Impaired activation of Notch-1 signaling hinders repair processes of bronchial epithelial cells exposed to cigarette smoke.

Notch-1 intervenes in the reparative processes of mucosa by controlling cell proliferation, differentiation and stem cell maintenance. Cigarette smoke alters airway epithelial homeostasis. The present study explored whether: Smokers showed altered Notch-1 expression; and whether in bronchial epithelial cells (16HBE): a) cigarette smoke extracts (CSE) altered the expression of Notch-1, of its ligand Jagged-1 (Jag-1) and the nuclear translocation of Notch-1; b) Notch-1 signaling activation as well as CSE modified Ki67, PCNA, p21, IL-33 expression, cell proliferation and repair processes. Notch-1 expression was assessed in the epithelium from large airway surgical samples from non-smoker and smoker subjects by immunohistochemistry.16HBE were cultured with/without CSE and Jag-1. A Notch-1 inhibitor (DAPT) was used as control. The expression of Notch-1, Jag-1, Ki67, PCNA, p21, IL-33 and cell proliferation (by CFSE) were all assessed by flow cytometry. Notch-1 nuclear expression was evaluated by immunofluorescence and western blot analysis. Repair processes were assessed by wound assay. Smokers had cytoplasmic but not nuclear Notch-1 expression. Although CSE increased Notch-1 expression, it counteracted Notch-1 signaling activation since it reduced Jag-1 expression and Notch-1 nuclear translocation. Notch-1 signaling activation by Jag-1 increased Ki67, PCNA and repair processes but reduced intracellular IL-33 and p21 expression without affecting cell proliferation. DAPT counteracted the effects of Notch-1 activation on PCNA and IL-33. CSE increased Ki67, PCNA, p21 and IL-33 expression but reduced cell proliferation and repair processes. In conclusion, cigarette smoke exposure, limiting Notch-1 signaling activation and hindering repair processes, amplifies injury processes in bronchial epithelial cells.

Outcome of Transplantation Using Organs From Donors Infected or Colonized With Carbapenem-Resistant Gram-Negative Bacteria.

Donor-derived infections due to multidrug-resistant bacteria are a growing problem in solid organ transplantation, and optimal management options are not clear. In a 2-year period, 30/214 (14%) recipients received an organ from 18/170 (10.5%) deceased donors with infection or colonization caused by a carbapenem-resistant gram-negative bacteria that was unknown at the time of transplantation. Among them, 14/30 recipients (47%) received a transplant from a donor with bacteremia or with infection/colonization of the transplanted organ and were considered at high risk of donor-derived infection transmission. The remaining 16/30 (53%) recipients received an organ from a nonbacteremic donor with colonization of a nontransplanted organ and were considered at low risk of infection transmission. Proven transmission occurred in 4 of the 14 high-risk recipients because donor infection was either not recognized, underestimated, or not communicated. These recipients received late, short or inappropriate posttransplant antibiotic therapy. Transmission did not occur in high-risk recipients who received appropriate and prompt antibiotic therapy for at least 7 days. The safe use of organs from donors with multidrug-resistant bacteria requires intra- and inter-institutional communication to allow appropriate management and prompt treatment of recipients in order to avoid transmission of infection.

Cigarette smoke alters cell cycle and induces inflammation in lung fibroblasts.

BACKGROUND: Lung fibroblasts are crucial for the integrity of alveolar structure. Cigarette smoking, the major risk factor for chronic obstructive pulmonary disease, impairs the repair functions of lung fibroblasts. AIMS: The study simultaneously assessed for the first time cell cycle, p53, p21, p38, ERK 1/2 and IL-8. MAIN METHODS: Primary foetal lung fibroblasts (HFL-1) and primary lung fibroblasts from former (n = 5) and current (n = 5) smokers with/without cigarette smoke extracts (CSEs) and inhibitors of p38 and ERK1/2 were studied for cell cycle events and for marker expression by flow-cytometry, western-blot analysis and ELISA. KEY FINDINGS: CSE exposure did not induce caspase 3 cleavage or DNA laddering but reduced S phase, and increased G1 and G2/M in HFL-1. Furthermore CSE increased: p53 and p21 expression; p38 and ERK 1/2 pathway activation; and IL-8 release. Inhibitors of p38 and ERK 1/2 reversed the effects of CSE on cell cycle and on IL-8 release. ERK 1/2 inhibitor was able to reverse the effects of CSE on p21 expression. Primary lung fibroblasts from current smokers had higher ERK 1/2 activation in comparison to normal primary fibroblasts and higher percentage of cells in G1 phase and lower percentage of cells in S phase in comparison to former smoker fibroblasts. SIGNIFICANCE: Cigarette smoke may affect the reparative potential of lung fibroblasts altering the expression of p53 and p21 and the progression of the cell cycle to S phase. All these events are promoted by the activation of pro-inflammatory pathways.

