IL-15-dependent induction of 4-1BB promotes antigen-independent CD8 memory T cell survival

Mice lacking CD137L (4-1BBL) show normal primary expansion and contraction of the CD8+ T cell response to influenza virus, but exhibit a defect in Ag-specific CD8+ T cell numbers at 3-6 wk postinfection. Previous results showed that the decrease in CD8+ T cell numbers in this model is not due to a programming defect during primary expansion. Thus, it appears that 4-1BB/4-1BBL interactions control the number of surviving CD8+ effector memory cells, late in the primary response. In this report, we asked how 4-1BB on T cells could play a role after Ag has apparently been cleared from the host. We show that IL-15, a cytokine involved in regulation of CD8+ memory T cell survival, induces the expression of 4-1BB on CD8+CD44(high) memory phenotype T cells, but not on CD4+ T cells. The Ag-independent induction of 4-1BB by IL-15 was dependent on MAPK p38 and ERK activation. Transfer of in vitro-generated OT-I CD8+ memory T cells into unimmunized wild-type or 4-1BBL-deficient hosts revealed a 2- to 3-fold survival advantage when 4-1BBL was present, recapitulating the effect seen in the endogenous response to influenza in mice. Decreases in the overall number of memory CD8+ T cells were also observed in the bone marrow of unmanipulated 4-1BBL-deficient mice. These data suggest a model whereby 4-1BB expression on memory CD8+ T cells, perhaps due to encounter with IL-15 in the bone marrow, allows 4-1BB/4-1BBL interactions to maintain memory CD8 T cell survival in the absence of Ag.     

Intimate social behavior in infant interactions in Cebus apella

The development and individual stability of three intimate social behaviors (Lipsmacking, Carrying Attempts, and Facial Inspection) were examined for 43 group-housed Cebus apella infants from birth to 2 years of age. Occurrence of these behaviors was scored from 10-min videotape samples recorded three times a week over that time. Frequency of Lipsmacking and Carrying Attempts by adult males, adult females, and juveniles were all highest in early months and decreased to low levels by the end of the first year. Facial Inspection of partners by infants, in contrast, first began at 3-4 months and increased over time, at least to adult males and juveniles. Correlational analyses indicated stable individual differences in these interactions with infants and outlined a relationship between these intimate behaviors and more general social patterns reported previously for these animals. Results suggest that adult males may play a special role in affording juveniles opportunities for social learning of foraging and manipulative skills.     

Early Evolution of Conserved Regulatory Sequences Associated with Development in Vertebrates

Comparisons between diverse vertebrate genomes have uncovered thousands of highly conserved non-coding sequences, an increasing number of which have been shown to function as enhancers during early development. Despite their extreme conservation over 500 million years from humans to cartilaginous fish, these elements appear to be largely absent in invertebrates, and, to date, there has been little understanding of their mode of action or the evolutionary processes that have modelled them. We have now exploited emerging genomic sequence data for the sea lamprey, Petromyzon marinus, to explore the depth of conservation of this type of element in the earliest diverging extant vertebrate lineage, the jawless fish (agnathans). We searched for conserved non-coding elements (CNEs) at 13 human gene loci and identified lamprey elements associated with all but two of these gene regions. Although markedly shorter and less well conserved than within jawed vertebrates, identified lamprey CNEs are able to drive specific patterns of expression in zebrafish embryos, which are almost identical to those driven by the equivalent human elements. These CNEs are therefore a unique and defining characteristic of all vertebrates. Furthermore, alignment of lamprey and other vertebrate CNEs should permit the identification of persistent sequence signatures that are responsible for common patterns of expression and contribute to the elucidation of the regulatory language in CNEs. Identifying the core regulatory code for development, common to all vertebrates, provides a foundation upon which regulatory networks can be constructed and might also illuminate how large conserved regulatory sequence blocks evolve and become fixed in genomic DNA.     

Variation in the daily rhythm of body temperature of free-living Arabian oryx (Oryx leucoryx): does water limitation drive heterothermy?

