Debby Thomas

Long-term nose-only cigarette smoke exposure induces emphysema and mild skeletal muscle dysfunction in mice.

SUMMARY Mouse models of chronic obstructive pulmonary disease (COPD) focus on airway inflammation and lung histology, but their use has been hampered by the lack of pulmonary function data in their assessment. Systemic effects such as muscle dysfunction are also poorly modeled in emphysematous mice. We aimed to develop a cigarette-smoke-induced emphysema mouse model in which serial lung function and muscular dysfunction could be assessed, allowing the disease to be monitored more appropriately. C57Bl6 mice were nose-only exposed to cigarette smoke or filtered air for 3–6 months. Lung function tests were repeated in the same mice after 3 and 6 months of cigarette smoke or air exposure and compared with lung histological changes. Contractile properties of skeletal muscles and muscle histology were also determined at similar time points in separate groups of mice. Serial lung function measurements documented hyperinflation after 3 and 6 months of cigarette smoke exposure, with a significant 31–37% increase in total lung capacity (TLC) and a significant 26–35% increase in compliance (Cchord) when compared with animals exposed to filtered air only (P<0.001 after 3 and after 6 months). These functional changes preceded the changes in mean linear intercept, which became only significant after 6 months of cigarette smoke exposure and which correlated very well with TLC (r=0.74, P=0.004) and Cchord (r=0.79, P=0.001). After 6 months of cigarette smoke exposure, a significant fiber-type shift from IIa to IIx/b was also observed in the soleus muscle (P<0.05), whereas a 20% reduction of force was present at high stimulation frequencies (80 Hz; P=0.09). The extensor digitorum longus (EDL) muscle was not affected by cigarette smoke exposure. These serial pulmonary function variables are sensitive outcomes to detect emphysema progression in a nose-only cigarette-smoke-exposed animal model of COPD. In this model, muscular changes became apparent only after 6 months, particularly in muscles with a mixed fiber-type composition.

Bortezomib partially protects the rat diaphragm from ventilator-induced diaphragm dysfunction.

Objective: Controlled mechanical ventilation leads to diaphragmatic contractile dysfunction and atrophy. Since proteolysis is enhanced in the diaphragm during controlled mechanical ventilation, we examined whether the administration of a proteasome inhibitor, bortezomib, would have a protective effect against ventilator-induced diaphragm dysfunction. Design: Randomized, controlled experiment. Settings: Basic science animal laboratory. Interventions: Anesthetized rats were submitted for 24 hrs to controlled mechanical ventilation while receiving 0.05 mg/kg bortezomib or saline. Control rats were acutely anesthetized. Measurements and Main Results: After 24 hrs, diaphragm force production was significantly lower in mechanically ventilated animals receiving an injection of saline compared to control animals (−36%, p < .001). Importantly, administration of bortezomib improved the diaphragmatic force compared to mechanically ventilated animals receiving an injection of saline (+15%, p < .01), but force did not return to control levels. Compared to control animals, diaphragm cross-sectional area of the type IIx/b fibers was significantly decreased by 28% in mechanically ventilated animals receiving an injection of saline (p < .01) and by 16% in mechanically ventilated animals receiving an injection of bortezomib (p < .05). Diaphragmatic calpain activity was significantly increased in mechanically ventilated animals receiving an injection of saline (+52%, p < .05) and in mechanically ventilated animals receiving an injection of bortezomib (+36%, p < .05). Caspase-3 activity was increased after controlled mechanical ventilation with saline by 55% (p < .05), while it remained similar to control animals in mechanically ventilated animals receiving an injection of bortezomib. Diaphragm 20S proteasome activity was slightly increased in both ventilated groups, and the amount of ubiquitinated proteins was significantly and similarly enhanced in mechanically ventilated animals receiving an injection of saline and mechanically ventilated animals receiving an injection of bortezomib. Conclusions: These data show that the administration of bortezomib partially protects the diaphragm from controlled mechanical ventilation–induced diaphragm contractile dysfunction without preventing atrophy. The fact that calpain activity was still increased after bortezomib treatment may explain the persistence of atrophy. Part of bortezomib effects might have been due to its ability to inhibit caspase-3 in this model.

Effects of controlled mechanical ventilation on sepsis-induced diaphragm dysfunction in rats.

