Synergistic effect of cold gas plasma and experimental drug exposure exhibits skin cancer toxicity in vitro and in vivo.

INTRODUCTION: Skin cancer is often fatal, which motivates new therapy avenues. Recent advances in cancer treatment are indicative of the importance of combination treatments in oncology. Previous studies have identified small molecule-based therapies and redox-based technologies, including photodynamic therapy or medical gas plasma, as promising candidates to target skin cancer. OBJECTIVE: We aimed to identify effective combinations of experimental small molecules with cold gas plasma for therapy in dermato-oncology. METHODS: Promising drug candidates were identified after screening an in-house 155-compound library using 3D skin cancer spheroids and high content imaging. Combination effects of selected drugs and cold gas plasma were investigated with respect to oxidative stress, invasion, and viability. Drugs that had combined well with cold gas plasma were further investigated in vascularized tumor organoids in ovo and a xenograft mouse melanoma model in vivo. RESULTS: The two chromone derivatives Sm837 and IS112 enhanced cold gas plasma-induced oxidative stress, including histone 2A.X phosphorylation, and further reduced proliferation and skin cancer cell viability. Combination treatments of tumor organoids grown in ovo confirmed the principal anti-cancer effect of the selected drugs. While one of the two compounds exerted severe toxicity in vivo, the other (Sm837) resulted in a significant synergistic anti-tumor toxicity at good tolerability. Principal component analysis of protein phosphorylation profiles confirmed profound combination treatment effects in contrast to the monotherapies. CONCLUSION: We identified a novel compound that, combined with topical cold gas plasma-induced oxidative stress, represents a novel and promising treatment approach to target skin cancer.

Quality of Life after Desarda Technique for Inguinal Hernia Repair-A Comparative Retrospective Multicenter Study of 120 Patients.

Inguinal hernia repair, according to Desarda, is a pure tissue surgical technique using external oblique fascia to reinforce the posterior wall of the inguinal canal. This has provided an impetus for the rethinking of guideline adherence toward minimally invasive and mesh-based surgery of inguinal hernia. In this study, a retrospective analysis of this technique was conducted in two German hospitals. Between 6/2013 and 12/2020, 120 operations were performed. Analysis included patient characteristics, duration of operation, length of hospital stay, and perioperative complications. Data were used to achieve a matched-pair analysis comparing Desarda to laparoscopic transabdominal preperitoneal (TAPP) hernia repair. Propensity scores were calculated based on five preoperative variables, including sex, age, American Society of Anesthesiology classification, localization, and width of the inguinal hernia in order to achieve comparability. Additionally, we assessed pain level and quality of life (QoL) 12 months postoperatively. The focus of our study was a comparison of QoL to a reference population and TAPP cohort. The study population consisted of 106 male and 14 female patients, and the median age was 37.5 years. The median operation time was 50 min, and the median length of hospital stay was 2 days. At a follow-up of 17 months, the median recurrence rate was 0.8%, and two cases of chronic postoperative pain were recorded. Postoperative QoL does not significantly differ between Desarda and TAPP. In contrast, Desarda patients had a significantly higher QoL compared with the reference population. In summary, Desarda's procedure is a good option as a pure tissue method for inguinal hernia repair.

The Dorsal Skinfold Chamber as a New Tympanic Membrane Wound Healing Model: Intravital Insights into the Pathophysiology of Epithelialized Wounds.

