Pressure-Enabled Drug Delivery (PEDD) of a class C TLR9 agonist in combination with checkpoint inhibitor therapy in a murine pancreatic cancer model.

BACKGROUND: Systemic immunotherapy has had limited clinical benefit in pancreatic ductal adenocarcinoma. This is thought to be due to its desmoplastic immunosuppressive tumor microenvironment in addition to high intratumoral pressures that limit drug delivery. Recent preclinical cancer models and early-phase clinical trials have demonstrated the potential of toll-like receptor 9 agonists, including the synthetic CpG oligonucleotide SD-101, to stimulate a wide range of immune cells and eliminate suppressive myeloid cells. We hypothesized that Pressure-Enabled Drug Delivery via Pancreatic Retrograde Venous Infusion of toll-like receptor 9 agonist would improve responsiveness to systemic anti-programmed death receptor-1 checkpoint inhibitor therapy in a murine orthotopic pancreatic ductal adenocarcinoma model. METHODS: Murine pancreatic ductal adenocarcinoma (KPC4580P) tumors were implanted into the pancreatic tails of C57BL/6J mice and treated 8 days after implantation. Mice were assigned to one of the following treatment groups: Pancreatic Retrograde Venous Infusion delivery of saline, Pancreatic Retrograde Venous Infusion delivery of toll-like receptor 9 agonist, systemic anti-programmed death receptor-1, systemic toll-like receptor 9 agonist, or the combination of Pancreatic Retrograde Venous Infusion delivery of toll-like receptor 9 agonist and systemic anti-programmed death receptor-1 (Combo). Fluorescently labeled toll-like receptor 9 agonist (radiant efficiency) was used to measure uptake of the drug on day 1. Changes in tumor burden were evaluated by necropsy at 2 different time points, 7 and 10 days after toll-like receptor 9 agonist treatment. Blood and tumors were collected at necropsy 10 days after toll-like receptor 9 agonist treatment for flow cytometric analysis of tumor-infiltrating leukocytes and plasma cytokines. RESULTS: All mice analyzed survived to necropsy. Site of tumor fluorescence measurements revealed 3-fold higher intensity fluorescence in Pancreatic Retrograde Venous Infusion delivery of toll-like receptor 9 agonist compared to systemic toll-like receptor 9 agonist mice. Tumor weights were significantly lower in the Combo group compared to Pancreatic Retrograde Venous Infusion delivery of saline. Flow cytometry of the Combo group demonstrated significantly increased overall T-cell number, specifically CD4+ T-cells, and a trend toward increased CD8+ T-cells. Cytokine analysis showed significantly decreased IL-6 and CXCL1. CONCLUSION: Pressure-Enabled Drug Delivery of toll-like receptor 9 agonist by Pancreatic Retrograde Venous Infusion with systemic anti-programmed death receptor-1 demonstrated improved pancreatic ductal adenocarcinoma tumor control in a murine pancreatic ductal adenocarcinoma model. These results support study of this combination therapy in pancreatic ductal adenocarcinoma patients and expansion of ongoing Pressure-Enabled Drug Delivery clinical trials.

Cellular microRNAs correlate with clinical parameters in multiple injury patients.

