Persistence and Sexual Dimorphism of Gut Dysbiosis and Pathobiome after Sepsis and Trauma.

OBJECTIVE: To evaluate the persistence of intestinal microbiome dysbiosis and gut-plasma metabolomic perturbations following severe trauma or sepsis weeks after admission in patients experiencing chronic critical illness (CCI). SUMMARY: Trauma and sepsis can lead to gut dysbiosis and alterations in the plasma and fecal metabolome. However, the impact of these perturbations and correlations between gut dysbiosis and the plasma metabolome in chronic critical illness have not been studied. METHODS: A prospective observational cohort study was performed with healthy subjects, severe trauma patients, patients with sepsis residing in an intensive care unit (ICU) for 2-3 weeks. A high-throughput multi-omics approach was utilized to evaluate the gut microbial and gut-plasma metabolite responses in critically ill trauma and sepsis patients 14-21 days after ICU admission. RESULTS: Patients in the sepsis and trauma cohorts demonstrated strikingly depleted gut microbiome diversity, with significant alterations and specific pathobiome patterns in the microbiota composition compared to healthy subjects. Further subgroup analyses based on sex revealed resistance to changes in microbiome diversity among female trauma patients compared to healthy counterparts. Sex-specific changes in fecal metabolites were also observed after trauma and sepsis, while plasma metabolite changes were similar in both males and females. CONCLUSIONS: Dysbiosis induced by trauma and sepsis persists up to 14-21 days after onset and is sex-specific, underscoring the implication of pathobiome and entero-septic microbial-metabolite perturbations in post-sepsis and post-trauma CCI. This indicates resilience to infection or injury in females' microbiome and should inform and facilitate future precision/personalized medicine strategies in the intensive care unit.

Gut mycobiome dysbiosis after sepsis and trauma.

BACKGROUND: Sepsis and trauma are known to disrupt gut bacterial microbiome communities, but the impacts and perturbations in the fungal (mycobiome) community after severe infection or injury, particularly in patients experiencing chronic critical illness (CCI), remain unstudied. METHODS: We assess persistence of the gut mycobiome perturbation (dysbiosis) in patients experiencing CCI following sepsis or trauma for up to two-to-three weeks after intensive care unit hospitalization. RESULTS: We show that the dysbiotic mycobiome arrays shift toward a pathobiome state, which is more susceptible to infection, in CCI patients compared to age-matched healthy subjects. The fungal community in CCI patients is largely dominated by Candida spp; while, the commensal fungal species are depleted. Additionally, these myco-pathobiome arrays correlate with alterations in micro-ecological niche involving specific gut bacteria and gut-blood metabolites. CONCLUSIONS: The findings reveal the persistence of mycobiome dysbiosis in both sepsis and trauma settings, even up to two weeks post-sepsis and trauma, highlighting the need to assess and address the increased risk of fungal infections in CCI patients.

Characterization of the Basal and mTOR-Dependent Acute Pulmonary and Systemic Immune Response in a Murine Model of Combined Burn and Inhalation Injury.

Severe burn injury leads to a cascade of local and systemic immune responses that trigger an extreme state of immune dysfunction, leaving the patient highly susceptible to acute and chronic infection. When combined with inhalation injury, burn patients have higher mortality and a greater chance of developing secondary respiratory complications including infection. No animal model of combined burn and inhalation injury (B+I) exists that accurately mirrors the human clinical picture, nor are there any effective immunotherapies or predictive models of the risk of immune dysfunction. Our earlier work showed that the mechanistic/mammalian target of rapamycin (mTOR) pathway is activated early after burn injury, and its chemical blockade at injury reduced subsequent chronic bacterial susceptibility. It is unclear if mTOR plays a role in the exacerbated immune dysfunction seen after B+I injury. We aimed to: (1) characterize a novel murine model of B+I injury, and (2) investigate the role of mTOR in the immune response after B+I injury. Pulmonary and systemic immune responses to B+I were characterized in the absence or presence of mTOR inhibition at the time of injury. Data describe a murine model of B+I with inhalation-specific immune phenotypes and implicate mTOR in the acute immune dysfunction observed.

