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Irritable bowel syndrome (IBS) is a functional gastrointestinal (GI) disorder characterized by chronic abdominal pain associated with alterations in bowel function. Given the heterogeneity of the symptoms, multiple pathophysiologic factors are suspected to play a role. We classified women with IBS into four subgroups based on distinct symptom profiles. In-depth shotgun proteomic analysis was carried out to profile the urinary proteomes to identify possible proteins associated with these subgroups. First void urine samples with urine creatinine level ≥ 100 mg/dL were used after excluding samples that tested positive for blood. Urine from ten subjects representing each symptom subgroup was pooled for proteomic analysis. The urine proteome was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a data-independent method known as Precursor Acquisition Independent From Ion Count (PAcIFIC) that allowed extended detectable dynamic range. Differences in protein quantities were determined by peptide spectral counting followed by validation of select proteins with ELISA or a targeted single reaction monitoring (LC-SRM/MS) approach. Four IBS symptom subgroups were selected: 1) Constipation, 2) Diarrhea + Low Pain, 3) Diarrhea + High Pain, and 4) High Pain + High Pychological Distress. A fifth group consisted of Healthy Control subjects. From comparisons of quantitative spectral counting data among the symptom subgroups and controls, a total of 18 proteins that showed quantitative differences in relative abundance and possible physiological relevance to IBS were selected for further investigation. Three of the 18 proteins were chosen for validation by either ELISA or SRM. An elevated expression of gelsolin (GSN) was associated with the high pain groups. Trefoil Factor 3 (TFF3) levels were higher in IBS groups compared to controls. In this study the IBS patients subclassified by predominant symptoms showed differences in urine proteome levels. Proteins showing distinctive changes are involved in homeostasis of intestinal function and inflammatory response. These findings warrant future studies with larger, independent cohorts to enable more extensive assessment and validation of urinary protein markers as a diagnostic tool in adult with IBS.

Recently, biobehavioral nursing scientists have focused their attention on the search for biomarkers or biological signatures to identify patients at risk for various health problems and poor disease outcomes. In response to the national impetus for biomarker discovery, the measurement of biological fluids and tissues has become increasingly sophisticated. Urine proteomics, in particular, may hold great promise for biobehavioral focused nursing scientists for examination of symptom-and syndrome-related research questions. Urine proteins are easily accessible secreted proteins that provide direct and indirect windows into bodily functions. Advances in proteomics and biomarker discovery provide new opportunities to conduct research studies with banked and fresh urine to benefit diagnosis, prognosis, and evaluation of outcomes in various disease populations. This article provides a review of proteomics and a rationale for utilizing urine proteomics in biobehavioral research. It addresses as well some of the challenges involved in data collection and sample preparation.

Ehlers-Danlos syndrome (EDS) type I (the classical variety) is a dominantly inherited, genetically heterogeneous connective-tissue disorder. Mutations in the COL5A1 and COL5A2 genes, which encode type V collagen, have been identified in several individuals. Most mutations affect either the triple-helical domain of the protein or the expression of one COL5A1 allele. We identified a novel splice-acceptor mutation (IVS4-2A--G) in the N-propeptide-encoding region of COL5A1, in one patient with EDS type I. The outcome of this mutation was complex: In the major product, both exons 5 and 6 were skipped; other products included a small amount in which only exon 5 was skipped and an even smaller amount in which cryptic acceptor sites within exon 5 were used. All products were in frame. Pro-alpha1(V) chains with abnormal N-propeptides were secreted and were incorporated into extracellular matrix, and the mutation resulted in dramatic alterations in collagen fibril structure. The two-exon skip occurred in transcripts in which intron 5 was removed rapidly relative to introns 4 and 6, leaving a large (270 nt) composite exon that can be skipped in its entirety. The transcripts in which only exon 5 was skipped were derived from those in which intron 6 was removed prior to intron 5. The use of cryptic acceptor sites in exon 5 occurred in transcripts in which intron 4 was removed subsequent to introns 5 and 6. These findings suggest that the order of intron removal plays an important role in the outcome of splice-site mutations and provide a model that explains why multiple products derive from a mutation at a single splice site.

Evidence suggests that patients with irritable bowel syndrome (IBS) are more vigilant to pain-associated stimuli. The aims of this study were to compare women with IBS ( n = 20) to healthy control (HC, n = 20) women on pain sensitivity, conditioned pain modulation (CPM) efficiency, and salivary cortisol levels before and after the CPM test and to examine the relationship of CPM efficiency with gastrointestinal pain, somatic pain, psychological distress symptoms, and salivary cortisol levels in each group. Women, aged 20–42 years, gave consent, completed questionnaires, and kept a symptom diary for 2 weeks. CPM efficiency was tested with a heat test stimulus and cold water condition stimulus in a laboratory between 8 and 10 a.m. on a follicular phase day. Salivary cortisol samples were collected just before and after the experimental testing. Compared to the HC group, women with IBS reported more days with gastrointestinal and somatic pain/discomfort, psychological distress, fatigue, and feeling stressed. During the CPM baseline testing, women with IBS reported greater pain sensitivity compared to the HC group. There was no significant group difference in salivary cortisol levels nor in CPM efficiency, though a post-hoc analysis showed a higher prevalence of impaired CPM efficiency among IBS subjects with more severe lower-GI symptoms. In the IBS group, reduced CPM efficiency was associated with daily abdominal pain/discomfort and psychological distress. Overall, women with IBS exhibited an increased sensitivity to thermal stimuli. Impaired CPM was present in a subset of women with IBS.