INTRODUCTION: The pathophysiology of the inflammatory response after major trauma is complex, and the magnitude correlates with severity of tissue injury and outcomes. Study of infection-mediated immune pathways has demonstrated that cellular microRNAs may modulate the inflammatory response. The authors hypothesize that the expression of microRNAs would correlate to complicated recoveries in polytrauma patients (PtPs). METHODS: Polytrauma patients enrolled in the prospective observational Tissue and Data Acquisition Protocol with Injury Severity Score of >15 were selected for this study. Polytrauma patients were divided into complicated recoveries and uncomplicated recovery groups. Polytrauma patients' blood samples were obtained at the time of admission (T0). Established biomarkers of systemic inflammation, including cytokines and chemokines, were measured using multiplexed Luminex-based methods, and novel microRNAs were measured in plasma samples using multiplex RNA hybridization. RESULTS: Polytrauma patients (n = 180) had high Injury Severity Score (26 [20-34]) and complicated recovery rate of 33%. MicroRNAs were lower in PtPs at T0 compared with healthy controls, and bivariate analysis demonstrated that variations of microRNAs correlated with age, race, comorbidities, venous thromboembolism, pulmonary complications, complicated recovery, and mortality. Positive correlations were noted between microRNAs and interleukin 10, vascular endothelial growth factor, Acute Physiology and Chronic Health Evaluation, and Sequential Organ Failure Assessment scores. Multivariable Lasso regression analysis of predictors of complicated recovery based on microRNAs, cytokines, and chemokines revealed that miR-21-3p and monocyte chemoattractant protein-1 were predictive of complicated recovery with an area under the curve of 0.78. CONCLUSION: Systemic microRNAs were associated with poor outcomes in PtPs, and results are consistent with previously described trends in critically ill patients. These early biomarkers of inflammation might provide predictive utility in early complicated recovery diagnosis and prognosis. Because of their potential to regulate immune responses, microRNAs may provide therapeutic targets for immunomodulation. LEVEL OF EVIDENCE: Diagnostic Tests/Criteria; Level II.

Tumor or Inflammatory Myofibroblastic Reaction in an Adolescent With an Abdominal Lymphatic Malformation?

We report the case of a 17-year-old male who presented with intractable nausea and vomiting. Cross-sectional imaging revealed a large retrogastric abdominal mass. Fine needle aspiration done via endoscopic ultrasound (EUS) was nondiagnostic. Exploratory laparotomy revealed a large inflammatory mass densely adherent to the stomach and retroperitoneum. Incisional biopsy frozen section revealed spindle cells, and subsequent resection of the mass with en-bloc subtotal gastrectomy with Roux-en-y gastrojejunostomy reconstruction was performed. Final pathology demonstrated a lymphatic malformation with reactive myofibroblastic proliferation. Inflammatory abdominal lymphatic malformations are especially rare and not well described in the literature. These masses may present diagnostic challenges until the specimen is sent for pathologic analysis.

Preparing Austere Maritime Surgical Teams for Deployment During the COVID-19 Global Pandemic: Is It Time to Change the Training Pipeline?

INTRODUCTION: Navy Medicine's Role 2 Light Maneuver (R2LM) Emergency Resuscitative Surgical Systems (ERSS) are austere surgical teams manned, trained, and equipped to provide life-saving damage control resuscitation and surgery in any environment on land or sea. Given the restrictions related to the COVID-19 pandemic, the previously established pre-deployment training pipeline for was modified to prepare a new R2LM team augmenting a Role 1 shipboard medical department. METHODS: The modified curriculum created in response to COVID-19 related restriction is compared and contrasted to the established pre-deployment R2LM ERSS curriculum. Subject Matter Experts and currently deployed R2LM members critically evaluate the two curricula. RESULTS: Both curricula included the team R2LM platform training and exposure to cadaver based team trauma skills training. The modified curriculum included didactics on shipboard resuscitation, anesthesia and surgery, shipboard COVID-19 management, and prolonged field care in austere maritime environments. CONCLUSIONS: We describe Navy Medicines R2LM ERSS capability and compare and contrast the standard R2LM pre-COVID-19 curriculum to the modified curriculum. Central to both curricula, the standard R2LM platform training is important for developing and honing team dynamics, communication skills and fluid leadership; important for the successful function austere surgical teams. Several opportunities for improvement in the pre-deployment training were identified for R2LM teams augmenting shipboard Role 1 medical departments.

Pressure-enabled delivery of gemcitabine in an orthotopic pancreatic cancer mouse model.

