Small interfering RNA delivery by polyethylenimine-functionalised porous silicon nanoparticles.

In this study, thermally hydrocarbonised porous silicon nanoparticles (THCpSiNPs) capped with polyethylenimine (PEI) were fabricated, and their potential for small interfering RNA (siRNA) delivery was investigated in an in vitro glioblastoma model. PEI coating following siRNA loading enhanced the sustained release of siRNA, and suppressed burst release effects. The positively-charged surface improved the internalisation of the nanoparticles across the cell membrane. THCpSiNP-mediated siRNA delivery reduced mRNA expression of the MRP1 gene, linked to the resistence of glioblastoma to chemotherapy, by 63% and reduced MRP1-protein levels by 70%. MRP1 siRNA loaded nanoparticles did not induce cytotoxicity in glioblastoma cells, but markedly reduced cell proliferation. In summary, the results demonstrated that non-cytotoxic cationic THCpSiNPs are promising vehicles for therapeutic siRNA delivery.

Clinical utility of molecular and flow cytometric markers in chronic lymphocytic leukaemia.

BACKGROUND: Chronic lymphocytic leukaemia (CLL) is a clinically heterogeneous disease. While immunoglobulin variable region heavy chain (IgVH) mutational status remains the 'gold standard' in molecular prognostication, a range of additional markers is increasingly being used in clinical trials. As awareness of trial data increases, requests to determine these prognostic markers for new CLL patients are becoming more prevalent in Australia. AIM: To explore the clinical utility of currently available prognostic markers for CLL in an Australian cohort. METHODS: IgVH mutational status and gene usage was determined and compared with other reported immunophenotypic markers, cytogenetics and clinical outcome as defined by treatment-free survival (TFS), lymphocyte doubling time and clinical stage in a cohort of 65 CLL patients. RESULTS: An unmutated IgVH gene, high expression of CD38, ZAP-70, CD25, CD49d, CD54 or low expression of CD49c was associated with shorter TFS indicating an adverse clinical prognosis in our cohort. High expression of each of CD38, ZAP-70, CD49d and CD54 was significantly associated with an unmutated IgVH gene; however, associations were not absolute. IgVH and CD25 expression retained their significance in multivariate analysis. Concordant CD25(high) /IgVH unmutated CLL patients had the shortest median TFS interval (40 months) in our cohort. CONCLUSIONS: Molecular and immunophenotypic markers remain useful as adjuncts to clinical prognostication; however, as single parameters they are unable to dictate the timing of therapeutic intervention. The combined use of CD25 and IgVH mutational status may be clinically relevant to CLL prognostication while also providing insight into the biological pathways involved in disease progression.

ARA, a novel ABC transporter, is located at 16p13.1, is deleted in inv(16) leukemias, and is shown to be expressed in primitive hematopoietic precursors.

ATP-binding cassette (ABC), ATP-dependent transporters are a large superfamily of proteins that include the multidrug resistance proteins, P-glycoprotein and MRP (multidrug resistance protein). The ARA (anthracycline resistance-associated) gene that codes for a putative member of the ABC transporters has recently been cloned and shown to have high sequence homology to the gene for MRP. We have previously shown MRP to be deleted in a subset of inv(16) leukemic patients. The deletion of MRP was associated with an improved patient survival compared with inv(16) patients who did not have such a deletion. In this study, the ARA gene is mapped to 16p13.1, in the same physical interval as the inv(16) short-arm breakpoint. It is shown to be situated proximal to both MYH11, the gene involved in the primary breakpoint on the short arm of the inv(16), and MRP. A YAC clone has been isolated containing both MRP and ARA. FISH analysis of metaphase chromosomes from inv(16) patients has established the gene order as telomere-MYH11-MRP-ARA-centromere and demonstrated that both ARA and MRP are deleted in a subgroup of the inv(16) leukemias. ARA and MRP are both shown to be expressed in normal hematopoietic precursors including CD34(+) cells. The mapping of ARA to this region and its homology to MRP raises questions about its potential role in the biology of the inv(16) leukemias.

The biological significance of the multidrug resistance gene MRP in inversion 16 leukemias.

