Pharmacodynamic monitoring by residual gene expression of the nuclear factor of activated T cell-regulated genes in lung transplant recipients and its correlation with tacrolimus blood levels.

Introduction: Trough blood levels (C0) of tacrolimus are used to adjust drug dosage, but they do not consistently correlate with clinical outcomes. Measurement of residual gene expression of nuclear factor of activated T cell (NFAT)-regulated genes (NFAT-RGE) has been proposed as a pharmacodynamic biomarker to assess the degree of immunosuppression in certain solid organ transplantations, but little is known regarding lung transplant recipients (LTR). Our primary objective is to correlate tacrolimus blood levels with NFAT-RGE. Methods: NFAT-RGE and tacrolimus C0 and peak (C1.5) levels were determined in 42 patients at three, six and 12 months post-transplantation. Results: Tacrolimus C0 did not exhibit a correlation with NFAT-RGE, whereas C1.5 did. Besides, over 20% of measurements indicated high levels of immunosuppression based on the below 30% NFAT-RGE threshold observed in many studies. Among those measurements within the therapeutic range, 19% had an NFAT-RGE<30%. Conclusion: Consequently, a subset of patients within the tacrolimus therapeutic range may be more susceptible to infection or cancer, potentially benefiting from NFAT-RGE and tacrolimus peak level monitoring to tailor their dosage. Further quantitative risk assessment studies are needed to elucidate the relationship between NFAT-RGE and the risk of infection, cancer, or rejection.

Lymphangioleiomyomatosis: Searching for potential biomarkers.

Background: Vascular endothelial growth factor-D (VEGF-D) is the most commonly used biomarker for diagnosing lymphangioleiomyomatosis (LAM). However, lung biopsy is often necessary as well; therefore, defining new biomarkers for LAM is crucial. The aim of this study was to describe the diagnostic accuracy of a variety of biomarkers. Methods: We assessed 13 analytes in serum related to extracellular matrix remodeling, lymphatic involvement and angiogenesis in a cohort of patients with LAM, comparing them with patients with other cystic lung diseases (OCLD) and healthy women. A scoring method based on the cut-point of each VEGF-D and metalloproteinase-2 (MMP-2) was used to evaluate the diagnostic performance of the marker combination. Results: A total of 97 subjects were recruited: 59 (61%) LAM patients, 18 (19%) OCLD patients, and 20 (20%) healthy female controls. MMP-2 was the only extracellular matrix remodeling biomarker able to differentiate LAM patients from OCLD and healthy patients. Serum MMP-2 was higher in LAM patients [median 578 (465-832) ng/ml] than in patients with OCLD and healthy controls [medians 360 (314-546) and 427 (365-513) ng/ml, respectively (p < 0.0001)]. The area under ROC curve (AUC) of MMP-2 was 0.785 and that of VEGF-D 0.815 (p = 0.6214). The sensitivity/specificity profiles of each biomarker (54/92% for MMP-2, 59/95% for VEGF-D) yielded a composite score (-6.36 + 0.0059 × VEGF-D + 0.0069 × MMP-2) with higher accuracy than each component alone (AUC 0.88 and sensitivity/specificity 79/87%). Conclusion: Combining MMP-2 and VEGF-D may increase diagnostic accuracy for LAM.

YKL-40 and KL-6 Levels in Serum and Sputum of Patients Diagnosed With Hypersensitivity Pneumonitis.

