Electrophoretic α1-globulin for screening of α1-antitrypsin deficient variants.

Background Available screening procedures for the detection of α1-antitrypsin-deficient (AATD) mutations have suboptimal cost-effectiveness ratios. The aim in this study was to evaluate and compare the viability of a composite approach, primarily based on the α1-globulin fraction, in identifying AAT genetic analysis eligible patients against standard screening procedures, based on clinically compatible profiling and circulating AAT < 1 g/L. Methods A total of 21,094 subjects were screened for AATD and deemed eligible when meeting one of these criteria: α1-globulin ≤2.6%; α1-globulin 2.6%-2.9% and AST: >37 U/L and ALT: > 78 U/L; α1-globulin %: 2.9-4.6% and AST: >37 U/L and ALT: >78 U/L and erythrocyte sedimentation rate (ESR) >34 mm/h and C-reactive protein (CRP) >3 mg/L. Subjects were genotyped for the AAT gene mutation. Detection rates, including those of the rarest variants, were compared with results from standard clinical screenings. Siblings of mutated subjects were included in the study, and their results compared. Results Eighty-two subjects were identified. Among these, 51.2% were found to carry some Pi*M variant versus 15.9% who were clinically screened. The detection rates of the screening, including relatives, were: 50.5% for the proposed algorithm and 18.9% for the clinically-based screening. Pi*M variant prevalence in the screened population was in line with previous studies. Interestingly, 46% of subjects with Pi*M variants had an AAT plasma level above the 1 g/L threshold. Conclusions A composite algorithm primarily based on the α1-globulin fraction could effectively identify carriers of Pi*M gene mutation. This approach, not requiring clinical evaluation or AAT serum determination, seems suitable for clinical and epidemiological purposes.

The Extract of Leonurus sibiricus Transgenic Roots with AtPAP1 Transcriptional Factor Induces Apoptosis via DNA Damage and Down Regulation of Selected Epigenetic Factors in Human Cancer Cells.

The aim of this study was to determine the anticancer potential of Leonurus sibiricus extract derived from in vitro transgenic roots transformed by Agrobacetrium rhizogenes with AtPAP1 transcriptional factor, and that of transformed roots without construct, on grade IV human glioma cells and the U87MG cell line, and attempt to characterize the mechanism involved in this process. The anticancer effect induced by the tested extracts was associated with DNA damage, PARP cleavage/increased H2A.X histone levels and UHRF-1/DNMT1 down-regulation of mRNA levels. Additionally, we demonstrated differences in the content of compounds in the tested extracts by HPLC analysis with ATPAP1 construct and without. Both the tested extracts showed anticancer properties and the better results were observed for AtPAP1 with transcriptional factor root extract; this effect could be ascribed to the presence of higher condensed phenolic acids such as neochlorogenic acid, chlorogenic acids, ferulic acid, caffeic acid and p-coumaric acid. Further studies with AtPAP1 (with the transcriptional factor from Arabidopisi thaliana) root extract which showed better activities in combination with anticancer drugs are needed.

Relationship between FEV1 and arterial stiffness in elderly people with chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is highly prevalent in the elderly, and both COPD and age per se are associated with cardiovascular morbidity. AIMS: We tested the hypothesis that in elderly COPD patients airflow limitation is associated with arterial stiffness and the relationship, if any, is related to endothelial function and systemic inflammation. METHODS: We evaluated lung function, augmentation index (AIx), flow-mediated dilation (FMD), Interleukin-6 (IL-6), and asymmetric dymethilarginine (ADMA) levels in 76 subjects (mean age 73.9 years, SD 6.2) attending a geriatric outpatient clinic. RESULTS: Participants with COPD (N = 41) and controls (N = 35) did not differ in terms of AIx (30 vs 28.2 %, P = 0.30) and FMD (14.2 vs 12.3 %, P = 0.10). Similarly, the two groups did not differ with respect to mean concentrations of inflammation markers (IL-6 and C-reactive protein) and ADMA. Among COPD participants there was an inverse correlation between AIx and Forced Expiratory Volume in the first second (r = -0.349, P = 0.02). This relationship remained significant after correction for potential confounders, including markers of inflammation and ADMA levels (ß = -0.194, P = 0.001). DISCUSSION: According to the results of this study, among COPD patients, bronchial patency and AIx are inversely related, and the relationship is explained neither by endothelial function nor by systemic inflammation. CONCLUSIONS: In elderly COPD people, increased arterial stiffness is related to reduced pulmonary function and it seems worth testing as a potential marker of higher cardiovascular risk.

