Neuroprotection is improved by watertightness of fetal spina bifida repair in the sheep model.

OBJECTIVES: A contributing factor to unsuccessful prenatal spina bifida aperta (SBA) repair via an open approach may be incomplete neurosurgical repair causing persistent in-utero leakage of cerebrospinal fluid (CSF) and exposure of the fetal spinal cord to amniotic fluid. We aimed to investigate the neurostructural and neurofunctional efficacy of watertight prenatal SBA repair in a validated SBA fetal lamb model. METHODS: A well-powered superiority study was conducted in the validated SBA fetal lamb model (n = 7 per group). The outcomes of lambs which underwent watertight or non-watertight multilayer repair through an open approach were compared to those of unrepaired SBA lambs (historical controls) at delivery (term = 145 days). At ∼75 days, fetal lambs underwent standardized induction of lumbar SBA. At ∼100 days, they were assigned to an either watertight or non-watertight layered repair group based on an intraoperative watertightness test using subcutaneous fluorescein injection. At 1-2 days postnatally, as primary outcome, we assessed reversal of hindbrain herniation using magnetic resonance imaging (MRI). Secondary proxies of neuroprotection were: absence of CSF leakage at the repair site; hindlimb motor function based on joint-movement score, locomotor grade and Motor Evoked Potential (MEP); four-score neuroprotection scale, encompassing live birth, complete hindbrain herniation reversal, absence of CSF leakage and joint-movement score ≥ 9/15; and brain and spinal cord histology and immunohistochemistry. As the watertightness test cannot be used clinically due to its invasiveness, we developed a potential surrogate intraoperative three-score skin-repair-quality scale based on visual assessment of the quality of the skin repair (suture inter-run distance ≤ 3 mm, absence of tear and absence of ischemia), with high quality defined by a score ≥ 2/3 and low quality by a score < 2/3, and assessed its relationship with improved outcome. RESULTS: Compared with unrepaired lambs, lambs with watertight repair achieved a high level of neuroprotection (neuroprotection score of 4/4 in 5/7 vs 0/7 lambs) as evidenced by: a significant 100% (vs 14%) reversal of hindbrain herniation on MRI; low CSF leakage (14% vs 100%); better hindlimb motor function, with higher joint-movement score, locomotor grade and MEP area under the curve and peak-to-peak amplitude; higher neuronal density in the hippocampus and corpus callosum; and higher reactive astrogliosis at the SBA lesion epicenter. Conversely, lambs with non-watertight SBA repair did not achieve the same level of neuroprotection (score of 4/4 in 1/7 lambs) compared with unrepaired lambs, with: a non-significant 86% (vs 14%) reversal of hindbrain herniation; high CSF leakage (43% vs 100%); no improvement in motor function; low brain neuron count in both the hippocampus and corpus callosum; and small spinal astroglial cell area at the epicenter. Both watertight layered repair and high (≥ 2/3) intraoperative skin-repair-quality score were associated with improved outcome, but the watertightness test and skin-repair-quality scale could not be used interchangeably due to result discrepancies. CONCLUSIONS: Watertight layered fetal SBA repair is neuroprotective since it improves brain and spinal-cord structure and function in the fetal lamb model. This translational research has important clinical implications. A neurosurgical technique that achieves watertightness should be adopted in all fetal centers to improve neuroprotection. Future clinical studies could assess whether a high skin-repair-quality score (≥ 2/3) correlates with neuroprotection. © 2022 International Society of Ultrasound in Obstetrics and Gynecology.

A randomised controlled trial of azithromycin to prevent chronic rejection after lung transplantation.