BACKGROUND: YKL-40 (chitinase 3-like-1) and Krebs von den Lungen-6 (KL-6) are 2 promising biomarkers that may have an important role in the management of interstitial lung diseases (ILD). OBJECTIVE: The aim of this study was to investigate the values of KL-6 and YKL-40 as biomarkers in the diagnosis and prognosis of patients with hypersensitivity pneumonitis (HP). METHODS: A cross-sectional study conducted in 49 patients diagnosed with HP due to exposure to birds (n = 32) or fungi (n = 17), 48 patients with other ILD, and 67 healthy volunteers. Patients with HP were divided into fibrotic and nonfibrotic. Serum and sputum YKL-40 and KL-6 levels were determined using commercial enzyme-linked immunosorbent assay kits. Receiver operating characteristic (ROC) curves were used to determine the sensitivity and specificity of both biomarkers for the diagnosis of HP. Pulmonary function tests were performed in patients during follow-up. RESULTS: KL-6 and YKL-40 levels were significantly higher in serum of patients with HP exposed to birds with a fibrotic pattern than in controls (P < .0001 and .0055, respectively). Serum KL-6 levels were also significantly higher in patients with fibrotic HP exposed to fungi compared with the control group (P = .0001). In patients with HP exposed to fungi, sputum KL-6 and YKL-40 levels were higher in those with a fibrotic pattern (P = .0289 and .016, respectively). ROC analysis showed that the range between 55-121 ng/mL for serum YKL-40 levels and 346-1441 U/mL for serum KL-6 levels had the best sensitivity and specificity for discriminating between patients with HP, healthy controls, and patients with idiopathic pulmonary fibrosis (IPF). In patients with HP, serum KL-6 levels correlated negatively with total lung capacity (r = -0.485; P = .0103) and diffusing capacity of the lungs for carbon monoxide (r = -0.534; P = .0002) at 12 months. CONCLUSIONS: Both KL-6 and YKL-40 proteins seem to be capable of distinguishing patients with HP from healthy individuals and from patients with IPF. Their sensitivity and specificity confirm their potential role as biomarkers. KL-6 may also be a predictor of disease progression.

Histamine signaling and metabolism identify potential biomarkers and therapies for lymphangioleiomyomatosis.

Inhibition of mTOR is the standard of care for lymphangioleiomyomatosis (LAM). However, this therapy has variable tolerability and some patients show progressive decline of lung function despite treatment. LAM diagnosis and monitoring can also be challenging due to the heterogeneity of symptoms and insufficiency of non-invasive tests. Here, we propose monoamine-derived biomarkers that provide preclinical evidence for novel therapeutic approaches. The major histamine-derived metabolite methylimidazoleacetic acid (MIAA) is relatively more abundant in LAM plasma, and MIAA values are independent of VEGF-D. Higher levels of histamine are associated with poorer lung function and greater disease burden. Molecular and cellular analyses, and metabolic profiling confirmed active histamine signaling and metabolism. LAM tumorigenesis is reduced using approved drugs targeting monoamine oxidases A/B (clorgyline and rasagiline) or histamine H1 receptor (loratadine), and loratadine synergizes with rapamycin. Depletion of Maoa or Hrh1 expression, and administration of an L-histidine analog, or a low L-histidine diet, also reduce LAM tumorigenesis. These findings extend our knowledge of LAM biology and suggest possible ways of improving disease management.

A rapid test for the environmental detection of pigeon antigen.

INTRODUCTION: Avoidance of inhaled bird antigens is essential to prevent hypersensitivity pneumonitis disease progression. The aim of the present study was to develop a sandwich enzyme link immunoassay (ELISA) and an immunochromatographic test (ICT) and compare their ability to detect pigeon antigens in environmental samples. METHODS: An amplified sandwich ELISA using pigeon serum as a calibration standard and a ICT using gold-labeled anti-pigeon serum antibodies for the rapid detection of pigeon antigens in environmental samples were developed. Twenty-two different airborne samples were collected and analysed using both methods. Strip density values obtained with ICT were calculated and compared with the concentrations determined by the ELISA method for pigeon antigens. Strips results were also visually analysed by five independent evaluators. RESULTS: The ELISA method to quantify pigeon antigen had a broader range (58.4 and 10,112.2 ng/ml), compared to the ICT assay (420 to 3360 ng/ml). A kappa index of 0.736 (p < 0.0001) was obtained between the observers evaluating the ICT strips. The results of the ELISA and the relative density of the ICT showed a highly significant correlation (rs:0.935; p < 0.0001). Bland-Altman plot also confirmed excellent agreement between the two methods (mean difference: -1.626; p < 0.0001). CONCLUSIONS: Since there was a good correlation between both assays, we can conclude that the rapid and simple ICT assay is a good and valid alternative, which does not require expensive equipment, for the validated ELISA technique.

Long-term results of sirolimus treatment in lymphangioleiomyomatosis: a single referral centre experience.