Objectives:Diaphragm dysfunction develops during severe sepsis as a consequence of hemodynamic, metabolic, and intrinsic abnormalities. Similarly, 12 hours of controlled mechanical ventilation also promotes diaphragm dysfunction. Importantly, patients with sepsis are often treated with mechanical ventilation for several days. It is unknown if controlled mechanical ventilation exacerbates sepsis-induced diaphragm dysfunction, and this forms the basis for these experiments. We investigate the effects of 12-hour controlled mechanical ventilation on contractile function, fiber dimension, cytokine production, proteolysis, autophagy, and oxidative stress in the diaphragm of septic rats. Design:Randomized controlled experiment. Setting:Animal research laboratory. Subjects:Adult male Wistar rats. Interventions:Treatment with a single intraperitoneal injection of either saline or Escherichia coli lipopolysaccharide (5 mg/kg). After 12 hours, the saline-treated animals (controlled mechanical ventilation) and half of the septic animals (lipopolysaccharide + controlled mechanical ventilation) were submitted to 12 hours of controlled mechanical ventilation while the remaining septic animals (lipopolysaccharide) were breathing spontaneously for 12 hours. They were compared to a control group. All animals were studied 24 hours after saline or lipopolysaccharide administration. Measurements and Main Results:Twenty-four hours after saline or lipopolysaccharide administration, diaphragm contractility was measured in vitro. We also measured diaphragm muscle fiber dimensions from stained cross sections, and inflammatory cytokines were determined by proteome array. Activities of calpain, caspase-3, and proteasome, expression of 20S-proteasome &agr; subunits, E2 conjugases, E3 ligases, and autophagy were measured with immunoblotting and quantitative polymerase chain reaction. Lipopolysaccharide and/or controlled mechanical ventilation independently decreased diaphragm contractility and fiber dimensions and increased diaphragm interleukin-6 production, protein ubiquitination, expression of Atrogin-1 and Murf-1, calpain and caspase-3 activities, autophagy, and protein oxidation. Compared with lipopolysaccharide alone, lipopolysaccharide + controlled mechanical ventilation worsened diaphragm contractile dysfunction, augmented diaphragm interleukin-6 levels, autophagy, and protein oxidation, but exerted no exacerbating effects on diaphragm fiber dimensions, calpain, caspase-3, or proteasome activation. Conclusions:Twelve hours of controlled mechanical ventilation potentiates sepsis-induced diaphragm dysfunction, possibly due to increased proinflammatory cytokine production and autophagy and worsening of oxidative stress.

Characterization of patient-derived tumor xenograft models of endometrial cancer for preclinical evaluation of targeted therapies.

OBJECTIVE Endometrial carcinoma (EC) is the sixth most common cancer in women and therapies are limited for advanced and recurrent disease. Patient-derived tumor xenograft (PDTX) models are becoming popular tools in translational research because of their histological and genetic similarity to the original tumors and the ability to predict therapeutic response to treatments. Here, we established and characterized a panel of 24 EC PDTX models which includes the major histological and genetic subtypes observed in patients. METHODS Fresh tumor tissues collected from primary, metastatic and recurrent type I and type II EC patients were engrafted in immunocompromised mice. Histology, vimentin, and cytokeratin expression were evaluated, together with Microsatellite instability (MSI), mutation profiling by Whole Exome Sequencing and copy number profiling by Whole Genome Low Coverage Sequencing. The efficacy of both PI3K and MEK inhibitors was evaluated in a model of endometrioid carcinoma harboring PTEN, PIK3CA and KRAS mutations. RESULTS We observed good similarity between primary tumors and the corresponding xenografts, at histological and genetic level. Among the engrafted endometrioid models, we found a significant enrichment of MSI and POLE mutated tumors, compared to non-engrafted samples. Combination treatment with NVP-BEZ235 and AZD6244 showed the possibility to stabilize the tumor growth in one model originated from a patient who already received several lines of chemotherapy. CONCLUSION The established EC PDTX models, resembling the original human tumors, promise to be useful for preclinical evaluation of novel combination and targeted therapies in specific EC subgroups.

