The choroid plexus stroma constitutes a sanctuary for paediatric B-cell precursor acute lymphoblastic leukaemia in the central nervous system.

Despite current central nervous system-directed therapies for childhood B-cell precursor acute lymphoblastic leukaemia, relapse at this anatomical site still remains a challenging issue. Few reports have addressed the study of the specific cellular microenvironments which can promote the survival, quiescence, and therefore chemoresistance of B-cell precursor acute lymphoblastic leukaemia cells in the central nervous system. Herein, we showed by immunofluorescence and electron microscopy that in xenotransplanted mice, leukaemic cells infiltrate the connective tissue stroma of the choroid plexus, the brain structure responsible for the production of cerebrospinal fluid. The ultrastructural study also showed that leukaemia cells are able to migrate through blood vessels located in the choroid plexus stroma. In short-term co-cultures, leukaemic cells established strong interactions with human choroid plexus fibroblasts, mediated by an increased expression of ITGA4 (VLA-4)/ITGAL (LFA-1) and their ligands VCAM1/ICAM1. Upon contact with leukaemia cells, human choroid plexus fibroblasts acquired a cancer-associated fibroblast phenotype, with an increased expression of α-SMA and vimentin as well as pro-inflammatory factors. Human choroid plexus fibroblasts also have the capacity to reduce the proliferative index of leukaemic blasts and promote their survival and chemoresistance to methotrexate and cytarabine. The inhibition of VLA-4/VCAM-1 interactions using anti-VLA-4 antibodies, and the blockade of Notch signalling pathway by using a γ-secretase inhibitor partially restored chemotherapy sensitivity of leukaemia cells. We propose that the choroid plexus stroma constitutes a sanctuary for B-cell precursor acute lymphoblastic leukaemia cells in the central nervous system. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Cloning, overexpression, and purification of glucose-6-phosphate dehydrogenase of Pseudomonas aeruginosa.

Glucose-6-phosphate dehydrogenase (G6PDH) (EC 1.1.1.363) plays an important role in the human pathogen Pseudomonas aeruginosa because it generates NADPH, an essential cofactor for several biosynthetic pathways and antioxidant enzymes. P. aeruginosa G6PDH is also a key enzyme in the metabolism of various carbon sources, such as glucose, glycerol, fructose, and mannitol. Understanding the kinetic characteristics and mechanisms that control the activity of this enzyme is crucial for future studies in this context. However, one of the impediments to achieving this goal is the limited amount of protein obtained when current purification protocols are implemented, a factor curtailing its biochemical characterization. In this study, we report a fast, efficient and reproducible procedure for the purification of P. aeruginosa G6PDH that can be implemented in a short period (2 days). In order to establish this protocol, the zwf gene, which encodes for this enzyme, was cloned and overexpressed in Escherichia coli cells. In contrast to other procedures, our method is based on protein precipitation with CaCl2 and further purification by ion exchange chromatography. Using this protocol, we were able to obtain 31 mg/L of pure protein that manifested specific activity of 145.7 U/mg. The recombinant enzyme obtained in this study manifested similar physicochemical and kinetic properties to those reported in previous works for this molecule. The large quantities of active enzyme obtained using this procedure will facilitate its structural characterization and identify differences between P. aeruginosa- and human G6PDH, thus contributing to the search for selective inhibitors against the bacterial enzyme.

Fibroblast growth factor and vascular endothelial growth factor play a critical role in endotheliogenesis from human adipose-derived stem cells.

