Peripheral intravenous catheter-induced phlebitis in a tertiary hospital of Karachi: a cohort study.

PURPOSE: This study aimed to determine the incidence of peripheral intravenous catheter (PIVC)-induced phlebitis and its predictors among adult patients hospitalized at Dow University Hospital, Karachi, Pakistan. METHODS: A sample of 258 adult patients admitted in the selected wards and planned for peripheral intravenous catheter insertion were recruited through consecutive sampling during March to May 2019. Daily follow-ups were performed to observe signs of phlebitis using a validated tool. The cohort was followed until discharge, removal of peripheral intravenous catheter, or study conclusion. RESULTS: Of 258 patients studied, 139 (53.9%) were females. A significant number of the participants 104 (40.3%) were young adults of age 20-40 years. The incidence of phlebitis was 39.1%. Tuberculosis (TB), peripheral intravenous catheter dwell time before initial assessment, administration of IV fluids, and dissatisfactory nursing care at Day 1 were associated significantly with the development of phlebitis. There was a doseresponse relationship between the catheter dwell time in hours before initial assessment and the development of phlebitis. CONCLUSION: This study found an increased incidence (39.1%) in three months of PIVC-induced phlebitis among adult patients. In addition to patient-related and PIVC-related risk factors considered in this study, PIVC-induced phlebitis is found to be significantly associated with the level of PIVC care provided by nurses. Continuous nursing education, developing standard care plans for PIVCs, and proper documentation of care are recommended.

Maintenance treatment with azacytidine for patients with high-risk myelodysplastic syndromes (MDS) or acute myeloid leukaemia following MDS in complete remission after induction chemotherapy.

This prospective Phase II study is the first to assess the feasibility and efficacy of maintenance 5-azacytidine for older patients with high-risk myelodysplastic syndrome (MDS), chronic myelomonocytic leukaemia and MDS-acute myeloid leukaemia syndromes in complete remission (CR) after induction chemotherapy. Sixty patients were enrolled and treated by standard induction chemotherapy. Patients that reached CR started maintenance therapy with subcutaneous azacytidine, 5/28 d until relapse. Promoter-methylation status of CDKN2B (P15 ink4b), CDH1 and HIC1 was examined pre-induction, in CR and 6, 12 and 24 months post CR. Twenty-four (40%) patients achieved CR after induction chemotherapy and 23 started maintenance treatment with azacytidine. Median CR duration was 13.5 months, >24 months in 17% of the patients, and 18-30.5 months in the four patients with trisomy 8. CR duration was not associated with CDKN2B methylation status or karyotype. Median overall survival was 20 months. Hypermethylation of CDH1 was significantly associated with low CR rate, early relapse, and short overall survival (P = 0.003). 5-azacytidine treatment, at a dose of 60 mg/m(2) was well tolerated. Grade III-IV thrombocytopenia and neutropenia occurred after 9.5 and 30% of the cycles, respectively, while haemoglobin levels increased during treatment. 5-azacytidine treatment is safe, feasible and may be of benefit in a subset of patients.

A role of Gab2 association in Flt3 ITD mediated Stat5 phosphorylation and cell survival.

The haematopoietic growth factor receptor Flt3 has been implicated as major cause of transformation in acute myeloid leukaemia. Intracellular signals mediated by wild-type Flt3 are involved in cell differentiation and survival whereas signalling via the mutant Flt3 ITD (internal tandem duplication) promotes enhanced cell growth. In this study, we identified tyrosines 768, 955 and 969 of Flt3 as phosphorylation sites and mediators of growth factor receptor binding protein 2 (Grb2) interaction, leading to the association of Grb2 associated binder 2 (Gab2) and contributing to proliferation and survival. Ba/F3 cells were transfected with either the wild-type Flt3 or the ITD, with or without a triple mutation of the Grb2 binding sites, and characterised in terms of proliferation and viability. Interestingly, the Flt3 ITD promoted increased survival but after introducing the triple mutation, this phenotype was lost. When looking into different downstream pathways, this effect was mainly caused by decreased phosphoinositide 3-kinase and Stat5 signalling, and the Flt3 ITD carrying the Grb2 binding mutations showed less Akt and Stat5 activation compared to the regular Flt3 ITD receptor. These findings not only reveal novel phosphorylation sites in Flt3 but contribute to the understanding of the molecular mechanism by which Flt3 ITD functions in pathological conditions.

