Sensitizing tumors to anti-PD-1 therapy by promoting NK and CD8+ T cells via pharmacological activation of FOXO3.

BACKGROUND: Stimulating antitumor immunity by blocking programmed death-1 (PD-1) or its ligand (programmed death-ligand 1 (PD-L1) is a promising antitumor therapy. However, numerous patients respond poorly to PD-1/PD-L1 blockade. Unresponsiveness to immune-checkpoint blockade (ICB) can cast significant challenges to the therapeutic options for patients with hard-to-treat tumors. There is an unmet clinical need to establish new therapeutic approaches for mitigating ICB unresponsiveness in patients. In this study, we investigated the efficacy and role of low-dose antineoplastic agent SN-38 or metformin in sensitizing unresponsive tumors to respond to ICB therapy. METHODS: We assessed the significant pathological relationships between PD-L1 and FOXO3 expression and between PD-L1 and c-Myc or STAT3 expression in patients with various tumors. We determined the efficacy of low-dose SN-38 or metformin in sensitizing unresponsive tumors to respond to anti-PD-1 therapy in a syngeneic tumor system. We deciphered novel therapeutic mechanisms underlying the SN-38 and anti-PD-1 therapy-mediated engagement of natural killer (NK) or CD8+ T cells to infiltrate tumors and boost antitumor immunity. RESULTS: We showed that PD-L1 protein level was inversely associated with FOXO3 protein level in patients with ovarian, breast, and hepatocellular tumors. Low-dose SN-38 or metformin abrogated PD-L1 protein expression, promoted FOXO3 protein level, and significantly increased the animal survival rate in syngeneic mouse tumor models. SN-38 or metformin sensitized unresponsive tumors responding to anti-PD-1 therapy by engaging NK or CD8+ T cells to infiltrate the tumor microenvironment (TME) and secret interferon-γ and granzyme B to kill tumors. SN-38 suppressed the levels of c-Myc and STAT3 proteins, which controlled PD-L1 expression. FOXO3 was essential for SN38-mediated PD-L1 suppression. The expression of PD-L1 was compellingly linked to that of c-Myc or STAT3 in patients with the indicated tumors. CONCLUSION: We show that SN-38 or metformin can boost antitumor immunity in the TME by inhibiting c-Myc and STAT3 through FOXO3 activation. These results may provide novel insight into ameliorating patient response to overarching immunotherapy for tumors.

Minimally invasive video-assisted double-valve replacement through right anterolateral Minithoracotomy.

OBJECTIVE: This study aimed to investigate feasibility and safety of minimally invasive video-assisted surgery for double-valve (mitral and aortic) replacement through right anterolateral minithoracotomy. METHODS: Between February 2011 and April 2013, 60 patients with combined valvular disease underwent double valve replacement, 26 of them by minimally invasive video-assisted surgery through right anterolateral minithoracotomy (study group) and 34 by median sternotomy (control group). Peripheral cardiopulmonary bypass (CPB) was established through right femoral artery and vein. The incision was made around the right breast approximately 5 cm in length. Pericardiotomy, bicaval occlusion, atriotomy and aortotomy, and double valve replacement were performed with thoracoscope. RESULTS: In the study group, times of CPB and aortic cross-clamp were 146.5 ± 40.5 min and 91.5 ± 23.4 min, respectively, which were significantly different from those in the control group, 115.4 ± 26.5 min and 75.4 ± 16.5 min (P<0.05). Thoracic drainage in the study group was significantly lower than the control group, 587 ± 245 ml (study group) versus 756 ± 267 ml (control group) (P<0.05). Length of ICU and postoperative hospital stay were shorter in the study group, 1.9 ± 0.8 and 8.7 ± 4.5 days versus 2.8 ± 1.3 and 11.2 ± 5.6 days in the control group (P<0.05), respectively. There was no statistical difference in the postoperative results of TTE (transthoracic echocardiography) (P>0.05). All patients recovered smoothly with follow-up of six months to two years, with no severe complications. CONCLUSIONS: Minimally invasive video-assisted procedure through right anterolateral minithoracotomy is a new promising approach for double valve replacement. Our study suggested that this approach was feasible, safe and had cosmetic effects.

