IVIg treatment increases thrombin activation of platelets and thrombin generation in paediatric patients with immune thrombocytopenia.

Clinical manifestations and laboratory parameters of haemostasis were investigated in 23 children with newly diagnosed immune thrombocytopenia (ITP) before and after intravenous immunoglobulin (IVIg) treatment. ITP patients with platelet counts of less than 20 × 109 /L and mild bleeding symptoms, graded by a standardized bleeding score (BS), were compared with healthy children with normal platelet counts and children with chemotherapy-related thrombocytopenia. Markers of platelet activation and platelet apoptosis in the absence and presence of platelet activators were analysed by flow cytometry; thrombin generation in plasma was determined. ITP patients at diagnosis presented with increased proportions of platelets expressing CD62P and CD63 and activated caspases, and with decreased thrombin generation. Thrombin-induced activation of platelets was reduced in ITP compared with controls, while increased proportions of platelets with activated caspases were observed. Children with a higher BS had lower proportions of CD62P-expressing platelets compared with those with a lower BS. IVIg treatment increased the number of reticulated platelets, the platelet count to more than 20 × 109 /L and improved bleeding in all patients. Decreased thrombin-induced platelet activation, as well as thrombin generation, were ameliorated. Our results indicate that IVIg treatment helps to counteract diminished platelet function and coagulation in children with newly diagnosed ITP.

A longitudinal study of gene expression in healthy individuals.

BACKGROUND: The use of gene expression in venous blood either as a pharmacodynamic marker in clinical trials of drugs or as a diagnostic test requires knowledge of the variability in expression over time in healthy volunteers. Here we defined a normal range of gene expression over 6 months in the blood of four cohorts of healthy men and women who were stratified by age (22-55 years and > 55 years) and gender. METHODS: Eleven immunomodulatory genes likely to play important roles in inflammatory conditions such as rheumatoid arthritis and infection in addition to four genes typically used as reference genes were examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), as well as the full genome as represented by Affymetrix HG U133 Plus 2.0 microarrays. RESULTS: Gene expression levels as assessed by qRT-PCR and microarray were relatively stable over time with approximately 2% of genes as measured by microarray showing intra-subject differences over time periods longer than one month. Fifteen genes varied by gender. The eleven genes examined by qRT-PCR remained within a limited dynamic range for all individuals. Specifically, for the seven most stably expressed genes (CXCL1, HMOX1, IL1RN, IL1B, IL6R, PTGS2, and TNF), 95% of all samples profiled fell within 1.5-2.5 Ct, the equivalent of a 4- to 6-fold dynamic range. Two subjects who experienced severe adverse events of cancer and anemia, had microarray gene expression profiles that were distinct from normal while subjects who experienced an infection had only slightly elevated levels of inflammatory markers. CONCLUSION: This study defines the range and variability of gene expression in healthy men and women over a six-month period. These parameters can be used to estimate the number of subjects needed to observe significant differences from normal gene expression in clinical studies. A set of genes that varied by gender was also identified as were a set of genes with elevated expression in a subject with iron deficiency anemia and another subject being treated for lung cancer.

Expression modes of interferon-alpha inducible genes in sensitive and resistant human melanoma cells stimulated with regular and pegylated interferon-alpha.

Interferon-alpha with its antiproliferative activity is widely used for the treatment of viral infections and tumor therapy such as melanoma. Naturally occurring resistance to recombinant interferon alpha-2a (IFN-alpha) and severe side effects limit the therapeutic efficacy. Understanding of the molecular mechanisms involved in unresponsiveness may therefore lead to the development of novel formulations that overcome resistance. Here, we have applied oligonucleotide DNA microarrays with probe sets for about 11,400 human transcripts to study the expression of interferon-alpha inducible genes in a sensitive and resistant melanoma cell line over multiple time points and two interferon formulations. We identified two major groups of genes with termed interferon primary response genes (IPRGs) or interferon secondary response genes (ISRGs). IPRGs are upregulated early after interferon stimulation in both the sensitive and the resistant line and they contain IREs in the noncoding regulatory region. In contrast, ISRG expression occurs preferentially in the sensitive line ME15 at late time points, and this group of genes lacks typically IREs. In addition to these two major interferon response gene classes, we identified a relatively small number of genes with complex kinetic expression modes. In addition, we show for the first time that regular and pegylated recombinant interferons are equally potent activators of interferon (IFN) gene expression. Finally, we propose that the ISRGs are activated downstream of the primary response genes by a molecule or pathway, which awaits identification, and interferon inducible gene expression is thus more complicated than previously thought.

Interferon-a sensitivity in melanoma cells: detection of potential response marker genes.

Interferon alpha (IFN-alpha) represents an adjuvant therapy of proven effectiveness in increasing disease-free interval and survival in subgroups of melanoma patients. Since high doses of cytokine are required, the treatment is often accompanied by toxic side effects. In addition, naturally occurring insensitivity to IFN-alpha may hamper its therapeutic efficacy. Clinical, molecular or immunological markers enabling the selection of potential responders have not so far been identified. To explore the molecular basis of IFN-alpha responsiveness, we analyzed the expression pattern of about 7000 genes in IFN-alpha-sensitive and IFN-alpha-resistant cell lines using high-density oligonucleotide arrays. Melanoma cell lines were screened for their sensitivity to proliferation inhibition and HLA class I induction by IFN-alpha by standard 3H-thymidine incorporation and flow cytometry. Total cellular RNA from four sensitive and two resistant cell lines was extracted, reverse-transcribed and hybridized to high-density oligonucleotide arrays. The comparative analysis of gene expression in either set of cell lines allowed the identification of four genes (RCCl, IFI16, hox2 and h19) preferentially transcribed in sensitive cells and two (SHB and PKC-zeta) preferentially expressed in resistant cells. These data may provide a useful basis for the development of diagnostic tools to select potential IFN-alpha responders as eligible for treatment, while avoiding unnecessary toxicity to nonresponders.

Determination of fexofenadine in human plasma and urine by liquid chromatography-mass spectrometry.

A sensitive method was developed to determine fexofenadine in human plasma and urine by HPLC-electrospray mass spectrometry with MDL 026042 as internal standard. Extraction was carried out on C18 solid-phase extraction cartridges. The mobile phases used for HPLC were: (A) 12 mM ammonium acetate in water and (B) acetonitrile. Chromatographic separation was achieved on a LUNA CN column (10 cm x 2.0 mm I.D., particle size 3 microm) using a linear gradient from 40% B to 60% B in 10 min. The mass spectrometer was operated in the selected ion monitoring mode using the respective MH+ ions, m/z 502.3 for fexofenadine and m/z 530.3 for the internal standard. The limit of quantification achieved with this method was 0.5 ng/ml in plasma and 1.0 ng in 50 microl of urine. The method described was successfully applied to the determination of fexofenadine in human plasma and urine in pharmacokinetic studies.