OBSERVE: guidelines for the refinement of rodent cancer models.

Existing guidelines on the preparation (Planning Research and Experimental Procedures on Animals: Recommendations for Excellence (PREPARE)) and reporting (Animal Research: Reporting of In Vivo Experiments (ARRIVE)) of animal experiments do not provide a clear and standardized approach for refinement during in vivo cancer studies, resulting in the publication of generic methodological sections that poorly reflect the attempts made at accurately monitoring different pathologies. Compliance with the 3Rs guidelines has mainly focused on reduction and replacement; however, refinement has been harder to implement. The Oncology Best-practices: Signs, Endpoints and Refinements for in Vivo Experiments (OBSERVE) guidelines are the result of a European initiative supported by EurOPDX and INFRAFRONTIER, and aim to facilitate the refinement of studies using in vivo cancer models by offering robust and practical recommendations on approaches to research scientists and animal care staff. We listed cancer-specific clinical signs as a reference point and from there developed sets of guidelines for a wide variety of rodent models, including genetically engineered models and patient derived xenografts. In this Consensus Statement, we systematically and comprehensively address refinement and monitoring approaches during the design and execution of murine cancer studies. We elaborate on the appropriate preparation of tumor-initiating biologicals and the refinement of tumor-implantation methods. We describe the clinical signs to monitor associated with tumor growth, the appropriate follow-up of animals tailored to varying clinical signs and humane endpoints, and an overview of severity assessment in relation to clinical signs, implantation method and tumor characteristics. The guidelines provide oncology researchers clear and robust guidance for the refinement of in vivo cancer models.

Severity Classification of Laboratory Animal Procedures in Two Belgian Academic Institutions.

According to the EU Directive 2010/63, all animal procedures must be classified as non-recovery, mild, moderate or severe. Several examples are included in the Directive to help in severity classification. Since the implementation of the Directive, different publications and guidelines have been disseminated on the topic. However, due to the large variety of disease models and animal procedures carried out in many different animal species, guidance on the severity classification of specific procedures or models is often lacking or not specific enough. The latter is especially the case in disease models where the level of pain, suffering, distress and lasting harm depends on the duration of the study (for progressive disease models) or the dosage given (for infectious or chemically induced disease models). This, in turn, may lead to inconsistencies in severity classification between countries, within countries and even within institutions. To overcome this, two Belgian academic institutions with a focus on biomedical research collaborated to develop a severity classification for all the procedures performed. This work started with listing all in-house procedures and assigning them to 16 (sub)categories. First, we determined which parameters, such as clinical signs, dosage or duration, were crucial for severity classification within a specific (sub)category. Next, a severity classification was assigned to the different procedures, which was based on professional judgment by the designated veterinarians, members of the animal welfare body (AWB) and institutional animal ethics committee (AEC), integrating the available literature and guidelines. During the classification process, the use of vague terminology, such as 'minor impact', was avoided as much as possible. Instead, well-defined cut-offs between severity levels were used. Furthermore, we sought to define common denominators to group procedures and to be able to classify new procedures more easily. Although the primary aim is to address prospective severity, this can also be used to assess actual severity. In summary, we developed a severity classification for all procedures performed in two academic, biomedical institutions. These include many procedures and disease models in a variety of animal species for which a severity classification was not reported so far, or the terms that assign them to a different severity were too vague.

Clinically relevant dosing and pharmacokinetics of DNA-encoded antibody therapeutics in a sheep model.

