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Label-Retaining Cells in Human Embryonic and Fetal Epidermis

Human embryonic and fetal epidermis was examined and labeling indices (LIs) for basal, intermediate, and periderm cells were determined. The LI for fetal basal cells was 8–11% and the LI for fetal intermediate cells was 7.5–9%. The total fetal epidermal LI was 16–20%, which equaled the basal LI for embryonic epidermis. After 21 days in organ culture, only basal cells in the fetal epidermis labeled with tritiated thymidine, while both basal and intermediate cells in the embryonic epidermis labeled and the total LI for fetal and embryonic epidermal cells was the same as the adult epidermal LI (7%). The LI for periderm decreased with increasing estimated gestational age (EGA) from 9.5% at 49 days EGA to 0.54% at 85 days EGA. A subpopulation of epithelial cells that retained tritiated thymidine label and that have some of the attributes associated with stem cells has been previously demonstrated in rodents. In order to examine human embryonic and fetal epidermis for the presence of such cells, epidermis from various gestational ages were labeled and grown in organ culture for 21 days. The mean percent label-retaining cells (LRCs) for embryonic and fetal epidermis was determined. Approximately 4% of the embryonic and 2% of the fetal epidermal cells retained label for 21 days in organ culture. Embryonic LRCs were found in the only in the basal layer. The presence of LRCs in human embryonic and fetal epidermis suggests that epithelial cell proliferation in these tissues may be regulated via a stem cell pattern of proliferation

Growth Factor and Growth Factor Receptor Localization in the Hair Follicle Bulge and Associated Tissue in Human Fetus

The bulge region of the hair follicle has been thought to contain follicular stem cells. The bulge in the human follicle is a collection of undifferentiated cells that is prominent only in the fetal period. Antibodies that recognize epidermal growth factor (EGF), transforming growth factor-α (TGF-α), EGF receptor, platelet-derived growth factor (PDGF) A and B chains, PDGF α and β receptors, and the low-affinity nerve growth factor receptor (p75) were used to study the bulge and associated mesenchymal cells in this fetal period. Weak EGF and TGF-α immunoreactivities were seen in the bulge. Confocal laser scanning microscopic images revealed intracytoplasmic and intranuclear punctate patterns of immunoreactivities in the bulge cells labeled by anti-EGF and anti-TGF-α antibodies. All the bulge cells stained strongly for EGF receptor. Cells within the bulge were labeled both with PDGF A chain and with PDGF B chain, although the immunoreactivities were weak in the outermost layer of cells. The follicular sheath was strongly immunoreactive with antibodies against both PDGF α and β receptors. p75 was expressed in mesenchymal cells around the hair follicle and in the lower portion of the bulge. These differential labeling patterns suggested that EGF, TGF-α, and nerve growth factor may be involved in regulation of the growth and differentiation of bulge cells and that PDGFS may have related functions in the interaction arising between the bulge and associated tissue during follicle morphogenesis

The Appearance of Four Basement Membrane Zone Antigens in Developing Human Fetal Skin

In order to study the ontogeny of various structural and antigenic components of the basement membrane zone of human skin, we have examined skin specimens from 20 aborted fetuses ranging in gestational ages from 6 to 25 weeks, utilizing light microscopy, transmission electron microscopy, and indirect immunofluorescence with antibodies to bullous pemphigoid antigen, laminin, type IV collagen, and to the antigen defined by KF-1 monoclonal antibody. Both laminin and type IV collagen were detectable as early as 6 weeks of gestational age. In contrast, bullous pemphigoid antigen and the antigen defined by KF- 1 antibody were not detectable before 10 weeks and 16 weeks, respectively. The appearance of bullous pemphigoid antigen correlated with stratification of the epidermis and the formation of hemidesmosomes and anchoring fibrils at the basement membrane zone. KF- 1 antigen is first expressed when the epidermis is further stratified, hemidesmosomes and anchoring fibrils are present in greater numbers and with increased frequency at the dermal-epidermal junction, and hair follicles have begun to bud downward from the basal layer of the epidermis. Our findings suggest an orderly sequence to the appearance of these basement membrane zone components within human skin

Periderm Cells Form Cornified Cell Envelope in Their Regression Process During Human Epidermal Development

