Children's emerging receptive, positive orientation toward their parents in the network of early attachment relationships.

Early security plays a major role in inaugurating the child's receptive, positive orientation - a foundation for cooperative parent-child relationships and successful socialization. However, few studies have considered the association between children's attachments with both mothers and fathers and multiple aspects of children's receptive, positive orientation, or compared all four attachment groups (secure, avoidant, resistant, and disorganized). In 192 mother-child and 186 father-child dyads from community families, children's attachment was assessed at 15-17 months in Strange Situation Paradigm. Aspects of receptive, positive orientation toward each parent - positive affect, committed compliance, empathic concern, and restraint in response to parental prohibition - were observed in naturalistic laboratory contexts. Generally, securely attached children were more receptive and positive than insecure, although specific effects depended on the measure, comparison group (avoidant, resistant, disorganized), and the relationship (mother- or father-child). For positive orientation in the father-child dyads, being secure with both parents conferred a modest additional benefit.

Functional recreation of age-related CD8 T cells in young mice identifies drivers of aging- and human-specific tissue pathology.

Mitigating effects of aging on human health remains elusive because aging impacts multiple systems simultaneously, and because experimental animals exhibit critical aging differences relative to humans. Separation of aging into discrete processes may identify targetable drivers of pathology, particularly when applied to human-specific features. Gradual homeostatic expansion of CD8 T cells dominantly alters their function in aging humans but not in mice. Injecting T cells into athymic mice induces rapid homeostatic expansion, but its relevance to aging remains uncertain. We hypothesized that homeostatic expansion of T cells injected into T-deficient hosts models physiologically relevant CD8 T cell aging in young mice, and aimed to analyze age-related T cell phenotype and tissue pathology in such animals. Indeed, we found that such injection conferred uniform age-related phenotype, genotype, and function to mouse CD8 T cells, heightened age-associated tissue pathology in young athymic hosts, and humanized amyloidosis after brain injury in secondary wild-type recipients. This validates a model conferring a human-specific aging feature to mice that identifies targetable drivers of tissue pathology. Similar examination of independent aging features should promote systematic understanding of aging and identify additional targets to mitigate its effects on human health.

CD103 Deficiency Promotes Autism (ASD) and Attention-Deficit Hyperactivity Disorder (ADHD) Behavioral Spectra and Reduces Age-Related Cognitive Decline.

The incidence of autism spectrum disorders (ASD) and attention deficit hyperactivity disorder (ADHD), which frequently co-occur, are both rising. The causes of ASD and ADHD remain elusive, even as both appear to involve perturbation of the gut-brain-immune axis. CD103 is an integrin and E-cadherin receptor most prominently expressed on CD8 T cells that reside in gut, brain, and other tissues. CD103 deficiency is well-known to impair gut immunity and resident T cell function, but it's impact on neurodevelopmental disorders has not been examined. We show here that CD8 T cells influence neural progenitor cell function, and that CD103 modulates this impact both directly and potentially by controlling CD8 levels in brain. CD103 knockout (CD103KO) mice exhibited a variety of behavioral abnormalities, including superior cognitive performance coupled with repetitive behavior, aversion to novelty and social impairment in females, with hyperactivity with delayed learning in males. Brain protein markers in female and male CD103KOs coincided with known aspects of ASD and ADHD in humans, respectively. Surprisingly, CD103 deficiency also decreased age-related cognitive decline in both sexes, albeit by distinct means. Together, our findings reveal a novel role for CD103 in brain developmental function, and identify it as a unique factor linking ASD and ADHD etiology. Our data also introduce a new animal model of combined ASD and ADHD with associated cognitive benefits, and reveal potential therapeutic targets for these disorders and age-related cognitive decline.

Reliable generation of induced pluripotent stem cells from human lymphoblastoid cell lines.

