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Continuous turnover of epithelia is ensured by the extensive self-renewal capacity of tissue-specific stem cells. Similarly, epithelial tumour maintenance relies on cancer stem cells (CSCs), which co-opt stem cell properties. For most tumours, the cellular origin of these CSCs and regulatory pathways essential for sustaining stemness have not been identified. In murine skin, follicular morphogenesis is driven by bulge stem cells that specifically express CD34. Here we identify a population of cells in early epidermal tumours characterized by phenotypic and functional similarities to normal bulge skin stem cells. This population contains CSCs, which are the only cells with tumour initiation properties. Transplants derived from these CSCs preserve the hierarchical organization of the primary tumour. We describe β-catenin signalling as being essential in sustaining the CSC phenotype. Ablation of the β-catenin gene results in the loss of CSCs and complete tumour regression. In addition, we provide evidence for the involvement of increased β-catenin signalling in malignant human squamous cell carcinomas. Because Wnt/β-catenin signalling is not essential for normal epidermal homeostasis, such a mechanistic difference may thus be targeted to eliminate CSCs and consequently eradicate squamous cell carcinomas. ©2008 Nature Publishing Group., Work in Madrid was supported in part by the Spanish Ministry of Education and Science to A.C.

Members of the transforming growth factor beta (TGF-beta) superfamily of signaling molecules are involved in the regulation of many developmental processes that involve the interaction between mesenchymal and epithelial tissues. Smad7 is a potent inhibitor of many members of the TGF-beta family, notably TGF-beta and activin. In this study, we show that embryonic overexpression of Smad7 in stratified epithelia using a keratin 5 promoter, results in severe morphogenetic defects in skin and teeth and leads to embryonic and perinatal lethality. To further analyze the functions of Smad7 in epithelial tissues of adult mice, we used an expression system that allowed a controlled overexpression of Smad7 in terms of both space and time. Skin defects in adult mice overexpressing Smad7 were characterized by hyper-proliferation and missing expression of early markers of keratinocyte differentiation. Upon Smad7-mediated blockade of TGF-beta superfamily signaling, ameloblasts failed to produce an enamel layer in incisor teeth. In addition, TGF-beta blockade in adult mice altered the pattern of thymic T cell differentiation and the number of thymic T cells was significantly reduced. This study shows that TGF-beta superfamily signaling is essential for development of hair, tooth and T-cells as well as differentiation and proliferation control in adult tissues.

Retinoid-X-receptor alpha (RXRalpha), a member of the nuclear receptor (NR) superfamily, is a ligand-dependent transcriptional regulatory factor. It plays a crucial role in NR signalling through heterodimerization with some 15 NRs. We investigated the role of RXRalpha and its partners on mouse skin tumor formation and malignant progression upon topical DMBA/TPA treatment. In mutants selectively ablated for RXRalpha in keratinocytes, epidermal tumors increased in size and number, and frequently progressed to carcinomas. As keratinocyte-selective peroxisome proliferator-activated receptor gamma (PPARgamma) ablation had similar effects, RXRalpha/PPARgamma heterodimers most probably mediate epidermal tumor suppression. Keratinocyte-selective RXRalpha-null and vitamin-D-receptor null mice also exhibited more numerous dermal melanocytic growths (nevi) than control mice, but only nevi from RXRalpha mutant mice progressed to invasive human-melanoma-like tumors. Distinct RXRalpha-mediated molecular events appear therefore to be involved, in keratinocytes, in cell-autonomous suppression of epidermal tumorigenesis and malignant progression, and in non-cell-autonomous suppression of nevi formation and progression. Our study emphasizes the crucial role of keratinocytes in chemically induced epidermal and melanocytic tumorigenesis, and raises the possibility that they could play a similar role in UV-induced tumorigenesis, notably in nevi formation and progression to melanoma.

Progressive tissue fibrosis is a major cause of the morbidity and mortality associated with repeated epithelial injuries and accumulation of myofibroblasts. Successful treatment options are limited by an incomplete understanding of the molecular mechanisms that regulate myofibroblast accumulation. Here, we employed in vivo lineage tracing and real-time gene expression transgenic reporting methods to analyze the early embryonic transcription factor T-box gene 4 (TBX4), and determined that TBX4-lineage mesenchymal progenitors are the predominant source of myofibroblasts in injured adult lung. In a murine model, ablation of TBX4-expressing cells or disruption of TBX4 signaling attenuated lung fibrosis after bleomycin-induced injury. Furthermore, TBX4 regulated hyaluronan synthase 2 production to enable fibroblast invasion of matrix both in murine models and in fibroblasts from patients with severe pulmonary fibrosis. These data identify TBX4 as a mesenchymal transcription factor that drives accumulation of myofibroblasts and the development of lung fibrosis. Targeting TBX4 and downstream factors that regulate fibroblast invasiveness could lead to therapeutic approaches in lung fibrosis.

The generation of ligand-activated site-specific Cre recombinases has led to the development of cell type-specific temporally controlled targeted somatic mutagenesis in the mouse. We illustrate this technique using K14-Cre-ER(T2) transgenic mice that express the tamoxifen (tam)-activatable Cre-ER(T2) recombinase in epidermal basal keratinocytes to induce mutations in epidermal keratinocytes of adult mice. Our highly reproducible technique, based on induction of Cre-ER(T2) recombinase activity by tamoxifen administration at low doses (once daily 100-µg intraperitoneal injection for 5 days), has allowed the generation of site-directed somatic mutations of numerous genes in mouse epidermal keratinocytes, and several mouse models of human diseases. The present step-by-step protocol describes how to introduce temporally controlled targeted mutations in epidermal keratinocytes of adult mice. Curr. Protoc. Mouse Biol. 1:55-70. © 2011 by John WileySons, Inc.

X-linked acrogigantism (X-LAG) is a new syndrome of pituitary gigantism, caused by microduplications on chromosome Xq26.3, encompassing the geneGPR101, which is highly upregulated in pituitary tumors. We conducted this study to explore the clinical, radiological, and hormonal phenotype and responses to therapy in patients with X-LAG syndrome. The study included 18 patients (13 sporadic) with X-LAG and microduplication of chromosome Xq26.3. All sporadic cases had unique duplications and the inheritance pattern in two families was dominant, with all Xq26.3 duplication carriers being affected. Patients began to grow rapidly as early as 2–3 months of age (median 12 months). At diagnosis (median delay 27 months), patients had a median height and weight standard deviation scores (SDS) of >+3.9 SDS. Apart from the increased overall body size, the children had acromegalic symptoms including acral enlargement and facial coarsening. More than a third of cases had increased appetite. Patients had marked hypersecretion of GH/IGF1 and usually prolactin, due to a pituitary macroadenoma or hyperplasia. Primary neurosurgical control was achieved with extensive anterior pituitary resection, but postoperative hypopituitarism was frequent. Control with somatostatin analogs was not readily achieved despite moderate to high levels of expression of somatostatin receptor subtype-2 in tumor tissue. Postoperative use of adjuvant pegvisomant resulted in control of IGF1 in all five cases where it was employed. X-LAG is a new infant-onset gigantism syndrome that has a severe clinical phenotype leading to challenging disease management.

Relative pay in the financial sector has experienced an extraordinary increase over the last few decades. A proposed explanation for this pattern has been that the demand for skilled workers in finance has risen more than in other sectors. We use Swedish administrative data, which include detailed cognitive and non-cognitive test scores as well as performance in high-school and university, to examine the implications of this hypothesis for talent allocation and relative wages in the financial sector. We find no evidence that the selection of talent into finance increased or improved, neither on average nor at the top of the talent distribution. A changing composition of talent or their returns cannot account for the surge in the finance wage premium. These findings alleviate concerns about a “brain drain” into finance at the expense of other sectors, but they also suggest that rents in finance are high, increasing, and largely unexplained.

