T Cells Promote Bronchial Epithelial Cell Secretion of Matrix Metalloproteinase-9 via a C-C Chemokine Receptor Type 2 Pathway: Implications for Chronic Lung Allograft Dysfunction.

Chronic lung allograft dysfunction (CLAD) is the major limitation of long-term survival after lung transplantation. CLAD manifests as bronchiolitis obliterans syndrome (BOS) or restrictive allograft syndrome (RAS). Alloimmune reactions and epithelial-to-mesenchymal transition have been suggested in BOS. However, little is known regarding the role of allogenicity in epithelial cell differentiation. Primary human bronchial epithelial cells (BECs) were treated with activated T cells in the presence or absence of transforming growth factor (TGF)-ß. The expression of epithelial and mesenchymal markers was investigated. The secretion of inflammatory cytokines and matrix metalloproteinase (MMP)-9 was measured in culture supernatants and in plasma from lung transplant recipients (LTRs): 49 stable, 29 with BOS, and 16 with RAS. We demonstrated that C-C motif chemokine 2 secreted by T cells supports TGF-ß-induced MMP-9 production by BECs after binding to C-C chemokine receptor type 2. Longitudinal investigation in LTRs revealed a rise in plasma MMP-9 before CLAD onset. Multivariate analysis showed that plasma MMP-9 was independently associated with BOS (odds ratio [OR] = 6.19, p = 0.002) or RAS (OR = 3.9, p = 0.024) and predicted the occurrence of CLAD 12 months before the functional diagnosis. Thus, immune cells support airway remodeling through the production of MMP-9. Plasma MMP-9 is a potential predictive biomarker of CLAD.

Culture-Based Methods and Molecular Tools for Azole-Resistant Aspergillus fumigatus Detection in a Belgian University Hospital.

Azole-resistant Aspergillus fumigatus is an increasing worldwide problem with major clinical implications. Surveillance is warranted to guide clinicians to provide optimal treatment to patients. To investigate azole resistance in clinical Aspergillus isolates in our institution, a Belgian university hospital, we conducted a laboratory-based surveillance between June 2015 and October 2016. Two different approaches were used: a prospective culture-based surveillance using VIPcheck on unselected A. fumigatus (n = 109 patients, including 19 patients with proven or probable invasive aspergillosis [IA]), followed by molecular detection of mutations conferring azole resistance, and a retrospective detection of azole-resistant A. fumigatus in bronchoalveolar lavage fluid using the commercially available AsperGenius PCR (n = 100 patients, including 29 patients with proven or probable IA). By VIPcheck, 25 azole-resistant A. fumigatus specimens were isolated from 14 patients (12.8%). Of these 14 patients, only 2 had proven or probable IA (10.5%). Mutations at the cyp51A gene were observed in 23 of the 25 A. fumigatus isolates; TR34/L98H was the most prevalent mutation (46.7%), followed by TR46/Y121F/T289A (26.7%). Twenty-seven (27%) patients were positive for the presence of Aspergillus species by AsperGenius PCR. A. fumigatus was detected by AsperGenius in 20 patients, and 3 of these patients carried cyp51A mutations. Two patients had proven or probable IA and cyp51A mutation (11.7%). Our study has shown that the detection of azole-resistant A. fumigatus in clinical isolates was a frequent finding in our institution. Hence, a rapid method for resistance detection may be useful to improve patient management. Centers that care for immunocompromised patients should perform routine surveillance to determine their local epidemiology.

Difference in release kinetics of unwashed and washed platelet-released supernatants from bone substitute materials: the impact of platelet preparation modalities.

