Pseudomonas aeruginosa ExoT induces G1 cell cycle arrest in melanoma cells.

Recently, we demonstrated that Pseudomonas aeruginosa Exotoxin T (ExoT) employs two distinct mechanisms to induce potent apoptotic cytotoxicity in a variety of cancer cell lines. We further demonstrated that it can significantly reduce tumour growth in an animal model for melanoma. During these studies, we observed that melanoma cells that were transfected with ExoT failed to undergo mitosis, regardless of whether they eventually succumbed to ExoT-induced apoptosis or survived in ExoTs presence. In this report, we sought to investigate ExoTs antiproliferative activity in melanoma. We delivered ExoT into B16 melanoma cells by bacteria (to show necessity) and by transfection (to show sufficiency). Our data indicate that ExoT exerts a potent antiproliferative function in melanoma cells. We show that ExoT causes cell cycle arrest in G1 interphase in melanoma cells by dampening the G1/S checkpoint proteins. Our data demonstrate that both domains of ExoT; (the ADP-ribosyltransferase (ADPRT) domain and the GTPase activating protein (GAP) domain); contribute to ExoT-induced G1 cell cycle arrest in melanoma. Finally, we show that the ADPRT-induced G1 cell cycle arrest in melanoma cells likely involves the Crk adaptor protein. Our data reveal a novel virulence function for ExoT and further highlight the therapeutic potential of ExoT against cancer

CrkII/Abl phosphorylation cascade is critical for NLRC4 inflammasome activity and is blocked by Pseudomonas aeruginosa ExoT.

Type 3 Secretion System (T3SS) is a highly conserved virulence structure that plays an essential role in the pathogenesis of many Gram-negative pathogenic bacteria, including Pseudomonas aeruginosa. Exotoxin T (ExoT) is the only T3SS effector protein that is expressed in all T3SS-expressing P. aeruginosa strains. Here we show that T3SS recognition leads to a rapid phosphorylation cascade involving Abl / PKCδ / NLRC4, which results in NLRC4 inflammasome activation, culminating in inflammatory responses that limit P. aeruginosa infection in wounds. We further show that ExoT functions as the main anti-inflammatory agent for P. aeruginosa in that it blocks the phosphorylation cascade through Abl / PKCδ / NLRC4 by targeting CrkII, which we further demonstrate to be important for Abl transactivation and NLRC4 inflammasome activation in response to T3SS and P. aeruginosa infection

Interleukin-2 fusion protein: an investigational therapy for interleukin-2 receptor expressing malignancies

DAB3s91L-2 is an interleukin-2 receptor (IL-2R) specific fusion protein with a molecular weight of 58 kD containing the enzymatic and translocation domains of diphtheria toxin (DT) and human IL-2. This fusion protein is able to direct the cytocidal action of the DT enzymatic region only to cells which bear the IL-2R. The human IL-2R exists in three forms: low, intermediate and high affinity. The high-affinity form is believed to be the biologically relevant form on mature, activated T-lymphocytes, B-lymphocytes and monocytes. DAB3sgIL-2 is able to bind selectively to the high-affinity IL-2R in a concentration-dependent manner, and once bound is internalised via receptor-mediated endocytosis. Upon acidification of the formed vesicle, the enzymatic portion of the fusion protein is believed to pass into the cytosol where it ultimately inhibits protein synthesis by inactivation of elongation factor-2, resulting in cell death. The constitutive expression of the IL-2R on certain leukaemic and lymphomatous cells of T and B cell origin has been reported to occur in patients with chronic lymphocytic ieukaemia, cutaneous T cell lymphoma (CTCL), Hodgkin\u27s disease and non-Hodgkin\u27s lymphomas (NHLs). A multicentre DAB3s91L-2 dose-escalation study of patients with IL-2R expressing lymphomas has been conducted. A 10-fold range of doses were evaluated on a five-daily dose schedule. Patients received up to six courses, with an additional two courses permitted for patients with partial responses that appeared to be still improving after six courses. Most adverse experiences were transient and mild. Preliminary assessment of response indicated five complete responses (CR, duration ongoing at 20, 11, 7, 5 and 4 months) and seven partial responses (PR, duration 3-20 months) in the 35 patients with CTCL. One CR (duration \u3e 20 months) in a patient with NHL (Lennett\u27s lymphoma) and two PR (duration 9 and 2 months) in 17 patients with B-cell NHL have been observed. Based on the mode of action of DAB3s9IL-2, its safety profile, and the patient responses associated with the phase I/II clinical trials, a phase III programme in CTCL patients has been initiated and plans for additional trials in NHL patients are targeted for 1996. © 1997 Elsevier Science Ltd. All rights reserved. Key words: clinical trial, CTCL, DAB3s9IL-2, fusion protein, IL-2, IL-2R, lymphoma

Therapeutic evaluation of immunomodulators in reducing surgical wound infection.

