Acute Neuronal Injury and Blood Genomic Profiles in a Nonhuman Primate Model for Ischemic Stroke

The goal of this study was to characterize acute neuronal injury in a novel nonhuman primate (NHP) ischemic stroke model by using multiple outcome measures. Silk sutures were inserted into the M1 segment of the middle cerebral artery of rhesus macaques to achieve permanent occlusion of the vessel. The sutures were introduced via the femoral artery by using endovascular microcatheterization techniques. Within hours after middle cerebral artery occlusion (MCAO), infarction was detectable by using diffusion-weighted MRI imaging. The infarcts expanded by 24 h after MCAO and then were detectable on T2-weighted images. The infarcts seen by MRI were consistent with neuronal injury demonstrated histologically. Neurobehavioral function after MCAO was determined by using 2 neurologic testing scales. Neurologic assessments indicated that impairment after ischemia was limited to motor function in the contralateral arm; other neurologic and behavioral parameters were largely unaffected. We also used microarrays to examine gene expression profiles in peripheral blood mononuclear cells after MCAO-induced ischemia. Several genes were altered in a time-dependent manner after MCAO, suggesting that this ischemia model may be suitable for identifying blood biomarkers associated with the presence and severity of ischemia. This NHP stroke model likely will facilitate the elucidation of mechanisms associated with acute neuronal injury after ischemia. In addition, the ability to identify candidate blood biomarkers in NHP after ischemia may prompt the development of new strategies for the diagnosis and treatment of ischemic stroke in humans.Abbreviations: MCAO, middle cerebral artery occlusion; NHP, nonhuman primate; PBMC, peripheral blood mononuclear cellsStroke is a debilitating neurologic condition, and little progress has been made in the development of neuroprotective treatments for acute stroke. The Stroke Therapy Academic Industry Roundtable (STAIR) report suggested that preclinical candidates for stroke therapy should be validated by testing in large animals with similarities to humans, such as nonhuman primates (NHP).²⁶ NHP stroke models have been developed in several species, including rhesus monkeys, marmosets, and baboons, by using a variety of techniques for middle cerebral artery occlusion (MCAO).^{4,10,12,13,14,25,32} The rhesus macaque is ideal for stroke studies because of its structural similarities to human brain. The rhesus brain is gyrencephalic, which makes it preferable to those of lissencephalic primates (for example, marmosets) and is functionally similar to human brain.⁶ In addition, the immunologic profile of rhesus macaques is similar to that of humans; therefore these animals are the preferred model for the study of immune responses to infectious diseases such as HIV/SIV, Dengue virus, and others.^17,23,30In addition to their use for neuroprotection assessment, NHP stroke models can facilitate efforts to develop diagnostic tools for identifying and treating stroke symptoms. The use of genomics in peripheral blood cells has been shown to be an excellent method to identify candidate biomarkers and cellular mechanisms associated with stroke.^28,29 Blood biomarkers can be used to rapidly determine the occurrence, timing, subtype, and severity of stroke.^11,15 One possible reason for the lack of viable stroke biomarkers may be the research models used to search for these markers. Although rodent stroke models have yielded a wealth of information on the mechanisms associated with brain ischemia, the findings have not translated well to human clinical trials.²⁶ Recent studies in human patients showed promising results when genomic tools have been used to screen for novel stroke biomarkers.^3,16,27 However, validation of human studies is limited by the need for large data sets in light of heterogeneity in stroke onset, subtype, comorbidities, and other factors. In addition, it is also impossible to know the exact time of stroke onset in most patients.Here we characterized acute neuronal injury in a novel, minimally invasive permanent ischemic stroke model involving rhesus macaques. Using endovascular catheterization techniques, we introduced silk sutures into the M1 segment of the middle cerebral artery and permanently occluded it. This procedure reliably produced infarcts that could be measured by MRI of the macaque brains during the acute phase period. The procedure resulted in discrete and limited neurobehavioral deficits, indicating the potential of this stroke model for chronic neuroprotection studies in the future. In addition, we used microarrays to identify blood genomic profiles that were altered in a time-dependent manner after ischemia. These studies characterize a preclinical model that is suitable for elucidating the mechanisms associated with cerebral ischemia and that may aid in identifying strategies for the diagnosis and treatment of stroke in humans.     

