Ex Vivo Irradiation of Lung Cancer Stem Cells Identifies the Lowest Therapeutic Dose Needed for Tumor Growth Arrest and Mass Reduction In Vivo.

Radiotherapy represents a first-line treatment for many inoperable lung tumors. New technologies offer novel opportunities for the treatment of lung cancer with the administration of higher doses of radiation in smaller volumes. Because both therapeutic and toxic treatment effects are dose-dependent, it is important to identify a minimal dose protocol for each individual patient that maintains efficacy while decreasing toxicity. Cancer stem cells sustain tumor growth, promote metastatic dissemination, and may give rise to secondary resistance. The identification of effective protocols targeting these cells may improve disease-free survival of treated patients. In this work, we evaluated the existence of individual profiles of sensitivity to radiotherapy in patient-derived cancer stem cells (CSCs) using both in vitro and in vivo models. Both CSCs in vitro and mice implanted with CSCs were treated with radiotherapy at different dose intensities and rates. CSC response to different radiation doses greatly varied among patients. In vitro radiation sensitivity of CSCs corresponded to the therapeutic outcome in the corresponding mouse tumor model. On the other side, the dose administration rate did not affect the response. These findings suggest that in vitro evaluation of CSCs may potentially predict patients' response, thus guiding clinical decision.

Engineering new metabolic pathways in isolated cells for the degradation of guanidinoacetic acid and simultaneous production of creatine.

Here we report, for the first time, the engineering of human red blood cells (RBCs) with an entire metabolic pathway as a potential strategy to treat patients with guanidinoacetate methyltransferase (GAMT) deficiency, capable of reducing the high toxic levels of guanidinoacetate acid (GAA) and restoring proper creatine levels in blood and tissues. We first produced a recombinant form of native human GAMT without any tags to encapsulate into RBCs. Due to the poor solubility and stability features of the recombinant enzyme, both bioinformatics studies and extensive optimization work were performed to select a mutant GAMT enzyme, where only four critical residues were replaced, as a lead candidate. However, GAMT-loaded RBCs were ineffective in GAA consumption and creatine production because of the limiting intra-erythrocytic S-adenosyl methionine (SAM) content unable to support GAMT activity. Therefore, a recombinant form of human methionine adenosyl transferase (MAT) was developed. RBCs co-entrapped with both GAMT and MAT enzymes performed, in vitro, as a competent cellular bioreactor to remove GAA and produce creatine, fueled by physiological concentrations of methionine and the ATP generated by glycolysis. Our results highlight that metabolic engineering of RBCs is possible and represents proof of concept for the design of novel therapeutic approaches.

Airborne Microorganism Inactivation by a UV-C LED and Ionizer-Based Continuous Sanitation Air (CSA) System in Train Environments.

Improving indoor air quality present in environments where people live is important to protect human health. This particularly applies to public transportation, where air quality may affect the health and safety of passengers, workers and staff. To provide better air quality, many buildings and transports are provided with heating, ventilation and air conditioning (HVAC) systems, which are always equipped with filters to retain the particulate present in the airflow, but they lack continuous air sanitization systems. In this study, a new UV-C LED and ionizer-based continuous sanitation air (CSA) system to be installed in a train HVAC was developed (international patent: N.PCT/IB2021/054194) and its sanitation efficacy against various microbial species (bacteria and fungi) was assessed. The device proved to be very effective at the microbial killing of aerodispersed microorganisms, both in its experimental configuration (ISO 15714:2019) and in a train setting. The installation of this CSA system on public transportation appears to be a promising solution to guarantee high microbiological air quality with a very low environmental impact due to its eco-friendly components.

Functional Classification of the ATM Variant c.7157C>A and In Vitro Effects of Dexamethasone.