Epidermolysis bullosa simplex are dominant disorders of skin fragility characterized by intraepidermal blistering upon mild mechanical trauma. Skin fragility is caused by expression of either an abnormal keratin 5 or an abnormal keratin 14 protein, which compromises the structure and function of the keratin cytoskeleton of basal cells. We report an epidermolysis bullosa simplex patient with a novel single base substitution (A--T1414) that changes the lysine residue at amino acid 472 to a non-sense codon (K472X). This change predicts the synthesis of a truncated keratin 5, missing 119 amino acids, including the entire tail domain and the highly conserved KLLEGE motif at the carboxy terminus of the 2B domain of the central rod. Expression of an altered keratin 5, of predicted mass and pI for the product of the K472X allele, was documented by one- and two-dimensional western blots of protein extracts from patient skin. Ultrastructural analysis of the patient's nonhyperkeratotic skin was remarkable for basal keratinocytes with dense and irregular keratin filaments proximal to the basement membrane. Keratinocytes, transfected with a cDNA carrying the A--T1414 non-sense mutation, overexpressed a truncated keratin 5, and showed a disorganized and collapsed keratin filament cytoskeleton. This is the second epidermolysis bullosa simplex patient reported with a premature termination mutation in the KLLEGE motif. The remarkable occurrence of severe palmar--plantar hyperkeratosis in both patients suggests that the keratin 5 tail domain may have unrecognized, but important, normal functions in palmar-plantar tissues.

The transcription factor retinoic acid receptor β 2 (RARβ 2 ) is a potent inhibitor of breast cancer cells in vitro , and studies suggest that RARβ expression is lost in primary breast cancer. Although RARβ 2 is selectively down-regulated at the mRNA level in breast tumor cells, we show that expression of an RARβ protein is elevated in five of five breast tumor cell lines relative to normal human mammary epithelial cells. Subsequent analysis identified this protein as the translation product of the human RARβ 4 transcript. Unlike the previously characterized mouse RARβ 4 isoform, the human RARβ 4 retains only half of a DNA-binding domain and lacks a ligand-independent transactivation domain at its N terminus. The RARβ 4 protein localizes to the cytoplasm and to subnuclear compartments that resemble nuclear bodies. The structure and preliminary characterizations of human RARβ 4 , coupled with the observation that its expression is greatly elevated in breast tumor cell lines, support the hypothesis that RARβ 4 functions as a dominant-negative repressor of RAR-mediated growth suppression.

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.

Epidermolysis bullosa with pyloric atresia (EB-PA), an autosomal recessive genodermatosis, manifests with neonatal cutaneous blistering associated with congenital pyloric atresia. The disease is frequently lethal, but nonlethal cases have also been reported. Expression of the alpha6 beta4 integrin is altered at the dermal-epidermal basement-membrane zone; recently, mutations in the corresponding genes (ITGA6 and ITGB4) have been disclosed in a limited number of patients, premature termination codons in both alleles being characteristic of lethal variants. In this study, we have examined the molecular basis of EB-PA in five families, two of them with lethal and three of them with nonlethal variants of the disease. Mutation analysis disclosed novel lesions in both ITGB4 alleles of each proband. One of the patients with lethal EB-PA was a compound heterozygote for premature termination-codon mutations (C738X/4791delCA), whereas the other patient with a lethal variant was homozygous for a missense mutation involving a cysteine residue (C61Y). The three nonlethal cases had missense mutations in both alleles (C562R/C562R, R1281W/R252C, and R1281W/R1281W). Immunofluorescence staining of skin in two of the nonlethal patients and in one of the lethal cases was positive, yet attenuated, for alpha6 and beta4 integrins. These results confirm that ITGB4 mutations underlie EB-PA and show that missense mutations may lead to nonlethal phenotypes.

The tenascins are a family of large extracellular matrix proteins with at least three members: tenascin-X (TNX), tenascin-C (TNC, or cytotactin) and tenascin-R (TN-R, or restrictin). Although the tenascins have been implicated in a number of important cellular processes, no function has been clearly established for any tenascin. We describe a new contiguous-gene syndrome, involving the CYP21B and TNX genes, that results in 21-hydroxylase deficiency and a connective-tissue disorder consisting of skin and joint hyperextensibility, vascular fragility and poor wound healing. The connective tissue findings are typical of the Ehlers-Danlos syndrome (EDS). The abundant expression of TNX in connective tissues is consistent with a role in EDS, and our patient's skin fibroblasts do not synthesize TNX protein in vitro or in vivo. His paternal allele carries a novel deletion arising from recombination between TNX and its partial duplicate gene, XA, which precludes TNX synthesis. Absence of TNX mRNA and protein in the proband, mapping of the TNX gene and HLA typing of this family suggest recessive inheritance of TNX deficiency and connective-tissue disease. Although the precise role of TNX in the pathogenesis of EDS is uncertain, this patient's findings suggest a unique and essential role for TNX in connective-tissue structure and function.

Epidermolysis bullosa simplex is a disease in which keratin gene mutations cause the production of defective intermediate filaments, which leads in turn to epidermal basal cell fragility and blistering. The inheritance in nearly all kindreds is autosomal dominant, most kindreds have missense mutations, and the encoded proteins appear to exert a dominant negative function. One previously reported patient with generalized blistering had a fully dominant mutation of keratin 5; in that kindred a homozygote was affected no more severely than the heterozygotes. By contrast we report here a keratin 14 mutation that causes blistering limited to the hands and feet in heterozygotes, but homozygotes have more severe, widespread blistering of the skin and mucous membranes. Thus keratin gene mutations may be not only fully recessive or fully dominant but also partially dominant as well.

