Risk factors and preventive strategies for post-traumatic stress disorder in neonatal intensive care unit.

Background: Preterm birth and admission to the neonatal intensive care unit (NICU) could induce post-traumatic stress disorder (PTSD). PTSD is an important factor to focus on, as it is associated with parental mental health difficulties and with changes in caregiving quality such as increased intrusiveness, reduced sensitivity, and increased attachment insecurity for the child.

Aims: We aimed to study the main risk factors, in the early life of newborns, and preventive measures for PTSD in parents of neonates hospitalized in the NICU.

Clinical outcomes of oral metronomic vinorelbine in advanced non-small cell lung cancer: correlations with pharmacokinetics and MDR1 polymorphisms.

Purpose: This study investigated correlations of the clinical outcomes of oral metronomic vinorelbine (VNR) with VNR pharmacokinetics and MDR1 polymorphisms.

Methods: Eighty-two patients with metastatic non-small cell lung cancer (NSCLC) unfit for standard chemotherapy were treated with VNR at the oral doses of 20-30 mg every other day or 50 mg three times a week. They had a performance status (PS) ≤ 3, were > 70-year-old and drug-naïve or cisplatin-pretreated.

Rapid LC-MS/MS method for quantification of vinorelbine and 4-O-deacetylvinorelbine in human whole blood suitable to monitoring oral metronomic anticancer therapy.

A rapid and sensitive LC-MS/MS method for therapeutic drug monitoring oral vinorelbine (VRL) metronomic anticancer chemotherapy has been developed and validated. Analysis of VRL and its main active metabolite 4-O-deacetylvinorelbine (M1) was performed in whole blood matrix. Both analytes were extracted by protein precipitation and separated on an Onyx monolith C , 50 × 2 mm column then quantified by positive electrospray ionization and multiple reaction monitoring mode.

Plasma matrix metalloprotease 9 correlates with blood lymphocytosis, leukemic cell invasiveness, and prognosis in B-cell chronic lymphocytic leukemia.

The complex biology underlying chronic lymphocytic leukemia cell migration and tissue invasiveness is not yet completely understood and might provide novel predictive markers and therapeutic targets. A total of 36 patients out of treatment from at least 3 months were enrolled and followed up for a median period of 44.2 months (range: 4.

Genetic prediction of long-term survival after neoadjuvant chemoradiation in locally advanced esophageal cancer.

Candidate genes involved in DNA repair, 5-fluorouracil metabolism and drug detoxification were genotyped in 124 patients receiving neoadjuvant chemoradiation treatment for locally advanced esophageal cancer and their predictive role for long-term relapse-free survival (RFS) and cancer-specific survival (CSS) were evaluated. A panel including MTHFR 677TT, MDR1 2677GT, GSTP1 114CC, XPC 499CC and XPC 939AC+CC, defined as high-risk genotypes, discriminated subgroups with significantly different outcomes. When the panel was combined with histology, patients split into two subsets with 5-year RFS and CSS rates of 65% vs 27% (hazard ratio (HR) 3.

Age affects pegylated liposomal doxorubicin elimination and tolerability in patients over 70 years old.

Purpose: Pegylated liposomal doxorubicin (PLD) is often used in elderly people, due to its improved tolerability. However, clinical and pharmacological data in the subset of patients over 70 are scanty.

Methods: PLD safety was evaluated in 35 patients (aged ≥70 years) who were treated with PLD as a single agent for 165 cycles.

The association of polymorphisms in 5-fluorouracil metabolism genes with outcome in adjuvant treatment of colorectal cancer.

Aim: The purpose of this study was to investigate whether specific combinations of polymorphisms in 5-fluorouracil (5-FU) metabolism-related genes were associated with outcome in 5-FU-based adjuvant treatment of colorectal cancer.

Methods: We analyzed two cohorts of 302 and 290 patients, respectively, one cohort for exploratory analyses and another cohort for validating the exploratory analyses. A total of ten polymorphisms in genes involved in 5-FU pharmacodynamics and pharmacokinetics were studied.

Frequency of uridine monophosphate synthase Gly(213)Ala polymorphism in Caucasian gastrointestinal cancer patients and healthy subjects, investigated by means of new, rapid genotyping assays.

Aims: Uridine monophosphate synthase (UMPS) is a fundamental enzyme in pyrimidine synthesis. A single-nucleotide polymorphism, a G-C transversion at the 638th nucleotide, was demonstrated to increase UMPS activity and suggested to have clinical effects. The aims of this study were to set up simple genotyping methods and investigate the UMPS 638G>C polymorphism in the Caucasian population.

Combinations of polymorphisms in genes involved in the 5-Fluorouracil metabolism pathway are associated with gastrointestinal toxicity in chemotherapy-treated colorectal cancer patients.

Purpose: The purpose of this study was to investigate whether specific combinations of polymorphisms in genes encoding proteins involved in 5-fluorouracil (5-FU) pharmacokinetics and pharmacodynamics are associated with increased risk of treatment-induced toxicity.