BACKGROUND: We describe the use of pancreatic retrograde venous infusion in an orthotopic murine model of pancreatic ductal adenocarcinoma and hypothesize that pancreatic retrograde venous infusion delivery of gemcitabine will increase concentrations of gemcitabine in the tumor and the subsequent tumor response to treatment. METHODS: Murine pancreatic ductal adenocarcinoma (KPC4580P) was transplanted onto the pancreatic tail of C57BL/6J mice. Groups (n = 15) of mice were assigned to sham laparotomy and 100 mg/kg intraperitoneal infusion of gemcitabine (systemic gemcitabine), pancreatic venous isolation with pancreatic retrograde venous infusion of 100 mg/kg gemcitabine, or pancreatic retrograde venous infusion with saline infusion. Tumor pressures were recorded during pancreatic retrograde venous infusion. Mice were killed at 1 hour or 7 days after infusion. RESULTS: Baseline tumor pressures were 45 ± 8 mm Hg, and pancreatic retrograde venous infusion increased tumor pressures by 29 ± 6 mm Hg (P < .01). Pancreatic retrograde venous infusion gemcitabine mice had greater tumor gemcitabine concentrations compared with systemic gemcitabine (127 vs 19 ng/mg; P < .01) and lesser tumor volumes compared with both systemic gem and pancreatic retrograde venous infusion with saline (274 vs 857 vs 629 mm3; P < .01). CONCLUSION: Pancreatic retrograde venous infusion increased tumor pressures greater than baseline, improved gemcitabine delivery, and increased the treatment response. These findings suggest that pressurized, regional delivery overcomes the increased pressure barrier in pancreatic ductal adenocarcinoma. Additional preclinical studies with cytotoxic and immunotherapeutic agents and clinical trials using pressure-enabled drug delivery with pancreatic retrograde venous infusion devices are underway.

The impact of septic stimuli on the systemic inflammatory response and physiologic insult in a preclinical non-human primate model of polytraumatic injury.

BACKGROUND: Established animal trauma models are limited in recapitulating the pathophysiology of human traumatic injury. Herein, we characterize the physiologic insult and inflammatory response in two clinically relevant non-human primate (NHP) trauma models. METHODS: Mauritian Cynomolgus Macaques underwent either a laparoscopic closed abdomen liver injury (laparoscopic 60% left-lobe hepatectomy) in an established uncontrolled severe hemorrhage model (THM), or a polytrauma hemorrhage model (PHM) involving combined liver and bowel injury, uncontrolled severe hemorrhage as well as an open full-thickness cutaneous flank wound. Fixed volume resuscitation strategies were employed in the THM and goal directed resuscitation was used in the PHM. Complete peripheral blood and critical clinical chemistry parameters, serum biomarkers of systemic inflammation, tissue perfusion parameters, as well as survival, were compared between the models throughout the 2-week study period. RESULTS: NHPs in both the THM (n = 7) and the PHM (n = 21) demonstrated tissue hypoperfusion (peak lactate 6.3 ± 0.71 mmol/L) with end organ injury (peak creatinine 3.08 ± 0.69 mg/dL) from a similar liver injury (60% left hemi-hepatectomy), though the PHM NHPs had a significantly higher blood loss (68.1% ± 12.7% vs. 34.3% ± 2.3%, p = 0.02), lower platelet counts (59 ± 25 vs. 205 ± 46 K/uL, p = 0.03) and a trend towards higher mortality (90.5% vs. 33.3%, p = 0.09). The inflammatory response was robust in both models with peak cytokine (IL-6 > 6000-fold above baseline) and peak leukocyte values (WBC 27 K/uL) typically occurring around t = 240 min from the time of hepatic injury. A more robust systemic inflammatory response was appreciated in the PHM resulting in marked elevations in peak serum IL-6 (7887 ± 2521 pg/mL vs.1076 ± 4833 pg/mL, p = 0.02), IL-1ra (34,499 ± 5987 pg/mL vs. 2511 ± 1228 pg/mL, p < 0.00), and IL-10 (13,411 pg/mL ± 5598 pg/mL vs. 617 pg/mL ± 252 pg/mL, p = 0.03). CONCLUSION: This comparative analysis provides a unique longitudinal perspective on the post-injury inflammatory response in two clinically relevant models, and demonstrates that the addition of septic stimuli to solid organ injury increases both the hemorrhagic insult and inflammatory response.

