Elevated level of metabotropic glutamate receptor 2/3 in the prefrontal cortex in major depression

Clinical, postmortem and preclinical research strongly implicates dysregulation of glutamatergic neurotransmission in major depressive disorder (MDD). Recently, metabotropic glutamate receptors (mGluRs) have been proposed as attractive targets for the discovery of novel therapeutic approaches against depression. The aim of this study was to examine mGluR2/3 protein levels in the prefrontal cortex (PFC) from depressed subjects. In addition, to test whether antidepressants influence mGluR2/3 expression we also studied levels of mGluR2/3 in fluoxetine-treated monkeys. Postmortem human prefrontal samples containing Brodmann's area 10 (BA10) were obtained from 11 depressed and 11 psychiatrically healthy controls. Male rhesus monkeys were treated chronically with fluoxetine (dose escalated to 3mg/kg, p.o.; n=7) or placebo (n=6) for 39 weeks. The mGluR2/3 immunoreactivity was investigated using Western blot method. There was a robust (+67%) increase in the expression of the mGlu2/3 protein in the PFC of depressed subjects relative to healthy controls. The expression of mGlu2/3 was unchanged in the PFC of monkeys treated with fluoxetine. Our findings provide the first evidence that mGluR2/3 is elevated in the PFC in MDD. This observation is consistent with reports showing that mGluR2/3 antagonists exhibit antidepressant-like activity in animal models and demonstrates that these receptors are promising targets for the discovery of novel antidepressants.

Metabolomics reveals attenuation of the SLC6A20 kidney transporter in nonhuman primate and mouse models of type 2 diabetes mellitus

To enhance understanding of the metabolic indicators of type 2 diabetes mellitus (T2DM) disease pathogenesis and progression, the urinary metabolomes of well characterized rhesus macaques (normal or spontaneously and naturally diabetic) were examined. High-resolution ultra-performance liquid chromatography coupled with the accurate mass determination of time-of-flight mass spectrometry was used to analyze spot urine samples from normal (n = 10) and T2DM (n = 11) male monkeys. The machine-learning algorithm random forests classified urine samples as either from normal or T2DM monkeys. The metabolites important for developing the classifier were further examined for their biological significance. Random forests models had a misclassification error of less than 5%. Metabolites were identified based on accurate masses (<10 ppm) and confirmed by tandem mass spectrometry of authentic compounds. Urinary compounds significantly increased (p < 0.05) in the T2DM when compared with the normal group included glycine betaine (9-fold), citric acid (2.8-fold), kynurenic acid (1.8-fold), glucose (68-fold), and pipecolic acid (6.5-fold). When compared with the conventional definition of T2DM, the metabolites were also useful in defining the T2DM condition, and the urinary elevations in glycine betaine and pipecolic acid (as well as proline) indicated defective re-absorption in the kidney proximal tubules by SLC6A20, a Na(+)-dependent transporter. The mRNA levels of SLC6A20 were significantly reduced in the kidneys of monkeys with T2DM. These observations were validated in the db/db mouse model of T2DM. This study provides convincing evidence of the power of metabolomics for identifying functional changes at many levels in the omics pipeline.

Comparative transcriptomics of extreme phenotypes of human HIV-1 infection and SIV infection in sooty mangabey and rhesus macaque

High levels of HIV-1 replication during the chronic phase of infection usually correlate with rapid progression to severe immunodeficiency. However, a minority of highly viremic individuals remains asymptomatic and maintains high CD4⁺ T cell counts. This tolerant profile is poorly understood and reminiscent of the widely studied nonprogressive disease model of SIV infection in natural hosts. Here, we identify transcriptome differences between rapid progressors (RPs) and viremic nonprogressors (VNPs) and highlight several genes relevant for the understanding of HIV-1-induced immunosuppression. RPs were characterized by a specific transcriptome profile of CD4⁺ and CD8⁺ T cells similar to that observed in pathogenic SIV-infected rhesus macaques. In contrast, VNPs exhibited lower expression of interferon-stimulated genes and shared a common gene regulation profile with nonpathogenic SIV-infected sooty mangabeys. A short list of genes associated with VNP, including CASP1, CD38, LAG3, TNFSF13B, SOCS1, and EEF1D, showed significant correlation with time to disease progression when evaluated in an independent set of CD4⁺ T cell expression data. This work characterizes 2 minimally studied clinical patterns of progression to AIDS, whose analysis may inform our understanding of HIV pathogenesis.