Totally thoracoscopic surgery for the treatment of atrial septal defect without of the robotic Da Vinci surgical system.

BACKGROUND: More and more surgeons and patients focus on the minimally invasive surgical techniques in the 21st century. Totally thoracoscopic operation provides another minimal invasive surgical option for patients with ASD (atrial septal defect). In this study, we reported our experience of 61 patients with atrial septal defect who underwent totally thoracoscopic operation and discussed the feasibility and safety of the new technique. METHODS: From January 2010 to October 2012, 61 patients with atrial septal defect underwent totally thoracoscopic closure but not traditional median sternotomy surgery. We divided the 61 patients into two groups based on the operation sequence. The data of group A (the first 30 cases) and group B (the last 31 cases). The mean age of the patients was 35.1 ± 12.8 years (range, 6.3 to 63.5 years), and mean weight was 52.7 ± 11.9 kg (range, 30.5 to 80 kg). Mean size of the atrial septal defect was 16.8 ± 11.3 mm (range, 13 to 39 mm) based on the description of the echocardiography. RESULTS: All patients underwent totally thoracoscopy successfully, 36 patients with pericardium patch and 25 patients were sutured directly. 7 patients underwent concomitant tricuspid valvuloplasty with Key technique. No death, reoperation or complete atrioventricular block occurred. The mean time of cardiopulmonary bypass was 68.5 ± 19.1 min (range, 31.0 to 153.0 min), the mean time of aortic cross-clamp was 27.2 ± 11.3 min (range, 0.0 to 80.0 min) and the mean time of operation was 149.8 ± 35.7 min (range, 63.0 to 300.0 min). Postoperative mechanical ventilation averaged 4.9 ± 2.5 hours (range, 3.5 to 12.6 hours), and the duration of intensive care unit stay 20.0 ± 4.8 hours (range, 15.5 to 25 hours). The mean volume of blood drainage was 158 ± 38 ml (range, 51 to 800 ml). No death, residual shunt, lung atelectasis or moderate tricuspid regurgitation was found at 3-month follow-up. CONCLUSION: The totally thoracoscopic operation is feasible and safe for patients with ASD, even with or without tricuspid regurgitation. This technique provides another minimal invasive surgical option for patients with atrial septal defect.

Totally thoracoscopic surgical treatment for atrial septal defect: mid-term follow-up results in 45 consecutive patients.

BACKGROUND: Totally thoracoscopic operation provides minimally invasive alternative for patients with atrial septal defect. In this study, we report the mid-term follow-up results of 45 patients with atrial septal defect who underwent totally thoracoscopic operation and discuss the feasibility and safety of this new technique. METHODS: From January 2010 to February 2012, 45 patients with atrial septal defect underwent totally thoracoscopic closure as an alternative to traditional median sternotomy surgery. The mean age of the patients was 33.2±12.5 years (range 6.3-61.5 years), and mean weight was 55.7±11.1 kg (range 30.5-80 kg). Based on echocardiography the mean size of the atrial septal defect was 16.0±10.8mm (range 13-39 mm). RESULTS: All patients underwent totally thoracoscopic repair. Twenty-five patients with a pericardial patch and 20 patients were sutured directly. Five patients underwent concomitant tricuspid valvuloplasty with Kay technique. No death, reoperation or complete atrioventricular block occurred. The mean time of cardiopulmonary bypass was 70.5±20.6 min (range 31.0-153.0 min), the mean time of aortic cross-clamp was 28.8±13.3 min (range 0.0-80.0 min) and the mean time of operation was 155.8±36.8 min (range 65.0-300.0 min). Postoperative mechanical ventilation averaged 5.1±2.8h (range 3.6-12.6h), and the duration of intensive care unit stay 20.0±5.6h (range 16.2-25 h). The mean volume of blood drainage was 156±36 ml (range 51-800 ml). No death, residual shunt, lung atelectasis or moderate tricuspid regurgitation was found at three-month follow-up. CONCLUSION: Totally thoracoscopic repair is feasible and safe for patients with ASD, even with or without tricuspid regurgitation however more clinical data is needed in the future study.

