Thickness of rectus abdominis measured by ultrasound in critically ill patients after abdominal surgery: A retrospective cohort study.

BACKGROUND: Early identification of patients at high risk of prolonged mechanical ventilation is important in critical care. Sarcopenia, the loss of muscle mass and function, has been reported to be associated with extended mechanical ventilation and prolonged ICU stay. Although ultrasound is noninvasive and widely used in critical care, there is no standard method of using it to assess sarcopenia. OBJECTIVES: The study aims to investigate the relationship between outcomes of critically ill patients and the ratio of BMI to the thickness of rectus abdominis measured by a standardised ultrasound examination. DESIGN: A retrospective cohort study. SETTING: Surgical ICU of a tertiary referral hospital, from October 2017 to June 2018. The thickness of rectus abdominis (RA) was measured while performing extended focused assessment sonography for trauma. BMI was divided by the thickness of rectus abdominis over the upper abdomen to derive the BMI-RA thickness ratio. PATIENTS: Sixteen male and 11 female patients admitted to ICU after major abdominal surgery. MAIN OUTCOME MEASURES: The primary outcome was in-hospital mortality, and the secondary outcomes were durations of mechanical ventilation, ICU stay and hospital stay. The disease severity, serum albumin level and BMI-RA thickness ratio were also analysed. RESULTS: Ultrasound measurement was easy to perform without adverse effects. The BMI-RA thickness ratio was significantly higher in nonsurvivors and was associated with ICU stay, hospital stay and duration of mechanical ventilation. Multivariable logistic regression showed that the BMI-RA thickness ratio was a predictor of in-hospital mortality. CONCLUSION: The BMI-RA thickness ratio is related to the outcomes of patients transferred to ICU after major abdominal surgery. Measuring the thickness of rectus abdominis by ultrasound is well tolerated and easy to perform in surgical ICU. Larger prospective studies are required to confirm current findings.

Lactobacillus salivarius reverse antibiotic-induced lung defense impairment in a ventilator model.

BACKGROUND: Widespread use of antibiotics in the intensive care unit is a potential cause of the emergence of hospital-acquired pneumonia. This study determined whether Lactobacillus salivarius feeding could reverse antibiotic-induced lung defense impairment in a ventilator model. METHODS: C57BL/6 wild-type (WT) mice received mechanical ventilation for 3 h after intramuscular antibiotic treatment for 6 days. Treatment with dead Lactobacillus salivarius and fructo-oligosaccharides (FOS) feeding were used to stimulate antibacterial protein expression in the intestine. Reactive oxygen species (ROS) in the intestinal mucosa was detected using 2'7'-dichlorofluorescein diacetate. The peroxynitrite production of alveolar macrophages (AMs) was measured using dihydrorhodamine 123 oxidation assay. N-acetylcysteine (NAC), an ROS scavenger, was orally administered to mice receiving antibiotics with FOS feeding. RESULTS: Antibiotic treatment decreased Pseudomonas aeruginosa (PA) phagocytic activity and activity of AMs and protein expression of regenerating islet-derived protein 3ß (Reg3ß) as well as Toll-like receptor 4 (TLR4) in the intestinal mucosa in the ventilator model. Antibiotic treatment also decreased ROS production in the intestinal mucosa, peroxynitrite production of AMs, and RELMß expression as well as NF-κB DNA binding activity of the intestinal mucosa in WT mice but not in MyD88-/- mice. Treatment with dead L. salivarius or FOS feeding increased ROS production, bacterial killing activity, and protein expression of Reg3ß as well as TLR4 in the intestinal mucosa and reversed the inhibitory effects of antibiotics on PA phagocytic activity of AMs. CONCLUSION: Taken together with the finding that ablation of FOS-induced intestinal ROS using NAC decreased peroxynitrite production as well as PA phagocytic activity of AMs and protein expression of CRP-ductin, IL-17, Reg3ß, and RELMß in the intestinal mucosa, we conclude that commensal microflora plays a key role in stimulating lung immunity. Intestinal ROS plays a role as a predictive indicator and modulator of pulmonary defense mechanisms. Antibiotic treatment reduces lung defense against PA infection through the decrease in intestinal Reg3ß and TLR4 expression. Treatment with dead L. salivarius or FOS feeding reverses the antibiotic-induced lung defense impairment through the intestinal ROS/MyD88 pathways.