Multiplexed Human Gene Expression Analysis Reveals a Central Role of the TLR/mTOR/PPARγ and NFkB Axes in Burn and Inhalation Injury-Induced Changes in Systemic Immunometabolism and Long-Term Patient Outcomes.

Burn patients are subject to significant acute immune and metabolic dysfunction. Concomitant inhalation injury increases mortality by 20%. In order to identify specific immune and metabolic signaling pathways in burn (B), inhalation (I), and combined burn-inhalation (BI) injury, unbiased nanoString multiplex technology was used to investigate gene expression within peripheral blood mononuclear cells (PBMCs) from burn patients, with and without inhalation injury. PBMCs were collected from 36 injured patients and 12 healthy, non-burned controls within 72 h of injury. mRNA was isolated and hybridized with probes for 1342 genes related to general immunology and cellular metabolism. From these specific gene patterns, specific cellular perturbations and signaling pathways were inferred using robust bioinformatic tools. In both B and BI injuries, elements of mTOR, PPARγ, TLR, and NF-kB signaling pathways were significantly altered within PBMC after injury compared to PBMC from the healthy control group. Using linear regression modeling, (1) DEPTOR, LAMTOR5, PPARγ, and RPTOR significantly correlated with patient BMI; (2) RPTOR significantly correlated with patient length of stay, and (3) MRC1 significantly correlated with the eventual risk of patient mortality. Identification of mediators of this immunometabolic response that can act as biomarkers and/or therapeutic targets could ultimately aid the management of burn patients.

Mechanisms of Foreign Body Response Mitigation by Nitric Oxide Release.

Implantable glucose biosensors provide real-time information about blood glucose fluctuations, but their utility and accuracy are time-limited due to the foreign body response (FBR) following their insertion beneath the skin. The slow release of nitric oxide (NO), a gasotransmitter with inflammation regulatory properties, from a sensor surface has been shown to dramatically improve sensors' analytical biocompatibility by reducing the overall FBR response. Indeed, work in a porcine model suggests that as long as the implants (sensors) continue to release NO, even at low levels, the inflammatory cell infiltration and resulting collagen density are lessened. While these studies strongly support the benefits of NO release in mitigating the FBR, the mechanisms through which exogenous NO acts on the surrounding tissue, especially under the condition of hyperglycemia, remain vague. Such knowledge would inform strategies to refine appropriate NO dosage and release kinetics for optimal therapeutic activity. In this study, we evaluated mediator, immune cell, and mRNA expression profiles in the local tissue microenvironment surrounding implanted sensors as a function of NO release, diabetes, and implantation duration. A custom porcine wound healing-centric multiplex gene array was developed for nanoString barcoding analysis. Tissues adjacent to sensors with sustained NO release abrogated the implant-induced acute and chronic FBR through modulation of the tissue-specific immune chemokine and cytokine microenvironment, resulting in decreased cellular recruitment, proliferation, and activation at both the acute (7-d) and chronic (14-d) phases of the FBR. Further, we found that sustained NO release abrogated the implant-induced acute and chronic foreign body response through modulation of mRNA encoding for key immunological signaling molecules and pathways, including STAT1 and multiple STAT1 targets including MAPK14, IRAK4, MMP2, and CXCL10. The condition of diabetes promoted a more robust FBR to the implants, which was also controlled by sustained NO release.

Performance and efficacy of a recently introduced diamond rotary instrument: Cutting, surface preparation, and cleanability.

