The Neonatal Innate Immune Response to Sepsis: Checkpoint Proteins as Novel Mediators of This Response and as Possible Therapeutic/Diagnostic Levers.

Sepsis, a dysfunctional immune response to infection leading to life-threatening organ injury, represents a significant global health issue. Neonatal sepsis is disproportionately prevalent and has a cost burden of 2-3 times that of adult patients. Despite this, no widely accepted definition for neonatal sepsis or recommendations for management exist and those created for pediatric patients are significantly limited in their applicability to this unique population. This is in part due to neonates' reliance on an innate immune response (which is developmentally more prominent in the neonate than the immature adaptive immune response) carried out by dysfunctional immune cells, including neutrophils, antigen-presenting cells such as macrophages/monocytes, dendritic cells, etc., natural killer cells, and innate lymphoid regulatory cell sub-sets like iNKT cells, γδ T-cells, etc. Immune checkpoint inhibitors are a family of proteins with primarily suppressive/inhibitory effects on immune and tumor cells and allow for the maintenance of self-tolerance. During sepsis, these proteins are often upregulated and are thought to contribute to the long-term immunosuppression seen in adult patients. Several drugs targeting checkpoint inhibitors, including PD-1 and PD-L1, have been developed and approved for the treatment of various cancers, but no such therapeutics have been approved for the management of sepsis. In this review, we will comparatively discuss the role of several checkpoint inhibitor proteins, including PD-1, PD-L1, VISTA, and HVEM, in the immune response to sepsis in both adults and neonates, as well as posit how they may uniquely propagate their actions through the neonatal innate immune response. We will also consider the possibility of leveraging these proteins in the clinical setting as potential therapeutics/diagnostics that might aid in mitigating neonatal septic morbidity/mortality.

Survival and Pulmonary Injury After Neonatal Sepsis: PD1/PDL1's Contributions to Mouse and Human Immunopathology.

Morbidity and mortality associated with neonatal sepsis remains a healthcare crisis. PD1-/- neonatal mice endured experimental sepsis, in the form of cecal slurry (CS), and showed improved rates of survival compared to wildtype (WT) counterparts. End-organ injury, particularly of the lung, contributes to the devastation set forth by neonatal sepsis. PDL1-/- neonatal mice, in contrast to PD1-/- neonatal mice did not have a significant improvement in survival after CS. Because of this, we focused subsequent studies on the impact of PD1 gene deficiency on lung injury. Here, we observed that at 24 h post-CS (but not at 4 or 12 h) there was a marked increase in pulmonary edema (PE), neutrophil influx, myeloperoxidase (MPO) levels, and cytokine expression sham (Sh) WT mice. Regarding pulmonary endothelial cell (EC) adhesion molecule expression, we observed that Zona occludens-1 (ZO-1) within the cell shifted from a membranous location to a peri-nuclear location after CS in WT murine cultured ECs at 24hrs, but remained membranous among PD1-/- lungs. To expand the scope of this inquiry, we investigated human neonatal lung tissue. We observed that the lungs of human newborns exposed to intrauterine infection had significantly higher numbers of PD1+ cells compared to specimens who died from non-infectious causes. Together, these data suggest that PD1/PDL1, a pathway typically thought to govern adaptive immune processes in adult animals, can modulate the largely innate neonatal pulmonary immune response to experimental septic insult. The potential future significance of this area of study includes that PD1/PDL1 checkpoint proteins may be viable therapeutic targets in the septic neonate.

Improving the value of palliative surgery by optimizing patient selection: The role of long-term survival on high impact palliative intent operations.

