Increasing maternal glucose concentrations is insufficient to restore placental glucose transfer in chorionic somatomammotropin RNA interference pregnancies.

We previously demonstrated impaired placental nutrient transfer in chorionic somatomammotropin (CSH) RNA interference (RNAi) pregnancies, with glucose transfer being the most impacted. Thus, we hypothesized that despite experimentally elevating maternal glucose, diminished umbilical glucose uptake would persist in CSH RNAi pregnancies, demonstrating the necessity of CSH for adequate placental glucose transfer. Trophectoderm of sheep blastocysts (9 days of gestational age; dGA) were infected with a lentivirus expressing either nontargeting control (CON RNAi; n = 5) or CSH-specific shRNA (CSH RNAi; n = 7) before transfer into recipient sheep. At 126 dGA, pregnancies were fitted with vascular catheters and underwent steady-state metabolic studies (3H2O transplacental diffusion) at 137 ± 0 dGA, before and during a maternal hyperglycemic clamp. Umbilical glucose and oxygen uptakes, as well as insulin and IGF1 concentrations, were impaired (P ≤ 0.01) in CSH RNAi fetuses and were not rescued by elevated maternal glucose. This is partially due to impaired uterine and umbilical blood flow (P ≤ 0.01). However, uteroplacental oxygen utilization was greater (P ≤ 0.05) during the maternal hyperglycemic clamp, consistent with greater placental oxidation of substrates. The relationship between umbilical glucose uptake and the maternal-fetal glucose gradient was analyzed, and while the slope (CON RNAi, Y = 29.54X +74.15; CSH RNAi, Y = 19.05X + 52.40) was not different, the y-intercepts and elevation were (P = 0.003), indicating reduced maximal glucose transport during maternal hyperglycemia. Together, these data suggested that CSH plays a key role in modulating placental metabolism that ultimately promotes maximal placental glucose transfer.NEW & NOTEWORTHY The current study demonstrated a novel, critical autocrine role for chorionic somatomammotropin in augmenting placental glucose transfer and maintaining placental oxidative metabolism. In pregnancies with CSH deficiency, excess glucose in maternal circulation is insufficient to overcome fetal hypoglycemia due to impaired placental glucose transfer and elevated placental metabolic demands. This suggests that perturbations in glucose transfer in CSH RNAi pregnancies are due to compromised metabolic efficiency along with reduced placental mass.

Fetal Hypoglycemia Induced by Placental SLC2A3-RNA Interference Alters Fetal Pancreas Development and Transcriptome at Mid-Gestation.

Glucose, the primary energy substrate for fetal oxidative processes and growth, is transferred from maternal to fetal circulation down a concentration gradient by placental facilitative glucose transporters. In sheep, SLC2A1 and SLC2A3 are the primary transporters available in the placental epithelium, with SLC2A3 located on the maternal-facing apical trophoblast membrane and SLC2A1 located on the fetal-facing basolateral trophoblast membrane. We have previously reported that impaired placental SLC2A3 glucose transport resulted in smaller, hypoglycemic fetuses with reduced umbilical artery insulin and glucagon concentrations, in addition to diminished pancreas weights. These findings led us to subject RNA derived from SLC2A3-RNAi (RNA interference) and NTS-RNAi (non-targeting sequence) fetal pancreases to qPCR followed by transcriptomic analysis. We identified a total of 771 differentially expressed genes (DEGs). Upregulated pathways were associated with fat digestion and absorption, particularly fatty acid transport, lipid metabolism, and cholesterol biosynthesis, suggesting a potential switch in energetic substrates due to hypoglycemia. Pathways related to molecular transport and cell signaling in addition to pathways influencing growth and metabolism of the developing pancreas were also impacted. A few genes directly related to gluconeogenesis were also differentially expressed. Our results suggest that fetal hypoglycemia during the first half of gestation impacts fetal pancreas development and function that is not limited to ß cell activity.

Impact of Placental SLC2A3 Deficiency during the First-Half of Gestation.