Stereotyped subset #1 chronic lymphocytic leukemia: a direct link between B-cell receptor structure, function, and patients' prognosis.

Chronic lymphocytic leukemia (CLL) with stereotyped B-cell receptor (BCR) belonging to subset #1 (IGHV1-5-7/ IGKV1-39) display a poor outcome. To characterize their genetic and genomic features and BCR function, we selected 20 subset #1 CLL from a series of 579 cases. Subset #1 CLL, all showing unmutated IGHV, were associated with the presence of del(11q) (50%) in comparison with unmutated CLL, unmutated stereotyped CLL other than subset #1 and with cases using the same IGHV genes but a heterogeneous VH CDR3 (non-subset #1 CLL). There were no distinctive features regarding CD38, ZAP-70, and TP53 disruption. NOTCH1, SF3B1, and BIRC3 were mutated in 15%, 0%, and 5% of cases, respectively, while BIRC3 was deleted in 22% of cases. Microarray unsupervised analysis on 80 unmutated/mutated/stereotyped/non-stereotyped CLL showed a tight clustering of subset #1 cases. Their genomic signature exhibited several differentially expressed transcripts involved in BCR signal transduction, apoptosis regulation, cell proliferation, and oxidative processes, regardless of del(11q). Accordingly, BCR ligation with anti-IgM revealed a significant higher proliferation of subset #1 versus unmutated non-subset #1 CLL, both at baseline and after 24­48 hr stimulation. Subset #1 CLL represent a paradigmatic example of the direct link between BCR structure, function, and patients prognosis.

Behind the scenes of non-nodal MCL: downmodulation of genes involved in actin cytoskeleton organization, cell projection, cell adhesion, tumour invasion, TP53 pathway and mutated status of immunoglobulin heavy chain genes.

Mantle cell lymphoma (MCL) is an aggressive neoplasm with a short survival. Cases with leukaemic MCL and splenomegaly without adenopathies (non-nodal MCL) may have a more indolent course. To gain insights into the biological features underlying this presentation, we investigated the gene expression profile (GEP) and the IGHV mutational status in a cohort of leukaemic MCL cases. Comparison of MCL with other lymphoproliferative disorders (i.e. splenic marginal zone lymphoma, follicular lymphoma, chronic lymphocytic leukaemia) revealed a MCL signature enriched for the following gene categories: mitochondrion, oxidoreductase activity, response to stress, to DNA damage and TP53-pathway. Furthermore, GEP analysis revealed that non-nodal MCL cases were characterized by the down-modulation of the following gene categories: cell projection, actin cytoskeleton organization, cell adhesion (ITGAE, CELSR1, PCDH9) and tumour invasion/progression (PGF, ST14, ETS1, OCIAD1, EZR). Many down-modulated genes were related to the TP53-pathway and to DNA damage response. IGHV status proved unmutated in all nodal and mutated in all non-nodal MCL. Non-nodal leukaemic MCLs display a peculiar clinical presentation, with distinctive biological features, such as mutated IGHV and a transcriptional profile lacking tumour invasion properties, that might contribute to the absence of nodal involvement and to the less aggressive clinical course.

ATM gene alterations in chronic lymphocytic leukemia patients induce a distinct gene expression profile and predict disease progression.