STATEMENT OF PROBLEM: Evaluation of the cutting efficiency and effectiveness, surface roughness, and cleanability of a novel rotary instrument is lacking. PURPOSE: The purpose of this in vitro study was to compare the cutting efficiency and effectiveness of a recently introduced diamond rotary instrument containing corundum microspheres with conventional instruments by evaluating the heat generated, surface roughness, and cleanability of each instrument after tooth preparations. MATERIAL AND METHODS: Sound molars (n=225) were used to evaluate cutting efficiency and effectiveness by measuring the heat generated by 3 diamond dental rotary instruments: test instrument (TI), reference instrument (RI), and NTI instrument (NI). Thirty cavity preparations (27 mm3) were prepared, and the thermal change (ΔT) was determined from a thermocouple inserted in the pulp chamber. The surface roughness of the dentin substrate was determined after veneer preparations using scanning white-light interferometry and scanning electron microscope imaging. The cleanability of TI and RI was also determined by comparing the efficacy of 3 conventional disinfection protocols after contaminating the instrument with Gram-positive or Gram-negative oral pathogens. The mean and standard deviation values for thermal change, surface roughness, and colony forming units were calculated at a 95% confidence level, and 1-way ANOVA was used to determine statistical significance (α=.05). RESULTS: The NI instrument had the lowest mean ΔT (1.47 °C). The TI (1.77 °C) and RI (1.85 °C) groups showed statistically similar means (P>.05). The TI presented the lowest surface roughness (1.68 µm), followed by the RI (1.87 µm) (P<.001). The NI resulted in the highest surface roughness (2.17 µm) (P<.001). The disinfection protocols used were more effective on the TI group than on the RI group regardless of organisms and time exposed to the cleaning solution (P<.001). CONCLUSIONS: The novel diamond instrument demonstrated similar cutting efficiency and effectiveness when compared with conventional diamond instruments. However, the novel instrument produced smoother tooth preparations and was easier to clean than the conventional diamond rotary instruments.

Characterization of extracellular vesicle miRNA identified in peripheral blood of chronic pancreatitis patients.

Plasma-derived extracellular vesicles (EV) can serve as markers of cell damage/disease but can also have therapeutic utility depending on the nature of their cargo, such as miRNA. Currently, there are challenges and lack of innovations regarding early diagnosis and therapeutic options within different aspects of management of patients suffering from chronic pancreatitis (CP). Use of EV as biomarkers for pancreatic health and/or as adjuvant therapy would make a difference in management of these patients. The aim of this study was to characterize the miRNA cargo of EV purified from the plasma of CP patients and compared to those of healthy participants. EVs were isolated from plasma of 15 CP patients and 10 healthy controls. Nanoparticle tracking analysis was used to determine frequency and size, while NanoString technology was used to characterize the miRNA cargo. Relevant clinical parameters were correlated with EV miRNA cargo. ~ 30 miRNA species were identified to have significantly (p < 0.05) different expression in EV from individuals with CP compared to healthy individuals; ~ 40 miRNA were differentially expressed in EV from pre-diabetic versus non-diabetic CP patients. miR-579-3p, while exhibiting significantly lower (~ 16-fold) expression in CP compared to healthy and lower (~ 24-fold) in CP narcotic users compared to the non-users, is actually enriched (~ 32-fold) within EV in pre-diabetic CP patients compared to non-diabetic CP patients. A unique pattern was identified in female CP patients. These data support the prospect of using a plasma-derived EV cargo to assess pancreatic health and its therapeutic potential in CP patients.

Nitric Oxide-Releasing Hyaluronic Acid as an Antibacterial Agent for Wound Therapy.

