Multi-omics analyses of airway host-microbe interactions in chronic obstructive pulmonary disease identify potential therapeutic interventions.

The mechanistic role of the airway microbiome in chronic obstructive pulmonary disease (COPD) remains largely unexplored. We present a landscape of airway microbe-host interactions in COPD through an in-depth profiling of the sputum metagenome, metabolome, host transcriptome and proteome from 99 patients with COPD and 36 healthy individuals in China. Multi-omics data were integrated using sequential mediation analysis, to assess in silico associations of the microbiome with two primary COPD inflammatory endotypes, neutrophilic or eosinophilic inflammation, mediated through microbial metabolic interaction with host gene expression. Hypotheses of microbiome-metabolite-host interaction were identified by leveraging microbial genetic information and established metabolite-human gene pairs. A prominent hypothesis for neutrophil-predominant COPD was altered tryptophan metabolism in airway lactobacilli associated with reduced indole-3-acetic acid (IAA), which was in turn linked to perturbed host interleukin-22 signalling and epithelial cell apoptosis pathways. In vivo and in vitro studies showed that airway microbiome-derived IAA mitigates neutrophilic inflammation, apoptosis, emphysema and lung function decline, via macrophage-epithelial cell cross-talk mediated by interleukin-22. Intranasal inoculation of two airway lactobacilli restored IAA and recapitulated its protective effects in mice. These findings provide the rationale for therapeutically targeting microbe-host interaction in COPD.

Pharmacomicrobiomics: The Holy Grail to Variability in Drug Response?

The human body, with 3.0 × 1013 cells and more than 3.8 × 1013 microorganisms, has nearly a one-to-one ratio of resident microbes to human cells. Initiatives like the Human Microbiome Project, American Gut, and Flemish Gut have identified associations between microbial taxa and human health. The study of interactions between microbiome and pharmaceutical agents, i.e., pharmacomicrobiomics, has revealed an instrumental role of the microbiome in modulating drug response that alters the therapeutic outcomes. In this review, we present our current comprehension of the relationship of the microbiome, host biology, and pharmaceutical agents such as cardiovascular drugs, analgesics, and chemotherapeutic agents to human disease and treatment outcomes. We also discuss the significance of studying diet-gene-drug interactions and further address the key challenges associated with pharmacomicrobiomics. Finally, we examine proposed models employing systems biology for the application of pharmacomicrobiomics and other -omics data, and provide approaches to elucidate microbiome-drug interactions to improve future translation to personalized medicine.

Human Organoids Share Structural and Genetic Features with Primary Pancreatic Adenocarcinoma Tumors.

Patient-derived pancreatic ductal adenocarcinoma (PDAC) organoid systems show great promise for understanding the biological underpinnings of disease and advancing therapeutic precision medicine. Despite the increased use of organoids, the fidelity of molecular features, genetic heterogeneity, and drug response to the tumor of origin remain important unanswered questions limiting their utility. To address this gap in knowledge, primary tumor- and patient-derived xenograft (PDX)-derived organoids, and 2D cultures for in-depth genomic and histopathologic comparisons with the primary tumor were created. Histopathologic features and PDAC representative protein markers (e.g., claudin 4 and CA19-9) showed strong concordance. DNA- and RNA-sequencing (RNAseq) of single organoids revealed patient-specific genomic and transcriptomic consistency. Single-cell RNAseq demonstrated that organoids are primarily a clonal population. In drug response assays, organoids displayed patient-specific sensitivities. In addition, the in vivo PDX response to FOLFIRINOX and gemcitabine/abraxane treatments were examined, which was recapitulated in vitro with organoids. This study has demonstrated that organoids are potentially invaluable for precision medicine as well as preclinical drug treatment studies because they maintain distinct patient phenotypes and respond differently to drug combinations and dosage. IMPLICATIONS: The patient-specific molecular and histopathologic fidelity of organoids indicate that they can be used to understand the etiology of the patient's tumor and the differential response to therapies and suggests utility for predicting drug responses.

The impact of changes in radiographic sarcopenia on overall survival in older adults undergoing different treatment pathways for pancreatic cancer.