Preprotachykinin-A gene disruption attenuates nociceptive sensitivity after opioid administration and incision by peripheral and spinal mechanisms in mice.

UNLABELLED: The preprotachykinin A gene (ppt-A) codes for Substance P (SP), supports nociceptive sensitization, and modulates inflammatory responses after incision. Repeated opioid use produces paradoxical pain sensitization-termed opioid-induced hyperalgesia (OIH) -which can exacerbate pain after incision. Here the contribution of SP to peri-incisional nociceptive sensitization and nociceptive mediator production after opioid treatment was examined utilizing ppt-A knockout (-/-) mice and the neurokinin (NK1) receptor antagonist LY303870. Less mechanical allodynia was observed in ppt-A(-/-) mice compared to wild types (wt) after morphine treatment both before and after incision. Moreover, LY303870 administered with morphine reduced incisional hyperalgesia in wt mice. Incision after saline or escalating morphine treatment upregulated skin IL-1ß, IL-6, G-CSF and MIP-1α levels in ppt-A(-/-) and wt mice similarly. However, chronic morphine treatment greatly exacerbated increases in skin nerve growth factor levels after incision, an effect entirely dependent upon intact SP signaling. Additionally, SP dependent upregulation of prodynorphin, NMDA1 and NK1 receptor expression in spinal cord was seen after morphine treatment and incision. A similar pattern was seen for 5-HT3 receptor expression in tissue from dorsal root ganglia. Therefore, SP may work at both central and peripheral sites to enhance nociceptive sensitization after morphine treatment and incision. PERSPECTIVE: These studies show that SP signaling modulates enhanced nerve growth factor production and changes in neuronal gene expression seen after incision in mice previously exposed to morphine.

The role of interleukin-1 in wound biology. Part I: Murine in silico and in vitro experimental analysis.

BACKGROUND: Wound healing is a multistep, complex process that involves the coordinated action of multiple cell types. Conflicting results have been obtained when conventional methods have been used to study wound biology. Therefore, we analyzed the wound response in a mouse genetic model. METHODS: We analyzed inflammatory mediators produced within incisional wounds induced in 16 inbred mouse strains. Computational haplotype-based genetic analysis of inter-strain differences in the level of production of 2 chemokines in wounds was performed. An in vitro experimental analysis system was developed to investigate whether interleukin (IL)-1 could affect chemokine production by 2 different types of cells that are present within wounds. RESULTS: The level of 2 chemokines, keratinocyte-derived chemokine (KC) and macrophage inflammatory protein 1α, exhibited very large (75- and 463-fold, respectively) interstrain differences within wound tissue across this inbred strain panel. Genetic variation within Nalp1, an inflammasome component that regulates IL-1 production, correlated with the interstrain differences in KC and macrophage inhibitory protein 1α production. Consistent with the genetic correlation, IL-1ß was shown to stimulate KC production by murine keratinocyte and fibroblast cell lines in vitro. CONCLUSIONS: Genetic variation within Nalp1 could contribute to interstrain differences in wound chemokine production by altering the amount of IL-1 produced.

The role of interleukin-1 in wound biology. Part II: In vivo and human translational studies.