Taking advantage of their respective wound-healing roles in physiology, the dual activity of hyaluronic acid (HA) and nitric oxide (NO) was combined to create a single-agent wound therapeutic. Carboxylic acid groups of HA (6 and 90 kDa) were chemically modified with a series of alkylamines via carbodiimide chemistry to provide secondary amines for subsequent N-diazeniumdiolate NO donor formation. The resulting NO-releasing HA derivatives stored 0.3-0.6 µmol NO mg-1 and displayed diverse release kinetics (5-75 min NO-release half-lives) under physiological conditions. The 6 kDa HA with terminal primary amines and intermediate release kinetics exhibited broad-spectrum bactericidal activity against common wound pathogens, including planktonic methicillin-resistant Staphylococcus aureus as well as planktonic and biofilm-based multidrug-resistant Pseudomonas aeruginosa. The treatment of infected murine wounds with NO-releasing HA facilitated more rapid wound closure and decreased the quantity of the P. aeruginosa genetic material in the remaining wound tissue. Hyaluronidase readily degraded the HA derivatives, indicating that NO donor modification did not prohibit endogenous biodegradation pathways.

Oral epithelial membrane-associated mucins and transcriptional changes with Sjögren's syndrome.

OBJECTIVES: To determine expression and localization of membrane-associated mucins within human keratinized and non-keratinized oral epithelia, and to explore transcriptional changes associated with primary Sjögren's syndrome. SUBJECTS AND METHODS: Mucin transcripts and glycoproteins were determined by RT-PCR and immunohistochemistry, respectively, in oral keratinized (hard palate) and non-keratinized (buccal) epithelia obtained from three cadavers. Mucin transcripts assessed by quantitative PCR were compared between cells harvested by brushing buccal and palatal epithelia of 25 female primary Sjögren's syndrome patients vs 25 healthy age-matched female control subjects. RESULTS: In hard palate, MUC4 is absent and MUC1 localized to deeper cell layers. Both mucins are within the apical layers of buccal epithelium. MUC15 is localized throughout all palatal cell layers and in all but the basal layer of buccal epithelia. MUC16, MUC20, and MUC21 glycoproteins are localized within all but the basal cell layer of both tissue types. In buccal cells of primary Sjögren's patients, MUC21 transcripts are down-regulated 3.4-fold and MUC20 2.6-fold. Dysregulation of select epithelial mucins may therefore contribute to xerostomia. CONCLUSIONS: Differential expression of multiple mucins and down-regulation in Sjögren's syndrome support further study of oral epithelial mucin physiology and pathophysiology, including their functions in hydration and lubrication of the oral mucosal pellicle.

Clastic cells in orthodontic treatment: Translational challenges and recent advances.

OBJECTIVES: Orthodontic treatment consists of numerous appliance activations that rely on stimulation of osteoclasts at alveolar bone sites. However, the action of osteoclast-like cells on dentin ("odontoclasts") is a pathological side effect of orthodontic treatment. The aim of this article is twofold: (a) To report preliminary results from ongoing cell culture experiments to identify unique markers of dentin resorption, and (b) To discuss our work using nanoparticle tracking analysis (NTA) and exosomes for developing biological fluid-based biopsies to monitor clastic cell activity. SETTING AND SAMPLE POPULATION: Twelve healthy volunteers in permanent dentition. MATERIAL AND METHODS: For the in vitro experiments, murine clastic cell precursors were cultured on dentin or bone slices for 7 days and phage-display biopanning was used to identify molecular surface differences between osteoclasts and odontoclasts. In the human study, gingival crevicular fluid (GCF) samples were collected using different tools and analysed for protein and exosome recovery. RESULTS: Biopanning generated antibody fragments that were uniquely reactive to odontoclasts. Numerous nanoparticles in the size range of exosomes were detected in all of the human GCF samples. CONCLUSIONS: Our results support that there are molecular differences between osteoclasts and odontoclasts. Emerging technologies may allow the use of exosomes in GCF as a clinical tool to detect markers of root resorption.

Local and Systemic Cytokine Profiling for Pancreatic Ductal Adenocarcinoma to Study Cancer Cachexia in an Era of Precision Medicine.