There are few published data on long-term treatment with sirolimus in lymphangioleiomyomatosis (LAM). The objective of this study was to describe the long-term effect of sirolimus in a series of LAM patients followed up in a referral centre, focusing on pulmonary function. We retrospectively reviewed a series of 48 patients with LAM diagnosed, followed up and treated with sirolimus in a single centre. Response to sirolimus was evaluated at 1 and 5 years. A negative sirolimus response was defined as an FEV1 decline greater than - 75 ml/year. A mixed-effects model was used to estimate the longitudinal changes in FEV1 (average slope), both as absolute (ml/year) and as predicted values (%predicted/year). From a total of 48 patients, 9 patients underwent lung transplantation and 4 died during the study. Mean (95% CI) FEV1 slope over 5 years was - 0.14 (- 26.13 to 25.85) ml/year in the whole LAM group, 42.55 (14.87 to 70.22) ml/year in the responder group, - 54.00 (- 71.60 to - 36.39) ml/year in the partial responder group and - 84.19 (- 113.5 to - 54.0) ml/year in the non-responder group. After 5 years of sirolimus treatment 59% had a positive response, 30% had a partial response and 11% had a negative response. Our study found that sirolimus treatment had a positive long-term effect on most LAM patients.

Pilot Trial of Extended Hypothermic Lung Preservation to Analyze Ischemia-reperfusion Injury in Pigs.

BACKGROUND: In lung transplantation (LT), the length of ischemia time is controversial as it was arbitrarily stablished. We ought to explore the impact of extended cold-ischemia time (CIT) on ischemia-reperfusion injury in an experimental model. METHODS: Experimental, randomized pilot trial of parallel groups and final blind analysis using a swine model of LT. Donor animals (n=8) were submitted to organ procurement. Lungs were subjected to 6h (n=4) or 12h (n=4) aerobic hypothermic preservation. The left lung was transplanted and re-perfused for 4h. Lung biopsies were obtained at (i) the beginning of CIT, (ii) the end of CIT, (iii) 30min after reperfusion, and (iv) 4h after reperfusion. Lung-grafts were histologically assessed by microscopic lung injury score and wet-to-dry ratio. Inflammatory response was measured by determination of inflammatory cytokines. Caspase-3 activity was determined as apoptosis marker. RESULTS: We observed no differences on lung injury score or wet-to-dry ratio any given time between lungs subjected to 6h-CIT or 12h-CIT. IL-1ß and IL6 showed an upward trend during reperfusion in both groups. TNF-α was peaked within 30min of reperfusion. IFN-γ was hardly detected. Caspase-3 immunoexpression was graded semiquantitatively by the percentage of stained cells. Twenty percent of apoptotic cells were observed 30min after reperfusion. CONCLUSIONS: We observed that 6 and 12h of CIT were equivalent in terms of microscopic lung injury, inflammatory profile and apoptosis in a LT swine model. The extent of lung injury measured by microscopic lung injury score, proinflammatory cytokines and caspase-3 determination was mild.

Predictive Value of Immune Cell Functional Assay for Non-Cytomegalovirus Infection in Lung Transplant Recipients: A Multicenter Prospective Observational Study.

INTRODUCTION: Immune cell functional assay (ImmuKnow®) is a non-invasive method that measures the state of cellular immunity in immunosuppressed patients. We studied the prognostic value of the assay for predicting non-cytomegalovirus (CMV) infections in lung transplant recipients. METHODS: A multicenter prospective observational study of 92 patients followed up from 6 to 12 months after transplantation was performed. Immune cell functional assay was carried out at 6, 8, 10, and 12 months. RESULTS: Twenty-three patients (25%) developed 29 non-CMV infections between 6 and 12 months post-transplant. At 6 months, the immune response was moderate (ATP 225-525ng/mL) in 14 (15.2%) patients and low (ATP<225ng/mL) in 78 (84.8%); no patients had a strong response (ATP≥525ng/mL). Only 1 of 14 (7.1%) patients with a moderate response developed non-CMV infection in the following 6 months compared with 22 of 78 (28.2%) patients with low response, indicating sensitivity of 95.7%, specificity of 18.8%, positive predictive value (PPV) of 28.2%, and negative predictive value (NPV) of 92.9% (AUC 0.64; p=0.043). Similar acute rejection rates were recorded in patients with mean ATP≥225 vs. <225ng/mL during the study period (7.1% vs. 9.1%, p=0.81). CONCLUSION: Although ImmuKnow® does not seem useful to predict non-CMV infection, it could identify patients with a very low risk and help us define a target for an optimal immunosuppression.

Pharmacokinetic Study of Conversion Between 2 Formulations of Once-daily Extended-release Tacrolimus in Stable Lung Transplant Patients.