OBJECTIVE: Adipose-derived stem cells (ASCs) are a potential adult mesenchymal stem cell source for restoring endothelial function in patients with critical limb ischemia. Fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF) play a major role in angiogenesis and wound healing. This study evaluated the effects of FGF and VEGF on the proliferation, migration, and potential endothelial differentiation of human ASCs with regards to their use as endothelial cell substitutes. METHODS: ASCs were isolated from clinical lipoaspirates and cultured in M199 medium with fetal bovine serum (10%), FGF2 (10 ng/mL), VEGF (50 ng/mL), or combinations of FGF2 and VEGF. Cell proliferation rates, viability, and migration were measured by growth curves, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), and scratch assays. For cell attachment determinations, ASCs were seeded onto a scaffold of small intestinal submucosa for 5 days. Endothelial differentiation capabilities of ASCs were confirmed by expression of endothelial cell-specific markers using quantitative polymerase chain reaction, immunofluorescence staining, and cord formation on Matrigel (BD Biosciences, San Jose, Calif). PD173074, a selective inhibitor of FGF receptor, was used to confirm the importance of FGF signaling. RESULTS: ASCs treated with FGF or combinations of FGF and VEGF showed increased proliferation rates and consistent differentiation toward an endothelial cell lineage increase in platelet endothelial cell adhesion molecule (CD31), von Willebrand factor, endothelial nitric oxide synthase, and vascular endothelial cadherin message, and in protein and cord formation on Matrigel. FGF and VEGF stimulated ASC migration and increased the attachment and retention after seeding onto a matrix graft of small intestinal submucosa. Blockade of FGF signaling with PD173074 abrogated ASC endothelial cell differentiation potential. CONCLUSIONS: These results indicate that FGF and VEGF are ASC promoters for proliferation, migration, attachment, and endothelial differentiation. FGF and VEGF have a costimulatory effect on ASC endotheliogenesis. These results further suggest that ASCs with enhanced FGF signaling may potentially be used for tissue engineering and cell-based therapies in patients with critical limb ischemia.

Paclitaxel impairs adipose stem cell proliferation and differentiation.

BACKGROUND: Cancer patients with chemotherapy-induced immunosuppression have poor surgical site wound healing. Prior literature supports the use of human adipose-derived stem cell (hASC) lipoinjection to improve wound healing. It has been established that multipotent hASCs facilitate neovascularization, accelerate epithelialization, and quicken wound closure in animal models. Although hASC wound therapy may benefit surgical cancer patients, the chemotherapeutic effects on hASCs are unknown. We hypothesized that paclitaxel, a chemotherapeutic agent, impairs hASC growth, multipotency, and induces apoptosis. METHODS: hASCs were isolated and harvested from consented, chemotherapy and radiation naive patients. Growth curves, MTT (3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide), and EdU (5-ethynyl-2-deoxyguridine) assays measured cytotoxicity and proliferation. Oil Red O stain, Alizarin Red stain, matrigel tube formation assay, and quantitative polymerase chain reaction analyzed hASC differentiation. Annexin V assay measured apoptosis. Immunostaining and Western blot determined tumor necrosis factor α (TNF-α) expression. RESULTS: hASCs were selectively more sensitive to paclitaxel (0.01-30 µM) than fibroblasts (P < 0.05). After 12 d, paclitaxel caused hASC growth arrest, whereas control hASCs proliferated (P = 0.006). Paclitaxel caused an 80.6% reduction in new DNA synthesis (P < 0.001). Paclitaxel severely inhibited endothelial differentiation and capillary-like tube formation. Differentiation markers, lipoprotein lipase (adipogenic), alkaline phosphatase (osteogenic), CD31, and van Willebrand factor (endothelial), were significantly decreased (all P < 0.05) confirming paclitaxel impaired differentiation. Paclitaxel was also found to induce apoptosis and TNF-α was upregulated in paclitaxel-treated hASCs (P < 0.001). CONCLUSIONS: Paclitaxel is more cytotoxic to hASCs than fibroblasts. Paclitaxel inhibits hASC proliferation, differentiation, and induces apoptosis, possibly through the TNF-α pathway. Paclitaxel's severe inhibition of endothelial differentiation indicates neovascularization disruption, possibly causing poor wound healing in cancer patients receiving chemotherapy.

Inferior Vena Cava Filter Occlusion Causing Phlegmasia Alba Dolens.

Phlegmasia alba dolens is a rare sequela of acute extensive venothrombus of the iliofemoral segments. Rarely, phlegmasia alba dolens can also result from clotted inferior vena cava filter. A 39-year-old with protein S deficiency, and prior inferior vena cava filter placement after remote trauma presented to the emergency department with progressive bilateral lower extremity pain and swelling. Venous duplex revealed extensive bilateral deep vein thromboses from the external iliac veins to popliteal veins, as well as thrombophlebitis of the left great saphenous vein. Venography confirmed patency of the suprarenal vena cava with abrupt occlusion of the infrarenal segment at the level of the inferior vena cava filter. The filter was removed followed by endovascular thrombectomy and adjunctive venoplasty. The patient progressed well and discharged on therapeutic anticoagulation. This case illustrates that a staged endovascular approach may be utilized for acute on chronic caval thrombosis and filter removal.