Previously we identified a transcription factor, LPS-Induced TNF-α Factor (LITAF), mediating inflammatory cytokine expression in LPS-induced processes. To characterize the role of LITAF in vivo , we generated a macrophage-specific LITAF-deficient mouse (macLITAF −/− ). Our data demonstrate that in macrophages ( i ) several cytokines (such as TNF-α, IL-6, sTNF-RII, and CXCL16) are induced at lower levels in macLITAF −/− compared with LITAF +/+ control macrophages; ( ii ) macLITAF −/− mice are more resistant to LPS-induced lethality. To further identify LITAF signaling pathways, we tested mouse TLR-2 −/− , -4 −/− , and -9 −/− and WT peritoneal macrophages exposed to LPS. Using these cells, we now show that LITAF expression can be induced after challenge either with LPS from Porphyromonas gingivalis via agonism at TLR-2, or with LPS from Escherichia coli via agonism at TLR-4, both requiring functional MyD88. We also show that, in response to LPS, the MyD88-dependent LITAF pathway differs from the NF-κB pathway. Furthermore, using a kinase array, p38α was found to mediate LITAF phosphorylation and the inhibition of p38α with a p38-specific inhibitor (SB203580) blocked LITAF nuclear translocation and reduced LPS-induced TNF-α protein levels. Finally, macLITAF −/− macrophages rescued by LITAF cDNA transfection restored levels of TNF-α similar to those observed in WT cells. We conclude that LITAF is an important mediator of the LPS-induced inflammatory response that can be distinguished from NF-κB pathway and that p38α is the specific kinase involved in the pathway linking LPS/MyD88/LITAF to TNF.

International audience; Skin inflammation is a complex process that involves interactions between various cell types residing in different skin compartments. Using mice with conditionally targeted I kappa B kinase 2 (IKK2) alleles, we have previously shown that epidermal keratinocytes can play a dominant role in the initiation of an inflammatory reaction. In order to investigate long-term consequences of IKK2 deletion in adult skin, we have generated mice with floxed IKK2 alleles in which expression of a Tamoxifen-inducible Cre recombinase construct is targeted to epidermal keratinocytes (K14-Cre-ER(T2)IKK2(fl/fl) mice). K14-Cre-ER(T2)IKK2(fl/fl) mice are born normally and do not show signs of a skin disease until the age of 6 months. Deletion of IKK2 can be observed after Tamoxifen application to the back skin or spontaneously, without Tamoxifen application, in mice older than 6 months. This deletion is accompanied by dramatic, localized skin changes that are characterized by invasion of inflammatory cells, hair follicle disruption, and pseudoepitheliomatous hyperplasia of the epidermis, but not by tumor formation. The hyperplastic epithelium shows increased phosphorylation of signal transducer and activator of transcription 3 and extracellular signal-regulated protein kinase 1/2, typical features of psoriatic epidermis. Our results identify a primary role for IKK2 in the development of skin inflammation and confirm its requirement for the maintenance of skin homeostasis.

Purpose Breast cancer subtype correlates with response to systemic therapy and overall survival (OS), but its impact on lymphatic spread is incompletely understood. In this study, we used the Surveillance, Epidemiology, and End Results registry to assess whether the subtype can predict the presence of nodal metastasis or advanced nodal stage in breast cancer. Methods A total of 7,274 eligible patients diagnosed with T1-3 infiltrating ductal carcinoma with known estrogen or progesterone hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) status, who underwent surgical excision of the primary tumor and pathologic lymph node evaluation, were included in this analysis. Patients were categorized into four breast cancer subtypes: HR+/HER2-; HR+/HER2+; HR-/HER2+; and HR-/HER2-. Binary logistic regression analysis was used to determine whether breast cancer subtype, tumor size, tumor grade, patient race, and patient age at diagnosis are independently predictive of lymph node positivity or advanced nodal stage. The Pearson chi-square test was used to determine whether progesterone receptor (PR) status had an impact on the incidence of lymph node positivity in estrogen receptor (ER) positive patients. Results Independent predictors of nodal positivity included breast cancer subtype (p=0.040), tumor size (p

The first aim of this study was to examine the role of myofiber androgen receptor (AR) in male mice on muscle performance gain and remodeling-induced muscle mechanical overloading (OVL) that mimics resistance training. The response of OVL in mice in which AR is selectively ablated in myofibers (ARskm−/y) was compared with that of wild-type (WT) mice. In addition, we determined whether the synthetic anabolic androgen nandrolone administration affects the OVL response. We found that OVL increased absolute maximal force and fatigue resistance in both mouse genotypes (P < .05). However, the absolute maximal force increased more in ARskm−/y mice as compared with WT mice (+88% vs +63%) (P < .05). Muscle weight increased less in response to OVL in ARskm−/y mice (+54%) than in WT mice (+115%) (P < .05). The fiber number per cross-section similarly increased in both mouse genotypes after OVL (P < .05). In contrast to WT mice, the diameter of the fibers expressing myosin heavy chain (MHC)-2x decreased after OVL in ARskm−/y mice (P < .05). The MHC-2b to MHC-2a fiber type transition in response to OVL was reduced in ARskm−/y mice as compared with WT mice (P < .05). Finally, nandrolone administration during OVL did not further improve absolute maximal force and fatigue resistance and markedly alter muscle remodeling in both mouse genotypes. Together, our results indicate that myofiber AR is required for a complete response to OVL and that exogenous androgens do not increase muscle performance during intensive remodeling in male mice.

To investigate the role of retinoid X receptors (RXRs) in epidermal homeostasis, we generated RXRαβ ep-/- somatic mutants in which both RXRα and RXRβ are selectively ablated in epidermal keratinocytes of adult mice. These mice develop a chronic dermatitis mimicking that observed in atopic dermatitis (AD) patients. In addition, they exhibit immunological abnormalities including elevated serum levels of IgE and IgG, associated with blood and tissue eosinophilia, indicating that keratinocyte-selective ablation of RXRs also generates a systemic syndrome similar to that found in AD patients. Furthermore, the profile of increased expression of cytokines and chemokines in skin of keratinocyte-selective RXRαβ-ablated mutants was typical of a T helper 2-type inflammation, known to be crucially involved in human AD pathogenesis. Finally, we demonstrate that thymic stromal lymphopoietin, whose expression is rapidly and strongly induced in RXRαβ-ablated keratinocytes, plays a key role in initiating the skin and systemic AD-like pathologies.

The Canadian Pediatric Endocrinology Group (CPEG) has produced complementary growth curves based on the 2010 'WHO Growth Charts for Canada'. In response to concerns from CPEG members and the general paediatric community regarding the presentation of the WHO data, complementary curves were generated, which the authors believe will enhance clarity, reduce potential errors in classification and enable users to better track short-term changes, particularly for weight in older children. Specifically, these curves extend weight-for-age beyond 10 years of age, restore additional percentiles within the normal range, remove extreme percentiles and harmonize the choice of body mass index percentiles with adult definitions of overweight and obesity. All modifications followed strict WHO methodology and used core data from the United States National Center for Health Statistics. The curves retain the clean appearance of the 2010 Canadian curves and are available from the CPEG website (http://cpeg-gcep.net).