In the ruminant placenta, glucose uptake and transfer are mediated by facilitative glucose transporters SLC2A1 (GLUT1) and SLC2A3 (GLUT3). SLC2A1 is located on the basolateral trophoblast membrane, whereas SLC2A3 is located solely on the maternal-facing, apical trophoblast membrane. While SLC2A3 is less abundant than SLC2A1, SLC2A3 has a five-fold greater affinity and transport capacity. Based on its location, SLC2A3 likely plays a significant role in the uptake of glucose into the trophoblast. Fetal hypoglycemia is a hallmark of fetal growth restriction (FGR), and as such, any deficiency in SLC2A3 could impact trophoblast glucose uptake and transfer to the fetus, thus potentially setting the stage for FGR. By utilizing in vivo placenta-specific lentiviral-mediated RNA interference (RNAi) in sheep, we were able to significantly diminish (p ≤ 0.05) placental SLC2A3 concentration, and determine the impact at mid-gestation (75 dGA). In response to SLC2A3 RNAi (n = 6), the fetuses were hypoglycemic (p ≤ 0.05), exhibited reduced fetal growth, including reduced fetal pancreas weight (p ≤ 0.05), which was associated with reduced umbilical artery insulin and glucagon concentrations, when compared to the non-targeting sequence (NTS) RNAi controls (n = 6). By contrast, fetal liver weights were not impacted, nor were umbilical artery concentrations of IGF1, possibly resulting from a 70% increase (p ≤ 0.05) in umbilical vein chorionic somatomammotropin (CSH) concentrations. Thus, during the first half of gestation, a deficiency in SLC2A3 results in fetal hypoglycemia, reduced fetal development, and altered metabolic hormone concentrations. These results suggest that SLC2A3 may be the rate-limiting placental glucose transporter during the first-half of gestation in sheep.

CSH RNA Interference Reduces Global Nutrient Uptake and Umbilical Blood Flow Resulting in Intrauterine Growth Restriction.

Deficiency of the placental hormone chorionic somatomammotropin (CSH) can lead to the development of intrauterine growth restriction (IUGR). To gain insight into the physiological consequences of CSH RNA interference (RNAi), the trophectoderm of hatched blastocysts (nine days of gestational age; dGA) was infected with a lentivirus expressing either a scrambled control or CSH-specific shRNA, prior to transfer into synchronized recipient sheep. At 90 dGA, umbilical hemodynamics and fetal measurements were assessed by Doppler ultrasonography. At 120 dGA, pregnancies were fitted with vascular catheters to undergo steady-state metabolic studies with the 3H2O transplacental diffusion technique at 130 dGA. Nutrient uptake rates were determined and tissues were subsequently harvested at necropsy. CSH RNAi reduced (p ≤ 0.05) both fetal and uterine weights as well as umbilical blood flow (mL/min). This ultimately resulted in reduced (p ≤ 0.01) umbilical IGF1 concentrations, as well as reduced umbilical nutrient uptakes (p ≤ 0.05) in CSH RNAi pregnancies. CSH RNAi also reduced (p ≤ 0.05) uterine nutrient uptakes as well as uteroplacental glucose utilization. These data suggest that CSH is necessary to facilitate adequate blood flow for the uptake of oxygen, oxidative substrates, and hormones essential to support fetal and uterine growth.

2-deoxy D-glucose treatment does not elicit a hair growth response in alopecia areata.

2-deoxy D-glucose (2DG) was tested for efficacy in treating alopecia areata using the C3H/HeJ skin graft model. 2DG has proven to be efficacious in treatment of various mouse models of autoimmunity with minimal serious side effects noted. This agent has been shown to normalize abnormally activated T-cell populations while also preventing cell surface expression of NKG2D; key factors defining alopecia areata disease progression. Daily oral ingestion of 2DG via drinking water to mice with patchy or diffuse alopecia areata for 16 weeks failed to prevent expansion of alopecia or cause regrowth of hair in treated mice. Histologically, there were no differences between treated and control groups. These results indicate that, while 2DG is effective for some autoimmune diseases, it was not efficacious for the cell-mediated autoimmune mouse disease, alopecia areata.

Nail abnormalities identified in an ageing study of 30 inbred mouse strains.