Patient-specific induced pluripotent stem cells (iPSCs) hold great promise for many applications, including disease modeling to elucidate mechanisms involved in disease pathogenesis, drug screening, and ultimately regenerative medicine therapies. A frequently used starting source of cells for reprogramming has been dermal fibroblasts isolated from skin biopsies. However, numerous repositories containing lymphoblastoid cell lines (LCLs) generated from a wide array of patients also exist in abundance. To date, this rich bioresource has been severely underused for iPSC generation. We first attempted to create iPSCs from LCLs using two existing methods but were unsuccessful. Here we report a new and more reliable method for LCL reprogramming using episomal plasmids expressing pluripotency factors and p53 shRNA in combination with small molecules. The LCL-derived iPSCs (LCL-iPSCs) exhibited identical characteristics to fibroblast-derived iPSCs (fib-iPSCs), wherein they retained their genotype, exhibited a normal pluripotency profile, and readily differentiated into all three germ-layer cell types. As expected, they also maintained rearrangement of the heavy chain immunoglobulin locus. Importantly, we also show efficient iPSC generation from LCLs of patients with spinal muscular atrophy and inflammatory bowel disease. These LCL-iPSCs retained the disease mutation and could differentiate into neurons, spinal motor neurons, and intestinal organoids, all of which were virtually indistinguishable from differentiated cells derived from fib-iPSCs. This method for reliably deriving iPSCs from patient LCLs paves the way for using invaluable worldwide LCL repositories to generate new human iPSC lines, thus providing an enormous bioresource for disease modeling, drug discovery, and regenerative medicine applications.

Constitutive TL1A expression under colitogenic conditions modulates the severity and location of gut mucosal inflammation and induces fibrostenosis.

Intestinal fibrostenosis is a hallmark of severe Crohn's disease and can lead to multiple surgeries. Patients with certain TNFSF15 variants overexpress TL1A. The aim of this study was to determine the effect of TL1A overexpression on intestinal inflammation and the development of fibrostenosis. We assessed the in vivo consequences of constitutive TL1A expression on gut mucosal inflammation and fibrostenosis using two murine models of chronic colitis. In the dextran sodium sulfate (DSS) and adoptive T-cell transfer models, there was proximal migration of colonic inflammation, worsened patchy intestinal inflammation, and long gross intestinal strictures in Tl1a transgenic compared to wild-type littermates. In the DSS model, myeloid- and T-cell-expressing Tl1a transgenic mice had increased T-cell activation markers and interleukin-17 expression compared to wild-type mice. In the T-cell transfer model, Rag1(-/-) mice receiving Tl1a transgenic T cells had increased interferon-γ expression but reduced T-helper 17 cells and IL-17 production. Narrowed ureters with hydronephrosis were found only in the Tl1a transgenic mice in all chronic colitis models. In human translational studies, Crohn's disease patients with higher peripheral TL1A expression also exhibited intestinal fibrostenosis and worsened ileocecal inflammation with relative sparing of rectosigmoid inflammation. These data show that TL1A is an important cytokine that not only modulates the location and severity of mucosal inflammation, but also induces fibrostenosis.

Constitutive TL1A (TNFSF15) expression on lymphoid or myeloid cells leads to mild intestinal inflammation and fibrosis.

TL1A is a member of the TNF superfamily and its expression is increased in the mucosa of inflammatory bowel disease patients. Moreover, a subset of Crohn's disease (CD) patients with the risk TL1A haplotype is associated with elevated TL1A expression and a more severe disease course. To investigate the in vivo role of elevated TL1A expression, we generated two transgenic (Tg) murine models with constitutive Tl1a expression in either lymphoid or myeloid cells. Compared to wildtype (WT) mice, constitutive expression of Tl1a in either lymphoid or myeloid cells showed mild patchy inflammation in the small intestine, which was more prominent in the ileum. In addition, mice with constitutive Tl1a expression exhibited enhanced intestinal and colonic fibrosis compared to WT littermates. The percentage of T cells expressing the gut homing chemokine receptors CCR9 and CCR10 was higher in the Tl1a Tg mice compared to WT littermates. Sustained expression of Tl1A in T cells also lead to increased Foxp3+ Treg cells. T cells or antigen presenting cells (APC) with constitutive expression of Tl1a were found to have a more activated phenotype and mucosal mononuclear cells exhibit enhanced Th1 cytokine activity. These results indicated an important role of TL1A in mucosal T cells and APC function and showed that up-regulation of TL1A expression can promote mucosal inflammation and gut fibrosis.

Oncogenic Kras requires simultaneous PI3K signaling to induce ERK activation and transform thyroid epithelial cells in vivo.

Thyroid tumors arising from the follicular cells often harbor mutations leading to the constitutive activation of the PI3K and Ras signaling cascades. However, it is still unclear what their respective contribution to the neoplastic process is, as well as to what extent they interact. We have used mice harboring a Kras oncogenic mutation and a Pten deletion targeted to the thyroid epithelium to address in vivo these questions. Here, we show that although each of these two pathways, alone, is unable to transform thyroid follicular cells, their simultaneous activation is highly oncogenic, leading to invasive and metastatic follicular carcinomas. In particular, phosphatidylinositol-3-kinase (PI3K) activation suppressed Kras-initiated feedback signals that uncouple mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) and ERK activation, thus stunting MAPK activity; in addition, PI3K and Kras cooperated to drastically up-regulate cyclin D1 mRNA levels. Finally, combined pharmacologic inhibition of PI3K and MAPK completely inhibited the growth of double-mutant cancer cell lines, providing a compelling rationale for the dual targeting of these pathways in thyroid cancer.