Saddle Pulmonary Embolus and Simultaneous Common Carotid and Subclavian Artery Emboli.

A patent foramen ovale (PFO) is present in 27-35% of the population. Right to left cardiac shunts predispose patients to arterial emboli in the presence of venous thromboembolisms. Paradoxical embolus should be suspected in patients with deep venous thrombosis (DVT) and arterial emboli. A 45-year-old man with hypercoagulability and history of DVT presented with a week-long history of chest pain, shortness of breath, and left arm numbness. Imaging showed a saddle pulmonary embolus (PE) and emboli involving the aortic arch, left common carotid, and left subclavian artery. The patient proceeded with an endovascular thrombectomy of the pulmonary artery, followed by open thrombectomy. Echocardiogram confirmed a right to left intra-cardiac shunt consistent with a PFO. Paradoxical emboli are rare manifestations of venous thromboemboli in patients with right to left intra-cardiac shunts. Patients should be evaluated for these to help prevent further manifestations.

Effects of radiation and chemotherapy on adipose stem cells: Implications for use in fat grafting in cancer patients.

Autologous fat transplantation is a versatile tool in reconstructive surgery. Adipose-derived stem cells (ASCs) increase survival of fat grafts and thus are increasingly used for breast reconstruction in breast cancer patients. However, radiation and/or chemotherapy have been proposed to inhibit soft tissue regeneration in wound healing thus suggesting alteration in stem cell pathways. Therefore, elucidating effects of radiation and chemotherapy on ASCs is critical if one desires to enhance the survival of fat grafts in patients. This review outlines our work evaluating the function and recoverability of ASCs from radiation or chemotherapy patients, focusing specifically on their availability as a source of autologous stem cells for fat grafting and breast reconstruction in cancer patients. Even though evidence suggests radiation and chemotherapy negatively influence ASCs at the cellular level, the efficiency of the isolation and differentiation capacity did not appear influenced in patients after receiving chemotherapy treatment, although fat from radiated patients exhibited significantly altered ASC differentiation into endothelial-like cells. Further, the in vitro growth rates of patient's ASCs do not differ significantly before or after treatment. Taken together, these studies suggest ASCs as an important new tool for grafting and reconstruction even when radiation and chemotherapy treatment are involved.

An empirical analysis of homicides in Mexico through Machine Learning and statistical design of experiments

Homicide is one of the most important mortality causes that has reduced the Mexican life expectancy. That is why the aim of this work is to identify some sociodemographic and economic factors that can help explain homicides in Mexico and measure their impact, assuming the current conditions prevail. To do that, several Machine Learning (ML) methods were evaluated. The C5.0 model is best suited for the data at hand. After fine-tuning the algorithm, we used the estimated model to identify the main factors that explain homicides. Among these factors, eleven were selected that can be influenced by direct changes in domestic public policy, laws and/or regulations. These were used as input in a two-level fractional factorial Statistical Design of Experiments (DOE) to estimate their main effects and possible interactions. Although several of these factors had statistically significant effects on homicide rate, the one that had the biggest and direct impact from a practical perspective, was the Rule of Law Index (RLI). In fact, if we assumed that all states had the median RLI of 0.37, implementing domestic policies and procedures to move them all to the best RLI level could significantly reduce homicide rates.