Experimental Design: We analyzed two cohorts of 161 and 340 patients, the exploration and validation cohort, respectively. All patients were treated similarly with 5-FU-based adjuvant chemotherapy.

Cochlear repair by transplantation of human cord blood CD133+ cells to nod-scid mice made deaf with kanamycin and noise.

We investigated the fate of human cord blood CD133+ hematopoietic stem cells (HSC) transplanted intravenously (IV) into irradiated nod-scid mice previously made deaf by ototoxic treatment with kanamycin and/ or intense noise, to verify whether HSC engraft the cochlea and contribute to inner ear restoration, in vivo. We tested the presence of HLA.DQalpha1 by PCR, used for traceability of engrafted cells, finding evidence that HSC migrated to various host tissues, including the organ of Corti (OC).

Gentamicin-induced cytotoxicity involves protein kinase C activation, glutathione extrusion and malondialdehyde production in an immortalized cell line from the organ of corti.

The objective of the present study was to investigate the biochemical mechanisms underlying gentamicin cytotoxicity in OC-k3 cells derived from an immortalized cell line developed from the organ of Corti of transgenic mice. Administration of 50 microM gentamicin significantly reduced cell proliferation and viability, as well as initiating morphological changes associated with apoptosis. Protein kinase C (PKC) alpha activity was increased in gentamicin-treated cells, peaking 15 min after dosing (+138.

Determination of histamine in the whole blood of colon cancer patients.

The aim of the present work is to investigate whether histamine assay could be useful in detecting the presence of primary cancer. The high-performance liquid chromatographic (HPLC)-based o-phthalaldialdehyde (OPA) histamine derivatization assay was investigated with respect to several variables, dramatization reagent concentration, organic solvent requirement, derivatization time and counter-ion effect on chromatographic separation. The OPA histamine assay, in the absence of added -SH groups, was found to detect histamine in whole blood samples with relative standard deviations <14% and recoveries not less than 90%.

Apoptosis in the OC-k3 immortalized cell line treated with different agents.

The aim of this study is to outline the mechanisms leading cochlear cells to die. We utilized an immortalized cell line (OC-k3 cells) derived from the organ of Corti of transgenic mice in order to perform in-depth biochemical studies with no limitations on sample size and number. We probed these cells with cisplatin and gentamicin, two drugs which display in vivo undesired ototoxic side-effects.

Human herpesvirus 7 infection induces profound cell cycle perturbations coupled to disregulation of cdc2 and cyclin B and polyploidization of CD4(+) T cells.

Human herpesvirus 7 (HHV-7) infection of both primary CD4(+) T lymphocytes and SupT1 lymphoblastoid T-cell line induced a progressive accumulation of cells exibiting a gap 2/mitosis (G2/M) and polyploid content coupled to an increased cell size. The expression of both cyclin-dependent kinase cdc2 and cyclin B was increased in HHV-7-infected cells with respect to the uninfected ones. Moreover, the simultaneous flow cytometric analysis of cyclin B and DNA content showed that cyclin B expression was not only increased but also unscheduled with respect to its usual cell cycle pattern.

High levels of HIV-1 replication show a clear correlation with downmodulation of Bcl-2 protein in peripheral blood lymphocytes of HIV-1-seropositive subjects.

Peripheral blood lymphocytes (PBLs) from 51 HIV-1-seropositive subjects with different levels of HIV-1 replication and 20 healthy blood donors were examined for the expression of the antiapoptotic Bcl-2 protein. All the plasma samples from HIV-1 patients were characterized for the presence of HIV-1 p24 and HIV, RNA viral load. Bcl-2 protein expression in fresh peripheral blood lymphocytes was studied by different tests, including Western blot and indirect immunofluorescence techniques.

Enforced expression of human bcl-2 in CD4+ T cells enhances human herpesvirus 7 replication and induction of cytopathic effects.

The cytopathic effects (CPE) resulting from the infection of CD4+ T cells by human herpes-virus 7 (HHV-7) comprises two major mechanisms: generation of large polyploid cells, which eventually undergo necrotic lysis, and apoptosis, predominantly occurring in small mononucleated cells. To dissect the relative contribution of these two phenomena to the overall cytopathicity of HHV-7 in vitro, we have investigated the effect of acute HHV-7 infection on SupT1 CD4+ T cell lines stably transfected either with the bcl-2 anti-apoptotic gene or with the control vector. Overexpression of Bcl-2 protein by these cells was associated with a progressive decline of the total number of viable cells, and a relative increase of enlarged polyploid cell.

Extracellular HIV-1 Tat protein induces the rapid Ser133 phosphorylation and activation of CREB transcription factor in both Jurkat lymphoblastoid T cells and primary peripheral blood mononuclear cells.