In a large-scale ageing study, 30 inbred mouse strains were systematically screened for histologic evidence of lesions in all organ systems. Ten strains were diagnosed with similar nail abnormalities. The highest frequency was noted in NON/ShiLtJ mice. Lesions identified fell into two main categories: acute to chronic penetration of the third phalangeal bone through the hyponychium with associated inflammation and bone remodelling or metaplasia of the nail matrix and nail bed associated with severe orthokeratotic hyperkeratosis replacing the nail plate. Penetration of the distal phalanx through the hyponychium appeared to be the initiating feature resulting in nail abnormalities. The accompanying acute to subacute inflammatory response was associated with osteolysis of the distal phalanx. Evaluation of young NON/ShiLtJ mice revealed that these lesions were not often found, or affected only one digit. The only other nail unit abnormality identified was sporadic subungual epidermoid inclusion cysts which closely resembled similar lesions in human patients. These abnormalities, being age-related developments, may have contributed to weight loss due to impacts upon feeding and should be a consideration for future research due to the potential to interact with other experimental factors in ageing studies using the affected strains of mice.

Gain of function p.E138A alteration in Card14 leads to psoriasiform skin inflammation and implicates genetic modifiers in disease severity.

Psoriasis (PS) is a common inflammatory and incurable skin disease affecting 2-3% of the human population. Although genome-wide association studies implicate more than 60 loci, the full complement of genetic factors leading to disease is not known. Rare, highly penetrant, gain-of-function, dominantly acting mutations within the human caspase recruitment domain family, member 14 (CARD14) gene lead to the development of PS and psoriatic arthritis (PSA) (a familial p.G117S and de-novo p.E138A alteration). These residues are conserved in mouse and orthologous Knock-In (KI) mutations within Card14 were created. The Card14tm.1.1Sun allele (G117S) resulted in no clinically or histologically evident phenotype of the skin or joints in young adult or old mice. However, mice carrying the Card14tm2.1Sun mutant allele (E138A) were runted and developed thick, white, scaly skin soon after birth, dying within two weeks or less. The skin hyperplasia and inflammation was remarkable similarity to human PS at the clinical, histological, and transcriptomic levels. For example, the skin was markedly acanthotic and exhibited orthokeratotic hyperkeratosis with minimal inflammation and no pustules and transcripts affecting critical pathways of epidermal differentiation and components of the IL17 axis (IL23, IL17A, IL17C, TNF and IL22) were altered. Similar changes were seen in a set of orthologous microRNAs previously associated with PS suggesting conservation across species. Crossing the Card14tm2.1Sun/WT mice to C57BL/6NJ, FVB/NJ, CBA/J, C3H/HeJ, and 129S1/SvImJ generated progeny with epidermal acanthosis and marked orthokeratotic hyperkeratosis regardless of the hybrid strain. Of these hybrid lines, only the FVB;B6N(129S4) mice survived to 250 days of age or older and has led to recombinant inbred lines homozygous for Card14E138A that are fecund and have scaly skin disease. This implicates that modifiers of PS severity exist in mice, as in the familial forms of the disease in humans.

Hyaline Arteriolosclerosis in 30 Strains of Aged Inbred Mice.

During a screen for vascular phenotypes in aged laboratory mice, a unique discrete phenotype of hyaline arteriolosclerosis of the intertubular arteries and arterioles of the testes was identified in several inbred strains. Lesions were limited to the testes and did not occur as part of any renal, systemic, or pulmonary arteriopathy or vasculitis phenotype. There was no evidence of systemic or pulmonary hypertension, and lesions did not occur in ovaries of females. Frequency was highest in males of the SM/J (27/30, 90%) and WSB/EiJ (19/26, 73%) strains, aged 383 to 847 days. Lesions were sporadically present in males from several other inbred strains at a much lower (<20%) frequency. The risk of testicular hyaline arteriolosclerosis is at least partially underpinned by a genetic predisposition that is not associated with other vascular lesions (including vasculitis), separating out the etiology of this form and site of arteriolosclerosis from other related conditions that often co-occur in other strains of mice and in humans. Because of their genetic uniformity and controlled dietary and environmental conditions, mice are an excellent model to dissect the pathogenesis of human disease conditions. In this study, a discrete genetically driven phenotype of testicular hyaline arteriolosclerosis in aging mice was identified. These observations open the possibility of identifying the underlying genetic variant(s) associated with the predisposition and therefore allowing future interrogation of the pathogenesis of this condition.

Loss of FAS/FASL signalling does not reduce apoptosis in Sharpin null mice.