BACKGROUND AND OBJECTIVE: In regenerative dentistry, platelet preparations are applied to stimulate bone healing and periodontal regeneration. Here, we pursue a strategy where bone substitutes are used as carriers for platelet-released supernatants. The mitogenic capacity and release kinetics of loaded bone substitutes were assessed. MATERIAL AND METHODS: Platelet-released supernatants of washed platelets (washed PRS) and platelet-released supernatants of unwashed platelets (unwashed PRS) were lyophilized onto the bone substitutes deproteinized bovine bone mineral, hydroxyapatite and ß-tricalcium phosphate. Scanning electron microscopy images were taken. Supernatants of bone substitutes were collected at hours 1, 3, 6, 24, and 48 and medium was replaced. We evaluated the protein content with the bicinchoninic acid assay and the effect on proliferation using bioassays with human periodontal fibroblasts. Release of growth factors from the loaded bone substitutes was measured based on the platelet-derived growth factor isoform (PDGF-BB) and thrombin immunoassays. Furthermore, we assessed DNA and RNA content of washed PRS and unwashed PRS. RESULTS: Unwashed PRS showed higher total protein concentrations than washed PRS, while the concentration of PDGF-BB, thrombin, DNA, RNA and their mitogenic effect was not significantly different. The bone substitute materials adsorbed protein over time but no significant changes in overall appearance was found. Supernatants collected from unwashed PRS-loaded bone substitute after 1 h induced a potent mitogenic response in periodontal fibroblasts. This pro-mitogenic capacity of the supernatants decreased over the observation period. Supernatants of washed PRS-loaded bone substitutes did not induce a substantial mitogenic effect. Levels of PDGF-BB, thrombin and protein were higher in supernatants of unwashed PRS-loaded bone substitutes than of washed PRS-loaded bone substitutes. CONCLUSION: Bone substitutes loaded with unwashed PRS, but not bone substitutes loaded with washed PRS show continuously declining release kinetics. These data suggest that plasma components in platelet preparations can modify the release kinetics profile.

Percutaneous endoscopic jejunostomy in patients with gastroparesis following lung transplantation: feasibility and clinical outcome.

The aim of the present study was to describe success rates, complications, and outcome in patients who underwent percutaneous endoscopic jejunostomy (PEJ) because of gastroparesis due to previous lung transplantation. Between October 2008 and May 2011, 14 attempts at PEJ placement were made in 12 patients in our center. Of the 14 attempts, 11 were successful, giving a technical success rate of 78.6 %. Median duration of followup was8.5 months (2­15 months). No immediate complications were reported. Two severe complications occurred during follow up (one volvulus and one jejunocolic fistula). Jejunal nutrition was well tolerated in most of patients (9 /10). PEJ insertion is a feasible technique, which could help to provide nutritional support for patients with gastroparesis and previous lung transplantation.

[Impact of the transition period from childhood to adulthood in cystic fibrosis]. / Impact de la période de transition de l'enfance à l'âge adulte dans la mucoviscidose.

INTRODUCTION: The aim of this study was to evaluate the impact of the transition period from childhood to adulthood in patients with cystic fibrosis (CF) being followed up in our reference center. METHODS: The clinical, functional, inflammatory and microbiological parameters of all transition patients were compared two years before (T-2) and two years after the transfer (T+2) from paediatric to adult centers and further analysed according to whether the transition conditions were optimal or suboptimal. RESULTS: Twenty-eight patients were included. The mean age at the transfer visit was 19.5 years (±3.5). There were no deaths during the study period. Consultations were more numerous at T-2 [14.5 (±5.9) vs. 12.0 (±5.1), P<0.004]. Chronic colonization with Pseudomonas aeruginosa was more frequent at T+2 (46.4% vs. 17.9%, P=0.021). A progressive decrease in FEV1 and FVC was observed between T-2 and T+2. The number of pulmonary exacerbations was lower in the optimal transition group. CONCLUSION: The period of transition from childhood to adulthood in patients with CF appears to be associated with functional and microbiological changes.

Influence of lung transplantation on the essential fatty acid profile in cystic fibrosis.

Lung transplantation is assumed to normalize essential fatty acid (EFA) profile in the plasma, described as abnormal in patients with cystic fibrosis (CF). This study sought to evaluate the EFA profile in both the plasma and erythrocyte membrane according to lung status by comparing CF patients with or without a lung transplant. A total of 50 homozygous F508del patients (33 CF patients [CF group] and 17 CF patients with a lung transplant [TX CF group]) were included. In comparison with the CF group, in the plasma, the levels of total n-3, α-linolenic, eicosapentaenoic, and docosahexaenoic acids were higher and the n-6/n-3 ratio was lower in the TX CF group. Yet, these differences were not observed in the erythrocyte membrane. This study supports that lung transplantation improves the EFA profile in the plasma but not in the erythrocyte membrane by means of the different mechanisms suggested in this article.

