Finally, our study underlines the dual role of IL-2 in the context of spontaneous and Fas-induced apoptosis. Several groups have already investigated the effect of IL-2 immunotherapy on apoptosis but results were quite discordant. Indeed, it was shown that, in HIV-infected patients, IL-2 treatment was associated with increased apoptosis rates . On the opposite, some studies showed that IL-2 could reduce lymphocyte apoptosis in HIV infected patients . Here, we report that IL-2 can effectively play both role depending on the dose used, high dose having a proapoptotic effect on both CD4 + and CD8 + T cells and low dose being antiapoptotic in CD4 + T cells only. This modulating effect on cell death seems to be restricted to CD4 + central memory T cells while it exerts a global effect on each CD8 + T cell sub- sets. Excessive apoptosis observed in high dose treated animals could be due to higher expression of Fas recep- tor on CM CD4 + T lymphocytes cell surface, indeed we observed that Fas expression on CD4 + T lymphocytes was increased after rIL-2 treatment (data not shown). We and others have reported that T cells from HIV- infected individuals or SIV-infected macaques are highly prone to undergo apoptosis after Fas-ligation [6-8,27]. A recent study showed that Rhesusmacaques treated with neutralizing anti-FasL antibody preserved higher levels of central memory CD4 + T cells  thereby supporting a critical role for Fas in the CD4 + T cell compartment. Herein, we found that in vivo IL-2 treatment has a detri- mental effect on the susceptibility of cells to undergo apoptosis after Fas ligation. However, we cannot exclude a role for other death molecules like PD-1 or TRAIL that have been proposed to participate in the death of T cells during HIV/SIV infection [46,53-56]. Further inves- tigation of these cell death pathways among various lymphoid populations should provide new insights about immunological mechanisms implicated in IL-2 immunotherapy.
Animals were housed in social cages with their mothers under controlled conditions of humidity, temperature, and light (12-h light/12-h dark cycle, lights on at 8.00 am); food and water were available ad libitum. Experiments were carried out in accordance with European Communities Council Directive of 3 June 2010 (2010/6106/EU) for care of laboratory animals in an AAALAC-accredited facility following acceptance of study design by the Institute of Lab Animal Science (Chinese Academy of Science, Beijing, China) IACUC. One day-old male rhesusmacaques received intravenous injections of an AAV9 vector that expresses green fluorescent protein (GFP) under the control of cytomegalovirus immediate early synapsin intron promoter (AAV9- CMVie-GFP). Four newborn male monkeys (C1 to C4) were injected with 1 ml with 4 different AAV9 viral titers (C1: 4.00310 14 vg/ml [1.33310 12 particles/g of body
depression of the cardiovascular and respiratory systems. For some procedures, a greater degree of analgesia and muscle relaxation would be advantageous. Although this can be achieved by the addition of medetomidine, as in other spe- cies, recovery is still relatively prolonged even after reversal of the medetomidine with atipamezole . Replacement of ketamine with agents that have less prolonged depressant ef- fects, or are reversible with specific antagonists could there- fore be advantageous. In human medicine, the combination of fentanyl and midazolam has been widely used for con- scious sedation for minor procedures, and at higher dose rates for surgery [12–15]. Combinations of opioids and ben- zodiazepines seem to be less effective in Rhesusmacaques , however in other species, addition of medetomidine to these combinations produces fully reversible anaesthe- sia [17–19] and an initial report suggests this combination can be used successfully in non-human primates .