Ehlers-Danlos syndrome type IV (EDS type IV) results from heterozygosity for mutations in the COL3A1 gene that encodes the chains of type III procollagen. By using light, transmission, and scanning electron microscopy, we examined skin biopsies from 22 individuals with EDS type IV in whom the COL3A1 mutations had been identified. The most striking changes in EDS type IV were correlated with point mutations that substituted a residue for a glycine near the carboxyl-terminal end of the triple-helical domain of pro alpha1(III). In three cases with the mutation G1012R, G1018V, or G1021E, cells in the dermis had extremely dilated rough endoplasmic reticulum (RER), the dermis was thin, and there was a reduced proportion of collagen although the proportion of elastic fibers appeared increased. In these tissues, collagen fibrils were small (65-80 nm) compared to normal (95-110 nm). Fibrils 80-90 nm in diameter and moderately dilated RER were found with mutations G769R, G373R, and G061E and with exon-skipping mutations of exons 34 and 45. With mutations G034R and G016C and exon-skipping mutations that deleted the sequences of exons 7, 8, 14, 18, 24, and 27, fibrils were more variable in size (85-120 nm). The composite collagen fibrils characteristic of EDS types I and II were not found in EDS type IV. These findings indicate that mutations in the COL3A1 gene have effects on secretion, fibrillogenesis, and skin architecture that reflect the position and nature of the mutation.

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-alpha (TGF-alpha), EGF receptor, platelet-derived growth factor (PDGF) A and B chains, PDGF alpha and beta 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-alpha 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-alpha 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 alpha and beta 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-alpha, 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.

Previous studies have indicated that amphiregulin is a major autocrine factor for human keratinocytes. To evaluate the possibilities that amphiregulin could function in fetal skin morphogenesis and contribute to the growth regulation of epidermis, immunostaining with a specific anti-amphiregulin monoclonal antibody was observed at different stages of fetal skin development, and the results were compared with neonatal and adult skin specimens and cultured neonatal keratinocytes. Immunoreactive amphiregulin was readily detected in the periderm and basal epidermal layers of embryonic epidermis but became gradually less detectable in the periderm concurrent with an increase in staining of the spinous layer as it developed during the fetal period. Basal and spinous keratinocyte expression of amphiregulin was predominantly cytoplasmic, but with punctate nuclear foci, and this pattern persisted into the neonatal period. At all developmental stages, epithelial and mesenchymal cells of the follicle were reactive, often in a nuclear pattern. Dermal mesenchymal cell were increasingly reactive in late fetal skin, but the staining decreased postnatally. In adult skin only randomly scattered nuclei of spinous keratinocytes and follicular structures such as the inner root sheath were stained. Examination by scanning laser confocal microscopy of cultured neonatal keratinocytes showed a nonrandom distribution of amphiregulin to the peripheral cytoplasm and plasma membranes at the outer perimeter of cell colonies, with much less reactivity of apposed keratinocyte membranes at interior sites. Nuclei were heterogeneously stained. Amphiregulin reactivity declined at higher cell densities. These data indicate that expression of amphiregulin is regulated in vitro and developmentally during cutaneous morphogenesis.

Herlitz junctional epidermolysis bullosa (HJEB) is a severe blistering disorder which usually results in death during infancy. We have previously shown that the anchoring filament protein laminin-5 (kalinin/nicein), which mediates keratinocyte attachment and dermal-epidermal cohesion, is abnormally expressed in individuals with HJEB. Laminin-5 was detected by Western blot analysis in amniotic fluid from 44 consecutive normal second-trimester control pregnancies, but was undetectable in second-trimester amniotic fluid from four pregnancies with fetuses affected by HJEB. In one case of severe non-Herlitz JEB, laminin-5 was detected in both amniotic fluid and skin. In human amniotic fluid, the laminin-5 a3 subunit was processed to a major 165 kD species and a minor 145 kD species and the beta 2 subunit was partially processed to 105 kD. Although laminin-5 was covalently associated with laminin-6 (K-laminin) in amniotic membrane, no covalent interaction was detected in amniotic fluid. Laminin-5 from amniotic fluid strongly supported keratinocyte attachment. These results suggest that Western blot analysis of second-trimester amniotic fluid is useful in determining the prenatal diagnosis of HJEB and that laminin-5 may serve a physiologically important function in amniotic fluid.

Dystrophic epidermolysis bullosa inversa is a rare form of epidermolysis bullosa characterized by blister formation in flexural skin areas and by marked oral and esophageal involvement. Recognition of this subset of dystrophic epidermolysis bullosa is important, because its prognosis differs from all other forms of dystrophic epidermolysis bullosa. Investigators recently reported normal staining with antibodies directed against type VII collagen in 14 patients, but electron microscopy in eight patients showed diminished or absent anchoring fibrils. We report here the cases of two additional patients with dystrophic epidermolysis bullosa inversa. One patient had finger web space scarring that required surgical correction and mild syndactyly of toes. Both patients had normal staining with LH 7:2, but electron microscopy showed diminished and rudimentary anchoring fibrils. These findings support the possibility that dystrophic epidermolysis bullosa inversa may be caused by a structural abnormality of type VII collagen that prevents proper assembly of collagen into distinct anchoring fibrils.

We report on the prenatal diagnosis of epidermolysis bullosa letalis with pyloric atresia in a pregnancy not known to be at risk for this condition. Elevated maternal serum alpha-fetoprotein levels led to ultrasonography which demonstrated gastric dilatation, consistent with pyloric atresia, and echogenic particles in the amniotic fluid, the "snowflake sign," previously described in two pregnancies of fetuses with disorders of skin sloughing. Amniotic fluid alpha-fetoprotein was markedly elevated and the acetylcholinesterase was positive. The diagnosis of epidermolysis bullosa letalis with pyloric atresia was confirmed after delivery by electron microscopy of fetal skin which showed typical changes of hypoplastic absent hemidesmosomes and separation along the dermal-epidermal junction. None of these abnormal prenatal findings are consistently present in pregnancies with epidermolysis bullosa with pyloric atresia. Thus, although useful when abnormal, when the test results are normal, the need for confirmatory fetoscopy and fetal skin biopsy remains.