DNA-encoded delivery and in vivo expression of antibody therapeutics presents an innovative alternative to conventional protein production and administration, including for cancer treatment. To support clinical translation, we evaluated this approach in 18 40-45 kg sheep, using a clinical-matched intramuscular electroporation (IM EP) and hyaluronidase-plasmid DNA (pDNA) coformulation setup. Two cohorts of eight sheep received either 1 or 4 mg pDNA encoding an ovine anti-cancer embryonic antigen (CEA) monoclonal antibody (mAb; OVAC). Results showed a dose-response with average maximum serum concentrations of respectively 0.3 and 0.7 µg/ml OVAC, 4-6 weeks after IM EP. OVAC was detected in all 16 sheep throughout the 6-week follow-up, and no anti-OVAC antibodies were observed. Another, more exploratory, cohort of two sheep received a 12 mg pOVAC dose. Both animals displayed a similar dose-dependent mAb increase and expression profile in the first two weeks. However, in one animal, an anti-OVAC antibody response led to loss of mAb detection four weeks after IM EP. In the other animal, no anti-drug antibodies were observed. Serum OVAC concentrations peaked at 4.9 µg/ml 6 weeks after IM EP, after which levels gradually decreased but remained detectable around 0.2 to 0.3 µg/ml throughout a 13-month follow-up. In conclusion, using a delivery protocol that is currently employed in clinical Phase 1 studies of DNA-based antibodies, we achieved robust and prolonged in vivo production of anti-cancer DNA-encoded antibody therapeutics in sheep. The learnings from this large-animal model regarding the impact of pDNA dose and host immune response on the expressed mAb pharmacokinetics can contribute to advancing clinical translation.

Injectable phosphopullulan-functionalized calcium-silicate cement for pulp-tissue engineering: An in-vivo and ex-vivo study.

OBJECTIVE: To evaluate, by means of an ex-vivo human tooth-culture model and in-vivo minipig animal study, the pulpal inflammatory reaction and reparative dentin-formation capacity of an injectable phosphopullulan-based calcium-silicate cement (GC, Tokyo, Japan) upon pulp capping, this in comparison with the commercial reference material Biodentine (Septodont). METHODS: For the ex-vivo tooth model, 9 freshly-extracted teeth from 3 different patients were pulp-capped with the experimental biomaterial (n = 3), Biodentine (n = 3) or left uncapped (control; n = 3). The teeth were kept in fresh culture medium for 4 weeks and, upon fixation three-dimensional Micro-CT and histology were performed. For the in-vivo animal study, 40 teeth from 3 minipigs were exposed and pulp capped with the experimental biomaterial containing phosphopullulan (n = 24) or Biodentine (n = 16) for 7 or 70 days. The inflammatory reaction and the tissue-regenerative potential was qualitatively and semi-quantitatively characterized using three-dimensional micro-CT and histology. RESULTS: Ex vivo, the treatment with the experimental phosphopullulan-based calcium-silicate cement and Biodentine stimulated the formation of fibrous tissue and mineralized foci. In vivo, early inflammatory reaction and regeneration of the pulp-tissue interface was promoted by both bioceramic materials after 7 and 70 days, respectively. SIGNIFICANCE: Our findings bring new insights into calcium-silicate-mediated dental pulp repair and regeneration. The novel ready-to-use and self-adhering functionalized calcium-silicate cement revealed effective pulpal repair potential.

Bridging the Clinical Gap for DNA-Based Antibody Therapy Through Translational Studies in Sheep.

Clinical translation of DNA-based administration of monoclonal antibodies (mAbs) is uncertain due to lack of large animal data. To bridge the clinical gap, we evaluated a panel of novel plasmid DNA (pDNA)-encoded mAbs in 40-70 kg sheep with a clinical intramuscular electroporation protocol. Injection of 4.8 mg of pDNA, encoding ovine anti-human CEA mAb (OVAC), led to peak plasma mAb titers of 300 ng/mL. OVAC remained detectable for 3 months and was boosted by a second pOVAC administration. Hyaluronidase muscle pretreatment increased OVAC concentrations up to 10-fold. These higher plasma titers, however, led to anti-drug antibodies (ADAs) toward the OVAC variable regions, resulting in loss of mAb detection and of adequate redosing. Transient immune suppression avoided ADA formation, with OVAC peaking at 3.5 µg/mL and remaining detectable for 11 months after pOVAC injection. DNA-based delivery of ovine anti-human EGFR mAb (OVAE), identical to OVAC except for the variable regions, preceded by hyaluronidase, allowed for at least three consecutive administrations in an immune-competent sheep, without ADA response. When tripling the pOVAE dose to 15 mg, transient ADAs of limited impact were observed; plasma OVAE peaked at 2.6 µg/mL and was detected up to 7 months. DNA-based anti-HER2 trastuzumab in sheep gave no detectable mAb concentrations despite previous validation in mice, highlighting the limitations of relying on small-rodent data only. In conclusion, our results highlight the potential and caveats of clinical DNA-based antibody therapy, can expedite preclinical and clinical development, and benefit the field of gene transfer as a whole.