[Complications of lung transplantation: perioperative complications, acute and chronic rejection]. / Complications de la transplantation pulmonaire: complications péri-opératoires, rejet aigu et chronique.

In 2009 lung transplantation is a valuable therapeutic option for a spectrum of end-stage pulmonary diseases. To many patients who are dying, lung transplantation offers a new and normal life for several years. However, lung transplantation is a major surgical intervention associated with a significant early mortality. Moreover, matching according to the major human histocompatibilty antigens is impossible, exposing the recipient to an increased risk of acute and chronic rejection. Chronic rejection and its clinical corollary the bronchiolitis obliterans syndrome, is the main cause of death medium and long term. The immunosuppressive treatment administered in order to prevent or stabilize this complication induces a number of potentially severe complications including infection, malignancies, and cardio-vascular, metabolic and renal complications which not only limit autonomy and quality of life, but also cause death in a number of long term survivors. A better understanding of the precise mechanisms underlying the development of the bronchiolitis obliterans syndrome and the development of specific preventive or therapeutic strategies will be key elements for the improvement of long term survival. The control of this main cause of death will allow individual tailoring of the immunosuppressive therapy and decrease the incidence of infectious and metabolic complications.

Emergence of livestock-associated MRSA isolated from cystic fibrosis patients: Result of a Belgian national survey.

BACKGROUND: This study aims to determine the prevalence and characteristics of Staphylococcus aureus in Belgian cystic fibrosis (CF) patients. METHODS: Non-duplicate respiratory samples from 510 CF-patients (2012-2013) were examined. One isolate per patient was analysed unless different phenotypes were recovered. Isolates were investigated for mecA/mecC, toxins presence, spa-typing, MLST and SCCmec-typing. Potential livestock-associated (LA) isolates were examined for their immune-evasion-cluster (IEC) genes. RESULTS: S. aureus (n = 380), including 41 small-colony variants (SCVs), were isolated from 66.7% patients. The prevalence of methicillin-resistant S. aureus (MRSA) colonization was 4.9%. Two MRSA isolates carried toxic shock syndrome toxin 1 (TSST-1). Most MRSA (65%) belonged to two nosocomial epidemic clones (CC5, CC8) widespread in Belgium. Methicillin susceptible S. aureus (MSSA) showed great genetic diversity. Five of 33 isolates belonging to potential LA-lineages were IEC negative, including three methicillin-resistant isolates, suggesting an animal origin. CONCLUSIONS: The MRSA-prevalence in Belgian CF-patients remained constant (2001-2013), but SCV-prevalence increased. Most MRSA belonged to health-care-associated clones. Three patients carrying LA-MRSA were found, requiring further investigation to determine the risk factors for LA-MRSA acquisition.

Clostridium difficile colitis in cystic fibrosis patients with and without lung transplantation.

BACKGROUND: Despite a large carriage rate of Clostridium difficile among cystic fibrosis (CF) patients, C. difficile-associated disease (CDAD) is rather rare. In case of lung transplantation, the incidence and clinical aspects of CDAD in this patient population are not well known. METHODS: We reviewed the medical files of all CF patients who presented with symptomatic C. difficile infection from January 1998 to December 2004 and compared the incidence, clinical aspects, severity of disease, and clinical outcome between non-transplanted and transplanted CF patients. RESULTS: Between 1998 and 2004, 106 adult CF patients were followed at our clinic. Forty-nine patients underwent lung transplantation; 15 before 1998 and 34 after 1998. The incidence density of CDAD was higher in transplanted CF patients as compared with non-transplanted CF patients (24.2 vs. 9.5 episodes/100,000 patient-days; risk ratio: 2.93 [1.41-6.08]; P=0.0044). Diarrhea was a very frequent feature, but was notably absent in 20% of the cases. Rates of moderate and severe colitis were similar in both groups. However, only transplanted patients developed complicated colitis. CT scan and endoscopy were performed more frequently in the transplant group. Two transplant recipients died because of CDAD. CONCLUSION: CF patients who undergo lung transplantation are at a higher risk of developing CDAD and seem to present more often atypical and/or complicated disease. CDAD should be part of the differential diagnosis in case of digestive symptoms, even in the absence of diarrhea, and requires early treatment.