Mice with mutations in SHANK-associated RH domain interactor (Sharpin) develop a hypereosinophilic auto-inflammatory disease known as chronic proliferative dermatitis. Affected mice have increased apoptosis in the keratinocytes of the skin, oesophagus and forestomach driven by extrinsic TNF receptor-mediated apoptotic signalling pathways. FAS receptor signalling is an extrinsic apoptotic signalling mechanism frequently involved in inflammatory skin diseases. Compound mutations in Sharpin and Fas or Fasl were created to determine whether these death domain proteins influenced the cutaneous phenotype in Sharpin null mice. Both Sharpin/Fas and Sharpin/Fasl compound mutant mice developed an auto-inflammatory phenotype similar to that seen in Sharpin null mice, indicating that initiation of apoptosis by FAS signalling is likely not involved in the pathogenesis of this disease.

Mouse genome-wide association study identifies polymorphisms on chromosomes 4, 11, and 15 for age-related cardiac fibrosis.

Dystrophic cardiac calcinosis (DCC), also called epicardial and myocardial fibrosis and mineralization, has been detected in mice of a number of laboratory inbred strains, most commonly C3H/HeJ and DBA/2J. In previous mouse breeding studies between these DCC susceptible and the DCC-resistant strain C57BL/6J, 4 genetic loci harboring genes involved in DCC inheritance were identified and subsequently termed Dyscalc loci 1 through 4. Here, we report susceptibility to cardiac fibrosis, a sub-phenotype of DCC, at 12 and 20 months of age and close to natural death in a survey of 28 inbred mouse strains. Eight strains showed cardiac fibrosis with highest frequency and severity in the moribund mice. Using genotype and phenotype information of the 28 investigated strains, we performed genome-wide association studies (GWAS) and identified the most significant associations on chromosome (Chr) 15 at 72 million base pairs (Mb) (P < 10(-13)) and Chr 4 at 122 Mb (P < 10(-11)) and 134 Mb (P < 10(-7)). At the Chr 15 locus, Col22a1 and Kcnk9 were identified. Both have been reported to be morphologically and functionally important in the heart muscle. The strongest Chr 4 associations were located approximately 6 Mb away from the Dyscalc 2 quantitative trait locus peak within the boundaries of the Extl1 gene and in close proximity to the Trim63 and Cap1 genes. In addition, a single-nucleotide polymorphism association was found on chromosome 11. This study provides evidence for more than the previously reported 4 genetic loci determining cardiac fibrosis and DCC. The study also highlights the power of GWAS in the mouse for dissecting complex genetic traits.

Angiogenesis in the skin of SHARPIN-deficient mice with chronic proliferative dermatitis.

Angiogenesis is a common feature of pathological processes including wound healing, tumor formation, and chronic inflammation. Chronic inflammation can also be associated with dilation or proliferation of lymph vessels. We examined blood vessels and lymphatics and the expression of pro- and anti-angiogenic genes in the skin of SHARPIN-deficient mice which spontaneously develop a chronic proliferative dermatitis (cpdm). The number of blood vessels in the dermis of cpdm mice increased with age as the inflammation progressed. Lymphatics identified by labeling for LYVE1 and podoplanin were moderately dilated, but they were not increased in number. The expression of proangiogenic Vegfa, Flt1 and anti-angiogenic Sema3a mRNA was increased. VEGFA was primarily localized in keratinocytes of cpdm skin. There was also increased expression of Ece1 and Pdpn mRNA. Podoplanin was restricted to lymphatic endothelial cells in normal skin, but fibroblasts in cpdm skin also reacted with anti-podoplanin antibodies indicating that they were activated. The expression of other angiogenic and lymphangiogenic factors was not altered or decreased. These results indicate that cpdm mice may be a useful model to study the pathogenesis of angiogenesis in chronic inflammation.

Dermal lymphatic dilation in a mouse model of alopecia areata.

Mouse models of various types of inflammatory skin disease are often accompanied by increased dermal angiogenesis. The C3H/HeJ inbred strain spontaneously develops alopecia areata (AA), a cell mediated autoimmune disorder that can be controllably expanded using full thickness skin grafts to young unaffected mice. This provides a reproducible and progressive model for AA in which the vascularization of the skin can be examined. Mice receiving skin grafts from AA or normal mice were evaluated at 5, 10, 15, and 20 weeks after engraftment. Lymphatics are often overlooked as they are small slit-like structures above the hair follicle that resemble artifact-like separation of collagen bundles with some fixatives. Lymphatics are easily detected using lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) by immunohistochemistry to label their endothelial cells. Using LYVE1, there were no changes in distribution or numbers of lymphatics although they were more prominent (dilated) in the mice with AA. Lyve1 transcripts were not significantly upregulated except at 10 weeks after skin grafting when clinical signs of AA first become apparent. Other genes involved with vascular growth and dilation or movement of immune cells were dysregulated, mostly upregulated. These findings emphasize aspects of AA not commonly considered and provide potential targets for therapeutic intervention.