BACKGROUND: The aim of this study was to compare the pharmacokinetic profile, tolerability, and safety of a novel once-daily extended-release formulation of tacrolimus (LCPT) with that of once-daily prolonged-release tacrolimus (ODT) in stable adult lung transplant (LT) recipients. METHODS: Phase II, open-label, single-arm, single-center, prospective pilot pharmacokinetic study. Study population comprised 20 stable LT recipients receiving ODT, mean age 55.9 years (range, 38-67 years), 13 (65%) men. Patients were switched to LCPT in a 1:0.7 (mg/mg) conversion dose. Follow-up was 6 months, and cystic fibrosis patients were excluded. Two 24-hour pharmacokinetic profiles were obtained for each patient, the first on day -14 and the second on day +14 after switching to LCPT. Pharmacokinetic parameters and safety were compared. RESULTS: Mean (SD) area under the concentration-time curve from 0 to 24 hours was 253.97 (61.90) ng/mL per hour for ODT and 282.44 (68.2) ng/mL per hour for LCPT. Systemic exposure was similar in both (Schuirmann two 1-sided test). Mean (SD) dose was 5.05 (1.67) mg in ODT and 3.36 (1.03) mg in LCPT (P = 0.0002). Time to maximum concentration was 125 minutes for ODT and 325 minutes for LCPT (P < 0.001). Correlation between area under the concentration-time curve from 0 to 24 hours and C24 was 0.896 (r) for ODT and 0.893 (r) for LCPT. There were no differences in adverse effects. At 6 months, conversion dose was 1:0.59 (mg/mg) in patients with unchanged minimum plasma concentration target levels. CONCLUSIONS: Switching from ODT to LCPT was safe and well tolerated in stable LT recipients without cystic fibrosis. A significantly lower dose of LCPT allows similar bioavailability. A conversion ratio 1:0.6 could be enough to maintain similar target levels.

BALF cytokines in different phenotypes of chronic lung allograft dysfunction in lung transplant patients.

The long-term success of lung transplantation (LT) is limited by chronic lung allograft dysfunction (CLAD). Different phenotypes of CLAD have been described, such as bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS). The purpose of this study was to investigate the levels of cytokines and chemokines in bronchoalveolar lavage fluid (BALF) as markers of these CLAD phenotypes. BALF was collected from 51 recipients who underwent (bilateral and unilateral) LT. The study population was divided into three groups: stable (ST), BOS, and RAS. Levels of interleukin (IL)-4, IL-5, IL-6, IL-10, IL-13, tumor necrosis factor alpha (TNF-α), interferon-gamma (IFN-γ), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were measured using the multiplex technology. BALF neutrophilia medians were higher in BOS (38%) and RAS (30%) than in ST (8%) (P=.008; P=.012). Regarding BALF cytokines, BOS and RAS patients showed higher levels of INF-γ than ST (P=.02; P=.008). Only IL-5 presented significant differences between BOS and RAS (P=.001). BALF neutrophilia is as a marker for both CLAD phenotypes, BOS and RAS, and IL-5 seems to be a potential biomarker for the RAS phenotype.

Bronchial inflammation in hypersensitivity pneumonitis after antigen-specific inhalation challenge.

BACKGROUND AND OBJECTIVE: The aim of this study is to compare the inflammatory profile before and after specific inhalation challenge (SIC) in induced sputum from patients with hypersensitivity pneumonitis (HP) and to investigate whether different causal antigens define the resulting profile. METHODS: A prospective study was conducted in 27 patients with HP: 15 patients due to exposure to birds (BHP) and 12 due to exposure to fungi (FHP), confirmed by SIC. Induced sputum was obtained before and/or 24 h after SIC. Cell types were determined by differential cell count using optical microscopy. Interferon-γ, interleukin (IL)-12p70, IL-2, IL-10, IL-8, IL-6, IL-4, IL-5, IL-1ß, tumour necrosis factor (TNF)-α and TNF-ß levels were measured in the supernatants. RESULTS: Following SIC, higher sputum neutrophilia levels (P = 0.048) and an increase in IL-8 levels (P = 0.017) were found in patients with FHP than in those with BHP. FHP patients also showed increased IL-1ß, IL12-p70 and IL5 levels (P = 0.011, P = 0.036 and P = 0.018, respectively) after SIC. In BHP, a trend towards increases in sputum eosinophils and TH2 cytokines (IL4, IL5) was seen following SIC (P = 0.059, P = 0.068 and P = 0.075 respectively). CONCLUSIONS: This study shows that bronchial inflammation is present in patients with HP evidenced by increases in sputum neutrophils and eosinophils following exposure to the offending antigen during SIC.