Successful lung transplantation in an HIV- and HBV-positive patient with cystic fibrosis.

Prior to the advent of highly active antiretroviral therapy (HAART), HIV-infected patients were usually not considered as transplant candidates because of the poor prognosis of their underlying disease and concerns regarding the potential detrimental effects of immunosuppression on viral load and immune status. However, with the significant HAART-associated improvements in morbidity and mortality, good short-term outcomes after liver and kidney transplantation for patients with HIV infection have been reported. Nevertheless, HIV infection is currently considered a contraindication to lung transplantation in most transplant centers worldwide. The results of a double lung transplant performed in an HIV and HBV co-infected patient with cystic fibrosis (CF) and end-stage respiratory failure (ESRF) are presented after a 2-year follow-up. Approval of and recommendations for the management of this patient were obtained from the Italian National Center for Transplantation as an extension of the ongoing Italian protocol for liver and kidney transplantation in HIV-infected individuals. The operation was successful and the patient recovered rapidly after surgery. A cautious infectious and immunosuppressive management allowed so far the avoidance of major infectious complications and rejection. To the best of our knowledge, this is the first report of lung transplantation in an HIV and HBV co-infected patient.

Peripheral CD4(+)CD25(+) TREG cell counts and the response to extracorporeal photopheresis in lung transplant recipients.

Extracorporeal photopheresis (ECP) has been proposed as a possible alternative therapy for patients with bronchiolitis obliterans syndrome (BOS), with some evidence of efficacy. Although the mechanism by which ECP exerts its protective effects remains to be determined, two recent studies suggest that the modulation of transplant immune rejection may depend on the capacity to increase the number of peripheral T-regulatory (Treg) cells. We evaluated the effect of ECP treatment on the number of naturally occurring CD4(+)CD25(+) Treg cells in the peripheral blood of six lung transplant recipients: in five cases after failure of augmented or changed immunosuppression for BOS, and in one case owing to persistent acute rejection in a patient who contracted chronic hepatitis C viral infection after lung transplant. A functional stabilization was observed in three of our five patients with BOS, which was accompanied by a slight increase or stabilization of the number of peripheral blood CD4(+)CD25(high) cells with in vitro features of Treg cells. On the contrary, two patients with BOS who did not experience graft functional stabilization also showed a decline in the peripheral Treg subset. In the last patient Treg cell kinetics showed stabilization during the first 5 months of ECP treatment when lung function remained stable and graft histology normalized but showed a subsequent decrease, predating BOS diagnosis. In all, our results indicate that ECP may modulate peripheral Treg cell number but the time course of peripheral Treg cells varies according to graft function.

Peripheral CD4+ CD25+ Treg cell expansion in lung transplant recipients is not affected by calcineurin inhibitors.