Extracellular HIV-1 Tat protein (0.1-100 ng/ml) induced a rapid (peak at 30 min) increase in the Ser133 phosphorylation levels of the transcription factor CREB in serum-starved Jurkat cells, as revealed by Western blot and indirect immunofluorescence analyses. Nuclear cAMP-responsive element (CRE) binding activity in electrophoretic mobility shift assays was constitutive in unstimulated Jurkat cells, showing only a small increase upon Tat treatment.

Accumulation of catalytically active PKC-zeta into the nucleus of HL-60 cell line plays a key role in the induction of granulocytic differentiation mediated by all-trans retinoic acid.

The effect of differentiating doses of all-trans retinoic acid (ATRA, 10(-6) M) and vitamin D3 (10(-7) M) was investigated on the nuclear levels of endogenous ceramide and protein kinase C-zeta (PKC-zeta) catalytic activity in HL-60 myeloid cells. ATRA induced a parallel increase of ceramide and catalytically active PKC-zeta into the nuclear compartment of HL-60 cells (peak at 72 h). On the other hand, vitamin D3 increased the levels of nuclear ceramide and PKC-zeta activity to a lesser extent and with a delayed kinetics compared to ATRA (peak at 96 h).

Nuclear translocation of protein kinase C-alpha and -zeta isoforms in HL-60 cells induced to differentiate along the granulocytic lineage by all-trans retinoic acid.

We investigated whether members of the protein kinase C (PKC) family of enzymes were involved in the nuclear events underlying granulocytic differentiation induced by 10(-6) M all-trans retinoic acid (ATRA) in HL-60 cells. PKC activity was analysed by using a serine substituted specific peptide which enabled the evaluation of the whole catalytic activity of both Ca2+ -dependent and Ca2+ -independent PKC isoforms. In parallel, the subcellular distribution of various PKC isoforms was evaluated by Western blot, immunoprecipitation and in situ immunocytochemistry analyses.

Changes of nuclear protein kinase C activity and isotype composition in PC12 cell proliferation and differentiation.

To establish whether protein kinase C was involved in the nuclear events underlying cell differentiation and proliferation, rat pheochromocytoma PC12 cells, serum-starved for 24 h, were treated with either differentiating doses of nerve growth factor or high serum concentrations, which represented a powerful mitogenic stimulus. Western blot analysis with isoform-specific antibodies, performed on whole cell homogenates, cytoplasms, and purified nuclei, showed that PKC isotypes alpha, beta I, beta II, delta, epsilon, eta, and zeta were expressed in PC12 cells and that all of them, except for beta I, were found at the nuclear level, variably modulated depending on the cell treatment. Compared to serum-stimulated cells, in which an early (1 day) and marked rise of protein kinase C activity was followed by a plateau, nerve growth factor-treated cells showed a progressive increase of protein kinase C activity coincident with the onset and maintenance of the differentiated phenotype.

In vivo and in vitro modulatory effect of human immunodeficiency virus type-1 (HIV-1) Tat protein on protein kinase C activity.

Extracellular HIV-1 Tat protein shows a pleiotropic activity on the survival/proliferation of different cell types, which may be relevant to the pathogenesis of the immune suppression as well as of the frequent neoplastic disorders observed during the course of HIV-1 disease. Therefore, we investigated the effect of recombinant Tat on the protein kinase C (PKC) activity in Jurkat CD4(+) T lymphoma cells by using a serine substituted specific PKC peptide substrate, which allowed the evaluation of the whole catalytic activity of both Ca++-dependent and Ca++-independent PKC isoforms. High concentrations of recombinant Tat (1 mu g/ml) induced an early (5 min) stimulation followed by a secondary (30-60 min) inhibition of PKC in whole Jurkat cell homogenates.

Low nanogram range quantitation of diglycerides and ceramide by high-performance liquid chromatography.

A method for ceramide (CER) and diradylglycerol (DG) determination after normal-phase HPLC separation was developed. The free oxydril group of ceramide and diradylglycerol is coupled to the carboxylic group of the fluorescent label (+)-6-methoxy-alpha-methyl-2 naphthaleneacetic acid (NAP), using as catalytic agents 4-dimethylaminopyridine and N,N'-dicyclohexylcarbodiimide. The use of NAP-free acid instead of the halide-activated form ensures higher stability of the reagent, lower reaction temperatures, and improved yield and reproducibility.

Exogenous human immunodeficiency virus type-1 Tat protein selectively stimulates a phosphatidylinositol-specific phospholipase C nuclear pathway in the Jurkat T cell line.

We investigated the effect of extracellular Tat protein of human immunodeficiency virus-type 1 (HIV-1) on the phosphatidylinositol (PI) cycle, which represents a major signal transduction pathway in lymphoid cells. Recombinant Tat, recombinant HIV-1 p24 and cross-linked anti-CD3 monoclonal antibody (mAb) were added in culture for 1-60 min to Jurkat lymphoblastoid CD4+ T cells. The stimulation of T cell receptor by cross-linked anti-CD3 mAb resulted in a rapid increase of the phosphatidylinositol-specific phospholipase C (PI-PLC) activity in whole cell lysates.