Genetic determinants of fibro-osseous lesions in aged inbred mice.

Fibro-osseous lesions in mice are progressive aging changes in which the bone marrow is replaced to various degrees by fibrovascular stroma and bony trabeculae in a wide variety of bones. The frequency and severity varied greatly among 28 different inbred mouse stains, predominantly affecting females, ranging from 0% for 10 strains to 100% for KK/HlJ and NZW/LacJ female mice. Few lesions were observed in male mice and for 23 of the strains, no lesions were observed in males for any of the cohorts. There were no significant correlations between strain-specific severities of fibro-osseous lesions and ovarian (r=0.11; P=0.57) or endometrial (r=0.03; P=0.89) cyst formation frequency or abnormalities in parathyroid glands. Frequency of fibro-osseous lesions was most strongly associated (P<10(-6)) with genome variations on chromosome (Chr) 8 at 90.6 and 90.8Mb (rs33108071, rs33500669; P=5.0·10(-10), 1.3·10(-6)), Chr 15 at 23.6 and 23.8Mb (rs32087871, rs45770368; P=7.3·10(-7), 2.7·10(-6)), and Chr 19 at 33.2, 33.4, and 33.6Mb (rs311004232, rs30524929, rs30448815; P=2.8·10(-6), 2.8·10(-6), 2.8·10(-6)) in genome-wide association studies (GWAS). The relatively large number of candidate genes identified in the GWAS analyses suggests that this may be an extremely complex polygenic disease. These results indicate that fibro-osseous lesions are surprisingly common in many inbred strains of laboratory mice as they age. While this presents little problem in most studies that utilize young animals, it may complicate aging studies, particularly those focused on bone.

Excavating the Genome: Large-Scale Mutagenesis Screening for the Discovery of New Mouse Models.

Technology now exists for rapid screening of mutated laboratory mice to identify phenotypes associated with specific genetic mutations. Large repositories exist for spontaneous mutants and those induced by chemical mutagenesis, many of which have never been fully studied or comprehensively evaluated. To supplement these resources, a variety of techniques have been consolidated in an international effort to create mutations in all known protein coding genes in the mouse. With targeted embryonic stem cell lines now available for almost all protein coding genes and more recently CRISPR/Cas9 technology, large-scale efforts are underway to create further novel mutant mouse strains and to characterize their phenotypes. However, accurate diagnosis of skin, hair, and nail diseases still relies on careful gross and histological analysis, and while not automated to the level of the physiological phenotyping, histopathology still provides the most direct and accurate diagnosis and correlation with human diseases. As a result of these efforts, many new mouse dermatological disease models are being characterized and developed.

The pathogenesis of chronic eosinophilic esophagitis in SHARPIN-deficient mice.

Increased numbers of eosinophils in the esophagus are common in several esophageal and systemic diseases, and a prominent feature of eosinophilic esophagitis. Mouse models can provide insight into the mechanisms of eosinophil infiltration and their pathogenic role. SHARPIN-deficient cpdm mice develop a chronic proliferative dermatitis and an esophagitis characterized by epithelial hyperplasia and the accumulation of eosinophils in the serosa, submucosa, lamina propria and epithelium of the esophagus. We conducted a detailed investigation of the pathogenesis of the esophagitis by light microscopy, immunohistochemistry, and gene expression as the mice aged from 4 to 10 weeks. The thickness of the esophageal epithelium and the number of eosinophils in the esophagus both increased with age. There were scattered apoptotic epithelial cells in mice at 6-10 weeks of age that reacted with antibodies to activated caspase 3 and caspase 9. The expression of CCL11 (eotaxin-1), IL4, IL13 and TSLP was increased in cpdm mice compared with wild type (WT) mice, and there was no change in the expression of CCL24 (eotaxin-2), IL5 and IL33. The expression of chitinase-like 3 and 4 (YM1 and YM2) proteins, markers of type 2 inflammation, was greatly increased in cpdm mice, and this was replicated in vitro by incubation of WT esophagus in the presence of IL4 and IL13. Immunohistochemistry showed that these proteins were localized in esophageal epithelial cells. The severity of the esophagitis was not affected by crossing SHARPIN-deficient mice with lymphocyte-deficient Rag1 null mice indicating that the inflammation is independent of B and T lymphocytes.