El homicidio es una de las principales causas de muerte que ha reducido la esperanza de vida de los mexicanos. El objetivo de este trabajo es identificar algunos factores sociodemográficos y económicos que puedan ayudar a explicar homicidios en México y medir su impacto, suponiendo que las condiciones actuales permanecen. Para lograrlo, comparamos diferentes métodos de Aprendizaje de Máquina (AM). Para tal fin, se encuentra que el modelo C5.0 es el más adecuado. Después de hacer una calibración final del modelo, lo utilizamos para determinar los veinticinco principales factores que explican el fenómeno de homicidios. Se seleccionan 11 factores que se consideran pueden ser influenciados directamente por cambios en políticas públicas, leyes y/o regulaciones. Estos predictores fueron utilizados como entrada en un diseño de experimentos factorial fraccionado con dos niveles para estimar los principales efectos principales e interacciones posibles. A pesar de que varios de estos factores tuvieron impactos estadísticamente significativos, el que mostró tener el mayor impacto directo desde una perspectiva práctica fue el Índice de Estado de Derecho (IED). De hecho, asumiendo que todos los estados tuvieran el valor de IED de 0.37, correspondiente a la mediana en todo el país, si se implementaran políticas y procedimientos para ubicar a todos los estados al nivel del mejor estado en términos de IED, se lograría una reducción altamente significativa en la incidencia de homicidios en México.

Caring for critically injured children: An analysis of 56 pediatric damage control laparotomies.

BACKGROUND: Injury is the leading cause of death in children under 18 years. Damage control principles have been extensively studied in adults but remain relatively unstudied in children. Our primary study objective was to evaluate the use of damage control laparotomy (DCL) in critically injured children. METHODS: An American College of Surgeons-verified Level 1 trauma center review (1996-2013) of pediatric trauma laparotomies was undertaken. Exclusion criteria included: age older than 18 years, laparotomy for abdominal compartment syndrome or delayed longer than 2 hours after admission. Demographics, mechanism, resuscitation variables, injuries, need for DCL, and outcomes were evaluated. Independent t test, Mann-Whitney U test, Fisher's exact test, and single-factor analysis of variance assessed statistical significance. Study endpoints were hospital survival and DCL complications. RESULTS: Of 371 children who underwent trauma laparotomy, the median age (IQR; LQ-UQ) age was 16 (5; 11-17) years. Most (73%) were male injured by blunt mechanism (65%). Fifty-six (15%) children (Injury Severity Score [ISS], 33 (25; 17-42), pediatric trauma score 5 (6; 2-8), penetrating abdominal trauma index score [PATI] 29 (32; 12-44)) underwent DCL after major solid organ (63%), vascular (36%), thoracic (38%) and pelvic (36%) injury. DCL patients were older (16.5 (4; 14-18) vs. 16 (7; 10-17)) and were more severely injured (ISS, 33 [25; 17-42] vs. 16 [16; 9-25]), requiring greater intraoperative packed red blood cell transfusion (8 [13; 3.5-16.5] vs. 1 (0; [0-1] units) than definitive laparotomy counterparts. Nonsurvivors arrived in severe physiologic compromise (base deficit, 17 [17; 8-25] vs. 7 [4; 4-8]), requiring more frequent preoperative blood product transfusion (67% vs. 10%) after comparable injury (ISS survivors, 36 [23; 18-41] vs. nonsurvivors 26 (7; 25-32), p = 0.8880). Fifty-five percent of DCL patients survived (length of stay, 26 [21; 18-39] days) requiring 3 (2; 2-4) laparotomies during 4 (6; 2-8) days until closure (fascial, 90%; vicryl/split thickness skin grafting, 10%). DCL complications (surgical site infection, 18%; dehiscence, 2%; enterocutaneous fistula, 2%) were analyzed. When stratified by age (<15 years vs. 15-18 years) and period (1996-2006 vs. 2007-2013), no differences were found in injury severity or DCL outcomes (p > 0.05). After controlling for DCL, age, and gender, multivariate analysis indicated only ISS (odds ratio, 1.10 [95% confidence interval, 1.01 - 1.19], p = 0.0218) and arrival systolic blood pressure (odds ratio, 0.96 [95% confidence interval, 0.93-0.99], p = 0.0254) predicted mortality after severe injury. CONCLUSION: DCL is a proven, lifesaving surgical technique in adults. This report is the first to analyze the use of DCL in children with critical abdominal injuries. With similar survival and morbidity rates as critically injured adults, DCL merits careful consideration in children with critical abdominal injuries. LEVEL OF EVIDENCE: Therapeutic study, level IV.