Longbao Lv

Maternal separation produces lasting changes in cortisol and behavior in rhesus monkeys.

Maternal separation (MS), which can lead to hypothalamic pituitary adrenal axis dysfunction and behavioral abnormalities in rhesus monkeys, is frequently used to model early adversity. Whether this deleterious effect on monkeys is reversible by later experience is unknown. In this study, we assessed the basal hair cortisol in rhesus monkeys after 1.5 and 3 y of normal social life following an early separation. These results showed that peer-reared monkeys had significantly lower basal hair cortisol levels than the mother-reared monkeys at both years examined. The plasma cortisol was assessed in the monkeys after 1.5 y of normal social life, and the results indicated that the peak in the peer-reared cortisol response to acute stressors was substantially delayed. In addition, after 3 y of normal social life, abnormal behavioral patterns were identified in the peer-reared monkeys. They showed decreases in locomotion and initiated sitting together, as well as increases in stereotypical behaviors compared with the mother-reared monkeys. These results demonstrate that the deleterious effects of MS on rhesus monkeys cannot be compensated by a later normal social life, suggesting that the effects of MS are long-lasting and that the maternal-separated rhesus monkeys are a good animal model to study early adversity and to investigate the development of psychiatric disorders induced by exposure to early adversity.

Chronic Clomipramine Treatment Reverses Core Symptom of Depression in Subordinate Tree Shrews

Chronic stress is the major cause of clinical depression. The behavioral signs of depression, including anhedonia, learning and memory deficits, and sleep disruption, result from the damaging effects of stress hormones on specific neural pathways. The Chinese tree shrew (Tupaia belangeri chinensis) is an aggressive non-human primate with a hierarchical social structure that has become a well-established model of the behavioral, endocrine, and neurobiological changes associated with stress-induced depression. The tricyclic antidepressant clomipramine treats many of the core symptoms of depression in humans. To further test the validity of the tree shrew model of depression, we examined the effects of clomipramine on depression-like behaviors and physiological stress responses induced by social defeat in subordinate tree shrews. Social defeat led to weight loss, anhedonia (as measured by sucrose preference), unstable fluctuations in locomotor activity, sustained urinary cortisol elevation, irregular cortisol rhythms, and deficient hippocampal long-term potentiation (LTP). Clomipramine ameliorated anhedonia and irregular locomotor activity, and partially rescued the irregular cortisol rhythm. In contrast, weight loss increased, cortisol levels were even higher, and in vitro LTP was still impaired in the clomipramine treatment group. These results demonstrate the unique advantage of the tree shrew social defeat model of depression.

Association of the LRRK2 genetic polymorphisms with leprosy in Han Chinese from Southwest China

Leprosy is a chronic infectious and neurological disease that is caused by infection of Mycobacterium leprae (M. leprae). A recent genome-wide association study indicated a suggestive association of LRRK2 genetic variant rs1873613 with leprosy in Chinese population. To validate this association and further identify potential causal variants of LRRK2 with leprosy, we genotyped 13 LRRK2 variants in 548 leprosy patients and 1078 healthy individuals from Yunnan Province and (re-)analyzed 3225 Han Chinese across China. Variants rs1427267, rs3761863, rs1873613, rs732374 and rs7298930 were significantly associated with leprosy per se and/or paucibacillary leprosy (PB). Haplotype A-G-A-C-A was significantly associated with leprosy per se (P=0.018) and PB (P=0.020). Overexpression of the protective allele (Thr2397) of rs3761863 in HEK293 cells led to a significantly increased nuclear factor of activated T-cells’ activity compared with allele Met2397 after lipopolysaccharides stimulation. Allele Thr2397 could attenuate 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine-induced autophagic activity in U251 cells. These data suggest that the protective effect of LRRK2 variant p.M2397T on leprosy might be mediated by increasing immune response and decreasing neurotoxicity after M. leprae loading. Our findings confirm that LRRK2 is a susceptible gene to leprosy in Han Chinese population.

Evidence That Rhesus Macaques Self-Cure from a Schistosoma japonicum Infection by Disrupting Worm Esophageal Function: A New Route to an Effective Vaccine?