Despite many advances in infection control practices, including prophylactic antibiotics, surgical site infections (SSIs) remain a significant cause of morbidity, prolonged hospitalization, and death worldwide. Our innate immune system possesses a multitude of powerful antimicrobial strategies which make it highly effective in combating bacterial, fungal, and viral infections. However, pathogens use various stealth mechanisms to avoid the innate immune system, which in turn buy them time to colonize wounds and damage tissues at surgical sites. We hypothesized that immunomodulators that can jumpstart and activate innate immune responses at surgical sites, would likely reduce infection at surgical sites. We used three immunomodulators; fMLP (formyl-Methionine-Lysine-Proline), CCL3 (MIP-1α), and LPS (Lipopolysaccharide), based on their documented ability to elicit strong inflammatory responses; in a surgical wound infection model with Pseudomonas aeruginosa to evaluate our hypothesis. Our data indicate that one-time topical treatment with these immunomodulators at low doses significantly increased proinflammatory responses in infected and uninfected surgical wounds and were as effective, (or even better), than a potent prophylactic antibiotic (Tobramycin) in reducing P. aeruginosa infection in wounds. Our data further show that immunomodulators did not have adverse effects on tissue repair and wound healing processes. Rather, they enhanced healing in both infected and uninfected wounds. Collectively, our data demonstrate that harnessing the power of the innate immune system by immunomodulators can significantly boost infection control and potentially stimulate healing. We propose that topical treatment with these immunomodulators at the time of surgery may have therapeutic potential in combating SSI, alone or in combination with prophylactic antibiotics

IL-10 Dysregulation Underlies Chemokine Insufficiency, Delayed Macrophage Response, and Impaired Healing in Diabetic Wounds.

Persistent inflammation is a major contributor to healing impairment in diabetic chronic wounds. Paradoxically, diabetic wound environment during the acute phase of healing is completely different because it exhibits a reduced macrophage response owing to inadequate expression of CCL2 proinflammatory cytokine. What causes a reduction in CCL2 expression in diabetic wounds early after injury remains unknown. In this study, we report that in contrast to prolonged exposure to high glucose, which makes monocytes proinflammatory, short-term exposure to high glucose causes a rapid monocyte reprogramming, manifested by increased expression and secretion of IL-10, which in an autocrine/paracrine fashion reduces glucose uptake and transforms monocytes into an anti-inflammatory phenotype by dampening signaling through toll-like receptors. We show that IL-10 expression is significantly increased in diabetic wounds during the acute phase of healing, causing significant reductions in toll-like receptor signaling and proinflammatory cytokine production, delaying macrophage and leukocyte responses, and underlying healing impairment in diabetic wounds. Importantly, blocking IL-10 signaling during the acute phase of healing improves toll-like receptor signaling, increases proinflammatory cytokine production, enhances macrophage and leukocyte responses, and stimulates healing in diabetic wounds. We posit that anti-IL-10 strategies have therapeutic potential if added topically after surgical debridement, which resets chronic wounds into acute fresh wounds

Presence of CrkI-containing microvesicles in squamous cell carcinomas could have ramifications on tumor biology and cancer therapeutics.

Recently, we described a phenomenon whereby apoptotic cells generate and release CrkI-containing microvesicles, which stimulate proliferation in surrounding cells upon contact to compensate for their own demise. We termed these microvesicles ACPSVs for Apoptotic Compensatory Proliferation Signaling microvesicles. As immune cells and a majority of current cancer therapeutics destroy tumor cells primarily by apoptosis, we conducted a small pilot study to assess the possibility that ACPSVs may also be generated in squamous cell carcinomas. We first evaluated a primary and a metastatic squamous cell carcinoma cancer cell lines for their ability to produce ACPSVs under normal and apoptotic conditions. We next conducted a pilot study to assess the occurrence of ACPSVs in solid tumors extracted from 20 cancer patients with squamous cell carcinomas. Both cancer cell lines produced copious amounts of ACPSVs under apoptotic conditions. Interestingly, the metastatic squamous cell carcinoma cancer cell line also produced high levels of ACPSVs under healthy condition, suggesting that the ability to generate ACPSVs may be hijacked by these cells. Importantly, ACPSVs were also abundant in the solid tumors of all squamous cell carcinoma cancer patients. Detection of ACPSVs in cancer has potentially important ramifications in tumor biology and cancer therapeutics which warrants further investigation