Squirrel Monkeys (Saimiri sciureus) Infected with the Agent of Bovine Spongiform Encephalopathy Develop Tau Pathology

Squirrel monkeys (Saimiri sciureus) were infected experimentally with the agent of classical bovine spongiform encephalopathy (BSE). Two to four years later, six of the monkeys developed alterations in interactive behaviour and cognition and other neurological signs typical of transmissible spongiform encephalopathy (TSE). At necropsy examination, the brains from all of the monkeys showed pathological changes similar to those described in variant Creutzfeldt-Jakob disease (vCJD) of man, except that the squirrel monkey brains contained no PrP-amyloid plaques typical of that disease. Constant neuropathological features included spongiform degeneration, gliosis, deposition of abnormal prion protein (PrP(TSE)) and many deposits of abnormally phosphorylated tau protein (p-Tau) in several areas of the cerebrum and cerebellum. Western blots showed large amounts of proteinase K-resistant prion protein in the central nervous system. The striking absence of PrP plaques (prominent in brains of cynomolgus macaques [Macaca fascicularis] with experimentally-induced BSE and vCJD and in human patients with vCJD) reinforces the conclusion that the host plays a major role in determining the neuropathology of TSEs. Results of this study suggest that p-Tau, found in the brains of all BSE-infected monkeys, might play a role in the pathogenesis of TSEs. Whether p-Tau contributes to development of disease or appears as a secondary change late in the course of illness remains to be determined.

Effect of mechanical and antiseptic therapy on peri-implant mucositis: an experimental study in monkeys

OBJECTIVES: This experiment was performed to evaluate clinically and histologically the effect of mechanical therapy with or without antiseptic therapy on peri-implant mucositis lesions in nine cynomolgus monkeys. MATERIAL AND METHODS: Two ITI titanium implants were inserted into each side of the mandibles. After 90 days of plaque control and soft tissue healing, a baseline clinical examination was completed. Peri-implant lesions were induced by placing silk ligatures and allowing plaque to accumulate for 6 weeks. The clinical examination was then repeated, and the monkeys were randomly assigned to three treatment groups: group A, mechanical cleansing only; group B, mechanical cleansing and local irrigation with 0.12% chlorhexidine (CHX) and application of 0.2% CHX gel; and group C, control, no treatment. The implants in treatment groups A and B were treated and maintained according to the assigned treatment for two additional months. At the end of the maintenance period, a final clinical examination was performed and the animals were sacrificed for biopsies. RESULTS: The mean probing depths (PD) values at mucositis were: 3.5, 3.7, and 3.4 mm, and clinical attachment level (CAL) = 3.8, 4.1, and 3.9 mm for treatment groups A, B and C, respectively. The corresponding values after treatment were: PD = 1.7, 2.1, and 2.5 mm, and CAL=2.6, 2.6, and 3.1 mm. ANOVA of mean changes (Delta) in PD and CAL after treatment showed no statistical difference between the treatment groups. Comparison of the mean changes in PD and CAL after treatment yielded statistical differences between the control and treatment groups P < 0.01. According to the t-test, no statistical difference was found between treatment groups A and B for the PD reduction but there was a significant difference for the CAL change, P < 0.03. Group A had significantly more recession and less CAL gain than group B. Non-parametric tests yielded no significant differences in modified plaque index (mPlI) and gingival index (GI) after treatment between both treatment groups. Frequencies and percent distributions of the mPlI and GI scores changed considerably for both treatment groups when compared with the changes in the control group after treatment. With regard to the histological evaluation, no statistical differences existed between the treatments for any linear measurement. The proportion of inflammation found in the mucosal tissues of the control implants was greater than the one found for both treatment groups, P < 0.01. More importantly, both treatment groups showed a similar low proportion of inflammation after 2 months of treatment. CONCLUSIONS: Within the limitations of this experiment, and considering the supportive plaque control rendered, it can be concluded that for pockets of 3-4 mm: (1) mechanical therapy alone or combined with CHX results in the clinical resolution of peri-implant mucositis lesions, (2) histologically, both treatments result in minimal inflammation compatible with health, and (3) the mechanical effect alone is sufficient to achieve clinical and histologic resolution of mucositis lesions.