Validation of the Fetal Lamb Model of Spina Bifida.

A randomized trial demonstrated that fetal spina bifida (SB) repair is safe and effective yet invasive. New less invasive techniques are proposed but are not supported by adequate experimental studies. A validated animal model is needed to bridge the translational gap to the clinic and should mimic the human condition. Introducing a standardized method, we comprehensively and reliably characterize the SB phenotype in two lamb surgical models with and without myelotomy as compared to normal lambs. Hindbrain herniation measured on brain magnetic resonance imaging (MRI) was the primary outcome. Secondary outcomes included gross examination with cerebrospinal fluid (CSF) leakage test, neurological examination with locomotor assessment, whole-body MRI, motor and somatosensory evoked potentials; brain, spinal cord, hindlimb muscles, bladder and rectum histology and/or immunohistochemistry. We show that the myelotomy model best phenocopies the anatomy, etiopathophysiology and symptomatology of non-cystic SB. This encompasses hindbrain herniation, ventriculomegaly, posterior fossa anomalies, loss of brain neurons; lumbar CSF leakage, hindlimb somatosensory-motor deficit with absence of motor and somatosensory evoked potentials due to loss of spinal cord neurons, astroglial cells and myelin; urinary incontinence. This model obtains the highest validity score for SB animal models and is adequate to assess the efficacy of novel fetal therapies.

Experimental tricalcium silicate cement induces reparative dentinogenesis.

OBJECTIVES: To overcome shortcomings of hydraulic calcium-silicate cements (hCSCs), an experimental tricalcium silicate (TCS) cement, named 'TCS 50', was developed. In vitro research showed that TCS 50 played no negative effect on the viability and proliferation of human dental pulp cells, and it induced cell odontogenic differentiation. The objective was to evaluate the pulpal repair potential of TCS 50 applied onto exposed minipig pulps. METHODS: Twenty permanent teeth from three minipigs were mechanically exposed and capped using TCS 50; half of the teeth were scheduled for 7-day and the other half for 70-day examination (n=10). Commercial hCSCs ProRoot MTA and TheraCal LC were tested as references (n=8). Tooth discoloration was examined visually. After animal sacrifice, the teeth were scanned using micro-computed tomography; inflammatory response at day 7 and day 70, mineralized tissue formation at day 70 were assessed histologically. RESULTS: Up to 70 days, TCS 50 induced no discoloration, ProRoot MTA generated gray/black discoloration in all teeth. For TCS 50, 40.0% pulps exhibited a mild/moderate inflammation at day 7. No inflammation was detected and complete reparative dentin with tubular structures was formed in all pulps after 70 days. ProRoot MTA induced a similar response, TheraCal LC generated a less favorable response in terms of initial inflammation and reparative dentin formation; however, these differences were not significant (Chi-square test of independence: p>0.05). SIGNIFICANCE: TCS 50 induced reparative dentinogenesis in minipig pulps. It can be considered as a promising pulp-capping agent, also for aesthetic areas.

Brain perfusion fixation in male pigs using a safer closed system.

Tissue fixation methods are well established for rodents, but not for large animals. We present a simple technique for in situ brain perfusion fixation in a male porcine model, using cervical vessels for inflow and outflow and achieving a closed system. Thirty-four pigs, aged 4.7 ± 0.6 months and weighing 60.7 ± 10.9 kg, were anaesthetised and mechanically ventilated. The ipsilateral common carotid artery and external jugular vein were dissected and constituted the inflow and outflow access, respectively. The brains were perfused and fixed in situ with heparinised saline followed by buffered formaldehyde. Then, specimens (brain, cerebellum and brainstem) were extracted and processed for histology. Fixative fluid leakage was avoided, achieving a closed system. This technique minimises the exposure to toxic chemicals such as formaldehyde and associated hazards (inherent toxicity, eye irritation), thereby increasing operators' safety. Perfusion was performed with a peristaltic pump for 20-30 minutes at an optimum rate of 0.20 l/min and required only 5 litres of the fixative. The specimens were sufficiently hardened to be extracted. High-quality tissues were available for histology analysis. This technique offers a user-friendly closed system for brain perfusion fixation which can be adapted for other tissues of the head, face and neck.