Most of the ATM variants associated with Ataxia Telangiectasia are still classified as variants with uncertain significance. Ataxia Telangiectasia is a multisystemic disorder characterized by "typical" and "atypical" phenotypes, with early-onset and severe symptoms or with late-onset and mild symptoms, respectively. Here we classified the c.7157C > A ATM variant found in homozygosity in two brothers of Lebanese ethnicity. The brothers presented with an atypical phenotype, showing less than 50% of the positive criteria considered for classification. We performed several in silico analyses to predict the effect of c.7157C > A at the DNA, mRNA and protein levels, revealing that the alteration causes a missense substitution in a highly conserved alpha helix in the FAT domain. 3D structural analyses suggested that the variant might be pathogenic due to either loss of activity or to a structural damage affecting protein stability. Our subsequent in vitro studies showed that the second hypothesis is the most likely, as indicated by the reduced protein abundance found in the cells carrying the variant. Moreover, two different functional assays showed that the mutant protein partially retains its kinase activity. Finally, we investigated the in vitro effect of Dexamethasone showing that the drug is able to increase both protein abundance and activity. In conclusion, our results suggest that the c.7157C > A variant is pathogenic, although it causes an atypical phenotype, and that dexamethasone could be therapeutically effective on this and possibly other missense ATM variants.

Endomyometriosis of the Rectum With Disseminated Peritoneal Leiomyomatosis 8 Years After Laparoscopic Myomectomy: A Case Report.

Endomyometriosis is a rare finding and it can be challenging to diagnose and to treat. It can arise in the uterus, in the ovary, in the broad ligament, in the peritoneal surface and in other pelvic structures. Usually patients with endomyometriosis are asymptomatic, but symptoms could occur due to large dimensions or site of the mass. We present a case of a 49-year-old woman with a symptomatic pelvic mass in the rectal wall, with no history of endometriosis, who underwent laparoscopic myomectomy 8 years earlier.

Percutaneous vs. surgical revascularization for patients with unprotected left main stenosis: a meta-analysis of 5-year follow-up randomized controlled trials.

AIMS: A 5-year survival of patients with unprotected left main (ULM) stenosis according to the choice of revascularization (percutaneous vs. surgical) remains to be defined. METHODS AND RESULTS: Randomized controlled trials (RCTs) comparing percutaneous coronary intervention (PCI) vs. coronary artery bypass graft (CABG) with a follow-up of at least 5 years were included. All-cause death was the primary endpoint. MACCE [a composite endpoint of all-cause mortality, myocardial infarction (MI), stroke, and repeat revascularization] along with its single components and cardiovascular (CV) death were the secondary ones. Analyses were stratified according to the use of first- vs. last-generation coronary stents. Subgroup comparisons were performed according to SYNTAX score (below or above 33) and to age (using cut-offs of each trial's subgroup analysis). Four RCTs with 4394 patients were identified: 2197 were treated with CABG, 657 with first generation, and 1540 with last-generation stents. At 5-year rates of all-cause death did not differ [odds ratio (OR) 0.93, 95% confidence interval (CI) 0.71-1.21], as those of CV death and stroke. Coronary artery bypass graft reduced rates of MACCE (OR 0.69, 95% CI 0.60-0.79), mainly driven by MI (OR 0.48, 95% CI 0.36-0.65) and revascularization (OR 0.53, 95% CI 0.45-0.64). Benefit of CABG for MACCE was consistent, although with different extent, across values of SYNTAX score (OR 0.76, 95% CI 0.59-0.97 for values < 32 and OR 0.63, 95% CI 0.47-0.84 for values ≥ 33) while was not evident for 'younger' patients (OR 0.83, 95% CI 0.65-1.07 vs. OR 0.65, 95% CI 0.51-0.84 for 'older' patients). CONCLUSION: For patients with ULM disease followed-up for 5 years, no significant difference was observed in all-cause and cardiovascular death between PCI and CABG. Coronary artery bypass graft reduced risk of MI, revascularization, and MACCE especially in older patients and in those with complex coronary disease and a high SYNTAX score.

Radiosensitivity of Cancer Stem Cells Has Potential Predictive Value for Individual Responses to Radiotherapy in Locally Advanced Rectal Cancer.

Neo-adjuvant radiotherapy is frequently employed in the therapeutic management of locally advanced rectal cancer (LARC). Radiotherapy can both reduce local recurrence and improve the success of surgical procedures by reducing tumor mass size. However, some patients show a poor response to treatment, which results in primary resistance or relapse after apparent curative surgery. In this work, we report in vitro and in vivo models based on patient-derived cancer stem cells (CSCs); these models are able to predict individual responses to radiotherapy in LARC. CSCs isolated from colorectal cancer biopsies were subjected to in vitro irradiation with the same clinical protocol used for LARC patients. Animal models, generated by CSC xenotransplantation, were also obtained and treated with the same radiotherapy protocol. The results indicate that CSCs isolated from rectal cancer needle biopsies possess an intrinsic grade of sensitivity to treatment, which is also maintained in the animal model. Notably, the specific CSCs' in vitro and in vivo sensitivity values correspond to patients' responses to radiotherapy. This evidence suggests that an in vitro radiotherapy response predictivity assay could support clinical decisions for the management of LARC patients, thus avoiding radiation toxicity to resistant patients and reducing the treatment costs.

