Psycho-Emotional and Behavioral Problems in Children With Growth Hormone Deficiency.

Objective: To identify psychosocial problems and self-esteem in children with growth hormone deficiency (GHD) and define the role of some clinical and sociodemographic determinants in the conceptualization of internalizing and externalizing problems as criteria for psychosocial functioning. Materials and Methods: A GHD sample (46 prepubescent children) was selected and compared to a matched control group (80 healthy children). Psychosocial functioning in children with GHD was investigated using Goodman's "Strengths and Difficulties Questionnaire (SDQ)." The study of children's self-esteem was carried out by the Dembo-Rubinstein method. Results: This study reveals that the GHD sample has more internalizing problems and lower self-esteem. Higher score and frequency of assessment in the abnormal score for "total difficulties," "emotional problem," and "peer problem" were found in children with GHD. The SDQ score and the frequency of assessment in the abnormal score for all SDQ scales in children with more pronounced growth deficit (height SDS < -3) did not exceed the same indicators in children with less growth retardation (-3 < height SDS < -2). A comparison of psychosocial features in children with isolated growth hormone deficiency and multiple pituitary hormones deficiency did not reveal differences in SDQ score and the frequency of assessment in the abnormal score for all SDQ scales. It was found that children with GHD have a reduced level of assertions, low self-esteem, and a weak discrepancy between the level of assertions and self-esteem. Some sociodemographic determinants (male gender, age < 9 years, and low family income) and clinical determinants (low compliance and suboptimal growth response after 1 year of rGHh therapy) have an impact on the overall assessment of psychological problems in children with GHD. The internalizing difficulties are associated with certain clinical determinants (growth status and treatment status) and sociodemographic determinants (female gender, age < 9 years). Conclusions: The identification of low self-esteem and the high SDQ score for scales "total difficulties," "emotional problems," and "peer problems" indicates psychosocial maladjustment and conceptualization of internalizing problems in children with GHD.

A method for manual and automated multiplex RNAscope in situ hybridization and immunocytochemistry on cytospin samples.

In situ analysis of biomarkers is essential for clinical diagnosis and research purposes. The increasing need to understand the molecular signature of pathologies has led to the blooming of ultrasensitive and multiplexable techniques that combine in situ hybridization (ISH) and immunohistochemistry or immunocytochemistry (IHC or ICC). Most protocols are tailored to formalin-fixed paraffin embedded (FFPE) tissue sections. However, methods to perform such assays on non-adherent cell samples, such as patient blood-derived PBMCs, rare tumor samples, effusions or other body fluids, dissociated or sorted cells, are limited. Typically, a laboratory would need to invest a significant amount of time and resources to establish one such assay. Here, we describe a method that combines ultrasensitive RNAscope-ISH with ICC on cytospin cell preparations. This method allows automated, sensitive, multiplex ISH-ICC on small numbers of non-adherent cells. We provide guidelines for both chromogenic and fluorescent ISH/ICC combinations that can be performed either in fully automated or in manual settings. By using a CD8+ T cells in vitro stimulation paradigm, we demonstrate that this protocol is sensitive enough to detect subtle differences in gene expression and compares well to commonly used methods such as RT-qPCR and flow cytometry with the added benefit of visualization at the cellular level.

FX knockout CHO hosts can express desired ratios of fucosylated or afucosylated antibodies with high titers and comparable product quality.

During antibody dependent cell cytotoxicity (ADCC) the target cells are killed by monocytes and natural killer cells. ADCC is enhanced when the antibody heavy chain's core N-linked glycan lacks the fucose molecule(s). Several strategies have been utilized to generate fully afucosylated antibodies. A commonly used and efficient approach has been knocking out the FUT8 gene of the Chinese hamster ovary (CHO) host cells, which results in expression of antibody molecules with fully afucosylated glycans. However, a major drawback of the FUT8-KO host is the requirement for undertaking two separate cell line development (CLD) efforts in order to obtain both primarily fucosylated and fully afucosylated antibody species for comparative studies in vitro and in vivo. Even more challenging is obtaining primarily fucosylated and FUT8-KO clones with similar enough product quality attributes to ensure that any observed ADCC advantage(s) can be strictly attributed to afucosylation. Here, we report generation and use of a FX knockout (FXKO) CHO host cell line that is capable of expressing antibody molecules with either primarily fucosylated or fully afucosylated glycan profiles with otherwise similar product quality attributes, depending on addition of fucose to the cell culture media. Hence, the FXKO host not only obviates the requirement for undertaking two separate CLD efforts, but it also averts the need for screening many colonies to identify clones with comparable product qualities. Finally, FXKO clones can express antibodies with the desired ratio of primarily fucosylated to afucosylated glycans when fucose is titrated into the production media, to allow achieving intended levels of FcγRIII-binding and ADCC for an antibody. Biotechnol. Bioeng. 2017;114: 632-644. © 2016 Wiley Periodicals, Inc.