Quantitative analysis of motor evoked potentials in the neonatal lamb.

Evoking motor potentials are an objective assessment method for neuromotor function, yet this was to our knowledge never done in neonatal lambs. There is neither a method for standardized quantification of motor evoked potentials (MEPs). We first aimed to evaluate the feasibility of MEP recording in neonatal lambs and test its validity. Second we aimed to develop an algorithm for its quantification and test its reliability since manual input is required. We recorded myogenic MEPs after transcranial motor cortex stimulation in 6 lambs aged 1-2 days. MEPs were also measured in one lamb undergoing Neuro-Muscular Blockade (NMB) and another undergoing lumbar spinal cord (SC) transection, both serving as controls. We computed 5 parameters using a custom-made algorithm: motor threshold, latency, area-under-the-curve, peak-to-peak amplitude and duration. Intra- and inter-observer reliability was analyzed. MEPs could be easily recorded, disappearing after NMB and SC transection. The algorithm allowed for analysis, hence physiologic readings of the parameters in all 4 limbs of all lambs were obtained. Our method was shown to have high intra- and inter-observer ( ≥70%) reliability for latency, area-under-the-curve and peak-to-peak amplitude. These results suggest that standardized MEP recording and analysis in neonatal lambs is feasible, and can reliably assess neuromotor function.

A growing animal model for neonatal repair of large diaphragmatic defects to evaluate patch function and outcome.

OBJECTIVES: We aimed to develop a more representative model for neonatal congenital diaphragmatic hernia repair in a large animal model, by creating a large defect in a fast-growing pup, using functional pulmonary and diaphragmatic read outs. BACKGROUND: Grafts are increasingly used to repair congenital diaphragmatic hernia with the risk of local complications. Growing animal models have been used to test novel materials. METHODS: 6-week-old rabbits underwent fiberoptic intubation, left subcostal laparotomy and hemi-diaphragmatic excision (either nearly complete (n = 13) or 3*3cm (n = 9)) and primary closure (Gore-Tex patch). Survival was further increased by moving to laryngeal mask airway ventilation (n = 15). Sham operated animals were used as controls (n = 6). Survivors (90 days) underwent chest X-Ray (scoliosis), measurements of maximum transdiaphragmatic pressure and breathing pattern (tidal volume, Pdi). Rates of herniation, lung histology and right hemi-diaphragmatic fiber cross-sectional area was measured. RESULTS: Rabbits surviving 90 days doubled their weight. Only one (8%) with a complete defect survived to 90 days. In the 3*3cm defect group all survived to 48 hours, however seven (78%) died later (16-49 days) from respiratory failure secondary to tracheal stricture formation. Use of a laryngeal mask airway doubled 90-day survival, one pup displaying herniation (17%). Cobb angel measurements, breathing pattern, and lung histology were comparable to sham. Under exertion, sham animals increased their maximum transdiaphragmatic pressure 134% compared to a 71% increase in patched animals (p<0.05). Patched animals had a compensatory increase in their right hemi-diaphragmatic fiber cross-sectional area (p<0.0001). CONCLUSIONS: A primarily patched 3*3cm defect in growing rabbits, under laryngeal mask airway ventilation, enables adequate survival with normal lung function and reduced maximum transdiaphragmatic pressure compared to controls.

Trileaflet aortic valve reconstruction with a decellularized pericardial patch in a sheep model.