Multidrug resistance represents an important mechanism by which leukaemic and solid tumour cells escape cell death after exposure to anthracyclines and other natural products. Acute myeloid leukaemia (AML) associated with the inversion chromosome 16: inv(16)(p13q22) has a favourable prognosis and is known to be chemosensitive. The inversion chromosome is seen in a number of FAB subclasses but is most commonly associated with acute myelomonocytic leukaemia with abnormal eosinophils, M4Eo. It results in the creation of a fusion between the myosin heavy chain gene (MYH11) on the short arm and the gene for a transcription factor, core binding factor beta (CBFB) on the long arm. In a subset of these inv(16) AML patients, inversion also results in loss of the gene for the multidrug resistance protein (MRP) at the short arm breakpoint. This gene maps to 16p13.13, centromeric to the primary short arm breakpoint, separated from MYH11 by a distance of approximately 150kb. Deletion of the MRP gene has been demonstrated by in situ hybridisation, gene dosage studies and by loss of heterozygosity of a flanking microsatellite marker (D16S405). Twenty two patients with inv(16) leukaemia were analysed for deletion of the MRP gene. Deletion of the gene was detected in seven patients, fourteen patients showed retention of the gene and in one case the findings were indeterminate. Clinical data from 13 of these patients were analysed revealing deletion of the MRP gene to be significantly associated with longer time from diagnosis until failure (death or relapse from complete remission) in these patients (p = 0.007). From this work and the growing literature concerning MRP, it appears likely that the deletion of an MRP allele, may favourably affect the biology of inv(16) AML and may have important prognostic implications.

Deletion of gene for multidrug resistance in acute myeloid leukaemia with inversion in chromosome 16: prognostic implications.

Acute myeloid leukaemia (AML) associated with an inversion in chromosome 16 has a relatively favourable prognosis. The AML subclass most commonly associated with this chromosomal abnormality is acute myelomonocytic leukaemia with abnormal eosinophils. In some AML patients with inversion 16 the chromosomal lesion results in deletion of MRP, the gene for multidrug resistance associated protein. This gene is proximal to the primary breakpoint and loss of its function may play a key role in determining the favourable outcome in inversion 16 AML. We have demonstrated deletion of MRP by in situ hybridisation, by gene dosage studies and by studying loss of heterogeneity of a flanking microsatellite marker. Among 13 AML patients with inversion 16 MRP deletion was detected in 5 while 7 had no deletion. Deletion of MRP gene was associated with longer time from diagnosis until death or relapse from complete remission (p = 0.007). These findings provide important insight into the biology of inversion 16 leukaemia and suggest that MRP deletion, as detected by molecular analysis, may have a key role in determining outcome in patients with inversion 16 AML.

Identification of an inversion 16 coexisting with an isochromosome 22q by in situ hybridization in a case of childhood AML M4e.

Rearrangements involving chromosome 16, including inv(16) (p13q22), del(16)(q22), and t(16;16)(p13;q22), are frequent findings in acute myeloblastic leukemia (AML). Each of these rearrangements can occur as the sole karyotypic change or in association with additional chromosomal abnormalities, including in decreasing order of frequency: trisomy 22, trisomy 8, and deletion of the long arm of chromosome 7. We report a pediatric case of de novo AML, M4e subtype, with a unique combination of inv(16) (p13q22) and i(22q) occurring within the same leukemic clone. The inv(16) was detected by fluorescence in situ hybridization (FISH) analysis with two cosmid probes specific for sequences flanking the inv(16) breakpoint on the long arm of chromosome 16. Use of a chromosome-22-specific painting probe unequivocally identified a small metacentric chromosome as an i(22q). This case illustrates a variation in the association of trisomy 22 with inv(16) and suggests that duplication of the long arm of chromosome 22 may contain critical gene(s) involved in the multistep process of evolution of leukemia with 16q22 abnormalities.

High dose hydroxyurea in collection of Philadelphia chromosome-negative stem cells in chronic myeloid leukaemia.

