Generation of an HIV-1-resistant immune system with CD34(+) hematopoietic stem cells transduced with a triple-combination anti-HIV lentiviral vector

HIV gene therapy has the potential to offer an alternative to the use of current small-molecule antiretroviral drugs as a treatment strategy for HIV-infected individuals. Therapies designed to administer HIV-resistant stem cells to an infected patient may also provide a functional cure, as observed in a bone marrow transplant performed with hematopoietic stem cells (HSCs) homozygous for the CCR5-Δ32-bp allele. In our current studies, preclinical evaluation of a combination anti-HIV lentiviral vector was performed, in vivo, in humanized NOD-RAG1(-/-) IL2rγ(-/-) knockout mice. This combination vector, which displays strong preintegration inhibition of HIV-1 infection in vitro, contains a human/rhesus macaque TRIM5α isoform, a CCR5 short hairpin RNA (shRNA), and a TAR decoy. Multilineage hematopoiesis from anti-HIV lentiviral vector-transduced human CD34(+) HSCs was observed in the peripheral blood and in various lymphoid organs, including the thymus, spleen, and bone marrow, of engrafted mice. Anti-HIV vector-transduced CD34(+) cells displayed normal development of immune cells, including T cells, B cells, and macrophages. The anti-HIV vector-transduced cells also displayed knockdown of cell surface CCR5 due to the expression of the CCR5 shRNA. After in vivo challenge with either an R5-tropic BaL-1 or X4-tropic NL4-3 strain of HIV-1, maintenance of human CD4(+) cell levels and a selective survival advantage of anti-HIV gene-modified cells were observed in engrafted mice. The data provided from our study confirm the safety and efficacy of this combination anti-HIV lentiviral vector in a hematopoietic stem cell gene therapy setting for HIV and validates its potential application in future clinical trials.     

Antibody-dependent cellular cytotoxicity against primary HIV-infected CD4+ T cells is directly associated with the magnitude of surface IgG binding

Antibody (Ab)-dependent cellular cytotoxicity (ADCC) is thought to potentially play a role in vaccine-induced protection from HIV-1. The characteristics of such antibodies remain incompletely understood. Furthermore, correlates between ADCC and HIV-1 immune status are not clearly defined. We screened the sera of 20 HIV-1-positive (HIV-1(+)) patients for ADCC. Normal human peripheral blood mononuclear cells were used to derive HIV-infected CD4(+) T cell targets and autologous, freshly isolated, natural killer (NK) cells in a novel assay that measures granzyme B (GrB) and HIV-1-infected CD4(+) T cell elimination (ICE) by flow cytometry. We observed that complex sera mediated greater levels of ADCC than anti-HIV-1 envelope glycoprotein (Env)-specific monoclonal antibodies and serum-mediated ADCC correlated with the amount of IgG and IgG1 bound to HIV-1-infected CD4(+) T cells. No correlation between ADCC and viral load, CD4(+) T cell count, or neutralization of HIV-1(SF162) or other primary viral isolates was detected. Sera pooled from clade B HIV-1(+) individuals exhibited breadth in killing targets infected with HIV-1 from clades A/E, B, and C. Taken together, these data suggest that the total amount of IgG bound to an HIV-1-infected cell is an important determinant of ADCC and that polyvalent antigen-specific Abs are required for a robust ADCC response. In addition, Abs elicited by a vaccine formulated with immunogens from a single clade may generate a protective ADCC response in vivo against a variety of HIV-1 species. Increased understanding of the parameters that dictate ADCC against HIV-1-infected cells will inform efforts to stimulate ADCC activity and improve its potency in vaccinees.     

