Pharmacological cyclin-dependent kinase (cdk) inhibitors (PCIs) block replication of several viruses, including herpes simplex virus type 1 (HSV-1) and human immunodeficiency virus type 1 (HIV-1). Yet, these antiviral effects could result from inhibition of either cellular cdks or viral enzymes. For example, in addition to cellular cdks, PCIs could inhibit any of the herpesvirus-encoded kinases, DNA replication proteins, or proteins involved in nucleotide metabolism. To address this issue, we asked whether purine-derived PCIs (P-PCIs) inhibit HSV and HIV-1 replication by targeting cellular or viral proteins. P-PCIs inhibited replication of HSV-1 and -2 and HIV-1, which require cellular cdks to replicate, but not vaccinia virus or lymphocytic choriomeningitis virus, which are not known to require cdks to replicate. P-PCIs also inhibited strains of HSV-1 and HIV-1 that are resistant to conventional antiviral drugs, which target viral proteins. In addition, the anti-HSV effects of P-PCIs and a conventional antiherpesvirus drug, acyclovir, were additive, demonstrating that the two drugs act by distinct mechanisms. Lastly, the spectrum of proteins that bound to P-PCIs in extracts of mock-and HSV-infected cells was the same. Based on these observations, we conclude that P-PCIs inhibit virus replication by targeting cellular, not viral, proteins.To ensure specificity and avoid toxicity, most antiviral drugs are designed to target viral proteins. Such drugs, however, select for drug-resistant viral mutants. Moreover, these drugs exhibit activity against only a few closely related viruses. In contrast, antiviral drugs that target cellular proteins required for viral replication would not be constrained by these limitations. In the past several years, pharmacological cyclin-dependent kinase inhibitors (PCIs) have been shown to inhibit the replication of four clinically important viruses: human cytomegalovirus (HCMV) (6), herpes simplex virus type 1 (HSV-1) (56-58), human immunodeficiency virus type 1 (HIV-1) (9, 47, 69), and varicella-zoster virus (J. Moffat, State University of New York, Upstate Medical University, personal communication). However, it is as yet unclear whether the antiviral effects of these drugs are mediated exclusively by inhibition of their known cellular targets, or by inhibition of yet-unknown viral targets.Of the PCIs developed to date, the 2,6,9-trisubstituted purines (P-PCIs), such as Roscovitine (Rosco) (45) and Purvalanol (Purv) (26), are the most specific and best characterized. Rosco and Purv differ in potency (Purv is more potent than Rosco [26,45]) but not in selectivity or mechanism of action. Both drugs inhibit cdk1, -2, and -5 and erk1 and -2 (at Ϸ50-to 1,000-fold higher concentrations than are needed to inhibit cdks), but they do not inhibit cdk4 or -6 or a large number of other kinases (26,36,45). Mechanistically, Rosco and Purv compete with ATP for binding to the ATP-binding pocket of the target cdks (16,26,45,68). All known effects of Rosco and Purv on cells can be attributed to inhi...

Key Points Infant acute lymphoblastic leukemia is sensitive to therapeutic targeting by apoptosis, necoptosis, and autophagy activation whether MLL is rearranged or germline. The disease-specific form of triple death mode killing by obatoclax overcomes the intrinsic resistance of MLL-rearranged infant acute lymphoblastic to cell death.