Ultrastructure of inclusion bodies in annulus cells in the degenerating human intervertebral disc

The rough endoplasmic reticulum (rER) of the cell has an architectural editing function that checks whether protein structure and three-dimensional assembly have occurred properly prior to export of newly synthesized material out of the cell. If these have been faulty, the material is retained within the rER as an inclusion body. Inclusion bodies have been identified previously in chondrocytes and osteoblasts in chondrodysplasias and osteogenesis imperfecta. Inclusion bodies in intervertebral disc cells, however, have only recently been recognized. Our objectives were to use transmission electron microscopy to analyze more fully inclusion bodies in the annulus pulposus and to study the extracellular matrix (ECM) surrounding cells containing inclusion bodies. ECM frequently encapsulated cells with inclusion bodies, and commonly contained prominent banded aggregates of Type VI collagen. Inclusion body material had several morphologies, including relatively smooth, homogeneous material, or a rougher, less homogeneous feature. Such findings expand our knowledge of the fine structure of the human disc cell and ECM during disc degeneration, and indicate the potential utility of ultrastructural identification of discs with intracellular inclusion bodies as a screening method for molecular studies directed toward identification of defective gene products in degenerating discs.     

Horizontal transmission, vertical inactivation, and stochastic loss of mariner-like transposable elements

Horizontal transmission has been well documented as a major mechanism for the dissemination of mariner-like elements (MLEs) among species. Less well understood are mechanisms that limit vertical transmission of MLEs resulting in the "spotty" or discontinuous distribution observed in closely related species. In this article we present evidence that the genome of the common ancestor of the melanogaster species subgroup of Drosophila contained an MLE related to the mellifera (honey bee) subfamily. Horizontal transmission, approximately 3-10 MYA, is strongly suggested by the observation that the sequence of the MLE in Drosophila erecta is 97% identical in nucleotide sequence with that of an MLE in the cat flea, Ctenocephalides felis. The D. erecta MLE has a spotty distribution among species in the melanogaster subgroup. The element has a high copy number in D. erecta and D. orena, a moderate copy number in D. teissieri and D. yakuba, and was apparently lost ("stochastic loss") in the lineage leading to D. melanogaster, D. simulans, D. mauritiana, and D. sechellia. In D. erecta, most copies are concentrated in the heterochromatin. Two copies from D. erecta, denoted De12 and De19, were cloned and sequenced, and they appear to be nonfunctional ("vertical inactivation"). It therefore appears that the predominant mode of MLE evolution is vertical inactivation and stochastic loss balanced against occasional reinvasion of lineages by horizontal transmission.      

Morphologic features of spontaneous annular tears and disc degeneration in the aging sand rat (Psammomys obesus obesus)

The sand rat, a member of the gerbil family, is a valuable small animal model in which intervertebral disc degeneration occurs spontaneously as the animal ages. Radiographic features of cervical and lumbar degeneration resemble those in human spines. We conducted a retrospective analysis of spines of 140 animals 3?41 months old focusing specifically on the presence of annular tears that are not visible by radiography and have not been described previously in the sand rat disc. During degeneration of the nucleus pulposus, notochordal cell death occurs and granular material, which stains with Alcian blue for proteoglycans, accumulates. Lamellar architecture also deteriorates and annular tears occur that are morphologically similar to the concentric, radiating and transdiscal annular tears in human discs. These tears contain granular material that provides a “marker” that can be used to distinguish the annular tears from artefactual separations during sectioning. We observed lamellar degeneration and separation in the annulus fibrosus at 4 months with associated tears that contained granular material in the nucleus. Tears that contained granular material and displacement of the degenerating nucleus were common in cervical and lumbar discs of animals older than 9 months; some specimens showed tears at 4 and 5 months. With advanced degeneration, granular globules were displaced dorsally adjacent to and into the spinal cord area and also ventrally into regions where osteophytes formed. We present morphologic data that expand the utility of this rodent model of spontaneous age-related disc degeneration and provide novel information on annular tears and disc degeneration.     