Fucosylation Deficiency in Mice Leads to Colitis and Adenocarcinoma.

BACKGROUND & AIMS: De novo synthesis of guanosine diphosphate (GDP)-fucose, a substrate for fucosylglycans, requires sequential reactions mediated by GDP-mannose 4,6-dehydratase (GMDS) and GDP-4-keto-6-deoxymannose 3,5-epimerase-4-reductase (FX or tissue specific transplantation antigen P35B [TSTA3]). GMDS deletions and mutations are found in 6%-13% of colorectal cancers; these mostly affect the ascending and transverse colon. We investigated whether a lack of fucosylation consequent to loss of GDP-fucose synthesis contributes to colon carcinogenesis. METHODS: FX deficiency and GMDS deletion produce the same biochemical phenotype of GDP-fucose deficiency. We studied a mouse model of fucosylation deficiency (Fx-/- mice) and mice with the full-length Fx gene (controls). Mice were placed on standard chow or fucose-containing diet (equivalent to a control fucosylglycan phenotype). Colon tissues were collected and analyzed histologically or by enzyme-linked immunosorbent assays to measure cytokine levels; T cells also were collected and analyzed. Fecal samples were analyzed by 16s ribosomal RNA sequencing. Mucosal barrier function was measured by uptake of fluorescent dextran. We transplanted bone marrow cells from Fx-/- or control mice (Ly5.2) into irradiated 8-week-old Fx-/- or control mice (Ly5.1). We performed immunohistochemical analyses for expression of Notch and the hes family bHLH transcription factor (HES1) in colon tissues from mice and a panel of 60 human colorectal cancer specimens (27 left-sided, 33 right-sided). RESULTS: Fx-/- mice developed colitis and serrated-like lesions. The intestinal pathology of Fx-/- mice was reversed by addition of fucose to the diet, which restored fucosylation via a salvage pathway. In the absence of fucosylation, dysplasia appeared and progressed to adenocarcinoma in up to 40% of mice, affecting mainly the right colon and cecum. Notch was not activated in Fx-/- mice fed standard chow, leading to decreased expression of its target Hes1. Fucosylation deficiency altered the composition of the fecal microbiota, reduced mucosal barrier function, and altered epithelial proliferation marked by Ki67. Fx-/- mice receiving control bone marrow cells had intestinal inflammation and dysplasia, and reduced expression of cytokines produced by cytotoxic T cells. Human sessile serrated adenomas and right-sided colorectal tumors with epigenetic loss of MutL homolog 1 (MLH1) had lost or had lower levels of HES1 than other colorectal tumor types or nontumor tissues. CONCLUSIONS: In mice, fucosylation deficiency leads to colitis and adenocarcinoma, loss of Notch activation, and down-regulation of Hes1. HES1 loss correlates with the development of human right-sided colorectal tumors with epigenetic loss of MLH1. These findings indicate that carcinogenesis in a subset of colon cancer is consequent to a molecular mechanism driven by fucosylation deficiency and/or HES1-loss.

The stigmatisation of pregnancy: societal influences on pregnant women's physical activity Behaviour.

Many women going through the major life transition of pregnancy experience decreases in physical activity behaviour, which may compromise maternal and infant health and wellbeing. Although research suggests that the social environment plays a large role in influencing women's physical activity behaviour, little is known about the association between societal attitudes and physical activity behaviour during the course of pregnancy. Through a qualitative longitudinal study, we explored women's physical activity experiences throughout pregnancy and how these were formed, supported and/or opposed by their social environment. This research included telephone interviews with 30 pregnant participants, recruited via a regional public hospital. Using a feminist standpoint analysis incorporating modern dialectics, three major tensions were identified, reflecting dominant societal discourses around physical activity and pregnancy: (1) engaging in physical activity and keeping the baby safe, (2) engaging in physical activity and obtaining social approval and (3) listening to oneself and to others. These findings present previously unrecognised opportunities for developing tailored and effective physical activity interventions among pregnant women.