Animals and materials. Rhesusmacaques were trained to sit
in a primate chair in front of a tangent touch-screen (Microtouch System, Methuen, USA) coupled to a display monitor located at arm’s reach. A computer controlled the presentation on the monitor of visual stimuli, which served as light targets, and recorded the position and correctness of each touch (CORTEX software, NIMH Laboratory of Neuropsychology, Bethesda, Maryland). Eye movements were recorded using the scleral search coil technique. The position of gaze was controlled by a moving eye-position window (12° ´ 12°) centered on the fixation point (FP) or on the different targets. The size of the moving eye fixation window took into account the size of the targets (6° ´ 6°) and discrepancies between actual direction of gaze (on the targets) and its measurement by the search-coil technique. A recording cylinder was implanted over the anterior cingulate cortex. Previously described surgical procedures and electrophysiological techniques 21 were carried out according to the 1986 European Communities Council Directive (Ministère de l’Agriculture et de la Forêt, Commission Nationale de l’Expérimentation Animale). Behavioral protocol. The task
Materials and Methods
Monkeys. Five adult female rhesusmacaques ( Macaca mulatta) were housed at the California
National Primate Research Center (CNPRC). Animals were caged individually with a 0600– 1800 hours light cycle and at a temperature maintained between 25–27˚C. Cage size depends on animal weight. Animals weighing less than 10kg were in a 4.3 square foot cage measuring 24 (L) x 27 (D) x 32 (H). Animals that were between 10kg and 15 kg were housed in a 6.4 square foot cage measuring 34 x 27 x 32. No animals exceeded 15 kg. Animals were fed a diet of Purina Monkey Chow and water ad libitum. Seasonal produce, seeds, and cereal were offered as sup- plements for environmental enrichment. Cages were made of stainless steel, and water was delivered to each cage by rigid PVC pipes and a “lixit” device. A wide array of enrichment is available at CNPRC including manipulation, food and visual/auditory activities. All food items were fresh and cage enrichment devices were determined to not be made of BPA-containing plastic to minimize any possible BPA exposure other than treatments. Only females with a his- tory of normal menstrual cycles were selected for this study. Females ranged in age from 6 to 13 years. Four females were time-mated according to standard CNPRC procedures. Pregnancy was detected by ultrasound and an estimated day of conception (day 0) was assigned at the time of pregnancy detection based on the timed mating schedule [ 29 ]. At approximately 40 days gestation the sex of the conceptus was determined using established protocols and those dams with female fetuses were selected for the study (N = 4). Animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Cali- fornia, Davis prior to study initiation; all studies were conducted in accordance with the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals.
term reconstitution with lentiviral vector-transduced cells of different lineages, its proportion remained below 1%. Hanawa et al., provided the first evidence that SIV-based vectors can successfully transduce rhesus macaque repop- ulating hematopoietic stem cells, with an average of 16% of peripheral blood leukocytes containing the SIV vector genome. However, this study was carried out with HSC from mobilized peripheral blood cells, making it possible to obtain larger numbers of HSC than can be harvested from bone marrow. Nevertheless theoretically, these cells contained more progenitors that were already committed and fewer pluripotent stem cells capable of long-term reconstitution than medullary HSC. The small num- bers of eGFP-producing cells observed in our study may be due to an anti-eGFP immune response. Some reports have suggested that such reactions do not generally occur after irradiation, but two reports described the induc- tion of cytotoxic T-lymphocyte responses to enhanced green (GFP) or yellow (YFP) fluorescent proteins after myeloablative conditioning. One of these reports con- cerned baboons that had received primitive hematopoi- etic cells transduced with HIV-1-based lentiviral vectors and the other concerned rhesusmacaques that had received CD34 + stem cells transduced with a retroviral
Macaques (Macaca mulatta and M. fascicularis), origi- nally received from three US-based vendors, received physical examinations and routine diagnostic evaluations during quarantine and at quarterly intervals in 2012, 2014 and 2016. The colony contained 84 animals in 2012 (4 cynomolgus macaques and 80 rhesusmacaques, of which 23 were female), 85 animals in 2014 (4 cynomol- gus macaques and 81 rhesusmacaques, of which 24 were female) and 97 animals in 2016 (2 cynomolgus macaques and 95 rhesusmacaques, of which 25 were female). They ranged in age from 4 to 20 years. Animals were routinely pair-housed and maintained in an animal facility accred- ited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International. They were fed specified amounts of primate chow (Purina Lab Diet 5038) twice a day and supplemented daily with treats, seasonal fruits and vegetables. Water was provided ad libitum when animals were not on studies requiring water regulation. Housing conditions were maintained at 20–22 °C, 30–70 % humidity, 10–15 non-recirculated air changes per hour and a light cycle of 12 h light:12 h dark. Microbiological analysis
32 transporter) [156,157]. Consequently, testis must be taken into consideration for effective HIV-1 therapies.