Azithromycin reduces airway inflammation and improves forced expiratory volume in 1 s (FEV1) in chronic rejection or bronchiolitis obliterans syndrome (BOS) after lung transplantation (LTx). Azithromycin prophylaxis might prevent BOS. A double-blind randomised controlled trial of azithromycin (n = 40) or placebo (n = 43), initiated at discharge and administered three times a week for 2 yrs, was performed in 2005-2009 at the Leuven University Hospital (Leuven, Belgium). Primary end-points were BOS-free and overall survival 2 yrs after LTx; secondary end-points were acute rejection, lymphocytic bronchiolitis and pneumonitis rate, prevalence of pseudomonal airway colonisation or gastro-oesophageal reflux, and change in FEV1, airway and systemic inflammation over time. Patients developing BOS were assessed for change in FEV1 with open-label azithromycin. BOS occurred less in patients receiving azithromycin: 12.5 versus 44.2% (p = 0.0017). BOS-free survival was better with azithromycin (hazard ratio 0.27, 95% CI 0.092-0.816; p = 0.020). Overall survival, acute rejection, lymphocytic bronchiolitis, pneumonitis, colonisation and reflux were comparable between groups. Patients receiving azithromycin demonstrated better FEV1 (p = 0.028), and lower airway neutrophilia (p = 0.015) and systemic C-reactive protein levels (p = 0.050) over time. Open-label azithromycin for BOS improved FEV1 in 52.2% patients. No serious adverse events were noted. Azithromycin prophylaxis attenuates local and systemic inflammation, improves FEV1 and reduces BOS 2 yrs after LTx.

Azithromycin reduces pulmonary fibrosis in a bleomycin mouse model.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease without proper treatment. Despite intensive research, the exact underlying pathogenesis remains elusive. It is regarded as a continuous injury, resulting in inflammation, infiltration, and proliferation of fibroblasts and extracellular matrix deposition, leading to an irreversible restrictive lung function deterioration and death. In this study the effect of azithromycin, a macrolide antibiotic on bleomycin-induced pulmonary fibrosis was investigated. C57BL/6 mice were intratracheally instilled with bleomycin (0.5 mg/kg) or saline. In the bleomycin group, half of the animals received azithromycin every other day from day 1 on. Bronchoalveolar lavage and histology were performed at days 7 and 35, and pulmonary function tests on day 35. At day 35, fibrotic lesions (spindle cell proliferation/collagen I deposition) were paralleled by a restrictive lung function pattern. Alterations were found in neutrophils and macrophages (innate immunity) and in T(H)2, T(H)17, and Treg cytokines (adaptive immunity). Azithromycin significantly reduced both fibrosis and the restrictive lung function pattern. This study demonstrated a beneficial effect of azithromycin on bleomycin-induced pulmonary fibrosis. A possible mechanism could be a modulation of both innate immunity and adaptive immunity. These findings might suggest a potential role for azithromycin in the treatment of IPF.

Follicular bronchiolitis: a rare cause of bronchiolitis obliterans syndrome after lung transplantation: a case report.

This case report is the first confirmed case of follicular bronchiolitis (FB), a rare bronchiolar disorder characterized by peribronchiolar lymphoid follicles, in a series of over 400 lung transplantations performed in our center. It is to our knowledge, the first publication describing FB after lung transplantation (LTx), presenting as chronic allograft dysfunction or bronchiolitis obliterans syndrome (BOS).

Plasma C-reactive protein levels correlate with markers of airway inflammation after lung transplantation: a role for systemic inflammation in bronchiolitis obliterans syndrome?

OBJECTIVE: Plasma C-reactive protein (CRP) concentration has been associated with allograft dysfunction in cardiac and renal transplantation; data in lung transplantation (LTx), however, are lacking. We hypothesized that in Ltx, systemic inflammation may be associated with airway inflammation, which has an important role in the development of chronic allograft dysfunction or bronchiolitis obliterans syndrome after LTx. METHODS: In this retrospective, longitudinal, cohort study, plasma CRP concentration, bronchoalveolar lavage (BAL) inflammatory markers (interleukin [IL]-6 and IL-8 protein levels and cell differentials), and pulmonary function (forced expiratory volume in 1 second) were evaluated in 100 LTx recipients at discharge and at 3-, 6-, and 12-month follow-up. The Spearman rank test was used to determine a possible relationship between these parameters at each routine follow-up visit. RESULTS: Plasma CRP concentration positively correlated with BAL total cell count and neutrophilia, whereas there was a negative correlation with pulmonary function at discharge and at 3 and 6 months after LTx. A correlation between plasma CRP concentration and BAL interleukin levels was present at discharge (IL-6 and IL-8) and at 6 months (IL-8) after LTx. CONCLUSION: Systemic inflammation and IL-8-mediated neutrophilic airway inflammation seem to be associated after LTx. Therefore, systemic inflammation has a possible role in the development of bronchiolitis obliterans syndrome after LTx.

The role of the IL23/IL17 axis in bronchiolitis obliterans syndrome after lung transplantation.

