Clinical and biological role of secretory phospholipase A2 in acute respiratory distress syndrome infants.

INTRODUCTION: Secretory phospholipase A2 is supposed to play a role in acute lung injury but no data are available for pediatric acute respiratory distress syndrome (ARDS). It is not clear which enzyme subtypes are secreted and what the relationships are between enzyme activity, biophysical and biochemical parameters, and clinical outcomes. We aimed to measure the enzyme and identify its subtypes and to study its biochemical and biophysical effect. The secondary aim was to correlate enzyme activity with clinical outcome. METHODS: Bronchoalveolar lavage was performed in 24 infants with ARDS and 14 controls with no lung disease. Samples were assayed for secretory phospholipase A2 and molecules related to its activity and expression. Western blotting and captive bubble surfactometry were also performed. Clinical data were real time downloaded. RESULTS: Tumor necrosis factor-α (814 (506-2,499) vs. 287 (111-1,315) pg/mL; P = 0.04), enzyme activity (430 (253-600) vs. 149 (61-387) IU/mL; P = 0.01), free fatty acids (4.3 (2.8-8.6) vs. 2 (0.8-4.6) mM; P = 0.026), and minimum surface tension (25.6 ± 6.1 vs. 18 ± 1.8 mN/m; P = 0.006) were higher in ARDS than in controls. Phospholipids are lower in ARDS than in controls (76.5 (54-100) vs. 1,094 (536-2,907) µg/mL; P = 0.0001). Three enzyme subtypes were identified (-IIA, -V, -X), although in lower quantities in controls; another subtype (-IB) was mainly detected in ARDS. Significant correlations exist between enzyme activity, free fatty acids (ρ = 0.823; P < 0.001), and surface tension (ρ = 0.55; P < 0.028). Correlations also exist with intensive care stay (ρ = 0.54; P = 0.001), PRISM-III24 (ρ = 0.79; P< 0.001), duration of ventilation (ρ = 0.53; P = 0.002), and oxygen therapy (ρ = 0.54; P = 0.001). CONCLUSIONS: Secretory phospholipase A2 activity is raised in pediatric ARDS and constituted of four subtypes. Enzyme correlates with some inflammatory mediators, surface tension, and major clinical outcomes. Secretory phospholipase A2 may be a clinically relevant target in pediatric ARDS.

Surfactant and varespladib co-administration in stimulated rat alveolar macrophages culture.

Acute lung injury is a life-threatening condition characterized by surfactant dysfunction and raised secretory phospholipase A2 (sPLA2) activity. Varespladib is a sPLA2 inhibitor shown to be effective in animal models of acute lung injury. We aimed at investigating the effect of co-administration of surfactant and varespladib on sPLA2 activity. Alveolar macrophages were cultured and stimulated with lipopolysaccharide and then treated with either varespladib, surfactant, varespladib followed by surfactant or nothing. sPLA2 activity, free fatty acids, tumour necrosis factor-α (TNF-α) and protein concentrations were measured in culture supernatants. Treatment with varespladib (p=0.019) and varespladib + surfactant (p=0.013), reduced the enzyme activity by approximately 15% from the basal level measured in the untreated cultures. Surfactant, varespladib and varespladib + surfactant, respectively decreased free fatty acids by -45% (p=0.045), - 62% (p=0.009) and -48% (p=0.015), from the baseline concentration of the untreated cultures. Varespladib and poractant- α co-administration reduces sPLA2 activity and free fatty acids release in cultured rat alveolar macrophages, although a clear drug synergy was not evident. Since co-administration may be useful to reduce inflammation and surfactant inactivation in acute lung injury, further in vivo studies are warranted to verify its clinical usefulness.

Myocardial performance index and biochemical markers for early detection of doxorubicin-induced cardiotoxicity in children with acute lymphoblastic leukaemia.

BACKGROUND: Despite significant improvements in the prognosis of childhood acute lymphoblastic leukaemia (ALL), the risk of anthracycline-induced cardiovascular disease remains a major concern. This study was designed to investigate the role of the myocardial performance index (MPI) and serum concentrations of biomarkers (cTnT and NT-pro-BNP) in the early detection of subclinical anthracycline-induced functional alterations in children with ALL. METHODS: All children consecutively admitted to our Pediatric Oncologic Department from January 2009 to October 2010 with a diagnosis of ALL were enrolled in this study. cTnT and NT-pro-BNP were evaluated in all patients at diagnosis, before doxorubicin therapy and 2 and 24 h following each anthracycline administration. ECG and echocardiography were performed at diagnosis and 24 h after each anthracycline course. RESULTS: Nineteen children with standard-risk ALL were evaluated. The mean age was 6 years. The cumulative doxorubicin dosage was 240 mg/m(2) according to the AIEOP (Associazione Italiana Ematologia Oncologia Pediatrica) ALL 2000 protocol. None of the 19 patients developed congestive heart failure. With increasing cumulative dosages of anthracyclines a significant increase was observed in MPI. This increase was statistically significant starting from the cumulative dosage of 120 mg/m(2) compared to baseline, while the median NT-pro-BNP level did not change significantly during treatment and cTnT levels never exceeded the cut-off value for cardiac injury. CONCLUSION: MPI value is a sensitive and accurate parameter, allowing subclinical cardiac dysfunction to be detected in children receiving anthracyclines. Lifelong cardiac surveillance of these patients is warranted in order to determine the clinical implications of increased MPI on long-term cardiac status.

