Hepatocytes coordinate immune evasion in cancer via release of serum amyloid A proteins.

T cell infiltration into tumors is a favorable prognostic feature, but most solid tumors lack productive T cell responses. Mechanisms that coordinate T cell exclusion are incompletely understood. Here we identify hepatocyte activation via interleukin-6/STAT3 and secretion of serum amyloid A (SAA) proteins 1 and 2 as important regulators of T cell surveillance of extrahepatic tumors. Loss of STAT3 in hepatocytes or SAA remodeled the tumor microenvironment with infiltration by CD8+ T cells, while interleukin-6 overexpression in hepatocytes and SAA signaling via Toll-like receptor 2 reduced the number of intratumoral dendritic cells and, in doing so, inhibited T cell tumor infiltration. Genetic ablation of SAA enhanced survival after tumor resection in a T cell-dependent manner. Likewise, in individuals with pancreatic ductal adenocarcinoma, long-term survivors after surgery demonstrated lower serum SAA levels than short-term survivors. Taken together, these data define a fundamental link between liver and tumor immunobiology wherein hepatocytes govern productive T cell surveillance in cancer.

Multimodal immune phenotyping reveals microbial-T cell interactions that shape pancreatic cancer.

Microbes are an integral component of the tumor microenvironment. However, determinants of microbial presence remain ill-defined. Here, using spatial-profiling technologies, we show that bacterial and immune cell heterogeneity are spatially coupled. Mouse models of pancreatic cancer recapitulate the immune-microbial spatial coupling seen in humans. Distinct intra-tumoral niches are defined by T cells, with T cell-enriched and T cell-poor regions displaying unique bacterial communities that are associated with immunologically active and quiescent phenotypes, respectively, but are independent of the gut microbiome. Depletion of intra-tumoral bacteria slows tumor growth in T cell-poor tumors and alters the phenotype and presence of myeloid and B cells in T cell-enriched tumors but does not affect T cell infiltration. In contrast, T cell depletion disrupts the immunological state of tumors and reduces intra-tumoral bacteria. Our results establish a coupling between microbes and T cells in cancer wherein spatially defined immune-microbial communities differentially influence tumor biology.

Intratumoral Cell Neighborhoods Coordinate Outcomes in Pancreatic Ductal Adenocarcinoma.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease characterized by a spatially heterogeneous tumor microenvironment. Within the PDA microenvironment, cells organize into communities where cell fate is influenced by neighboring cells of diverse ontogeny and function. However, it remains unclear how cell neighborhoods in the tumor microenvironment evolve with treatment and impact clinical outcomes. METHODS: Here, using automated chromogenic multiplex immunohistochemistry and unsupervised computational image analysis of human PDA tumors, we investigated cell neighborhoods in surgically resected tumors from patients with chemotherapy-naïve PDA (n = 59) and neoadjuvant chemotherapy-treated PDA (n = 57). Single cells were defined by lineage markers (CD3, CD8, Foxp3, CD68, CK19), proliferation (Ki67), and neighboring cells. RESULTS: Distinct intratumoral immune and tumor cell subsets were defined by neighboring cells. Higher content of stromal-associated macrophages was seen in chemotherapy-naïve tumors from long-term survivors (overall survival >3 years) compared with short-term survivors (overall survival <1 year), whereas immune-excluded tumor cells were higher in short-term survivors. Chemotherapy-treated vs -naïve tumors showed lower content of tumor-associated T cells and macrophages but similar densities of stromal-associated immune cells. However, proliferating tumor cell subsets with immune-rich neighborhoods were higher in chemotherapy-treated tumors. In a blinded analysis of tumors from patients treated with neoadjuvant chemotherapy, a composite index comprising lower quantities of immune-excluded tumor cells and higher spatially distinct immune cell subsets was associated with prolonged survival. CONCLUSIONS: Together, these data provide new insights into discrete cell communities in PDA and show their clinical relevance.

Improved classification and pathogenicity assessment by comprehensive functional studies in a large data set of KCNQ2 variants.