BACKGROUND: The genetic characterization of chronic lymphocytic leukemia cells correlates with the behavior, progression and response to treatment of the disease. DESIGN AND METHODS: Our aim was to investigate the role of ATM gene alterations, their biological consequences and their value in predicting disease progression. The ATM gene was analyzed by denaturing high performance liquid chromatography and multiplex ligation probe amplification in a series of patients at diagnosis. The results were correlated with immunoglobulin gene mutations, cytogenetic abnormalities, ZAP-70 and CD38 expression, TP53 mutations, gene expression profile and treatment-free interval. RESULTS: Mutational screening of the ATM gene identified point mutations in 8/57 cases (14%). Multiplex ligation probe amplification analysis identified six patients with 11q deletion: all of them had at least 20% of deleted cells, analyzed by fluorescent in situ hybridization. Overall, ATM point mutations and deletions were detected in 14/57 (24.6%) cases at presentation, representing the most common unfavorable genetic anomalies in chronic lymphocytic leukemia, also in stage A patients. Patients with deleted or mutated ATM had a significantly shorter treatment-free interval compared to patients without ATM alterations. ATM-mutated cases had a peculiar gene expression profile characterized by the deregulation of genes involved in apoptosis and DNA repair. Finally, definition of the structure of the ATM-mutated protein led to a hypothesis that functional abnormalities are responsible for the unfavorable clinical course of patients carrying these point mutations. CONCLUSIONS: ATM alterations are present at diagnosis in about 25% of individuals with chronic lymphocytic leukemia; these alterations are associated with a peculiar gene expression pattern and a shorter treatment-free interval.

Evaluation of TP53 mutations with the AmpliChip p53 research test in chronic lymphocytic leukemia: correlation with clinical outcome and gene expression profiling.

Given that TP53 alterations predict prognosis and response to therapy in chronic lymphocytic leukemia (CLL), screening for TP53 mutations has an increasing role in patient management. TP53 direct sequencing is a time-consuming method, while the AmpliChip p53 Research Test is a novel non time-consuming microarray-based resequencing assay and queries Exons 2-11. We evaluated the impact of TP53 mutations on clinical outcome by analyzing 98 untreated CLL using the AmpliChip p53 Research Test and direct sequencing and performed microarrays analysis on TP53 mutated and/or deleted cases. The AmpliChip p53 Research Test detected 17 mutations in 14 patients (17.3%); a significant association between TP53 mutations and del(17p) was recorded. From a clinical standpoint, a higher percentage of mutation was found in CLL with unfavorable outcome (17.2% vs. 7.1% in progressive vs. stable cases). Detection of TP53 mutations by the AmpliChip p53 Research Test was associated with a significantly worse survival (P = 0.0002). Comparison of the array and direct sequencing tests showed that the p53 Research Test detected more mutations, although it failed to identify two microdeletions. Finally, microarrays analysis showed a more distinctive signature associated with del(17p) than with TP53 mutations, likely due to a concomitant gene dosage effect. The AmpliChip p53 Research Test is a straightforward method that bears prognostic value. This study confirms a high percentage of TP53 mutations in CLL with unfavorable outcome and a significant association between TP53 aberrations and del(17p). Finally, specific gene expression profiles are recognized for TP53 alterations.

AICDA expression in BCR/ABL1-positive acute lymphoblastic leukaemia is associated with a peculiar gene expression profile.

Activation-induced cytidine deaminase (AICDA) initiates somatic hypermutation and class-switch recombination of immunoglobulin (Ig) genes and induces mutations also in non-Ig genes. AICDA aberrant expression was detected in B-lineage acute lymphoblastic leukaemia (B-ALL), particularly BCR/ABL1+ B-ALL; patients expressing AICDA carried more copy number alterations than 'AICDA-negative' cases. To determine the role of AICDA, AICDA expression and gene expression profiling were studied in adult BCR/ABL1+ B-ALL. Patients displaying the full-length isoform AICDA are characterized by up-regulation of DNA repair/replication and cell cycle genes, suggesting their involvement in the genetic instability of BCR/ABL1+ B-ALL.

Spontaneous regression of chronic lymphocytic leukemia: clinical and biologic features of 9 cases.