Oxygenation of carcinomas of the uterine cervix: evaluation by computerized O2 tension measurements.

Direct oxygen partial pressure (pO2) readings in cancers of the cervix and in the normal cervix of nulliparous or parous women were obtained using a computerized pO2 histography system. The oxygenation status of the tumors was evaluated as a function of clinical staging and histological grading. pO2 measurements were performed with a customized electrode system in conscious pre- and postmenopausal, untreated patients with well-defined arterial blood gas status. With this technique, pO2 measurements in the normal cervix of nulliparous women resulted in oxygenation patterns which were characteristic for normal, adequately supplied tissues (median pO2, 48 mm Hg) with approximately 1% of the pO2 values grouped between zero and 2.5 mm Hg, i.e., in a range with less than half-maximum radiosensitivity. As a rule, the mean (and median) pO2 values were distinctly lower in the normal cervix of parous women (most probably due to scar formation following vaginal delivery) and in malignancies. In the normal cervix of parous women the median pO2 value was 13 mm Hg (with approximately 14% of the pO2 readings in the lowest class), 14 mm Hg in International Federation of Gynecologists and Obstetricians I/II tumors (2% of the readings in the lowest pO2 class), and 11 mm Hg in International Federation of Gynecologists and Obstetricians III/IV cancers (1% of the pO2 data in the lowest class). To date, 5 of 18 cervical cancers exhibited pO2 values between zero and 2.5 mm Hg. The oxygenation pattern in cervical cancers and the occurrence of hypoxia and/or anoxia did not correlate with either the clinical stages and histological grades or with a series of clinically relevant parameters (e.g., tumor size). No significant differences were found between pre- and postmenopausal tumors, between squamous cell carcinomas and adenocarcinomas, and between endophytic or exophytic tumors. From these studies there is clear indication that the oxygenation status of individual tumors cannot be predicted on the basis of staging and/or grading, predominantly because of the pronounced tumor-to-tumor variabilities. Evaluation of the tissue oxygenation of individual tumors is thus mandatory to prove that tumor oxygenation can predict the overall prognosis and/or treatment outcome.

Oxygenation of human tumors: evaluation of tissue oxygen distribution in breast cancers by computerized O2 tension measurements.

Direct oxygen partial pressure (pO2) readings in breast cancers, in fibrocystic disease, and in the normal breast have been obtained using a novel technique which allows for the systematic evaluation of the oxygenation status as a function of pathological staging and histological grading. Measurements were performed in awake pre- and postmenopausal patients with well-defined arterial blood gas status. The measuring procedure encompasses a computerized electrode movement in the tissue which avoids significant compression artifacts and allows routine measurement in human tumors before, during, and after treatment. Using this reliable technique, pO2 measurements in the normal breast and in fibrocystic disease resulted in oxygenation patterns which were characteristic for normal, adequately supplied tissues. The median pO2 values were 65 and 67 mm Hg, respectively, with no pO2 readings below 12.5 mm Hg in the normal breast, and less than or equal to 5 mm Hg in fibrocystic disease, respectively. In contrast, in breast cancers the median pO2 value was 30 mm Hg (pooled data for pathological stages T1-T4). To date, 6 of 15 breast cancers exhibited pO2 values between zero and 2.5 mm Hg, i.e., tissue areas with less than half-maximum radiosensitivity. The oxygenation pattern in breast cancers and the occurrence of hypoxia and/or anoxia did not correlate with either the pathological stages and histological grades or with a series of clinically relevant parameters. No significant differences were found between pre- and postmenopausal tumors and between lobular and ductal carcinomas. Tumor-to-tumor variability in the oxygenation pattern was more pronounced than intra-tumor heterogeneity. pO2 variations within a tumor cannot be predicted, e.g., as a function of the measuring site (tumor center versus periphery).

[Small airway diseases and immune deficiency]. / Atteinte des voies aériennes distales et immunodépression.