Construct and criterion-related validation of nutrient profiling models: A systematic review of the literature.

Nutrient profiling (NP) is defined as the science of ranking foods according to their nutritional composition for the purpose of preventing disease or promoting health. The application of NP is ultimately to assist consumers to make healthier food choices, and thus provide a cost effective public health strategy to reduce the incidence of diet-related chronic disease. To our knowledge, no review has assessed the evidence to confirm the validity of NP models. We conducted a systematic review to investigate the construct and criterion-related validity of NP models in ranking food according to their nutritional composition for the purpose of preventing disease and promoting health. We searched peer-reviewed research published to 30 June 2015 and used PUBMED, Global Health (CABI), and SCOPUS databases. Within study bias was assessed using an adapted version of the QUADAS-2 (Quality Assessment of Diagnostic Accuracy Studies -2) tool for all diagnostic studies and the Cochrane Collaboration's Risk of Bias tool for all non-diagnostic studies. The GRADE (Grades of Recommendation, Assessment, Development, and Evaluation) approach was used to guide our judgement of the quality of the body of evidence for each outcome measure. From a total of 83 studies, 69 confirmed the construct validity of NP models; however most of these studies contained methodological weaknesses. Six studies used objective external measures to confirm the criterion-related validity of NP models; which inherently improved quality. The overall quality of evidence on the accuracy of NP models was judged to be very low to moderate using the GRADE approach. Many carefully designed studies to establish both construct and criterion-related validity are necessary to authenticate the application of NP models and provide the evidence to support the current definition of NP.

Therapeutic antibodies reveal Notch control of transdifferentiation in the adult lung.

Prevailing dogma holds that cell-cell communication through Notch ligands and receptors determines binary cell fate decisions during progenitor cell divisions, with differentiated lineages remaining fixed. Mucociliary clearance in mammalian respiratory airways depends on secretory cells (club and goblet) and ciliated cells to produce and transport mucus. During development or repair, the closely related Jagged ligands (JAG1 and JAG2) induce Notch signalling to determine the fate of these lineages as they descend from a common proliferating progenitor. In contrast to such situations in which cell fate decisions are made in rapidly dividing populations, cells of the homeostatic adult airway epithelium are long-lived, and little is known about the role of active Notch signalling under such conditions. To disrupt Jagged signalling acutely in adult mammals, here we generate antibody antagonists that selectively target each Jagged paralogue, and determine a crystal structure that explains selectivity. We show that acute Jagged blockade induces a rapid and near-complete loss of club cells, with a concomitant gain in ciliated cells, under homeostatic conditions without increased cell death or division. Fate analyses demonstrate a direct conversion of club cells to ciliated cells without proliferation, meeting a conservative definition of direct transdifferentiation. Jagged inhibition also reversed goblet cell metaplasia in a preclinical asthma model, providing a therapeutic foundation. Our discovery that Jagged antagonism relieves a blockade of cell-to-cell conversion unveils unexpected plasticity, and establishes a model for Notch regulation of transdifferentiation.

Glucagon Couples Hepatic Amino Acid Catabolism to mTOR-Dependent Regulation of α-Cell Mass.

Understanding the regulation of islet cell mass has important implications for the discovery of regenerative therapies for diabetes. The liver plays a central role in metabolism and the regulation of endocrine cell number, but liver-derived factors that regulate α-cell and ß-cell mass remain unidentified. We propose a nutrient-sensing circuit between liver and pancreas in which glucagon-dependent control of hepatic amino acid metabolism regulates α-cell mass. We found that glucagon receptor inhibition reduced hepatic amino acid catabolism, increased serum amino acids, and induced α-cell proliferation in an mTOR-dependent manner. In addition, mTOR inhibition blocked amino-acid-dependent α-cell replication ex vivo and enabled conversion of α-cells into ß-like cells in vivo. Serum amino acids and α-cell proliferation were increased in neonatal mice but fell throughout postnatal development in a glucagon-dependent manner. These data reveal that amino acids act as sensors of glucagon signaling and can function as growth factors that increase α-cell proliferation.

Implications of understanding the signaling, cellular, and cytotoxic mechanisms afforded by afucosylated antibodies.