BACKGROUND: Tympanic membrane perforations (TMPs) are a common complication of trauma and infection. Persisting perforations result from the unique location of the tympanic membrane. The wound is surrounded by air of the middle ear and the external auditory canal. The inadequate wound bed, growth factor, and blood supply lead to circular epithelialization of the perforation's edge and premature interruption of defect closure. Orthotopic animal models use mechanical or chemical tympanic membrane laceration to identify bioactive wound dressings and overcome premature epithelialization. However, all orthotopic models essentially lack repetitive visualization of the biomaterial-wound interface. Therefore, recent progress in 3D printing of customized wound dressings has not yet been transferred to the unique wound setup of the TMP. Here, we present a novel application for the mice dorsal skinfold chamber (DSC) with an epithelialized full-thickness defect as TMP model. METHODS: A circular 2-mm defect was cut into the extended dorsal skinfold using a biopsy punch. The skinfold was either perforated through both skin layers without prior preparation or perforated on 1 side, following resection of the opposing skin layer. In both groups, the wound was sealed with a coverslip or left unclosed (n = 4). All animals were examined for epithelialization of the edge (histology), size of the perforation (planimetry), neovascularization (repetitive intravital fluorescence microscopy), and inflammation (immunohistology). RESULTS: The edge of the perforation was overgrown by the cornified squamous epithelium in all pre-parations. Reduction in the perforation's size was enhanced by application of a coverslip. Microsurgical preparation before biopsy punch perforation and sealing with a coverslip enabled repetitive high-quality intravital fluorescence microscopy. However, spontaneous reduction of the perforation occurred frequently. Therefore, the direct biopsy punch perforation without microsurgical preparation was favorable: spontaneous reduction did not occur throughout 21 days. Moreover, the visualization of the neovascularization was sufficient in intravital microscopy. CONCLUSIONS: The DSC full-thickness defect is a valuable supplement to orthotopic TMP models. Repetitive intravital microscopy of the epithelialized edge enables investigation of the underlying pathophysiology during the transition from the inflammation to the proliferation phase of wound healing. Using established analysis procedures, the present model provides an effective platform for the screening of bioactive materials and transferring progress in tissue engineering to the special conditions of tympanic membrane wound healing.

Treatment of an Infected TEVAR with Extra- and Endovascular Bacteriophage Application.

Introduction: Graft infections are severe complications. Surgical resection of infected aortic stent grafts is associated with high mortality and morbidity. Therefore, alternatives or adjuncts to antibiotic treatment and extensive surgery are urgently needed. Report: A 67 year old woman was admitted with a methicillin sensitive Staphylococcus aureus infected stent graft in the thoracic aorta. Local infection was confirmed by PET-CT imaging. Surgical resection of the stent graft was not feasible because of comorbidities. Therefore, a three step approach for local bacteriophage treatment was performed as a last resort treatment. Firstly, the para-aortic tissue was debrided via left thoracotomy, a bacteriophage suspension was applied on the outer surface of the aorta, and a vacuum irrigation system was installed. After repeated alternating instillation of the bacteriophage suspension for three days, as a second step, the vacuum sponges were removed and a bacteriophage containing gel was applied locally on the outer surface of the aorta. In the third step, the bacteriophage containing gel was applied to a thoracic stent graft, which in turn was placed endovascularly into the infected stent. Discussion: After 28 days, the patient was discharged from hospital with normalised infection parameters. PET-CT imaging at three and 12 months post-intervention did not show signs of infection in or around the thoracic aorta. This Case demonstrates successful treatment of an infected endovascular stent graft by application of bacteriophages both to extravascular and, as a novel approach, endovascular sites using a bacteriophage coated stent graft.

Assessment of Quality of Life after Endovascular and Open Abdominal Aortic Aneurysm Repair: A Retrospective Single-Center Study.

Postoperative quality of life is an important outcome parameter after treatment of abdominal aortic aneurysms. The aim of this retrospective single-center study was to assess and compare the health-related quality of life (HRQoL) of patients after open repair (OR) or endovascular treatment (EVAR), and furthermore to investigate the effect of incisional hernia (IH) formation on HRQoL. Patients who underwent OR or EVAR for treatment of an abdominal aortic aneurysm between 2008 and 2016 at a University Medical Center were included. HRQoL was assessed using the SF-36 questionnaire. The incidence of IH was recorded from patient files and by telephone contact. SF-36 scores of 83 patients (OR: n = 36; EVAR: n = 47) were obtained. The mean follow-up period was 7.1 years. When comparing HRQoL between OR and EVAR, patients in both groups scored higher in one of the eight categories of the SF36 questionnaires. The incidence of IH after OR was 30.6%. In patients with postoperative IH, HRQoL was significantly reduced in the dimensions "physical functioning", "role physical" and "role emotional" of the SF-36. Based on this data, it can be concluded that neither OR nor EVAR supply a significant advantage regarding HRQoL. In contrast, the occurrence of IH has a relevant impact on the HRQoL of patients after OR.