Rapid diagnosis and quantification of Francisella tularensis in organs of naturally infected common squirrel monkeys (Saimiri sciureus)

Francisella tularensis, a small Gram-negative facultative intracellular bacterium, is the causative agent of tularaemia, a severe zoonotic disease transmitted to humans mostly by vectors such as ticks, flies and mosquitoes. The disease is endemic in many parts of the northern hemisphere. Among animals, the most affected species belong to rodents and lagomorphs, in particular hares. However, in the recent years, many cases of tularaemia among small monkeys in zoos were reported. We have developed a real-time PCR that allows to quantify F. tularensis in tissue samples. Using this method, we identified the spleen and the kidney as the most heavily infected organ containing up to 400 F. tularensis bacteria per simian host cell in two common squirrel monkeys (Saimiri sciureus) from a zoo that died of tularaemia. In other organs such as the brain, F. tularensis was detected at much lower titres. The strain that caused the infection was identified as F. tularensis subsp. holarctica biovar I, which is susceptible to erythromycin. The high number of F. tularensis present in soft organs such as spleen, liver and kidney represents a high risk for persons handling such carcasses and explains the transmission of the disease to a pathologist during post-mortem analysis. Herein, we show that real-time PCR allows a reliable and rapid diagnosis of F. tularensis directly from tissue samples of infected animals, which is crucial in order to attempt accurate prophylactic measures, especially in cases where humans or other animals have been exposed to this highly contagious pathogen.

Rhesus factors and ammonium: a function in efflux?

Completion of fungal, plant and human genomes paved the way to the identification of erythrocytic rhesus proteins and their kidney homologs as ammonium transporters. Ammonium is the preferred nitrogen source of bacteria and fungi, and plants acquire nitrogen from the soil in the form of ammonium [1]. In animals and humans, assimilated forms of nitrogen - amino acids - are much preferred for nutrition, and, in the case of ammonotelic animals, ammonium is used for the excretion of nitrogen instead. In the human kidney, ammonium is produced, reabsorbed and excreted as a means to maintain pH balance and to get rid of surplus inorganic nitrogen. Whether ammonium transport also has a role in the pH regulation of other organs is not known and the molecular mechanisms were not, up to now, understood.

Retrospective comparison of maternal vs. HPA-matched donor platelets for treatment of fetal alloimmune thrombocytopenia

In fetal alloimmune thrombocytopenia (FAIT), transplacental maternal antibodies cause destruction of fetal platelets. FAIT is similar to fetal Rhesus haemolytic disease, but half of the affected fetuses are born to primiparous women. In 10-20% of cases, prenatal and perinatal intracranial haemorrhages are reported. Different therapeutic approaches have been described, including maternally administered high-dose intravenous immunoglobulin (high dose IVIG) without or with steroids or intrauterine transfusion (IUT) of compatible platelets. For the latter, the use of plasma-free maternal and donor platelets has been described, but a comparison of these two sources of platelets has not been reported.

Spatio-temporal credit assignment in population learning

Learning by reinforcement is important in shaping animal behavior, and in particular in behavioral decision making. Such decision making is likely to involve the integration of many synaptic events in space and time. However, using a single reinforcement signal to modulate synaptic plasticity, as suggested in classical reinforcement learning algorithms, a twofold problem arises. Different synapses will have contributed differently to the behavioral decision, and even for one and the same synapse, releases at different times may have had different effects. Here we present a plasticity rule which solves this spatio-temporal credit assignment problem in a population of spiking neurons. The learning rule is spike-time dependent and maximizes the expected reward by following its stochastic gradient. Synaptic plasticity is modulated not only by the reward, but also by a population feedback signal. While this additional signal solves the spatial component of the problem, the temporal one is solved by means of synaptic eligibility traces. In contrast to temporal difference (TD) based approaches to reinforcement learning, our rule is explicit with regard to the assumed biophysical mechanisms. Neurotransmitter concentrations determine plasticity and learning occurs fully online. Further, it works even if the task to be learned is non-Markovian, i.e. when reinforcement is not determined by the current state of the system but may also depend on past events. The performance of the model is assessed by studying three non-Markovian tasks. In the first task, the reward is delayed beyond the last action with non-related stimuli and actions appearing in between. The second task involves an action sequence which is itself extended in time and reward is only delivered at the last action, as it is the case in any type of board-game. The third task is the inspection game that has been studied in neuroeconomics, where an inspector tries to prevent a worker from shirking. Applying our algorithm to this game yields a learning behavior which is consistent with behavioral data from humans and monkeys, revealing themselves properties of a mixed Nash equilibrium. The examples show that our neuronal implementation of reward based learning copes with delayed and stochastic reward delivery, and also with the learning of mixed strategies in two-opponent games.