Influence of rest and exercise at a simulated altitude of 4,000 m on appetite, energy intake, and plasma concentrations of acylated ghrelin and peptide YY

The reason for high altitude anorexia is unclear but could involve alterations in the appetite hormones ghrelin and peptide YY (PYY). This study examined the effect of resting and exercising in hypoxia (12.7% O(2); ～4,000 m) on appetite, energy intake, and plasma concentrations of acylated ghrelin and PYY. Ten healthy males completed four, 7-h trials in an environmental chamber in a random order. The four trials were control-normoxia, control-hypoxia, exercise-normoxia, and exercise-hypoxia. During exercise trials, participants ran for 60 min at 70% of altitude-specific maximal oxygen consumption (Vo(2max)) and then rested. Participants rested throughout control trials. A standardized meal was consumed at 2 h and an ad libitum buffet meal at 5.5 h. Area under the curve values for hunger (assessed using visual analog scales) tended to be lower during hypoxic trials than normoxic trials (repeated-measures ANOVA, P = 0.07). Ad libitum energy intake was lower (P = 0.001) in hypoxia (5,291 ± 2,189 kJ) than normoxia (7,718 ± 2,356 kJ; means ± SD). Mean plasma acylated ghrelin concentrations were lower in hypoxia than normoxia (82 ± 66 vs. 100 ± 69 pg/ml; P = 0.005) while PYY concentrations tended to be higher in normoxia (32 ± 4 vs. 30 ± 3 pmol/l; P = 0.059). Exercise suppressed hunger and acylated ghrelin and increased PYY but did not influence ad libitum energy intake. These findings confirm that hypoxia suppresses hunger and food intake. Further research is required to determine if decreased concentrations of acylated ghrelin orchestrate this suppression.     

Split decisions: oesophageal progenitor cell behaviour

Science advance online publication July192012; doi:10.1126/science.1218835The maintenance and regeneration of continually shedding epithelial tissues that make up the linings and barriers of our bodies requires rapid and continual input of proliferative progenitor cells for tissue homeostasis. The mechanisms by which epithelial progenitors cells maintain tissues remain controversial. In a recent Science paper, Doupé et al (2012) demonstrate that a population of equivalent progenitor cells support tissue homeostasis of the oesophagus without the need for slow cycling cells as described in other rapidly dividing epithelia.In tissues such as blood and skin in which differentiated cells constantly turnover, proliferative progenitor populations are required to continually produce lost differentiated cells. Several models have been proposed to explain mechanisms by which progenitor cells contribute to tissue maintenance (Figure 1). A hierarchical model has been suggested in which longer lived stem cells, which may also cycle slowly, produce highly proliferative cells with less self-renewal potential that differentiate into a restricted number of cells. Following proliferative cells in pulse-chase experiments and genetic lineage tracing has supported a hierarchical model in the blood, epidermis and intestine (Fuchs, 2009). Alternatively, an equivalency model has been proposed in which all proliferative progenitor cells are equally able to produce proliferative and differentiated progeny in a stochastic manner. Analysis of labelled clones has supported an equivalency model for progenitors in the interfollicular epidermis and intestine (Clayton et al, 2007; Doupé et al, 2010; Snippert et al, 2010).Open in a separate windowFigure 1Two types of models have been put forward to describe the pattern of progenitor behaviour within mammalian tissues. In the hierarchical model, a stem cell can produce proliferative progenitors with less self-renewal potential that differentiate into lineage-specific cells. Alternatively, an equivalency model has been proposed that assumes equal behaviour of progenitor cells to maintain tissue homeostasis.An elevated interest in understanding the dynamics of oesophageal epithelium has resulted, in part, from the rapid increase in the incidence of oesophageal adenocarcinoma (Devesa et al, 1998). The oesophagus is a stratified epithelium that lacks any appendages or glands, and thus consists of a basal layer of proliferative keratinocytes and several suprabasal layers of differentiated cells, which are continually shed. Previously, labelling of proliferative cells with DNA analogues has demonstrated that proliferation is restricted to the basal cells, which all proliferate in 5 days seemingly stochastically, supporting an equivalency model (Marques-Periera and Leblond, 1965). In contrast, studies using chimeric mice have suggested that proliferation of labelled progenitor cells occurs in a hierarchical manner (Thomas et al, 1988; Croagh et al, 2008).To address this controversy, a recent study in Science uses several genetic mouse models to define the contribution of proliferative basal cells to oesophageal homeostasis (Doupé et al, 2012). In one mouse model, the authors utilized a genetic pulse-chase system based on the tetracycline-regulated expression of the histone H2B-GFP (Tumbar et al, 2004). They find that the rapidly dividing epithelial cells of the oesophagus lose H2B-GFP expression after 4 weeks. These data suggest that either H2B-GFP is degraded (Waghmare et al, 2008) or oesophageal progenitor cells proliferate faster than their counterparts in skin epithelial appendages or blood lineages, which retain H2B-GFP after 4 weeks (Tumbar et al, 2004; Foudi et al, 2009).To analyse the properties of oesophageal progenitor cells in more detail, the authors label single cells using an inducible cre-lox genetic system and followed clones for a year. Similar to their results with this system in the tail and ear epidermis (Clayton et al, 2007; Doupé et al, 2010), the authors find that the size of the persistent clones is linear with time. Statistical analysis of the clone size data supports the ability of the cells to contribute to proliferative and non-proliferative (i.e., differentiated) progeny with equal probability. Thus, these data support a model in which all of the labelled cells are equivalent.In addition to homeostasis, the authors explore how proliferative progenitors contribute to alterations in tissue homeostasis. After inflicting wounds by biopsy, marked clones span both proliferative and non-proliferative zones of the healing oesophageal epithelium, suggesting that they maintain a progenitor fate with distinct phenotypes. With atRA treatment, the authors show that suprabasal cell formation increases, which is consistent with the known effect of atRA on the oesophagus (Lasnitzki, 1963). Statistical analysis reveals that the probability of forming basal and suprabasal cells was not altered with atRA administration. However, since proliferative cells exist in suprabasal layers during epithelial hyperplasia, additional analyses of cell state are required to determine if atRA maintains stochastic fate decisions of progenitor cells. Furthermore, the progenitor response to atRA treatment might be limited by niche space along the basement membrane like in intestinal crypt progenitor cells (Snippert et al, 2010).In summary, this study together with the authors'' previous work provides additional support for the existence of equivalent progenitor cells within stratified epithelium in several tissues. Additional studies revealing how epithelial progenitor cells behave when proliferation and differentiation are altered in the oesophagus could shed light on mechanisms for the pathogenesis of oesophageal tumours or diseases such as Barrett''s oesophagus.     