A five-year-four-month-old Caucasian boy of nonconsanguineous parents presented with a one-year history of papules over the cheeks, glabella and ears. His mother noted that he experienced several episodes of body odour that resolved spontaneously. He also experiences occasional headaches. He had no history of salt-craving, axillary or pubic hair, or change in phallus size. Rapid growth was noted by the mother in the past six months. His medical history was unremarkable, and the family history only revealed that the paternal grandmother’s first child died shortly after birth. No autopsy results were available. On examination, the patient was in no distress. His blood pressure was 94/53 mmHg, and his pulse was 87 beats/min. His height was 120.7 cm and his weight was 21.3 kg, at the 97th and 80th percentiles, respectively. He had no body odour, his penile length was 7 cm (>2 SD) and his testes were descended and 3 mL in volume. His cutaneous examination revealed multiple keratotic papules on his cheeks, as well as open comedones around the inner and outer ears and nose. He exhibited no cutaneous hyperpigmentation. Partial initial laboratory results demonstrated a normal complete blood count, electrolyte levels, and normal tests of liver and renal function. Follicle-stimulating and luteinizing hormone levels were also within normal limits. His bone age was nine years. Additional investigations revealed the diagnosis.

We have generated mice with a floxed fak allele under the control of keratin-14-driven Cre fused to a modified estrogen receptor (CreERT2). 4-Hydroxy-tamoxifen treatment induced fak deletion in the epidermis, and suppressed chemically induced skin tumor formation. Loss of fak induced once benign tumors had formed inhibited malignant progression. Although fak deletion was associated with reduced migration of keratinocytes in vitro, we found no effect on wound re-epithelialization in vivo. However, increased keratinocyte cell death was observed after fak deletion in vitro and in vivo. Our work provides the first experimental proof implicating FAK in tumorigenesis, and this is associated with enhanced apoptosis.

The recent development of conditional gene-targeting techniques in the mouse, based mainly on the bacteriophage P1 Cre/LoxP system, enable spatio- and/or temporal-controlled target gene ablation in the liver. Transgenic mouse lines with liver-selective gene ablation established during the past few years are presented. They illustrate how these new genetic approaches improve the understanding of homeostatic liver functions, and the establishment of animal models for liver diseases.

Acquired lengthening of the QT interval due to hypocalcaemia is a rare cause of arrhythmia in childhood. Early recognition, rapid institution of appropriate cardiac monitoring, and replacement therapy are essential. An endocrinal work-up may be necessary to exclude primary disorders of calcium metabolism. We report four cases documenting the varied clinical spectrum in which hypocalcaemic-induced prolongation of the QT interval and arrhythmia can occur in childhood.

The peroxisome proliferator-activated receptor γ (PPARγ) plays a major role in fat tissue development and physiology. Mutations in the gene encoding this receptor have been associated to disorders in lipid metabolism. A thorough investigation of mice in which one PPARγ allele has been mutated reveals that male PPARγ heterozygous (PPARγ +/−) mice exhibit a reduced body size associated with decreased body weight, reflecting lean mass reduction. This phenotype is reproduced when treating the mice with a PPARγ- specific antagonist. Monosodium glutamate treatment, which induces weight gain and alters body growth in wild-type mice, further aggravates the growth defect of PPARγ +/− mice. The levels of circulating GH and that of its downstream effector, IGF-I, are not altered in mutant mice. However, the IGF-I mRNA level is decreased in white adipose tissue (WAT) of PPARγ +/− mice and is not changed by acute administration of recombinant human GH, suggesting an altered GH action in the mutant animals. Importantly, expression of the gene encoding the suppressor of cytokine signaling-2, which is an essential negative regulator of GH signaling, is strongly increased in the WAT of PPARγ +/− mice. Although the relationship between the altered GH signaling in WAT and reduced body size remains unclear, our results suggest a novel role of PPARγ in GH signaling, which might contribute to the metabolic disorder affecting insulin signaling in PPARγ mutant mice.

Bone morphogenetic protein (BMP) signaling is thought to perform multiple functions in the regulation of skin appendage morphogenesis and the postnatal growth of hair follicles. However, definitive genetic evidence for these roles has been lacking. Here, we show that Cre-mediated mutation of the gene encoding BMP receptor 1A in the surface epithelium and its derivatives causes arrest of tooth morphogenesis and lack of external hair. The hair shaft and hair follicle inner root sheath (IRS) fail to differentiate, and expression of the known transcriptional regulators of follicular differentiation Msx1,Msx2, Foxn1 and Gata3 is markedly downregulated or absent in mutant follicles. Lef1 expression is maintained, but nuclearβ-catenin is absent from the epithelium of severely affected mutant follicles, indicating that activation of the WNT pathway lies downstream of BMPR1A signaling in postnatal follicles. Mutant hair follicles fail to undergo programmed regression, and instead continue to proliferate, producing follicular cysts and matricomas. These results provide definitive genetic evidence that epithelial Bmpr1a is required for completion of tooth morphogenesis, and regulates terminal differentiation and proliferation in postnatal hair follicles.

The peroxisome proliferator-activated receptor γ (PPARγ) mediates the activity of the insulin-sensitizing thiazolidinediones and plays an important role in adipocyte differentiation and fat accretion. The analysis of PPARγ functions in mature adipocytes is precluded by lethality of PPARγ –/– fetuses and tetraploid-rescued pups. Therefore we have selectively ablated PPARγ in adipocytes of adult mice by using the tamoxifen-dependent Cre-ER T2 recombination system. We show that mature PPARγ-null white and brown adipocytes die within a few days and are replaced by newly formed PPARγ-positive adipocytes, demonstrating that PPARγ is essential for the in vivo survival of mature adipocytes, in addition to its well established requirement for their differentiation. Our data suggest that potent PPARγ antagonists could be used to acutely reduce obesity.

Friedreich ataxia (FRDA), the most common recessive ataxia, is characterized by degeneration of the large sensory neurons of the spinal cord and cardiomyopathy. It is caused by severely reduced levels of frataxin, a mitochondrial protein involved in iron-sulfur cluster (ISC) biosynthesis. Through a spatiotemporally controlled conditional gene-targeting approach, we have generated two mouse models for FRDA that specifically develop progressive mixed cerebellar and sensory ataxia, the most prominent neurological features of FRDA. Histological studies showed both spinal cord and dorsal root ganglia (DRG) anomalies with absence of motor neuropathy, a hallmark of the human disease. In addition, one line revealed a cerebellar granule cell loss, whereas both lines had Purkinje cell arborization defects. These lines represent the first FRDA models with a slowly progressive neurological degeneration. We identified an autophagic process as the causative pathological mechanism in the DRG, leading to removal of mitochondrial debris and apparition of lipofuscin deposits. These mice therefore represent excellent models for FRDA to unravel the pathological cascade and to test compounds that interfere with the degenerative process.

We generated two complementary systems for Cre-mediated recombination of target genes in the mouse digestive epithelium and tested them with a Cre-reporter mouse strain. Cre was expressed under the control of a 9 kb regulatory region of the murine villin gene (vil-Cre). Genetic recombination was initiated at embryonic day (E) 9 in the visceral endoderm, and by E12.5 in the entire intestinal epithelium, but not in other tissues. Cre expression was maintained throughout adulthood. Furthermore, transgenic mice bearing a tamoxifen-dependent Cre recombinase (vil-Cre-ERT2) expressed under the control of the villin promoter were created to perform targeted spatiotemporally controlled somatic recombination. After tamoxifen treatment, recombination was detectable throughout the digestive epithelium. The recombined locus persisted for 60 days after tamoxifen administration, despite rapid intestinal cell renewal, indicating that epithelial progenitor cells had been targeted. The villin-Cre and villin-Cre-ERT2 mice provide valuable tools for studies of cell lineage allocation and gene function in the developing and adult intestine.