OBJECTIVE: Sarcopenia is associated with poor outcomes in patients undergoing surgery for pancreatic ductal adenocarcinoma (PDAC). However, few studies have assessed changes in sarcopenia during multimodality therapy or its effect on overall survival (OS). METHODS: Computed tomography (CT) total psoas area index (TPAI) and weighted average Hounsfield units (HU) were measured at each treatment interval in patients with resectable PDAC. Four cohorts were compared: 1. Neoadjuvant chemotherapy plus surgery plus adjuvant chemotherapy ("NSA"; n = 20); 2. surgery plus adjuvant chemotherapy ("SA"; n = 20); 3. neoadjuvant chemotherapy with intent to perform surgery ("Chemotherapy"; n = 24); and 4. treated with palliative intent ("Palliative"; n = 21). RESULTS: Fifty-nine deaths were identified. Median OS was 15.7 months (95% Confidence Interval (CI) 12.7-20.2). Patients who underwent surgery had a higher OS (p < 0.001), with the SA group having a longer OS than the NSA group. Cox regression models identified baseline TPAI (Hazard Ratio (HR) = 0.82; p = 0.04), but not psoas HU, as a significant predictor of OS. The mean decrease in TPAI following neoadjuvant chemotherapy was 0.6 cm2/m2 (p < 0.001; 95% CI -0.8--0.3) and the mean decrease in HU was 2.7 (p = 0.04, 95% CI -5.4--0.1). For patients who underwent surgery (NSA and SA cohorts), a decrease in TPAI was associated with worse OS (HR 0.52; p = 0.05). In contrast, decreased HU was associated with worse OS in patients who did not undergo surgery (HR 0.93; p = 0.01). CONCLUSIONS: In patients who received neoadjuvant chemotherapy, there was a significant decrease in TPAI and HU during treatment. Prospective studies are warranted to assess the impact of TPAI loss and HU changes on clinical outcomes to better individualize treatment pathways based on a patient's fitness.

Research priorities in cancer cachexia: The University of Rochester Cancer Center NCI Community Oncology Research Program Research Base Symposium on Cancer Cachexia and Sarcopenia.

PURPOSE OF REVIEW: Cancer cachexia remains understudied and there are no standard treatments available despite the publication of an international consensus definition and the completion of several large phase III intervention trials in the past 6 years. In September 2015, The University of Rochester Cancer Center NCORP Research Base led a Symposium on Cancer Cachexia and Sarcopenia with goals of reviewing the state of the science, identifying knowledge gaps, and formulating research priorities in cancer cachexia through active discussion and consensus. RECENT FINDINGS: Research priorities that emerged from the discussion included the implementation of morphometrics into clinical decision making, establishing specific diagnostic criteria for the stages of cachexia, expanding patient selection in intervention trials, identifying clinically meaningful trial endpoints, and the investigation of exercise as an intervention for cancer cachexia. SUMMARY: Standardizing how we define and measure cancer cachexia, targeting its complex biologic mechanisms, enrolling patients early in their disease course, and evaluating exercise, either alone or in combination, were proposed as initiatives that may ultimately result in the improved design of cancer cachexia therapeutic trials.

Can Comprehensive Imaging Analysis with Analytic Morphomics and Geriatric Assessment Predict Serious Complications in Patients Undergoing Pancreatic Surgery?

We aimed to determine whether comprehensive imaging analysis with analytic morphomics (AM) enhances or replaces geriatric assessment (GA) in risk-stratifying pancreatic surgery patients. One hundred thirty-four pancreatic surgery patients were identified from a prospective cohort. Sixty-three patients in the cohort had preoperative CT scans in addition to comprehensive geriatric assessments. CT scans were processed using AM. Associations with National Surgical Quality Improvement Program (NSQIP) serious complications were evaluated using univariate analysis and robust elastic net modeling to obtain AUROC curves by adding AM and GA measures to our previously defined clinical base risk model (age, body mass index, American Society of Anesthesiologists classification, and Charlson comorbidity index). NSQIP serious complications were associated with low psoas Hounsfield units (HUs) (p = 0.002), low-density (0 to 30 HU) psoas area (p = 0.01), visceral fat HU (p ≤ 0.001), visceral fat area (p = 0.009), subcutaneous fat HU (p = 0.023), and total body area (p = 0.012) on univariate analysis. Elastic net models incorporating the base model with geriatric assessment and psoas HU (AUC = 0.751), and AM alone (AUC = 0.739) have greater predictive value than the base model alone (AUC = 0.601). The model utilizing AM and GA in combination had the highest predictive value (AUC = 0.841). When combined, AM and GA improve prediction of NSQIP serious complications compared to either technique alone. The additive nature of these two modalities suggests they likely capture unique aspects of a patient's fitness for surgery.

ZO-1 interactions with F-actin and occludin direct epithelial polarization and single lumen specification in 3D culture.

