Morphometric analysis of Macaca arctoides and M-thibetana in relation to other macaque species

As a first step in reviewing the classification of the two stump-tailed macaque species, Macaca arctoides and M. thibetana, as compared with other species of the genus Macaca, 72 linear dental and cranial variables of 11 macaque species were examined by morphometric analyses. The results indicate that the two stump-tailed species are the largest of the macaques and although rather similar overall, they exhibit significant differences in the pattern of variation in most of the five skull regions as shown by Principal Components and Canonical Variate Analyses. Euclidean Distances based on Canonical Variate scores indicate that the females of M. arctoides and M. thibetana are more widely separated than eight other pairs of macaque species, and that the separations of the respective males are greater than those of three other pairs of species. These findings are consistent with FOODEN's classification of the stump-tailed macaques as two separate species (FOODEN, 1976; FOODEN et al., 1985). The present results suggest, as other researchers have proposed on the basis of external features, biochemistry and genetics, that the two stump-tailed macaque species and M. assamensis are closely related. The results also tentatively imply associations with M. fuscata and M. sylvanus but these require further study. The findings have implications for the assessment of the various Chinese Pleistocene macaque fossils.

猕猴属的分子系统学与遗传多样性研究

用六个线粒体基因片段序列数据，对称猴属的分子系统发育关系进行了分析。8种称猴：台湾猴，北平顶猴，藏酋猴，熊猴，红面猴，J厦河猴，狮尾猴和食蟹猴共40个样本用于本研究。结合从GenBank里下载的雯猴的相应序列，用拂拂作为外群构建了系统发育树。由于六个目的片段均为线粒体基因片段，缺乏重组且作为单一位点遗传，因此我们将六个数据集合并为一个数据集进行分析。我们获得了比前人置信度更高的线粒体系统树。除了红面猴，我们的结果支持Delson（1980）的分组。与前人研究结果一致，红面猴与食蟹猴组成员关系较近（尤其是食蟹猴）。恒河猴（相对于台湾猴），熊猴（相对于藏酋猴）和北平顶猴（相对于狮尾猴）都形成了并系。通过线粒体控制区序列和20个微卫星位点对14个恒河猴地理群体的群体结构进行了研究。微卫星数据和线粒体控制区数据均显示恒河猴有较高的遗传多样度。线粒体数据表明各地区的恒河猴群体间的差异显著大于微卫星数据显示的结果。也就是说，基于微卫星数据所得到的群体间基因流远大于基于线粒体数据所得到的群体间基因流。两种分子标记所得到的结果差异应归因为雌性恒河猴的归家冲动和雄性恒河猴的扩散。线粒体数据显示所有恒河猴群体近期没有发生过快速扩张。微卫星数据显示大部分恒河猴群体近期没有经历过瓶颈效应。恒河猴是分布较广的非人灵长类动物，但它的地理差异研究一直颇有争论且不足以划分其亚种。我们对采自包括印度、越南、缅甸和18个中国地区在内的21个地区共35个恒河猴样品的四个线粒体基因片段（COI，Cofl，COlll和12srRNA）进行了研究。食蟹猴、红面猴、台湾猴和史猴的相应序列作为外群。大约长为100bp的序列用于构建最大似然树和贝叶斯树。与前人结果一致（彭燕章等，1993；蒋学龙等，1995；Groves，2001），西部恒河猴（印度）．相当于M.m.mulatta。根据王应祥（2003）的分类，我们的东部恒河猴可分为四组：M．最早分离出来且一个来自缅甸的恒河猴位于该组基部；M.m.brachyurus和M.m．littoralis组成一个大枝，这个大枝与M.m，lasiotus构成姐妹群。