This study was undertaken to identify the extent of involvement of cutaneous and noncutaneous epithelia during expression in utero of junctional epidermolysis bullosa. Skin and other organs from a 19-wk estimated gestational age fetus affected with junctional epidermolysis bullosa and from age-matched controls were examined by light and transmission electron microscopy. In the affected fetus, skin samples from different body regions including trunk, leg, arm, and finger all showed some separation at the dermal-epidermal junction in the plane of the lamina lucida. Hemidesmosomes were absent or hypoplastic, whereas anchoring fibrils appeared normal in structure and number. Interfollicular epidermis appeared to have separated easily, whereas some follicles remained anchored in the dermis. Areas of epithelium in the trachea and bronchi had separated, but within the lung parenchyma the epithelium of smaller bronchioles and alveoli remained attached to supporting connective tissue. The transitional epithelium of the urinary bladder showed small areas of separation compared with the gall bladder epithelium, which showed extensive separation. Gall bladder epithelium in several control fetuses also was consistently separated. In the affected fetus as well as in controls, gall bladder had multiple layers of basal lamina, a previously unrecognized structural feature of human fetal gall bladder. Epithelia that remained intact included the linings of the stomach and small and large intestines and endothelium of large and small vessels in all organs. Kidney, spleen, liver, and lymph nodes appeared normal in all aspects. In control samples, neither skin nor most of the noncutaneous epithelia had separated from the underlying connective tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

An infant born with severe blisters on the limbs, face, trunk, and oral mucosa was diagnosed by light and electron microscopy to have recessive dystrophic epidermolysis bullosa. Transmission electron microscopy showed that the basal lamina remained with the epidermis and that the floor of the blister was exposed collagen of the papillary dermis. No banded anchoring fibrils were observed along either the roof or the floor of the blister; however, small filamentous structures, possibly immature anchoring fibrils, extended down from the lamina densa along the blister roof. Some basal and suprabasal keratinocytes contained large vesicles filled with filamentous matrix of variable electron density. Immunofluorescent staining of skin for type VII collagen showed sparse and irregular staining of type VII collagen along the blister roof, and intense intracellular labeling for type VII collagen in clusters of epidermal cells in basal and suprabasal layers. Type VII collagen appeared to be synthesized by keratinocytes but not secreted.

Background The Washington State Nutrition and Physical Activity Plan provides a framework in which policy makers can work together to build and support healthy environments for nutrition and physical activity. The city of Moses Lake, Wash, was chosen to serve as a pilot site to test the conceptual approaches and recommendations of the plan and to develop a model for healthy communities elsewhere in the state. Context Moses Lake is an ethnically diverse, geographically isolated town with a population of about 15,000. Methods An advisory committee used data from an inventory of local policies and environments, along with the recommendations from the state plan, to develop a plan for Healthy Communities Moses Lake. Three initiatives were chosen for the first actions: a connected system of trails and paths, enhanced facilities for breastfeeding in the community, and a community garden. Consequences Records of cumulative actions demonstrated that Healthy Communities Moses Lake continued to be an active and productive project. Initial measures of success were collected by each of the three first action teams. Environmental changes will be monitored by comparison with the initial inventory of local policies. Long-term health outcomes in Moses Lake will be monitored by the Washington State Department of Health. Interpretation Healthy Communities Moses Lake was successful because the city had leaders and volunteers who were committed to making the city a healthier place. Lessons learned about community-based planning and evaluation are now being applied to Healthy Communities initiatives.

Providing small grants to community organizations can be an effective way to encourage changes in the environment that support better nutrition. This is effective because these organizations can provide insights into their communities, ready-made relationships with community members, and the trust of the community. Small-grants programs are more likely to be successful when they are tailored to the needs of individual communities, led by organizations that have established reputations with the community, fully supported by the lead community organization, and engage local partners that complement the skills and resources of the lead organization. An evaluation of a small-grants program, Grants for Healthy Youth, found that grantees developed unique approaches to improving their community nutrition environments, gained experience and skills in program development, built partnerships, and received recognition for their project work. Grantees faced some common barriers, especially with program evaluation. Small-grants programs can be an effective way to improve community nutrition environments, but granting agencies need to provide effective technical assistance to communities throughout the process.

Information on the morphology of mitochondria during embryogenesis is scattered in the literature, but there appears to be a consistent pattern. During early organogenesis, the embryo is in a state of relative hypoxia associated with a major decrease in terminal electron transport system activity and a marked increase in anaerobic glycolysis. Ultrastructural studies of a 14-somite monkey embryo and day 10 and 12 rat embryos, together with a review of the literature, led us to determine that this hypoxic stage is characterized by vesiculation of the mitochondrial inner membranes, or cristae. Starting in the late morula stage and continuing during early postimplantation embryogenesis, the cristae increase but appear tubular or vesicular. After the end of neurulation, and with the onset of vascular perfusion of embryonic tissues, the cristae gradually become lamellated; by the limb bud stage they appear more mature. We suggest that new cristae derive from blebs of the inner mitochondrial membrane and that with maturation these blebs collapse, giving them a lamelliform appearance. The delamellated state of the cristae might inactivate oxidative phosphorylation to protect the embryo from toxic respiratory end-products that could accumulate in an embryo before there is vascular perfusion. Consistent with this hypothesis, mitochondrial diameters in the developing heart of monkey and rat embryos were approximately twice those found in skin and neural tube.