BACKGROUND: In order to better characterize outcomes of palliative surgery (PS), we evaluated patients that experienced top quartile survival to elucidate predictors of high impact PS. METHODS: All PS performed on advanced cancer patients from 2003 to 2017 were identified from a PS database. RESULTS: 167 patients were identified. Multivariate analysis demonstrated the ability to rise from a chair was independently associated with top quartile survival (HR 7.61, 95% CI 2.12-48.82, p=0.008) as was the need for re-operation (HR 2.81, 95% CI 1.26-6.30, p=0.0012). Patients who were able to rise from a chair had significantly prolonged overall survival (320 vs 87 days, p < 0.001). CONCLUSIONS: Although not the primary goal, long-term survival can be achieved following PS and is associated with re-operation and the ability to rise from a chair. These patients experience the benefits of PS for a longer period of time, which in turn maximizes value and positive impact. SUMMARY: Long-term survival and symptom control can be achieved in highly selected advanced cancer patients following palliative surgery. The ability of the patient to independently rise from a chair and the provider to offer a re-operation when indicated are associated with long-term survival following a palliative operation.

Malignant Bowel Obstruction in the Time of the COVID-19 Pandemic.

Regardless of the anatomic site of malignant bowel obstruction leading to the need for palliative intervention, decisions must consider the natural history of the disease, the availability and success of nonsurgical treatments, the individual patient's symptom severity, goals, preferences, quality, and expectancy of life. Therapy for symptoms must remain flexible and individualized because the specific needs of the patient will change as disease progresses. Because strangulation is uncommon, malignant bowel obstruction is usually not a surgical emergency. There is usually time to proceed with deliberate and thoughtful decisions on how best to meet the needs and expectations of the individual patient and family. Providers must be well versed in both surgical and nonsurgical therapeutic options, the natural history of disease, and be active and compassionate providers to foster meaningful ongoing dialogue focused on excellent care even after cure is no longer possible. The palliative triangle not only allows patient, family, and surgeon to effectively utilize the full continuum of care that can be delivered, but also it supports end-of-life decisions when continuity in care matters most. Due to social distancing requirements, the dynamics of communication between patient, family, and surgeon have changed. Zoom, Skype, and FaceTime have become tools in our communication armamentarium.

Blockade of endothelial, but not epithelial, cell expression of PD-L1 following severe shock attenuates the development of indirect acute lung injury in mice.

This study sets out to establish the comparative contribution of PD-L1 expression by pulmonary endothelial cells (ECs) and/or epithelial cells (EpiCs) to the development of indirect acute lung injury (iALI) by taking advantage of the observation that treatment with naked siRNA by intratracheal delivery in mice primarily affects lung EpiCs, but not lung ECs, while intravenous delivery of liposomal-encapsulated siRNA largely targets vascular ECs including the lung, but not pulmonary EpiCs. We showed that using a mouse model of iALI [induced by hemorrhagic shock followed by septic challenge (Hem-CLP)], PD-L1 expression on pulmonary ECs or EpiCs was significantly upregulated in the iALI mice at 24 h post-septic insult. After documenting the selective ability of intratracheal versus intravenous delivery of PD-L1 siRNA to inhibit PD-L1 expression on EpiCs versus ECs, respectively, we observed that the iALI-induced elevation of cytokine/chemokine levels (in the bronchoalveolar lavage fluid, lung lysates, or plasma), lung myeloperoxidase and caspase-3 activities could largely only be inhibited by intravenous, but not intratracheal, delivery of PD-L1 siRNA. Moreover, intravenous, but not intratracheal, delivery led to a preservation of normal tissue architecture, lessened pulmonary edema, and reduced neutrophils influx induced by iALI. In addition, in vitro mouse endothelial cell line studies showed that PD-L1 gene knockdown by siRNA or knockout by CRISPR/Cas9-mediated gene manipulation, reduced monolayer permeability, and maintained tight junction protein levels upon recombinant IFN-γ stimulation. Together, these data imply a critical role for pulmonary vascular ECs in mediating PD-1:PD-L1-driven pathological changes resulting from systemic stimuli such as Hem-CLP.

A novel role for programmed cell death receptor ligand 2 in sepsis-induced hepatic dysfunction.

