockwave lithotripsy (ESWL), there were minimal complications observed and no difference in clinical success in the elderly. All surgical techniques were presumed to be safe in the elderly and most found no difference in stone-free rates. CONCLUSIONS: Unique attributes of the geriatric population contribute to stone formation and must be considered when determining appropriate management modalities. This review provides an overview of the utility and efficacy of PCNL, URS and ESWL in the elderly, as well as a porposed algorithm for management in this population.

Neonatal outcomes following introduction of routine probiotic supplementation to very preterm infants.

AIM: To evaluate the combined outcome of death and/or severe grade necrotising enterocolitis (NEC) in very preterm infants admitted to Cork University Maternity Hospital, Ireland, before and after introduction of routine supplementation with Bifidobacterium bifidum and Lactobacillus acidophilus probiotics (Infloran®). METHODS: A retrospective study of infants <32 weeks gestation and < 1500 g surviving beyond 72 h of life was performed. Two 6-year epochs; pre-probiotics (Epoch 1: 2008-2013) and with probiotics (Epoch 2: 2015-2020), were evaluated. The primary outcome was defined as death after 72 h or NEC Bell stage 2a or greater. RESULTS: Seven-hundred-and-forty-four infants were included (Epoch 1: 391, Epoch 2: 353). The primary outcome occurred in 67 infants (Epoch 1: 37, Epoch 2: 30, p = 0.646). After adjustment, the difference was significant (OR [95% CI]: 0.53 [0.29 to 0.97], p = 0.038). Differences between epochs did not depend on gestational age group (<28 weeks; ≥28 weeks). CONCLUSION: There was an associated reduction of the composite outcome of severe grade NEC and/or death, after adjustment for confounding variables, with introduction of routine administration of a B. bifidum and L. acidophilus probiotic at our institution.

Metformin treatment rescues CD8+ T-cell response to immune checkpoint inhibitor therapy in mice with NAFLD.

BACKGROUND & AIMS: Non-alcoholic steatohepatitis (NASH) represents the fastest growing underlying cause of hepatocellular carcinoma (HCC) and has been shown to impact immune effector cell function. The standard of care for the treatment of advanced HCC is immune checkpoint inhibitor (ICI) therapy, yet NASH may negatively affect the efficacy of ICI therapy in HCC. The immunologic mechanisms underlying the impact of NASH on ICI therapy remain unclear. METHODS: Herein, using multiple murine NASH models, we analysed the influence of NASH on the CD8+ T-cell-dependent anti-PD-1 responses against liver cancer. We characterised CD8+ T cells' transcriptomic, functional, and motility changes in mice receiving a normal diet (ND) or a NASH diet. RESULTS: NASH blunted the effect of anti-PD-1 therapy against liver cancers in multiple murine models. NASH caused a proinflammatory phenotypic change of hepatic CD8+ T cells. Transcriptomic analysis revealed changes related to NASH-dependent impairment of hepatic CD8+ T-cell metabolism. In vivo imaging analysis showed reduced motility of intratumoural CD8+ T cells. Metformin treatment rescued the efficacy of anti-PD-1 therapy against liver tumours in NASH. CONCLUSIONS: We discovered that CD8+ T-cell metabolism is critically altered in the context of NASH-related liver cancer, impacting the effectiveness of ICI therapy - a finding which has therapeutic implications in patients with NASH-related liver cancer. LAY SUMMARY: Non-alcoholic steatohepatitis represents the fastest growing cause of hepatocellular carcinoma. It is also associated with reduced efficacy of immunotherapy, which is the standard of care for advanced hepatocellular carcinoma. Herein, we show that non-alcoholic steatohepatitis is associated with impaired motility, metabolic function, and response to anti-PD-1 treatment in hepatic CD8+ T cells, which can be rescued by metformin treatment.

Evaluating the association between food insecurity and risk of nephrolithiasis: an analysis of the National Health and Nutrition Examination Survey.

PURPOSE: This study aimed to investigate the relationship between self-reported food security and kidney stone formation. METHODS: Data were collected from the National Health and Nutrition Examination Survey (NHANES), a database representative of the United States population. Food security status was assessed using the US Household Food Security Survey Module: Six-Item Short Form. Characteristics of patients were compared using the Chi-square test and the student t-test. Multivariate logistic regression was performed using a multi-model approach. RESULTS: We analyzed 6,800 NHANES survey respondents. 37.2% of respondents were categorized as having "low food security" (scores 2-4) and 24.0% having "very low food security" (scores 5-6). 8.4% of respondents had a history of kidney stones. We found that people with very low food security had a 42% increased likelihood of developing kidney stones compared to those with high or marginal food security, after controlling for race, age, and comorbidities (OR 1.42; 95% CI 1.01-1.99). Between the different food security groups, no significant differences were observed in age, race/ethnicity, body mass index, gout history, osteoporosis history, or coronary artery disease history. Lower food security was associated with slightly younger age (< 1 year difference, p = 0.001), higher poverty-income ratio (p = 0.001), and many comorbidities, including kidney stones (p = 0.007). CONCLUSION: Our study provides evidence for an association between food access and the risk of kidney stone disease. Given these findings, food insecurity should be investigated as a modifiable risk factor for the development of kidney stone disease.