The ability of a retinoid X receptor (RXR) to heterodimerize with many nuclear receptors, including LXR, PPAR, NGF1B and RAR, underscores its pivotal role within the nuclear receptor superfamily. Among these heterodimers, PPAR: RXR is considered an important signalling mediator of both PPAR ligands, such as fatty acids, and 9-cis retinoic acid (9-cis RA), an RXR ligand. In contrast, the existence of an RXR/9-cis RA signalling pathway independent of PPAR or any other dimerization partner remains disputed. Using in vivo chromatin immunoprecipitation, we now show that RXR homodimers can selectively bind to functional PPREs and induce transactivation. At the molecular level, this pathway requires stabilization of the homodimer-DNA complexes through ligand-dependent interaction with the coactivator SRC1 or TIF2. This pathway operates both in the absence and in the presence of PPAR, as assessed in cells carrying inactivating mutations in PPAR genes and in wildtype cells. In addition, this signalling pathway via PPREs is fully functional and can rescue the severe hypothermia phenotype observed in fasted PPARalpha(-/-) mice. These observations have important pharmacological implications for the development of new rexinoid-based treatments.

To generate temporally controlled targeted somatic mutations selectively and efficiently in hepatocytes, we established SA+/CreERT2 mice in which the tamoxifen-dependent Cre-ERT2 recombinase coding sequence preceded by an internal ribosomal entry site was inserted in the 3′ untranslated region of the serum albumin (SA) gene. Whereas the wildtype SA allele was strongly expressed in the liver and at lower levels in some extrahepatic tissues, SA-Cre-ERT2 fusion transcripts were only detected in the liver. The Cre-ERT2 protein was expressed in most if not all hepatocytes, and tamoxifen (Tam) treatments of adult mice at various ages efficiently induced Cre-mediated recombination of LoxP flanked (floxed) alleles in these cells, but none in other cell types or tissues. Thus, SA+/CreERT2 mice should be of great value to analyze gene function in the liver and to establish animal models of human diseases. genesis 39:167–172, 2004. © 2004 Wiley-Liss, Inc.

T cells develop through distinct stages directed by a series of signals. We explored the roles of SWI/SNF-like BAF chromatin remodeling complexes in this process by progressive deletion of the ATPase subunit, Brg, through successive stages of early T cell development. Brg-deficient cells were blocked at each of the developmental transitions examined. Bcl-xL overexpression suppressed cell death without relieving the developmental blockades, leading to the accumulation of Brg-deleted cells that were unexpectedly cell cycle arrested. These defects resulted partly from the disruptions of pre-TCR and potentially Wnt signaling pathways controlling the expression of genes such as c-Kit and c-Myc critical for continued development. Our studies indicate that BAF complexes dynamically remodel chromatin to propel sequential developmental transitions in response to external signals.

As locally converted estrogen from testicular testosterone contributes to apparent androgen activity, the physiological significance of androgen receptor (AR) function in the beneficial effects of androgens on skeletal tissues has remained unclear. We show here that inactivation of AR in mice using a Cre-loxP system-mediated gene-targeting technique caused bone loss in males but not in females. Histomorphometric analyses of 8-week-old male AR knockout (ARKO) mice showed high bone turnover with increased bone resorption that resulted in reduced trabecular and cortical bone mass without affecting bone shape. Bone loss in orchidectomized male ARKO mice was only partially prevented by treatment with aromatizable testosterone. Analysis of primary osteoblasts and osteoclasts from ARKO mice revealed that AR function was required for the suppressive effects of androgens on osteoclastogenesis supporting activity of osteoblasts but not on osteoclasts. Furthermore, expression of the receptor activator of NF-κB ligand ( RANKL ) gene, which encodes a major osteoclastogenesis inducer, was found to be up-regulated in osteoblasts from AR-deficient mice. Our results indicate that AR function is indispensable for male-type bone formation and remodeling.

TAF10 (formerly TAFII30), is a component of TFIID and the TATA box-binding protein (TBP)-free TAFcontaining complexes (TFTC/PCAF/STAGA). To investigate the physiological function of TAF10, we disrupted its gene in mice by using a Cre recombinase/LoxP strategy. Interestingly, no TAF10 / animals were born from intercrosses of TAF10 / mice, indicating that TAF10 is required for embryogenesis. TAF10 / embryos developed to the blastocyst stage, implanted, but died shortly after ca. 5.5 days postcoitus. Surprisingly, trophoblast cells from TAF10 / blastocysts were viable, whereas inner cell mass cells failed to survive, highlighting that TAF10 is not generally required for transcription in all cells. TAF10-deficient cells express normal levels of TBP and TAFs other than TAF10 but contain only partially formed TFIID, are endocycle arrested, and have undetectable levels of transcription. Thus, our results demonstrate that TAF10 is required for TFIID stability, cell cycle progression, and transcription in the early mouse embryo. Initiation of transcription at eukaryotic class II promoters is a multistep process requiring the coordinated action of many proteins. In general, RNA polymerase II (Pol II) and a host of other factors, including the general transcription factors TFIIA, -B, -D, -E, -F, and -H, work together to form a preinitiation complex (PIC) and allow subsequent transcription. The TATA-box-binding protein (TBP) and 14 evolutionarily conserved TBP-associated factors (TAFs) form TFIID (15). Multiple TFIID complexes exist, indeed nuclear extracts contain at least two subpopulations of TFIID: those that contain TAF10 and those that do not contain TAF10 (39). Electron microscopy has shown that TFIID contains surfaces that could mediate both extensive core promoter and protein-protein interactions (1, 4). Given its early requirement in vitro, as well as its capacity to direct PIC assembly on both TATA-containing and TATA-less promoters, TFIID was proposed to be a central component of the PIC. At present, the role played by TAFs in transcription is not fully understood. Nonetheless, in vitro transcription experiments have suggested that TAFs within TFIID function as coactivators by engaging in direct and selective interactions with transactivators and/or core promoter sequences (2). Furthermore, TAF1 (formerly TAFII250) (39) possesses kinase, histone acetyltransferase (HAT), and ubiquitin conjugating/ligating enzymatic activities (44), suggesting that TAFs can affect transcription at multiple levels. In yeast TAFs are required for viability, but strains lacking functional TAFs can activate transcription from a variety of inducible genes (38). Moreover, gene expression studies have demonstrated that TAFs have gene-selective effects, with histone fold domain (HFD)-containing TAFs having wide-ranging effects on transcription, whereas the effects of other TAFs appear to be more restricted (18, 23, 24). Taken together with experiments in Drosophila melanogaster and mammalian systems, these results suggest that TAFs, rather than being necessary for all activated transcription, have gene selective effects and therefore play specialized roles at certain subsets of promoters.

Inducible transgenesis provides a valuable technique for the analysis of gene function in vivo. We report the generation and characterization of mouse lines carrying glia lineage-specific transgenes expressing an improved variant of the tamoxifen-inducible Cre recombinase, CreERT2, where the recombinase is fused to a mutated ligand binding domain of the human estrogen receptor. Using a PLP-CreERT2 transgene, we have generated mice that show specific inducible Cre function, as analyzed by cross-breeding experiments into the Rosa26 Cre-LacZ reporter line, in developing and adult Schwann cells, in mature myelinating oligodendrocytes, and in undifferentiated NG2-positive oligodendrocyte precursors in the adult. Using a P0Cx-CreERT2 transgene, we have also established mouse lines with inducible Cre function specifically in the Schwann cell lineage. These tamoxifen-inducible CreERT2 lines will allow detailed spatiotemporally controlled analysis of gene functions in loxP-based conditional mutant mice in both developing and adult Schwann cells and in the oligodendrocyte lineage.