Afucosylated antibodies potentiate natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) by enhancing signaling pathways and cellular processes, which in turn, increases cytotoxic potential. Importantly, a better understanding of these processes and properties will aid in exploiting them to help design therapeutic antibodies and strategies that may be of the greatest benefit to patients.

Notch Receptor-Ligand Engagement Maintains Hematopoietic Stem Cell Quiescence and Niche Retention.

Notch is long recognized as a signaling molecule important for stem cell self-renewal and fate determination. Here, we reveal a novel adhesive role of Notch-ligand engagement in hematopoietic stem and progenitor cells (HSPCs). Using mice with conditional loss of O-fucosylglycans on Notch EGF-like repeats important for the binding of Notch ligands, we report that HSPCs with faulty ligand binding ability display enhanced cycling accompanied by increased egress from the marrow, a phenotype mainly attributed to their reduced adhesion to Notch ligand-expressing stromal cells and osteoblastic cells and their altered occupation in osteoblastic niches. Adhesion to Notch ligand-bearing osteoblastic or stromal cells inhibits wild type but not O-fucosylglycan-deficient HSPC cycling, independent of RBP-JK -mediated canonical Notch signaling. Furthermore, Notch-ligand neutralizing antibodies induce RBP-JK -independent HSPC egress and enhanced HSPC mobilization. We, therefore, conclude that Notch receptor-ligand engagement controls HSPC quiescence and retention in the marrow niche that is dependent on O-fucosylglycans on Notch.

Afucosylated antibodies increase activation of FcγRIIIa-dependent signaling components to intensify processes promoting ADCC.

Antibody-dependent cellular cytotoxicity (ADCC) is a key mechanism by which therapeutic antibodies mediate their antitumor effects. The absence of fucose on the heavy chain of the antibody increases the affinity between the antibody and FcγRIIIa, which results in increased in vitro and in vivo ADCC compared with the fucosylated form. However, the cellular and molecular mechanisms responsible for increased ADCC are unknown. Through a series of biochemical and cellular studies, we find that human natural killer (NK) cells stimulated with afucosylated antibody exhibit enhanced activation of proximal FcγRIIIa signaling and downstream pathways, as well as enhanced cytoskeletal rearrangement and degranulation, relative to stimulation with fucosylated antibody. Furthermore, analysis of the interaction between human NK cells and targets using a high-throughput microscope-based antibody-dependent cytotoxicity assay shows that afucosylated antibodies increase the number of NK cells capable of killing multiple targets and the rate with which targets are killed. We conclude that the increase in affinity between afucosylated antibodies and FcγRIIIa enhances activation of signaling molecules, promoting cytoskeletal rearrangement and degranulation, which, in turn, potentiates the cytotoxic characteristics of NK cells to increase efficiency of ADCC.

The prevalence of and knowledge about tobacco use among physicians in the Odessa region, Ukraine.

We investigated prevalence of and knowledge about tobacco use among physicians, and their counselling of patients in the Odessa region (Ukraine). Paediatricians (40), family doctors (40) and interns (70) were selected from the physician population of the Odessa region. The proportion of smokers was unacceptably high for health care professionals: paediatricians, 32.5%; family doctors, 37.5%; and interns, 50%. Majority of smokers were men. Less than half of smokers had considered quitting or seriously attempted to quit. Interns least frequently asked their patients about smoking (52.5 vs. 80% paediatricians and 72.5% family doctors). Ukrainian universities need to better educate medical students on tobacco control measures.

Multiplexed ion beam imaging of human breast tumors.

Immunohistochemistry (IHC) is a tool for visualizing protein expression that is employed as part of the diagnostic workup for the majority of solid tissue malignancies. Existing IHC methods use antibodies tagged with fluorophores or enzyme reporters that generate colored pigments. Because these reporters exhibit spectral and spatial overlap when used simultaneously, multiplexed IHC is not routinely used in clinical settings. We have developed a method that uses secondary ion mass spectrometry to image antibodies tagged with isotopically pure elemental metal reporters. Multiplexed ion beam imaging (MIBI) is capable of analyzing up to 100 targets simultaneously over a five-log dynamic range. Here, we used MIBI to analyze formalin-fixed, paraffin-embedded human breast tumor tissue sections stained with ten labels simultaneously. The resulting data suggest that MIBI can provide new insights into disease pathogenesis that will be valuable for basic research, drug discovery and clinical diagnostics.