Bacteriophages for the Treatment of Graft Infections in Cardiovascular Medicine.

Bacterial infections of vascular grafts represent a major burden in cardiovascular medicine, which is related to an increase in morbidity and mortality. Different factors that are associated with this medical field such as patient frailty, biofilm formation, or immunosuppression negatively influence antibiotic treatment, inhibiting therapy success. Thus, further treatment strategies are required. Bacteriophage antibacterial properties were discovered 100 years ago, but the focus on antibiotics in Western medicine since the mid-20th century slowed the further development of bacteriophage therapy. Therefore, the experience and knowledge gained until then in bacteriophage mechanisms of action, handling, clinical uses, and limitations were largely lost. However, the parallel emergence of antimicrobial resistance and individualized medicine has provoked a radical reassessment of this approach and cardiovascular surgery is one area in which phages may play an important role to cope with this new scenario. In this context, bacteriophages might be applicable for both prophylactic and therapeutic use, serving as a stand-alone therapy or in combination with antibiotics. From another perspective, standardization of phage application is also required. The ideal surgical bacteriophage application method should be less invasive, enabling highly localized concentrations, and limiting bacteriophage distribution to the infection site during a prolonged time lapse. This review describes the latest reports of phage therapy in cardiovascular surgery and discusses options for their use in implant and vascular graft infections.

Distress Analysis of Mice with Cervical Arteriovenous Fistulas.

The welfare of laboratory animals is a consistent concern for researchers. Its evaluation not only fosters ethical responsibility and addresses legal requirements, but also provides a solid basis for a high quality of research. Recently, a new cervical arteriovenous model was created in mice to understand the pathophysiology of arteriovenous fistula, which is the most commonly used access for hemodialysis. This study evaluates the distress caused by this new animal model. Ten male C57B6/J mice with cervical arteriovenous fistula were observed for 21 days. Non-invasive parameters, such as body weight, faecal corticosterone metabolites, burrowing activity, nesting activity and distress scores were evaluated at each time point. Six out of ten created arteriovenous fistula matured within the observation time as defined by an increased diameter. The body weight of all animals was reduced after surgery but recovered within five days. In addition, the distress score was significantly increased during the early time point but not at the late time point after arteriovenous fistula creation. Neither burrowing activity nor nesting behaviour were significantly reduced after surgical intervention. Moreover, faecal corticosterone metabolite concentrations did not significantly increase. Therefore, the cervical murine arteriovenous fistula model induced moderate distress in mice and revealed an appropriate maturation rate of the fistulas.

Diagnostic Ability of Methods Depicting Distress of Tumor-Bearing Mice.

Subcutaneous tumor models in mice are the most commonly used experimental animal models in cancer research. To improve animal welfare and the quality of scientific studies, the distress of experimental animals needs to be minimized. For this purpose, one must assess the diagnostic ability of readout parameters to evaluate distress. In this study, we evaluated different noninvasive readout parameters such as body weight change, adjusted body weight change, faecal corticosterone metabolites concentration, burrowing activity and a distress score by utilising receiver operating characteristic curves. Eighteen immunocompromised NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice were used for this study; half were subcutaneously injected with A-375 cells (human malignant melanoma cells) that resulted in large tumors. The remaining mice were inoculated with SCL-2 cells (cutaneous squamous cell carcinoma cells), which resulted in small tumors. The adjusted body weight and faecal corticosterone metabolites concentration had a high diagnostic ability in distinguishing between mice before cancer cell injection and mice bearing large tumors. All other readout parameters had a low diagnostic ability. These results suggest that adjusted body weight and faecal corticosterone metabolites are useful to depict the distress of mice bearing large subcutaneous tumors.

