Flow Cytometric Assessment of AKT Signaling in Platelet Activation: An Alternative Diagnostic Tool for Small Volumes of Blood.

INTRODUCTION: The diagnosis of platelet function disorder in children is challenging. Light transmission aggregometry is the gold standard for platelet function disorders. However, large blood volumes are required. Currently, there are no existing tools for the diagnosis of platelet function disorders that use small blood volumes. AKT signaling plays a central role in platelet activation during hemostasis and might be visualized by flow cytometry. METHODS: Platelet-rich plasma obtained by centrifugation of citrated blood from healthy volunteers was activated with arachidonic acid, thrombin receptor activating peptide-6 (TRAP-6), collagen, adenosine diphosphate ADP, collagen-related peptide (CRP), and epinephrine. After platelet activation, the phosphorylation of AKT was assessed by flow cytometer using a Navios cytometer. RESULTS: Healthy volunteers showed a reproducible phosphorylation of AKT upon activation. In comparison to nonactivated platelets, we documented an increase in pAKT expression with all agonists. Especially TRAP-6 and CRP caused considerable increase in percentage of pAKT expression throughout all the tested healthy volunteers. CONCLUSION: An activation of the AKT-signal pathway by different agonists can clearly be detected on the flow cytometer, indicating that the visualization of signaling in platelets by flow cytometry might be an efficient alternative for light transmission aggregometry to test platelet function in children.

Caspofungin infusion solutions (50 mg/100 mL): chemical stability and antifungal activity against Candida spp.

BACKGROUND: Ready to use caspofungin infusion bags are centrally prepared in the Hospital Pharmacy, University Hospital of Heidelberg, for economic reasons and possibly occurring problems with drug shortages. The aim of this study was a quality control of the in-house preparation of caspofungin infusion bags and the preparation process. Caspofungin concentration with regard to chemical stability and antifungal activity of caspofungin preparations were defined as quality parameters. METHODS: Three caspofungin infusion bags (50 mg in 100 mL 0.9% sodium chloride) were examined every seven days for a total of four weeks. Chemical stability of caspofungin solutions was analyzed using a validated high performance liquid chromatography (HPLC) method. Antifungal activity was assessed by microdilution tests according to the EUCAST protocol. Additionally, concentration and sterility were determined in returned caspofungin infusion bags. RESULTS: The amount of caspofungin in the infusion solutions still exceeded 90% after four weeks (2-8 °C). Antifungal activity was consistent over 28 days with a MIC ≤2 mg/L for different Candida spp. In returned infusion bags, caspofungin concentration was found to be ≥90% in 12 out of 13 bags and sterility was given in all preparations. CONCLUSION: These results show that chemical stability of caspofungin infusion solutions (50 mg/100 mL) can be guaranteed for four weeks at 2-8 °C and are confirmed by corresponding results regarding sterility and antifungal activity.

Antagonism between granulocytic maturation and deacetylase inhibitor-induced apoptosis in acute promyelocytic leukaemia cells.

BACKGROUND: Transcriptional repression is a key mechanism driving leukaemogenesis. In acute promyelocytic leukaemia (APL), the fusion protein promyelocytic leukaemia-retinoic acid receptor-α fusion (PML-RARα) recruits transcriptional repressors to myeloid differentiation genes. All-trans-retinoic acid (ATRA) induces the proteasomal degradation of PML-RARα and granulocytic differentiation. Histone deacetylases (HDACs) fall into four classes (I-IV) and contribute to the transcription block caused by PML-RARα. METHODS: Immunoblot, flow cytometry, and May-Grünwald-Giemsa staining were used to analyze differentiation and induction of apoptosis. RESULTS: A PML-RARα- and ATRA-dependent differentiation programme induces granulocytic maturation associated with an accumulation of the myeloid transcription factor CCAAT/enhancer binding protein (C/EBP)ɛ and of the surface protein CD11b. While this process protects APL cells from inhibitors of class I HDAC activity, inhibition of all Zinc-dependent HDACs (classes I, II, and IV) with the pan-HDACi (histone deacetylase inhibitor(s)) LBH589 induces apoptosis of immature and differentiated APL cells. LBH589 can eliminate C/EBPɛ and the mitochondrial apoptosis regulator B-cell lymphoma (BCL)-xL in immature and differentiated NB4 cells. Thus, BCL-xL and C/EBPɛ are newly identified molecular markers for the efficacy of HDACi against APL cells. CONCLUSIONS: Our results could explain the therapeutic limitations occurring with ATRA and class I HDACi combinations. Pro-apoptotic effects caused by pan-HDAC inhibition are not blunted by ATRA-induced differentiation and may provide a clinically interesting alternative.