Blocking TNF-α enhances Pseudomonas aeruginosa-induced mortality in burn mice through induction of IL-1ß.

PURPOSE: Tumor necrosis factor (TNFα) is a proinflammatory cytokine and has been a target for intervention in human sepsis. However, inhibition of TNF-α with a high dose of a TNF-receptor fusion protein in patients with septic shock worsened patient survival. This study was designed to investigate whether blocking TNF-α enhances mortality in infected burn mice through the induction of IL-1ß. METHODS: WT or Tnfrsf1a(-/-) mice received Pseudomonas aeruginosa injection in the back at 8h after burn injury. The animals were sacrificed at 24h after burn and lung tissues were harvested and examined for determining myeloperoxidase (MPO) activity, pulmonary microvascular dysfunction, NF-κB DNA binding activity, and IL-1ß expression. Also, the lung and blood were harvested for bacterial count assay. RESULT: Thermal injury alone induced NF-κB DNA binding activity and neutrophil infiltration in the lung in WT but not in Tnfrsf1a(-/-) mice. A 50% total body surface area (TBSA) burn induced a significant increase of mortality in WT compared with Tnfrsf1a(-/-) mice. In contrast, P. aeruginosa injection with a 30% TBSA burn pretreatment enhanced IL-1ß expression, bacterial counts in lung and blood, pulmonary microvascular dysfunction, and mortality in Tnfrsf1a(-/-) mice compared with WT mice. Injection of the IL-1 receptor antagonist, Anakinra, reduced P. aeruginosa infection with burn pretreatment-induced blood bacterial counts, IL-1ß levels as well as permeability of lung, and mortality in Tnfrsf1a(-/-) mice. CONCLUSIONS: Our findings suggest that thermal injury induces lung NF-κB activation and neutrophil sequestration through TNFα signaling. However, blocking TNF-α enhances P. aeruginosa infection-induced lung damage in burn mice via induction of IL-1ß. Using an IL-1 receptor antagonist combined with the neutralization of TNF-α could be a useful strategy for decreasing P. aeruginosa infection-induced mortality in burn patients.

NF-κB activation in myeloid cells mediates ventilator-induced lung injury.

BACKGROUND: Although use of the mechanical ventilator is a life-saving intervention, excessive tidal volumes will activate NF-κB in the lung with subsequent induction of lung edema formation, neutrophil infiltration and proinflammatory cytokine/chemokine release. The roles of NF-κB and IL-6 in ventilator-induced lung injury (VILI) remain widely debated. METHODS: To study the molecular mechanisms of the pathogenesis of VILI, mice with a deletion of IкB kinase in the myeloid cells (IKKß(Δmye)), IL-6(-/-) to WT chimeric mice, and C57BL/6 mice (WT) were placed on a ventilator for 6 hr.WT mice were also given an IL-6-blocking antibody to examine the role of IL-6 in VILI. RESULTS: Our results revealed that high tidal volume ventilation induced pulmonary capillary permeability, neutrophil sequestration, macrophage drifting as well as increased protein in bronchoalveolar lavage fluid (BALF). IL-6 production and IL-1ß, CXCR2, and MIP2 expression were also increased in WT lungs but not in those pretreated with IL-6-blocking antibodies. Further, ventilator-induced protein concentrations and total cells in BALF, as well as lung permeability, were all significantly decreased in IKKß(Δmye) mice as well as in IL6(-/-) to WT chimeric mice. CONCLUSION: Given that IKKß(Δmye) mice demonstrated a significant decrease in ventilator-induced IL-6 production, we conclude that NF-κB-IL-6 signaling pathways induce inflammation, contributing to VILI, and IкB kinase in the myeloid cells mediates ventilator-induced IL-6 production, inflammation, and lung injury.

TNF-alpha decreases infection-induced lung injury in burn through negative regulation of TLR4/iNOS.