猕猴结肠镜下活检组织病理学观察

目的通过对猕猴结肠镜检及活检取材，对结肠镜检测方法应用于非人灵长类动物予以评价。方法 实验猴在麻醉状态下接受结肠镜检查和活检标本取材，对活检标本进行固定和病理切片观察，并对所检猴进行解 剖和组织学观察，比较各取材点的情况。结果结肠镜下及大体标本观察见猕猴肠壁厚度明显小于人体，活检取 材部位见黏膜破损、肠出血，个别部位见肠穿孔。病理切片HE 染色观察发现，与人体比较，猕猴大肠腺较小而表 浅，固有层内淋巴小结数量较少，肌层、黏膜下层明显较薄。结论对猕猴行结肠镜检查及镜下活检取材是可行 的，但因猕猴肠壁较人体薄而极易穿孔，故需尽量应用活检杯小的活检钳，并需要充分的肠道准备和有经验的肠镜 操作。

树突状细胞亚群在SIVmac239感染中国恒河猴中数量、表型和功能变化及其机制的研究

本论文主要由3 个相对独立的部分组成：中国恒河猴单核细胞来源的树突 状细胞的表型及功能研究；外周血DC 亚群在SIVmac239 感染的中国恒河猴中 数量及细胞因子的变化以及急性感染期SIVmac239 对中国恒河猴外周血DC 亚 群的凋亡和免疫表型的影响。 非人灵长类动物是人类的近亲，由于在组织结构、免疫、生理和代谢等诸 多方面与人类高度近似，科学界较普遍地利用非人灵长类作为动物模型来进行 艾滋病（AIDS）的发病机制和疫苗研究。中国恒河猴发病缓慢，更适合于HIV 感染的相关研究。在本研究中，我们在体外成功培养了中国恒河猴单核细胞来 源的树突状细胞（monocyte derived dendritic cells，MDDC），并测定其表型和免 疫刺激功能。通过GM-CSF 和IL-4 共同刺激培养单核细胞6 天以后便获得了未 成熟MDDC，随后加入IL-1β、PGE2、LPS 和TNF-α 联合刺激MDDC 成熟。 成熟的MDDC 上调了共刺激分子和CD83 的表达，具有很强的刺激T 淋巴细胞 增殖的能力并分泌大量的IL-12。本研究为后续的DC 疫苗研究奠定了基础。 我们实验室建立了SIVmac239 感染的中国恒河猴动物模型。以该模型为依 托，我们研究了外周血中DC 亚群在急性感染期以及慢性感染期的数量、表型 及功能变化。DC 作为最重要的连接先天免疫与获得性免疫的抗原递呈细胞， 在AIDS 发病进程中扮演着重要的角色。研究发现AIDS 患者血液和淋巴结中 髓样DC（myeloid DC，mDC）和浆细胞样DC（plasmacytoid DC，pDC）会随 着感染的进程而减少，并且伴随着功能损伤。本论文通过研究发现，中国恒河 猴的DC 亚群数量在感染后尽管波动十分剧烈，但并没有显著性地增加或减少， 中文摘要 2 在后期DC 数量能够回升到正常的范围之类，这种回升不同于印度恒河猴，很 可能是中国恒河猴缓慢发病的原因之一。进一步通过研究体外刺激DC 亚群分 泌的细胞因子，我们发现在急性感染期，pDC 分泌的IFN-α 显著提高，并很可 能刺激mDC 成熟并促进了IL-12 的分泌。早期大量细胞因子的分泌有助于控制 病毒复制，但同时也激起了整个免疫系统的活化，促进了疾病进程。而在整个 感染阶段，IFN-α 与CD4+ T 细胞呈正相关，而与病毒载量呈负相关，表明了 IFN-α 对于延缓疾病进程具有重要的意义。 我们测定了急性感染期DC 亚群受病毒影响而发生的表型变化，发现pDC 更容易受到病毒影响而发生凋亡，这可能与pDC 高表达SIV 受体CD4 和CCR5 有关。在感染过程中，尽管mDC 和pDC 都显著地下调了CD4 表达，而上调了 CCR5 的表达，不过仅发现pDC CD4 的表达与病毒载量呈负相关，而CCR5 的 表达与病毒载量呈正相关。在此过程中DC 亚群都会因为病毒的影响而活化， 继而提高CCR7 的表达。同时无论mDC 还是pDC，其表达的CD80 和CD86 都与病毒载量呈正相关。在早期感染中，DC 的活化促使整个免疫系统针对病 毒发挥免疫反应，对于控制疾病发展具有重要意义。