Potential usefulness of CTC detection in follow up of prostate cancer patients. A preliminary report obtained by using Adnagene platform.

OBJECTIVE: Prostate cancer (PCa) represents one of the most important medical problems for males, being the second major cause of cancer death. Routinely, PCa patients are followed up with both periodic evaluation of serum PSA levels and imaging. Recently, alternative laboratory methods were proposed for PCa patients' monitoring, with contrasting results. Aim of the present study was to evaluate the usefulness of a new commercially CE-IVD kit for detection of prostate circulating tumour cells. Our intention was to verify the Adnagene platform usefulness to identify patients with disease progression, whatever treatment ongoing, in order to modify the therapeutic process even before treatment failure is evident with imaging methods. MATERIALS AND METHODS: Twenty-one patients were enrolled and subdivided into three groups: n = 10 high risk tumor PCa patients; n = 6 low risk PCa patients; n = 5 sbjects without any signs of PCa. AdnaTest Prostate Cancer kit was used for enrichment and molecular characterization of prostate circulating tumour cells. RESULTS: Healthy subjects (with BPH) and patients without metastases resulted as negative, while 3 out of 10 high risk PCa patients were positive at least for one molecular marker like PSA, while only two showed positivity for PSMA mRNA. Our results indicate that the test specificity is 100% and the sensitivity is 100%; of course the sample is too small to give it statistical validity. In detail we verified that only the "not responder" patients resulted positive for AdnaTest. CONCLUSIONS: The present preliminary report provides evidence that isolation and detection of circulating tumour cells (CTCs) is feasible and it may be useful in the follow-up of patients with advanced prostate cancer. If the results of this preliminary study would be confirmed by a large prospective cohort study, it could be demonstrated that this test is a rapid diagnostic method, based on the analysis of a blood sample and useful to the clinician to decide when to change therapy for patients resistant to castration or able to confirm that, at that time, the therapy is effective.

Ex vivo effect of varespladib on secretory phospholipase A2 alveolar activity in infants with ARDS.

BACKGROUND: Secretory phospholipase A2 (sPLA2) plays a pivotal role in acute respiratory distress syndrome (ARDS). This enzyme seems an interesting target to reduce surfactant catabolism and lung tissue inflammation. Varespladib is a specifically designed indolic sPLA2 inhibitor, which has shown promising results in animals and adults. No specific data in pediatric ARDS patients are yet available. METHODS: We studied varespladib in broncho-alveolar lavage (BAL) fluids obtained ex vivo from pediatric ARDS patients. Clinical data and worst gas exchange values during the ARDS course were recorded. Samples were treated with saline or 10-40-100 µM varespladib and incubated at 37°C. Total sPLA2 activity was measured by non-radioactive method. BAL samples were subjected to western blotting to identify the main sPLA isotypes with different sensitivity to varespladib. Results was corrected for lavage dilution using the serum-to-BAL urea ratio and for varespladib absorbance. RESULTS: Varespladib reduces sPLA2 activity (p<0.0001) at 10,40 and 100 µM; both sPLA2 activity reduction and its ratio to total proteins significantly raise with increasing varespladib concentrations (p<0.001). IC(50) was 80 µM. Western blotting revealed the presence of sPLA2-IIA and -IB isotypes in BAL samples. Significant correlations exist between the sPLA2 activity reduction/proteins ratio and PaO(2) (rho = 0.63;p<0.001), PaO(2)/FiO(2) (rho = 0.7; p<0.001), oxygenation (rho = -0.6; p<0.001) and ventilation (rho = -0.4;p = 0.038) indexes. CONCLUSIONS: Varespladib significantly inhibits sPLA2 in BAL of infants affected by post-neonatal ARDS. Inhibition seems to be inversely related to the severity of gas exchange impairment.

Circulating endothelial cells as marker of endothelial damage in male hypogonadism.