Intraoperative Assessment of Gastric Sleeve Oxygenation Using Hyperspectral Imaging in Esophageal Resection: A Feasibility Study.

INTRODUCTION: Sufficient tissue oxygenation is essential for anastomotic healing in visceral surgery. Hyperspectral imaging (HSI) is a noncontact, noninvasive technique for clinical assessment of tissue oxygenation in real time. METHODS: In this case series, HSI was used in 4 patients who were admitted for either esophageal cancer or cardiac carcinoma (AEG type I or II). Thoraco-abdominal surgical esophageal resection was performed after staging and neoadjuvant therapy. Intraoperative oxygenation of superficial (StO2) and underlying tissue (NIR perfusion index) of the gastric sleeve were studied intrathoracic by means of the TIVITA® Tissue HSI camera. This was performed prior to esophagogastric anastomosis. The postoperative course, especially in view of surgical complications, was recorded. RESULTS: Assessment of StO2 and NIR perfusion index was performed in 4 regions of interest per gastric sleeve, aboral and oral of the clinically determined resection line. It allowed the fast quantification of gastric oxygenation prior gastroesophageal anastomosis. Median StO2 aboral of the determined resection line was 69%, while median StO2 in the oral part of the gastric sleeve was found at 53%. In contrast, the median NIR perfusion index was similar aboral (80) and oral (82) of the resection line. In none of the 4 studied patients, an anastomotic failure appeared. DISCUSSION/CONCLUSION: This report suggests that HSI is a feasible technique for intraoperative assessment of tissue oxygenation before gastroesophageal anastomosis and might reduce the incidence of anastomotic failure in the gastrointestinal tract.

Multimodal Imaging Techniques to Evaluate the Anticancer Effect of Cold Atmospheric Pressure Plasma.

BACKGROUND: Skin cancer is the most frequent cancer worldwide and is divided into non-melanoma skin cancer, including basal cell carcinoma, as well as squamous cell carcinoma (SCC) and malignant melanoma (MM). METHODS: This study evaluates the effects of cold atmospheric pressure plasma (CAP) on SCC and MM in vivo, employing a comprehensive approach using multimodal imaging techniques. Longitudinal MR and PET/CT imaging were performed to determine the anatomic and metabolic tumour volume over three-weeks in vivo. Additionally, the formation of reactive species after CAP treatment was assessed by non-invasive chemiluminescence imaging of L-012. Histological analysis and immunohistochemical staining for Ki-67, ApopTag®, F4/80, CAE, and CD31, as well as protein expression of PCNA, caspase-3 and cleaved-caspase-3, were performed to study proliferation, apoptosis, inflammation, and angiogenesis in CAP-treated tumours. RESULTS: As the main result, multimodal in vivo imaging revealed a substantial reduction in tumour growth and an increase in reactive species after CAP treatment, in comparison to untreated tumours. In contrast, neither the markers for apoptosis, nor the metabolic activity of both tumour entities was affected by CAP. CONCLUSIONS: These findings propose CAP as a potential adjuvant therapy option to established standard therapies of skin cancer.

Molecular Mechanisms of the Efficacy of Cold Atmospheric Pressure Plasma (CAP) in Cancer Treatment.

Recently, the potential use of cold atmospheric pressure plasma (CAP) in cancer treatment has gained increasing interest. Especially the enhanced selective killing of tumor cells compared to normal cells has prompted researchers to elucidate the molecular mechanisms for the efficacy of CAP in cancer treatment. This review summarizes the current understanding of how CAP triggers intracellular pathways that induce growth inhibition or cell death. We discuss what factors may contribute to the potential selectivity of CAP towards cancer cells compared to their non-malignant counterparts. Furthermore, the potential of CAP to trigger an immune response is briefly discussed. Finally, this overview demonstrates how these concepts bear first fruits in clinical applications applying CAP treatment in head and neck squamous cell cancer as well as actinic keratosis. Although significant progress towards understanding the underlying mechanisms regarding the efficacy of CAP in cancer treatment has been made, much still needs to be done with respect to different treatment conditions and comparison of malignant and non-malignant cells of the same cell type and same donor. Furthermore, clinical pilot studies and the assessment of systemic effects will be of tremendous importance towards bringing this innovative technology into clinical practice.