Ongoing Developments and Clinical Progress in Drug-Loaded Red Blood Cell Technologies.

Engineered red blood cells (RBCs) appear to be a promising method for therapeutic drug and protein delivery. With a number of agents in clinical trials (e.g., dexamethasone 21-phosphate in ataxia telangiectasia, asparaginase in pancreatic cancer/acute lymphoblastic leukemia, thymidine phosphorylase in mitochondrial neurogastrointestinal encephalomyopathy, RTX-134 in phenylketonuria, etc.), this leading article summarizes the ongoing efforts in developing these agents, focuses on the clinical progress, and provides a brief background into engineered RBCs and the different ways in which they can be exploited for therapeutic/diagnostic purposes. References to available data on safety, efficacy, and tolerability are reported. Due to the continuous progress in this field, the information is updated as of January 2020 from databases, websites, and press releases of the involved companies and information that is in the public domain.

Managing the incidentally detected gallbladder cancer: algorithms and controversies.

INTRODUCTION: Gallbladder cancer (GBC) is the fifth most common neoplasm of the gastrointestinal tract and the most common cancer of the biliary tract. GBC is suspected preoperatively in only 30-40% of patients. The other 60-70% are discovered incidentally (IGBC) by the pathologist on a gallbladder specimen following cholecystectomy for benign diseases such as polyps, gallstones, and cholecystitis. MATERIALS AND METHODS: Between 1995 and 2011, 30 cases of GBC, who underwent resection with curative intent in our institutions, were retrospectively reviewed. They were analyzed for demographic data, and type of operation, surgical morbidity and mortality, histopathological classification, and survival. Incidental GBC was compared with suspected or preoperatively diagnosed GBC. Overall survival, disease-free survival (DFS) and the difference in DFS between patients previously treated with laparoscopic cholecystectomy and those who had oncological resection as first intervention were analyzed. The authors also present a systematic review to evaluate the role of extended surgery in the treatment of the incidental GBC. RESULTS: GBC was diagnosed in 30 patients, 16 women and 14 men. The M/F ratio was 1:1.14 and the mean age was 69.4 years (range 45-83 years). A preoperative diagnosis was possible only in 14 cases; fourteen of the incidental cases were diagnosed postoperatively after the pathological examination; two were suspected intraoperatively at the opening of the surgical specimen and then confirmed by frozen sections. The ratio between incidental and nonincidental cases was 1, 14/1, with twelve cases discovered after laparoscopic cholecystectomy. Eighty-one per cent of the incidental cases were discovered at an early stage (≤II). The preoperative diagnosis of the 30 patients with GBC was: GBC with liver invasion diagnosed by preoperative CT (nine cases); gallbladder abscess perforated into hepatic parenchyma and involving the transversal mesocolon and hepatic hilum (one case); porcelain gallbladder (three cases); gallbladder adenoma (four cases); and chronic cholecystolithiasis (thirteen cases). Every case, except one, with a T1b or more advanced invasion underwent IVb + V wedge liver resection and pericholedochic/hepatoduodenal lymphoadenectomy. One patient refused further surgery. Cases with Tis and T1a involvement were treated with cholecystectomy alone. Nine of the sixteen patients with incidental diagnosis reached 5-year DFS (56.25%) and eight of them are recurrence free. Surprisingly, one patient reached 38 mo survival despite a port-site recurrence (the only one in our experience) 2 years after the original surgery requiring further resection. Cases with non incidental diagnosis were more locally advanced and only two patients experienced 5 years DFS (Tables 2 and 3). CONCLUSION: Laparoscopic cholecystectomy does not affect survival if implemented properly. Reoperation should have two objectives: R0 resection and clearance of the lymph nodes.

The role of 68-Ga-DOTATOC CT-PET in surgical tactic for gastric neuroendocrine tumors treatment: our experience: a case report.