In chronic lymphocytic leukemia (CLL), spontaneous regressions are an exceptional phenomenon, whose biologic features are unknown. We describe 9 CLL patients who underwent a spontaneous clinical regression over an 11-year follow-up, despite a residual neoplastic clone detected by flow cytometry. CD38 and ZAP-70 were negative in all cases. Immunoglobulin heavy chain variable region (IgVH) genes, mutated in all 7 evaluable patients, were restricted to the VH3 family in 6, with the usage of V(H)3-30 gene in 2. The light chain variable region genes were mutated in 6 of 8 cases, with the use of V(kappa)4-1 gene in 3. Microarray analysis of CLL cells showed a distinctive genomic profile with an overrepresentation of BCR-related and ribosomal genes, regulators of signal transduction and transcription. The number of activated T lymphocytes expressing IFN-gamma, TNF-alpha, and IL-4 was similar between CLL in spontaneous regression and healthy persons. In conclusion, spontaneous clinical regressions can occur in CLL despite the persistence of the neoplastic clone, and the biologic features include negative CD38 and ZAP-70, mutated V(H)3-30 and V(kappa)4-1 genes. The peculiar gene profile suggests that BCR signaling may play an important role in this scenario as the most significant feature of the leukemic clone in regression.

White blood cell count at diagnosis and immunoglobulin variable region gene mutations are independent predictors of treatment-free survival in young patients with stage A chronic lymphocytic leukemia.

A comprehensive panel of clinical-biological parameters was prospectively evaluated at presentation in 112 patients with chronic lymphocytic leukemia (<65 years), to predict the risk of progression in early stage disease. Eighty-one percent were in Binet stage A, 19% in stages B/C. Treatment-free survival was evaluated as the time from diagnosis to first treatment, death or last follow up. In univariate analysis, advanced stage, hemoglobin, platelets, white blood cell, leukemic lymphocyte count, raised beta 2-microglobulin and LDH, unmutated immunoglobulin variable region genes, CD38, del(17p), del(11q) and +12, were significantly associated with a short treatment-free survival; the T/leukemic lymphocyte ratio was associated with a better outcome. Multivariate analysis of treatment-free survival in stage A patients selected a high white blood cell count and unmutated immunoglobulin variable region genes as unfavorable prognostic factors and a high T/leukemic lymphocyte ratio as a favorable one. At diagnosis, these parameters independently predict the risk of progression in stage A chronic lymphocytic leukemia patients.

BCR ligation induced by IgM stimulation results in gene expression and functional changes only in IgV H unmutated chronic lymphocytic leukemia (CLL) cells.

Chronic lymphocytic leukemia (CLL) patients exhibit a variable clinical course. To investigate the association between clinicobiologic features and responsiveness of CLL cells to anti-IgM stimulation, we evaluated gene expression changes and modifications in cell-cycle distribution, proliferation, and apoptosis of IgV(H) mutated (M) and unmutated (UM) samples upon BCR cross-linking. Unsupervised analysis highlighted a different response profile to BCR stimulation between UM and M samples. Supervised analysis identified several genes modulated exclusively in the UM cases upon BCR cross-linking. Functional gene groups, including signal transduction, transcription, cell-cycle regulation, and cytoskeleton organization, were up-regulated upon stimulation in UM cases. Cell-cycle and proliferation analyses confirmed that IgM cross-linking induced a significant progression into the G(1) phase and a moderate increase of proliferative activity exclusively in UM patients. Moreover, we observed only a small reduction in the percentage of subG(0/1) cells, without changes in apoptosis, in UM cases; contrariwise, a significant increase of apoptotic levels was observed in stimulated cells from M cases. These results document that a differential genotypic and functional response to BCR ligation between IgV(H) M and UM cases is operational in CLL, indicating that response to antigenic stimulation plays a pivotal role in disease progression.