Effects of Aging and Moderate Calorie Restriction on the Reproductive Axis of the Male Rhesus Macaque (Macaca mulatta)

Calorie restriction (CR) has been established as the only non-genetic method of altering longevity and attenuating biological changes associated with aging. This nutritional paradigm has been effective in nematodes, flies, rodents, dogs and possibly non-human primates. Its long history notwithstanding, little is known regarding the exact mechanism(s) of CR action or its potential impact on the hypothalamic-pituitary-gonadal (HPG) axis. The objectives of this project were to: 1) analyze neuroendocrine changes to the HPG axis that occur with aging and 2) evaluate the effects of moderate CR on reproductive function in male rhesus macaques. Pituitary gene expression profiling, semi-quantitative RT-PCR (sqRT-PCR) and immunohistochemistry showed circadian clock mechanism components present in three age categories of macaques, demonstrated age differences in expression for Per2, indicated differential expression of Per2 and Bmal1 at opposing time points and revealed daily rhythmic expression of REV-ERBα protein. These data indicate the ability of the macaque pituitary to express core-clock genes, their protein products, and to do so in a 24-hour rhythm. Young Adult CON and CR pituitary gene expression profiles detected potential differential expression in <150 probesets. A decline in>TSHR and CGA was detected in CR macaques as measured by sqRT-PCR. Other genes investigated showed no diet-induced changes. Young Adult CON and CR testicular gene expression profiles detected potential differential expression in <300 probesets although mRNA expression was not altered based on sqRT-PCR and real-time RT-PCR. Age-related>and/or diet-induced changes in HSD17β3, INSL3, CSNK1E and CGA were observed in a separate experiment with CGA in Old Adult CR subjects returning to youthful levels. Semen samples were collected from Young Adult CON and CR macaques. Normal spermiogram measures, ZP-binding, AR assay and SCSA^® were conducted and indicated no differences between CON and CR-treated animals. Both groups exhibited similar daily testosterone profiles with no differences in mean or maximum levels; however, daily minimum testosterone levels were lower in CON animals. It appears that moderate CR had limited impact on neuroendocrine or reproductive function in male rhesus macaques based on our selected endpoints. Thus, advantageous CR health benefits can be achieved without obvious negative consequences to the HPG axis.

Convergent differential regulation of parvalbumin in the brains of vocal learners.

Spoken language and learned song are complex communication behaviors found in only a few species, including humans and three groups of distantly related birds--songbirds, parrots, and hummingbirds. Despite their large phylogenetic distances, these vocal learners show convergent behaviors and associated brain pathways for vocal communication. However, it is not clear whether this behavioral and anatomical convergence is associated with molecular convergence. Here we used oligo microarrays to screen for genes differentially regulated in brain nuclei necessary for producing learned vocalizations relative to adjacent brain areas that control other behaviors in avian vocal learners versus vocal non-learners. A top candidate gene in our screen was a calcium-binding protein, parvalbumin (PV). In situ hybridization verification revealed that PV was expressed significantly higher throughout the song motor pathway, including brainstem vocal motor neurons relative to the surrounding brain regions of all distantly related avian vocal learners. This differential expression was specific to PV and vocal learners, as it was not found in avian vocal non-learners nor for control genes in learners and non-learners. Similar to the vocal learning birds, higher PV up-regulation was found in the brainstem tongue motor neurons used for speech production in humans relative to a non-human primate, macaques. These results suggest repeated convergent evolution of differential PV up-regulation in the brains of vocal learners separated by more than 65-300 million years from a common ancestor and that the specialized behaviors of learned song and speech may require extra calcium buffering and signaling.