Several evidences indicate that viral strains and infected cells present in semen do not originate solely from blood but would originate at least in part within the MGT. During the acute phase of infection, viral RNA (corresponding to free virus) and viral DNA (corresponding to infected leucocytes) in semen have sequences extremely similar to those from virus present in the blood (review in ). However, during the chronic phase genetic differences between HIV strains in the blood and in the sperm emerge and free viral particles present in the semen constitute a population distinct from that found in the blood [134,158]. This indicates the existence of an independent local viral replication, as well as a restricted exchange of virions and infected cells between the two compartments, allowing the parallel evolution of various virus populations within the body. In addition, the sequences of DNA and viral RNA in infected sperm cells may differ from those of virions, suggesting a different origin of virions and infected cells (review in ). Whittney et al. (2011) reported that in SIV infected rhesusmacaques the viruses in blood and semen were similar during early infection then were distinguished after the peak of viremia, indicating that anatomic compartmentalization occurred at early time point . Anderson et al., proposes a number of non-exclusive and variable mechanisms allowing sperm contamination by HIV over time, namely: (i) direct importation of virus from blood; (ii) clonal amplification of viral blood strains in infected cells infiltrating the male genital tract; (iii) local replication in cells resident in the MGT leading to a distinct viral evolution .
challenged with a DENV1 variant. We hypothesized that the monkey would be positive for anti-DENV1 NS1 and potentially anti-DENV2-4 NS1 antibodies, as anti-DENV antibodies have been shown to cross-react with other DENV serotypes. 8 Briefly, 96-well plates were pre-coated with 100ng/mL recombinant NS1 from DENV serotypes 1-4. The samples were then incubated in the wells for one h at 37°C, washed, and then incubated with 100ng/mL biotinylated goat anti-Rhesus IgG (H+L) (SouthernBiotech, Birmingham, AL) for one h at 37°C. The plate was then washed and read with a standard Horse radish peroxidase (HRP) - conjugated streptavidin protocol. The positive control was serum from previously confirmed DENV-1 infected Rhesusmacaques (provided by Dr. I. Bosch), and the negative controls were age- and sex-matched macaques from the subject’s colony. This ELISA confirmed the presence of anti-DENV1 and DENV4 NS1 antibodies (Figure 6B). Although anti-DENV1 NS1 antibodies were present during both normal and clinical episodes, anti-DENV4 NS1 antibodies appeared to be higher during episodes. This could suggest that the anti-DENV4 NS1 antibody was related to the clinical bleeding episodes, with the antibody being cross- reactive to a native antigen, such as platelet surface antigens.
Au sein de leur troupe, les macaques respectent une hiérarchie stricte reposant sur des rapports de force et des relations de « dominance / subordination ». De plus, les liens de parenté sont très importants puisqu’un jeune né d’une mère dominante sera lui-même dominant, et donc prioritaire en ce qui concerne les aliments et la boisson. On observe ainsi, au sein d’une même tribu, plusieurs clans qui se forment autour des femelles reproductrices de grade différent. Par ailleurs, des études ont montré qu’une femelle qui met bas, atteint un rang de plus haut grade et accède donc à de nouveaux privilèges. Cela lui permet de répondre à ses besoins nutritionnels devenus plus importants en période de lactation (Weisbard et Goy, 1975). Les jeunes sont respectés et protégés par tout le groupe. Les mâles adultes, par exemple, ne s’approchent pas d’un jeune qui veut venir boire. Dans le cas contraire, les femelles du groupe, solidaires, peuvent attaquer le mâle et protéger le jeune.
DisRev. Unlike in many natural environments, in DisRev, animals must learn to assign more prominence to specific choice–reward associations at the expense of the general reward history.
