Urokinase-type plasminogen activator (uPA) is critical for progression of tuberous sclerosis complex 2 (TSC2)-deficient tumors.

Lymphangioleiomyomatosis (LAM) is a fatal lung disease associated with germline or somatic inactivating mutations in tuberous sclerosis complex genes (TSC1 or TSC2). LAM is characterized by neoplastic growth of smooth muscle-α-actin-positive cells that destroy lung parenchyma and by the formation of benign renal neoplasms called angiolipomas. The mammalian target of rapamycin complex 1 (mTORC1) inhibitor rapamycin slows progression of these diseases but is not curative and associated with notable toxicity at clinically effective doses, highlighting the need for better understanding LAM's molecular etiology. We report here that LAM lesions and angiomyolipomas overexpress urokinase-type plasminogen activator (uPA). Tsc1-/- and Tsc2-/- mouse embryonic fibroblasts expressed higher uPA levels than their WT counterparts, resulting from the TSC inactivation. Inhibition of uPA expression in Tsc2-null cells reduced the growth and invasiveness and increased susceptibility to apoptosis. However, rapamycin further increased uPA expression in TSC2-null tumor cells and immortalized TSC2-null angiomyolipoma cells, but not in cells with intact TSC. Induction of glucocorticoid receptor signaling or forkhead box (FOXO) 1/3 inhibition abolished the rapamycin-induced uPA expression in TSC-compromised cells. Moreover, rapamycin-enhanced migration of TSC2-null cells was inhibited by the uPA inhibitor UK122, dexamethasone, and a FOXO inhibitor. uPA-knock-out mice developed fewer and smaller TSC2-null lung tumors, and introduction of uPA shRNA in tumor cells or amiloride-induced uPA inhibition reduced tumorigenesis in vivo These findings suggest that interference with the uPA-dependent pathway, when used along with rapamycin, might attenuate LAM progression and potentially other TSC-related disorders.

Comparative study of ex vivo probe-based confocal laser endomicroscopy and light microscopy in lung cancer diagnostics.

BACKGROUND AND OBJECTIVE: Probe-based confocal laser endoscopy (pCLE) allows for real-time non-invasive histological imaging via bronchoscopy. Interpreting CLE images and correlating with traditional histopathology remains challenging. We performed an ex vivo study to evaluate the correlation between light microscopy findings and pCLE imaging of primary lung carcinoma. METHODS: Post-lobectomy specimens for lung cancer nodules were examined ex vivo by pCLE. The examined areas were marked with brilliant green dye, and the surrounding tissues were stained by methylene blue dye. Lung tissue segments were resected and histopathological specimens were generated with 50-µm thickness from the marked areas and stained with haematoxylin and eosin. Pathologists and pulmonologists reviewed the images for correlating features. RESULTS: Eighteen lobectomy specimens from 18 different patients were collected. Three primary features were observed in all samples using pCLE in the cancer surroundings: alveolar dystelectasis with thickening of alveolar walls, alveolar edema and a large amount of macrophages. The stromal and parenchymal components of the studied subtypes of non-small-cell lung cancer differed from each other. The stromal component for all nine adenocarcinoma specimens had a highly fluorescent field penetrated by dark hollows. All six squamous cell carcinoma specimens had the stromal component appeared as 'biparously' branching, highly fluorescent fibres. No stromal component was observed in any small-cell carcinoma specimen, and at low power field, the cellular component was dominant with an observed light scattering pattern. CONCLUSIONS: pCLE can identify lung carcinoma in ex vivo samples. Certain light microscopy features of lung carcinoma can be visualized with pCLE.

The Technology of Homogeneous Scar Tissue Creating as a Result of Ablation of the Atrial Wall with a Radiofrequency Bipolar Clamp: an Experimental and Clinical Study.