In rodents, the hair follicle stem cells lie in a well-defined bulge in the outer root sheath; however, the bulge as a stem cell site of human hair follicle epithelium is still controversial. Epidermal stem cells are thought to express high levels of beta1 integrin and low levels of E-cadherin and beta- and gamma-catenin. In order to clarify the ontogenic distribution of possible stem cells during hair follicle development, the expression patterns of beta1 integrin subunits, E-cadherin, and beta- and gamma-catenins in the skin samples from human fetuses of a series of estimated gestational ages (EGA) were examined. beta1 integrin-rich, E-cadherin-, and beta- and gamma-catenin-poor cells, possible stem cells, were localized to the entire hair germ (65-84 d EGA) and later to the outermost cells of hair peg (85-104 d EGA). In the bulbous hair peg (105-135 d EGA) and in the differentiated lanugo hair follicle (135 d EGA), they were settled in the bulge and the outermost layer of the outer root sheath. This sequential localization was similar to that of cells rich in epidermal growth factor receptor expression and positive with keratin 19, a putative marker of epidermal stem cells. In addition, these beta1 integrin-rich, E-cadherin-, and beta- and gamma-catenin-poor cells showed similar, undifferentiated morphologic features by electron microscopy. This information of ontogenic localization of possible hair follicle stem cells contributes to the further understanding of mechanisms of human hair follicle morphogenesis and supports the idea that the human fetal hair follicle bulge is a site of stem cells for follicular epithelium.

Calcium concentration is a critical factor for epidermal differentiation and is implicated in the expression and post-translational modification of numerous proteins in suprabasal cells of the epidermis. Calpains (calcium-activated neutral proteases) are believed to participate in signal transduction via highly regulated cytoplasmic protease activity. Here we investigate the expression of calpain I in normal human skin development and in neonatal harlequin ichthyosis (HI), a disorder of altered epidermal differentiation, especially the transition from the granular to the fully differentiated cornified layer. Calpain I was detected in developing foetal epidermis at 54 days estimated gestational age in the basal layer and the periderm of the developing foetal epidermis. By 125 days, calpain I was also detected in the granular layer. This pattern was maintained in newborn skin, but expression was significantly weaker in HI biopsies (n = 7). Reduced expression of calpain was specific to HI and was not observed in other skin diseases. Calpain was also normally expressed in the outer root sheath of hair follicles, in sebaceous glands and in sweat ducts and glands. Immunoblots of epidermal and keratinocyte extracts showed that the 78-kDa and 76-kDa active forms were generated via limited proteolysis of the 80-kDa inactive subunit; however, all forms were diminished in HI, consistent with findings in tissue sections. Our results show that calpain is present throughout the epidermis and is expressed from the early stages of development. These findings implicate calcium-mediated signalling events in the alteration of differentiation that occurs in HI.

Background Junctional epidermolysis bullosa–pyloric atresia syndrome is recognized as a distinct autosomal recessive entity. Affected infants present with skin fragility and inability to feed due to intestinal obstruction. Despite successful surgical repair of the anatomical defect, the outcome is poor owing to poor feeding, malabsorption, failure to thrive, and sepsis. Observations In 70 cases of intestinal obstruction and epidermolysis bullosa reported in the medical literature and the 3 reported here, surgical intervention was attempted 51 times. In all except 16 infants, death occurred before age 11 months (mean age, 70 days). Renal involvement and continued failure to thrive accompanied the skin disease in survivors, who ranged in age from 30 days to 16 years (mean age, 4.0 years). Conclusions The poor prognosis of this condition must be considered when decisions are made regarding surgical correction. Attempting surgical correction may be warranted in individual circumstances, but withholding surgical intervention and providing palliative support is an acceptable alternative.

Keratin 2e (K2e) is expressed in the upper spinous and granular cells of adult epidermis. A highly specific polyclonal antibody was made against a C-terminal peptide of K2e and used to observe K2e expression at different developmental stages. At 12.5 weeks estimated gestational age (EGA) K2e was detected in trunk skin in scattered cells in the intermediate layer. At 13.5 weeks EGA, greater numbers of intermediate cells were stained with variable intensity, and staining in this pattern increased with age. Epidermal sheets from 14 weeks EGA showed that K2e + cells were excluded from developing hair follicles. At 135 days EGA, the following regional patterns were observed: in cheek, trunk, dorsal and ventral knee, elbow and dorsal hand there was moderate to intense staining of upper intermediate keratinocytes excluding cells of the hair canals and sweat ducts. The periumbilical region distinctly lacked K2e staining, while more distal areas showed increasing numbers of K2e + cells. The earliest expression of K2e was at 10 weeks EGA in the presumptive nail bed of developing digits. By 13.5 weeks EGA this pattern had shifted to the proximal nail fold, and K2e was absent in the nail bed. K2e was excluded from developing sweat glands and ducts and from developing hair follicles at the hair germ and early peg stages. By 15 weeks EGA in the fetal hair follicle small groups of cells were K2e + and by 19 weeks K2e + cells were seen at the level of the matrix. Some overlap in staining was detected for K2e with K10, and in palmar skin with K9; however, mostly the filamentous staining patterns for these keratins were distinctive. This study shows that the complex patterns of temporal and regional expression of K2e differ from known patterns for other epidermal keratins and suggest different regulation and function for this epidermal keratin.