INTRODUCTION: Innate or acquired immune deficiency may show respiratory manifestations, often characterized by small airway involvement. The purpose of this article is to provide an overview of small airway disease across the major causes of immune deficiency. BACKGROUND: In patients with common variable immune deficiency, recurrent lower airway infections may lead to bronchiolitis and bronchiectasis. Follicular and/or granulomatous bronchiolitis of unknown origin may also occur. Bronchiolitis obliterans is the leading cause of death after the first year in patients with lung transplantation. Bronchiolitis obliterans also occurs in patients with allogeneic haematopoietic stem cell transplantation, especially in the context of systemic graft-versus-host disease. VIEWPOINT AND CONCLUSION: Small airway diseases have different clinical expression and pathophysiology across various causes of immune deficiency. A better understanding of small airways disease pathogenesis in these settings may lead to the development of novel targeted therapies.

The clinical benefits of long-term supplementation with omega-3 fatty acids in cystic fibrosis patients - A pilot study.

Effectiveness of omega-3 supplementation in cystic fibrosis (CF) remains controversial. This study sought to evaluate clinical status, exercise tolerance, inflammatory parameters, and erythrocyte fatty acid profile after 1 year of oral omega-3 supplementation in CF patients. Fifteen ΔF508-homozygous patients undergoing chronic azithromycin were randomized to receive omega-3 fish oil supplementation at a dose of 60mg/Kg/day or placebo. In comparison with the previous year, in the supplemented group, the number of pulmonary exacerbations decreased at 12 months (1.7 vs. 3.0, p<0.01), as did the duration of antibiotic therapy (26.5 days vs. 60.0 days, p<0.025). Supplementation significantly increased the levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as early as <3 months of administration, with concomitant decreases in arachidonic acid (AA) levels. This pilot study suggests that long-term omega-3 supplementation offers several clinical benefits as to the number of exacerbations and duration of antibiotic therapy in CF patients.

Erratum to "Practical guidelines: lung transplantation in patients with cystic fibrosis".

[This corrects the article DOI: 10.1155/2014/621342.].

Oxygen tension distributions are sufficient to explain the local response of human breast tumors treated with radiation alone.

PURPOSE: Several factors are known to influence the probability of tumor control after radiation. These include tumor oxygen tension distribution, glutathione content, intrinsic radiation sensitivity, rate of repopulation, tumor size, physician skill, etc. The relative impact of oxygen on human tumor response is unknown. The purpose of this analysis is to determine to what extent the observed shape of the radiation response curve for human tumors can be predicted by the tumor oxygenation status. METHODS AND MATERIALS: The radiation dose response curve for patients treated with radiation alone for breast cancer was calculated based on pooled data. Tumor control rates as a function of radiation dose were fitted to a probit curve. Twenty-two women with breast cancer in Mainz (Germany) and at Stanford University had pO2 measurements made of their tumors. An average of 87 +/- 58 (range 21 to 300) measurements were made from each patient. Hypoxia was assumed to be a purely dose modifying factor with a maximum oxygen enhancement ratio of 2.5. Assuming patients are treated with daily radiation doses of 2 Gy, the breast cancer alpha/beta ratio is 10 Gy, tumors have a mean of 10(8) stem cells, and using the linear quadratic formula for modelling surviving fraction, it was possible to estimate tumor control probability. RESULTS: Tumor oxygenation was an extremely important modifier of the shape of the dose response curve and alone was sufficient to account for the slope of the observed dose response curve for human breast carcinoma. Tumor size distribution had a smaller effect on the shape and the slope of the dose response curve. Two models of radiation induced reoxygenation were tested, one that allowed full reoxygenation to the baseline state between the daily radiation fractions and another with no reoxygenation between fractions. The clinical data fell between these two models in accordance with the expected incomplete reoxygenation between treatments. CONCLUSION: The results support the conclusion that in human breast carcinoma, oxygen tension distribution is a critical modifier of radiation treatment response.

Detection of blood chimerism after lung and heart-lung transplantation. The superiority of nested as compared with standard polymerase chain reaction amplification.