BACKGROUND: In the accompanying paper, we demonstrate that genetic variation within Nalp1 could contribute to interstrain differences in wound chemokine production through altering the amount of interleukin (IL)-1 produced. We further investigate the role of IL-1 in incisional wound biology and its effect on wound chemokine production in vivo and whether this mechanism could be active in human subjects. METHODS: A well-characterized murine model of incisional wounding was used to assess the in vivo role of IL-1 in wound biology. The amount of 7 different cytokines/chemokines produced within an experimentally induced skin incision on a mouse paw and the nociceptive response was analyzed in mice treated with an IL-1 inhibitor. We also investigated whether human IL-1ß or IL-1α stimulated the production of chemokines by primary human keratinocytes in vitro, and whether there was a correlation between IL-1ß and chemokine levels in 2 experimental human wound paradigms. RESULTS: Administration of an IL-1 receptor antagonist to mice decreased the nociceptive response to an incisional wound, and reduced the production of multiple inflammatory mediators, including keratinocyte-derived chemokine (KC) and macrophage inhibitory protein (MIP)-1α, within the wounds. IL-1α and IL-1ß stimulated IL-8 and GRO-α (human homologues of murine keratinocyte-derived chemokine) production by primary human keratinocytes in vitro. IL-1ß levels were highly correlated with IL-8 in human surgical wounds, and at cutaneous sites of human ultraviolet B-induced sunburn injury. CONCLUSIONS: IL-1 plays a major role in regulating inflammatory mediator production in wounds through a novel mechanism; by stimulating the production of multiple cytokines and chemokines, it impacts clinically important aspects of wound biology. These data suggest that administration of an IL-1 receptor antagonist within the perioperative period could decrease postsurgical wound pain.

Caspase-1 modulates incisional sensitization and inflammation.

BACKGROUND: Surgical injury induces production and release of inflammatory mediators in the vicinity of the wound. They in turn trigger nociceptive signaling to produce hyperalgesia and pain. Interleukin-1ß plays a crucial role in this process. The mechanism regulating production of this cytokine after incision is, however, unknown. Caspase-1 is a key enzyme that cleaves prointerleukin-1ß to its active form. We hypothesized that caspase-1 is a crucial regulator of incisional interleukin-1ß levels, nociceptive sensitization, and inflammation. METHODS: These studies employed a mouse hind paw incisional model. Caspase-1 was blocked using the selective inhibitors Ac-YVAD-CMK and VRTXSD727. Nociceptive sensitization, edema, and hind paw warmth were followed in intact animals whereas caspase-1 activity, cytokine, and prostaglandin E2 levels were assessed in homogenized skin. Confocal microscopy was used to detect the expression of caspase-1 near the wounds. RESULTS: Analysis of enzyme activity demonstrated that caspase-1 activity was significantly increased in periincisional skin. Pretreatment with Ac-YVAD-CMK significantly reduced mechanical allodynia and thermal hyperalgesia. Repeated administration of this inhibitor produced robust analgesia, especially to mechanical stimulation. Administration of VRTXSD727 provided qualitatively similar results. Caspase-1 inhibition also reduced edema and the normally observed increase in paw warmth around the wound site. Correspondingly, caspase-1 inhibition significantly reduced interleukin-1ß as well as macrophage-inflammatory protein 1α, granulocyte colony-stimulating factor, and prostaglandin E2 levels near the wound. The expression of caspase-1 was primarily observed in keratinocytes in the epidermal layer and in neutrophils deeper in the wounds. CONCLUSIONS: The current study demonstrates that the inhibition of caspase-1 reduces postsurgical sensitization and inflammation, likely through a caspase-1/interleukin-1ß-dependent mechanism.

Chronic morphine administration enhances nociceptive sensitivity and local cytokine production after incision.