AIMS: The paucity of functional annotations on hundreds of KCNQ2 variants impedes the diagnosis and treatment of KCNQ2-related disorders. The aims of this work were to determine the functional properties of 331 clinical KCNQ2 variants, interpreted the pathogenicity of 331 variants using functional data，and explored the association between homomeric channel functions and phenotypes. MAIN METHODS: We collected 145 KCNQ2 variants from 232 epilepsy patients and 186 KCNQ2 missense variants from the ClinVar database. Whole-cell patch-clamp recording was used to classify the function of 331 variants. Subsequently, we proposed 24 criteria for the pathogenicity interpretation of KCNQ2 variants and used them to assess pathogenicity of 331 variants. Finally, we analyzed the clinical phenotypes of patients carrying these variants, and explored the correlations between functional mechanisms and phenotypes. KEY FINDINGS: In the homozygous state, 287 were classified as loss-of-function and 14 as gain-of-function. In the more clinically relative heterozygous state, 200 variants exhibited functional impairment, 121 of which showed dominant-negative effects on wild-type KCNQ2 subunits. After introducing functional data as strong-level evidence to interpret pathogenicity, over half of variants (169/331) were reclassified and 254 were classified as pathogenic/likely pathogenic. Moreover, dominant-negative effect and haploinsufficiency were identified as primary mechanisms in DEE/ID and SeLNE, respectively. The degree of impairment of channel function correlated with the phenotype severity. SIGNIFICANCE: Our study reveals the possible cause of KCNQ2-related disorders at the molecular level, provides compelling evidence for clinical classification of KCNQ2 variants, and expands the knowledge of correlations between functional mechanisms and phenotypes.

Cancer immunotherapy via synergistic coactivation of myeloid receptors CD40 and Dectin-1.

Myeloid cells facilitate T cell immune evasion in cancer yet are pliable and have antitumor potential. Here, by cotargeting myeloid activation molecules, we leveraged the myeloid compartment as a therapeutic vulnerability in mouse models of pancreatic cancer. Myeloid cells in solid tumors expressed activation receptors including the pattern recognition receptor Dectin-1 and the TNF receptor superfamily member CD40. In mouse models of checkpoint inhibitor-resistant pancreatic cancer, coactivation of Dectin-1, via systemic ß-glucan therapy, and CD40, with agonist antibody treatment, eradicated established tumors and induced immunological memory. Antitumor activity was dependent on cDC1s and T cells but did not require classical T cell-mediated cytotoxicity or blockade of checkpoint molecules. Rather, targeting CD40 drove T cell-mediated IFN-γ signaling, which converged with Dectin-1 activation to program distinct macrophage subsets to facilitate tumor responses. Thus, productive cancer immune surveillance in pancreatic tumors resistant to checkpoint inhibition can be invoked by coactivation of complementary myeloid signaling pathways.

Potentially Inappropriate Medication Use and Hard Braking Events in Older Drivers.

Potentially inappropriate medications (PIMs) identified by the American Geriatrics Society should generally be avoided by older adults because of ineffectiveness or excess risk of adverse effects. Few studies have examined the effects of PIMs on driving safety measured by prospectively and objectively collected driving data. Data for this study came from the Longitudinal Research on Aging Drivers study, a multisite naturalistic driving study of older adults. Multivariable negative binominal modeling was used to estimate incidence rate ratios and 95% confidence intervals of hard braking events (proxies for unsafe driving behavior defined as events with a deceleration rate ≥0.4 g) associated with PIM use among older drivers. The study sample consisted of 2932 drivers aged 65-79 years at baseline, including 542 (18.5%) who used at least one PIM. These drivers were followed through an in-vehicle recording device for up to 44 months. The overall incidence of hard braking events was 1.16 per 1000 miles. Use of PIMs was associated with a 10% increased risk of hard braking events. Compared to drivers who were not using PIMs, the risk of hard braking events increased 6% for those using one PIM, and 24% for those using two or more PIMs. Use of PIMs by older adult drivers is associated in a dose-response fashion with elevated risks of hard braking events. Reducing PIM use in older adults might help improve driving safety as well as health outcomes.

Interval estimation of proportion ratios for stratified bilateral correlated binary data.