CD4+CD25+ regulatory T (Treg) cells have been shown to play a role in allograft tolerance and their peripheral counts vary according to the degree of graft acceptance in lung transplant recipients (LTR). Recent studies demonstrate that certain drugs might modulate generation, expansion and activity of Treg cells. Aim of this study was to evaluate the effect of therapeutic regimens used in our institution on peripheral CD4+CD25(high)CD69- Treg cell numbers in a group of 51 LTR with stable clinical conditions. They were treated with standard immunosuppression: calcineurin inhibitor (CNI)+azathioprine (AZA)+steroids (n=28) or with CNI+mycophenolate mofetil (MMF)+steroids (n=11) or with CNI+steroids (n=12). These stable LTR were compared with age-matched healthy controls (n=35) and with 19 LTR who developed bronchiolitis obliterans syndrome (BOS) and were treated analogously. Stable LTR showed higher peripheral Treg cell counts with respect to age-matched healthy controls (59.9+/-31.8/mul versus 42.1+/-16.9/mul, respectively; p<0.05). This increase was detectable in all patients treated with CNI either in association with AZA or MMF. During these treatments a significant expansion of Treg cell counts was detectable during acute rejection (AR) episodes (86.03+/-26.6/mul during AR versus 36.34+/-7.6 before AR; p<0,05). Moreover, the development of BOS was associated to a significant decrease of Treg cell counts irrespective to the immunosuppressive regimen used. In conclusion, therapeutic regimens based on CNI seem to allow a certain degree of peripheral Treg cell expansion in stable LTR.

Determinants of quality of life after lung transplant: an Italian collaborative study.

BACKGROUND: With the improvement in survival rates after lung transplantation, concern has arisen about evaluating quality of life (QoL). This multicenter cross-sectional study aimed at describing QoL and identifying factors associated with it. METHODS: We assessed QoL in 129 lung transplant recipients from 5 centres in Italy, during scheduled follow-up visits, using the SF-36, GHQ and St George's respiratory questionnaires (SGRQ). RESULTS: The SF-36 elicited impaired QoL in the physical, but not in the mental domains (PCS = 44; MCS = 53). The GHQ identified 29 patients (23%) with psychological discomfort and the SGRQ scores were significantly better than those of patients with chronic respiratory disease. On multivariate analysis, exertional dyspnea was an independent predictor of the PCS (adjusted delta -6.3 (p < 0.001), while osteoporosis (delta = -3.1), BOS (delta = -4.3), acute rejection (delta = -3.9) and heart and lung transplant (delta = +6.4) were only marginally associated. Dyspnea was also related to a GHQ score > 5. CONCLUSIONS: The study identified exertional dyspnea as the main determinant of QoL as measured both by SF36 (PCS) and GHQ. Other objective measures contributed only to the PCS. Thus, the SF-36 (PCS) and GHQ were useful in identifying patients who needed treatment not only for complications but also psychological support and continued physical rehabilitation.

Everolimus versus azathioprine in maintenance lung transplant recipients: an international, randomized, double-blind clinical trial.

Everolimus is a proliferation signal inhibitor with immunosuppressive activity that may reduce the rate of progression of chronic rejection, bronchiolitis obliterans syndrome (BOS), after lung transplantation. In a randomized, double-blind clinical trial, 213 BOS-free maintenance patients received everolimus (3 mg/day) or azathioprine (AZA, 1-3 mg/kg/day) in combination with cyclosporine and corticosteroids. The prospectively defined primary endpoint was the incidence of efficacy failure (decline in FEV1 >15%[deltaFEV1 >15%], graft loss, death or loss to follow-up) at 12 months. Incidence of efficacy failure at 12 months was significantly lower in the everolimus group than AZA (21.8% vs. 33.9%; p = 0.046); at 24 months, rates of efficacy failure became similar between the groups. At 12 months, the everolimus group had significantly reduced incidences of deltaFEV1 >15%, deltaFEV1 >15% with BOS, and acute rejection. At 24 months, only incidence of acute rejection remained significantly less in the everolimus group. Treatment discontinuations (particularly due to adverse events), serious adverse events and high serum creatinine values were more common with everolimus. For the first time, a drug has demonstrated significant slowing of loss in lung function, suggesting that patients kept on prolonged maintenance treatment with everolimus may benefit from replacing AZA with everolimus 3 months after lung transplantation.

Time course of matrix metalloproteases and tissue inhibitors in bleomycin-induced pulmonary fibrosis.