Cluster analysis on breath print of newly diagnosed COPD patients: effects of therapy.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a highly heterogeneous disease and airflow limitation and symptoms only partially capture such heterogeneity. Since COPD is known to affect the production of volatile organic compounds (VOCs), we aimed to verify to which extent exhaled VOCs can characterize newly diagnosed COPD patients and changes in response to inhaled therapy. MATERIALS AND METHODS: Fifty newly diagnosed COPD patients were consecutively recruited among those attending the pulmonary medicine outpatient clinic at 'Campus Bio-Medico' University Hospital. VOCs were collected using the Pneumopipe® and analysed by the BIONOTE electronic nose both at baseline and after 12 weeks of inhaled therapy. Patients were grouped using K-mean cluster analysis on BIONOTE responses and the obtained clusters were compared via non-parametric tests. RESULTS: We identified three clusters of subjects: (a) without remarkable comorbidities; (b) with air trapping and higher BODE index score (mean 1.2); (c) without air trapping and with a lower BODE index. Inhaled bronchodilators caused a quantitative reduction in the amound of VOCs, while inhaled steroids provided a qualitative modification of the breath profile. CONCLUSION: VOCs patterns categorize newly diagnosed COPD subjects. VOCs production declines after bronchodilators administration and changes in quality after topic steroid treatment.

Chest ultrasonography in health surveillance of asbestos related pleural disease.

High resolution computed tomography, (HRCT), is currently considered the diagnostic gold standard to diagnose early stage malignant pleural mesothelioma and other non-malignant pleural conditions, but it is expensive and exposes the patient to radiation dose. In a screening and population medicine perspective, Thoracic Ultrasounds may become a valuable alternative because it can detect minimal changes in pleural surface, is widely available and safe. On these bases, we therefore validated thoracic US in subjects with history of exposure to asbestos, having HRCT as the reference standard. One hundred-fifty subjects were screened and 117 were recruited. Pleural abnormalities at US and/or HRCT were detected in 66 out of 117 subjects (prevalence=57%), and their prevalence was unrelated to both mansion and smoking habit, while mean age and mean length of exposure were higher in those having pleural abnormalities (age=47±5 vs 44±6years, p<0.05;years of exposure=20±7 vs 17±5, p<0.05). Thirteen out of 19 subjects with pleural abnormalities at HRCT were also identified by thoracic US, whereas 47 participants had lesions seen at US, but not at the HRCT scan. Positive and negative percent agreement were 66.6% and 51.8%, respectively; the McNemar's test for equality showed a p-value <0.001. In conclusion, chest US might complement HRCT in the health surveillance of asbestos exposed population to detect earlier lesions or to follow up US approachable lesions. Further research is needed to clarify whether this approach may enhance early recognition of pleural mesothelioma and ameliorate prognosis.

Alpha-1 Antitrypsin Deficiency: Current Perspective from Genetics to Diagnosis and Therapeutic Approaches.

Alpha-1 antitrypsin (A1AT) is a 52-kDa, acute phase glycoprotein encoded by the protease inhibitor (PI) locus, located on the long arm of chromosome 14 (14q31-32.3). Its structure is composed of a total of 7 exons, 4 coding (II, III, IV, and V) and 3 non-coding (IA, IB, and IC). A1AT is produced primarily by hepatocytes and acts as a serine protease inhibitor with antiprotease and immunoregulatory activities. The main target of A1AT is neutrophil elastase (NE), an enzyme released during a neutrophil-mediated inflammatory process. When the enzyme is not adequately balanced by A1AT activity, it can cause tissue injury and destruction. A1AT deficiency (A1ATD) is a genetic autosomal recessive disease, characterized by low serum levels of A1AT. The condition may lead to liver disease, early-onset pulmonary emphysema and, rare multi-organ vasculitis, necrotizing panniculitis and fibromyalgia. At least 100 allelic variants of the polymorphic PI locus have been described with groups including associations with different A1AT plasma levels and functions. Treatments with purified A1AT preparations, obtained through pooled human plasma (augmentation therapy), have been proven to improve survival and disease-related quality of life, as well as, slow down the progression of organ damage. Furthermore, ongoing research is now focusing on the development of specifically targeted, new medications. The aim of this review is to summarize our knowledge of the genetic A1AT variants, focusing on their variable clinical manifestation, report routine and recently updated laboratory diagnostic techniques, and to highlight the relevance of early diagnosis of A1ATD. Moreover, we will review the role of augmentation therapy recommendations and future perspectives focusing on a personalized treatment of A1ATD.