Sedation using propofol induces similar diaphragm dysfunction and atrophy during spontaneous breathing and mechanical ventilation in rats

Background:Mechanical ventilation is crucial for patients with respiratory failure. The mechanical takeover of diaphragm function leads to diaphragm dysfunction and atrophy (ventilator-induced diaphragmatic dysfunction), with an increase in oxidative stress as a major contributor. In most patients, a sedative regimen has to be initiated to allow tube tolerance and ventilator synchrony. Clinical data imply a correlation between cumulative propofol dosage and diaphragm dysfunction, whereas laboratory investigations have revealed that propofol has some antioxidant properties. The authors hypothesized that propofol reduces markers of oxidative stress, atrophy, and contractile dysfunction in the diaphragm. Methods:Male Wistar rats (n = 8 per group) were subjected to either 24 h of mechanical ventilation or were undergone breathing spontaneously for 24 h under propofol sedation to test for drug effects. Another acutely sacrificed group served as controls. After sacrifice, diaphragm tissue was removed, and contractile properties, cross-sectional areas, oxidative stress, and proteolysis were examined. The gastrocnemius served as internal control. Results:Propofol did not protect against diaphragm atrophy, oxidative stress, and protease activation. The decrease in tetanic force compared with controls was similar in the spontaneous breathing group (31%) and in the ventilated group (34%), and both groups showed the same amount of muscle atrophy. The gastrocnemius muscle fibers did not show atrophy. Conclusions:Propofol does not protect against ventilator-induced diaphragmatic dysfunction or oxidative injury. Notably, spontaneous breathing under propofol sedation resulted in the same amount of diaphragm atrophy and dysfunction although diaphragm activation per se protects against ventilator-induced diaphragmatic dysfunction. This makes a drug effect of propofol likely.

Time course of diaphragm function recovery after controlled mechanical ventilation in rats

Controlled mechanical ventilation (CMV) is known to result in rapid and severe diaphragmatic dysfunction, but the recovery response of the diaphragm to normal function after CMV is unknown. Therefore, we examined the time course of diaphragm function recovery in an animal model of CMV. Healthy rats were submitted to CMV for 24-27 h (n = 16), or to 24-h CMV followed by either 1 h (CMV + 1 h SB, n = 9), 2 h (CMV + 2 h SB, n = 9), 3 h (CMV + 3 h SB, n = 9), or 4-7 h (CMV + 4-7 h SB, n = 9) of spontaneous breathing (SB). At the end of the experiment, the diaphragm muscle was excised for functional and biochemical analysis. The in vitro diaphragm force was significantly improved in the CMV + 3 h SB and CMV + 4-7 h SB groups compared with CMV (maximal tetanic force: +27%, P < 0.05, and +59%, P < 0.001, respectively). This was associated with an increase in the type IIx/b fiber dimensions (P < 0.05). Neutrophil influx was increased in the CMV + 4-7 h SB group (P < 0.05), while macrophage numbers remained unchanged. Markers of protein synthesis (phosphorylated Akt and eukaryotic initiation factor 4E binding protein 1) were significantly increased (±40%, P < 0.001, and ±52%, P < 0.01, respectively) in the CMV + 3 h SB and CMV + 4-7 h SB groups and were positively correlated with diaphragm force (P < 0.05). Finally, also the maximal specific force generation of skinned single diaphragm fibers was increased in the CMV + 4-7 h SB group compared with CMV (+45%, P < 0.05). In rats, reloading the diaphragm for 3 h after CMV is sufficient to improve diaphragm function, while complete recovery occurs after longer periods of reloading. Enhanced muscle fiber dimensions, increased protein synthesis, and improved intrinsic contractile properties of diaphragm muscle fibers may have contributed to diaphragm function recovery.

Prolonged mechanical ventilation alters the expression pattern of angio-neogenetic factors in a pre-clinical rat model.

Objective Mechanical ventilation (MV) is a life saving intervention for patients with respiratory failure. Even after 6 hours of MV, diaphragm atrophy and dysfunction (collectively referred to as ventilator-induced diaphragmatic dysfunction, VIDD) occurs in concert with a blunted blood flow and oxygen delivery. The regulation of hypoxia sensitive factors (i.e. hypoxia inducible factor 1α, 2α (HIF-1α,–2α), vascular endothelial growth factor (VEGF)) and angio-neogenetic factors (angiopoietin 1–3, Ang) might contribute to reactive and compensatory alterations in diaphragm muscle. Methods Male Wistar rats (n = 8) were ventilated for 24 hours or directly sacrificed (n = 8), diaphragm and mixed gastrocnemius muscle tissue was removed. Quantitative real time PCR and western blot analyses were performed to detect changes in angio-neogenetic factors and inflammatory markers. Tissues were stained using Isolectin (IB 4) to determine capillarity and calculate the capillary/fiber ratio. Results MV resulted in up-regulation of Ang 2 and HIF-1α mRNA in both diaphragm and gastrocnemius, while VEGF mRNA was down-regulated in both tissues. HIF-2α mRNA was reduced in both tissues, while GLUT 4 mRNA was increased in gastrocnemius and reduced in diaphragm samples. Protein levels of VEGF, HIF-1α, -2α and 4 did not change significantly. Additionally, inflammatory cytokine mRNA (Interleukin (IL)-6, IL-1β and TNF α) were elevated in diaphragm tissue. Conclusion The results demonstrate that 24 hrs of MV and the associated limb disuse induce an up-regulation of angio-neogenetic factors that are connected to HIF-1α. Changes in HIF-1α expression may be due to several interactions occurring during MV.