Infection of monkeys by simian-human immunodeficiency viruses with transmitted/founder clade C HIV-1 envelopes.

Simian-human immunodeficiency viruses (SHIVs) that mirror natural transmitted/founder (T/F) viruses in man are needed for evaluation of HIV-1 vaccine candidates in nonhuman primates. Currently available SHIVs contain HIV-1 env genes from chronically-infected individuals and do not reflect the characteristics of biologically relevant HIV-1 strains that mediate human transmission. We chose to develop clade C SHIVs, as clade C is the major infecting subtype of HIV-1 in the world. We constructed 10 clade C SHIVs expressing Env proteins from T/F viruses. Three of these ten clade C SHIVs (SHIV KB9 C3, SHIV KB9 C4 and SHIV KB9 C5) replicated in naïve rhesus monkeys. These three SHIVs are mucosally transmissible and are neutralized by sCD4 and several HIV-1 broadly neutralizing antibodies. However, like natural T/F viruses, they exhibit low Env reactivity and a Tier 2 neutralization sensitivity. Of note, none of the clade C T/F SHIVs elicited detectable autologous neutralizing antibodies in the infected monkeys, even though antibodies that neutralized a heterologous Tier 1 HIV-1 were generated. Challenge with these three new clade C SHIVs will provide biologically relevant tests for vaccine protection in rhesus macaques.

Spatial and temporal scales of neuronal correlation in visual area V4.

The spiking activity of nearby cortical neurons is correlated on both short and long time scales. Understanding this shared variability in firing patterns is critical for appreciating the representation of sensory stimuli in ensembles of neurons, the coincident influences of neurons on common targets, and the functional implications of microcircuitry. Our knowledge about neuronal correlations, however, derives largely from experiments that used different recording methods, analysis techniques, and cortical regions. Here we studied the structure of neuronal correlation in area V4 of alert macaques using recording and analysis procedures designed to match those used previously in primary visual cortex (V1), the major input to V4. We found that the spatial and temporal properties of correlations in V4 were remarkably similar to those of V1, with two notable differences: correlated variability in V4 was approximately one-third the magnitude of that in V1 and synchrony in V4 was less temporally precise than in V1. In both areas, spontaneous activity (measured during fixation while viewing a blank screen) was approximately twice as correlated as visual-evoked activity. The results provide a foundation for understanding how the structure of neuronal correlation differs among brain regions and stages in cortical processing and suggest that it is likely governed by features of neuronal circuits that are shared across the visual cortex.

Development and evaluation of a vaginal ring device for sustained delivery of HIV microbicides to non-human primate

Background There is considerable interest in developing coitally indepen- dent, sustained release formulations for long-term administration of HIV microbicides. Vaginal ring devices are at the forefront of this formulation strategy. Methods Non-medicated silicone elastomer vaginal rings were prepared having a range of appropriate dimensions for testing vaginal ?t in pig- tailed and Chinese rhesus macaques. Cervicovaginal proin?ammatory markers were evaluated. Compression testing was performed to compare the relative ?exibility of various macaque and commercial human rings. Results All rings remained in place during the study period and no tissue irritation or signi?cant induction of cervicovaginal proin?ammatory mark- ers or signs of physical discomfort were observed during the 8-week study period. Conclusions Qualitative evaluation suggests that the 25 · 5-mm ring pro- vided optimal ?t in both macaque species. Based on the results presented here, low-consistency silicone elastomers do not cause irritation in maca-

Non-aqueous silicone elastomer gels as a vaginal microbicide delivery system for the HIV-1 entry inhibitor maraviroc