Screening of Obstructive Sleep Apnea Syndrome by Electronic-Nose Analysis of Volatile Organic Compounds.

Obstructive Sleep Apnea Syndrome (OSAS) carries important social and economic implications. Once the suspicion of OSAS has arisen, Polysomnography (PSG) represents the diagnostic gold standard. However, about 45% of people who have undergone PSG are free from OSAS. Thus, efforts should be made to improve the selection of subjects. We verified whether the pattern of Volatile Organic Compounds (VOCs) helps to select patients amenable to PSG. We studied 136 subjects (20 obese non-OSAS, 20 hypoxic OSAS, 20 non-hypoxic OSAS, and 20 non-hypoxic Chronic Obstructive Pulmonary Disease (COPD) vs 56 healthy controls) without any criteria of exclusion for comorbidity to deal with a real-life population. VOCs patterns were analyzed using electronic-nose (e-nose) technology. A Discriminant Analysis (Partial Least Square-Discriminant Analysis) was performed to predict respiratory functions and PSG parameters. E-nose distinguished controls (100% correct classification) from others and identified 60% of hypoxic, and 35% of non-hypoxic OSAS patients. Similarly, it identified 60% of COPD patients. One-by-one group comparison yielded optimal discrimination of OSAS vs controls and of COPD vs controls (100% correct classification). In conclusion, e-nose technology applied to breath-analysis can discriminate non-respiratory from respiratory diseased populations in real-life multimorbid populations and exclude OSAS. If confirmed, this evidence may become pivotal for screening purposes.

Short-term effects of a vibrotactile neck-based treatment device for positional obstructive sleep apnea: preliminary data on tolerability and efficacy.

BACKGROUND: Positional supine obstructive sleep apnea syndrome (OSAS) characterizes a subgroup of patients suffering from OSAS. Several devices designed to limit supine position have been developed, but evidences of their efficacy and safety are lacking. It is unclear whether a neck-worn vibrating device could induce positional change in patients with positional OSAS. We evaluated the efficacy of a neck-worn device to induce supine avoidance positional feedback over a short-term trial in OSAS patients and its impact on sleep quality and polysomnographyc indexes. METHODS: Twenty patients with positional apneas/hypopneas were prospectively studied. Baseline characteristics of daytime somnolence and risk of sleep apnea were screened and the efficacy of a 3-day trial of supine-avoidance therapy by vibrotactile neck worn device assessed by reporting the self-perceived change in quality of sleep and performing cardio-respiratory polysomnography. Comparison between baseline and treatment results was performed. RESULTS: The neck device produced a reduction in overall apnea-hypopnea index (AHI) (mean AHI pre =16.8/h and post =4.4/h, P<0.0001), oxygen desaturation (pre =13.7/h and post =3.8/h, P<0.0001) and Respiratory Disturbance Indexes (RDI) (20.0/h vs. 5.2/h; P<0.0001).The time spent in supine position decreased from 62.1% to 33.7% of the total (P<0.001). However, the impact on the perceived quality of sleep was unpredictable. CONCLUSIONS: The neck position therapy device is effective in restricting supine sleep, improving AHI and related polysomnographic indexes. However, at least in a short-term trial, it seems unable to improve the patient's sleep quality.

Genetic background of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease characterized by progressive fibrosing interstitial pneumonia. The histological pattern, which displays dense fibrosis with active areas of fibroblastic proliferation, suggests a pathogenetic role of aberrant response to healing of multiple microscopic, repeated alveolar epithelial injuries. Although the exact etiology of the disease is still under investigation, several studies suggest that a combination of genetic and environmental factors may play a causal role. The aim of this review is to describe the genetic background of IPF, reporting the latest advancements made possible by genomic techniques that allow a high-throughput analysis and the identification of target genes implicated in IPF. This information may help to clarify pivotal aspects on prognosis and diagnosis, and may help to identify potential targets for future therapies.