BACKGROUND: Sepsis is an infectious process-induced generalized inflammatory response that mediates the excessive production of cytokines. However, anti-tumor necrosis factor (TNF)-α therapy has failed in decreasing mortality of sepsis patients due to undefined mechanisms. This study was designed to investigate whether absence of TNF receptor enhanced lung damage and mortality through toll-like receptors (TLRs) and inducible nitric oxide synthase (iNOS). MATERIALS AND METHODS: We injected Pseudomonas aeruginosa or lipopolysaccharide in the backs of wild-type, Tnfrsf1a(-/-) (deficient of TNF-α receptor 1), and TLR4(-/-) mice at 8 h after 30% total body surface area burn. The animals were sacrificed at 16 h after burn and lung tissues were harvested and examined for determining pulmonary microvascular dysfunction and interleukin (IL)-1ß, iNOS, and TLR4 expression. The blood of animals was harvested for bacterial count assay. The effect of S-methylisothiourea, an iNOS inhibitor, on P aeruginosa infection with thermal injury pretreatment-induced lung damage was also examined. RESULTS: P aeruginosa or lipopolysaccharide injection with thermal injury pretreatment enhanced TLR4, iNOS, and IL-1ß expression and pulmonary microvascular dysfunction in Tnfrsf1a(-/-) mice compared with wild-type mice. P aeruginosa infection with thermal injury pretreatment did not induce IL-1ß or iNOS expression and mortality in TLR4(-/-) mice. S-methylisothiourea treatment significantly decreased P aeruginosa infection with thermal injury pretreatment-induced lung injury, blood bacterial counts, pulmonary IL-1ß expression, and mortality in Tnfrsf1a(-/-) mice. CONCLUSIONS: Given that absence of the TNF-α receptor 1 is associated with increased lung permeability, we conclude that TNF-α decreases P aeruginosa infection-induced lung damage in burn mice through negative regulation of TLR4 as well as iNOS expression, and iNOS inhibitor might be useful in reversing anti-TNF-α therapy-induced lung injury in burn.

Gut flora enhance bacterial clearance in lung through toll-like receptors 4.

BACKGROUND: The influence of the gut flora on lung inflammatory reaction against bacterial challenge remains undefined. This study was designed to investigate whether gut flora enhances lung defense against E.coli pneumonia through TLR4 signaling. METHODS: C3H/HeN (WT) mice and C3H/HeJ (TLR4 deficient) mice were treated with antibiotics in drinking water for 4 weeks to deplete gut commensal microflora. At week 3, drinking water was supplemented with lipopolysaccharide (LPS); a ligand for TLR4, to trigger TLRs in intestinal tract. At the end of 4th week, E.coli was injected to trachea to induce E.coli pneumonia. RESULTS: We found that commensal depletion by antibiotic pretreatment before E.coli pneumonia challenge induced a 30% decrease of MPO activity in the lung, a significant decrease of bacterial killing activity of alveolar macrophage, and bacterial counts in C3H/HeN mice but not in C3H/HeJ (TLR4 deficient) mice. LPS, a TLR4 ligand, supplementation during antibiotic pretreatment reversed these effects and decreased E.coli pneumonia-induced mortality in C3H/HeN mice. Furthermore, commensal depletion induced a suppression of NF-κB DNA binding activity and an increase of KC, MIP-2, IL-1ß expression in the lung in C3H/HeN mice but not in C3H/HeJ mice. CONCLUSIONS: Taken together with that commensal depletion increased E.coli pneumonia-induced mortality and LPS supplementation decreased it, we conclude that gut flora enhances bacterial clearance against E.coli pneumonia through TLR4.

Lactobacillus plantarum reverse diabetes-induced Fmo3 and ICAM expression in mice through enteric dysbiosis-related c-Jun NH2-terminal kinase pathways.