Potential Targets' Analysis Reveals Dual PI3K/mTOR Pathway Inhibition as a Promising Therapeutic Strategy for Uterine Leiomyosarcomas-an ENITEC Group Initiative

Purpose: Uterine sarcomas are rare and heterogeneous tumors characterized by an aggressive clinical behavior. Their high rates of recurrence and mortality point to the urgent need for novel targeted therapies and alternative treatment strategies. However, no molecular prognostic or predictive biomarkers are available so far to guide choice and modality of treatment. Experimental Design: We investigated the expression of several druggable targets (phospho-S6S240 ribosomal protein, PTEN, PDGFR-α, ERBB2, and EGFR) in a large cohort of human uterine sarcoma samples (288), including leiomyosarcomas, low-grade and high-grade endometrial stromal sarcomas, undifferentiated uterine sarcomas, and adenosarcomas, together with 15 smooth muscle tumors of uncertain malignant potential (STUMP), 52 benign uterine stromal tumors, and 41 normal uterine tissues. The potential therapeutic value of the most promising target, p-S6S240, was tested in patient-derived xenograft (PDX) leiomyosarcoma models. Results: In uterine sarcomas and STUMPs, S6S240 phosphorylation (reflecting mTOR pathway activation) was associated with higher grade (P = 0.001) and recurrence (P = 0.019), as shown by logistic regression. In addition, p-S6S240 correlated with shorter progression-free survival (P = 0.034). Treatment with a dual PI3K/mTOR inhibitor significantly reduced tumor growth in 4 of 5 leiomyosarcoma PDX models (with tumor shrinkage in 2 models). Remarkably, the 4 responding models showed basal p-S6S240 expression, whereas the nonresponding model was scored as negative, suggesting a role for p-S6S240 in response prediction to PI3K/mTOR inhibition. Conclusions: Dual PI3K/mTOR inhibition represents an effective therapeutic strategy in uterine leiomyosarcoma, and p-S6S240 expression is a potential predictive biomarker for response to treatment. Clin Cancer Res; 23(5); 1274–85. ©2017 AACR.

Abdominal distension as the first echographic sign of hydrometrocolpos in a female fetus

We report a 31 weeks gestation female fetus with marked abdominal distension on echography as the first sign of hydrometrocolpos. Postmortem examination revealed a massive plastic peritonitis due to the spillage of genital secretions into the peritoneal cavity with secondary lung hypoplasia and obstructive renal dysplasia type IV.

Establishment and characterization of uterine sarcoma and carcinosarcoma patient-derived xenograft models

OBJECTIVE Uterine sarcomas (US) and carcinosarcomas (CS) are rare, aggressive cancers. The lack of reliable preclinical models hampers the search for new treatment strategies and predictive biomarkers. To this end, we established and characterized US and CS patient-derived xenograft (PDX) models. METHODS Tumor fragments of US and CS were subcutaneously implanted into immunocompromised mice. Engrafted xenograft and original tumors were compared by means of histology, immunohistochemistry, whole-genome low-coverage sequencing for copy number variations, and RNA sequencing. RESULTS Of 13 implanted leiomyosarcomas (LMS), 10 engrafted (engraftment rate of 77%). Also 2 out of 7 CS (29%) and one high-grade US (not otherwise specified) models were successfully established. LMS xenografts showed high histological similarity to their corresponding human tumors. Expression of desmin and/or H-caldesmon was detected in 8/10 LMS PDX models. We noticed that in CS models, characterized by the concomitant presence of a mesenchymal and an epithelial component, the relative distribution of the components is varying over the generations, as confirmed by changes in vimentin and cytokeratin expression. The similarity in copy number profiles between original and xenograft tumors ranged from 57.7% to 98.2% for LMS models and from 47.4 to 65.8% for CS models. Expression pattern stability was assessed by clustering RNA expression levels of original and xenograft tumors. Six xenografts clustered together with their original tumor, while 3 (all LMS) clustered apart. CONCLUSIONS We present here a panel of clinically annotated uterine sarcoma and carcinosarcoma PDX models, which will be a useful tool for preclinical testing of new therapies.