To investigate simultaneously localization and relative activity of MMPs during extracellular matrix (ECM) remodeling in bleomycin-induced pulmonary fibrosis in rat, we analyzed the time course of the expression, activity and/or concentration of gelatinases MMP-2 and MMP-9, collagenase MMP-1, matrylisin MMP-7, TIMP-1 and TIMP-2, both in alveolar space (cellular and extracellular compartments) and in lung tissue. MMP and TIMP expression was detected (immunohistochemistry) in lung tissue. MMP activity (zymography) and TIMP concentration (ELISA) were evaluated in lung tissue homogenate (LTH), BAL supernatant (BALs) and BAL cell pellet (BALp) 3, 7, 14, and 28 days after bleomycin intratracheal instillation. Immunohistochemistry showed an extensive MMP and TIMP expression from day 7 in a wide range of structural and inflammatory cells in treated rats. MMP-2 was present mainly in epithelia, MMP-9 in inflammatory cells. MMP-2 and MMP-9 activity was increased respectively in BAL fluid and BAL cells, with a peak at day 7. TIMP-1 and TIMP-2 concentration (ELISA) enhancement was delayed at day 14. In conclusion gelatinases and their inhibitors are significantly activated during bleomycin-induced pulmonary fibrosis. Marked changes in gelatinases activity are observed early in the alveolar compartment, with a prevailing extracellular activity of MMP-2 and a predominant intracellular distribution of MMP-9, while enzyme activity changes in lung parenchyma were less evident. In the repairing phase the reduction of gelatinases activity is synchronous with a peak of alveolar concentration of their inhibitors.

The frequency of interleukin-10- and interleukin-5-secreting CD4+ T cells correlates to tolerance of transplanted lung.

Posttransplant bronchiolitis obliterans syndrome (BOS) results from a chronic immunological/inflammatory insult that leads to fibro-obliteration of the lumen of the allograft airways. The functional T-cell response that is associated with graft acceptance needs to be further clarified in humans. The aim of the present study was to assess the functional activity of peripheral CD4+ T lymphocytes in nine lung transplant recipients with BOS stage II or III (mean 5.4 years after transplant), in seven lung patients with stable clinical conditions (3.4 years posttransplant); and in six normal controls. Peripheral CD4+ T cells, obtained by magnetic bead vs negative purification, were studied using a computer-assisted enzyme-linked immunospot assay (ELISPOT) to assess the number of IFN-gamma-, interleukin (IL)5-, and IL10-gamma-producing cells (no./10(6) CD4+ cells) after allogeneic stimulation. The frequencies of IFN-gamma-producing CD4+ cells did not change significantly in stable patients compared to those with BOS. Interestingly in BOS, the number of IL5- and IL10-producing cells was significantly lower than in stable patients (P < or = .05), suggesting a possible role of these Th2 cytokines in the modulation of graft tolerance.

Monocyte chemoattractant protein-1 levels in bronchoalveolar lavage fluid of lung-transplanted patients treated with tacrolimus as rescue treatment for refractory acute rejection.

BACKGROUND: Cytokines are important mediators of the complex process of extravasation and influx of peripheral mononuclear cells into a site of graft injury, an action that may be affected by the immunosuppressive regimen. The aim of this study was to compare the effect of different immunosuppressive regimens on cytokine expression in the grafted lung. METHODS: We analyzed the cytokine profiles in broncho-alveolar lavage fluid (BAL-F) from 18 lung transplanted patients undergoing a shift from a cyclosporine- to a tacrolimus-based triple therapy regimen due to refractory acute rejection. RESULTS: Three months after the conversion to tacrolimus, BAL-F levels of interleukin 8 (IL8), IL18, IL12 and IL10 were not significantly different than those measured before conversion. In contrast, monocyte chemoattractant protein-1 (MCP-1) levels showed a significant and sustained decrease in BAL-F during tacrolimus therapy. In addition the levels of gamma interferon (IFN-gamma) in the BAL-F were decreased albeit not significantly. CONCLUSIONS: These findings suggest that the clinical and functional stabilization of patients observed after conversion to a tacrolimus based regimen, may be due, at least in part, to the induced down-regulation of MCP-1 production.

Successful therapy with high-dose steroids and cyclosporine for the treatment of carmustine-mediated lung injury.