INTRODUCTION: The objective of this study was to develop a radiofrequency ablation technique to create a homogeneous scar tissue in the atrial myocardium. METHODS: In the double-blinded morphological stage of the study, the left atrial appendage was used as an anatomical model to investigate the efficacy of one experimental and two conventional techniques to create ablation lines. Then, these lines were studied by morphologists. The clinical stage involved investigation of the outcomes of the developed technique for creation of ablation lines. During thoracoscopic radiofrequency fragmentation of the left atrium, all ablation lines were created using the experimental radiofrequency technique. RESULTS: In all histological sections of ablation lines created using the criterion of "steady decrease in the time to transmurality", there were no intact (viable) cells, in contrast to the other two conventional methods, i.e., a homogeneous scar of the atrial wall. Investigation of clinical efficacy of this developed technique revealed recurrent atrial fibrillation only in six of 137 patients (4.4%) at median follow-up time of 36 (10; 58) months. None of the patients developed specific complications (wall perforation or bleeding). According to intracardiac mapping performed after the end of the blind period, the sources of atrial fibrillation in these six patients were outside the radiofrequency ablation zone (perimitral or in the right atrium). CONCLUSION: A steady decrease in the time to transmurality should be considered as the priority intraoperative criterion for the formation of a homogeneous scar during radiofrequency ablation of the left atrium wall using a bipolar ablation clamp.

Oncolytic therapy with recombinant vaccinia viruses targeting the interleukin-15 pathway elicits a synergistic response.

We developed recombinant variants of oncolytic vaccinia virus LIVP strain expressing interleukin-15 (IL-15) or its receptor subunit alpha (IL-15Rα) to stimulate IL-15-dependent immune cells. We evaluated their oncolytic activity either alone or in combination with each other in vitro and in vivo using the murine CT26 colon carcinoma and 4T1 breast carcinoma models. We demonstrated that the admixture of these recombinant variants could promote the generation of the IL-15/IL-15Rα complex. In vitro studies indicated that 4T1 breast cancer cells were more susceptible to the developed recombinant viruses. In vivo studies showed significant survival benefits and tumor regression in 4T1 breast cancer syngeneic mice that received a combination of LIVP-IL15-RFP with LIVP-IL15Ra-RFP. Histological analysis showed recruited lymphocytes at the tumor region, while no harmful effects to the liver or spleen of the animals were detected. Evaluating tumor-infiltrated lymphocytes represented profound activation of cytotoxic T cells and macrophages in mice receiving combination therapy. Thus, our experiments showed superior oncolytic effectiveness of simultaneous injection of LIVP-IL15-RFP and LIVP-IL15Ra-RFP in breast cancer-bearing mice. The combined therapy by these recombinant variants represents a potent and versatile approach for developing new immunotherapies for breast cancer.

The Technology of Homogeneous Scar Tissue Creating as a Result of Ablation of the Atrial Wall with a Radiofrequency Bipolar Clamp: an Experimental and Clinical Study

ABSTRACT Introduction: The objective of this study was to develop a radiofrequency ablation technique to create a homogeneous scar tissue in the atrial myocardium. Methods: In the double-blinded morphological stage of the study, the left atrial appendage was used as an anatomical model to investigate the efficacy of one experimental and two conventional techniques to create ablation lines. Then, these lines were studied by morphologists. The clinical stage involved investigation of the outcomes of the developed technique for creation of ablation lines. During thoracoscopic radiofrequency fragmentation of the left atrium, all ablation lines were created using the experimental radiofrequency technique. Results: In all histological sections of ablation lines created using the criterion of "steady decrease in the time to transmurality", there were no intact (viable) cells, in contrast to the other two conventional methods, i.e., a homogeneous scar of the atrial wall. Investigation of clinical efficacy of this developed technique revealed recurrent atrial fibrillation only in six of 137 patients (4.4%) at median follow-up time of 36 (10; 58) months. None of the patients developed specific complications (wall perforation or bleeding). According to intracardiac mapping performed after the end of the blind period, the sources of atrial fibrillation in these six patients were outside the radiofrequency ablation zone (perimitral or in the right atrium). Conclusion: A steady decrease in the time to transmurality should be considered as the priority intraoperative criterion for the formation of a homogeneous scar during radiofrequency ablation of the left atrium wall using a bipolar ablation clamp.

The case of diagnostics of invasive pulmonary aspergillosis by in vivo probe-based confocal laser endomicroscopy of central and distal airways.

We present a case of 41-year-old patient with invasive pulmonary aspergillosis (IPA) in which probe-based confocal laser endomicroscopy (pCLE) imaging of central and distal airways was first performed in vivo. pCLE imaging showed the signs of complete or partial destruction of elastin network of alveolar wall with fibrillar branching fluorescent structures in the zone with typical IPA changes on HRCT.