Mammalian target of rapamycin is the key effector of phosphatidylinositol-3-OH-initiated proliferative signals in the thyroid follicular epithelium.

Activation of the phosphatidylinositol-3-OH kinase (PI3K) signaling cascade is becoming increasingly recognized as a common feature of thyroid follicular neoplasms. We have recently shown that conditional loss of Pten in the mouse thyroid follicular cells is sufficient to stimulate continuous autonomous growth, leading to a homogeneously hyperplastic gland and to the development of follicular adenomas. Because the PI3K/AKT cascade can activate a plethora of different signaling pathways, it is still unclear which of these may represent the key mitogenic output of PI3K-initiated signaling. Here, we show that the in vivo proliferative response to chronic PI3K activation profoundly relies on the activation of the mammalian target of rapamycin (mTOR)/S6K1 axis, and that mTOR inhibition in Pten mutant mice and cells restores virtually normal proliferation rates, despite the presence of still elevated Akt activity, at least in part by down-regulating cyclins D1 and D3, and without affecting cell survival.

Thyroid-stimulating hormone initiated proliferative signals converge in vivo on the mTOR kinase without activating AKT.

Thyroid-stimulating hormone (TSH) has long been recognized as the major proliferative and functional stimulus for thyroid follicular cells. TSH receptor (TSHR) engagement stimulates the production of cyclic AMP and the subsequent activation of downstream effector molecules, including protein kinase A, S6K1, and Rap1, whereas the role of the RAS and phosphatidylinositol-3-kinase signaling cascades downstream of TSHR is still controversial. Despite the abundance of candidates, it is still unclear which of these pathways represent(s) the key mitogenic output of TSH-initiated signaling. We have used an in vivo model of goitrogenesis to dissect the contribution of these pathways to TSH-induced thyrocyte proliferation and thyroid hyperplasia. We show that the in vivo proliferative response to chronic TSHR stimulation relies heavily on the activation of the mTOR/S6K1 axis, and that mTOR inhibition during goitrogenic stimulation abrogates the hyperplastic but not the hypertrophic thyrocyte responses to TSH, thus functionally uncoupling these two processes. Strikingly, goitrogenesis was not associated with an increase in AKT phosphorylation levels, underlining the existence of an AKT-independent pathway leading to mTOR activation upon TSH stimulation.

Pten loss in the mouse thyroid causes goiter and follicular adenomas: insights into thyroid function and Cowden disease pathogenesis.

Inactivation and silencing of the tumor suppressor PTEN are found in many different epithelial tumors, including thyroid neoplasia. Cowden Disease patients, who harbor germ-line PTEN mutations, often display thyroid abnormalities, including multinodular goiter and follicular adenomas, and are at increased risk of thyroid cancer. To gain insights into the role PTEN plays in thyroid function and disease, we have generated a mouse strain, in which Cre-mediated recombination is used to specifically delete Pten in the thyrocytes. We found that Pten mutant mice develop diffuse goiter characterized by extremely enlarged follicles, in the presence of normal thyroid-stimulating hormone and T4 hormone levels. Loss of Pten resulted in a significant increase in the thyrocyte proliferative index, which was more prominent in the female mice, and in increased cell density in the female thyroid glands. Surprisingly, goitrogen treatment did not cause a substantial increase of the mutant thyroid size and increased only to some extent the proliferation index of the female thyrocytes, suggesting that a relevant part of the thyroid-stimulating hormone-induced proliferation signals are funneled through the phosphatidylinositol-3-kinase (PI3K)/Akt cascade. Although complete loss of Pten was not sufficient to cause invasive tumors, over two thirds of the mutant females developed follicular adenomas by 10 months of age, showing that loss of Pten renders the thyroid highly susceptible to neoplastic transformation through mechanisms that include increased thyrocyte proliferation. Our findings show that constitutive activation of the PI3K/Akt cascade is sufficient to stimulate continuous autonomous growth and provide novel clues to the pathogenesis of Cowden Disease and sporadic nontoxic goiter.