Oncogenic Flt3 receptors display different specificity and kinetics of autophosphorylation.

OBJECTIVE: Fms-like tyrosine kinase-3 (Flt3), a growth factor receptor normally expressed in hematopoietic progenitor cells, has been shown to have an important role in development of acute myeloid leukemia (AML) due to activating mutations. Flt3 mutations are found in approximately one-third of AML patients and correlate with a poor prognosis, thus making the Flt3 receptor a potential therapeutic target. The aim of the investigation was to analyze the kinetics and specificity of Flt3 autophosphorylation in wild-type Flt3 as well as in oncogenic Flt3 mutants. MATERIALS AND METHODS: We have used Ba/F3 cells stably expressing either wild-type, internal tandem duplication, or D835Y mutants of Flt3 in order to compare the site selectivity of tyrosine phosphorylation sites. By the use of a panel of phosphospecific antibodies directed against potential tyrosine phosphorylation sites in Flt3, we identified several novel phosphorylation sites in Flt3 and studied the kinetics and specificity of ligand-induced phosphorylation in living cells. RESULTS: Eight phosphorylated tyrosines (pY589, pY591, pY599, pY726, pY768, pY793, pY842, and pY955) were investigated and shown to be differentially phosphorylated in the wild-type versus the mutated receptors. Furthermore, we show that tyrosines 726, 793, and 842 are novel phosphorylation sites of Flt3 in intact cells. CONCLUSION: In this study, we have looked at the site-specific phosphorylation in the wild-type Flt3 in comparison to the mutants found in AML. We observed not only quantitative changes but, more importantly, qualitative differences in the phosphorylation patterns of the wild-type and the mutated Flt3 receptors, which might enhance the understanding of the mechanisms by which Flt3 contributes to AML in patients with mutations in Flt3.

Hypomethylation and apoptosis in 5-azacytidine-treated myeloid cells.

OBJECTIVE: Although clinically approved for myelodysplastic syndromes (MDS), the mode of action of 5-azacytidine has not been well understood at the cellular level. The present study aimed at characterizing the mechanisms for 5-azacytidine-induced apoptosis, as well as the presence of a possible link between apoptosis and DNA hypomethylation. MATERIALS AND METHODS: We investigated the effects of 5-azacytidine on a spectrum of specific apoptotic pathways, as well as on global DNA methylation, assessed by luminometric methylation assay, in myeloid (P39, HL60) and T cells (Jurkat). RESULTS: 5-Azacytidine induced dose-dependent apoptosis as well as non-dose-dependent global DNA hypomethylation at concentrations >or=0.5 microM. Hypomethylation was observed in the sorted apoptotic fraction (41% decrease with 1 microM after 24 hours), while nonapoptotic cells retained a methylation pattern similar to untreated cells (+/-6%). The induced apoptotic pattern involved several pathways: cleavage of Bcl-2 family proteins, activation of caspase-2 and -3-like, mitochondrial involvement characterized by loss of transmembrane potential (tetramethylrhodamine ethyl ester [TMRE]) and cytochrome release, and acidification of cytosol. Selective inhibition of caspase-3-like, -2, -8, -9, and pan-caspase activity, as well as stabilization of cytosolic pH by monensin completely failed to block apoptosis. Poly(ADP-ribose) polymerase (PARP) inhibitors only partially inhibited loss of TMRE (32% reduction) and caspase-2 activity (38% reduction); indicative of PARP operation (or action) upstream of caspase-2. Moreover, cytosine arabinoside induced a similar degree of apoptosis, while leaving methylation status mainly unaffected. CONCLUSIONS: 5-Azacytidine acts via multiple and separately regulated pathways, including parallel induction of hypomethylation. The broad action of 5-azacytidine may explain its therapeutic effects in poor-prognostic MDS.