We have previously reported that the retinoic acid (RA) catabolizing enzyme CYP26A1 plays an important role in protecting tail bud tissues from inappropriate exposure to RA generated in the adjacent trunk tissues by RALDH2, and that Cyp26a1-null animals exhibit spina bifida and caudal agenesis. We now show that, in the absence of Cyp26a1, retinoic acid receptor gamma (RARγ) mediates ectopic RA-signaling in the tail bud. We also show that activated RARγ results in downregulation ofWnt3a and Fgf8, which integrate highly conserved signaling pathways known for their role in specifying caudal morphogenesis. Ablation of the gene for RARγ (Rarg) rescues Cyp26a1-null mutant animals from caudal regression and embryonic lethality, thus demonstrating that CYP26A1 suppresses the RA-mediated downregulation of WNT3A and FGF8 signaling pathways by eliminating ectopic RA in gastrulating tail bud mesoderm.

We have obtained a PrP-Cre-ER(T) transgenic mouse line (28.8) that selectively expresses in testis the tamoxifen-inducible Cre-ER(T) recombinase under the control of a mouse Prion protein (PrP) promoter-containing genomic fragment. Cre-ER(T) is expressed in spermatogonia and spermatocytes, but not in Sertoli and Leydig cells. We also established reporter PrP-L-EGFP-L transgenic mice harboring a LoxP-flanked enhanced green fluorescent protein (EGFP) Cre reporter cassette under the control of the same PrP promoter-containing genomic fragment that exhibits prominent EGFP expression in brain and testis. Using the PrP-L-EGFP-L as well as other Cre-reporter mice, we demonstrate that tamoxifen administration efficiently and selectively induces Cre-mediated recombination in the germ cell lineage. The established PrP-Cre-ER(T) line should provide a valuable tool for studying functions of germ cell-expressed genes involved in spermatogenesis.

Aims/hypothesis. The aim of this study is to investigate whether apoptosis in human beta cells can be related to the induction of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway. Methods. We examined the expression of TRAIL and TRAIL receptors in two human pancreatic beta-cell lines and in human primary islet cells using RT-PCR assays and flow cytometric analyses and tested TRAIL-mediated beta-cell destruction in 51Cr release cytotoxicity assays, Annexin-V and APO-DIREC assays. Results. Most of the human beta cells express TRAIL receptors-R1, -R2, -R3, -R4 and/or TRAIL. TRAIL induced much stronger cytotoxicity and apoptosis to beta-cell lines CM and HP62 than did FasL, TNF-α, LTα1β2, LTα2β1, LIGHT, and IFN-γ. The cytotoxicity and apoptosis induced by TRAIL to beta-cell lines CM were inhibited competitively by soluble TRAIL receptors, R1, R2, R3 or R4. Treatment of these beta cells with antibodies against TRAIL receptors was able to block the cytotoxicity of TRAIL to these cells. Beta-cell antigen-specific CTL (CD4+ and CD8+) clones express TRAIL, suggesting that these cells are potential sources of TRAIL-inducing beta-cell destruction. Normal primary islet cells from most donors are resistant to the cytotoxicity mediated by TRAIL. However, treatment with an inhibitor of protein synthesis (cycloheximide) or with an enzyme (PI-PLC) that can remove TRAIL-R3 from the islet-cell membrane was able to increase the susceptibility of TRAIL-resistant primary islet cells to the TRAIL death pathway. Conclusion/interpretation. The TRAIL death pathway is present and can function in human islet beta cells, but unidentified inhibitors of the TRAIL death pathway are present in normal islet cells.

Contractile myoepithelial cells dominate the basal layer of the mammary epithelium and are considered to be differentiated cells. However, we observe that up to 54% of single basal cells can form colonies when seeded into adherent culture in the presence of agents that disrupt actin-myosin interactions, and on average, 65% of the single-cell-derived basal colonies can repopulate a mammary gland when transplanted in vivo. This indicates that a high proportion of basal myoepithelial cells can give rise to a mammary repopulating unit (MRU). We demonstrate that myoepithelial cells, flow-sorted using two independent myoepithelial-specific reporter strategies, have MRU capacity. Using an inducible lineage-tracing approach we follow the progeny of myoepithelial cells that express α-smooth muscle actin and show that they function as long-lived lineage-restricted stem cells in the virgin state and during pregnancy.

Vascular endothelial growth factor (VEGF) is exercise responsive, pro-angiogenic, and expressed in several muscle cell types. We hypothesized that in adult mice, VEGF generated within skeletal myofibers (and not other cells within muscle) is necessary for the angiogenic response to exercise training. This was tested in adult conditional, skeletal myofiber-specific VEGF gene-deleted mice (skmVEGF-/-), with VEGF levels reduced by >80%. After 8 wk of daily treadmill training, speed and endurance were unaltered in skmVEGF-/- mice, but increased by 18% and 99% (P < 0.01), respectively, in controls trained at identical absolute speed, incline, and duration. In vitro, isolated soleus and extensor digitorum longus contractile function was not impaired in skmVEGF-/- mice. However, training-induced angiogenesis was inhibited in plantaris (wild type, 38%, skmVEGF-/- 18%, P < 0.01), and gastrocnemius (wild type, 43%, P < 0.01; skmVEGF-/-, 7%, not significant). Capillarity was maintained (different from VEGF gene deletion targeted to multiple cell types) in untrained skmVEGF-/- mice. Arteriogenesis (smooth muscle actin+, artery number, and diameter) and remodeling [vimentin+, 5′-bromodeoxycytidine (BrdU)+, and F4/80+ cells] occurred in skmVEGF-/- mice, even in the absence of training. skmVEGF-/- mice also displayed a limited oxidative enzyme [citrate synthase and β-hydroxyacyl CoA dehydrogenase (β-HAD)] training response; β-HAD activity levels were elevated in the untrained state. These data suggest that myofiber expressed VEGF is necessary for training responses in capillarity and oxidative capacity and for improved running speed and endurance. © 2014 the American Physiological Society.