Evaluation of peripheral artery disease with the TIVITA® Tissue hyperspectral imaging camera system.

BACKGROUND: Objective, reliable and easy screening for peripheral artery disease (PAD) is essential to confirm the diagnosis and initiate the respective treatment. Therefore, a new non-invasive hyperspectral camera (TIVITA® Tissue) was tested in patients with and without PAD. OBJECTIVE: It was hypothesized that the oxygenation parameters of the TIVITA® Tissue correlate to established modalities for detection of PAD and allow differentiation between individuals with and without PAD. METHODS: Evaluation of tissue oxygenation was performed in the angiosome of the medial plantar artery in 25 healthy young people and in 24 patients with and 25 patients without PAD in comparable age. Thereby, superficial oxygenation (StO2) and near-infrared (NIR) perfusion index were measured with the TIVITA® Tissue. Additionally, the ankle-brachial-index (ABI), the complaint free walking distance and the vascular quality of life were assessed and demographic data were obtained from all participants. RESULTS: TIVITA® Tissue analysis revealed significantly reduced StO2 and NIR perfusion index in PAD compared to healthy young participants and patients without PAD. StO2 and NIR perfusion index positively correlated with ABI, the complaint free walking distance and the vascular quality of life score. CONCLUSIONS: In summary, this new hyperspectral imaging camera bears great potential for PAD screening as well as for follow up.

Ectopic Spleen Tissue - an Underestimated Differential Diagnosis of a Hypervascularised Liver Tumour.

BACKGROUND: Patients with liver cirrhosis have an increased risk of developing hepatocellular carcinoma (HCC). Implantation metastasis following diagnostic biopsy is a well-known complication. Therefore, primary resection of a hypervascularised tumour suspicious for HCC is often performed with curative intent. CASE REPORT: An exophytically growing mass was diagnosed between liver segments III and IVb by means of ultrasound in a 53-year old male patient with decompensated liver cirrhosis. Computed tomography confirmed a 3.5 cm large hypervascularised tumour with given resectability. Intraoperatively, the tumour appeared like a HCC. Thus, an atypical resection was performed. Histopathology revealed ectopic spleen tissue without any signs of malignancy. As enquiries revealed, the patient had undergone splenectomy after a blunt abdominal trauma 9 years prior to admission. CONCLUSION: In the present patient, hepatic splenosis in a cirrhotic liver was misinterpreted as HCC. In patients with a history of traumatic rupture of the spleen or splenectomy, splenosis has to be considered as a potential differential diagnosis of a hypervascularised tumour. Specific diagnostics should be performed to rule out splenosis.

Contribution of protein Z and protein Z-dependent protease inhibitor in generalized Shwartzman reaction.