Aqueous semi-solid polymeric gels, such as those based on hydroxyethylcellulose (HEC) and polyacrylic acid (e.g. Carbopol®), have a long history of use in vaginal drug delivery. However, despite their ubiquity, they often provide sub-optimal clinical performance, due to poor mucosal retention and limited solubility for poorly water-soluble actives. These issues are particularly pertinent for vaginal HIV microbicides, since many lead candidates are poorly water-soluble and where a major goal is the development of a coitally independent, once daily gel product. In this study, we report the use of a non-aqueous silicone elastomer gel for vaginal delivery of the HIV-1 entry inhibitor maraviroc. In vitro rheological, syringeability and retention studies demonstrated enhanced performance for silicone gels compared with a conventional aqueous HEC gel, while testing of the gels in the slug model confirmed a lack of mucosal irritancy. Pharmacokinetic studies following single dose vaginal administration of a maraviroc silicone gel in rhesus macaques showed higher and sustained MVC levels in vaginal fluid, vaginal tissue and plasma compared with a HEC gel containing the same maraviroc loading. The results demonstrate that non-aqueous silicone gels have potential as a formulation platform for coitally independent vaginal HIV microbicides.

Early target cells of measles virus after aerosol infection of non-human primates

Measles virus (MV) is highly infectious, and has long been thought to enter the host by infecting epithelial cells of the respiratory tract. However, epithelial cells do not express signaling lymphocyte activation molecule (CD150), which is the high-affinity cellular receptor for wild-type MV strains. We have generated a new recombinant MV strain expressing enhanced green fluorescent protein (EGFP), based on a wild-type genotype B3 virus isolate from Khartoum, Sudan (KS). Cynomolgus macaques were infected with a high dose of rMV(KS)EGFP by aerosol inhalation to ensure that the virus could reach the full range of potential target cells throughout the entire respiratory tract. Animals were euthanized 2, 3, 4 or 5 days post-infection (d.p.i., n?=?3 per time point) and infected (EGFP(+)) cells were identified at all four time points, albeit at low levels 2 and 3 d.p.i. At these earliest time points, MV-infected cells were exclusively detected in the lungs by fluorescence microscopy, histopathology and/or virus isolation from broncho-alveolar lavage cells. On 2 d.p.i., EGFP(+) cells were phenotypically typed as large mononuclear cells present in the alveolar lumen or lining the alveolar epithelium. One to two days later, larger clusters of MV-infected cells were detected in bronchus-associated lymphoid tissue (BALT) and in the tracheo-bronchial lymph nodes. From 4 d.p.i. onward, MV-infected cells were detected in peripheral blood and various lymphoid tissues. In spite of the possibility for the aerosolized virus to infect cells and lymphoid tissues of the upper respiratory tract, MV-infected cells were not detected in either the tonsils or the adenoids until after onset of viremia. These data strongly suggest that in our model MV entered the host at the alveolar level by infecting macrophages or dendritic cells, which traffic the virus to BALT or regional lymph nodes, resulting in local amplification and subsequent systemic dissemination by viremia.

Pre-clinical development of a combination microbicide vaginal ring containing dapivirine and darunavir

Objectives: Combination microbicide vaginal rings may be more effective than single microbicide rings at reducing/preventing sexual transmission of HIV. Here, we report the preclinical development and macaque pharmacokinetics of matrix-type silicone elastomer vaginal rings containing dapivirine and darunavir.

Methods: Macaque rings containing 25 mg dapivirine, 300 mg darunavir and 100 mg dapivirine, and 300 mg darunavir were manufactured and characterised by differential scanning calorimetry. In vitro release was assessed into isopropanol/water and simulated vaginal fluid. Macaque vaginal fluid and blood serum concentrations for both antiretrovirals were measured during 28-day ring use. Tissue levels were measured on day 28. Ex vivo challenge studies were performed on vaginal fluid samples and IC50 values calculated.

Results: Darunavir caused a concentration-dependent reduction in the dapivirine melting temperature in both solid drug mixes and in the combination ring. In vitro release from rings was dependent on drug loading, the number of drugs present, and the release medium. In macaques, serum concentrations of both microbicides were maintained between 101–102 pg/mL. Vaginal fluid levels ranged between 103–104 ng/g and 104–105 ng/g for dapivirine and darunavir, respectively. Tissue concentrations ranges for each drug were: vagina (1.8×103–3.8×103 ng/g) > cervix (9.4×101–3.9×102 ng/g) > uterus (0–108 ng/g) > rectum (0–40 ng/g). Measured IC50 values were > 2 ng/mL for both compounds.