BACKGROUND: The chronic use of opioids prior to surgery leads to lowered pain thresholds and exaggerated pain levels after these procedures. Several mechanisms have been proposed to explain this heightened sensitivity commonly termed opioid-induced hyperalgesia (OIH). Most of these proposed mechanisms involve plastic events in the central or peripheral nervous systems. Alterations in the abundance of peripheral mediators of nociception have not previously been explored. RESULTS: In these experiments mice were treated with saline (control) or ascending daily doses of morphine to generate a state of OIH followed by hind paw incision. In other experiments morphine treatment was initiated at the time of incision. Both mechanical allodynia and peri-incisional skin cytokine levels were measured. Myeloperoxidase (MPO) assays were used to determine neutrophil activity near the wounds. The cytokine production inhibitor pentoxifylline was used to determine the functional significance of the excess cytokines in previously morphine treated animals. Mice treated chronically treated with morphine prior to incision were found to have enhanced skin levels of IL-1beta, IL-6, G-CSF, KC and TNFalpha after incision at one or more time points compared to saline pretreated controls. The time courses of individual cytokines followed different patterns. There was no discernable effect of chronic morphine treatment on wound area neutrophil infiltration. Pentoxifylline reduced cytokine levels and reversed the excess mechanical sensitization caused by chronic morphine administration prior to incision. Morphine treatment initiated at the time of incision did not lead to a generalized enhancement of cytokine production or nociceptive sensitization in excess of the levels observed after incision alone. CONCLUSION: The enhanced level of nociceptive sensitization seen after incision in animals chronically exposed to morphine is associated with elevated levels of several cytokines previously reported to be relevant to this incisional pain model. The cytokines may be functional in supporting nociceptive sensitization because pentoxifylline reverses both peri-incisional skin cytokine levels and OIH. Opioid administration beginning at the time of incision does not seem to have the same cytokine enhancing effect. Approaches to postoperative pain control involving a reduction of cytokines may be an effective way to control excessive pain in patients chronically using opioids prior to surgical procedures.

Morphine reduces local cytokine expression and neutrophil infiltration after incision.

BACKGROUND: Inflammation and nociceptive sensitization are hallmarks of tissue surrounding surgical incisions. Recent studies demonstrate that several cytokines may participate in the enhancement of nociception near these wounds. Since opioids like morphine interact with neutrophils and other immunocytes, it is possible that morphine exerts some of its antinociceptive action after surgical incision by altering the vigor of the inflammatory response. On the other hand, keratinocytes also express opioid receptors and have the capacity to produce cytokines after injury. Our studies were directed towards determining if opioids alter cytokine production near incisions and to identify cell populations responsible for producing these cytokines. RESULTS: A murine incisional model was used to measure the effects of acute morphine administration (0.1-10 mg/kg) on nociceptive thresholds, neutrophil infiltration and cytokine production in hind paw skin 30 minutes and 2 hours after incision. Incised hind paws displayed profound allodynia which was reduced by morphine (0.1-10 mg/kg) in the 2 hours following incision. Skin samples harvested from these mice showed enhanced levels of 5 cytokines: IL-1 beta, IL-6, tumor necrosis factor alpha (TNFalpha), granulocyte colony stimulating factor (G-CSF) and keratinocyte-derived cytokine (KC). Morphine reduced these incision-stimulated levels. Separate analyses measuring myeloperoxidase (MPO) and using immunohistochemistry demonstrated that morphine dose-dependently reduced the infiltration of neutrophils into the peri-incisional tissue. The dose of morphine required for reduction of cytokine accumulation, however, was below that required for inhibition of peri-incisional neutrophil infiltration. Additional immunohistochemical studies revealed wound edge keratinocytes as being an important source of cytokines in the acute phase after incision. CONCLUSION: Acute morphine administration of doses as low as 0.1 mg/kg reduces peri-incisional cytokine expression. A reduction in neutrophil infiltration does not provide a complete explanation for this effect, and keratinocytes may be responsible for some incision area cytokine production. These studies suggest that morphine may alter the inflammatory milieu of incisional wounds, but these alterations do not likely contribute significantly to analgesia in the acute setting.

Microglia potentiate damage to blood-brain barrier constituents: improvement by minocycline in vivo and in vitro.