BACKGROUND: The purpose of this study was to provide a preliminary assessment of the performance of a decellularized pericardial patch in a trileaflet aortic valve reconstruction in a long-term juvenile sheep model. METHODS: A sheep surgical model was used to perform a complete trileaflet reconstruction (Ozaki technique) of the aortic valve with 3 separate pericardial patches. Valve function was assessed 1 week, 3 months, and 6 months after surgery via transthoracic echocardiography. Calcification resistance and host cell infiltration of the pericardial material were assessed at 6 months after surgery. RESULTS: Three of 6 sheep with implanted pericardial neo-cusps survived until the planned time of sacrifice after surgery, whereas 3 animals had a successful implant but died shortly after the procedure as the result of a bad recovery from cardiopulmonary bypass. Echocardiography at 6 months revealed a high coaptation area with only minimal regurgitation. In all explanted leaflets, cusp tissue was soft. There was only minimal calcification in 8 of 9 leaflets. CONCLUSIONS: Aortic valves reconstructed with a decellularized pericardial patch demonstrated adequate diastolic function with minimal regurgitation and resistance to calcification. Combining the Ozaki technique with this decellularized pericardial scaffold showed adequate hemodynamics, sustained mechanical integrity of the patch and limited calcification of the material. These results, together with earlier experimental and clinical data, indicate the potential of this material for aortic valve repair.

Evaluating lung injury at increasing time intervals in a murine brain death model.

INTRODUCTION: Only 15%-25% of brain death (BD) donors match the ideal donor criteria for lung transplantation. Lung injury may evolve in the hours after onset of brain death, but the evolution over time has not been well studied in lung. The aim of this study was to evaluate lung injury at different time points after BD using a murine model. MATERIALS AND METHODS: Male C57BL6/J mice (8-10 wk) were anesthetized, tracheotomized, and mechanically ventilated. Mice were randomly assigned to six groups (n=8/group): 1 h, 3 h, and 6 h sham ([SH1], [SH3], [SH6]) and 1 h, 3 h, and 6 h brain death ([BD1], [BD3], [BD6]). BD was gradually induced by a subdural balloon catheter. Heart rate and mean arterial pressure were continuously monitored. At the end of the experiment, bronchoalveolar lavage was performed and the left lung was excised for histopathologic analysis. RESULTS: The Cushing reflex was characterized by a rapid increase in heart rate and mean arterial pressure after balloon inflation in BD animals. An increase in percentage of neutrophils was seen with a longer follow-up period (P<0.05). Interleukin 6 and interleukin 10 levels in bronchoalveolar lavage progressively increased with longer time intervals after BD ([BD1] versus [BD6]; P<0.01). Histologic signs of lung injury (congestion, hemorrhage, and neutrophilic influx) were more pronounced in [BD3] and [BD6] compared with the other groups; however, this difference did not reach statistical significance. CONCLUSION: Three hours after brain death, significant signs of inflammation and lung injury were seen compared with sham-operated animals. This murine BD model gives us opportunities for further mechanistic studies regarding treatment of BD-related donor lung injury.

Azithromycin attenuates fibroblast growth factors induced vascular endothelial growth factor via p38(MAPK) signaling in human airway smooth muscle cells.

The airways in asthma and COPD are characterized by an increase in airway smooth muscle (ASM) mass and bronchial vascular changes associated with increased expression of pro-angiogenic growth factors, such as fibroblast growth factors (FGF-1 and FGF-2) and vascular endothelial growth factor (VEGF). We investigated the contribution of FGF-1/-2 in VEGF production in ASM cells and assessed the influence of azithromycin and dexamethasone and their underlying signaling mechanisms. Growth-synchronized human ASM cells were pre-treated with MAPK inhibitors, U0126 for ERK1/2(MAPK) and SB239063 for p38(MAPK) as well as with dexamethasone or azithromycin, 30 min before incubation with FGF-1 or FGF-2. Expression of VEGF (VEGF-A, VEGF121, and VEGF165) was assessed by quantitative PCR, VEGF release by ELISA and MAPK phosphorylation by Western blotting. Both FGF-1 and FGF-2 significantly induced mRNA levels of VEGF-A, VEGF121, and VEGF165. The VEGF protein release was increased 1.8-fold (FGF-1) and 5.5-fold (FGF-2) as compared to controls. Rapid transient increase in ERK1/2(MAPK) and p38(MAPK) phosphorylation and subsequent release of VEGF from FGF-1 or FGF-2-treated ASM cells were inhibited by respective blockers. Furthermore, azithromycin and dexamethasone significantly reduced both the VEGF release and the activation of p38(MAPK) pathway in response to FGF-1 or FGF-2 treatment. Our Results demonstrate that FGF-1 and FGF-2 up-regulate VEGF production via ERK1/2(MAPK) and p38(MAPK) pathways. Both azithromycin and dexamethasone elicited their anti-angiogenic effects via p38(MAPK) pathway in vitro, thereby suggesting a possible therapeutic approach to tackle VEGF-mediated vascular remodeling.