Host responses to concurrent combined injuries in non-human primates.

BACKGROUND: Multi-organ failure (MOF) following trauma remains a significant cause of morbidity and mortality related to a poorly understood abnormal inflammatory response. We characterized the inflammatory response in a non-human primate soft tissue injury and closed abdomen hemorrhage and sepsis model developed to assess realistic injury patterns and induce MOF. METHODS: Adult male Mauritan Cynomolgus Macaques underwent laparoscopy to create a cecal perforation and non-anatomic liver resection along with a full-thickness flank soft tissue injury. Treatment consisted of a pre-hospital phase followed by a hospital phase after 120 minutes. Blood counts, chemistries, and cytokines/chemokines were measured throughout the study. Lung tissue inflammation/apoptosis was confirmed by mRNA quantitative real-time PCR (qPCR), H&E, myeloperoxidase (MPO) and TUNEL staining was performed comparing age-matched uninjured controls to experimental animals. RESULTS: Twenty-one animals underwent the protocol. Mean percent hepatectomy was 64.4 ± 5.6; percent blood loss was 69.0 ± 12.1. Clinical evidence of end-organ damage was reflected by a significant elevation in creatinine (1.1 ± 0.03 vs. 1.9 ± 0.4, p=0.026). Significant increases in systemic levels of IL-10, IL-1ra, IL-6, G-CSF, and MCP-1 occurred (11-2986-fold) by 240 minutes. Excessive pulmonary inflammation was evidenced by alveolar edema, congestion, and wall thickening (H&E staining). Concordantly, amplified accumulation of MPO leukocytes and significant pulmonary inflammation and pneumocyte apoptosis (TUNEL) was confirmed using qRT-PCR. CONCLUSION: We created a clinically relevant large animal multi-trauma model using laparoscopy that resulted in a significant systemic inflammatory response and MOF. With this model, we anticipate studying systemic inflammation and testing innovative therapeutic options.

An Evolving Uncontrolled Hemorrhage Model Using Cynomolgus Macaques.

BACKGROUND: Trauma-induced hemorrhagic shock produces hemodynamic changes that often result in a systemic inflammatory response that can lead to multiple organ failure and death. In this prospective study, the pathophysiology of a nonhuman primate uncontrolled hemorrhagic shock model is evaluated with the goal of creating an acute systemic inflammatory syndrome response and a reproducible hemorrhage. METHODS: Nonhuman primates were divided into 2 groups. A laparoscopic left hepatectomy was performed in groups A and B, 60% and 80%, respectively, resulting in uncontrolled hemorrhage. Resuscitation during the prehospital phase lasted 120 min and included a 0.9% saline bolus at 20 mL/kg. The hospital phase involved active warming, laparotomy, hepatorrhaphy for hemostasis, and transfusion of packed red blood cells (10 mL/kg). The animals were recovered and observed over a 14-day survival period with subsequent necropsy for histopathology. RESULTS: Baseline demographics and clinical parameters of the two groups were similar. Group A (n = 7) underwent a 57.7% ± 2.4% left hepatectomy with a 33.9% ± 4.0% blood loss and 57% survival. Group B (n = 4) underwent an 80.0% ± 6.0% left hepatectomy with 56.0% ± 3.2% blood loss and 75% survival. Group B had significantly lower hematocrit (P < 0.05) for all postinjury time points. Group A had significantly elevated creatinine on postoperative day 1. Nonsurvivors succumbed to an early death, averaging 36 h from the injury. Histopathologic evaluation of nonsurvivors demonstrated kidney tubular degeneration. CONCLUSIONS: Nonhuman primates displayed the expected physiologic response to hemorrhagic shock due to liver trauma as well as systemic inflammatory response syndrome with resultant multiple organ dysfunction syndrome and either early death or subsequent recovery. Our next step is to establish a clinically applicable nonhuman primate polytrauma model, which reproduces the prolonged maladaptive immunologic reactivity and end-organ dysfunction consistent with multiple organ failure found in the critically injured patient.