Identification of the protein kinases that activate the E3 ubiquitin ligase Pellino 1 in the innate immune system

The E3 ubiquitin ligase Pellino 1 can be interconverted between inactive and active forms by a reversible phosphorylation mechanism. In vitro, phosphorylation and activation can be catalysed by either the IRAKs [IL (interleukin)-1-receptor-associated kinases] IRAK1 and IRAK4, or the IKK {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase}-related kinases [IKK? and TBK1 (TANK {TRAF [TNF (tumour-necrosis-factor)-receptor-associated factor]-associated NF-κB activator}-binding kinase 1)]. In the present study we establish that IRAK1 is the major protein kinase that mediates the IL-1-stimulated activation of Pellino 1 in MEFs (mouse embryonic fibroblasts) or HEK (human embryonic kidney)-293 cells, whereas the IKK-related kinases activate Pellino 1 in TNFα-stimulated MEFs. The IKK-related kinases are also the major protein kinases that activate Pellino 1 in response to TLR (Toll-like receptor) ligands that signal via the adaptors MyD88 (myeloid differentiation primary response gene 88) and/or TRIF [TIR (Toll/IL-1 receptor) domain-containing adaptor protein inducing interferon β]. The present studies demonstrate that, surprisingly, the ligands that signal via MyD88 do not always employ the same protein kinase to activate Pellino 1. Our results also establish that neither the catalytic activity of IRAK1 nor the activation of Pellino 1 is required for the initial transient activation of NF-κB and MAPKs (mitogen-activated protein kinases) that is triggered by IL-1 or TNFα in MEFs, or by TLR ligands in macrophages. The activation of Pellino 1 provides the first direct readout for IRAK1 catalytic activity in cells.     