The treatment of the pulmonary toxicity induced by carmustine is nowadays based on the use of corticosteroids that generally lead to a rapid resolution of pneumonitis. On the contrary, no therapeutic alternatives are reported for those patients who do not respond to steroids. We describe a case of non-Hodgkin's lymphoma in a patient who developed a severe interstitial pneumonitis after an autologous transplantation including carmustine in the conditioning regimen. He was successfully treated with an association of steroids and cyclosporine A with a rapid improvement of symptoms and a complete resolution of pneumonitis. This is, to our knowledge, the first case of carmustine-induced pneumonitis, resistant to steroids alone, successfully treated with cyclosporine A. This suggests an immunoallergic mechanism in the pathogenesis of the damage, which can be reversed with prompt therapy.

Pulmonary toxicity following carmustine-based preparative regimens and autologous peripheral blood progenitor cell transplantation in hematological malignancies.

Sixty-five patients with hematological malignancies (25 multiple myeloma, 18 Hodgkin's disease, 22 non-Hodgkin's lymphomas) who received a carmustine-based regimen followed by autologous PBPC transplantation, were studied retrospectively to evaluate the incidence of post-transplant non-infective pulmonary complications (NIPCs), risk factors predictive of NIPCs, and response to steroids. Carmustine (BCNU) given i.v. at a dose of 600 mg/m2 was combined with etoposide and cyclophosphamide in 40 patients (BCV regimen) and with etoposide and melphalan in 25 patients (BEM regimen). Seventeen of 65 patients (26%) had one episode of NIPCs. The median time to NIPCs was 90 days (52-289). Factors that increased the risk of developing NIPCs on multivariate analysis were female sex (P < 0. 001) and BCV regimen (P < 0.05). All patients with NIPCs received prednisone at a dose of 1 mg/kg body weight for 10 days then tapered by 5 mg every two days; complete response to steroids was achieved in 15 of 17 patients; one unresponsive patient died of interstitial pneumonia. BCNU given at the dose of 600 mg/m2 is well tolerated when associated with melphalan and etoposide. In females and in patients receiving BCNU with cyclophosphamide, a BCNU dose reduction may be advisable. Bone Marrow Transplantation (2000) 25, 309-313.

Risk factors for early death in patients awaiting heart-lung or lung transplantation: experience at a single European center.

BACKGROUND: Our purpose was to establish whether patients on the waiting list for heart-lung or lung transplantation had different survival rates according to diagnosis and to determine the specific variables responsible for early death. METHODS: Between 1988 and 1996, 278 patients were placed on the waiting list for organ transplant. Diagnoses were pulmonary vascular disease in 128, parenchymal disease in 141, and retransplantation in 9 patients. Eighty patients received transplants, 100 patients died awaiting transplantation, and 98 patients are still awaiting transplantation. Univariate and multivariate analyses of risk factors for early death on the waiting list were performed. Patients still listed < or =6 months (n=24), transplanted < or =6 months (n=37), or in the retransplantation group (n=9) were excluded. Of the remaining 208 patients, 52 died < or =6 months and 156 survived >6 months. RESULTS: Patients with primary pulmonary hypertension, pulmonary fibrosis, or cystic fibrosis had statistically significantly lower survival rates at 6, 12, and 24 months (31%, 36% and 26%, respectively, at 24 months) than patients with Eisenmenger's syndrome and chronic obstructive pulmonary disease (76% and 71%). Patients with Eisenmenger's syndrome who died < or =6 months had significantly higher systolic pulmonary artery pressure (134+/-39 vs. 108+/-25 mmHg) and pulmonary vascular resistance (1928+/-1686 vs. 1191+/-730 dyn/sec/cm(-5)) than those who survived longer. Patients with pulmonary fibrosis who died < or =6 months had significantly lower forced vital capacity (36+/-15 vs. 47+/-13% predicted), forced expiratory volume (37+/-14 vs. 48+/-14% predicted), room air PO2 (42+/-11 vs. 50+/-11 mmHg), and room air O2-saturation (78+/-10 vs. 84+/-8%) than those who survived longer. In the multivariate analysis, only the type of pathology was a significant risk factor for death after being on the waiting list < or =6 months. CONCLUSIONS: Certain pathologies and variables are risk factors for early death in patients on the waiting list. This information may be used to allocate specific donor organs to patients in greater need.