Cancer cachexia is a debilitating condition seen frequently in patients with pancreatic ductal adenocarcinoma (PDAC). The underlying mechanisms driving cancer cachexia are not fully understood but are related, at least in part, to the immune response to the tumor both locally and systemically. We hypothesize that there are unique differences in cytokine levels in the tumor microenvironment and systemic circulation between PDAC tumors and that these varying profiles affect the degree of cancer cachexia observed. Patient demographics, operative factors, oncologic factors, and perioperative data were collected for the two patients in the patient derived xenograft (PDX) model. Human pancreatic cancer PDX were created by implanting fresh surgical pancreatic cancer tissues directly into immunodeficient mice. At PDX end point, mouse tumor, spleen and muscle tissues were collected and weighed, muscle atrophy related gene expression measured, and tumor and splenic soluble proteins were analyzed. PDX models were created from surgically resected patients who presented with different degrees of cachexia. Tumor free body weight and triceps surae weight differed significantly between the PDX models and control (P < 0.05). Both PDX groups had increased atrophy related gene expression in muscle compared to control (FoxO1, Socs3, STAT3, Acvr2b, Atrogin-1, MuRF1; P < 0.05). Significant differences were noted in splenic soluble protein concentrations in 14 of 15 detected proteins in tumor bearing mice when compared to controls. Eight splenic soluble proteins were significantly different between PDX groups (P < 0.05). Tumor soluble proteins were significantly different between the two PDX groups in 15 of 24 detected proteins (P < 0.05). PDX models preserve the cachectic heterogeneity found in patients and are associated with unique cytokine profiles in both the spleen and tumor between different PDX. These data support the use of PDX as a strategy to study soluble cachexia protein markers and also further efforts to elucidate which cytokines are most related to cachexia in order to provide potential targets for immunotherapy.

Isolation of Pancreatic Cancer Cells from a Patient-Derived Xenograft Model Allows for Practical Expansion and Preserved Heterogeneity in Culture.

Commercially available, highly passaged pancreatic cancer (PC) cell lines are of limited translational value. Attempts to overcome this limitation have primarily consisted of cancer cell isolation and culture directly from human PC specimens. However, these techniques are associated with exceedingly low success rates. Here, we demonstrate a highly reproducible culture of primary PC cell lines (PPCLs) from patient-derived xenografts, which preserve, in part, the intratumoral heterogeneity known to exist in PC. PPCL expansion from patient-derived xenografts was successful in 100% of attempts (5 of 5). Phenotypic analysis was evaluated with flow cytometry, immunofluorescence microscopy, and short tandem repeat profiling. Importantly, tumorigenicity of PPCLs expanded from patient-derived xenografts was assessed by subcutaneous injection into nonobese diabeteic.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ mice. Morphologically, subcutaneous injection of all PPCLs into mice yielded tumors with similar characteristics to the parent xenograft. PPCLs uniformly expressed class I human leukocyte antigen, epithelial cell adhesion molecule, and cytokeratin-19. Heterogeneity within each PPCL persisted in culture for the frequency of cells expressing the cancer stem cell markers CD44, CD133, and c-Met and the immunologic markers human leukocyte antigen class II and programmed death ligand 1. This work therefore presents a reliable method for the rapid expansion of primary human PC cells and, thereby, provides a platform for translational investigation and, importantly, potential personalized therapeutic approaches.

TRIF-dependent innate immune activation is critical for survival to neonatal gram-negative sepsis.

Current evidence suggests that neonatal immunity is functionally distinct from adults. Although TLR signaling through the adaptor protein, MyD88, has been shown to be critical for survival to sepsis in adults, little is known about the role of MyD88 or TRIF in neonatal sepsis. We demonstrate that TRIF(-/-) but not MyD88(-/-) neonates are highly susceptible to Escherichia coli peritonitis and bacteremia. This was associated with decreased innate immune recruitment and function. Importantly, we found that the reverse was true in adults that MyD88(-/-) but not TRIF(-/-) or wild-type adults are susceptible to E. coli peritonitis and bacteremia. In addition, we demonstrate that TRIF but not MyD88 signaling is critical for the TLR4 protective adjuvant effect we have previously demonstrated. These data suggest a differential requirement for the survival of neonates versus adults to Gram-negative infection, and that modulation of TRIF in neonates can be used to augment survival to neonatal sepsis.