Much of the autonomous growth of cultured keratinocytes is attributable to the signaling of amphiregulin, a heparin-binding autocrine growth factor, through the epidermal growth factor receptor. Emerging evidence suggests, moreover, that the membrane proteoglycan, CD44, is a cofactor for the interaction of heparin-binding ligands with their receptors. This model was evaluated by characterizing the patterns of the immunolabeled molecules in cultured human neonatal keratinocytes, to test the hypothesis that involvement in a common function results in coordinate segregation within or on the cell. The molecules were localized by double immunofluorescence labeling to detect amphiregulin and either the epidermal growth factor receptor or CD44, and the immunostained products were imaged by scanning laser confocal microscopy. Both amphiregulin and the epidermal growth factor receptor segregated to a perinuclear distribution and to intercellular contacts. In addition, amphiregulin localized to the outer leading edge of colonies and focally to intranuclear sites. Metabolic blockade of proteoglycan sulfation with sodium chlorate inhibited growth of the cells and concurrently enhanced the nuclear, but decreased the outer leading edge, labeling for amphiregulin. There was no nuclear or perimeter labeling for the epidermal growth factor receptor. Cultures co-immunolabeled for CD44 and amphiregulin exhibited variable perinuclear staining for both, but otherwise CD44 was distributed to intercellular contacts. The intercellular localizations of CD44 with amphiregulin and of amphiregulin with the epidermal growth factor receptor were strongly concordant. These data are consistent with a concerted function at intercellular contacts, where cytokine signaling is mediated via receptor binding and possibly regulated by the CD44 proteoglycan as cofactor. The intranuclear and perimeter labeling of amphiregulin, however, suggests that this cytokine has additional functions, both in the nucleus and as a matrix receptor.

OBJECTIVE: Although hypopigmented macules are an important manifestation of tuberous sclerosis (TS), the probability of TS in healthy individuals who have hypopigmented macules is unknown. The purpose of this study was to establish the prevalence of hypopigmented macules among a cross section of the general white population. STUDY DESIGN: The skin of 423 white individuals younger than 45 years of age was screened for hypopigmented macules with ambient incandescent and fluorescent light and a Wood lamp. Indirect ophthalmoscopy was performed in patients with unexplained hypopigmentation to screen for retinal manifestations of TS. RESULTS: Twenty individuals (4.7%) had at least one hypopigmented macule. Of these, four had more than one macule. None had more than three. Two (8%) of the 25 hypopigmented macules were identified only with a Wood lamp. Indirect ophthalmoscopy was performed in 13 (65%) of these 20 individuals. None showed the retinal findings of TS. CONCLUSIONS: The prevalence of hypopigmented macules in the general population has been underestimated. The presence of a few hypopigmented macules on the skin of an otherwise healthy individual without a family history of TS need not prompt an evaluation to rule out this disorder. (J P EDIATR 1996;129:355-61)

The distribution of type VI collagen was examined in human embryonic and fetal skin and in cultured cells and matrix from this tissue. Frozen sections were immunolabeled with primary antibodies against type VI collagen a types I, III or V collagen, and processed further for fluorescence microscopy and immunoelectron microscopy. At 6 weeks estimated gestational age (EGA), type VI collagen was identified by positive fluorescence and by immunogold staining of filaments and fibers beneath the dermal-epidermal junction (DEJ), weaker fluorescence in the fine matrix of the dermis, and stronger fluorescence in the subcutis. At progressive stages of gestation, immunolabeling for type VI collagen increased in the dermis in parallel with increased deposition of types I and III collagen. By 15 weeks EGA, type VI collagen stained intensely throughout the dermis. At 13 weeks EGA, type IV, type VI collagen appeared diminished from the growing tips of invaginating hair buds, but as the hair peg developed, type VI collagen accumulated in adnexal sheaths. Cell cultures were derived from fetal skin at 7.5 to 12 weeks EGA. In primary explant cultures containing both keratinocytes and fibroblasts, mats of type V collagen were present beneath keratinocytes and associated with dense spots that co-labeled for both type VI and type V collagen. In passaged cultures of fibroblasts, individual cells with or without pretreatment with monensin were positive for type VI and/or types I, III or V collagen. Fibrous matrix that was labeled for type VI collagen was also immunopositive for type I or III collagen, while filamentous matrix that was type VI collagen positive tender to exclude types I and III collagen but in some areas to overlap with type V collagen. These findings support the hypothesis that type VI collagen present in both filamentous and fibrous matrix and networks of type VI collagen may serve as a fine scaffolding that facilitates the integration of types I and III collagen into developing fibrous matrix.

We describe initial observations of an infant with dermatosparaxis (another form of Ehlers-Danlos syndrome, designated as type VIIC), an autosomal recessive disorder characterized by skin fragility and described in several species of domesticated animals. Electron microscopic examination of the skin shows collagen sheets rather than fibrils, and characteristic distortions resembling hieroglyphs. In addition to skin fragility, the disorder is characterized by redundant skin folds and edema, healing with minimal scar formation, large fontanels and wide sagittal and metopic sutures, blue scierae, micrognathia, and umbilical hernia; after the neonatal period there are joint laxity, growth failure, short limbs, and normal mineralization of the skeleton except for the cranial vault. This disorder may also be a cause of premature rupture of placental membranes and myopia.

Buschke-Ollendorff syndrome (BOS; McKusick 16670) is an autosomal dominant connective-tissue disorder characterized by uneven osseous formation in bone (osteopoikilosis) and fibrous skin papules (dermatofibrosis lenticularis disseminata). We describe two patients in whom BOS occurred in an autosomal dominant inheritance pattern. The connective tissue of the skin lesions showed both collagen and elastin abnormalities by electron microscopy. Cultured fibroblasts from both patients produced 2-8 times more tropoelastin than normal skin fibroblasts in the presence of 10% calf serum. Involved skin fibroblasts of one patient produced up to eight times normal levels, whereas apparently uninvolved skin was also elevated more than threefold. In a second patient, whose involvement was nearly complete, elastin production was high in involved areas and less so in completely involved skin. Transforming growth factor-beta 1 (TGF beta 1), a powerful stimulus for elastin production, brought about similar relative increases in normal and BOS strains. Basic fibroblast growth factor, an antagonist of TGF beta 1-stimulated elastin production, was able to reduce elastin production in basal and TGF beta 1 stimulated BOS strains. Elastin mRNA levels were elevated in all patient strains, suggesting that Buschke-Ollendorff syndrome may result, at least in part, from abnormal regulation of extracellular matrix metabolism that leads to increased steady-state levels of elastin mRNA and elastin accumulation in the dermis.