C-type Natriuretic Peptide: a novel biomarker of steroid induced bone toxicity in children with acute lymphoblastic leukemia (ALL)

Impaired bone growth and mineralization, and osteonecrosis are significant and common long-term sequelae of chemotherapy for childhood acute lymphoblastic leukemia (ALL). Here we have evaluated the relationship between linear bone growth during chemotherapy for ALL and bone derived C-type Natriuretic Peptide (CNP). CNP is known to be critical to normal endochondral bone growth in both rodents and humans, and plasma concentration of the amino terminal pro CNP (NTproCNP) is strongly correlated with concurrent height velocity in children. Plasma NTproCNP and CNP were measured by radio-immunoassay in 12 children aged 2-9 years during induction and maintenance chemotherapy for children with ALL. Height velocity was calculated from stadiometer readings at intervals of 3-12 months and related to plasma NTproCNP during each growth interval. Plasma NTproCNP was markedly suppressed in all subjects during induction chemotherapy. Brief periods of NTproCNP decline and rapid rebound during maintenance treatment coincided with the use of dexamethasone but not with other chemotherapeutics. Height velocity was markedly reduced during ALL induction but unaffected in maintenance phase, and these changes in growth were strongly correlated with plasma NTproCNP concentration. Plasma NTproCNP has potential use as a biomarker of glucocorticoid-induced bone toxicity.     

Ecology of Vibrio parahaemolyticus and Vibrio vulnificus in the Coastal and Estuarine Waters of Louisiana,Maryland, Mississippi,and Washington (United States)

Vibrio parahaemolyticus and Vibrio vulnificus, which are native to estuaries globally, are agents of seafood-borne or wound infections, both potentially fatal. Like all vibrios autochthonous to coastal regions, their abundance varies with changes in environmental parameters. Sea surface temperature (SST), sea surface height (SSH), and chlorophyll have been shown to be predictors of zooplankton and thus factors linked to vibrio populations. The contribution of salinity, conductivity, turbidity, and dissolved organic carbon to the incidence and distribution of Vibrio spp. has also been reported. Here, a multicoastal, 21-month study was conducted to determine relationships between environmental parameters and V. parahaemolyticus and V. vulnificus populations in water, oysters, and sediment in three coastal areas of the United States. Because ecologically unique sites were included in the study, it was possible to analyze individual parameters over wide ranges. Molecular methods were used to detect genes for thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and tdh-related hemolysin (trh) as indicators of V. parahaemolyticus and the hemolysin gene vvhA for V. vulnificus. SST and suspended particulate matter were found to be strong predictors of total and potentially pathogenic V. parahaemolyticus and V. vulnificus. Other predictors included chlorophyll a, salinity, and dissolved organic carbon. For the ecologically unique sites included in the study, SST was confirmed as an effective predictor of annual variation in vibrio abundance, with other parameters explaining a portion of the variation not attributable to SST.     

Defining the boundaries of species specificity for the Saccharomyces cerevisiae glycosylphosphatidylinositol transamidase using a quantitative in?vivo assay

In eukaryotes, GPI (glycosylphosphatidylinositol) lipid anchoring of proteins is an abundant post-translational modification. The attachment of the GPI anchor is mediated by GPI-T (GPI transamidase), a multimeric, membrane-bound enzyme located in the ER (endoplasmic reticulum). Upon modification, GPI-anchored proteins enter the secretory pathway and ultimately become tethered to the cell surface by association with the plasma membrane and, in yeast, by covalent attachment to the outer glucan layer. This work demonstrates a novel in vivo assay for GPI-T. Saccharomyces cerevisiae INV (invertase), a soluble secreted protein, was converted into a substrate for GPI-T by appending the C-terminal 21 amino acid GPI-T signal sequence from the S. cerevisiae Yapsin 2 [Mkc7p (Y21)] on to the C-terminus of INV. Using a colorimetric assay and biochemical partitioning, extracellular presentation of GPI-anchored INV was shown. Two human GPI-T signal sequences were also tested and each showed diminished extracellular INV activity, consistent with lower levels of GPI anchoring and species specificity. Human/fungal chimaeric signal sequences identified a small region of five amino acids that was predominantly responsible for this species specificity.