Terminally differentiated stratified squamous epithelium forms a lining of the plasma membrane called the cornified cell envelope, a thick layer of several covalently cross-linked precursor proteins including involucrin, small proline-rich proteins, and loricrin. Their cross-linking isodipeptide bonds are formed by epidermal transglutaminases 1–3. Material from lamellar granules is attached on the extracellular surface of corneocytes during the keratinization process. The formation of cornified cell envelope and sequential expression of major cornified cell envelope precursor proteins, transglutaminases, and 25 kDa lamellar granule-associated protein were studied in human embryonic and fetal skin. Ultrastructurally, membrane thickening has already started in periderm cells of the two-layered epidermis and an electron-dense, thickened cell envelope similar to cornified cell envelope in adult epidermis is observed in periderm cells at the three-layered and later stages of skin development. In the two-layered epidermis (49–65 d estimated gestational age), immunoreactivities of involucrin, small proline-rich proteins, all the transglutaminases, and lamellar granule-associated protein were present only in the periderm. In the three-layered epidermis and thereafter (66–160 d estimated gestational age), loricrin became positive in the periderm cells, transglutaminases extended to the entire epidermis, and lamellar granule-associated protein was detected in intermediate cells as well as periderm cells. Immunoelectron microscopy demonstrated that both major cornified cell envelope precursor proteins, involucrin and loricrin, were restricted to the cornified cell envelope in periderm cells at this stage of development. After 160 d estimated gestational age, the periderm had disappeared and cornified cell envelope proteins and lamellar granule-associated proteins were expressed in the spinous, granular, and cornified cells and transglutaminases were detected in the entire epidermis. These findings indicate that cornified cell envelope precursor proteins, transglutaminases, and lamellar granule-associated proteins are expressed in coordination in periderm cells during human epidermal development and suggest that periderm cells form cornified cell envelope in the process of regression

Morphogenesis and Malformations of the Skin NICHD/NIADDK Research Workshop

Developmentally caused skin malformations constitute a spectrum of birth defects, some of which can be recognized prenatally by morphologic or biochemical means. The number of prenatally diagnosable skin diseases could be greatly expanded with an increased understanding of the molecular and cellular bases of skin development and the mechanisms that result in the generation of skin defects. The National Institute of Child Health and Human Development and the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases, therefore, sponsored a workshop that recommended basic biologic studies combined with clinical investigations of normal and abnormal cutaneous development set forth in this article. Investigations resulting from these research recommendations are intended to contribute to the knowledge that should aid in the prevention of developmentally caused skin deformities

Ontogeny and characterization of Factor XIIIa + cells in developing human skin

Factor XIIIa (FXIIIa), a coagulation transglutaminase, is a cytoplasmic marker for dermal dendritic cells reported to be bone marrow-derived, phagocytic and antigen-presenting. In non-inflamed skin, these cells populate the papillary dermis in a perivascular distribution. They are increased in dermatoproliferative disorders and have been implicated as dermal stimulants for psoriatic hyperkeratosis. Since developing skin provides an example of dermal influence on the epidermis, we evaluated the presence of FXIIIa + cells in human fetal skin to determine whether their location would suggest a role in morphogenetic events in the skin. Embryonic and fetal skin of progressive estimated gestational ages (EGA) was examined using immunocytochemistry with a polyclonal antibody to FXIIIa. At 6 weeks EGA, globular FXIIIa + cells were present in the hypodermis. By 7–8 weeks, a compact sub-epidermal network of fusiform FXIIIa + cells was also evident. By 11–12 weeks, the sub-epidermal cellular network was no longer FXIIIa + , but discrete FXIIIa + dendritic cells were present in the reticular dermis. With advancing gestational age, FXIIIa + dendritic cells populated the papillary dermis in a perivascular distribution. This adult-like distribution persisted through 22 weeks EGA, the oldest specimen examined. Because FXIIIa + cells were evident in embryonic skin before the onset of bone marrow hematopoietic function, the skin was double-labeled with the FXIIIa antibody and with monoclonal antibodies to CD45 (marker for bone marrow-derived cells), CD68 (marker for macrophages) and HLA-DR (class II major histocompatibility antigen). Most of the FXIIIa + dendritic cells did not colocalize CD45, but were CD68 + ; some cells did react with the HLA-DR antibody. Notably, the FXIIIa + cells of the sub-epidermal network in the 7 weeks EGA specimens did not react with the other antibodies. We conclude that FXIIIa + cells are first present in embryonic hypodermis and sub-epidermal dermis and later they are distributed in the papillary dermis in a perivascular pattern. In embryonic skin FXIIIa + cells are not exclusively dendritic. Our data support the idea that cells that express FXIIIa do not constitute a unique bone marrow-derived cell type, but that multiple cell types produce FXIIIa.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47524/1/429_2004_Article_BF00186831.pd

Selecting an Anti-Malarial Clinical Candidate from Two Potent Dihydroisoquinolones