The potential nutrition-, physical- and health-related benefits of cow's milk for primary-school-aged children.

Cow's milk is a naturally nutrient-dense foodstuff. A significant source of many essential nutrients, its inclusion as a component of a healthy balanced diet has been long recommended. Beyond milk's nutritional value, an increasing body of evidence illustrates cow's milk may confer numerous benefits related to health. Evidence from adult populations suggests that cow's milk may have a role in overall dietary quality, appetite control, hydration and cognitive function. Although evidence is limited compared with the adult literature, these benefits may be echoed in recent paediatric studies. This article, therefore, reviews the scientific literature to provide an evidence-based evaluation of the associated health benefits of cow's milk consumption in primary-school-aged children (4-11 years). We focus on seven key areas related to nutrition and health comprising nutritional status, hydration, dental and bone health, physical stature, cognitive function, and appetite control. The evidence consistently demonstrates cow's milk (plain and flavoured) improves nutritional status in primary-school-aged children. With some confidence, cow's milk also appears beneficial for hydration, dental and bone health and beneficial to neutral concerning physical stature and appetite. Due to conflicting studies, reaching a conclusion has proven difficult concerning cow's milk and cognitive function; therefore, a level of caution should be exercised when interpreting these results. All areas, however, would benefit from further robust investigation, especially in free-living school settings, to verify conclusions. Nonetheless, when the nutritional-, physical- and health-related impact of cow's milk avoidance is considered, the evidence highlights the importance of increasing cow's milk consumption.

Is hyperkinetic gallbladder an indication for cholecystectomy?

BACKGROUND: The (99m) technetium-labelled hepato imino diacetic acid (HIDA) scan is widely used to evaluate patients with biliary colic with a normal trans-abdominal ultrasound scan. Most studies recommend cholecystectomy for patients with biliary dyskinesia, defined by gallbladder ejection fraction (GBEF) of less than 35-40% on HIDA scan. There are no recommendations regarding management of hyperkinetic gallbladder defined by GBEF of greater than 80% on HIDA scan. The aim of our study was to evaluate the outcomes following cholecystectomy on patients with biliary colic associated with hyperkinetic gallbladder. METHODS: We performed a retrospective chart review of all patients with biliary colic associated with hyperkinetic gallbladder that underwent cholecystectomy in our practice from July 2014 to February 2018. Data collection included age, gender, body mass index, preoperative symptoms, comorbidities, additional tests, ejection fraction, surgery, and histopathology of the gallbladder. Symptomatic improvement was assessed during routine 2-week postoperative visit and a follow-up phone interview. RESULTS: Thirty-two patients had undergone laparoscopic cholecystectomy during the study period for symptomatic hyperkinetic gallbladder. All the patients had abdominal pain related to food intake and 17 (53%) patients had worsening of symptoms with CCK infusion. The average GBEF was 92%. Chronic cholecystitis was seen in 29 (90%) patients on pathology. 23 (74%) patients had complete resolution of biliary symptoms, 5 (16%) had improved symptoms, and 3 (10%) had no change in symptoms. CONCLUSION: Patients with biliary colic and hyperkinetic gallbladder respond favorably to laparoscopic cholecystectomy. Our results suggest that patients with biliary colic in the setting of hyperkinetic gallbladder have symptomatic improvement following cholecystectomy.

STAT2 Is Required for TLR-Induced Murine Dendritic Cell Activation and Cross-Presentation.

TLR-stimulated cross-presentation by conventional dendritic cells (cDCs) is important in host defense and antitumor immunity. We recently reported that cDCs lacking the type I IFN signaling molecule STAT2 are impaired in cross-presenting tumor Ags to CD8(+) T cells. To investigate how STAT2 affects cross-presentation, we determined its requirements for dendritic cell activation. In this study, we report that STAT2 is essential for the activation of murine female cDCs upon TLR3, -4, -7, and -9 stimulation. In response to various TLR ligands, Stat2(-/-) cDCs displayed reduced expression of costimulatory molecules and type I IFN-stimulated genes. The cDC responses to exogenous IFN-α that we evaluated required STAT2 activation, indicating that the canonical STAT1-STAT2 heterodimers are the primary signaling transducers of type I IFNs in cDCs. Interestingly, LPS-induced production of IL-12 was STAT2 and type I IFN receptor (IFNAR) dependent, whereas LPS-induced production of TNF-α and IL-6 was STAT2 and IFNAR independent, suggesting a specific role of the IFNAR-STAT2 axis in the stimulation of proinflammatory cytokines by LPS in cDCs. In contrast, R848- and CpG-induced cytokine production was less influenced by the IFNAR-STAT2 axis. Short kinetics and IFNAR blockade studies showed that STAT2 main function is to transduce signals triggered by autocrine type I IFNs. Importantly, Stat2(-/-) cDCs were deficient in cross-presenting to CD8(+) T cells in vitro upon IFN-α, CpG, and LPS stimulation, and also in cross-priming and licensing cytotoxic T cell killers in vivo. We conclude that STAT2 plays a critical role in TLR-induced dendritic cell activation and cross-presentation, and thus is vital in host defense.