Heterothermy, a variability in body temperature beyond the limits of homeothermy, has been advanced as a key adaptation of Arabian oryx (Oryx leucoryx) to their arid-zone life. We measured body temperature using implanted data loggers, for a 1-year period, in five oryx free-living in the deserts of Saudi Arabia. As predicted for adaptive heterothermy, during hot months compared to cooler months, not only were maximum daily body temperatures higher (41.1 ± 0.3 vs. 39.7 ± 0.1°C, P = 0.0002) but minimum daily body temperatures also were lower (36.1 ± 0.3 vs. 36.8 ± 0.2°C, P = 0.04), resulting in a larger daily amplitude of the body temperature rhythm (5.0 ± 0.5 vs. 2.9 ± 0.2°C, P = 0.0007), while mean daily body temperature rose by only 0.4°C. The maximum daily amplitude of the body temperature rhythm reached 7.7°C for two of our oryx during the hot-dry period, the largest amplitude ever recorded for a large mammal. Body temperature variability was influenced not only by ambient temperature but also water availability, with oryx displaying larger daily amplitudes of the body temperature rhythm during warm-dry months compared to warm-wet months (3.6 ± 0.6 vs. 2.3 ± 0.3°C, P = 0.005), even though ambient temperatures were the same. Free-living Arabian oryx therefore employ heterothermy greater than that recorded in any other large mammal, but water limitation, rather than high ambient temperature, seems to be the primary driver of this heterothermy.     

A forward genetic screen to explore chloroplast protein import in vivo identifies Moco sulfurase,pivotal for ABA and IAA biosynthesis and purine turnover

A genetic screen in Arabidopsis was developed to explore the regulation of chloroplast protein import in vivo using two independent reporters representing housekeeping and photosynthetic pre‐proteins. We first used 5‐enolpyruvylshikimate 3‐phosphate synthase (EPSP synthase*), a key enzyme in the shikimic acid pathway, with a mutation that confers tolerance to the herbicide glyphosate. Because the EPSP synthase* pre‐protein must be imported for its function, the loss of glyphosate tolerance provided an initial indication of an import deficiency. Second, the fate of GFP fused to a ferredoxin transit peptide (FD5–GFP) was determined. A class of altered chloroplast import (aci) mutants showed both glyphosate sensitivity and FD5–GFP mislocalized to nuclei. aci2‐1 was selected for further study. Yellow fluorescent protein (YFP) fused to the transit peptide of EPSP synthase* or the small subunit of Rubisco was not imported into chloroplasts, but also localized to nuclei during protoplast transient expression. Isolated aci2‐1 chloroplasts showed a 50% reduction in pre‐protein import efficiency in an in vitro assay. Mutants did not grow photoautotrophically on media without sucrose and were small and dark green in soil. aci2‐1 and two alleles code for Moco‐sulfurase, which activates the aldehyde oxidases required for the biosynthesis of the plant hormones abscisic acid (ABA) and indole‐acetic acid (IAA) and controls purine nucleotide (ATP and GTP) turnover and nitrogen recycling via xanthine dehydrogenase. These enzyme activities were not detected in aci2‐1. ABA, IAA and/or purine turnover may play previously unrecognized roles in the regulation of chloroplast protein import in response to developmental, metabolic and environmental cues.     

The discovery and SAR of indoline-3-carboxamides—A new series of 5-HT6 antagonists

Antagonists of the 5-HT₆ receptor have been shown to improve cognitive function in a wide range of animal models and as such may prove to be attractive agents for the symptomatic treatment of cognitive disorders such as Alzheimer’s disease (AD) and schizophrenia. We report herein the identification and SAR around N-(2-aminoalkyl)-1-(arylsulfonyl)indoline-3-carboxamides—a novel chemotype of 5-HT₆ antagonists.     

Cell culture monitoring via an auto‐sampler and an integrated multi‐functional off‐line analyzer