OBJECTIVE: Sepsis, a leading cause of mortality in critically ill patients, is closely linked to the excessive activation of coagulation and inflammation. Protein Z, a cofactor for the protein Z-dependent protease inhibitor, enhances the inhibition of coagulation factor Xa, and protein Z-dependent protease inhibitor inhibits factor XIa in a protein Z-independent fashion. The functions of protein Z and protein Z-dependent protease inhibitor in the inflammatory and coagulant responses to septic illness have not been evaluated. DESIGN: For induction of generalized Shwartzman reaction, dorsal skinfold chamber-equipped mice were challenged twice with lipopolysaccharide (0.05 mg/kg on day -1 and 5 mg/kg body weight 24 hr later). Time-matched control animals received equal volumes of saline. SETTING: University research laboratory. SUBJECTS, INTERVENTIONS, AND MEASUREMENTS: Using intravital fluorescence microscopy in protein Z-dependent protease inhibitor deficient (ZPI) and protein Z deficient (PZ) mice, as well as their wild-type littermates (ZPI, PZ), kinetics of light/dye-induced thrombus formation and microhemodynamics were assessed in randomly chosen venules. Plasma concentrations of chemokine (C-X-C motif) ligand 1, interleukin-6, and interleukin-10 were measured. Liver and lung were harvested for quantitative analysis of leukocytic tissue infiltration and thrombus formation. MAIN RESULTS: After induction of generalized Shwartzman reaction, all mice showed significant impairment of microhemodynamics, including blood flow velocity, volumetric blood flow, and functional capillary density, as well as leukocytopenia and thrombocytopenia. Thrombus formation time was markedly prolonged after induction of generalized Shwartzman reaction in all mice, except of ZPI mice, which also had a significantly higher fraction of occluded vessels in liver sections. PZ mice developed the highest concentrations of interleukin-6 and interleukin-10 in response to generalized Shwartzman reaction and showed greater leukocytic tissue infiltration than their wild-type littermates. CONCLUSIONS: In this murine model of generalized Shwartzman reaction, protein Z-dependent protease inhibitor deficiency enhanced the thrombotic response to vascular injury, whereas protein Z deficiency increased inflammatory response.

The anti-thrombotic effect of hydrogen sulfide is partly mediated by an upregulation of nitric oxide synthases.

INTRODUCTION: Hydrogen sulfide (H2S) known as a gasotransmitter is increasingly recognized for its anti-adhesive, anti-inflammatory and vasoactive properties. Due to these properties, we analysed anti-thrombotic effects of H2S and the participation of the nitric oxide synthase (NOS)-pathway. MATERIALS AND METHODS: In individual venules of the ear of hairless SKH1-hr mice, thrombus formation was induced using a phototoxic light/dye-injury model and intravital fluorescence microscopy. Animals were treated intravenously with the H2S donor Na2S or NaCl as control. In a second setting, the NOS inhibitor L-NAME was applied intraperitoneally as a bolus 12h prior to Na2S treatment and thrombus induction. Blood and ear tissue were sampled after microscopy for assessment of plasma concentrations of soluble (s)P-selectin, sE-selectin, sVCAM-1 and sICAM-1 and expression of endothelial (e)NOS and inducible (i)NOS, respectively. RESULTS: When mice were treated with Na2S, venular thrombus formation was significantly delayed versus that in animals of the NaCl-treated control group. While plasma levels of pro-thrombotic adhesion molecules were not affected by Na2S, immunohistochemistry of the vessel walls showed a significant up-regulation of eNOS and iNOS expression within the Na2S-treated group. The delay of thrombus formation in the Na2S-group was partly but significantly reverted by application of L-NAME. CONCLUSIONS: The anti-thrombotic efficacy of H2S involves the NOS-pathway and may be of preventive and therapeutic value for clinical disorders with increased risk of thrombotic events.

Erythropoietin improves skin wound healing and activates the TGF-ß signaling pathway.

We could recently report that erythropoietin (EPO) accelerates skin wound healing in mice. Now, we provide insight into the molecular mechanisms of this non-hematopoietic property of EPO analyzing the transforming growth factor (TGF)-ß signaling pathway. EPO receptor was found expressed in both non-wounded and wounded skin tissue as well as in fibroblasts and keratinocytes. In saline-treated control animals, wounds exhibited a significant upregulation of TGF-ß1 and of α-smooth muscle actin (α-SMA) compared with non-wounded skin. EPO treatment accelerated wound epithelialization and induced mRNA expression of TGF-ß1 and α-SMA. In addition, EPO significantly enhanced phosphorylation of Smad2 and Smad3 in fibroblasts and also elevated phosphorylation of Smad3 in wound tissue. Blockade of TGF-ß using a neutralizing anti-TGF-ß antibody attenuated EPO-induced acceleration of wound epithelialization in vivo and markedly reversed EPO effects on mRNA expression of TGF-ß1 and α-SMA. In conclusion, EPO caused activation of the Smad-dependent TGF-ß signaling pathway, enhanced differentiation of myofibroblasts, and accelerated skin wound closure.