Hydroxymethylation is uniquely distributed within term placenta, and is associated with gene expression.

The conversion of cytosine to 5-methylcystosine (5mC) is an important regulator of gene expression. 5mC may be enzymatically converted to 5-hydroxymethylcytosine (5hmC), with a potentially distinct regulatory function. We sought to investigate these cytosine modifications and their effect on gene expression by parallel processing of genomic DNA using bisulfite and oxidative bisulfite conversion in conjunction with RNA sequencing. Although values of 5hmC across the placental genome were generally low, we identified ∼21,000 loci with consistently elevated levels of 5-hydroxymethycytosine. Absence of 5hmC was observed in CpG islands and, to a greater extent, in non-CpG island-associated regions. 5hmC was enriched within poised enhancers, and depleted within active enhancers, as defined by H3K27ac and H3K4me1 measurements. 5hmC and 5mC were significantly elevated in transcriptionally silent genes when compared with actively transcribed genes. 5hmC was positively associated with transcription in actively transcribed genes only. Our data suggest that dynamic cytosine regulation, associated with transcription, provides the most complete epigenomic landscape of the human placenta, and will be useful for future studies of the placental epigenome.-Green, B. B., Houseman, E. A., Johnson, K. C., Guerin, D. J., Armstrong, D. A., Christensen, B. C., Marsit, C. J. Hydroxymethylation is uniquely distributed within term placenta, and is associated with gene expression.

Metabolic, endocrine and appetite-related responses to acute and daily milk snack consumption in healthy, adolescent males.

Comprising of two experiments, this study assessed the metabolic, endocrine and appetite-related responses to acute and chronic milk consumption in adolescent males (15-18 y). Eleven adolescents [mean ± SD age: 16.5 ± 0.9 y; BMI: 23.3 ± 3.3 kg/m2] participated in the acute experiment and completed two laboratory visits (milk vs. fruit-juice) in a randomized crossover design, separated by 7-d. Seventeen adolescents [age: 16.1 ± 0.9 y; BMI: 21.8 ± 3.7 kg/m2] completed the chronic experiment. For the chronic experiment, a parallel design with two groups was used. Participants were randomly allocated and consumed milk (n = 9) or fruit-juice (n = 8) for 28-d, completing laboratory visits on the first (baseline, day-0) and last day (follow-up, day-28) of the intervention phase. On laboratory visits (for both experiments), measures of appetite, metabolism and endocrine responses were assessed at regular intervals. In addition, eating behavior was quantified by ad libitum assessment under laboratory conditions and in the free-living environment by weighed food record. Acute milk intake stimulated glucagon (P = 0.027 [16.8 pg mL; 95% CI: 2.4, 31.3]) and reduced ad libitum energy intake relative to fruit-juice (P = 0.048 [-651.3 kJ; 95% CI: -1294.1, -8.6]), but was comparable in the free-living environment. Chronic milk intake reduced free-living energy intake at the follow-up visit compared to baseline (P = 0.013 [-1910.9 kJ; 95% CI: -554.6, -3267.2]), whereas the opposite was apparent for fruit-juice. Relative to baseline, chronic milk intake increased the insulin response to both breakfast (P = 0.031) and mid-morning milk consumption (P = 0.050) whilst attenuating blood glucose (P = 0.025). Together, these findings suggest milk consumption impacts favorably on eating behavior in adolescent males, potentially through integrated endocrine responses.

Differential responsiveness of Holstein and Angus dermal fibroblasts to LPS challenge occurs without major differences in the methylome.

BACKGROUND: We have previously found substantial animal-to-animal and age-dependent variation in the response of Holstein fibroblast cultures challenged with LPS. To expand on this finding, fibroblast cultures were established from dairy (Holstein) and beef (Angus) cattle and challenged with LPS to examine breed-dependent differences in the innate immune response. Global gene expression was measured by RNA-Seq, while an epigenetic basis for expression differences was examined by methylated CpG island recovery assay sequencing (MIRA-Seq) analysis. RESULTS: The Holstein breed displayed a more robust response to LPS than the Angus breed based on RNA-Seq analysis of cultures challenged with LPS for 0, 2, and 8 h. Several immune-associated genes were expressed at greater levels (FDR < 0.05) in Holstein cultures including TLR4 at all time points and a number of pro-inflammatory genes such as IL8, CCL20, CCL5, and TNF following LPS exposure. Despite extensive breed differences in the transcriptome, MIRA-Seq unveiled relatively similar patterns of genome-wide DNA methylation between breeds, with an overall hypomethylation of gene promoters. However, by examining the genome in 3Kb windows, 49 regions of differential methylation were discovered between Holstein and Angus fibroblasts, and two of these regions fell within the promoter region (-2500 to +500 bp of the transcription start site) of the genes NTRK2 and ADAMTS5. CONCLUSIONS: Fibroblasts isolated from Holstein cattle display a more robust response to LPS in comparison to cultures from Angus cattle. Different selection strategies and management practices exist between these two breeds that likely give rise to genetic and epigenetic factors contributing to the different immune response phenotypes.