Background Rhesus macaques are unusual among schistosome hosts, self-curing from an established infection and thereafter manifesting solid immunity against a challenge, an ideal model for vaccine development. Previously, the immunological basis of self-cure was confirmed; surviving worms had ceased feeding but how immunological pressure achieved this was unclear. The schistosome esophagus is not simply a conduit for blood but plays a central role in its processing. Secretions from the anterior and posterior esophageal glands mix with incoming blood causing erythrocyte lysis and tethering and killing of leucocytes. Methodology/Principal Findings We have analysed the self-cure process in rhesus macaques infected with Schistosoma japonicum. Faecal egg output and circulating antigen levels were used to chart the establishment of a mature worm population and its subsequent demise. The physiological stress of surviving females at perfusion was especially evident from their pale, shrunken appearance, while changes in the structure and function of the esophagus were observed in both sexes. In the anterior region electron microscopy revealed that the vesicle secretory process was disrupted, the tips of lining corrugations being swollen by greatly enlarged vesicles and the putative sites of vesicle release obscured by intense deposits of IgG. The lumen of the posterior esophagus in starving worms was occluded by cellular debris and the lining cytoplasmic plates were closely adherent, also potentially preventing secretion. Seven proteins secreted by the posterior gland were identified and IgG responses were detected to some or all of them. Intrinsic rhesus IgG colocalized with secreted SjMEGs 4.1, 8.2, 9, 11 and VAL-7 on cryosections, suggesting they are potential targets for disruption of function. Conclusions/Significance Our data suggest that rhesus macaques self-cure by blocking esophagus function with antibody; the protein products of the glands provide a new class of potential vaccine targets.

Human β-Defensin 2 Is a Novel Opener of Ca2+-Activated Potassium Channels and Induces Vasodilation and Hypotension in Monkeys

Human &bgr;-defensin 2 (HBD2) is a cysteine-rich cationic antimicrobial peptide known for its important role in innate immune system. Intensive studies have demonstrated its antimicrobial and chemotactic activities in vitro. In this study, ELISA analysis showed that HBD2 was significantly downregulated in sera of patients with hypertension. It relaxed vessel smooth muscle by acting on the major regulatory pathways, contributing to vessel smooth muscle contraction. Electrophysiology analysis indicated that HBD2 acted as an opener of large-conductance Ca2+-activated potassium (BKCa)-mSlo+h&bgr;1 channels and increased BKCa currents. Mutation analysis revealed that HBD2 activated BKCa-mSlo+h&bgr;1 channels via interacting with Leu41 and Gln43 of &bgr;1-loop. In vivo experiments suggested that HBD2 at 4× to 6× of physiological concentration exerted hypotensive effect in monkeys significantly, whereas the selective blocker of BKCa channels, Paxilline, inhibited the effect. HBD2 is the first peptide opener of BKCa-mSlo+h&bgr;1 channels. It may be a novel regulator of blood pressure and provides a new therapeutic target for the treatment of hypertension. The HBD2 blockade of the BKCa channels may represent a new type of cross-talk between immune and cardiovascular systems.

Long-term propagation of tree shrew spermatogonial stem cells in culture and successful generation of transgenic offspring

Tree shrews have a close relationship to primates and have many advantages over rodents in biomedical research. However, the lack of gene manipulation methods has hindered the wider use of this animal. Spermatogonial stem cells (SSCs) have been successfully expanded in culture to permit sophisticated gene editing in the mouse and rat. Here, we describe a culture system for the long-term expansion of tree shrew SSCs without the loss of stem cell properties. In our study, thymus cell antigen 1 was used to enrich tree shrew SSCs. RNA-sequencing analysis revealed that the Wnt/β-catenin signaling pathway was active in undifferentiated SSCs, but was downregulated upon the initiation of SSC differentiation. Exposure of tree shrew primary SSCs to recombinant Wnt3a protein during the initial passages of culture enhanced the survival of SSCs. Use of tree shrew Sertoli cells, but not mouse embryonic fibroblasts, as feeder was found to be necessary for tree shrew SSC proliferation, leading to a robust cell expansion and long-term culture. The expanded tree shrew SSCs were transfected with enhanced green fluorescent protein (EGFP)-expressing lentiviral vectors. After transplantation into sterilized adult male tree shrews testes, the EGFP-tagged SSCs were able to restore spermatogenesis and successfully generate transgenic offspring. Moreover, these SSCs were suitable for the CRISPR/Cas9-mediated gene modification. The development of a culture system to expand tree shrew SSCs in combination with a gene editing approach paves the way for precise genome manipulation using the tree shrew.