Junctional epidermolysis bullosa, Herlitz variant (junctional EB-Herlitz) is a lethal autosomal recessive skin disorder currently amenable to prenatal diagnosis only by direct analysis of fetal skin. However, elevated levels of alpha-fetoprotein, as well as the presence of acetylcholinesterase in amniotic fluid, have been associated with other severe fetal genodermatoses. Fetal skin samplings were performed in ten pregnancies at risk for fetal junctional EB-Herlitz, with three fetuses affected on the basis of electron microscopic detection of blisters within the lamina lucida and abnormal hemidesmosomes. In neither affected nor unaffected pregnancies were maternal serum or amniotic fluid alpha-fetoprotein levels elevated. Moreover, alpha-fetoprotein levels in both maternal serum and amniotic fluid were not statistically different comparing affected and unaffected fetuses. Acetylcholinesterase was not present in the amniotic fluid samples of the three affected pregnancies. Unlike other severe fetal genodermatoses, neither alpha-fetoprotein nor acetylcholinesterase was predictive of junctional EB-Herlitz.

Cells in culture from a woman with a variety of the Marfan syndrome produce two species of the alpha 2 chains of type I collagen. One alpha 2 chain appears normal; the abnormal chain has a higher apparent molecular weight than normal and migrates more slowly during electrophoresis in sodium dodecyl sulfate/polyacrylamide gels. A similar change in electrophoretic behavior is seen in the prepro alpha 2 chain and the pN alpha 2 chain (which contains the amino-terminal extension). Asymmetric cleavage of the pepsin-treated procollagens with a fibroblast collagenase locates the abnormal segment amino terminal to the cleavage site, and analysis of cyanogen bromide peptides of collagenase cleavage peptides and of whole collagens indicates that the abnormal segment is in either the alpha 2CB3 peptide or the short segment of alpha 2CB5 amino terminal to the collagenase site of the altered alpha 2 chain. The higher apparent molecular weight is consistent with the insertion of a small peptide fragment of approximately 20 amino acids. This alteration in chain size has marked effects on crosslinking because collagen from the patient's skin was 5-10 times more extractable in nondenaturing solvents than that from control skins. Although the abnormal chain was not found in several other individuals with the Marfan syndrome, these findings suggest that the phenotype may be the expression of a variety of primary structure alterations in the chains of type I collagen that interfere with normal crosslink formation.

To describe a normal adult dermis is a seemingly simple task considering the diverse microscopic methods available for examination of the tissue, staining procedures to delineate the fibrous and cellular components, and immunolabeling techniques to identify precisely the various fibrous elements. Yet it is not simple because the range of normal in any of the dermal components has never been surveyed. There are well recognized age-related changes in the dermis; the tissue can be modified by environmental insults (e.g., actinic damage) and alterations can occur in tissue of individuals with inherited disorders of connective tissue metabolism, other metabolic diseases (e.g., diabetes) and in those receiving topically applied or systemic medication. From our own experience there is also marked individual variability (at any age) in the connective tissue architecture and its fibrous components. Thus, we are describing the structure of a normal dermis without demonstrating the range of normal in any one of its elements. Reference will occasionally be made to abnormal conditions of the matix since through these deviations it is possible to understand more about the normal. Structural and biochemical properties of the dermal connective tissue in human embryos and fetuses have been described in a number of studies, but in only a few instances was the goal of the research focused on this problem; instead, fetal tissue was used for comparative purposes in aging studies, or a certain characteristic of the fetal dermis was pointed out along with the description of another structure (e.g., hair follicle). In the few instances where a sequential study was carried out on one matrix component during development, an animal (pig, chick) was selected for the work.

The dermis of human skin develops embryonically from lateral plate mesoderm and is established in an adult-like pattern by the end of the first trimester of gestation. In this study the structure, biochemistry, and immunocytochemistry of collagenous matrix in embryonic and fetal dermis during the period of 5 to 26 weeks of gestation was investigated. The dermis at five weeks contains fine, individual collagen fibrils draped over the surfaces of mesenchymal cells. With increasing age, collagen matrix increases in abundance in the extracellular space. The size of fibril diameters increases, and greater numbers of fibrils associate into fiber bundles. By 15 weeks, papillary and reticular regions are recognized. Larger-diameter fibrils, larger fibers, denser accumulations of collagen, and fewer cells distinguish the deeper reticular region from the finer, more cellular papillary region located beneath the epidermis. The distribution of collagen types I, III, and V were studied at the light microscope level by immunoperoxidase staining and at the ultrastructural level by transmission (TEM) and scanning electron microscopy (SEM) with immunogold labeling. By immunoperoxidase, types I and III were found to be evenly distributed, regardless of fetal age, throughout the dermal and subdermal connective tissue with an intensification of staining at the dermal-epidermal junction (DEJ). Staining for types III and V collagen was concentrated around blood vessels. Type V collagen was also localized in basal and periderm cells of the epidermis. By immuno-SEM, types I and III were found associated with collagen fibrils, and type V was localized to dermal cell surfaces and to a more limited extent with fibrils. The results of biochemical analyses for relative amounts of types I, III, and V collagen in fetal skin extracts were consistent with immunoperoxidase data. Type I collagen was 70-75%, type III collagen was 18-21%, and type V was 6-8% of the total of these collagens at all gestational ages tested, compared to 85-90% type I, 8-11% type III, and 2-4% type V in adult skin. The enrichment of both types III and V collagen in fetal skin may reflect in part the proportion of vessel- and nerve-associated collagen versus dermal fibrillar collagen. The accumulation of dermal fibrillar collagen with increasing age would enhance the estimated proportion of type I collagen, even though the ratios of type III to I in dermal collagen fibrils may be similar at all ages.