The impact of elevated sulfur and nitrogen levels on cadmium tolerance in Euglena species.

Heavy metal (HM) pollution threatens human and ecosystem health. Current methods for remediating water contaminated with HMs are expensive and have limited effect. Therefore, bioremediation is being investigated as an environmentally and economically viable alternative. Freshwater protists Euglena gracilis and Euglena mutabilis were investigated for their tolerance to cadmium (Cd). A greater increase in cell numbers under Cd stress was noted for E. mutabilis but only E. gracilis showed an increase in Cd tolerance following pre-treatment with elevated concentrations of S or N. To gain insight regarding the nature of the increased tolerance RNA-sequencing was carried out on E. gracilis. This revealed transcript level changes among pretreated cells, and additional differences among cells exposed to CdCl2. Gene ontology (GO) enrichment analysis reflected changes in S and N metabolism, transmembrane transport, stress response, and physiological processes related to metal binding. Identifying these changes enhances our understanding of how these organisms adapt to HM polluted environments and allows us to target development of future pre-treatments to enhance the use of E. gracilis in bioremediation relating to heavy metals.

The Impact of SLC2A8 RNA Interference on Glucose Uptake and the Transcriptome of Human Trophoblast Cells.

While glucose is the primary fuel for fetal growth, the placenta utilizes the majority of glucose taken up from the maternal circulation. Of the facilitative glucose transporters in the placenta, SLC2A8 (GLUT8) is thought to primarily function as an intracellular glucose transporter; however, its function in trophoblast cells has not been determined. To gain insight into the function of SLC2A8 in the placenta, lentiviral-mediated RNA interference (RNAi) was performed in the human first-trimester trophoblast cell line ACH-3P. Non-targeting sequence controls (NTS RNAi; n = 4) and SLC2A8 RNAi (n = 4) infected ACH-3P cells were compared. A 79% reduction in SLC2A8 mRNA concentration was associated with an 11% reduction (p ≤ 0.05) in ACH-3P glucose uptake. NTS RNAi and SLC2A8 RNAi ACH-3P mRNA were subjected to RNAseq, identifying 1525 transcripts that were differentially expressed (|log2FC| > 1 and adjusted p-value < 0.05), with 273 transcripts derived from protein-coding genes, and the change in 10 of these mRNAs was validated by real-time qPCR. Additionally, there were 147 differentially expressed long non-coding RNAs. Functional analyses revealed differentially expressed genes involved in various metabolic pathways associated with cellular respiration, oxidative phosphorylation, and ATP synthesis. Collectively, these data indicate that SLC2A8 deficiency may impact placental uptake of glucose, but that its likely primary function in trophoblast cells is to support cellular respiration. Since the placenta oxidizes the majority of the glucose it takes up to support its own metabolic needs, impairment of SLC2A8 function could set the stage for functional placental insufficiency.

Alopecia areata: updates from the mouse perspective.

Alopecia areata (AA) is a cell-mediated autoimmune disease that targets actively growing hair follicles in mammals, including humans and mice. Development of the C3H/HeJ spontaneous mouse model AA nearly 20 years ago provided a much needed tool to test the hypotheses and ultimately serve as a preclinical model for drug testing. Discoveries in both human AA patients and the mouse model supported each other and lead to discoveries on the incredibly complex genetic basis of this disease. The discovery that A/J, MRL/MpJ, SJL/J, and SWR/J strains also develop AA now allows genome-wide association mapping studies to expand the list of genes underlying this disease. Potential new targets for unraveling the pathogenesis of AA include the role of retinoic acid metabolism in the severity of disease and hair shaft proteins that may be either the inciting antigen or ultimate target of the immune reaction leading to breakage of the shaft causing clinical alopecia. Comparing these model systems with human and mouse clinical disease, for both discovery and validation of the discoveries, continues to resolve the complex questions surrounding AA.

Demonstrating the psychometric properties of a problem-related distress screener in a community sample of 319 cancer survivors.