Diabetes mellitus (DM) is characterized by increased fatality associated with the atherogenetic process. Circulating trimethylamine-N-oxide (TMAO) levels are closely associated with atherosclerosis. The flavin mono-oxygenase family (Fmo) members oxidize trimethylamine (TMA) to TMAO. The effect and the regulatory mechanism of intestinal microflora on diabetes-induced Fmo3 and intercellular adhesion molecule (ICAM) expression were examined in streptozotocin-induced diabetic mice (STZDM) and Akita mice (C57BL/6J-Ins2Akita). STZDM-JNK1-/- and Ins2Akita-JNK1-/- mice were produced and used to study the role of pJNK in the regulatory mechanisms. Diabetic mice exhibited decreased Lactobacilli growth and reactive oxygen species (ROS) production in the intestinal mucosa; increased levels of pJNK and iNOS proteins in the intestinal mucosa; increased levels of serum nitrate, IL-1ß, and TNF-α expression in Kupffer cells; increased Fmo3 expression in the liver; and increased ICAM expression in the aorta. Reversal of diabetes-induced enteric dysbiosis by prebiotic (FOS) or probiotic (dead L. plantarum) treatment decreased diabetes-induced pJNK and iNOS expression in the intestine, Fmo3 expression in the liver, IL-1ß expression in Kupffer cells, and ICAM expression in the aorta and liver. Ins2Akita-JNK1-/- and STZDM-JNK1-/- mice demonstrated decreased levels of serum NO, IL-1ß expression in Kupffer cells, Fmo3 expression in the liver, and ICAM expression in the aorta. GF mice cohoused with DM mice demonstrated an increase in ICAM expression in the liver. In conclusion, diabetes induced the expression of both Fmo3 and ICAM expression and possible vascular impairment through enteric dysbiosis. Diabetes-induced Fmo3 and ICAM expression could be reversed by pJNK inhibition or by correcting enteric dysbiosis.

Integration of Oncogenes via Sleeping Beauty as a Mouse Model of HPV16+ Oral Tumors and Immunologic Control.

Human papillomavirus type 16 (HPV16) is the etiologic factor for cervical cancer and a subset of oropharyngeal cancers. Although several prophylactic HPV vaccines are available, no effective therapeutic strategies to control active HPV diseases exist. Tumor implantation models are traditionally used to study HPV-associated buccal tumors. However, they fail to address precancerous phases of disease progression and display tumor microenvironments distinct from those observed in patients. Previously, K14-E6/E7 transgenic mouse models have been used to generate spontaneous tumors. However, the rate of tumor formation is inconsistent, and the host often develops immune tolerance to the viral oncoproteins. We developed a preclinical, spontaneous, HPV16+ buccal tumor model using submucosal injection of oncogenic plasmids expressing HPV16-E6/E7, NRas G12V , luciferase, and sleeping beauty (SB) transposase, followed by electroporation in the buccal mucosa. We evaluated responses to immunization with a pNGVL4a-CRT/E7(detox) therapeutic HPV DNA vaccine and tumor cell migration to distant locations. Mice transfected with plasmids encoding HPV16-E6/E7, NRas G12V , luciferase, and SB transposase developed tumors within 3 weeks. We also found transient anti-CD3 administration is required to generate tumors in immunocompetent mice. Bioluminescence signals from luciferase correlated strongly with tumor growth, and tumors expressed HPV16-associated markers. We showed that pNGVL4a-CRT/E7(detox) administration resulted in antitumor immunity in tumor-bearing mice. Lastly, we demonstrated that the generated tumor could migrate to tumor-draining lymph nodes. Our model provides an efficient method to induce spontaneous HPV+ tumor formation, which can be used to identify effective therapeutic interventions, analyze tumor migration, and conduct tumor biology research. Cancer Immunol Res; 6(3); 305-19. ©2018 AACR.

Buccal injection of synthetic HPV long peptide vaccine induces local and systemic antigen-specific CD8+ T-cell immune responses and antitumor effects without adjuvant.

BACKGROUND: Human Papillomavirus is responsible for over 99 % of cervical cancers and is associated with cancers of the head and neck. The currently available prophylactic vaccines against HPV do not generate therapeutic effects against established HPV infections and associated lesions and disease. Thus, the need for a therapeutic vaccine capable of treating HPV-induced malignancies persists. Synthetic long peptides vaccination is a popular antigen delivery method because of its safety, stability, production feasibility, and its need to be processed by professional antigen presenting cells before it can be presented to cytotoxic CD8+ T lymphocytes. Cancers in the buccal mucosa have been shown to elicit cancer-related inflammations that are capable of recruiting inflammatory and immune cells to generate antitumor effects. In the current study, we evaluated the therapeutic potential of synthetic HPV long peptide vaccination in the absence of adjuvant in the TC-1 buccal tumor model. RESULT: We show that intratumoral vaccination with E7 long peptide alone effectively controls buccal TC-1 tumors in mice. Furthermore, we observed an increase in systemic as well as local E7-specific CD8+ T cells in buccal tumor-bearing mice following the vaccination. Finally, we show that induction of immune responses against buccal tumors by intratumoral E7 long peptide vaccination is independent of CD4+ T cells, and that the phenomenon may be related to the unique environment associated with mucosal tissues. CONCLUSION: Our results suggest the possibility for clinical translation of the administration of adjuvant free therapeutic long peptide vaccine as a potentially effective and safe strategy for mucosal HPV-associated tumor treatment.