Gastric neuroendocrine tumors (g-NETs), which originate from gastric enterochromaffin-like (ECL) mucosal cells and account for 2.4% of all carcinoids, are increasingly recognized due to expanding indications of upper gastrointestinal endoscopy. Often silent and benign, g-NETs may however, be aggressive and sometimes they mimic the course of gastric adenocarcinoma. Current nosography distinguishes those occurring in chronic conditions with hypergastrinemia, as the type 1 associated with chronic atrophic gastritis, and the type 2 associated with Zollinger-Ellison syndrome in MEN1. Conversely, type 3 and 4 (according to some authors) are unrelated to hypergastrinemia and are frequently malignant, with a propension to develop distant metastases. While there is a general agreement concerning the treatment of malignant gastric neuroendocrine tumors, for types 1 and 2, current possibilities include surveillance, endoscopic polypectomy, surgical excision, associated or not with surgical antrectomy, or total gastrectomy. This report, based on our clinical experience, discusses how the size, number, depth, histological grading, staging with CT, MRI, and the use of recently developed somatostatin receptor tracers (68Ga-DOTATATE, 68Ga-DOTA-TOC) could allow the correct identification of a benign or malignant propensity of an individual tumor, thus avoiding underestimation or overtreatment of these uncommon neoplasms.

The safety of the Harmonic® FOCUS in open thyroidectomy: a prospective, randomized study comparing the Harmonic® FOCUS and traditional suture ligation (knot and tie) technique.

Since Kocher and Billroth refined an acceptable technique, the thyroidectomy has become one of the most frequent procedures in endocrine surgery and bilateral total thyroidectomy is performed in the majority of thyroid diseases. This work evaluated the use of the Harmonic(®) FOCUS and traditional suture ligation (knot and tie) technique in a prospective, randomized study of open thyroidectomy. Eighty two patients were randomized and divided into two similarly sized groups: the Harmonic(®) FOCUS group (F group) and traditional group (T group). The use of the harmonic FOCUS shows some statistically significant advantages limited to a few intraoperative parameters: surgical time and volume of blood loss. The surgical time was significantly shorter in F group than in the T group (105 ± 27 min vs 143 ± 32 respectively; p < 0.05). Intraoperative volume blood loss was significantly more in the T group than in the F group (36 ± 23 ml vs. 24 ± 18; p < 0.05). The postoperative parameters (volume of drainage fluid, serum calcium at 12 and 48 h, hypocalcemia, wound complication, RLN palsy, postoperative pain and length of hospital stay) showed no statistical difference. The Harmonic Focus may provide a cost-effective option only in high volume centers where reducing operative time may balance the number of daily procedures.

Staple line as a cause of unusual early internal hernia after appendectomy.

The use of mechanical stapling devices in laparoscopic appendectomies has become a common practice. Occasionally, the retained staples have been described to cause adhesions that might result in bowel obstruction. Early bowel obstruction after routine abdominal surgery should be closely investigated and might warrant early re-exploration. We present a rare case of small bowel obstruction caused by a staple line adhesive band one week after appendectomy. A 46-year-old female underwent laparoscopic appendectomy for uncomplicated appendicitis. A linear endoscopic stapling device was utilized during the procedure. The patient was discharged without complication. One week later, the patient presented to the emergency room for abdominal pain and she was discharged after adequate pain control. Several hours later she returned with similar symptoms, and she was diagnosed with distal small bowel obstruction by computed tomography scan. During the diagnostic laparoscopy there was an internal hernia through a defect created by the appendiceal staple line and the adjacent small bowel mesentery. After reduction of the hernia, the small bowel venous drainage improved, and no intestinal resection was necessary. The offending staple was removed and the staple line covered with omentum. The patient had complete resolution of symptoms and she was discharged the following day. No perioperative complications occurred. Mechanical staplers are routinely used in laparoscopic appendectomy. The staple line should be inspected at the end of the procedure to confirm the absence of free, unformed staples that can generate adhesions and postoperative complications.

Hernioscopy: a simple application of single-port endoscopic surgery in acute inguinal hernias.