Importance The disease process leading to clinical type 1 diabetes often starts during the first years of life. Early exposure to complex dietary proteins may increase the risk of β-cell autoimmunity in children at genetic risk for type 1 diabetes. Extensively hydrolyzed formulas do not contain intact proteins. Objective To test the hypothesis that weaning to an extensively hydrolyzed formula decreases the cumulative incidence of diabetes-associated autoantibodies in young children. Design, Setting, and Participants A double-blind randomized clinical trial of 2159 infants with HLA-conferred disease susceptibility and a first-degree relative with type 1 diabetes recruited from May 2002 to January 2007 in 78 study centers in 15 countries; 1078 were randomized to be weaned to the extensively hydrolyzed casein formula and 1081 were randomized to be weaned to a conventional cows’ milk–based formula. The participants were observed to April 16, 2013. Interventions The participants received either a casein hydrolysate or a conventional cows’ milk formula supplemented with 20% of the casein hydrolysate. Main Outcomes and Measures Primary outcome was positivity for at least 2 diabetes-associated autoantibodies out of 4 analyzed. Autoantibodies to insulin, glutamic acid decarboxylase, and the insulinoma-associated–2 (IA-2) molecule were analyzed using radiobinding assays and islet cell antibodies with immunofluorescence during a median observation period of 7.0 years (mean, 6.3 years). Results The absolute risk of positivity for 2 or more islet autoantibodies was 13.4% among those randomized to the casein hydrolysate formula (n = 139) vs 11.4% among those randomized to the conventional formula (n = 117). The unadjusted hazard ratio for positivity for 2 or more autoantibodies among those randomized to be weaned to the casein hydrolysate was 1.21 (95% CI, 0.94-1.54), compared with those randomized to the conventional formula, while the hazard ratio adjusted for HLA risk, duration of breastfeeding, vitamin D use, study formula duration and consumption, and region was 1.23 (95% CI, 0.96-1.58). There were no clinically significant differences in the rate of reported adverse events between the 2 groups. Conclusions and Relevance Among infants at risk for type 1 diabetes, the use of a hydrolyzed formula, when compared with a conventional formula, did not reduce the incidence of diabetes-associated autoantibodies after 7 years. These findings do not support a benefit from hydrolyzed formula. Trial Registration clinicaltrials.gov Identifier: NCT00179777 Type 1 diabetes is characterized by selective loss of insulin-producing β cells in the pancreatic islets in genetically susceptible individuals. Overt clinical disease is preceded by an asymptomatic period of highly variable duration1 during which diabetes-associated autoantibodies appear in the peripheral circulation as markers of emerging β-cell autoimmunity. Several disease-related autoantibodies predict clinical type 1 diabetes including classical islet cell antibodies (ICA), insulin autoantibodies, autoantibodies to glutamic acid decarboxylase (GAD), and the tyrosine phosphatase-related insulinoma-associated 2 molecule (IA-2).2 In natural history studies from infancy, positivity for at least 2 autoantibodies signals a risk of approximately 60% for the development of clinical diabetes over 10 years, whereas the 10-year risk among those with a single autoantibody is about 15% and among those with no detectable autoantibodies less than 1%.3 Accumulating evidence suggests that β-cell autoimmunity emerges early in life.4,5 The incidence of type 1 diabetes is increasing among children in Europe and North America,6,7 although some studies suggest it may be stabilizing.8 This scenario implies that any measure aimed at primary prevention of type 1 diabetes, ie, prevention of the initiation of the diabetic disease process, has to be started in infancy. Early feeding may modify the risk of type 1 diabetes later in life. Some epidemiological and immunological studies suggest that exposure to complex foreign proteins in early infancy may increase the risk of β-cell autoimmunity and type 1 diabetes in genetically susceptible individuals,9- 11 although others do not.12,13 A pilot study suggested that weaning to an extensively hydrolyzed casein formula (99.7% of the generated peptides having a molecular weight of less than 2000 Da) decreased the cumulative incidence of diabetes-associated autoantibodies in children with an affected first-degree relative and a risk-associated HLA genotype.14,15 This led to TRIGR (Trial to Reduce IDDM in the Genetically at Risk), with the study powered to assess the effect of the intervention on the development of type 1 diabetes by age 10 years. A prior prespecified end point, early humoral β-cell autoimmunity, is reported herein.

To develop spatio-temporally controlled somatic mutagenesis in the adult mouse nervous system, we established transgenic mice expressing the tamoxifen-inducible Cre-ERT recombinase under the control of the mouse prion protein (PrP) promoter. Cre-ERT was expressed in most regions of the brain and in the retina of one transgenic line, whereas its expression was mostly restricted to the hippocampus and the cerebellum in another line. As tamoxifen efficiently induced Cre-mediated recombination in the various neuronal cell types expressing Cre-ERT in the brain of adult mice, the PrP-Cre-ERT lines should be valuable tools to study the functions of genes involved in neurodegenerative diseases or regeneration, and in complex processes such as behaviour, learning and memory. Some limitations of presently available reporter lines for Cre-mediated recombination in adult mouse CNS are discussed.

Familial glucocorticoid deficiency due to corticotropin (ACTH) resistance consists of two distinct genetic syndromes that are both inherited as autosomal recessive traits: isolated ACTH resistance (iACTHR), which may be caused by inactivating mutations of the ACTH receptor (the MC2R gene) or mutations in an as yet unknown gene(s), and Allgrove syndrome (AS). The latter is also known as triple-A syndrome (MIM 231550). In three large cohorts of AS kindreds, the disease has been mapped to chromosome 12; most recently, mutations in the AAAS gene on 12q13 were found in these AS families. AAAS codes for the WD-repeat containing ALADIN (for alacrima-achalasia-adrenal insufficiency-neurologic disorder) protein. We investigated families with iACTHR (n = 4) and AS (n = 6) and a Bedouin family with ACTHR and a known defect of the TSH receptor. Four AS families were of mixed extraction from Puerto Rico (PR); most of the remaining six families were Caucasian families from North America (NA). Sequencing analysis found no MC2R genetic defects in any of the kindreds. No iACTHR kindreds, but all of AS families, had AAAS mutations. The previously reported IVS14+1G-->A splice donor mutation was found in all PR families, apparently due to a founder effect; one NA kindred was heterozygous for this mutation. In the latter family, long-range PCR failed to identify a deletion or other rearrangements of the AAAS gene. No other heterozygote or transmitting parent had any phenotype that could be considered part of AS. The IVS14+1G-->A mutation results in a premature termination of the predicted protein; although it was present in all PR families (in the homozygote state in three of them), there was substantial clinical variation between them. One PR family also carried a novel splice donor mutation of the AAAS gene in exon 11, IVS11+1G-->A; the proband was a compound heterozygote. A novel point mutation, 43C-->A(Gln15Lys), in exon 1 of the AAAS gene was identified in the homozygote state in a Canadian AS kindred with a milder AS phenotype. The predicted amino acid substitution in this family is located in a sequence that may participate in the preservation of stability of ALADIN beta-strands, whereas the splicing mutation in exon 11 may interfere with the formation of WD repeats in this molecule. We conclude that 1) AAAS does not appear to be frequently mutated in families with iACTHR; 2) AAAS is mutated in AS families from PR (that had previously been mapped to 12q13) and NA; and, 3) there is significant clinical variability between patients with the same AAAS defect.

The efficient introduction of somatic mutations in a given gene, at a given time, in a specific cell type, will facilitate studies of gene function and the generation of animal models for human diseases. We have established a conditional site-specific recombination system in mice using a new version of the Cre/lox system. The Cre recombinase has been fused to a mutated ligand binding domain of the human estrogen receptor (ER), resulting in a tamoxifen-dependent Cre recombinase, Cre-ER(T), that is activated by tamoxifen, but not by estradiol. Transgenic mice were generated expressing Cre-ER(T) under the control of a cytomegalovirus promoter. Administration of tamoxifen to these transgenic mice induced excision of a chromosomally integrated gene flanked by loxP sites in a number of tissues, whereas no excision could be detected in untreated animals. However, the efficiency of excision varied between tissues, and the highest level (approximately 40%) was obtained in the skin. To determine the efficiency of excision mediated by Cre-ER(T) in a given cell type, Cre-ER(T)-expressing mice were crossed with reporter mice in which expression of Escherichia coli beta-galactosidase can be induced through Cre-mediated recombination. The efficiency and kinetics of this recombination were analyzed at the cellular level in the epidermis of 6- to 8-week-old double transgenic mice. Site-specific excision occurred within a few days of tamoxifen treatment in essentially all epidermis cells expressing Cre-ER(T). These results indicate that cell-specific expression of Cre-ER(T) in transgenic mice can be used for efficient tamoxifen-dependent Cre-mediated recombination at loci containing loxP sites, to generate site-specific somatic mutations in a spatiotemporally controlled manner. This conditional site-specific recombination system should allow the analysis of knockout phenotypes that cannot be addressed by conventional gene targeting.