Abstract:To determine whether exercise increases endothelial progenitor cells (EPCs) in patients with peripheral vascular disease, we developed a multi-parameter flow cytometry assay to rigorously assess EPCs and mature endothelial cells (ECs) in control subjects and patients with peripheral artery disease (PAD) subjected to graded exercise. Blood was collected from young healthy subjects (n ϭ 9, mean age 33 years), older healthy subjects (n ϭ 13, mean age 66 years), and older subjects with PAD (n ϭ 15, mean age 69 years) before and 10 minutes after exercise. White blood cells were isolated and stained with a five-color antibody panel: FITC-anti-CD31, PE-anti-CD146, PE-anti-CD133, PerCP-Cy5.5-anti-CD3,-CD19,-CD33, PE-Cy7-anti-CD34, and APC-anti-VEGF-R2. Viability was assessed by propidium iodide exclusion. Viable, low, side scatter singlets that were CD3-, 19-, and 33-negative were counted. While baseline levels of EPCs and ECs were similar among all subjects, young healthy subjects demonstrated significantly greater (p Ͻ 0.05) levels of progenitor cells (PCs) than older healthy and PAD subjects. Levels of EPCs and ECs tended to increase in all subjects after exercise; however, increases in PCs were only observed in young healthy and PAD subjects. Further, trends in the magnitude of change of subsets with exercise were most similar between young and PAD subjects. Our findings suggest that aging may reduce baseline circulating levels of PCs, but not EPCs or ECs, and that exercise-induced mobilization of subsets may differ depending on age and presence of PAD.

Methods: Blood was collected from young healthy subjects (N = 9, mean age 33 6 8 years), older healthy subjects (N = 13, mean age 66 6 8 years), and older subjects with PAD (N = 15, mean age 69 6 8 years). After ammonium chloride lysis, cells were stained and analyzed on a Becton-Dickinson LSR II with a 5-color antibody panel: FITC-anti-CD31, PE-anti-CD146, PE-anti-CD133, PerCP-Cy5.5-anti-CD3, -CD19,-CD33 (lineage panel), PE-Cy7-anti-CD34, and APC-anti-VEGF-R2. Viability was assessed by propidium iodide exclusion, and only viable, low to medium side scatter lineage-negative singlets were analyzed. In some studies, cells were sorted for morphological studies. Subsets were defined as indicated later.Results: Our results, using a comprehensive flow cytometric panel, indicate that CD133+, CD34+, and CD133+/CD34+ PCs are elevated in younger healthy individuals compared to older individuals, both healthy and with PAD. However, the number of EPCs and mature ECs did not significantly differ among the three groups. Assessment of endothelial colony forming units and dual acLDL-lectin staining supported the flow cytometric findings.Conclusions: We describe a comprehensive flow cytometric method to detect circulating mature and progenitor endothelial populations confirmed by conventional morphological and functional assays. Our findings suggest that aging may influence circulating levels of PCs, but not EPCs or ECs; PAD had no effect on baseline levels of any populations investigated. This study provides the basis for evaluating the potential effects of acute stress and therapeutic intervention on circulating progenitor and endothelial populations as a biomarker for cardiovascular status. q

Background: Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic disorders diagnosed using morphologic and clinical findings supported by cytogenetics. Because abnormalities may be subtle, diagnosis using these approaches can be challenging. Flow cytometric (FCM) approaches have been described; however the value of bone marrow immunophenotyping in MDS remains unclear due to the variability in detected abnormalities. We sought to refine the FCM approach by using peripheral blood (PB) to create a clinically useful tool for the diagnosis of MDS.Methods: PB from 15 patients with MDS was analyzed by multiparametric flow cytometry using an extensive panel of monoclonal antibodies. Patterns of neutrophil antigen expression were compared with those of normal controls (n ‫؍‬ 16) to establish light scatter and/or immunophenotypic abnormalities that correlated with MDS. A scoring algorithm was developed and validated prospectively on a blinded patient set.Results: PB neutrophils from patients with MDS had lower side scatter and higher expression of CD66 and CD11a than did controls. Some MDS PB neutrophils demonstrated abnormal CD116 and CD10 expression. Because none of these abnormalities proved consistently diagnostic, we sought to increase the power of the assay by devising a scoring system to allow the association of multiple abnormalities and account for phenotypic variations. The PB MDS score differentiated patients with MDS from controls (P < 0.0001) in the test set. In a prospective validation, the PB MDS score successfully identified patients with MDS (sensitivity 73%, specificity 90%).Conclusions: FCM analysis of side scatter and only four additional immunophenotypic parameters of PB neutrophils using the PB MDS score proved more sensitive than standard laboratory approaches and may provide an additional, more reliable diagnostic tool in the identification of MDS.