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

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.

Chronic rejection pathology after orthotopic lung transplantation in mice: the development of a murine BOS model and its drawbacks.

Almost all animal models for chronic rejection (CR) after lung transplantation (LTx) fail to resemble the human situation. It was our attempt to develop a representative model of CR in mice. Orthotopic LTx was performed in allografts receiving daily immunosuppression with steroids and cyclosporine. Controls included isografts and mice only undergoing thoracotomy (SHAM). Allografts were sacrificed 2, 4, 6, 8, 10 or 12 weeks after LTx. Pulmonary function was measured repeatedly in the 12w allografts, isografts and SHAM mice. Histologically, all allografts demonstrated acute rejection (AR) around the blood vessels and airways two weeks after LTx. This decreased to 50-75% up to 10 weeks and was absent after 12 weeks. Obliterative bronchiolitis (OB) lesions were observed in 25-50% of the mice from 4-12 weeks. Isografts and lungs of SHAM mice were normal after 12 weeks. Pulmonary function measurements showed a decline in FEV(0.1), TLC and compliance in the allografts postoperatively (2 weeks) with a slow recovery over time. After this initial decline, lung function of allografts increased more than in isografts and SHAM mice indicating that pulmonary function measurement is not a good tool to diagnose CR in a mouse. We conclude that a true model for CR, with clear OB lesions in about one third of the animals, but without a decline in lung function, is possible. This model is an important step forward in the development of an ideal model for CR which will open new perspectives in unraveling CR pathogenesis and exploring new treatment options.

Survival determinants in lung transplant patients with chronic allograft dysfunction.

BACKGROUND: Chronic lung allograft dysfunction (CLAD) remains the leading cause of mortality after lung transplantation. METHODS: In this retrospective single-center study, we aimed to identify different phenotypes of and risk factors for mortality after CLAD diagnosis using univariate and multivariate Cox proportional hazard survival regression analysis. RESULTS: CLAD was diagnosed in 71 of 294 patients (24.2%) at 30.9±22.8 months after transplantation. Pulmonary function was obstructive in 51 (71.8%) of the CLAD patients, restrictive in 20 (28.2%) patients, of whom 17 had persistent parenchymal infiltrates on pulmonary computer tomography (CAT) scan. In univariate analysis, previous development of neutrophilic reversible allograft dysfunction (NRAD, P=0.012) and a restrictive pulmonary function (P=0.0024) were associated with a worse survival, whereas there was a strong trend for early development of CLAD and persistent parenchymal infiltrates on CAT scan (P=0.067 and 0.056, respectively). In multivariate analysis, early development of CLAD (P=0.0067), previous development of NRAD (P=0.0016), and a restrictive pulmonary function pattern (P=0.0005) or persistent parenchymal infiltrates on CAT scan (P=0.0043) remained significant. CONCLUSION: Although most CLAD patients develop an obstructive pulmonary function, 28% develop a restrictive pulmonary function, compatible with the recently defined restrictive allograft syndrome phenotype. Early-onset CLAD, previous development of NRAD, and the development of restrictive allograft syndrome are associated with worse survival after CLAD has been diagnosed.

Medium-term outcome after lung transplantation is comparable between brain-dead and cardiac-dead donors.