Strangulated hernia remains one of the most common emergencies encountered in general surgery. During induction of general or spinal anesthesia, the potential self-reduction of a gangrenous bowel can occur in approximately 1% of cases. In these cases, bowel viability can no longer be directly assessed unless a more extensive operation (laparoscopy or laparotomy) is performed. A simple alternative to unnecessary laparotomy or to a standard laparoscopy is a hernia sac laparoscopy (hernioscopy). Here, we presented 4 patients with a diagnosis of small-bowel obstruction secondary to incarcerated inguinal hernias, in which the incarcerated hernia content was evaluated by hernioscopy. Only 1 case presented persistent signs of bowel ischemia after hernia reduction and required a small-bowel segmental resection. All hernias were repaired using prosthetic tension-free technique. Hernia sac laparoscopy (hernioscopy), the introduction of the laparoscope through an open inguinal hernia sac, can be useful to evaluate the viability of the incarcerated hernia content, to avoid unnecessary laparotomy.

Mechanical ventilation in patients with end-stage idiopathic pulmonary fibrosis.

BACKGROUND: Acute respiratory failure (ARF) occurring during idiopathic pulmonary fibrosis (IPF) is associated with a poor prognosis. In this subset of individuals, mechanical ventilation (MV) may be required. OBJECTIVES: We analysed the characteristics of a group of IPF patients undergoing MV for ARF in order to give some indications on the supposed prognosis. METHODS: Hospital records of 34 consecutive patients with IPF, who underwent MV for ARF, were retrospectively examined. Demographic data, time from diagnosis, gas exchange, Acute Physiology and Chronic Health Evaluation (APACHE) II score, ARF causes and MV failure were recorded. RESULTS: Fifteen subjects (group A) underwent invasive MV and 19 patients (group B) non-invasive ventilation (NIV). The 2 groups were different for disease severity (APACHE II score 24.2 +/- 6 vs. 19.5 +/- 5.9; p = 0.01). Both ventilatory strategies temporarily increased PaO2/FiO2 as compared with spontaneous breathing (group A: 148.5 +/- 52 vs. 99 +/- 39, p = 0.0004; group B: 134 +/- 36 vs. 89 +/- 26, p = 0.0004). NIV reduced the respiratory rate (26 +/- 7 vs. 36 +/- 9 with spontaneous breathing; p = 0.002). Duration of MV correlated with the time of evolution of IPF (r = 0.45; p = 0.018). The in-hospital mortality rate was 85% (100% for invasive MV, 74% for NIV). Four of the 5 survivors died within 6 months from hospital discharge (range 2-6 months). CONCLUSIONS: MV does not appear to have a significant impact on the survival of patients with end-stage IPF. NIV may be useful for compassionate use, providing relief from dyspnoea and avoiding aggressive approaches.

HRCT and histopathological evaluation of fibrosis and tissue destruction in IPF associated with pulmonary emphysema.

Idiopathic pulmonary fibrosis has been associated with emphysema in cigarette smokers as a new clinical entity: combined pulmonary fibrosis and emphysema (CPFE). In order to compare histomorphometrical, roentgenological and immunohistochemical aspects of usual interstitial pneumonia (UIP) with and without associated pulmonary emphysema, 17 patients with biopsy-proven UIP were evaluated. Morphometrical evaluation of lung parenchyma destruction was used to divide patients in two subgroups: emphysema/UIP (n=9) and UIP alone (n=8); four patients with biopsy-proven emphysema without fibrosis were also evaluated. At HRTC scan, emphysematous lesions were prevalent in the upper fields of both emphysema/UIP and emphysema groups and the distribution of fibrotic lesions was similar in emphysema/UIP compared to UIP alone. The semiquantitative histopathological fibrotic score was also similar in emphysema/UIP and UIP alone. In addition, the expression of tumor necrosis factor (TNF)-alpha, matrix metalloproteinase (MMP)-2, MMP-9, MMP-7 and membrane type 1-metalloproteinase (MT1-MMP) by fibroblasts of myofibroblastic foci was similar in emphysema/UIP and UIP alone patients. In contrast, fibroblasts in areas of parenchymal destruction of emphysema/UIP expressed MMP-2, MMP-9, MMP-7 and MT1-MMP at variable but significantly higher levels when compared to emphysema subjects, in the presence of similar levels of TIMP-1, TIMP-2 and TNF-alpha. Fibrotic and emphysematous lesions in emphysema/UIP patients appear to follow the roentgenological and histopathological patterns expected for either UIP or emphysema. Interstitial fibroblast activation is more pronounced in the areas of lung destruction in emphysema/UIP compared to those with emphysema alone, as for exaggerated tissue remodeling.