Epithelia within tubular organs form and expand lumens. Failure of these processes can result in serious developmental anomalies. Although tight junction assembly is crucial to epithelial polarization, the contribution of specific tight junction proteins to lumenogenesis is undefined. Here, we show that ZO-1 (also known as TJP1) is necessary for the formation of single lumens. Epithelia lacking this tight junction scaffolding protein form cysts with multiple lumens and are defective in the earliest phases of polarization, both in two and three dimensions. Expression of ZO-1 domain-deletion mutants demonstrated that the actin-binding region and U5-GuK domain are crucial to single lumen development. For actin-binding region, but not U5-GuK domain, mutants, this could be overcome by strong polarization cues from the extracellular matrix. Analysis of the U5-GuK binding partners shroom2, α-catenin and occludin showed that only occludin deletion led to multi-lumen cysts. Like ZO-1-deficiency, occludin deletion led to mitotic spindle orientation defects. Single lumen formation required the occludin OCEL domain, which binds to ZO-1. We conclude that ZO-1-occludin interactions regulate multiple phases of epithelial polarization by providing cell-intrinsic signals that are required for single lumen formation.

Radiographic Sarcopenia and Self-reported Exhaustion Independently Predict NSQIP Serious Complications After Pancreaticoduodenectomy in Older Adults.

INTRODUCTION: Sarcopenia is linked to poor outcomes after abdominal surgery. We hypothesized that radiographic sarcopenia metrics enhance prediction of complications after pancreaticoduodenectomy (PD) when combined with clinical and frailty data. METHODS: Preoperative geriatric assessments and CT scans of patients undergoing PD were reviewed. Sarcopenia was assessed at L3 using total psoas area index (TPAI) and weighted average Hounsfield units (HU), i.e., estimates of psoas muscle volume and density. Outcomes included 30-day American College of Surgeons National Surgical Quality Improvement Program (NSQIP) serious complications, Clavien-Dindo complications, unplanned intensive care unit (ICU) admission, hospital length of stay (LOS), non-home facility (NHF) discharge, and readmission rates. RESULTS: Low HU score correlated with NSQIP serious complications (r = -0.31, p = 0.0098), Clavien-Dindo complication grade (r = -0.29, p = 0.0183), unplanned ICU admission (r = -0.28, p = 0.0239), and NHF discharge (r = -0.25, p = 0.0426). Controlling for a "base model" of age, body mass index, American Society of Anesthesiologists score, and comorbidity burden, Fried's exhaustion (odds ratio [OR] 4.72 [1.23-17.71], p = 0.021), and HU (OR 0.88 [0.79-0.98], p = 0.024) predicted NSQIP serious complications. Area under the receiver-operator characteristic (AUC) curves demonstrated that the combination of the base model, exhaustion, and HU trended towards improving the prediction of NSQIP serious complications compared with the base model alone (AUC = 0.81 vs. 0.70; p = 0.09). Additionally, when controlling for the base model, TPAI (ß-coefficient = 0.55 [0.10-1.01], p = 0.018) and exhaustion (ß-coefficient = 2.47 [0.75-4.20], p = 0.005) predicted LOS and exhaustion (OR 4.14 [1.48-11.6], p = 0.007) predicted readmissions. CONCLUSIONS: When combined with clinical and frailty assessments, radiographic sarcopenia metrics enhance prediction of post-PD outcomes.

The role of molecular remodeling in differential regulation of tight junction permeability.

Tight junctions create a paracellular barrier that is essential for survival of complex organisms. In many cases tight junctions define separate, generally sterile, tissue compartments. In the skin and gut, tight junctions must also seal the paracellular space to prevent microbiota from accessing the internal milieu. This is a relatively simple task in the integument, where an absolute barrier is effective. However, intestinal epithelial tight junctions are charged with the far more complex task of supporting paracellular transport of water, ions, and nutrients while providing a barrier to microbial translocation. The delicate nature of this balance, which is disrupted in disease, makes the intestine a unique organ in which to explore the complexities of tight junction permeability and barrier regulation. Here we review recent progress in understanding the molecular determinants of barrier function and events responsible for regulation, and dysregulation, of tight junction permeability.

Occludin OCEL-domain interactions are required for maintenance and regulation of the tight junction barrier to macromolecular flux.

