Human herpesvirus 8 (HHV8, also known as Kaposi's sarcoma [KS]-associated herpesvirus) has been implicated as an etiologic agent for KS, an angiogenic tumor composed of endothelial, inflammatory, and spindle cells. Here, we report that transgenic mice expressing the HHV8-encoded chemokine receptor (viral G protein–coupled receptor) within hematopoietic cells develop angioproliferative lesions in multiple organs that morphologically resemble KS lesions. These lesions are characterized by a spectrum of changes ranging from erythematous maculae to vascular tumors, by the presence of spindle and inflammatory cells, and by expression of vGPCR, CD34, and vascular endothelial growth factor. We conclude that vGPCR contributes to the development of the angioproliferative lesions observed in these mice and suggest that this chemokine receptor may play a role in the pathogenesis of KS in humans.

Gfi-1 is a cellular proto-oncogene that was identified as a target of provirus integration in T-cell lymphoma lines selected for interleukin-2 (IL-2) independence in culture and in primary retrovirus-induced lymphomas. Gfi-1 encodes a zinc finger protein that functions as a transcriptional repressor. Here we show that Gfi-1B, a Gfi-1 related gene expressed in bone marrow and spleen, also encodes a transcriptional repressor. IL-6-induced G 1 arrest and differentiation of the myelomonocytic cell line M1 were linked to the downregulation of Gfi-1B and the parallel induction of the cyclin-dependent kinase inhibitor p21WAF1 . Experiments addressing the potential mechanism of the apparent coordinate regulation of these genes revealed that Gfi-1B represses p21 WAF1 directly by binding to a high-affinity site at ؊1518 to ؊1530 in the p21 WAF1 promoter. Forced expression of Gfi-1B, but not of Gfi-1B deletion mutants lacking the repressor domain, blocked the IL-6-mediated induction of p21 WAF1 and inhibited G 1 arrest and differentiation. We conclude that Gfi-1B is a direct repressor of the p21 WAF1 promoter, the first such repressor identified to date, and that sustained expression of Gfi-1B blocks IL-6-induced G 1 arrest and differentiation of M1 cells perhaps because it prevents p21 WAF1induction by IL-6.Hematopoiesis is a process that takes place in the bone marrow throughout the life of an individual. During this process a small number of hematopoietic stem cells respond to microenvironmental cues to either divide and self-renew or differentiate into hematopoietic progenitors committed to specic hematopoietic lineages. The committed hematopoietic progenitors, in turn, also undergo self-renewal or terminal differentiation. The maintenance of the hematopoietic stem cells and their selection, commitment, and maturation along different hematopoietic lineages depend on cell-to-cell and cell-tostroma interactions, secreted cytokines, and intracellular signaling molecules (45,46). The molecular mechanisms involved in regulating hematopoietic cell commitment and differentiation can be addressed in differentiating hematopoietic tissues in intact animals (64) and in cell lines that can be induced to differentiate (35,38). With both systems, a number of molecules, including growth factors, receptors, and transcription factors, have been identified and shown to contribute to hematopoiesis in a hierarchical order (10,29,42,65).The myelomonocytic cell line M1 undergoes G 1 arrest and differentiation following exposure to interleukin-6 (IL-6) or leukemia inhibitory factor (15, 50). During this process the expression of several signaling molecules is altered. c-myb is downregulated within 3 h from the start of the exposure to IL-6 or leukemia inhibitory factor (26). This is followed by the downregulation of c-myc (25). Overexpression of either c-myb or c-myc inhibits IL-6-induced differentiation of M1 cells (25,26,49), suggesting that the downregulation of these molecules is required for differentiation. Another molecule whose expre...

Chemokine receptors transduce signals important for the function and trafficking of leukocytes. Recently, it has been shown that CC chemokine receptor (CCR)8 is selectively expressed by Th2 subsets, but its functional relevance is unclear. To address the biological role of CCR8, we generated CCR8 deficient (−/−) mice. Here we report defective T helper type 2 (Th2) immune responses in vivo in CCR8−/− mice in models of Schistosoma mansoni soluble egg antigen (SEA)-induced granuloma formation as well as ovalbumin (OVA)- and cockroach antigen (CRA)-induced allergic airway inflammation. In these mice, the response to SEA, OVA, and CRA showed impaired Th2 cytokine production that was associated with aberrant type 2 inflammation displaying a 50 to 80% reduction in eosinophils. In contrast, a prototypical Th1 immune response, elicited by Mycobacteria bovis purified protein derivative (PPD) was unaffected by CCR8 deficiency. Mechanistic analyses indicated that Th2 cells developed normally and that the reduction in eosinophil recruitment was likely due to systemic reduction in interleukin 5. These results indicate an important role for CCR8 in Th2 functional responses in vivo.

The 2015 9th Workshop on Recent Issues in Bioanalysis (9th WRIB) took place in Miami, Florida with participation of 600 professionals from pharmaceutical and biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5 day, week-long event - A Full Immersion Bioanalytical Week - specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS and LBA approaches, including the focus on biomarkers and immunogenicity. This 2015 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2015 edition of this comprehensive White Paper has been divided into three parts. Part 3 discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Part 1 (small molecule bioanalysis using LCMS) and Part 2 (hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in volume 7, issues 22 and 23 of Bioanalysis, respectively.

In this study we used adenovirus vector-mediated transduction of either the p53 gene (rAd-p53) or the p21 WAF1⁄CIP1 gene (rAd-p21) to mimic both p53-dependent and -independent up-regulation of p21 WAF1⁄CIP1within a human ovarian cancer cell line, 2774, and the derivative cell lines, 2774qw1 and 2774qw2. We observed that rAd-p53 can induce apoptosis in both 2774 and 2774qw1 cells but not in 2774qw2 cells. Surprisingly, overexpression of p21 WAF1⁄CIP1 also triggered apoptosis within these two cell lines. Quantitative reverse transcription-PCR analysis revealed that the differential expression of BAX, BCL2, and caspase 3 genes, specific in rAd-p53-induced apoptotic cells, was not altered in rAd-p21-induced apoptotic cells, suggesting p21 WAF1⁄CIP1 -induced apoptosis through a pathway distinguishable from p53-induced apoptosis. Expression analysis of 2774qw1 cells infected with rAd-p21 on 60,000 cDNA microarrays identified 159 genes in response to p21 WAF1⁄CIP1 expression in at least one time point with 2.5-fold change as a cutoff. Integration of the data with the parallel microarray experiments with rAd-p53 infection allowed us to extract 66 genes downstream of both p53 and p21 WAF1⁄CIP1 and 93 genes in response to p21 WAF1⁄CIP1 expression in a p53-independent pathway. The genes in the former set may play a dual role in both p53-dependent and p53-independent pathways, and the genes in the latter set gave a mechanistic molecular explanation for p53-independent p21 WAF1⁄CIP1 -induced apoptosis. Furthermore, promoter sequence analysis suggested that transcription factor E2F family is partially responsible for the differential expression of genes following p21WAF1⁄CIP1 . This study has profound significance toward understanding the role of p21 WAF1⁄CIP1 in p53-independent apoptosis.