Confidence interval (CI) methods for the ratio of two proportions in the presence of correlated bilateral binary data are constructed for comparative clinical trials with stratified design. Simulations are conducted to evaluate the performance of the presented CIs with respect to mean coverage probability (MCP), mean interval width (MIW), and the ratio of mesial non-coverage probability to the distal non-coverage probability (RMNCP). Based on the empirical results, we suggest the use of the proposed CI method based on the complete score statistics (CS) for general applications. An example from a rheumatology study is used to demonstrate the proposed methodologies.

Exploring Medicaid claims data to understand predictors of healthcare utilization and mortality for Medicaid individuals with or without a diagnosis of lung cancer: a feasibility study.

Health disparities in low-income populations complicate care for at-risk individuals or those diagnosed with lung cancer and may influence their patterns of healthcare utilization. The purpose of this study is to examine whether age, sex, provider's affiliation, Medicare dual eligibility, and number of comorbidities can predict healthcare utilization, as well as to examine factors influencing mortality in lung biopsy patients. A retrospective review of de-identified Medicaid claims of adults having a lung biopsy in 2013 resulted in classification into lung cancer and non-lung cancer cases based on a lung cancer diagnostic code within 30 days after biopsy. Biopsy cases were further divided by whether or not the provider's institution was accredited by the Commission on Cancer (CoC). Inpatient (IP), outpatient (OP), and emergency department (ED) utilization was followed from initial date of biopsy through 2015, or to the earliest date of death, disenrollment, or study end for both groups. The result of Cox proportional hazards regression model indicated that age and the number of comorbidities significantly predicted OP use and the number of comorbidities significantly predicted ED use in patients with lung cancer. However, for non-lung cancer patients, only the number of comorbidities significantly predicted IP and ED uses. Furthermore, for patients with lung cancer, the significant factors of mortality included IP use per month and the number of comorbidities. Patients with lung cancer who received a lung biopsy by a CoC-accredited organization had a longer time of survival from the biopsy event. Our findings suggest that understanding predictors of healthcare utilization and mortality may create opportunities to improve health and quality of life through better healthcare coordination.

Integrating Social Determinants of Health into Primary Care Clinical and Informational Workflow during Care Transitions.

CONTEXT: Care continuity during transitions between the hospital and home requires reliable communication between providers and settings and an understanding of social determinants that influence recovery. CASE DESCRIPTION: The coordinating transitions intervention uses real time alerts, delivered directly to the primary care practice for complex chronically ill patients discharged from an acute care setting, to facilitate nurse care coordinator led telephone outreach. The intervention incorporates claims-based risk stratification to prioritize patients for follow-up and an assessment of social determinants of health using the Patient-centered Assessment Method (PCAM). Results from transitional care are stored and transmitted to qualified healthcare providers across the continuum. FINDINGS: Reliance on tools that incorporated interoperability standards facilitated exchange of health information between the hospital and primary care. The PCAM was incorporated into both the clinical and informational workflow through the collaboration of clinical, industry, and academic partners. Health outcomes improved at the study practice over their baseline and in comparison with control practices and the regional Medicaid population. MAJOR THEMES: Current research supports the potential impact of systems approaches to care coordination in improving utilization value after discharge. The project demonstrated that flexibility in developing the informational and clinical workflow was critical in developing a solution that improved continuity during transitions. There is additional work needed in developing managerial continuity across settings such as shared comprehensive care plans. CONCLUSIONS: New clinical and informational workflows which incorporate social determinant of health data into standard practice transformed clinical practice and improved outcomes for patients.

Lactobacillus acidophilus induces cytokine and chemokine production via NF-κB and p38 mitogen-activated protein kinase signaling pathways in intestinal epithelial cells.