BACKGROUND: Blood-brain barrier (BBB) disruption after stroke can worsen ischemic injury by increasing edema and causing hemorrhage. We determined the effect of microglia on the BBB and its primary constituents, endothelial cells (ECs) and astrocytes, after ischemia using in vivo and in vitro models. METHODS AND RESULTS: Primary astrocytes, ECs, or cocultures were prepared with or without added microglia. Primary ECs were more resistant to oxygen-glucose deprivation/reperfusion than astrocytes. ECs plus astrocytes showed intermediate vulnerability. Microglia added to cocultures nearly doubled cell death. This increase was prevented by minocycline and apocynin. In vivo, minocycline reduced infarct volume and neurological deficits and markedly reduced BBB disruption and hemorrhage in mice after experimental stroke. CONCLUSIONS: Inhibition of microglial activation may protect the brain after ischemic stroke by improving BBB viability and integrity. Microglial inhibitors may prove to be an important treatment adjunct to fibrinolysis.

Glycogen synthase kinase 3beta inhibitor Chir025 reduces neuronal death resulting from oxygen-glucose deprivation, glutamate excitotoxicity, and cerebral ischemia.

The serine/threonine kinase, glycogen synthase kinase 3beta (GSK3beta), is abundant in CNS and is neuron specific. GSK3beta plays a pivotal role in the regulation of numerous cellular functions. GSK3beta phosphorylates and thereby regulates many metabolic, signaling, and structural proteins which can influence cell survival. Increased GSK3beta correlates with increased cell death, whereas reduced GSK3beta expression correlates with increased cell survival. We report that the GSK3beta inhibitor Chir025 is neuroprotective in vitro and in vivo. First, Chir025 reduced cultured hippocampal neuron death following glutamate exposure by 15-20% versus vehicle-treated controls. Second, Chir025 significantly reduced cultured cortical neuron death following oxygen-glucose deprivation (OGD) by approximately 50%. Third, Chir025 reduced infarct size following focal cerebral ischemia by nearly 20%. There were no significant differences in the number of TUNEL-positive neurons or in caspase-3 and -9 activities between Chir025- and vehicle-treated rats, although Chir025 elevated cytosolic Bcl-2 expression. These data show that Chir025-mediated inhibition of GSK3beta is neuroprotective and that the mechanism is probably not anti-apoptotic.

Overexpression of HDJ-2 protects astrocytes from ischemia-like injury and reduces redistribution of ubiquitin staining in vitro.

HDJ-2, a member of the HSP40 family, functions as a cochaperone to promote protein folding both by binding to unfolded polypeptides and by regulating the activity of HSP70. This study tested whether HDJ-2 overexpression alone could provide significant protection from ischemia-like injury. Primary mouse astrocyte cultures were infected with an HDJ-2 encoding retroviral vector or control retrovirus lacking HDJ-2. Expression of HDJ-2 was confirmed by immunohistochemical staining and immunoblotting. Injury paradigms to mimic ischemia were glucose deprivation (GD) for 24 hours and oxygen-glucose deprivation (OGD) for 8 hours. Cell death was determined by trypan blue exclusion and cell counting. Overexpression of HDJ-2 alone significantly reduced astrocyte injury after both GD and OGD, independent of an elevation in HSP70. To further search for the mechanism of HDJ-2 protection, cultured astrocytes allowed to recover 16 hours after 8 hours GD were labeled with a monoclonal antiubiquitin antibody that recognizes both free ubiquitin and ubiquitinated proteins. The immunolabeling pattern changed from a relatively even distribution in both nuclei and cytoplasm in control cells to heterogeneous aggregates and marked reduction of nuclear staining in most of the cells after GD. When HDJ-2 was overexpressed, the number of cells with evidence of protein aggregation was significantly reduced. Thus, blocking protein aggregation may be an important mechanism by which HDJ-2 protects cells from damage.