A rapid test for soy aeroallergens exposure assessment.

BACKGROUND: Determining soy aeroallergens levels is extremely important in the assessment of health risks due to these airborne substances. Currently, soy aeroallergens exposure in the environment is monitored using enzyme immunoassays (EIA) which must be evaluated in a specialized laboratory by skilled personnel. OBJECTIVE: To describe the development and performance of a rapid immunochromatography assay for the detection of soy aeroallergens in environmental samples. METHODS: A test strip using gold labeled anti-soy hull low molecular weight extract (SHLMWE) antibody for the rapid detection of soy aeroallergens in environmental samples was developed. One hundred nineteen airborne samples were analysed in parallel by the strip assay and the anti-SHLMWE sandwich EIA. The assay results were visually analysed by three independent observers who ranked samples as: -, + or ++. Strips were also scanned and analysed by densitometry. RESULTS: The rapid test detected a range of concentrations from 6.25 to 25 ng/mL. Agreement in strip assay interpretations between evaluators was substantial (Kappa = 0.63; CI 0.544-0.715). Visual interpretation also gave a good concordance with EIA results, with sensitivity ranging from 77.3 to 100 and specificity from 65 to 83.5 depending on the observer. Furthermore, a strong correlation was observed between densitometry results of strip assay and EIA determinations. CONCLUSIONS: The strip assay developed is rapid, simple, and sensitive and does not require expensive equipment or specific skills. It has considerable potential in the environmental monitoring field for screening soy aeroallergens levels in port cities where allergen measurements are not currently performed. Due to its simplicity, the test will improve the management of soy allergic patients by controlling environmental allergen exposure without the need for apparatus or skilled personnel.

[Course of bronchial hyperresponsiveness in patients with occupational asthma caused by exposure to persulfate salts]. / Evolución de la hiperrespuesta bronquial en pacientes con asma ocupacional por exposición a sales de persulfato.

OBJECTIVE: Persulfate salts are among the most frequently implicated causes of occupational asthma. The aim of this study was to describe the course of bronchial hyperresponsiveness and immunologic test results in patients with occupational asthma due to persulfate salts. PATIENTS AND METHODS: Ten patients with occupational asthma due to persulfate salts were studied. Diagnosis was based on specific bronchial challenge tests performed at least 3 years before enrollment. An exhaustive medical and work history was taken during interviews with all patients, and all underwent spirometry and nonspecific bronchial challenge testing. Total immunoglobulin E levels were determined and skin prick tests to several persulfate salts were performed. RESULTS: At the time of evaluation, 7 patients had avoided workplace exposure to persulfate salts. The bronchial hyperresponsiveness of 3 of those 7 patients had improved significantly. No improvement was observed in patients who continued to be exposed. Specific skin prick tests became negative in 3 patients who were no longer exposed at the time of the follow-up evaluation. Most of the patients continued to report symptoms, although improvements were noted. One patient, however, reported worsening of symptoms in spite of avoidance of exposure. CONCLUSIONS: Although asthma symptoms and bronchial hyperresponsiveness may persist for patients with occupational asthma due to persulfate salts, their condition seems to improve if they avoid exposure. This course does not seem to differ from that reported for other cases of occupational asthma.

Effects of oral estrone on rat energy balance.

Oral doses of estrone from 10 nmol/(kg day) to 10 micromol/(kg day) were given to adult Wistar male rats for 10 days. Body composition, energy balance, total body estrone balance and plasma metabolites and hormones were measured at the end of the treatment. Body weight (as well as food intake, body energy, fat and water accrual) increased at doses in the 10--100 nmol/(kg day) range, but decreased at higher doses. Energy expenditure decreased with increasing doses of estrone. Plasma metabolite changes suggested the maintenance of energy homeostasis, and lipid parameters indicated that lipid mobilization increased with the increasing doses of estrone. Plasma estrone, acyl-estrone and estradiol levels decreased at low doses and increased at high doses of estrone. We conclude that: (a) repeated estrone gavages, even at very high doses, do not result in the accrual of estrone in the body; (b) low doses of estrone promote growth and high doses decrease body mass and fat accretion; (c) administration of estrone at low doses decreases its circulating levels and the levels of estradiol and acyl-estrone, a situation reverted at higher doses and (d) estrone administration induces a dose-dependent shift towards lower energy expenditure.