Bronchiolitis obliterans syndrome (BOS) is the leading cause of death after lung transplantation. Treatment is challenging, as the precise pathophysiology remains unclear. We hypothesize that T(H)17 lineage plays a key role in the pathophysiology of BOS by linking T-cell activation to neutrophil influx and chronic inflammation. In a cross-sectional study, bronchoalveolar lavage (BAL) samples of 132 lung transplant recipients were analyzed. Patients were divided in four groups: stable or suffering from infection (INF), acute rejection (AR) or BOS. The upstream T(H)17 skewing (TGF-beta/IL1beta/IL6/IL23), T(H)17 counteracting (IL2), T(H)17 effector cytokine (IL17) and the principal neutrophil-attracting chemokine (IL8), were quantified at the mRNA or protein level in combination with the cell profiles. The BOS group (n = 36) showed an increase in IL1beta protein (x1.5), IL6 protein (x3), transforming growth factor-beta (TGF-beta) mRNA (x3), IL17 mRNA (x20), IL23 mRNA (x10), IL8 protein (x2), IL8 mRNA (x3) and a decrease in IL2 protein (x0.8). The infection group (n = 11) demonstrated an increase in IL1beta protein (x5), IL6 protein (x20), TGF-beta mRNA (x10), IL17 mRNA (x300), IL23 mRNA (x200) and IL8 protein (x6). The acute rejection group (n = 43) only revealed an increase in IL6 protein (x6) and IL8 protein (x2) and a decrease in IL2 protein (x0.7). Lymphocytes and neutrophils were increased in all groups compared to the stable (n = 42). Our findings demonstrate the IL23/IL17 axis to be involved in the pathophysiology of BOS potentially triggering the IL8-mediated neutrophilia. IL6, IL1beta and IL23 seem to be skewing cytokines and IL2 a counteracting cytokine for T(H)17 alignment. The involvement of TGF-beta could not be confirmed, either as T(H)17 steering or as counteracting cytokine.

The role of recipient derived interleukin-17A in a murine orthotopic lung transplant model of restrictive chronic lung allograft dysfunction.

The single most important cause of late mortality after lung transplantation is chronic lung allograft dysfunction (CLAD). However, the pathological development of CLAD was not as simple as previously presumed and subclassification phenotypes, bronchiolitis obliterans syndrome (BOS) and restrictive CLAD (rCLAD), have been introduced. We want to re-investigate how CLAD manifests in the murine orthotopic lung transplant model and investigate the role of interleukin 17A (IL-17A) within this model. Orthotopic LTx was performed in CB57BL/6, IL-17 WT and IL-17 KO mice. In a first experiment, CB57BL/6 mice receiving an isograft (CB57BL/6) or allograft (BALB/C) were compared. In a second experiment IL-17 WT and IL-17 KO mice (both CB57BL/6 background) received an allograft (BALB/C). Mice received daily immunosuppression with steroids and cyclosporine and were sacrificed 10weeks after transplantation for histopathological analysis by an experienced lung pathologist. After murine orthotopic lung transplantation, the allograft histopathologically presented features of human rCLAD (i.e. overt inflammation, pleural/parenchymal fibrosis and obliterative bronchiolitis). In the IL-17A KO group, less inflammation in the bronchovascular axis (p=0.03) was observed and a non-significant trend towards less bronchovascular fibrosis, pleural/septal inflammation and fibrosis, and parenchymal inflammation and fibrosis when compared to WT mice. The major mismatch orthotopic lung transplant model resembles features of human rCLAD. IL-17A mediated immunity is involved in the inflammatory component, but had little influence on the degree of fibrosis. Further mechanistic and therapeutic studies in this mouse model are needed to fully understand the mechanisms in rCLAD.

Post-operative echocardiographic evaluation of bioprosthetic mitral valve implantation in sheep.