The liver is an organ that, when dysfunctional in a septic patient, is strongly associated with morbidity and mortality. Understanding the pathophysiology of liver failure during sepsis may lead to improved diagnostics and potential therapeutic targets. Historically, programmed cell death receptor (PD) ligand 1 (PD-L1) has been considered the primary ligand for its checkpoint molecule counterpart, PD-1, with PD-L2 rarely in the immunopathological spotlight. PD-1 and PD-L1 contribute to liver dysfunction in a murine cecal ligation and puncture (CLP) model of sepsis, but virtually nothing is known about PD-L2's role in sepsis. Therefore, our central hypothesis was that sepsis-induced changes in hepatic PD-L2 expression contributed to worsened liver function and, subsequently, more pronounced morbidity and mortality. We found that although PD-L1 gene deficiency attenuated the hepatic dysfunction seen in wild-type mice after CLP, the loss of PD-L2 appeared to actually worsen indices of liver function along with a trend toward higher liver tissue vascular permeability. Conversely, some protective effects of PD-L2 gene deletion were noted, such as reduced liver/peritoneal bacterial load and reduced IL-6, IL-10, and macrophage inflammatory protein 2 levels following CLP. These diverse actions, as well as the unique expression pattern of PD-L2, may explain why no overt survival advantage could be witnessed in the septic PD-L2-/- mice. Taken together, these data suggest that although PD-L2 has some selective effects on the hepatic response seen in the septic mouse, these factors are not sufficient to alter septic mortality in this adult murine model. NEW & NOTEWORTHY Our study shows not only that ligands of the checkpoint protein PD-1 respond inversely to a stressor such as septic challenge (PD-L2 declines, whereas PD-L1 rises) but also that aspects of liver dysfunction increase in septic mice lacking the PD-L2 gene. Furthermore, these differences in PD-L2 gene-deficient animals culminated in the abrogation of the survival advantage seen in the septic PD-L1-knockout mice, suggesting that PD-L2 may have roles beyond a simple immune tolerogen.

Herpes Virus Entry Mediator (HVEM) Expression Promotes Inflammation/ Organ Injury in Response to Experimental Indirect-Acute Lung Injury.

Therapeutic interventions to treat acute lung injury (ALI) remain largely limited to lung-protective strategies, as a real molecular pathophysiologically driven therapeutic intervention has yet to become available. While we have previously documented the expression of herpes virus entry mediator (HVEM) on leukocytes of septic mice and critically ill patients, its functional role in shock/sepsis-induced ALI has not yet been studied. Inasmuch, a murine model of indirect ALI (iALI) was induced by hemorrhagic shock (HEM) followed by cecal ligation and puncture (CLP), septic challenge and HVEM-siRNA or phosphate buffered saline was administrated by intratracheal instillation 2 h after hemorrhage to determine the role of HVEM in the development of experimental iALI. Indices of lung injury were measured. HVEM expression was significantly elevated in iALI mice. Compared with phosphate buffered saline treated iALI mice, HVEM knock-down by siRNA caused a reduction of cytokine/chemokine levels, myeloperoxidase activity, broncho-alveolar lavage fluid (BALF) cell count and protein concentration. HVEM-siRNA treatment reduced inflammation and attenuated pulmonary architecture destruction as well as provided an early (60 h post HEM-CLP) survival benefit in iALI mice. This ability of anti-HVEM treatment to prevent the development of iALI and provide a transient survival benefit implies that mitigating signaling through HVEM may be a novel target worth further investigation.

Group 2 Innate Lymphoid Cells (ILC2s) Are Key Mediators of the Inflammatory Response in Polymicrobial Sepsis.