OBJECTIVE: To establish the prevalence of celiac disease (CD) in children with type 1 diabetes in British Columbia.PATIENTS AND METHODS: Two hundred thirty-three children with type 1 diabetes were prospectively screened for CD using blind testing with the current 'gold standard', immunoglobulin A endomysium antibody (EmA), and the novel immunoglobulin A tissue transglutaminase (tTG) antibody. Those children with positive results were offered small bowel biopsy; a gluten-free diet was recommended if CD was confirmed.RESULTS: Nineteen children were positive for EmA and had an elevated tTG level. One patient from this group was already known to have CD, and the other 18 patients consented to biopsy. One biopsy was normal, three biopsies demonstrated elevated intraepithelial lymphocyte counts with normal morphology and 14 biopsies had morphological changes consistent with CD. Growth parameters were normal in all patients, and nine of 19 children who were positive for EmA were asymptomatic. Seven patients had mild elevation of tTG levels alone. Two children from this latter group had normal biopsies, and five declined biopsy.CONCLUSIONS: At least 14 new cases of CD were detected in addition to four known cases, yielding an overall biopsy-confirmed prevalence of CD of 7.7% (18 of 233). The present study confirms that CD is as prevalent in the pediatric type 1 diabetic population in British Columbia as it is in Europe. Serological screening of these children is important because many children have no symptoms or signs suggestive of CD. This study suggests that tTG serology may also be useful in monitoring response and compliance with a gluten-free diet.

The study of gene functions in complex genetic environments such as mammalian cells would greatly benefit from systems allowing a tight control of gene expression. The tetracycline-inducible gene expression system and the site-specific Cre/loxP recombination system have gained increasing popularity for conditional expression and gene disruption. To facilitate the analysis of gene functions in a cell autonomous system, we have established an F9 murine embryonal carcinoma cell line, constitutively expressing both the doxycycline-controlled transactivator rtTA and the tamoxifen-dependent Cre recombinase Cre-ERT. The expression of a reporter gene placed under the control of tetracycline operators was induced about 1000-fold by doxycycline, and tamoxifen-induced excision of a loxP-flanked DNA segment occurred in all cells. This genetically engineered cell line, which allows, upon simple ligand addition, sophisticated genetic manipulations, such as sequential inactivation of loxP-flanked genes, and tightly controlled reexpression of their cDNAs, should be a valuable tool for studying mammalian gene functions.

Estrogen receptor (ER) alpha and beta mediate estrogen actions in target cells through transcriptional control of target gene expression. For 17beta-estradiol-induced transactivation, the N-terminal A/B domain (AF-1) and the C-terminal E/F domain (AF-2) of ERs are required. Ligand binding is considered to induce functional synergism between AF-1 and AF-2, but the molecular mechanism remains unknown. To clarify this synergism, we studied the role of reported AF-2 coactivators, p300/CREB binding protein, steroid receptor coactivator-1/transcriptional intermediary factor-2 (SRC-1/TIF2) family proteins and thyroid hormone receptor-associated protein-220/(vitamin D3 receptor-interacting protein- 205-(TRAP220/DRIP205) on the AF-1 activity in terms of synergism with the AF-2 function. We found that neither any of the SRC-1/TIF2 family coactivators nor TRAP220/DRIP205 is potent, whereas p300 potentiates the AF-1 function of both human ERalpha and human ERbeta. Direct interactions of p300 with the A/B domains of ERalpha and ERbeta were observed in an in vitro glutathione S-transferase pull-down assay in accordance with the interactions in yeast and mammalian two-hybrid assays. Furthermore, mutations in the p300 binding sites (56-72 amino acids in ERalpha and 62-72 amino acids in ERbeta) in the A/B domains caused a reduction in ligand-induced transactivation functions of both ERalpha and ERbeta. Thus, these findings indicate that ligand-induced functional synergism between AF-1 and AF-2 is mediated through p300 by its direct binding to the A/B regions of ERalpha and ERbeta.

Ligand-dependent site-specific recombinases are powerful tools to engineer the mouse genome in specific somatic cell types at selected times during pre- and postnatal development. Current efforts are primarily directed towards increasing the efficiency of this recombination system in mice. We have generated transgenic mouse lines expressing a tamoxifen-activated Cre recombinase, CreER(T2), under the control of the smooth muscle-specific SM22 promoter. Both a randomly integrated transgene [SM-CreER(T2)(tg)] and a transgene that has been "knocked in" into the endogenous SM22 locus [SM-CreER(T2)(ki)] were expressed in smooth muscle-containing tissues. The level of CreER(T2) expression and tamoxifen-induced recombination was lower in SM-CreER(T2)(tg) mice compared with SM-CreER(T2)(ki) mice. Whereas no recombinase activity could be detected in vehicle-treated SM-CreER(T2)(ki) mice, administration of tamoxifen induced the excision of a loxP-flanked reporter transgene in up to 100% of smooth muscle cells. The recombined genome persisted for at least four months after tamoxifen treatment. SM-CreER(T2)(ki) transgenic mice should be useful to study the effects of various somatic mutations in smooth muscle.

OBJECTIVE: To establish the prevalence of celiac disease (CD) in girls with Turner syndrome (TS) in British Columbia.METHODS: Forty-five girls with TS were prospectively screened for CD using blinded testing with the current ’gold standard’ - immunoglobulin A (IgA) endomysium antibody (EmA) and the novel IgA tissue transglutaminase antibody (tTG). Those with positive results were offered small bowel biopsies, and a gluten-free diet was recommended if CD was confirmed.RESULTS: One asymptomatic prepubertal East Indian girl was positive for EmA, had an elevated tTG concentration of 560 U/mL and histological evidence of CD. Seven girls were negative for EmA but had elevated tTG concentrations (175 to 250 U/mL); five were white, one was Asian and one was East Indian. Small bowel biopsies were performed on three girls, and the histologies were normal. The remaining four patients declined biopsy.CONCLUSIONS: One girl with TS was identified with CD from 45 screened, giving an overall biopsy-confirmed prevalence of 2.2%. This study confirms previous observations placing girls with TS at higher risk for CD and suggests a similar high prevalence in British Columbia.

Conditional DNA excision between two LoxP sites can be achieved in the mouse using Cre-ER(T), a fusion protein between a mutated ligand binding domain of the human estrogen receptor (ER) and the Cre recombinase, the activity of which can be induced by 4-hydroxy-tamoxifen (OHT), but not natural ER ligands. We have recently characterized a new ligand-dependent recombinase, Cre-ER(T2), which was approximately 4-fold more efficiently induced by OHT than Cre-ER(T) in cultured cells. In order to compare the in vivo efficiency of these two ligand-inducible recombinases to generate temporally-controlled somatic mutations, we have engineered transgenic mice expressing a LoxP-flanked (floxed) transgene reporter and either Cre-ER(T) or Cre-ER(T2) under the control of the bovine keratin 5 promoter that is specifically active in the epidermis basal cell layer. No background recombinase activity could be detected, while recombination was induced in basal keratinocytes upon OHT administration. Interestingly, a dose-response study showed that Cre-ER(T2) was approximately 10-fold more sensitive to OHT induction than Cre-ER(T).