Patient-derived xenograft models for pancreatic adenocarcinoma demonstrate retention of tumor morphology through incorporation of murine stromal elements.

Direct implantation of viable surgical specimens provides a representative preclinical platform in pancreatic adenocarcinoma. Patient-derived xenografts consistently demonstrate retained tumor morphology and genetic stability. However, the evolution of the tumor microenvironment over time remains poorly characterized in these models. This work specifically addresses the recruitment and incorporation of murine stromal elements into expanding patient-derived pancreatic adenocarcinoma xenografts, establishing the integration of murine cells into networks of invading cancer cells. In addition, we provide methods and observations in the establishment and maintenance of a patient-derived pancreatic adenocarcinoma xenograft model. A total of 25 histologically confirmed pancreatic adenocarcinoma specimens were implanted subcutaneously into nonobese diabetic severe combined immunodeficiency mice. Patient demographics, staging, pathological analysis, and outcomes were analyzed. After successful engraftment of tumors, histological and immunofluorescence analyses were performed on explanted tumors. Pancreatic adenocarcinoma specimens were successfully engrafted in 15 (60%) of 25 attempts. Successful engraftment does not appear to correlate with clinicopathologic factors or patient survival. Tumor morphology is conserved through multiple passages, and tumors retain metastatic potential. Interestingly, despite morphological similarity between passages, human stromal elements do not appear to expand with invading cancer cells. Rather, desmoplastic murine stroma dominates the xenograft microenvironment after the initial implantation. Recruitment of stromal elements in this manner to support and maintain tumor growth represents a novel avenue for investigation into tumor-stromal interactions.

Aging, inflammation, immunity and periodontal disease.

The increased prevalence and severity of periodontal disease have long been associated with aging, such that this oral condition affects the majority of the adult population over 50 years of age. Although the immune system is a critical component for maintaining health, aging can be characterized by quantitative and qualitative modifications of the immune system. This process, termed 'immunosenescence', is a progressive modification of the immune system that leads to greater susceptibility to infections, neoplasia and autoimmunity, presumably reflecting the prolonged antigenic stimulation and/or stress responses that occur across the lifespan. Interestingly, the global reduction in the host capability to respond effectively to these challenges is coupled with a progressive increase in the general proinflammatory status, termed 'inflammaging'. Consistent with the definition of immunosenescence, it has been suggested that the cumulative effect of prolonged exposure of the periodontium to microbial challenge is, at least in part, a contributor to the effects of aging on these tissues. Thus, it has also been hypothesized that alterations in the function of resident immune and nonimmune cells of the periodontium contribute to the expression of inflammaging in periodontal disease. Although the majority of aging research has focused on the adaptive immune response, it is becoming increasingly clear that the innate immune compartment is also highly affected by aging. Thus, the phenomenon of immunosenescence and inflammaging, expressed as age-associated changes within the periodontium, needs to be more fully understood in this era of precision and personalized medicine and dentistry.

Localized aggressive periodontitis immune response to healthy and diseased subgingival plaque.

AIM: The objective of this case-control study was to compare the inflammatory response of peripheral blood from localized aggressive periodontitis (LAP) patients when stimulated with healthy or diseased plaque samples. MATERIALS AND METHODS: Whole blood and subgingival plaque samples were collected from 13 LAP subjects, 14 siblings of LAP subjects and six periodontally healthy individuals. Whole blood was stimulated for 24 h with plaque samples generated from healthy or diseased sites. The levels of 14 cyto/chemokines were detected using multiplex technology. RESULTS: Localized aggressive periodontitis-derived cultures displayed higher levels of G-CSF, INFγ, IL10, IL12p40, IL1ß, IL-6, IL-8, MCP-1, MIP-1α, and TNFα, than control cultures regardless of stimulus used. Whole blood from healthy siblings displayed higher levels of IL-6 compared to control subjects, but lower levels than those observed in cultures from LAP participants. CONCLUSIONS: This study suggests that although bacteria is an important factor in eliciting the hyper-inflammatory response observed in LAP patients, the predisposition of host's response to bacterial presence may play a more significant role than the components of the stimulatory plaque.