Sepsis remains a major public health concern, characterized by marked immune dysfunction. Innate lymphoid cells develop from a common lymphoid precursor but have a role in orchestrating inflammation during innate response to infection. Here, we investigate the pathologic contribution of the group 2 innate lymphoid cells (ILC2s) in a murine model of acute septic shock (cecal ligation and puncture). Flow cytometric data revealed that ILC2s increase in number and percentage in the small intestine and in the peritoneal cells and inversely decline in the liver at 24 hours after septic insult. Sepsis also resulted in changes in ILC2 effector cytokine (IL-13) and activating cytokine (IL-33) in the plasma of mice and human patients in septic shock. Of interest, the sepsis-induced changes in cytokines were abrogated in mice deficient in functionally invariant natural killer T cells. Mice deficient in IL-13-producing cells, including ILC2s, had a survival advantage after sepsis along with decreased morphologic evidence of tissue injury and reduced IL-10 levels in the peritoneal fluid. Administration of a suppressor of tumorigenicity 2 (IL-33R) receptor-blocking antibody led to a transient survival advantage. Taken together, these findings suggest that ILC2s may play an unappreciated role in mediating the inflammatory response in both mice and humans; further, modulating ILC2 response in vivo may allow development of immunomodulatory strategies directed against sepsis.

A novel role for coinhibitory receptors/checkpoint proteins in the immunopathology of sepsis.

Coinhibitory molecules, such as PD-1, CTLA-4, 2B4, and BTLA, are an important new family of mediators in the pathophysiology of severe bacterial and/or fungal infection, as well as the combined insults of shock and sepsis. Further, the expression of these molecules may serve as indicators of the immune status of the septic individual. Using PD-1:PD-L as an example, we discuss in this review how such checkpoint molecules may affect the host response to infection by mediating the balance between effective immune defense and immune-mediated tissue injury. Additionally, we explore how the up-regulation of PD-1 and/or PD-L1 expression on not only adaptive immune cells (e.g., T cells), but also on innate immune cells (e.g., macrophages, monocytes, and neutrophils), as well as nonimmune cells during sepsis and/or shock contributes to functional alterations often with detrimental sequelae.

PAD4 Deficiency Leads to Decreased Organ Dysfunction and Improved Survival in a Dual Insult Model of Hemorrhagic Shock and Sepsis.

Indirect acute respiratory distress syndrome (iARDS) is caused by a nonpulmonary inflammatory process resulting from insults such as nonpulmonary sepsis. Neutrophils are thought to play a significant role in mediating ARDS, with the development of iARDS being characterized by dysregulation and recruitment of activated neutrophils into the lung. Recently, a novel mechanism of microbial killing by neutrophils was identified through the formation of neutrophil extracellular traps (NETs). NETs are composed of large webs of decondensed chromatin released from activated neutrophils into the extracellular space; they are regulated by the enzyme peptidylarginine deiminase 4 (PAD4) through mediation of chromatin decondensation via citrullination of target histones. Components of NETs have been implicated in ARDS. However, it is unknown whether there is any pathological significance of NET formation in ARDS caused indirectly by nonpulmonary insult. We subjected PAD4-/- mice and wild-type mice to a "two-hit" model of hypovolemic shock (fixed-pressure hemorrhage [Hem]) followed by septic cecal ligation and puncture (CLP) insult (Hem/CLP). Mice were hemorrhaged and resuscitated; 24 h after Hem, mice were then subjected to CLP. Overall, PAD4 deletion led to an improved survival as compared with wild-type mice. PAD4-/- mice displayed a marked decrease in neutrophil influx into the lung, as well decreased presence of proinflammatory mediators. PAD4-/- mice were also able to maintain baseline kidney function after Hem/CLP. These data taken together suggest PAD4-mediated NET formation contributes to the mortality associated with shock/sepsis and may play a role in the pathobiology of end organ injury in response to combined hemorrhage plus sepsis.

Novel Role for PD-1:PD-L1 as Mediator of Pulmonary Vascular Endothelial Cell Functions in Pathogenesis of Indirect ARDS in Mice.