Conclusions: Based on these results, and in light of recent clinical progress of the 25mg dapivirine ring, a combination vaginal ring containing dapivirine and darunavir is a viable second-generation HIV microbicide candidate.

Development of a Temperature-Recording Vaginal Ring for Monitoring User Adherence

Background: Vaginal ring devices are being actively developed for controlled delivery of HIV microbicides and as multi-purpose prevention technology (MPT) products combining hormonal contraception with prevention of HIV and other sexually transmitted diseases. Presently, there is no reliable method for monitoring user adherence in HIV vaginal ring trials; previous acceptability studies have included some type of participant self-reporting mechanism, which have often been unreliable. More objective, quantitative and accurate methods for assessing adherence are needed.
Methods: A silicone elastomer vaginal ring containing an encapsulated miniature temperature recording device has been developed that can capture and store real-time temperature data during the period of designated use. Devices were tested in both simulated vaginal environments and following vaginal placement in cynomolgus macaques. Various use protocols and data sampling rates were tested to simulate typical patient usage scenarios. Results: The temperature logging devices accurately recorded vaginal temperature in macaques, clearly showing the regular diurnal temperature cycle. When environmental temperature and vaginal temperature was significantly different, the device was able to accurately pinpoint the insertion and removal times. Based on the data collected it was possible to infer removal periods as short as 5 min when the external environmental temperature was 25 °C. Accuracy increased with data sampling rate. Conclusions: This work provides proof-of-concept for monitoring adherence using a vaginal ring device containing an encapsulated temperature logger. The addition of one or more active agents into the ring body is not anticipated to affect the temperature monitoring function. A clinical study to compare self- reported user adherence data with that obtained by the device would be highly informative.

A Combination Vaginal Ring Releasing Dapivirine and Darunavir

Background: Combination microbicide vaginal rings, containing two or more antiretrovirals targeting different steps in the HIV replicative process, may be more effective than single microbicide products at preventing sexual transmission of HIV. Here, we report the preclinical development, including in vitro release and macaque pharmacokinetics, of matrix-type silicone elastomer rings containing dapivirine (DPV; an experimental non-nucleoside reverse transcriptase inhibitor) and darunavir (DRV; a marketed protease inhibitor). Methods: Macaque rings containing 25 mg DPV, 300 mg DRV and 100 mg DPV, and 300 mg DRV were manufactured and characterised by differential scanning calorimetry. In vitro release was assessed into isopropanol/water and simulated vaginal fluid. Macaque vaginal fluid and blood serum concentrations for both antiretrovirals were measured during 28-day ring use. Tissue levels were measured on day 28. Ex vivo challenge studies were performed on vaginal fluid samples and IC50 values calculated.
Results: DRV caused a concentration-dependent reduction in the DPV melting temperature in both solid drug mixes and in the combination ring. In vitro release from rings was dependent on drug loading, the number of drugs present, and the release medium. In macaques, serum concentrations of both
microbicides were maintained between 101-102 pg/mL. Vaginal fluid levels
ranged between 103-104 ng/g and 104-105 ng/g for DPV and DRV, respectively. Vaginal tissue concentrations decreased in rank order: vagina
(1.8×103-3.8×103 ng/g) > cervix (9.4×101-3.9×102 ng/g) > uterus (0-108 ng/g) > rectum (0-40 ng/g). Measured IC50 values (HIV-1 BaL) determined from macaque vaginal fluid samples were < 2 ng/mL for both compounds. Conclusions: Based on these results, and in light of the ongoing clinical progress of the 25mg DPV ring, a combination vaginal ring containing DPV and DRV is a viable second-generation HIV microbicide candidate.