Understanding community pharmacy intervention practice: lessons from intervention researchers.

BACKGROUND: Community pharmacy (CP) is a setting with health promotion and public health potential which could include strategies with a nutrition promotion focus. Research embedded in this setting has explored and produced evidence to inform practice change to develop this potential. The experience of undertaking research in this setting may provide insight into the challenges and key features of intervention research practice. Exploring experienced-based knowledge presents as a productive area of research, extending what can be known beyond the bounds of what is measurable. OBJECTIVES: This study aimed to understand the experience of intervention research in CP with a focus on nutrition and to develop guidance for future research practice (intervention design and implementation) in CP based on interventionists' reflections and practice wisdom. METHODS: Semi-structured interviews were conducted with 9 researchers with experience in undertaking intervention research in CP with a nutrition component. Content analysis, constant comparison and interpretive description were used in the analysis and interpretation of interview data. RESULTS: Five key lessons were identified - 1) utilize existing capacity; 2) navigate and utilize social power and interests; 3) personalize engagement and recruitment; 4) consider the logistics and 5) intervention type considerations. Key challenges for translating research into practice and sustaining change included financial sustainability, physical constraints, logistics, collaboration, and practice change enablers. Personal reflections on research practice identified qualities, such as determination and skills in networking, as key for researching in CP. CONCLUSIONS: CP-embedded research is challenging given the complexity of the practice environment. The social context of CP appears central to intervention research and a nuanced understanding of the social context needs to be the basis for intervention design to inform successful implementation. Experience-based and insider knowledge is useful and needed for nuanced design and development of intervention research in CP.

Little evidence of human infection with equine influenza during the 2007 epizootic, Queensland, Australia.

BACKGROUND: Equine influenza virus (EIV) is considered enzootic in Europe (except Iceland), Asia, North Africa, and North and South America. When EIV outbreaks occur they may severely impact the equine and tourist industries. Australia faced its first EIV outbreak beginning in August of 2007. The outbreak was concentrated in New South Wales and Queensland, with more than 1400 confirmed EIV infections in horses during the first month. Rapid response from the equine industry and the federal government was successful and Australia was declared free from EIV by the end of 2007. OBJECTIVES: This cross-sectional study was designed to examine associations between exposure to EIV-infected horses and evidence of EIV infection in humans. STUDY DESIGN: Employing informed consent, between October 2007 and April 2008, 100 subjects (89 with horse exposures and 11 non-exposed) were enrolled during equine events and at the University of the Sunshine Coast. All subjects provided a blood sample and were asked to complete an online questionnaire including health history, animal exposure and demographic information. Sera samples were tested for the presence of antibodies against two H3N8 EIV strains using microneutralization, hemagglutination inhibition, and enzyme-linked lectin assays. RESULTS: Evidence for H3N8 infection was sparse, with only 9 study participants having any indication of H3N8 infection and the seroreactivity seen was low and easily explained by cross-reactions against human influenza strains or vaccines. CONCLUSIONS: These data provide little evidence to support the premise that EIV infections occurred among humans exposed to EIV-infected horses during the 2007 Australian epizootic.

Does artificial UV use prior to spring break protect students from sunburns during spring break?

BACKGROUND: Dark-skinned individuals are less likely than light-skinned individuals to become sunburned or develop skin cancer. Some have extrapolated this relationship and surmised that developing and maintaining a tan will reduce the risk of sunburns and melanoma. In order to examine whether this strategy indeed protects against sunburns, we surveyed college students about both their tanning habits prior to spring break and their spring break activities. METHODS: Sorority and fraternity students were recruited after spring break. Analyses examined associations between potential risk factors and the development of one or more sunburns during spring break. RESULTS: As expected, the risk of obtaining a sunburn increased with: time spent in the sun during spring break; light complexion, as assessed by various sun-sensitivity factors; and lack of sunscreen use. We also found that tanning using an artificial UV source during the 10 weeks prior to spring break was not associated with reduced risk of sunburns during spring-break, but rather with a marginal increase in this risk. CONCLUSIONS: These data provide evidence that maintaining a tan may not provide protection from sunburns. Public health messages need to address this misconception, stating clearly that a tan does not protect against or reduce the chances of developing a sunburn.