Deficiency of the co-inhibitory receptor, Programmed cell death receptor (PD)-1, provides a survival benefit in our murine shock/sepsis model for the development of indirect acute respiratory distress syndrome (iARDS). Further, of clinical significance, patients that develop ARDS express increased PD-1 on their blood leukocytes. While PD-1 expression and its regulatory role have been associated with mainly T-cell responses, the contribution of its primary ligand, PD-L1, broadly expressed on non-immune cells such as lung endothelial cells (ECs) as well as immune cells, is less well-understood. Here we show that a "priming insult" for iARDS, such as non-lethal hemorrhagic shock alone, produced a marked increase in lung EC PD-L1 as well as blood leukocyte PD-1 expression, and when combined with a subsequent "trigger event" (polymicrobial sepsis), not only induced marked iARDS but significant mortality. These sequelae were both attenuated in the absence of PD-L1. Interestingly, we found that gene deficiency of both PD-1 and PD-L1 improved EC barrier function, as measured by decreased bronchoalveolar lavage fluid protein (i.e., lung leak). However, PD-L1 deficiency, unlike PD-1, significantly decreased EC activation through the Angiopoietin/Tie2 pathway in our iARDS mice. Additionally, while PD-1 gene deficiency was associated with decreased neutrophil influx in our iARDS mice, EC monolayers derived from PD-L1 deficient mice showed increased expression of EC junction proteins in response to ex vivo TNF-α stimulation. Together, these data suggest that ligation of PD-1:PD-L1 may play a novel role(s) in the maintenance of pulmonary EC barrier regulation, beyond that of the classic regulation of the leukocyte tolerogenic immune response, which may account for its pathogenic actions in iARDS.

Program Cell Death Receptor-1-Mediated Invariant Natural Killer T-Cell Control of Peritoneal Macrophage Modulates Survival in Neonatal Sepsis.

We have shown that invariant natural killer T (iNKT) cells mediate sepsis-induced end-organ changes and immune responses, including macrophage bacterial phagocytosis, a finding regulated by the check point protein program cell death receptor-1 (PD-1). Furthermore, PD-1 mediates mortality in both adult and neonatal murine sepsis as well as in surgical patients. Given our previous findings, we hypothesize that iNKT cells will also modulate neonatal sepsis survival, and that this effect is regulated in part through PD-1. We utilized a polymicrobial intra-peritoneal cecal slurry (CS) sepsis model in wild type (WT), iNKT-/- or PD-1-/- 5-7 day old neonatal pups. Typically, tissues were harvested at 24 h for various bioassays/histology and, in some cases, survival was assessed for up to 7 days. Interestingly, similar to what we recently reported for PD-1-/- mice following CS, iNKT-/--deficient animals exhibit a markedly improved survival vs. WT. Histologically, minor alterations in liver architectural, which were noted in WT pups, were attenuated in both iNKT-/- and PD-1-/- pups. Following CS, PECAM-1 expression was unchanged in the WT pups but increased in both iNKT-/- and PD-1-/- pups. In WT, following CS the emergence of a Ly6Clow subpopulation was noted among the influxed peritoneal macrophage population. Conversely, within iNKT-/- pups, there were fewer peritoneal macrophages and a greater percentage of Ly6Chigh macrophages. We show not only a key role for iNKT cells in affecting end-organ damage as well as alterations in phagocytes phenotypes in neonatal sepsis but that this iNKT cell mediated effect is driven by the central checkpoint protein PD-1.

Improved survival after induction of sepsis by cecal slurry in PD-1 knockout murine neonates.