Multiplex cytokine analysis of dermal interstitial blister fluid defines local disease mechanisms in systemic sclerosis

IntroductionClinical diversity in systemic sclerosis (SSc) reflects multifaceted pathogenesis and the effect of key growth factors or cytokines operating within a disease-specific microenvironment. Dermal interstitial fluid sampling offers the potential to examine local mechanisms and identify proteins expressed within lesional tissue. We used multiplex cytokine analysis to profile the inflammatory and immune activity in the lesions of SSc patients.MethodsDermal interstitial fluid sample from the involved forearm skin, and synchronous plasma samples were collected from SSc patients (n = 26, diffuse cutaneous SSc (DcSSc) n = 20, limited cutaneous SSc (LcSSc) n = 6), and healthy controls (HC) (n = 10) and profiled by Luminex® array for inflammatory cytokines, chemokines, and growth factors.ResultsLuminex® profiling of the dermal blister fluid showed increased inflammatory cytokines (median interleukin ( IL)-6 in SSc 39.78 pg/ml, HC 5.51 pg/ml, p = 0.01, median IL-15 in SSc 6.27 pg/ml, HC 4.38 pg/ml, p = 0.03), chemokines (monocyte chemotactic protein (MCP)-3 9.81 pg/ml in SSc, 7.18 pg/ml HC, p = 0.04), and profibrotic growth factors (platelet derived growth factor (PDGF)-AA 10.38 pg/ml versus 6.94 pg/ml in HC, p = 0.03). In general dermal fluid and plasma cytokine levels did not correlate, consistent with predominantly local production of these factors within the dermal lesions, rather than leakage from the serum. In hierarchical clustering and network analysis IL-6 emerged as a key central mediator.ConclusionsOur data confirm that an immuno-inflammatory environment and aberrant vascular repair are intimately linked to fibroblast activation in lesional skin in SSc. This non-invasive method could be used to profile disease activity in the clinic, and identifies key inflammatory or pro-fibrotic proteins that might be targeted therapeutically. Distinct subgroups of SSc may be defined that show innate or adaptive immune cytokine signatures.     

基于psbA trnH序列的十种棘豆属植物分子系统学研究

选择内蒙古28个样地采集的10种棘豆属植物56个单株,提取样品的基因组DNA,对其叶绿体psbA trnH序列进行扩增、测序,所得序列利用ClustalX软件进行对位排列,并用MEGA50软件采用最大似然法构建系统发育树。结果显示：（1）10种棘豆属psbA trnH序列的变异位点50个,信息位点36个,种间碱基差异百分率为34%,GC含量变化范围在2318%~2572%之间。（2）棘豆属与黄芪属各为一支,自展支持率达99%,这10种棘豆属植物可能为单系起源。（3）系统树中小叶小花棘豆与小花棘豆的样本独立成一支,支持将小叶小花棘豆作为小花棘豆的变种来处理。（4）多叶棘豆、砂珍棘豆和黄毛棘豆的样本相互混杂,表明亲缘关系很近,支持《内蒙古植物志》将三者归入真棘豆亚属轮叶棘豆组的观点。（5）缘毛棘豆与薄叶棘豆的样本聚成一支,支持将二者归入矮生棘豆组。研究表明,psbA trnH序列可为棘豆属下种间系统发育关系研究提供分子证据。     

Selective attention toward female secondary sexual color in male rhesus macaques

Pink-to-red anogenital and facial sexual skin occurs in females of many primate species. Since female sexual skin color varies with reproductive state, it has long been assumed that color acts to stimulate male sexual interest. Although there is supportive evidence for this as regards anogenital skin, it is unclear whether this is also the case for facial sexual skin. In this study we experimentally manipulated digital facial and hindquarter images of female rhesus macaques (Macaca mulatta) for color within the natural range of variation. The images were presented to adult male conspecifics to assess whether the males exhibited visual preferences for red vs. non-red female coloration, and whether preferences varied with anatomical region. The males displayed significantly longer gaze durations in response to reddened versions of female hindquarters, but not to reddened versions of faces. This suggests that female facial coloration may serve an alternative purpose to that of attracting males, and that the signal function of sexual skin and the intended recipients may vary across anatomical regions.     

Decreased dengue replication and an increased anti-viral humoral response with the use of combined Toll-like receptor 3 and 7/8 agonists in macaques

Background

Pathogenic versus protective outcomes to Dengue virus (DENV) infection are associated with innate immune function. This study aimed to determine the role of increased TLR3- and TLR7/8-mediated innate signaling after Dengue infection of rhesus macaques in vivo to evaluate its impact on disease and anti-DENV immune responses.