The ovine model is generally considered to be the best for testing bioprosthetic heart valve durability. Although echocardiography is the method of choice for the interim evaluation of the valve, literature on sheep echocardiography is scarce. Within the context of a study on treatment of pericardial heart valve prostheses, 19 adolescent sheep underwent transthoracic echocardiography six days after mitral implantation of bioprosthetic valves. Echocardiographic examination was performed under mild anesthesia and animals were put in a right lateral decubitus position. Four images were obtained: right parasternal long axis four and five chamber views, right parasternal long axis view with left ventricular outflow, and right parasternal short axis view through the mitral valve. We measured aortic annulus and velocity time integral over the aortic valve to determine stroke volume, cardiac output and cardiac index. The mitral valve was evaluated through color Doppler imaging for valvular and paravalvular leakages. Pulsed wave spectral Doppler was used for the measurement of velocities, pressures and velocity time integrals. For the evaluation of valve stenosis deceleration time and pressure half-time were determined. Effective orifice area of the mitral valve was derived. And, although not measured, other structures could clearly be visualized: right and left ventricle and atrium, wall thicknesses, tricuspid valve. This study shows that echocardiography in sheep is feasible, and that right parasternal images, obtained in animals in a right lateral decubitus position, are well qualified for the interim evaluation of bioprosthetic valves implanted in the mitral position. Besides the implanted valve, other cardiac structures like atria and ventricles can be visualized and evaluated.

Neutrophilic reversible airways dysfunction after liver transplantation: a case report.

In the present case report we have described a 46-year-old female patient who underwent a liver transplantation in 1998 for polycystic disease and developed a syndrome of increasing dyspnea, with sputum production and a progressive decline in pulmonary function [forced expiratory volume in one second (FEV(1)) (decreased from 153% predicted to 87% predicted). Further examination revealed an impressive tree in a bud pattern with diffuse peribronchiolar infiltrates on computed axial tomographic scan of the thorax. Sputum cultures remained negative. Bronchoscopic central airway biopsy specimens showed lymphocytic bronchitis; sputum induction showed 92% neutrophils. This condition was similar to the bronchiolitis obliterans syndrome after lung transplantation, although the specific neutrophilic phenotype of bronchiolitis obliterans syndrome has recently been renamed as neutrophilic reversible allograft/airway dysfunction, based on a progressive decline in FEV(1), neutrophilic airway inflammation and its response to neomacrolides. Additional azithromycin treatment resulted in complete recovery in our patient, with normalization of FEV(1) and computed axial tomographic scan of the thorax at 3 months after initiation. This case report suggests that neutrophilic reversible allograft airway dysfunction can no longer be diagnosed only after lung transplantation. Moreover, it demonstrates that this condition is not always related to allograft rejection, but rather may be induced by non-immunologic factors, which remain to be further investigated.

The need for a new animal model for chronic rejection after lung transplantation.

The single most important cause of late mortality after lung transplantation is obliterative bronchiolitis (OB), clinically characterized by a decrease in lung function and morphologically by characteristic changes. Recently, new insights into its pathogenesis have been acquired: risk factors have been identified and the use of azithromycin showed a dichotomy with at least 2 different phenotypes of bronchiolitis obliterans syndrome (BOS). It is clear that a good animal model is indispensable to further dissect and unravel the pathogenesis of BOS. Many animal models have been developed to study BOS but, so far, none of these models truly mimics the human situation. Looking at the definition of BOS, a good animal model implies histological OB lesions, possibility to measure lung function, and airway inflammation. This review sought to discuss, including pros and cons, all potential animal models that have been developed to study OB/BOS. It has become clear that a new animal model is needed; recent developments using an orthotopic mouse lung transplantation model may offer the answer because it mimics the human situation. The genetic variants among this species may open new perspectives for research into the pathogenesis of OB/BOS.

Diagnostic value of antibodies against Pseudomonas aeruginosa in bronchoalveolar lavage fluid after lung transplantation.