Protection of intestinal injury during heat stroke in mice by interleukin-6 pretreatment.

KEY POINTS: Heat stroke afflicts thousands of humans each year, worldwide. The immune system responds to hyperthermia exposure resulting in heat stroke by producing an array of immunological proteins, such as interleukin-6 (IL-6). However, the physiological functions of IL-6 and other cytokines in hyperthermia are poorly understood. We hypothesized that IL-6 plays a protective role in conditions of heat stroke. To test this, we gave small IL-6 supplements to mice prior to exposing them to hot environments sufficient to induce conditions of heat stroke. Pretreatment with IL-6 resulted in improved ability to withstand heat exposure in anaesthetized mice, it protected the intestine from injury, reducing the permeability of the intestinal barrier, and it attenuated the release of other cytokines involved in inflammation. The results support the hypothesis that IL-6 is a 'physiological stress hormone' that plays an important role in survival during acute life-threatening conditions such as heat stroke. ABSTRACT: The role of interleukin-6 (IL-6) in hyperthermia and heat stroke is poorly understood. Plasma IL-6 is elevated following hyperthermia in animals and humans, and IL-6 knockout mice are more intolerant of severe hyperthermia. We evaluated the effect of IL-6 supplementation on organ injury following severe hyperthermia exposure in anaesthetized mice. Two hours prior to hyperthermia, mice were treated with 0.6 µg intraperitoneal IL-6, or identical volumes of saline in controls. Mice were anaesthetized, gavaged with FITC-dextran for measures of gastrointestinal permeability, and exposed to incremental (0.5°C every 30 min) increases in temperature. Heating stopped when maximum core temperature (Tc) of 42.4°C was attained (Tc,max). The mice recovered at room temperature (≈22°C) for 30 or 120 min, at which time plasma and tissues were collected. IL-6-treated mice, on average, required ≈25 min longer to attain Tc,max . Injury and swelling of the villi in the duodenum was present in untreated mice after 30 min of recovery. These changes were blocked by IL-6 treatment. IL-6 also reduced gastrointestinal permeability, assayed by the accumulation of FITC-dextran in plasma. Plasma cytokines were also attenuated in IL-6-treated animals, including significant reductions in TNFα, MCP-1 (CXCL2), RANTES (CCL5) and KC (CCL5). The results demonstrate that IL-6 has a protective influence on the pattern of physiological responses to severe hyperthermia, suggesting that early endogenous expression of IL-6 may provide a protection from the development of organ damage and inflammation.

Primary outgrowth cultures are a reliable source of human pancreatic stellate cells.

Recent advances demonstrate a critical yet poorly understood role for the pancreatic stellate cell (PSC) in the pathogenesis of chronic pancreatitis (CP) and pancreatic cancer (PC). Progress in this area has been hampered by the availability, fidelity, and/or reliability of in vitro models of PSCs. We examined whether outgrowth cultures from human surgical specimens exhibited reproducible phenotypic and functional characteristics of PSCs. PSCs were cultured from surgical specimens of healthy pancreas, CP and PC. Growth dynamics, phenotypic characteristics, soluble mediator secretion profiles and co-culture with PC cells both in vitro and in vivo were assessed. Forty-seven primary cultures were established from 52 attempts, demonstrating universal α-smooth muscle actin and glial fibrillary acidic protein but negligible epithelial surface antigen expression. Modification of culture conditions consistently led to cytoplasmic lipid accumulation, suggesting induction of a quiescent phenotype. Secretion of growth factors, chemokines and cytokines did not significantly differ between donor pathologies, but did evolve over time in culture. Co-culture of PSCs with established PC cell lines resulted in significant changes in levels of multiple secreted mediators. Primary PSCs co-inoculated with PC cells in a xenograft model led to augmented tumor growth and metastasis. Therefore, regardless of donor pathology, outgrowth cultures produce PSCs that demonstrate consistent growth and protein secretion properties. Primary cultures from pancreatic surgical specimens, including malignancies, may represent a reliable source of human PSCs.