Local administration of granulocyte macrophage colony-stimulating factor induces local accumulation of dendritic cells and antigen-specific CD8+ T cells and enhances dendritic cell cross-presentation.

Immunotherapy has emerged as a promising treatment strategy for the control of HPV-associated malignancies. Various therapeutic HPV vaccines have elicited potent antigen-specific CD8+ T cell mediated antitumor immune responses in preclinical models and are currently being tested in several clinical trials. Recent evidence indicates the importance of local immune activation, and higher number of immune cells in the site of lesion correlates with positive prognosis. Granulocyte macrophage colony-stimulating factor (GMCSF) has been reported to posses the ability to induce migration of antigen presentation cells and CD8+ T cells. Therefore, in the current study, we employ a combination of systemic therapeutic HPV DNA vaccination with local GMCSF application in the TC-1 tumor model. We show that intramuscular vaccination with CRT/E7 DNA followed by GMCSF intravaginal administration effectively controls cervicovaginal TC-1 tumors in mice. Furthermore, we observe an increase in the accumulation of E7-specific CD8+ T cells and dendritic cells in vaginal tumors following the combination treatment. In addition, we show that GMCSF induces activation and maturation in dendritic cells and promotes antigen cross-presentation. Our results support the clinical translation of the combination treatment of systemic therapeutic vaccination followed by local GMCSF administration as an effective strategy for tumor treatment.

Cancer immunotherapy using a potent immunodominant CTL epitope.

Immunotherapy has emerged as a promising approach that can be used in conjunction with conventional chemotherapy and radiotherapy to further improve the survival rate of patients with advanced cancer. We have recently shown in previous studies that chemotherapy and radiation therapy can alter the tumor microenvironment and allow intratumoral vaccination to prime the adaptive immune system leading to the generation of antigen-specific cell-mediated immune responses. Here, we investigated whether intratumoral injection of a foreign immunodominant peptide (GP33) and the adjuvant CpG into tumors following cisplatin chemotherapy could lead to potent antitumor effects and antigen-specific cell-mediated immune responses. We observed that treatment with all three agents produced the most potent antitumor effects compared to pairwise combinations. Moreover, treatment with cisplatin, CpG and GP33 was able to control tumors at a distant site, indicating that our approach is able to induce cross-presentation of the tumor antigen. Treatment with cisplatin, CpG and GP33 also enhanced the generation of GP33-specific and E7-specific CD8+ T cells and decreased the number of MDSCs in tumor loci, a process found to be mediated by the Fas-FasL apoptosis pathway. The treatment regimen presented here represents a universal approach to cancer control.

Cancer immunotherapy employing an innovative strategy to enhance CD4+ T cell help in the tumor microenvironment.

Chemotherapy and/or radiation therapy are widely used as cancer treatments, but the antitumor effects they produce can be enhanced when combined with immunotherapies. Chemotherapy kills tumor cells, but it also releases tumor antigen and allows the cross-presentation of the tumor antigen to trigger antigen-specific cell-mediated immune responses. Promoting CD4+ T helper cell immune responses can be used to enhance the cross-presentation of the tumor antigen following chemotherapy. The pan HLA-DR binding epitope (PADRE peptide) is capable of generating antigen-specific CD4+ T cells that bind various MHC class II molecules with high affinity and has been widely used in conjunction with vaccines to improve their potency by enhancing CD4+ T cell responses. Here, we investigated whether intratumoral injection of PADRE and the adjuvant CpG into HPV16 E7-expressing TC-1 tumors following cisplatin chemotherapy could lead to potent antitumor effects and antigen-specific cell-mediated immune responses. We observed that treatment with all three agents produced the most potent antitumor effects compared to pairwise combinations. Moreover, treatment with cisplatin, CpG and PADRE was able to control tumors at a distant site, indicating that our approach is able to induce cross-presentation of the tumor antigen. Treatment with cisplatin, CpG and PADRE also enhanced the generation of PADRE-specific CD4+ T cells and E7-specific CD8+ T cells and decreased the number of MDSCs in tumor loci. The treatment regimen presented here represents a universal approach to cancer control.