BACKGROUND: The ongoing global malaria eradication campaign requires development of potent, safe, and cost-effective drugs lacking cross-resistance with existing chemotherapies. One critical step in drug development is selecting a suitable clinical candidate from late leads. The process used to select the clinical candidate SJ733 from two potent dihydroisoquinolone (DHIQ) late leads, SJ733 and SJ311, based on their physicochemical, pharmacokinetic (PK), and toxicity profiles is described. METHODS: The compounds were tested to define their physicochemical properties including kinetic and thermodynamic solubility, partition coefficient, permeability, ionization constant, and binding to plasma proteins. Metabolic stability was assessed in both microsomes and hepatocytes derived from mice, rats, dogs, and humans. Cytochrome P450 inhibition was assessed using recombinant human cytochrome enzymes. The pharmacokinetic profiles of single intravenous or oral doses were investigated in mice, rats, and dogs. RESULTS: Although both compounds displayed similar physicochemical properties, SJ733 was more permeable but metabolically less stable than SJ311 in vitro. Single dose PK studies of SJ733 in mice, rats, and dogs demonstrated appreciable oral bioavailability (60-100%), whereas SJ311 had lower oral bioavailability (mice 23%, rats 40%) and higher renal clearance (10-30 fold higher than SJ733 in rats and dogs), suggesting less favorable exposure in humans. SJ311 also displayed a narrower range of dose-proportional exposure, with plasma exposure flattening at doses above 200 mg/kg. CONCLUSION: SJ733 was chosen as the candidate based on a more favorable dose proportionality of exposure and stronger expectation of the ability to justify a strong therapeutic index to regulators

Epigenetic age acceleration in adolescence associates with BMI, inflammation and risk score for middle age cardiovascular disease

BACKGROUND: 'Accelerated ageing', assessed by adult DNA methylation predicts cardiovascular disease (CVD). Adolescent accelerated aging might predict CVD earlier. We investigated whether epigenetic age acceleration (assessed age 17-years) associated with adiposity/CVD-risk measured (ages 17, 20, 22-years), and projected CVD by middle-age. METHODS: DNA methylation measured in peripheral blood provided 2 estimates of epigenetic age acceleration; intrinsic (IEAA, (preserved across cell types) and extrinsic (EEAA, dependent on cell admixture and methylation levels within each cell type).Adiposity was assessed by anthropometry, ultrasound and DEXA (ages 17, 20, 22 years). CVD-risk factors (lipids, HOMA-IR, blood pressure, inflammatory markers) were assessed at age 17-years. CVD development by age 47 years was calculated by Framingham algorithms. Results are presented as regression coefficients/5-year epigenetic age acceleration (IEAA/EEAA) for adiposity, CVD-risk factors and CVD development. RESULTS: In 995 participants (49.6% female, age 17.3+/-0.6 years), EEAA (/5-years) was associated with increased BMI of 2.4% (95%CI 1.2-3.6%) and 2.4% (0.8-3.9%) at 17 and 22 years, respectively. EEAA was associated with increases of 23% (3-33%) in hsCRP, 10% (4-17%) in interferon-gamma induced protein (IP-10) and 4% (2-6%) in tumour necrosis factor receptor 2 (sTNFR2), adjusted for BMI and HOMA-IR. EEAA(/5-years) results in a 4% increase in hard endpoints of CVD by 47 years old and a 3% increase, after adjustment for conventional risk factors. CONCLUSIONS: Accelerated epigenetic age in adolescence was associated with inflammation, BMI measured 5 years later, and probability of middle-age CVD. Irrespective whether this is cause or effect, assessing epigenetic age might refine disease prediction

Quantitative conversations: the importance of developing rapport in standardised interviewing

© 2014, The Author(s). When developing household surveys, much emphasis is understandably placed on developing survey instruments that can elicit accurate and comparable responses. In order to ensure that carefully crafted questions are not undermined by ‘interviewer effects’, standardised interviewing tends to be utilised in preference to conversational techniques. However, by drawing on a behaviour coding analysis of survey paradata arising from the 2012 UK Poverty and Social Exclusion Survey we show that in practice standardised survey interviewing often involves extensive unscripted conversation between the interviewer and the respondent. Whilst these interactions can enhance response accuracy, cooperation and ethicality, unscripted conversations can also be problematic in terms of survey reliability and the ethical conduct of survey interviews, as well as raising more basic epistemological questions concerning the degree of standardisation typically assumed within survey research. We conclude that better training in conversational techniques is necessary, even when applying standardised interviewing methodologies. We also draw out some theoretical implications regarding the usefulness of the qualitative–quantitative dichotomy