Migration of donor cells from the graft to various tissues of the recipient has been demonstrated after different types of solid organ transplants. Currently, the detection of donor cells in the recipient's tissues is most simply performed by polymerase chain reaction (PCR) amplification of a donor-specific gene. In the present study, we first determined in vitro the sensitivity of standard and nested PCR amplification with sequence-specific primers (PCR-SSP) of a donor-specific allele of the HLA-DRB1 gene and then used this technique to assess prospectively blood chimerism in two single-lung (SLT) and one heart-lung (HLT) transplant recipients. Standard PCR-SSP consisted in a single amplification round with sequence-specific primers for the donor-specific DRB1 allele. Nested PCR-SSP consisted in a first round of generic amplification of exon 2 of the DRB1 gene, followed by a second amplification round with primers specific for the donor allele. In vitro, nested PCR-SSP of the donor-specific allele was 1000-fold more sensitive than standard PCR-SSP and allowed the detection of 1 donor cell in 10(5) recipient cells. In vivo, standard PCR-SSP detected donor cells among the recipients' peripheral blood mononuclear cells (PBMCs) only during the first postoperative days, whereas nested PCR-SSP demonstrated their presence until the end of the first postoperative month in patients 1 and 2 and until 3 months after transplantation in patient 3. We conclude that donor cells can be detected in the peripheral blood of SLT and HLT recipients during the first postoperative months and that nested PCR-SSP amplification of a donor-specific HLA-DRB1 allele is much more sensitive than standard PCR-SSP to demonstrate such chimerism.

Lung rejection occurs in lung transplant recipients with blood chimerism.

BACKGROUND: It has been postulated that chimerism after transplantation might promote graft acceptance. In the present study, we prospectively assessed blood chimerism in 10 lung transplant recipients during the first posttransplant year and investigated whether chimerism was associated with an immunologically stable situation of the graft. METHODS: The recipients' peripheral blood mononuclear cells were obtained before transplantation and at various time points during the first postoperative year. Donor cells were detected using nested polymerase chain reaction amplification of a donor-specific HLA-DRB1 allele. Clinical graft acceptance was determined by the number of rejection episodes. RESULTS: The incidence of blood chimerism was high during the first 3 postoperative months and then decreased over time. All patients experienced at least one acute rejection episode, and three patients developed chronic rejection. CONCLUSION: We, thus, conclude that rejection of the lung allograft may occur in the presence of blood chimerism.

Impaired antigen-presenting cell function contributes to T-cell hyporesponsiveness in stable lung transplant recipients.

BACKGROUND: Peripheral blood mononuclear cells (PBMC) of stable renal or cardiac transplant recipients were previously shown to respond to allogeneic cells but not to soluble protein antigens. The aim of the present study was to assess the T-cell and antigen-presenting cell (APC) functions of stable lung transplant (LT) recipients. METHODS: We obtained PBMC from 38 stable LT recipients. PBMC from healthy volunteers served as controls. PBMC were stimulated with either anti-CD3 monoclonal antibody, allogeneic PBMC, or tetanus toxoid (TT). T-cell activation was assessed by determination of interleukin (IL)-2 levels in culture supernatants; in some experiments, interferon-y levels were also determined. Patients' APC function was tested in a mixed leukocyte reaction using patients' PBMC as stimulators. The expression of class II MHC, B7.2, and CD40 molecules on patients' APC was determined by flow cytometry, and their production of IL-10 and IL-12 at the basal state and upon CD40 ligation was also measured. RESULTS: Patients' T cells produced normal amounts of IL-2 in response to anti-CD3 monoclonal antibody and allogeneic PBMC. In contrast, the response of memory T cells to TT was severely blunted both in terms of IL-2 and interferon-y production. Patients' PBMC were poor stimulators in mixed leukocyte reaction, and class II MHC expression on patients' monocytes was significantly reduced. Patients' APC presented a modest but significant increase in basal IL-10 production and produced significantly less IL-12 upon CD40 ligation than control APC. CONCLUSIONS: T cells from stable LT recipients respond normally to stimuli that do not depend on autologous APC. The major impairment in the T-cell response to TT is caused by APC dysfunction, which involves decreased class II MHC expression and deficient IL-12 synthesis.