Modeling acute traumatic injury.

Acute traumatic injury is a complex disease that has remained a leading cause of death, which affects all ages in our society. Direct mechanical insult to tissues may result in physiological and immunologic disturbances brought about by blood loss, coagulopathy, as well as ischemia and reperfusion insults. This inappropriate response leads to an abnormal release of endogenous mediators of inflammation that synergistically contribute to the incidence of morbidity and mortality. This aberrant activation and suppression of the immune system follows a bimodal pattern, wherein activation of the innate immune responses is followed by an anti-inflammatory response with suppression of the adaptive immunity, which can subsequently lead secondary insults and multiple organ dysfunction. Traumatic injury rodent and swine models have been used to describe many of the underlying pathologic mechanisms, which have led to an improved understanding of the morbidity and mortality associated with critically ill trauma patients. The enigmatic immunopathology of the human immunologic response after severe trauma, however, has never more been apparent and there grows a need for a clinically relevant animal model, which mimics this immune physiology to enhance the care of the most severely injured. This has necessitated preclinical studies in a more closely related model system, the nonhuman primate. In this review article, we summarize animal models of trauma that have provided insight into the clinical response and understanding of cellular mechanisms involved in the onset and progression of ischemia-reperfusion injury as well as describe future treatment options using immunomodulation-based strategies.

Voice outcomes after total thyroidectomy, partial thyroidectomy, or non-neck surgery using a prospective multifactorial assessment.

BACKGROUND: Voice alteration remains a significant complication of thyroid surgery. We present a comparison of voice outcomes between total thyroidectomy (TT), partial thyroidectomy (PT), and non-neck (NN) surgery using a multifactorial voice-outcomes classification tool. STUDY DESIGN: Patients with normal voice (n = 112) were enrolled between July 2004 and March 2009. The patients underwent TT (n = 54), PT (n = 35), or NN (n = 23) surgery under general endotracheal anesthesia as part of a prospective observational study involving serial multimodality voice evaluation preoperatively, and at 2 weeks, 3 months, and 6 months postoperatively. Patients with adverse voice outcomes were grouped into the negative voice outcomes (NegVO) category, including patients with objective (abnormality on videolaryngostroboscopy and substantial voice dysfunction) and subjective (normal videolaryngostroboscopy but with notable voice impairment) NegVO. Voice outcomes were compared among study groups. RESULTS: Negative voice outcomes occurred in 46% (95% CI, 34-59%) and 14% (95% CI, 6-30%) of TT and PT groups, respectively. No NegVOs were observed after NN surgery. Early NegVOs were more common in the TT group than in the NN or PT groups (p < 0.001). Most voice disturbances resolved by 6 months (TT 84%; PT 92%) with no difference in NegVO among all groups (p = 0.23). Black race and significant changes in certain voice outcomes measures at the 2-week follow-up visit were identified as predictors of late (3 to 6 months) NegVO. CONCLUSIONS: This comprehensive voice outcomes study revealed that the extent of thyroidectomy impacts voice outcomes in the early postoperative period, and identified risk factors for late NegVO in post-thyroidectomy patients who should be considered for early voice rehabilitation referral.

Axillary sentinel lymph node biopsy after mastectomy: a case report.

BACKGROUND: Sentinel lymph node biopsy has been established as the preferred method for staging early breast cancer. A prior history of mastectomy is felt to be a contraindication. CASE PRESENTATION: A patient with recurrent breast cancer in her skin flap was discovered to have positive axillary sentinel nodes by sentinel lymph node biopsy five years after mastectomy for ductal carcinoma in situ. CONCLUSION: A prior history of mastectomy may not be an absolute contraindication to sentinel lymph node biopsy.