Intestinal epithelial cells can respond to certain bacteria by producing an array of cytokines and chemokines which are associated with host immune responses. Lactobacillus acidophilus NCFM is a characterized probiotic, originally isolated from human feces. This study aimed to test the ability of L. acidophilus NCFM to stimulate cytokine and chemokine production in intestinal epithelial cells and to elucidate the mechanisms involved in their upregulation. In experiments using intestinal epithelial cell lines and mouse models, we observed that L. acidophilus NCFM could rapidly but transiently upregulate a number of effector genes encoding cytokines and chemokines such as interleukin 1α (IL-1α), IL-1ß, CCL2, and CCL20 and that cytokines showed lower expression levels with L. acidophilus NCFM treatment than chemokines. Moreover, L. acidophilus NCFM could activate a pathogen-associated molecular pattern receptor, Toll-like receptor 2 (TLR2), in intestinal epithelial cell lines. The phosphorylation of NF-κB p65 and p38 mitogen-activated protein kinase (MAPK) in intestinal epithelial cell lines was also enhanced by L. acidophilus NCFM. Furthermore, inhibitors of NF-κB (pyrrolidine dithiocarbamate [PDTC]) and p38 MAPK (SB203580) significantly reduced cytokine and chemokine production in the intestinal epithelial cell lines stimulated by L. acidophilus NCFM, suggesting that both NF-κB and p38 MAPK signaling pathways were important for the production of cytokines and chemokines induced by L. acidophilus NCFM.

[Lactobacillus plantarum NDC 75017 affects il-6 gene expression in Caco-2 cells].

OBJECTIVE: We tested the expression of immune-related gene interleukin 6 (il-6) in vitro to understand the influence from Lactobacillus plantarum NDC 75017 on host cells and further to reveal the regulatory mechanism. METHODS: Caco-2 cells were cocultured with L. plantarum NDC 75017 for 0, 2, 4, 6, 8, 10 and 12 h, the total RNA were extracted; then the expressions of il-6 and tlr2 genes were analyzed by Real Time RT-PCR. The phosphorylation level of NF-KB was analyzed by Western Blot after the Caco-2 cells stimulation with L. plantarum NDC 75017 at 0, 0.5, 1, 2 and 4 h. Caco-2 cells were pretreated with pyrrolidine dithiocarbamate for 30 min before being treated with L. plantarum NDC 75017 for 2 h, then the total RNA was extracted and the expressions of il-6 and tlr2 genes were analyzed by Real Time RT-PCR. RESULTS: Lactobacillus plantarum NDC 75017 could induce the expressions of il-6 and tlr2 in Caco-2 cells, the il-6 and tlr2 expressions peaked at 8 h and 6 h after cocultured with L. plantarum NDC 75017. L. plantarum NDC 75017 could rapidly activate the phosphorylation of NF-kappaB, and the expressions of il-6 and tlr2 were decreased notably after pretreated with pyrrolidine dithiocarbamate. CONCLUSION: L. plantarum NDC 75017 could up-regulate and then down-regulate the expression of il6 through rapidly activating tlr2-mediated NF-kappaB signaling pathway in Caco-2 cells.

[Expression of PTX3 gene in Caco-2 cells treated with Lactobacillus acidophilus NCFM].

OBJECTIVE: To study the expression of immunity and inflammatory mediator factor PTX3 in intestinal epithelial cells treated with Lactobacillus acidophilus NCFM and further to reveal the regulatory mechanism. METHODS: Caco-2 cells were cocultured with Lactobacillus acidophilus NCFM for 0, 2, 4, 8, 12 h and 0, 0.5, 1, 2, 4 h respectively, then the total RNA and protein were extracted. The expression of PTX3 gene was analyzed by Real Time RT-PCR. The phosphorylation levels of NF-kappaB was analyzed by Western Blot. Caco-2 cells were pretreated with PDTC for 30 min before cocultured with Lactobacillus acidophilus NCFM for 2 h, then the total RNA was extracted and the expression of PTX3 gene was analyzed by Real Time RT-PCR. RESULTS: Lactobacillus acidophilus NCFM could induce the expression of PTX3 in Caco-2 cells. The PTX3 expression peaked at 4 h after coculture. Then its expression gradually waned out. Lactobacillus acidophilus NCFM could rapidly activate the phosphorylation of NF-kappaB, and the expression of PTX3 was decreased notably after pretreated with PDTC for 30 min. CONCLUSION: Lactobacillus acidophilus NCFM could transiently regulate the immunity and inflammatory mediator factor PTX3 expression through rapidly activating NF-kappaB signaling pathway in Caco-2 cells.