In vitro and in vivo studies implicate occludin in the regulation of paracellular macromolecular flux at steady state and in response to tumor necrosis factor (TNF). To define the roles of occludin in these processes, we established intestinal epithelia with stable occludin knockdown. Knockdown monolayers had markedly enhanced tight junction permeability to large molecules that could be modeled by size-selective channels with radii of ~62.5 Å. TNF increased paracellular flux of large molecules in occludin-sufficient, but not occludin-deficient, monolayers. Complementation using full-length or C-terminal coiled-coil occludin/ELL domain (OCEL)-deficient enhanced green fluorescent protein (EGFP)-occludin showed that TNF-induced occludin endocytosis and barrier regulation both required the OCEL domain. Either TNF treatment or OCEL deletion accelerated EGFP-occludin fluorescence recovery after photobleaching, but TNF treatment did not affect behavior of EGFP-occludin(ΔOCEL). Further, the free OCEL domain prevented TNF-induced acceleration of occludin fluorescence recovery, occludin endocytosis, and barrier loss. OCEL mutated within a recently proposed ZO-1-binding domain (K433) could not inhibit TNF effects, but OCEL mutated within the ZO-1 SH3-GuK-binding region (K485/K488) remained functional. We conclude that OCEL-mediated occludin interactions are essential for limiting paracellular macromolecular flux. Moreover, our data implicate interactions mediated by the OCEL K433 region as an effector of TNF-induced barrier regulation.

Laminin 511 partners with laminin 332 to mediate directional migration of Madin-Darby canine kidney epithelial cells.

Sustained directional migration of epithelial cells is essential for regeneration of injured epithelia. Front-rear polarity of migrating cells is determined by local activation of a signaling network involving Cdc42 and other factors in response to spatial cues from the environment, the nature of which are obscure. We examined the roles of laminin (LM)-511 and LM-332, two structurally different laminin isoforms, in the migration of Madin-Darby canine kidney cells by suppressing expression of their α subunits using RNA interference. We determined that knockdown of LM-511 inhibits directional migration and destabilizes cell-cell contacts, in part by disturbing the localization and activity of the polarization machinery. Suppression of integrin α3, a laminin receptor subunit, in cells synthesizing normal amounts of both laminins has a similar effect as knockdown of LM-511. Surprisingly, simultaneous suppression of both laminin α5 and laminin α3 restores directional migration and cell-cell contact stability, suggesting that cells recognize a haptotactic gradient formed by a combination of laminins.

Autocrine transforming growth factor-{beta}1 activation mediated by integrin {alpha}V{beta}3 regulates transcriptional expression of laminin-332 in Madin-Darby canine kidney epithelial cells.

Laminin (LM)-332 is an extracellular matrix protein that plays a structural role in normal tissues and is also important in facilitating recovery of epithelia from injury. We have shown that expression of LM-332 is up-regulated during renal epithelial regeneration after ischemic injury, but the molecular signals that control expression are unknown. Here, we demonstrate that in Madin-Darby canine kidney (MDCK) epithelial cells LM-332 expression occurs only in subconfluent cultures and is turned-off after a polarized epithelium has formed. Addition of active transforming growth factor (TGF)-ß1 to confluent MDCK monolayers is sufficient to induce transcription of the LM α3 gene and LM-332 protein expression via the TGF-ß type I receptor (TßR-I) and the Smad2-Smad4 complex. Significantly, we show that expression of LM-332 in MDCK cells is an autocrine response to endogenous TGF-ß1 secretion and activation mediated by integrin αVß3 because neutralizing antibodies block LM-332 production in subconfluent cells. In confluent cells, latent TGF-ß1 is secreted apically, whereas TßR-I and integrin αVß3 are localized basolaterally. Disruption of the epithelial barrier by mechanical injury activates TGF-ß1, leading to LM-332 expression. Together, our data suggest a novel mechanism for triggering the production of LM-332 after epithelial injury.

Regulated synthesis and functions of laminin 5 in polarized madin-darby canine kidney epithelial cells.

Renal tubular epithelial cells synthesize laminin (LN)5 during regeneration of the epithelium after ischemic injury. LN5 is a truncated laminin isoform of particular importance in the epidermis, but it is also constitutively expressed in a number of other epithelia. To investigate the role of LN5 in morphogenesis of a simple renal epithelium, we examined the synthesis and function of LN5 in the spreading, proliferation, wound-edge migration, and apical-basal polarization of Madin-Darby canine kidney (MDCK) cells. MDCK cells synthesize LN5 only when subconfluent, and they degrade the existing LN5 matrix when confluent. Through the use of small-interfering RNA to knockdown the LN5 alpha3 subunit, we were able to demonstrate that LN5 is necessary for cell proliferation and efficient wound-edge migration, but not apical-basal polarization. Surprisingly, suppression of LN5 production caused cells to spread much more extensively than normal on uncoated surfaces, and exogenous keratinocyte LN5 was unable to rescue this phenotype. MDCK cells also synthesized laminin alpha5, a component of LN10, that independent studies suggest may form an assembled basal lamina important for polarization. Overall, our findings indicate that LN5 is likely to play an important role in regulating cell spreading, migration, and proliferation during reconstitution of a continuous epithelium.