Testosterone deficiency has become a frequently diagnosed condition in today's society affected by epidemic obesity, and is associated with cardiovascular risk. Recent studies have established the importance of altered vascular endothelium function in cardiovascular disease. The damage to the endothelium might also cause endothelial cell detachment, resulting in increased numbers of circulating endothelial cells (CEC) within the bloodstream. To evaluate whether hypogonadism could modify CEC count in peripheral bloodstream, we investigated peripheral blood CEC count using the CellSearch System, a semiautomatic method to accurately and reliably enumerate CECs, which are sorted based on a CD146(+), CD105(+), DAPI(+), CD45(-) phenotype, in a population of 20 patients with hypogonadism. The control group comprised 10 age- and sex-matched healthy participants. CEC count per milliliter was significantly increased in patients with hypogonadism vs the control group. In the group with hypogonadism, an inverse exponential correlation was present between testosterone levels and CEC count per milliliter. A direct linear correlation was present between waist circumference and CECs and between body mass index and CECs. The regression analysis showed that testosterone was the significant independent determinant of CECs. Our results underline that male hypogonadism is associated with endothelial dysfunction. The correlation between CEC and waist circumference underlines that visceral obesity may be synergically implicated in this regulation. Future studies are required to unveil the mechanisms involved in the pathogenesis of testosterone-induced endothelial disfunction, which may provide novel therapeutic targets to be incorporated in the management of hypogonadism.

Blood presence of circulating oncofetal fibronectin mRNA, by RT-PCR, does not represent a useful specific marker for the management and follow-up of thyroid cancer patients.

BACKGROUND: Recent studies strongly suggest the use of oncofetal fibronectin (onfFN) mRNA in diagnostic follow-up and staging due to its very high specificity for thyroid cancers. Since the use of this marker has not been well established yet, particularly in the monitoring of minimal residual disease, we have tried to verify the diagnostic power of onfFN and its usefulness as a prognostic molecular marker. For this reason, we evaluated (by RT-PCR) the presence of onfFN mRNAs, not only in blood samples and thyroid tissues (both normal and neoplastic), but also in different biological fluids (such as K3-EDTA blood samples, saliva and urine) belonging to healthy individuals. METHODS: Molecular investigations, such as RT-PCR protocol, and sequencing of onfFN cDNAs evaluation of the above-mentioned samples were performed. RESULTS: The onfFN transcript was largely expressed in all benign and malignant thyroid tissues [differentiated thyroid carcinomas (DTCs)] tested as well as in a large number of biological fluids; in particular, 100% urine samples were positive for onfFN transcript as compared to the thyroglobulin (Tg) mRNA (75%), while saliva was always positive for onfFN and never for Tg. These findings indicate that onfFN cannot be considered a marker specific for thyroid cancer presence. Finally, Tg results were positive in a large part of the samples, but not always in concomitance with onfFN. CONCLUSIONS: We underline how the complexity of onfFN transcripts could affect the RT-PCR procedure. In addition, the presence of onfFN transcripts in several normal and cancer tissues, along with non-thyroid biological fluids or cells, does not allow the use of this marker for cancer monitoring.

A new standardized absolute quantitative RT-PCR method for detection of tyrosinase mRNAs in melanoma patients: technical and operative instructions.

BACKGROUND: To develop a new absolute quantitative real-time PCR method for blood mRNA tyrosinase assay and to compare this new method with standard RT-PCR nested. METHODS: Ten blood of melanoma patients (stages I-III), 5 tissue samples, 2 surgical fresh metastatic skin and 3 lymph nodes paraffin-embedded slices were analysed, and 10 negative controls were used. Ten millilitres of blood was analysed for each individual. Three different protocols for RNA extraction and two reverse transcription methods were used. Specific human tyrosinase cDNA fragment was cloned into pcDNA3+ vector and then titrated for the standard curve construction (from 10(6) to 10(1)copies/microl). Recovery assays for RNA and cells were also performed. RESULTS: Our method was able to detect less than 5 cells/10(8) WBC and about 100 fg of tyrosinase RNA. Very low CVs (<1.5%) were obtained on all samples run in triplicate. Sensitivity and specificity were of 100%. The amount of starting volume of blood was crucial for the determination of copy number since large volumes are necessary for patient's monitoring. CONCLUSIONS: Our absolute qRT-PCR assay could be proposed as a new standardized molecular method for the management of melanoma patients, particularly for the follow up of the highest AJCC stages.

Two novel CYP21A2 missense mutations in Italian patients with 21-hydroxylase deficiency: Identification and functional characterisation.