Toward efficient photomodulation of conjugated polymer emission: optimizing differential energy transfer in azobenzene-substituted PPV derivatives.

We present fluorescence studies of quenching behavior in photoaddressable azobenzene-substituted derivatives of the fluorescent conjugated polymer poly(p-phenylenevinylene) (PPV). The azobenzene side chains partially quench the PPV fluorescence, and we have shown previously that the quenching efficiency is greater when the azobenzene side chains are cis than when they are trans. This effect provides a photoaddressable means of modulating the fluorescence intensity of PPV derivatives. To optimize the efficiency of photoinduced intensity modulation, it is important to understand the molecular nature of quenching by both trans- and cis-azobenzene side chains. Here we investigate the photophysical origins of quenching by the two isomers using steady-state and time-resolved fluorescence spectroscopy. We present results from the azobenzene-modified PPV derivative poly(2-methoxy-5-((10-(4-(phenylazo)phenoxy)decyl)oxy)-1,4-phenylenevinylene) (MPA-10-PPV) and two new related polymers, a copolymer lacking half of the azobenzene side chains and an analogue of MPA-10-PPV with a tert-butyl-substituted azobenzene. These studies reveal that steric interactions influence the extent of PPV emission quenching by trans-azobenzene but do not affect the efficient quenching by cis-azobenzene. The difference in dynamic quenching efficiencies between trans- and cis-azobenzene isomers is consistent with fluorescence resonance energy transfer. These results show that it is possible to control the efficiency of photoswitchable fluorescence modulation through specific structural variations designed to encourage or block quenching by trans-azobenzene. This is a promising approach to providing useful general guidelines for designing photomodulated PPV derivatives.

Phototriggered fluorescence color changes in azobenzene-functionalized conjugated polymers.

We report fluorescence studies of phototriggered changes in spectral position and shape for two azobenzene-functionalized poly(p-phenylenevinylene) derivatives, poly(2-methoxy-5-(4-phenylazophenyl-4'-(1,10-dioxydecyl))-1,4-phenylenevinylene) (MPA-10-PPV) and poly(2-hexyloxy-5-(4-phenylazophenyl-4'-(1,10-dioxydecyl))-1,4-phenylenevinylene) (HPA-10-PPV). Upon trans --> cis azobenzene photoisomerization, small (ca. 1 nm) blue shifts in spectral position are observed for MPA-10-PPV in 100% toluene, a good solvent for this polymer. These shifts are reversed upon visible irradiation and can be cycled many times. To probe the dependence of these shifts on initial polymer conformation, a dichloromethane-methanol cosolvent study was performed in which the solvent quality was decreased incrementally to induce a reduction in polymer coil dimensions. Unirradiated dichloromethane solutions of both MPA-10-PPV and HPA-10-PPV showed a red shift and reduction in quantum yield with increasing methanol concentration as expected based on literature results for other poly(p-phenylenevinylene) derivatives. These changes have been attributed to a dramatic conformational collapse by others and occur for these azo polymers over the 30-60% (v/v) methanol range. While little or no light-induced spectral shifting was observed at low (or=70%) methanol concentrations, significant spectral shifts were observed for both polymers upon azobenzene photoisomerization in solutions with 30-60% methanol, the same range over which the polymer undergoes collapse to a highly coiled state. The largest shifts are visible to the eye, with a 65:35 (v/v) dichloromethane-methanol solution of HPA-10-PPV showing yellow-orange fluorescence when the azobenzenes are trans, green fluorescence when they are cis, and yellow-orange again after the azobenzenes are returned to the trans state. We attribute these color changes to a reversible UV-phototriggered expansion of polymer coil dimensions that occurs as a result of trans --> cis azobenzene side chain isomerization and provide temperature data to support this conclusion.