BACKGROUND: Donation after cardiac death (DCD) to overcome the donor organ shortage is now moving into the clinical setting, but the medium-term outcome after DCD lung transplantation (LTx) remains largely unknown. METHODS: In this retrospective study, DCD LTx recipients (n = 21) were compared with a cohort of donation-after-brain-death (DBD) LTx recipients (n = 154) transplanted between February 2007 and July 2010. Immediate (post)operative outcome was evaluated by assessing need for peri-operative extracorporeal membrane oxygenation (ECMO), time to extubation, hospital discharge and primary graft dysfunction (PGD) within the first 48 hours. Survival, incidence of bronchiolitis obliterans syndrome (BOS), acute rejection (AR) and inflammatory markers in blood and in bronchoalveolar lavage (BAL) were assessed and compared over a median follow-up of 327 days for DCD and 531 days for DBD, showing no statistically significant difference (NS). RESULTS: There were no differences between groups with regard to patient characteristics except for a higher number of patients transplanted for obliterative bronchiolitis in the DCD group (4 of 21 vs 7 of 154; p < 0.05). In the DCD group, 2 of 21 patients died, vs 23 of 154 patients in the DBD group (NS). Actuarial survival rates at 6 months, 1 year and 3 years are 95%, 95% and 71% for the DCD group and 96%, 91% and 75% for the DBD group (NS). Three patients (14%) in the DCD group developed BOS vs 15 patients (10%) in the DBD group (NS). Survival and freedom from BOS were not different between the groups. AR, inflammatory markers and immediate (post)operative outcome also did not differ. CONCLUSIONS: In our experience, both early- and medium-term outcome in DCD lung recipients is comparable to that of DBD lung recipients. Use of lungs recovered from controlled donors after cardiac death is a safe option for expansion of the donor pool.

Montelukast for bronchiolitis obliterans syndrome after lung transplantation: a pilot study.

Bronchiolitis obliterans syndrome (BOS) remains the major hurdle to improve long-term survival after lung transplantation, as its treatment remains troublesome. In this pilot study, we investigated the effect of montelukast (a leukotriene receptor antagonist) on the FEV(1) decline after diagnosis of BOS and compared this with a control group. In both groups, 11 patients were included with BOS stage <3 and bronchoalveolar lavage (BAL) neutrophilia <15%, already being treated or concurrently being started on azithromycin. Control patients were selected retrospectively. After adding montelukast (10 mg/day) to the immunosuppressive regimen, the FEV(1) decline significantly decreased from 112 ± 26 ml/month before BOS diagnosis to 13 ± 13 ml/month after 6 months of montelukast therapy (P = 0.001). In the control group, there was no significant change in the rate of FEV(1) decline: 103 ± 20 ml/month before BOS diagnosis to 114 ± 27 ml/month (P = 0.55). Adding montelukast may be a promising treatment option in patients with low neutrophilic (<15%) BOS after lung transplantation, already or concurrently being treated with azithromycin.

Repeated invasive lung function measurements in intubated mice: an approach for longitudinal lung research.

Invasive lung function measurements are useful tools to describe respiratory disease models in mice but only result in one time-point measurements because of tracheostomy. We explored if intubation may overcome the need for tracheostomy thereby allowing invasive lung function monitoring of individual mice over time. Repeated invasive lung function measurements with Scireq(©) - FlexiVent or Buxco(©) - Forced Pulmonary Maneuvers(®) were performed three times in BALB/c mice with intervals of 10 days. Each lung function assessment following intubation was compared with a similar measurement in age-matched tracheostomized mice, the golden standard in lung function measurements. Tracheostomy and intubation gave similar results for resistance, elastance and compliance of the whole respiratory system as assessed by Flexivent. Likewise, Forced Pulmonary Maneuvers used to measure lung volumes such as total lung capacity, functional residual capacity, forced expiratory volume in 0.1 s and forced vital capacity, resulted in identical outcomes for both airway approaches. No interaction was found between the procedures for any of the pulmonary function variables. The observed changes over time were rather related to animal growth than to repetitive intubation. Eighty percent of the animals survived three consecutive intubations, which were hampered by transient breathing difficulties, weight loss and neutrophilic bronchoalveolar lavage immediately postextubation. Repetitive invasive lung function measurements by intubation are feasible and reproducible in healthy mice and results are comparable to the standard method. This may open new perspectives for longitudinal research in animal models of respiratory diseases.