In vivo evaluation in cynomolgus monkey brain and metabolism of [¹⁸F]fluorodeprenyl: a new MAO-B pet radioligand

In this study, we evaluated the in vivo characteristics of a new monoamine oxidase type B (MAO-B) radioligand, [¹⁸F]fluorodeprenyl, by positron emission tomography (PET) in two cynomolgus monkeys. The brain uptake of [¹⁸F]fluorodeprenyl was more than 7% (600% SUV) of the total injected radioactivity and similar to that of [¹¹C]deprenyl, an established MAO-B radioligand. The highest uptake was observed in the striatum, one of the MAO-B-rich regions, with a peak at approximately 2-3 min after injection, followed by lower uptake in the thalamus and the cortex and lowest uptake in the cerebellum. Brain uptake of [¹⁸F]fluorodeprenyl was largely inhibited by preadministration of the MAO-B inhibitor, L-deprenyl, whereas clorgyline, a MAO Type A blocker, had no significant inhibitory effect, thus demonstrating selectivity for MAO-B. [¹⁸F]Fluorodeprenyl showed relatively slow metabolism with the presence of two radiometabolite peaks with similar retention time as the labeled metabolites of [¹¹C]deprenyl. These results suggest that [¹⁸F]fluorodeprenyl is a potential PET radioligand for visualization of MAO-B activity.

Life stage differences in mammary gland gene expression profile in non-human primates

Breast cancer (BC) is the most common malignancy of women in the developed world. To better understand its pathogenesis, knowledge of normal breast development is crucial, as BC is the result of disregulation of physiologic processes. The aim of this study was to investigate the impact of reproductive life stages on the transcriptional profile of the mammary gland in a primate model. Comparative transcriptomic analyses were carried out using breast tissues from 28 female cynomolgus macaques (Macaca fascicularis) at the following life stages: prepubertal (n = 5), adolescent (n = 4), adult luteal (n = 5), pregnant (n = 6), lactating (n = 3), and postmenopausal (n = 5). Mammary gland RNA was hybridized to Affymetrix GeneChip(®) Rhesus Macaque Genome Arrays. Differential gene expression was analyzed using ANOVA and cluster analysis. Hierarchical cluster analysis revealed distinct separation of life stage groups. More than 2,225 differentially expressed mRNAs were identified. Gene families or pathways that changed across life stages included those related to estrogen and androgen (ESR1, PGR, TFF1, GREB1, AR, 17HSDB2, 17HSDB7, STS, HSD11B1, AKR1C4), prolactin (PRLR, ELF5, STAT5, CSN1S1), insulin-like growth factor signaling (IGF1, IGFBP1, IGFBP5), extracellular matrix (POSTN, TGFB1, COL5A2, COL12A1, FOXC1, LAMC1, PDGFRA, TGFB2), and differentiation (CD24, CD29, CD44, CD61, ALDH1, BRCA1, FOXA1, POSTN, DICER1, LIG4, KLF4, NOTCH2, RIF1, BMPR1A, TGFB2). Pregnancy and lactation displayed distinct patterns of gene expression. ESR1 and IGF1 were significantly higher in the adolescent compared to the adult animals, whereas differentiation pathways were overrepresented in adult animals and pregnancy-associated life stages. Few individual genes were distinctly different in postmenopausal animals. Our data demonstrate characteristic patterns of gene expression during breast development. Several of the pathways activated during pubertal development have been implicated in cancer development and metastasis, supporting the idea that other developmental markers may have application as biomarkers for BC.