BACKGROUND: Sepsis and the ensuing immune dysfunction continue to be major contributors to neonatal morbidity and mortality. Neonatal sepsis also is associated with profound immune dysfunction. We have recently identified a role for a family of coinhibitory molecules that are altered in murine sepsis and in critically ill adult patients, which may be a target for development of novel therapies. There is, however, a paucity of data pertaining to the role of coinhibitory checkpoint proteins in the control and modulation of neonatal sepsis. METHODS: The cecal slurry model consists of harvesting the cecal content of an adult, wild-type, male mouse and combining it with 5% dextrose to create a cecal slurry with a concentration of 80 mg/mL (LD70 at 7 days). Neonatal mice (5-7 days of age) underwent intraperitoneal injection of the cecal slurry or 0.9% saline for the sham procedure. Wild-type (C57BL/6) or PD-1-/- mice were used; a 7-day survival study was undertaken. Cytometric bead array was used for cytokine expression. Blood and peritoneal fluid was cultured for bacterial burden. Flow cytometry was used to assess the peritoneal cavity cell populations. RESULTS: There was no mortality after the sham procedure in either wild-type or PD-1-/- pups. PD-1 markedly affected sepsis survival with significantly improved survival in the PD-1-/- pups (40% vs 80%; P < .01). This survival improvement was not associated with any difference in bacterial clearance. The bacterial burden was equivalent between wild-type and PD-1-/- pups at 24 hours after cecal slurry. However, PD-1-/- pups did display an increased circulating cytokine response to the cecal slurry compared with wild type, with increased expression of IL-6, IL-10, and TNF-α levels. Within the peritoneal cavity, sepsis induced an influx of neutrophils, a finding that was increased in PD-1-/- pups. Although the T-cell response was unaffected by PD-1, it was noted that cecal slurry induced a loss of peritoneal B cells in WT, while the peritoneal B-cell population was preserved in PD-1-/- pups. CONCLUSION: Our data suggest that the checkpoint protein, PD-1, plays an important role in controlling the immune response to sepsis in the neonate, ultimately affecting sepsis-related mortality in this neonatal murine model of sepsis. Akin to adult studies, these data further emphasize the potential therapeutic target for PD-1 across a spectrum of septic patients.

Radiation-associated sarcoma after recurrent colorectal primary tumor: A complex surgical case.

Radiation associated sarcoma is a significant consequence of cancer therapy. Incidence of radiation associated sarcoma correlates with overall radiotherapy exposure. Prognosis is generally poor with 5 year survival rates lower than that for spontaneously occurring sarcomas. Surgical management presents many challenges including having to work in irradiated tissue planes while trying to achieve negative margins. We present a patient with a rare radiation associated pelvic sarcoma whose course illustrates the complexity of this problem.

Interactive Instrument-Driven Image Display in Laparoscopic Surgery.

BACKGROUND: A significant limitation of minimally invasive surgery is dependence of the entire surgical team on a single endoscopic viewpoint. An individualized, instrument-driven image display system that allows all operators to simultaneously define their viewing frame of the surgical field may be the solution. We tested the efficacy of such a system using a modified Fundamentals of Laparoscopic Surgery™ (Society of American Gastrointestinal and Endoscopic Surgeons, Los Angeles, CA) bead transfer task. MATERIALS AND METHODS: A program was custom-written to allow zooming and centering of the image window on specific color signals, each attached near the tip of a different laparoscopic instrument. Two controls were used for the bead transfer task: (1) a static, wide-angle view and (2) a single moving camera allowing close-up and tracking of the bead as it was transferred. Time to task completion and number of bead drops were recorded. RESULTS: Thirty-six sessions were performed by surgical residents. Average time for bead transfer was 127.3±21.3 seconds in the Experimental group, 139.1±27.8 seconds in the Control 1 group, and 186.2±18.5 seconds in the Control 2 group (P=.034, by analysis of variance). Paired analysis (the Wilcoxon Signed-Rank Test) showed that the Experimental group was significantly faster than the Control 1 group (P=.035) and the Control 2 group (P=.028). CONCLUSIONS: We have developed an image navigation system that allows intuitive and efficient laparoscopic performance compared with two controls. It offers high-resolution images and ability for multitasking. The tracking system centers close-up images on the laparoscopic target. Further development of robust prototypes will help transition this in vitro system into clinical application.