Downstream mediators of the intratumoral interferon response suppress antitumor immunity, induce gemcitabine resistance and associate with poor survival in human pancreatic cancer.

The cancer microenvironment allows tumor cells to evade immune surveillance through a variety of mechanisms. While interferon-γ (IFNγ) is central to effective antitumor immunity, its effects on the microenvironment are not as clear and have in some cancers been shown to induce immune checkpoint ligands. The heterogeneity of these responses to IFNγ remains poorly characterized in desmoplastic malignancies with minimal inflammatory cell infiltration, such as pancreatic cancer (PC). Thus, the IFNγ response within and on key cells of the PC microenvironment was evaluated. IFNγ induced expression of human leukocyte antigen (HLA) class I and II on PC cell lines, primary pancreatic cancer epithelial cells (PPCE) and patient-derived tumor-associated stroma, concomitant with an upregulation of PDL1 in the absence of CD80 and CD86 expression. As expected, IFNγ also induced high levels of CXCL10 from all cell types. In addition, significantly higher levels of CXCL10 were observed in PC specimens compared to those from chronic pancreatitis, whereby intratumoral CXCL10 concentration was an independent predictor of poor survival. Immunohistochemical analysis revealed a subset of CXCR3-positive cancer cells in over 90 % of PC specimens, as well as on a subset of cultured PC cell lines and PPCE, whereby exposure to CXCL10 induced resistance to the chemotherapeutic gemcitabine. These findings suggest that IFNγ has multiple effects on many cell types within the PC microenvironment that may lead to immune evasion, chemoresistance and shortened survival.

The inflammatory milieu within the pancreatic cancer microenvironment correlates with clinicopathologic parameters, chemoresistance and survival.

BACKGROUND: The tumor microenvironment impacts pancreatic cancer (PC) development, progression and metastasis. How intratumoral inflammatory mediators modulate this biology remains poorly understood. We hypothesized that the inflammatory milieu within the PC microenvironment would correlate with clinicopathologic findings and survival. METHODS: Pancreatic specimens from normal pancreas (n = 6), chronic pancreatitis (n = 9) and pancreatic adenocarcinoma (n = 36) were homogenized immediately upon resection. Homogenates were subjected to multiplex analysis of 41 inflammatory mediators. RESULTS: Twenty-three mediators were significantly elevated in adenocarcinoma specimens compared to nonmalignant controls. Increased intratumoral IL-8 concentrations associated with larger tumors (P = .045) and poor differentiation (P = .038); the administration of neoadjuvant chemotherapy associated with reduced IL-8 concentrations (P = .003). Neoadjuvant therapy was also associated with elevated concentrations of Flt-3 L (P = .005). Elevated levels of pro-inflammatory cytokines IL-1ß (P = .017) and TNFα (P = .033) were associated with a poor histopathologic response to neoadjuvant therapy. Elevated concentrations of G-CSF (P = .016) and PDGF-AA (P = .012) correlated with reduced overall survival. Conversely, elevated concentrations of FGF-2 (P = .038), TNFα (P = .031) and MIP-1α (P = .036) were associated with prolonged survival. CONCLUSION: The pancreatic cancer microenvironment harbors a unique inflammatory milieu with potential diagnostic and prognostic value.