BACKGROUND: Pseudomonal airway colonization is a risk factor for chronic allograft dysfunction after lung transplantation (LTx). Pseudomonas aeruginosa exoproteases are involved in initiating colonization, and immune complexes directed against these proteases may activate innate immune responses. OBJECTIVE: To investigate whether specific antibodies against pseudomonal proteases could be measured in bronchoalveolar lavage (BAL) fluid, whether they are associated with innate immune responses, and whether they could identify patients with chronic P. aeruginosa colonization after LTx. MATERIALS AND METHODS: BAL fluid from 40 noncolonized and 25 chronically colonized LTx recipients was retrospectively assayed for IgG antibodies against P. aeruginosa alkaline protease (AP), elastase (Ela), and exotoxin (Exo), and for BAL total and differential cell counts and IL-8 protein concentration. RESULTS: BAL anti-Ela and anti-Exo antibody titers were significantly increased in colonized compared with noncolonized patients (P = .009 and P = .02, respectively), whereas anti-AP titers were comparable (P = .79). Antibody titers strongly correlated with each other, and anti-Ela and anti-Exo titers, but not anti-AP titers, also correlated with BAL total cellularity, neutrophilia, and IL-8 protein concentration. Anti-Ela antibodies demonstrated the greatest diagnostic value in receiver operating characteristic analysis to detect chronic airway colonization (P = .009), followed by anti-Exo (P = .02) and anti-AP (P = .79). A combination of all 3 antibodies resulted in overall sensitivity of 45% (95% confidence interval [CI], 29.3-61.5), specificity of 88% (95% CI, 68.8-97.5), and positive predictive value of 55% (95% CI, 38.5-70.7). CONCLUSION: P. aeruginosa proteases in BAL may be associated with local innate immune responses, and could have the potential to enable detection of chronic colonization after LTx.

C-reactive protein in bronchoalveolar lavage fluid is associated with markers of airway inflammation after lung transplantation.

BACKGROUND: C-reactive protein (CRP), an acute-phase marker of systemic inflammation, may also be a local regulator of the pulmonary immune system. Its role in lung transplantation (LT), however, is unclear. We hypothesized that CRP in bronchoalveolar lavage (BAL) fluid might be associated with airway inflammation or remodeling. Therefore, it could play a role in the development of bronchiolitis obliterans syndrome (BOS). PATIENTS AND METHODS: A total of 100 LT recipients who had undergone transplantation between August 2001 and August 2005 were included in the current cross-sectional study. Patients who were evaluated at 90 days after LT were categorized as either stable (n = 36), colonized (n = 25), or suffering from infection (n = 16) or acute rejection (n = 23). BAL CRP, cell differentials, and interleukin (IL), IL8, transforming growth factor beta (TGFbeta), and vascular endothelial growth factor (VEGF) protein levels, as well as blood leukocytosis, plasma CRP, and forced expiratory value in 1 second (FEV(1); % predicted) were compared between groups. We analyzed the correlation of BAL CRP with inflammatory or remodeling markers and FEV(1). RESULTS: Compared with stable LT recipients, BAL CRP was significantly increased in patients with infection or acute rejection (P < .0001), but not in those with colonization. Generally, BAL CRP levels positively correlated with BAL total cell count, neutrophilia, and IL8 levels, as well as with plasma CRP levels (P < .0001). An inverse correlation was observed with BAL macrophages (P < .01), VEGF (P < .0001), and FEV(1) (P < .0001). Only a trend for a positive, respectively inverse correlation was seen for BAL IL6 and TGFbeta. CONCLUSIONS: The current data corroborate a possible role for CRP in airway inflammation after LT. Its importance for BOS should therefore be further elucidated.

Efficacy of total lymphoid irradiation in azithromycin nonresponsive chronic allograft rejection after lung transplantation.

Bronchiolitis obliterans syndrome (BOS) remains a major problem after lung transplantation. Azithromycin seems to be beneficial in some patients with established BOS. We investigated the efficacy of total lymphoid irradiation (TLI) in 6 BOS patients with a continuous decline in FEV(1), despite treatment with azithromycin for a mean of 12 +/- 13 (range, 1-35) months. A historical control group consisted of 5 patients with declining FEV(1), also nonresponders to azithromycin and those not treated with TLI. All 6 TLI patients received the total dose of 8 Gy in 10 sessions. There was a significant change in the decline of the FEV(1) after TLI treatment (from 221 +/- 107 to 94 +/- 79 mL/mo; P = .041). Three patients died, due to BOS progression, overwhelming pneumonia, and sudden cardiac arrest, respectively, 3.5, 11, and 26 months after TLI; two patients underwent retransplantation at 6 and 19 months after TLI, respectively. The sixth patient remains stable in BOS stage 3 after a follow-up period of 24 months. In the control group, there was no significant change in FEV(1) decline (209 +/- 97 mL/mo before versus 193 +/- 81 mL/mo after starting azithromycin; P = not significant). Two patients remain stable in BOS stage 3, 1 died of BOS progression, and the 5th patient is scheduled for retransplantation. We conclude that patients who do not or no longer respond to azithromycin may benefit from TLI, as suggested by a decreased rate in decline of the FEV(1).