Mammalian cell‐based bioprocesses are used extensively for production of therapeutic proteins. Off‐line monitoring of such cultivations via manual sampling is often labor‐intensive and can introduce operator‐dependent error into the process. An integrated multi‐functional off‐line analyzer, the BioProfile FLEX (NOVA Biomedical, Waltham MA) has been developed, which combines the functionality of three off‐line analyzers (a cell counter, an osmometer, and a gas/electrolyte & nutrient/metabolite bio‐profile analyzer) into one device. In addition, a novel automated sampling system has also been developed that allows the BioProfile FLEX to automatically analyze the culture conditions in as many as ten bioreactors. This is the first report on the development and function of this integrated analyzer and an auto‐sampler prototype for monitoring of mammalian cell cultures. Evaluation of the BioProfile FLEX was conducted in two separate laboratories and involved two BioProfile FLEX analyzers and two sets of reference analyzers (Nova BioProfile 400, Beckman‐Coulter Vi‐Cell AS, and Advanced Instruments Osmometer 3900), 13 CHO cell lines and over 20 operators. In general, BioProfile FLEX measurements were equivalent to those obtained using reference analyzers, and the auto‐sampler did not alter the samples it provided to the BioProfile FLEX. These results suggest that the system has the potential to dramatically reduce the manual labor involved in monitoring mammalian cell bioprocesses without altering the quality of the data obtained, and integration with a bioreactor control system will allow feedback control of parameters previously available only for off‐line monitoring. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health disorders such as chronic wounds. We report the development of a dual-mode imaging system for non-invasive and non-contact imaging of cutaneous tissue oxygenation and vascular function. The imaging system integrated an infrared camera, a CCD camera, a liquid crystal tunable filter and a high intensity fiber light source. A Labview interface was programmed for equipment control, synchronization, image acquisition, processing, and visualization. Multispectral images captured by the CCD camera were used to reconstruct the tissue oxygenation map. Dynamic thermographic images captured by the infrared camera were used to reconstruct the vascular function map. Cutaneous tissue oxygenation and vascular function images were co-registered through fiduciary markers. The performance characteristics of the dual-mode image system were tested in humans.     

Macrophage Dysfunction Impairs Resolution of Inflammation in the Wounds of Diabetic Mice

Background

Chronic inflammation is a characteristic feature of diabetic cutaneous wounds. We sought to delineate novel mechanisms involved in the impairment of resolution of inflammation in diabetic cutaneous wounds. At the wound-site, efficient dead cell clearance (efferocytosis) is a pre-requisite for the timely resolution of inflammation and successful healing.

Methodology/Principal Findings

Macrophages isolated from wounds of diabetic mice showed significant impairment in efferocytosis. Impaired efferocytosis was associated with significantly higher burden of apoptotic cells in wound tissue as well as higher expression of pro-inflammatory and lower expression of anti-inflammatory cytokines. Observations related to apoptotic cell load at the wound site in mice were validated in the wound tissue of diabetic and non-diabetic patients. Forced Fas ligand driven elevation of apoptotic cell burden at the wound site augmented pro-inflammatory and attenuated anti-inflammatory cytokine response. Furthermore, successful efferocytosis switched wound macrophages from pro-inflammatory to an anti-inflammatory mode.

Conclusions/Significance

Taken together, this study presents first evidence demonstrating that diabetic wounds suffer from dysfunctional macrophage efferocytosis resulting in increased apoptotic cell burden at the wound site. This burden, in turn, prolongs the inflammatory phase and complicates wound healing.     

Repeated lipopolysaccharide (LPS) exposure inhibits HIV replication in primary human macrophages

Repetitive exposure of macrophages to microbial antigen is known to tolerize them to further stimulation and to inhibit proinflammatory cytokine release. Using transgenic (Tg) mice that incorporate the entire HIV-1 genome we have previously shown that toll like receptor (TLR)-2, -4, and -9 ligands induced tolerance as assessed by decreased proinflammatory cytokine secretion and nuclear factor-kappa beta activation. Yet, despite cytokine modulation, HIV-1 p24 production was enhanced in tolerized cells in vitro and in vivo. Since mice are not natural hosts for HIV infection, in the following report we examined whether TLR2 and TLR4 ligands induced tolerance in human monocytic cell lines stably expressing the HIV-long terminal repeat (LTR) luciferase construct (THP-LTR-Luc) as well as in primary macrophages that had been infected with HIV(BAL)in vitro. In THP-LTR-luc, TLR2 and TLR4 tolerization suppressed tumor necrosis factor (TNF)-alpha release and HIV-LTR transactivation. In HIV(BAL) infected macrophages, repeated LPS exposure inhibited HIV replication as assessed by decreased genetic expression and protein production of HIV-1 p24, although TNF-alpha release was not inhibited. These observations may have important clinical implications in understanding the role of macrophages as HIV reservoirs at anatomical sites where there is repeated exposure to microbial antigens.