High dose hydroxyurea (HU) was used in a pilot study to assess its efficacy in mobilizing Philadelphia (Ph) chromosome negative progenitor cells in chronic myeloid leukaemia (CML). Five patients received 12 g/M2 oral HU in divided doses. Side effects were minimal, allowing outpatient administration. Nine to fourteen days later 4 patients achieved a mean leukocyte nadir of 3.5 x 10(9)/L (Range 2.4-4.9) and a mean platelet nadir of 99 x 10(9)/L (Range 95-108). Peripheral blood mononuclear cells (PB-MNC) sampled prior to the HU priming were 100% Ph positive. Between 10 and 18 days post HU, 3 patients achieved a marked reduction (80-100%) in the number of Ph positive metaphases in PB-MNC collected by apheresis. One patient failed to achieve any Ph suppression. Polymerase chain reaction analysis (PCR) for the bcr-abl fusion product remained positive in all samples. Rapid rises in CFU-GM numbers were associated with a return to 100% Ph positive metaphases however slower rises represented recovery with predominantly Ph negative cells, allowing apheresis collection of these cells. We conclude that HU induces a reproducible leukocyte nadir with sufficient stem cell mobilization for potential autologous transplantation. Higher doses of HU together with early and intensive apheresis is required to maximize Ph negative progenitor cell harvests.

The perivertebral collar--a new sign in lymphoproliferative malignancies.

Nine patients with lymphoproliferative malignancies, one of whom had not been previously diagnosed, were found on CT examination for back pain to have partial or complete soft tissue perivertebral collars. The thoracic and or lumbar regions were involved in all. Only 3 had gross bony changes at the time, and in others the changes appeared so innocuous that in combination with the vague clinical symptoms their significance was underestimated. Five patients ultimately had episodes of cord compression, and in all nine the appearance of this spinal lesion appeared to be of grave prognostic significance. All 9 were dead within 1 year of the presentation of their spinal lesions. The observation of a perivertebral collar in the context of a known or suspected lymphoproliferative malignancy should therefore raise the strong suspicion of spinal involvement. The vertebrae should be examined on bone windows and the contents of the spinal canal on narrow windows to assess bony and epidural spread.

Optimal oxygen delivery in critically ill patients.

Standard hemodynamic support in septic shock is to increase pulmonary capillary wedge pressure to above 15 mmHg by volume replacement and to give inotropic support if the mean arterial pressure (MAP) is not adequate. In an attempt to decrease mortality in critically ill patients, oxygen delivery (DO2) was increased by switching inotropic support from dobutamine alone or in combination with norepinephrine to dopamine alone, or by adding dopexamine, prostacyclin, or hypertonic saline to the treatment. DO2 increased significantly in all patients, but the increase in DO2 was accompanied by only a 10% increase in oxygen consumption (VO2). The increase in VO2 was similar in survivors and nonsurvivors and in patients with and without septic shock. The results indicate that if adequate volume and inotropic support is provided for critically ill patients, the detectable oxygen debt is small and has little effect on patient outcome. When DO2 is adequate, factors other than a tissue oxygen deficit seem to determine patient outcome.

[Incorrect placement of a vena cava catheter and its prevention by intra-atrial ECG]. / Cavakatheterfehllagen und ihre Vermeidung durch intraatriale EKG-Ableitung.

Up to now, radiological position control was obligatory due to the frequent complications arising from false cava catheter positioning. Apart from the radiological control, which is time consuming and expensive and involves the danger of allergic reactions due to contrast media, intraatrial ECG-recording can be applied to achieve correct positioning of the catheter tip. By this method exact placement of the catheter tip in the superior vena cava was possible in 98 of 100 cases examined. Only 2 catheters could not be placed: one was placed intra-arterially, and the other could not pass a venous valve. The method proved to be inexpensive, time saving and could even be applied in emergency situations (intraoperatively). Important prerequisites for the success of the procedure are a disturbance-free ECG-derivation, a sinus rhythm, and sufficient practical experience on the part of the examiner with regard to the assessment of intra-atrial ECG-alterations.

[Quantitative determination of tricyclic antidepressant drugs by high-pressure liquid chromatography (author's transl)]. / Quantitative Bestimmung trizyklischer Antidepressiva mit der Hochdruckflüssigkeitschromatographie.

With regard to the increasing importance of determining plasma levels of antidepressant drugs a procedure has been worked out which makes it possible to analyze these substances in a simple and sensitive way. 5 ng )ca. 20 pmol) amitriptyline, nortriptyline, imipramine, desmethylimipramine, chlorimipramine, desmethylchlorimipramine, and protriptyline can be detected with a high-pressure liquid chromatograph and a UV-monitor without prior derivatisation. The extraction is made from 1 ml serum. Because of their retention time the substances can be identified. This fact allows simultaneous analyzation of all tricyclic antidepressants.