[A case of occupational pulmonary siderosis: the pathogenetic and prognostic considerations]. / Un caso di siderosi polmonare professionale: considerazioni patogenetiche e prognostiche.

Pulmonary siderosis is a disease which can be considered with the pneumoconioses of the so-called inert dust (either by deposit or by accumulation) and, from the earliest nosographic descriptions dating back to the thirties and forties, it has been substantially considered a benign form because of the scarce sclerogenic evolution and the preservation of discrete respiratory function. We present a case of pulmonary siderosis characterized on one hand by the importance and clarity of the cytopathological picture and on the other by the presence of changes, although modest, in lung function which confirm the most recent hypotheses in the literature on the potential evolution of pneumosiderotic diseases and their relatively less benign prognosis. From a preventive point of view the case draws attention to the need for greater control of environmental hygiene conditions in small factories. From an insurance and medico-legal point of view siderosis has recently been explicitly added to the occupational diseases which can be declared to the Italian state department dealing with occupational disease and disability payments (I.N.A.I.L.) (DPR of the 13.04.1994, section 47).

Pulmonary function in patients with systemic sclerosis.

The lungs are frequently affected in systemic sclerosis (SSc), a generalized connective tissue disorder. We evaluated the prevalence of respiratory functional abnormalities and their correlation with symptoms and radiograph features in a group of 34 patients who fulfilled the American Rheumatism Association criteria for the diagnosis of systemic sclerosis. Patients were submitted to a specific respiratory questionnaire and to lung function tests. Measurements were performed according to the European Coal and Steel Community (ECSC) recommendations and results expressed as a SD score, an accurate method that, taking into account the dispersion of the parameters in the reference population, allows precise definition of pathological subjects. Of the patients examined, 38% reported dyspnoea at rest or on exertion. No other respiratory symptoms were reported. Fifty percent had a normal chest radiograph. This study documents the high prevalence of respiratory functional abnormalities in patients with SSc. A restrictive pattern was found in 41% and an isolated diffusion impairment in 18%. No significant relationship was found between the isolated impairment of transfer factor of the lungs for carbon monoxide (TL,CO) and the mean duration of the scleroderma: thus, it does not seem to represent an early sign of severe restrictive disease. No bronchial or bronchiolar obstructive patterns were observed: it can be stated that small airways dysfunction is not a characteristic manifestation of SSc as considered previously. A significant association was found between the group of subjects with chest radiographic abnormalities and that with a restrictive pattern or isolated TL,CO alteration (p = 0.018). Chest radiographic abnormalities were also found in 29% and dyspnoea in 35% of the patients with normal respiratory function. The mean duration of scleroderma was not significantly different between the groups with and without abnormalities on chest radiography, between the groups with and without a restrictive pattern or isolated diffusion impairment, and between the groups of patients with and without dyspnoea. In conclusion, an accurate evaluation of respiratory function is recommended in the assessment of patients with systemic sclerosis, since the functional involvement of the lung cannot be predicted on the basis of the chest radiograph and the respiratory symptoms.

Pulmonary changes in a man affected by von Recklinghausen's disease.

Lung involvement in von Recklinghausen's disease is very rare and only sporadic cases have been reported in the literature. We present the case of a man affected by neurofibromatosis type 1 (NF1). The man complained of shortness of breath of 3-4 years duration. Chest radiograph and computed tomographic (CT) scan showed the presence of severe bullous emphysema, with extensive widespread bulky subpleural bullae, involving mainly the upper lobes and apical segments of the lower lobes, bilaterally. Functional respiratory tests documented a picture of severe obstruction associated with marked alveolar hyperinflation (vital capacity (VC) 80% of predicted, forced expiratory volume in one second (FEV1) 49% pred, maximal mid-expiratory flow (MMEF) 22% pred, residual volume (RV) 151% pred). Capillary alveolar diffusion was also markedly altered (transfer factor of the lungs for carbon monoxide (TL,co) 41% pred). The eventual prospect of a lung transplant, never previously considered for this disease, has been advised by the thoracic surgeons. In the light of this possibility, multidisciplinary assessment and monitoring of respiratory function over time are indispensable, in order to identify the transplant window correctly.