The co-recruitment of plOFC/AI and another region near to, but outside, central OFC, lOFC/12o, may reflect direct connections between the areas [ 69 , 70 ]. In rat, pharmacological lesions of the lateral orbital and AI are associated with reversal learning deficits [ 20 ]. Future studies will aim at disentangling the specific roles of the two regions in primates—plOFC/AI and lOFC/12o—closest to central OFC in promoting behavioral flexibility. It is also possible to link the present results with fMRI activity recorded in a nearby region when monkeys learn to use specific choice–reward outcomes to guide decision-making [ 9 ] and reversal learning or task switching in humans [ 71 , 72 ], reinforcing the idea of a similar structural–functional orga- nization of the OFC and its subdivisions in macaques and humans [ 73 ]. Lesions that include this OFC region disrupt the process of reward credit assignment to specific choices [ 14 ], whereas AI reversible lesions affect retrieval of goal value to guide decisions [ 21 ].
ad libitum except when water regulation is required under approved protocols. Environmental
enrichment includes positive interaction with care staff, treats, food puzzles, toys, mirrors, and videos. Macaques are pair-housed, except when precluded by established behavioral incompat- ibility or for medical reasons, as determined by a veterinarian. The macaques sampled in this study were under investigation by four different cognitive neuroscience research laboratories (A-D; Table 1 ). For this study, 44 cephalic recording chambers with craniotomies, and 1 with- out a craniotomy, were sampled. The majority of macaques (11/25) had one cephalic recording chamber; 8/25 had two, and the remaining 6 macaques had three cephalic recording chambers ( Table 1 ). Most (26) of the recording chambers had been in place between one and three years, 12 had been implanted between three and five years, and 7 had been in place less than one year. To minimize discomfort and stress, sampling of cephalic chambers was performed under ketamine anesthesia (10mg/kg, intramuscular injection) during routine semi-annual physical examination. All animals remained on IACUC-approved protocols for cognitive neuroscience at the conclusion of sampling and no animals were euthanized for reasons related to this study.
CE: European Conformity; CHEERS: Consolidated Health Economic Evaluation Reporting Standards; CNGOF: French college of gynaecology and obstetrics; CNRHP: Centre National de Référence en Hémobiologie Périnatale; DNA: Deoxyribonucleic acid; EDTA: Ethylene diamine tetraacetic acid; ENC: French National Cost Study; FMH: Feto-maternal haemorrhages; IAT: Indirect Antiglobulin Test; ICER: Incremental cost-effectiveness ratio; IgRh: Anti-D specific immunoglobulin; INSERM: French National Institute of Health and Medical Research; INTS: National Institute of Blood Transfusion; RHD: Rhesus D; SD: Standard Deviation
Cellular immune response
The anti-GFP immune cellular response analysis was performed us- ing an ELISpot assay to measure IFN-g and IL-2 secretion in PBMCs isolated prior to dose and at necropsy and, in total splenocytes and CD4- or CD8-depleted splenocytes collected at day 60 post-injection. CD4+ and CD8+ T cells were depleted by positive selection using Spe- cial StemSep Rhesus CD4+ or CD8+ Tetramer kits (STEMCELL Technologies). Cell depletion efﬁciency was determined by ﬂow cy- tometry before (total splenocytes) and after magnetic separation (CD4- and CD8-depleted splenocytes). Brieﬂy, CD4+ T cell and CD8+ T cell populations were analyzed using CD3 (557749; BD Bio- sciences)/CD4 (556616; BD Biosciences) and CD3/CD8 (301035; Bio- Legend) markers, respectively. Extracellular staining consisted of cell incubation on ice for 30 min. Cells were then washed twice with 1 PBS and re-suspended in 1 PBS supplemented with 0.5% fetal bovine serum (FBS) and 2 mM EDTA. Cells were acquired using a BD FACS LSRII ﬂow cytometer (BD Biosciences) and analyzed with FlowJo software (v.10; BD Biosciences). IFN-g (MABTech) and IL-2 (Ucytech) ELISpot assays were performed according to the manufacturer’s recommendations (Monkey IFN-g ELISpot PLUS
However, a gene therapy strategy for HIV infection would only be possible during the chronic phase of infection. At this stage, the immune system, and particularly CD4 + T
cells – the major target of our gene therapy approach – may be strongly affected by the virus. We therefore inves- tigated the safety and efficacy of this strategy in macaques chronically infected with a primary, pathogenic isolate of SIVmac251, but still in an asymptomatic state. The effi- cacy of our strategy has been examined according to two parameters. The eventual survival advantage of IFN- β transduced cells has been monitored by following the presence of such transduced cells in the blood stream as well as in the lymph nodes of infused macaques. This group of animals was compared to a controlgroup having received cells transduced with a retrovirus carrying a mod- ified version of the MaIFN sequence with a deletion blocking mRNA translation. Animals were subjected to three infusions of autologous T lymphocytes transduced ex vivo with both constructs. The eventual clinical benefits of the presence of IFN- β-transduced cells have been mon- itored for two years, by examining, in both groups of ani- mals, the absolute number of circulating CD4+ lymphocytes, cell associated viral load and plasma vial load.