The estrogen receptor (ER) regulates the expression of target genes in a ligand-dependent manner. The ligand-dependent activation function AF-2 of the ER is located in the ligand binding domain (LBD), while the N-terminal A/B domain (AF-1) functions in a ligand-independent manner when isolated from the LBD. AF-1 and AF-2 exhibit cell type and promoter context specificity. Furthermore, the AF-1 activity of the human ERalpha (hERalpha) is enhanced through phosphorylation of the Ser(118) residue by mitogen-activated protein kinase (MAPK). From MCF-7 cells, we purified and cloned a 68-kDa protein (p68) which interacted with the A/B domain but not with the LBD of hERalpha. Phosphorylation of hERalpha Ser(118) potentiated the interaction with p68. We demonstrate that p68 enhanced the activity of AF-1 but not AF-2 and the estrogen-induced as well as the anti-estrogen-induced transcriptional activity of the full-length ERalpha in a cell-type-specific manner. However, it did not potentiate AF-1 or AF-2 of ERbeta, androgen receptor, retinoic acid receptor alpha, or mineralocorticoid receptor. We also show that the RNA helicase activity previously ascribed to p68 is dispensable for the ERalpha AF-1 coactivator activity and that p68 binds to CBP in vitro. Furthermore, the interaction region for p68 in the ERalpha A/B domain was essential for the full activity of hERalpha AF-1. Taken together, these findings show that p68 acts as a coactivator specific for the ERalpha AF-1 and strongly suggest that the interaction between p68 and the hERalpha A/B domain is regulated by MAPK-induced phosphorylation of Ser(118).

To fully characterize human glutamic acid decarboxylase (GAD)65 protein T-cell epitopes associated with insulin-dependent diabetes mellitus (IDDM), CTL clones specific to GAD65 protein antigens were isolated from two congenital rubella syndrome (CRS)-associated IDDM patients. Overlapping nonamer T-cell epitopes recognized by both CD4 + or CD8 + CTL clones within peptides GAD65(252-266) and GAD65(274-286) were identified as sequences bounded by GAD65(255-266) with 6/9 overlapping residues, and GAD65(276-285) with 8/9 overlapping residues, respectively, using two panels of overlapping peptide analogs in cytotoxicity assays. HLA restrictive elements of the T-cell clones were also identified using a panel of B cell lines with different HLA phenotypes as targets in cytotoxicity assays. The antigenic GAD65 peptides elicited cytotoxic responses of peptide-specific CD4 + T-cell clones in the context of HLA DRB1∗0404. The CD8 + T-cell clone specific to GAD65(255-263) was found to be restricted by HLA A3 and A11. Similarly, the CD8 + T-cell clone specific to GAD65(277-285) killed peptide-sensitized target cells expressing HLA B35 and B15. The observed HLA restriction of these overlapping epitopes implies that a tandem of [DRB1∗0404-A11(3)] and/or a tandem of [DRB1∗0404-B35(15)] might predispose CRS patients to development of IDDM.

Antenatal treatment for classic 21-hydroxylase forms of congenital adrenal hyperplasia and the issues

The active form of vitamin D, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3; calcitriol], plays a key role in mineral and bone homeostasis, and exerts potent anti-inflammatory and anti-proliferative activities [1]. It is thus a potential pharmacological agent to treat various diseases, including autoimmune disorders, infections and cancer [1]. However, the 1,25(OH)2D3 doses required to elicit such effects induce hypercalcemia, resulting in ectopic calcification of the vascular wall, kidney and other soft tissues, that can lead to organ failure and death [1]. 1,25(OH)2D3 activities are mediated by the Vitamin D receptor (VDR; NR1I1), a member of the nuclear receptor superfamily [1]. More than 3000 VDR ligands were synthesized using medicinal chemistry approaches, but all those exhibiting potent anti-inflammatory and/or anti-proliferative properties still have hypercalcemic activities, which limit their clinical use [1]. VDR loss-of-function mutations in humans, termed Vitamin D-Dependent Rickets type-II (VDDR-II), and VDR-null mice develop skeletal deformities, osteomalacia, hypocalcemia and hypophosphatemia [2]. In addition, VDR DNA-binding deficient patients and VDR-null mice display alopecia [2]. Hair follicle defects in VDR-null mice are prevented by transgenic expression of ligand-binding deficient VDR in keratinocytes [3]. In contrast, severe deficiencies in 1,25(OH)2D3 induced by dysfunctional 25(OH)-vitaminD3-1α-hydroxylase (Cyp27b1), the enzyme that converts 25(OH)D3 to 1,25(OH)2D3 in VDDR-I patients, as well as in Cyp27b1-null mice, do not induce alopecia, even though they lead to rickets, which can be treated by 1,25(OH)2D3 [2, 4]. Similarly, patients and mice expressing a mutated VDR with reduced affinity for 1,25(OH)2D3 without impairing DNA binding have skeletal but no hair defects [2]. Thus, it appears that liganded VDR is essential for mineral ion homeostasis and skeletal metabolism, whereas ligand-independent VDR activities control hair cycling. Phenotypic analyses of mutant mice suggested that bone and mineral homeostasis alterations might be more pronounced in Cyp27b1-null mice than in VDR-null [4, 5]. Moreover, data presented in a recent report indicated that the size of mice expressing VDRL233S, a VDR bearing a mutation in the ligand binding domain that impairs calcitriol binding, was reduced compared to VDR-null [3]. Taken together, these results indicated that unliganded VDR might induce more severe skeletal defects than VDR deficiency. To characterise VDR ligand-independent activities, we generated, based on VDR structural analyses, mice expressing a ligand binding domain point-mutated VDR (VDRgem) that is unresponsive to 1,25(OH)2D3 [6]. Phenotypic analyses showed that mineral ion and bone homeostasis was more impaired in VDRgem than in VDR-null mice, even though they had no hair defects [6]. Moreover, selective ablation of VDR in intestinal epithelial cells did not affect mineral ion homeostasis [7], while selective expression of VDRgem in such cells induced hypocalcemia (our unpublished data), thus demonstrating that intestinal unliganded VDR strongly impairs calcium homeostasis. Importantly, our study revealed that apo-VDRgem binds to VDR response elements of VDR target genes in the mouse duodenum and represses many genes [6]. Therefore, the repressive activity of unliganded VDR in intestinal epithelial cells accounts, at least in part, for increased bone and mineral defects, compared to VDR deficiency. Alopecia in VDDR-II patients is thought to be associated with the severity of rickets and metabolic abnormalities [2]. However, as our data show that mice expressing ligand-binding deficient VDR develop more severe skeletal defects than VDR-null mice, and that only the latter have hair defects, it will important to determine whether it is also the case in VDDR-II patients, to improve the diagnosis and treatment. The possibility to restore the transcriptional activity of VDR mutants that are unresponsive to natural ligands is instrumental to identify VDR target genes. However, amongst various VDR agonist tested, none induced VDRL233S transcriptional activity (our unpublished data). In contrast, we have shown that VDRgem transcriptional activity is efficiently induced by gemini ligands, and that they restore serum calcium levels of VDRgem mice, thus opening new avenues to characterize gene networks controlled by VDR ligands in the mouse [6]. Further phenotypic and molecular analyses of VDRgem, VDR-null and wild-type mice treated or not with 1,25(OH)2D3 or gemini ligands should allow to identify the signaling pathways controlled by unliganded and liganded VDR in various tissues, and thus facilitate the identification of new drug targets for various diseases, as well as VDR ligands with increased selectivity.