Steroid 21-hydroxylase deficiency is present in more than 90% of patients with congenital adrenal hyperplasia (CAH), an inherited metabolic disorder of adrenal steroidogenesis. Impaired enzymatic activity leads to the accumulation of metabolic intermediates (progesterone and 17-hydroxyprogesterone), which results in excessive androgen production and varied signs of virilisation. CYP21A2 is an active gene and encodes for the steroid 21-hydroxylase enzyme, whereas CYP21A1P is an inactive pseudogene that contains a series of deleterious mutations. The major part of disease- causing mutations in CYP21A2 alleles are CYP21A1P-derived sequence transferred to the active gene by macro or microconversion events. Only around 5% of all disease-causing CYP21A2 alleles harbour rare mutations that do not originate from the pseudogene. In this report, we report the characterisation of two novel CYP21A2 missense mutations (p.H119R and p.I194N) found in two unrelated Italian patients with nonclassic (NC) CAH clinical diagnosis. Functional in vitro assays for mutagenized CYP21 enzymes were performed in transiently transfected mammalian cells to test the residual enzyme activity and the apparent kinetic values. The residual activities obtained for p.H119R and p.I194N mutants allowed to classify them as NC-CAH associated mutations. These results correlate with the rate of severity of the patients' disease. Finally, the new p.H119R and p.I194N mutations should be included in the panel of those already listed for association with the NC form of 21-hydroxylase deficiency.

Functional analysis of two rare CYP21A2 mutations detected in Italian patients with a mildest form of congenital adrenal hyperplasia.

BACKGROUND: More than 90% of all cases of congenital adrenal hyperplasia (CAH) result from steroid 21-hydroxylase gene (CYP21A2) mutations. Most of these mutations result from intergenic recombinations between CYP21A2 and closely linked CYP21A1P pseudogene. Rare mutations not generated by gene conversion account for 5-10% of 21-hydroxylase deficiency alleles. OBJECTIVE: Functional analysis of two novel CYP21A2 missense mutations (p.R224W and p.D407N) was performed. DESIGN: Our study was composed of two Italian patients suffering from a very mild form of nonclassic CAH (NC-CAH). To assay the enzymatic activity of mutants, the in vitro analysis was performed in transiently transfected COS-1 cells. RESULTS: The residual activities obtained for p.R224W and p.D407N mutants allow their classification as NC-CAH mutations. These results correlate with the rate of severity of the patients' disease. CONCLUSIONS: In this paper, we report two novel CYP21A2 mutations in two Italian individuals affected by 21-hydroxylase deficiency. Based on the functional in vitro analysis we can classify these mutations as NC-CAH variants.

Glucose-6-phosphate dehydrogenase Buenos Aires: a novel de novo missense mutation associated with severe enzyme deficiency.

OBJECTIVE: : Glucose 6-phosphate dehydrogenase (G6PD) catalyzes the first committed steps in the pentose phosphate pathway: the generation of NADPH by this enzyme is essential for protection against oxidative stress. The human enzyme is in a dimer<-->tetramer equilibrium and its stability depends on NADP(+) concentration. Herein, we report a case of a symptomatic baby affected by severe deficiency of G6PD activity due to a novel de novo genetic mutation (g1465C>T) in the thirteenth exon of its gene. METHODS: : Clinical, biochemical and genetic evaluations of the affected baby and his mother were performed. RESULTS: : We found the g1465C>T novel mutation, in the thirteenth exon of G6PD gene (named "G6PD Buenos Aires variant"). This g1465C>T mutation produce a P489S substitution at protein level. The P489S mutation was absent in his mother, suggesting that G6PD Buenos Aires resulted from a de novo mutation. CONCLUSIONS: : The absence of mosaicism in the baby's DNA (from saliva and blood samples) suggests that a de novo mutation event may occur in the very early stages in embryogenesis or in the mother's germ cell lines.

GSTT1 and GSTM1 allelic polymorphisms in head and neck cancer patients from Italian Lazio Region.

BACKGROUND: The association between head and neck squamous cell carcinoma (HNSCC) and allelic variants of glutathione S-transferase M1 (GSTM1) and -T1 (GSTT1) is currently controversial. The present study investigates the prevalences of GSTT1 and GSTM1 polymorphism in a cohort of 100 head and neck cancer patients, 100 healthy donors and 200 controls with non-neoplastic head and neck diseases from Italian Lazio Region. METHODS: The patients with benign head and neck pathologies, as well as the healthy donors were matched for age, sex, cigarette smoke (yes/no) and alcohol consumption (yes/no). Molecular definition of GSTT1 and GSTM1 genotype has been performed by means of allele-specific PCR technique. RESULTS: A significant association between head and neck cancer and GSTM1 null genotype was observed both considering benign disease controls (p=0.001, OR=2.613; 95% C.I.=1.48-4.62), and healthy donors (p=0.0003, OR=3.35; 95% C.I. 1.69-6.67) while no significant association was found with GSTT1 null genotype (p>or=0.14). No interactive association was observed when combining the different genotypes of the two polymorphisms. These results were confirmed after correction for daily number of cigarettes and period of tobacco exposure. CONCLUSIONS: The present study confirms a role for genetic alterations of GSTM1 detoxifying enzyme as a risk factor for the development of HNSCC in patients from the Italian Lazio Region, independently of age, sex and other confounding variables.