of a chimeric antigen comprising gp120 linked to CD4 sequences did not elicit detectable autoantibody reactivity with cell surface CD4, despite a robust response to the antigen 56 .
Ethics statement. Adult cynomolgus macaques (Macaca fascicularis) were imported from Mauritius and housed in the facilities of the Commissariat à l’Energie Atomique et aux Energies Alternatives (CEA). Non- human primates (NHP) are used at the CEA in accordance with French national regulations and under national veterinary inspectors (CEA Permit Number B 92-032-02). The CEA complies with the Public Health Service Policy on Humane Care and Use of Laboratory Animals of the Office for Laboratory Animal Welfare (OLAW, USA) under OLAW Assurance number #A5826-01. All experimental procedures were conducted according to the newly published European directive (2010/63, recommendation N°9). The protocols employed were approved under statement number 14-060 by the ethics committee “Comité d’Ethique en Expérimentation Animale du CEA” registered with the French Ministry of Research under N°44. The animals were used under the supervision of the veterinarians in charge of the animal facility. Handling procedures were conducted after animal sedation with ketamine chlorhydrate (Rhone-Merieux, Lyon, France; 10 mg/kg body weight).
Key-words: Macaca fascicularis, density, overcrowding, aggression, coping.
A Bali, Indonésie, les groupes de Macaca fascicularis vivent parfois en situation de forte densité ou de surpopulation. De précédentes recherches ont proposé trois modèles afin d’expliquer comment les macaques s’adaptent à des conditions de fortes densités. Nous avons testé la validité de ces modèles pour des M. fascicularis en liberté, considérés comme moins despotiques que M. mulatta sur lesquels les modèles ont été testés à l’origine en comparant des populations captives à une population sauvage. Permettant une plus grande validité écologique pour nos conclusions, les macaques sauvages de cette étude ont eu suffisamment de temps pour établir des stratégies sociales d’ajustement efficaces et stables. Les sites d’études Ubud et Uluwatu sont respectivement constitués de six et cinq groupes de M. fascicularis. Nous avons collecté les données démographiques en utilisant une méthode de comptage par procession, ainsi que les données comportementales en utilisant des méthodes d’échantillonnage par focal et toute-occurrence. Nous avons évalué la taille des domaines vitaux par la localisation GPS journalière des groupes. Ubud est une zone en surpopulation alors qu’Uluwatu ne l’est pas mais il n’y a que peu de chevauchement entre les groupes en comparaison à Uluwatu. Bien qu’il n’y ait pas de différences en agression entre les deux populations, les temps d’agressivité et de soumission augmentent alors que le temps d’affiliation diminue quand la densité augmente. Selon le budget d’activité, bien que les temps de contacts affiliatifs soient plus courts, les comportements affiliatifs à distance sont plus longs. Les femelles passent plus de temps en agression que les mâles mais, bien qu’elles augmentent davantage le temps de soumission et diminuent le temps d’affiliation, elles augmentent moins le temps d’agressivité que les mâles ne le font quand la densité augmente. Un plateau dans l’agressivité apparait quand la densité augmente. Les macaques semblent devenir structurés plus hiérarchiquement que l’espèce ne l’est déjà. Cependant, certaines évidences semblent indiquer qu’ils peuvent être aussi moins despotiques, supportant le modèle d’ajustement testé originellement sur M. mulatta. Macaca fascicularis pourrait être susceptible de combiner deux stratégies d'ajustement des comportements pour s’adapter à de fortes densités.