OBJECTIVE: The purpose was to test the psychometric properties of a 36-item community-based problem-related distress screening tool, among 319 cancer survivors recruited across 14 affiliates of the Cancer Support Community. METHODS: Internal reliability was estimated using Cronbach's alpha coefficient. Test-retest reliability was assessed using the intra-class correlation coefficient (ICC). Concurrent validity was determined by correlations with the Functional Assessment of Cancer Therapy-General Well-Being Scale (FACT-G), the Center for Epidemiologic Studies Depression Scale (CES-D), and the Distress Thermometer (DT) and receiver operating characteristic (ROC) curve analysis using the CES-D (≥16) and DT (≥4) as the criterion. Non-parametric analysis of variance was used to establish discriminant validity. RESULTS: The distress screener demonstrated high internal consistency (Cronbach's alpha = 0.91) and strong test-retest reliability (ICC ≥ 0.75). Summary scores of the distress screener correlated substantially with the FACT-G (R(2) = 0.58, p < 0.001), CES-D (R(2) = 0.48, p < 0.001), and DT (R(2) = 0.35, p < 0.001) indicating strong concurrent validity and were able to discriminate groups of clinical relevance. ROC analyses showed a cutoff score of 8 for problem items rated ≥3 had optimal sensitivity and specificity relative to the CES-D and DT. CONCLUSIONS: The distress screener shows strong psychometric properties and can be considered a valuable community-based instrument to screen for psychological distress related to social, emotional, physical, and other patient-related symptoms and problems. This study is the first to address the chasm between hospital and community-based screening by validating a community-based instrument and has begun to demonstrate the feasibility of screening in the community.

Impact of Chorionic Somatomammotropin In Vivo RNA Interference Phenotype on Uteroplacental Expression of the IGF Axis.

While fetal growth is dependent on many factors, optimal placental function is a prerequisite for a normal pregnancy outcome. The majority of fetal growth-restricted (FGR) pregnancies result from placental insufficiency (PI). The insulin-like growth factors (IGF1 and IGF2) stimulate fetal growth and placental development and function. Previously, we demonstrated that in vivo RNA interference (RNAi) of the placental hormone, chorionic somatomammotropin (CSH), resulted in two phenotypes. One phenotype exhibits significant placental and fetal growth restriction (PI-FGR), impaired placental nutrient transport, and significant reductions in umbilical insulin and IGF1. The other phenotype does not exhibit statistically significant changes in placental or fetal growth (non-FGR). It was our objective to further characterize these two phenotypes by determining the impact of CSH RNAi on the placental (maternal caruncle and fetal cotyledon) expression of the IGF axis. The trophectoderm of hatched blastocysts (9 days of gestation, dGA) were infected with a lentivirus expressing either a non-targeting sequence (NTS RNAi) control or CSH-specific shRNA (CSH RNAi) prior to embryo transfer into synchronized recipient ewes. At ≈125 dGA, pregnancies were fitted with vascular catheters to undergo steady-state metabolic studies. Nutrient uptakes were determined, and tissues were harvested at necropsy. In both CSH RNAi non-FGR and PI-FGR pregnancies, uterine blood flow was significantly reduced (p ≤ 0.05), while umbilical blood flow (p ≤ 0.01), both uterine and umbilical glucose and oxygen uptakes (p ≤ 0.05), and umbilical concentrations of insulin and IGF1 (p ≤ 0.05) were reduced in CSH RNAi PI-FGR pregnancies. Fetal cotyledon IGF1 mRNA concentration was reduced (p ≤ 0.05) in CSH RNAi PI-FGR pregnancies, whereas neither IGF1 nor IGF2 mRNA concentrations were impacted in the maternal caruncles, and either placental tissue in the non-FGR pregnancies. Fetal cotyledon IGF1R and IGF2R mRNA concentrations were not impacted for either phenotype, yet IGF2R was increased (p ≤ 0.01) in the maternal caruncles of CSH RNAi PI-FGR pregnancies. For the IGF binding proteins (IGFBP1, IGFBP2, IGFBP3), only IGFBP2 mRNA concentrations were impacted, with elevated IGFBP2 mRNA in both the fetal cotyledon (p ≤ 0.01) and maternal caruncle (p = 0.08) of CSH RNAi non-FGR pregnancies. These data support the importance of IGF1 in placental growth and function but may also implicate IGFBP2 in salvaging placental growth in non-FGR pregnancies.