Wound healing following regenerative procedures in furcation degree III defects: histomorphometric outcomes

Degree III furcation involvements were surgically created at four first molars in each of three monkeys. Following 6 weeks of healing, full-thickness flaps were elevated. Following 24% EDTA gel conditioning, the defects were treated with one of the following: (1) enamel matrix proteins (EMD), (2) guided tissue regeneration (GTR) or (3) a combination EMD and GTR. The control defects did not receive any treatment. After 5 months of healing, the animals were sacrificed. Three 8 μm thick histological central sections, 100 μm apart, were used for histomorphometric analysis in six zones of each tooth either within the furcation area or on the pristine external surface of the root. In all specimens, new cementum with inserting collagen fibres was formed. Following GTR or GTR + EMD, cementum was formed up to and including the furcation fornix indicating complete regeneration on the defect periphery. Periodontal ligament fibres were less in all four modalities compared to pristine tissues. In the teeth treated with GTR and GTR + EMD a higher volume of bone and periodontal ligament tissues was observed compared to EMD. After 5 months of healing, regenerated tissues presented quantitative differences from the pristine tissues. In the two modalities where GTR alone or combined with EMD was used, the regenerated tissues differed in quantity from the EMD-treated sites.

Mandibular reconstruction using a combination graft of rhBMP-2 with bone marrow cells expanded in vitro

BACKGROUND: The aim of this study was to evaluate the efficacy of a combination graft, using recombinant human bone morphogenetic protein-2 (rhBMP-2) and culture-expanded cells derived from bone marrow, for bone regeneration in a nonhuman primate mandible. METHODS: Five Japanese monkeys were used. Three milliliters of bone marrow was obtained from the tibia and plated into culture flasks. Adherent cells were cultured until near confluence; then, the proliferated cells were transferred to a three-dimensional culture system using collagen beads as the cell carrier. The medium was supplemented with ascorbic acid, beta-glycerophosphate, and dexamethasone to promote osteoblastic differentiation. After further proliferation on beads, the cells were mixed with a collagen sponge that was impregnated with rhBMP-2 and grafted into surgically created segmental bone defects of the mandibles. Three animals received this treatment, and either culture-expanded cells alone or collagen beads without cells were implanted into the remaining two monkeys as controls. The animals were killed 24 weeks after surgery, and the results were assessed by radiographic and histologic evaluation. RESULTS: The combination graft of culture-expanded bone marrow cells with rhBMP-2 in a collagen sponge regenerated the mandibular bone completely. By contrast, the graft of culture-expanded cells alone resulted in only a small amount of bone formation, and the implantation of collagen beads alone led to no bone formation. CONCLUSION: The combination graft of rhBMP-2 and culture-expanded cells, which requires only a small amount of bone marrow, is a reliable method for the reconstruction of segmental bone defects of the mandible.

In-frame triplet deletions in RHD alter the D antigen phenotype

BACKGROUND: The deletion of three adjacent nucleotides in an exon may cause the lack of a single amino acid, while the protein sequence remains otherwise unchanged. Only one such in-frame deletion is known in the two RH genes, represented by the RHCE allele ceBP expressing a "very weak e antigen." STUDY DESIGN AND METHODS: Blood donor samples were recognized because of discrepant results of D phenotyping. Six samples came from Switzerland and one from Northern Germany. The molecular structures were determined by genomic DNA nucleotide sequencing of RHD. RESULTS: Two different variant D antigens were explained by RHD alleles harboring one in-frame triplet deletion each. Both single-amino-acid deletions led to partial D phenotypes with weak D antigen expression. Because of their D category V-like phenotypes, the RHD(Arg229del) allele was dubbed DVL-1 and the RHD(Lys235del) allele DVL-2. These in-frame triplet deletions are located in GAGAA or GAAGA repeats of the RHD exon 5. CONCLUSION: Partial D may be caused by a single-amino-acid deletion in RhD. The altered RhD protein segments in DVL types are adjacent to the extracellular loop 4, which constitutes one of the most immunogenic parts of the D antigen. These RhD protein segments are also altered in all DV, which may explain the similarity in phenotype. At the nucleotide level, the triplet deletions may have resulted from replication slippage. A total of nine amino acid positions in an Rhesus protein may be affected by this mechanism.