Overriding impaired FPR chemotaxis signaling in diabetic neutrophil stimulates infection control in murine diabetic wound.

Infection is a major co-morbidity that contributes to impaired healing in diabetic wounds. Although impairments in diabetic neutrophils have been blamed for this co-morbidity, what causes these impairments and whether they can be overcome, remain largely unclear. Diabetic neutrophils, isolated from diabetic individuals, exhibit chemotaxis impairment but this peculiar functional impairment has been largely ignored because it appears to contradict the clinical findings which blame excessive neutrophil influx as a major impediment to healing in chronic diabetic ulcers. Here, we report that exposure to glucose in diabetic range results in impaired chemotaxis signaling through the formyl peptide receptor (FPR) in neutrophils, culminating in reduced chemotaxis and delayed neutrophil trafficking in the wound of Leprdb (db/db) type two diabetic mice, rendering diabetic wound vulnerable to infection. We further show that at least some auxiliary receptors remain functional under diabetic conditions and their engagement by the pro-inflammatory cytokine CCL3, overrides the requirement for FPR signaling and substantially improves infection control by jumpstarting the neutrophil trafficking toward infection, and stimulates healing in diabetic wound. We posit that CCL3 may have therapeutic potential for the treatment of diabetic foot ulcers if it is applied topically after the surgical debridement process which is intended to reset chronic ulcers into acute fresh wounds

Phase II basket trial of Dual Anti-CTLA-4 and Anti-PD-1 blockade in Rare Tumors (DART) SWOG S1609: Adrenocortical carcinoma cohort

OBJECTIVES: Multiple common cancers benefit from immunotherapy; however, less is known about efficacy in rare tumors. We report the results of the adrenocortical carcinoma cohort of NCI/SWOG S1609 Dual Anti-CTLA-4 and Anti-PD-1 blockade in Rare Tumors. DESIGN/SETTING: A prospective, phase 2 clinical trial of ipilimumab plus nivolumab was conducted by the SWOG Early Therapeutics and Rare Cancers Committee for multiple rare tumor cohorts across \u3e1,000 National Clinical Trial Network sites. PARTICIPANTS: 21 eligible patients were registered. Median age was 53 years (range 26-69); 16 (76%) were women. INTERVENTIONS: Ipilimumab 1 mg/kg intravenously every 6 weeks with nivolumab 240 mg intravenously every 2 weeks was administered until disease progression, symptomatic deterioration, treatment delay for any reason \u3e56 days, unacceptable or immune-related toxicity with inability to decrease prednisone to \u3c10 mg daily, or per patient request. MAIN OUTCOME MEASURES: The primary endpoint was the overall response rate (ORR) (RECIST V.1.1). Secondary endpoints include clinical benefit rate (CBR) (includes stable disease (SD)\u3e6 months), progression-free survival (PFS), overall survival (OS), and toxicity. Immune-related outcomes included immune ORR (iORR), immune CBR (iCBR), and immune PFS (iPFS). A two-stage design was used assuming: null=5% alternative=30%, n=6 in the first stage, 16 max, one-sided alpha=13%. RESULTS: The median number of prior therapy lines was 2 (range: 1-9). 3 of 21 patients attained confirmed partial response (PR) (ORR=14%). In addition, one patient had an unconfirmed PR; one, stable disease (SD)\u3e6 months; one, immune-related RECIST (iRECIST) PR (iPR); and one patient attained iSD\u3e6 months: clinical benefit rate (response or SD\u3e6 months)=5/21 (24%), iORR=4/21 (19%), iCBR=7/21 (33%). The 6-month PFS was 24%; 6-month iPFS, 33%. The PFS for patients (N=7) with iRECIST clinical benefit were 57, 52, 18, 15, 13, 7, and 7 months. The 6-month OS was 76%; the median OS, was 15.8 months. The most common toxicities were fatigue (62%) and rash (38%), and the most common grade 3/4 immune-related adverse events were hepatic dysfunction (9.5%) and adrenal insufficiency (9.5%). Treatment-related adverse events leading to discontinuation of therapy in four patients (21%). There were no grade 5 adverse events. CONCLUSIONS: Ipilimumab plus nivolumab is active in refractory metastatic adrenocortical cancer meeting the primary endpoint of the study, with a 19% iORR and 33% iCBR (includes SD/iSD\u3e6 months) and with the longest PFS/iPFS of 52 and 57 months. TRIAL REGISTRATION NUMBER: NCT02834013 (registered 15 July, 2016; https://clinicaltrials.gov/ct2/show/NCT02834013)