Negative effect of DNA hypermethylation on the outcome of intensive chemotherapy in older patients with high-risk myelodysplastic syndromes and acute myeloid leukemia following myelodysplastic syndrome.

PURPOSE: Promoter hypermethylation of, for example, tumor-suppressor genes, is considered to be an important step in cancerogenesis and a negative risk factor for survival in patients with myelodysplastic syndromes (MDS); however, its role for response to therapy has not been determined. This study was designed to assess the effect of methylation status on the outcome of conventional induction chemotherapy. EXPERIMENTAL DESIGN: Sixty patients with high-risk MDS or acute myeloid leukemia following MDS were treated with standard doses of daunorubicin and 1-beta-d-arabinofuranosylcytosine. Standard prognostic variables and methylation status of the P15(ink4b) (P15), E-cadherin (CDH), and hypermethylated in cancer 1 (HIC) genes were analyzed before treatment. RESULTS: Forty percent of the patients achieved complete remission (CR). CR rate was lower in patients with high WBC counts (P = 0.03) and high CD34 expression on bone marrow cells (P = 0.02). Whereas P15 status alone was not significantly associated with CR rate (P = 0.25), no patient with hypermethylation of all three genes achieved CR (P = 0.03). Moreover, patients with CDH methylation showed a significantly lower CR rate (P = 0.008), and CDH methylation retained its prognostic value also in the multivariate analysis. Hypermethylation was associated with increased CD34 expression, but not with other known predictive factors for response, such as cytogenetic profile. CONCLUSIONS: We show for the first time a significant effect of methylation status on the outcome of conventional chemotherapy in high-risk MDS and acute myelogenous leukemia following MDS. Provided confirmed in an independent study, our results should be used as a basis for therapeutic decision-making in this patient group.

A pharmacodynamic study of 5-azacytidine in the P39 cell line.

OBJECTIVE: 5-azacytidine (azacytidine), a DNA hypomethylating agent, was recently approved as the first therapeutic agent for the treatment of myelodysplastic syndromes. The present subcutaneous dosing schedule, 75 mg/m(2) for 7/28 days, is based on early clinical studies and may constitute a practical problem for patients. The present in vitro study aimed at evaluating the pharmacodynamics of azacytidine, thereby providing a rationale for clinical dose-finding studies. METHODS: P39 cells were incubated with 0.1, 0.5, and 1 microM azacytidine daily for 24, 48, and 72 hours, followed by 48 hours in drug-free medium. The effects of azacytidine on cell growth, proliferation, apoptosis, cell cycle status, and promoter methylation of E-cadherin, ER, and HIC genes were studied. RESULTS: Azacytidine decreased cell growth and proliferation, increased apoptosis, and affected cell cycle status in a dose-dependent manner. However, the exposure time, 24 to 72 hours, at doses between 0.5 and 1 microM, did not significantly affect any of these variables. Using first-order exponential pharmacokinetic model, we found that the effect of 1, 2, or 3 microM over 24 hours did not differ from that of 0.5 to 1 microM given over 48 to 72 hours. Induction of promoter hypomethylation was observed already after 24 hours of exposure with >or=0.5 microM azacytidine with no clear dose-effect relationship. CONCLUSION: Our results indicate that optimal cellular effects of azacytidine might be achieved by shorter exposure times. The model provides information about the relation between azacytidine dose intensity and exposure time on malignant myeloid cells, which could serve as a rationale for further clinical development of practical, safe, and cost-effective dosing schedules.