Methodology/Principal Findings

TLR3 and TLR7/8 agonists (emulsified in Montanide) were administered subcutaneously to rhesus macaques at 48 hours and 7 days after DENV infection. The Frequency and activation of myeloid dendritic cells, plasmacytoid dendritic cells, and B cells were measured by flow cytometry while the serum levels of 14 different cytokines and chemokines were quantified. Adaptive immune responses were measured by DENV-specific antibody subtype measurements. Results showed that the combined TLR agonists reduced viral replication and induced the development of a proinflammatory reaction, otherwise absent in Dengue infection alone, without any clear signs of exacerbated disease. Specifically, the TLR-induced response was characterized by activation changes in mDC subsets concurrent with higher serum levels of CXCL-10 and IL-1Ra. TLR stimulation also induced higher titers of anti-DENV antibodies and acted to increase the IgG2/IgG1 ratio of anti-DENV to favor the subtype associated with DENV control. We also observed an effect of DENV-mediated suppression of mDC activation consistent with prior in vitro studies.

Conclusions/Significance

These data show that concurrent TLR3/7/8 activation of the innate immune response after DENV infection in vivo acts to increase antiviral mechanisms via increased inflammatory and humoral responses in rhesus macaques, resulting in decreased viremia and melioration of the infection. These findings underscore an in vivo protective rather than a pathogenic role for combined TLR3/7/8-mediated activation in Dengue infection of rhesus macaques. Our study provides definitive proof-of-concept into the mechanism by which DENV evades immune recognition and activation in vivo.     

Cancer-Related Causes of Death among HIV-Infected Patients in France in 2010: Evolution since 2000

Objectives

The current study aimed at describing the distribution and characteristics of malignancy related deaths in human immunodeficiency virus (HIV) infected patients in 2010 and at comparing them to those obtained in 2000 and 2005.

Methods

Data were obtained from three national surveys conducted in France in 2010, 2005 and 2000. The underlying cause of death was documented using a standardized questionnaire fulfilled in French hospital wards involved in the management of HIV infection.

Results

Among the 728 deaths reported in 2010, 262 were cancer-related (36%). After a significant increase from 28% in 2000 to 33% in 2005 and 36% in 2010, cancers represent the leading cause of mortality in HIV infected patients. The proportion of deaths attributed to non-AIDS/non-hepatitis-related cancers significantly increased from 2000 to 2010 (11% of the deaths in 2000, 17% in 2005 and 22% in 2010, p<0.001), while those attributed to AIDS-defining cancers decreased during the same period (16% in 2000, 13% in 2005 and 9% in 2010, p = 0.024). Particularly, the proportion of respiratory cancers significantly increased from 5% in 2000 to 6% in 2005 and 11% in 2010 (p = 0.004). Lung cancer was the most common cancer-related cause of death in 2010 (instead of non-Hodgkin lymphoma so far) and represented the leading cause of death in people living with HIV overall.

Conclusions

Cancer prevention (especially smoking cessation), screening strategies and therapeutic management need to be optimized in HIV-infected patients in order to reduce mortality, particularly in the field of respiratory cancers.     

Constitutive expression of IL-22 in the human intervertebral disc and its reduction by exposure to pro-inflammatory cytokines in vitro

The importance of cytokines in disc degeneration is well recognized. Little is known about IL-22 expression in the human intervertebral disc. We investigated IL-22 immuno-localization in disc tissue, and molecular expression and production of IL-22 by annulus cells cultured in three-dimensional (3D) culture. We examined human disc tissue using immunohistochemistry and we cultured isolated annulus cells in 3D to analyze IL-22 expression and production, and its receptor, IL-22R, in conditioned media. Ingenuity pathway analysis (IPA) also was used to identify significant gene expression networks within the molecular data. IL-22 and IL-22R were immunolocalized in many cells in the human outer and inner annulus; fewer cells exhibited localization in the nucleus. Three-dimensional culture of annulus cells demonstrated production of IL-22 in conditioned media; exposure to IL-1ß or TNF-α significantly reduced IL-22 levels. Significant decreases also were identified in conditioned media assayed for IL-22R in TNF-α treated cells. IPA analysis showed that IL-22 ranked among the top canonical pathways. We found constitutive expression and production of IL-22 and IL-22R in the disc, which expands our understanding of the effect of pro-inflammatory cytokines on IL-22 expression and production. Three-dimensional cultured annulus cells exposed to IL-1ß or TNF produced significantly lower levels of IL-22 into their conditioned media compared to levels produced by control cells. Our findings have clinical relevance because of the elevated pro-inflammatory milieu within the degenerating human disc.