The Impact of COVID-19 Pandemic on Spine Surgeons Worldwide : A One Year Prospective Comparative Study

Study Design: Survey Objective: In March of 2020, an original study by Louie et al investigated the impact of COVID-19 on 902 spine surgeons internationally. Since then, due to varying government responses and public health initiatives to the pandemic, individual countries and regions of the world have been affected differently. Therefore, this follow-up study aimed to assess how the COVID-19 impact on spine surgeons has changed 1 year later. Methods: A repeat, multi-dimensional, 90-item survey written in English was distributed to spine surgeons worldwide via email to the AO Spine membership who agreed to receive surveys. Questions were categorized into the following domains: demographics, COVID-19 observations, preparedness, personal impact, patient care, and future perceptions. Results: Basic respondent demographics, such as gender, age, home demographics, medical comorbidities, practice type, and years since training completion, were similar to those of the original 2020 survey. Significant differences between groups included reasons for COVID testing, opinions of media coverage, hospital unemployment, likelihood to be performing elective surgery, percentage of cases cancelled, percentage of personal income, sick leave, personal time allocation, stress coping mechanisms, and the belief that future guidelines were needed (P<.05). Conclusion: Compared to baseline results collected at the beginning of the COVID-19 pandemic in 2020, significant differences in various domains related to COVID-19 perceptions, hospital preparedness, practice impact, personal impact, and future perceptions have developed. Follow-up assessment of spine surgeons has further indicated that telemedicine and virtual education are mainstays. Such findings may help to inform and manage expectations and responses to any future outbreaks.publishedVersionPeer reviewe

TGF-β Regulates DNA Methyltransferase Expression in Prostate Cancer, Correlates with Aggressive Capabilities, and Predicts Disease Recurrence

DNA methyltransferase (DNMT) is one of the major factors mediating the methylation of cancer related genes such as TGF-β receptors (TβRs). This in turn may result in a loss of sensitivity to physiologic levels of TGF-β in aggressive prostate cancer (CaP). The specific mechanisms of DNMT's role in CaP remain undetermined. In this study, we describe the mechanism of TGF-β-mediated DNMT in CaP and its association with clinical outcomes following radical prostatectomy.We used human CaP cell lines with varying degrees of invasive capability to describe how TGF-β mediates the expression of DNMT in CaP, and its effects on methylation status of TGF-β receptors and the invasive capability of CaP in vitro and in vivo. Furthermore, we determined the association between DNMT expression and clinical outcome after radical prostatectomy. We found that more aggressive CaP cells had significantly higher TGF-β levels, increased expression of DNMT, but reduced TβRs when compared to benign prostate cells and less aggressive prostate cancer cells. Blockade of TGF-β signaling or ERK activation (p-ERK) was associated with a dramatic decrease in the expression of DNMT, which results in a coincident increase in the expression of TβRs. Blockade of either TGF-β signaling or DNMT dramatically decreased the invasive capabilities of CaP. Inhibition of TGF-β in an TRAMP-C2 CaP model in C57BL/6 mice using 1D11 was associated with downregulation of DNMTs and p-ERK and impairment in tumor growth. Finally, independent of Gleason grade, increased DNMT1 expression was associated with biochemical recurrence following surgical treatment for prostate cancer.Our findings demonstrate that CaP derived TGF-β may induce the expression of DNMTs in CaP which is associated with methylation of its receptors and the aggressive potential of CaP. In addition, DNMTs is an independent predictor for disease recurrence after prostatectomy, and may have clinical implications for CaP prognostication and therapy