PTEN/PIK3CA genes are frequently mutated in spontaneous and medroxyprogesterone acetate-accelerated 7,12-dimethylbenz(a)anthracene-induced mammary tumours of tree shrews

Tree shrew has increasingly become an attractive experimental animal model for human diseases, particularly for breast cancer due to spontaneous breast tumours and their close relationship to primates and by extension to humans. However, neither normal mammary glands nor breast tumours have been well characterised in the Chinese tree shrew (Tupaia belangeri chinensis). In this study, normal mammary glands from four different developmental stages and 18 spontaneous breast tumours were analysed. Haematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) showed that normal mammary gland morphology and structures of tree shrews were quite similar to those found in humans. Spontaneous breast tumours of tree shrews were identified as being intraductal papilloma, papillary carcinoma, and invasive ductal carcinoma with or without lung metastasis. To further analyse breast cancer tumours among tree shrews, 40 3-4 month-old female tree shrews were orally administrated 20 mg 7,12-dimethylbenz(a)anthracene (DMBA) or peanut oil thrice, and then, 15 of these DMBA administrated tree shrews were implanted with medroxyprogesterone acetate (MPA) pellets. DMBA was shown to induce breast tumours (12%) while the addition of MPA increased the tumour incidence (50%). Of these, three induced breast tumours were intraductal papillary carcinomas and one was invasive ductal carcinoma (IDC). The PTEN/PIK3CA (phosphatase and tensin homologue/phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha), but not TP53 and GATA3, genes are frequently mutated in breast tumours, and the PTEN/PIK3CA gene mutation status correlated with the expression of pAKT in tree shrew breast tumours. These results suggest that tree shrews may be a promising animal model for a subset of human breast cancers with PTEN/PIK3CA gene mutations.

CDKN2B deletion is essential for pancreatic cancer development instead of unmeaningful co-deletion due to juxtaposition to CDKN2A

Pancreatic cancer is among the deadliest malignancies; however, the genetic events that lead to pancreatic carcinogenesis in adults remain unclear. In vivo models in which these genetic alterations occur in adult animals may more accurately reflect the features of human cancer. In this study, we demonstrate that inactivation of Cdkn2b (p15ink4b) is necessary for induction of pancreatic cancer by oncogenic KRASG12D expression and inactivation of Tp53 and Cdkn2a in adult mouse pancreatic ductal cells (P60 or older). KRASG12D overexpression in these cells activated transforming growth factor-β signaling and expression of CDKN2B, which, along with CDKN2A, led to cellular senescence and protected cells from KRAS-mediated transformation via inhibition of retinoblastoma phosphorylation. These results show a critical role of CDKN2B inactivation in pancreatic carcinogenesis, and provide a useful adult animal model by genetic engineering via lentiviral delivery.

A tree shrew glioblastoma model recapitulates features of human glioblastoma

Tupaia belangeri (tree shrew), an animal species whose genome has significantly higher similarity to primates than rodents, has been used in biomedical research. To generate animal models that reproduce the human tumors more faithfully than rodents, we present the first report of a cancer model mimicking human tumor genetics in tree shrew. By engineering a lentiviral system for the transduction of mutant H-Ras and a shRNA against tree shrew p53, we successfully generated malignant glioma in tree shrew. The tree shrew glioma exhibited aggressive behavior and a relatively short latency, and markedly reduced animal survival. Remarkably, the biological features of human high-grade glioma (necrosis, microvascular proliferation, pseudopalisading) were all present in tree shrew glioma. Furthermore, genetic analysis of tree shrew glioma revealed that the tumors were clustered within the mesenchymal subgroup of human glioblastoma multiforme. Compared with the corresponding mouse glioma, tree shrew gliomas were markedly more similar to human glioblastoma at gene expression profile. The tree shrew glioma model provides colleagues working in the field of gliomas and cancer in general with a more accurate animal model.

Social correlates of the dominance rank and long-term cortisol levels in adolescent and adult male rhesus macaques (Macaca mulatta)

A common pattern in dominance hierarchies is that some ranks result in higher levels of psychosocial stress than others. Such stress can lead to negative health outcomes, possibly through altered levels of stress hormones. The dominance rank-stress physiology relationship is known to vary between species; sometimes dominants show higher levels of glucocorticoid stress hormones, whereas in other cases subordinates show higher levels. It is less clear how this relationship varies between groups of different ages or cultures. In this study, we used long-term cortisol measurement methods to compare the effect of rank on cortisol levels in adult and adolescent male rhesus macaques. In the adult groups, subordinates had significantly higher cortisol levels. In the adolescents, no significant correlation between cortisol and status was found. Further analysis demonstrated that the adult hierarchy was stricter than that of the adolescents. Adult subordinates received extreme aggression more frequently than dominants, and this class of behavior was positively correlated with cortisol; by contrast, adolescents showed neither trend. Together, these findings provide evidence for a cortisol-rank relationship determined by social factors, namely, despotism of the group, and highlight the importance of group-specific social analysis when comparing or combining results obtained from different groups of animals.