The ontogeny and composition of the dermal-epidermal junction (DEJ) in developing human embryonic and fetal skin was studied at progressive stages of gestation by immunofluorescence microscopy and immunocytochemistry using transmission electron microscopy (TEM). The DEJ of embryonic skin at 5 weeks estimated gestational age (EGA) was a simple basement membrane zone limited to the basal cell plasma membrane, lamina lucida, and lamina densa. A network of reticular collagen fibrils (reticular lamina) was deposited beneath the lamina densa by 6 weeks. Coincident with the onset of increased complexity in epidermal and dermal structure, at the time of the embryonic to fetal transition, the DEJ displayed additional components that were markers of maturation. At 7–8 weeks EGA, fine filamentous structures extended from the DEJ into the reticular lamina. By 9 weeks EGA hemidesmosomes and banded anchoring fibrils were recognizable, although distributed sparsely at the DEJ. With increasing gestational age, these structures displayed greater electron density and structural completeness. By the end of the first trimester, the DEJ appeared ultrastructurally similar to that of mature skin. Weak immunofluorescent labeling demonstrated the presence of type VII collagen at the DEJ by 8 weeks EGA. From 10–12 weeks EGA immunofluorescent labeling of the DEJ for type VII collagen was distinctly punctate, while immunoperoxidase labeling observed by TEM was linear, continuous, and sublamina densa in position. With ongoing gestation the immunofhiorescent labeling became increasingly stronger at the DEJ. Thus, type VII collagen was present at the DEJ in the zone immediately beneath the lamina densa, before the appearance of mature anchoring fibrils but coordinate with the appearance of fine filamentous, unbanded structures, and appeared to increase with the development and accumulation of anchoring fibrils.

The process of human dermal development has been examined from the stage of the embryonic mesenchymal dermis (5-8 weeks of gestation) through the formation of the fibrous dermal connective (end of the first trimester), and during the growth of the fetal dermis (second and third trimesters) until birth. The elaboration of dermal organization and the overall increase in dermal thickness, largely consequences of extracellular, fibrous matrix accumulation, parallel the development and growth of the fetus as a whole. Although the mechanisms that determine the formation of the connective tissue architecture are not fully understood, the structural, histochemical, and biochemical changes that occur throughout development suggest processes that may be important in human dermal embryogenesis.

Type I human skin collagenase (HSC-1) was localized in developing embryonic and fetal skin ranging from 6 to 20 weeks estimated gestational age using an antigen-specific, affinity-purified, polyclonal antiserum to HSC-1 and an avidin-biotin alkaline phosphatase procedure. Double immunolabeling with monoclonal antibodies for Factor VIII-related antigen, type IV collagen, and the 68-kilodalton neurofilament subunit was performed using a direct peroxidase procedure. By 8 weeks estimated gestational age, HSC-1 localized to the periderm, the basal cell epidermal keratinocytes, dermal fibroblasts, and surrounding extracellular matrix. At 12 weeks estimated gestational age, HSC-1 immunolabeling showed a continued association with the epidermis and dermis. Dermal and subcutaneous blood vessels and the surrounding extracellular matrix were positive for HSC-1 labeling. HSC-1 staining was also found around developing nerves and in association with dermal fibroblasts. In the developing hair follicle, HSC-1 was present in keratinocytes of the pre-germ, germ, hair peg, and bulbous hair peg. HSC-1 immunoreactivity was also found in association with the hair canal, the bulge, and the dermal papillae, but was absent from the fetal sebaceous gland. These data demonstrate the association of HSC-1 with the development of interfollicular epidermis, the dermal collagenous matrix, the process of angiogenesis, the development of nerves, and hair follicle morphogenesis.

Kinetic and pharmacologic properties of uptake of serotonin and dopamine by normal human platelets have been investigated to test whether platelets can be employed as a model system for the reuptake of serotonin and dopamine in brain. Uptake of serotonin into platelets closely resembles reuptake of serotonin into serotonergic neurons. In contrast, uptake of dopamine into platelets appears to be mediated inefficiently via the specific serotonin uptake mechanism, based upon several lines of evidence. Serotonin and dopamine compete with each other, Antidepressant drugs, which are competitive inhibitors of uptake of both of these neurotransmitters, act at the same concentration of drug despite large differences in the Km values. Serotonin antagonists inhibit both serotonin and dopamine uptake. Finally, a serotonin-specific uptake inhibitor (fluoxetine) blocks dopamine, as well as serotonin, uptake.

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.

Since skin collagenase is required for initiation of the degradation of types I and III collagens, the major collagens of the human dermis, we examined its expression during embryonic and fetal development. When using skin fibroblasts cultured from human embryos and fetuses, immunoreactive collagenase concentrations were strongly correlated with estimated gestational age (p less than 0.001), with levels at 7-8 weeks of gestation that were about one-twentieth of those in the 29-week cell cultures. In crude culture medium, the apparent catalytic efficiency (activity per unit immunoreactive protein) was variable, an observation attributable in part to variable expression of a collagenase-inhibitory protein. Following chromatographic purification, four of ten fetal collagenases were found to have greater than or equal to 4-fold decrease in specific activity, suggesting that these particular fetal collagenases may be structurally and/or catalytically altered. Since the decreased levels of immunoreactive protein suggested that decreased enzyme synthesis was the major mechanism, we examined collagenase synthesis in a cell-free translation system. Here, we quantitated collagenase expression in the culture medium of intact cells prior to harvesting mRNA. Compared with the intact adult cells, the fetal cells had 3-17 times less collagenase activity in the medium, while in cell-free translation there was a 2- to 3-fold decrease in collagenase synthesis. These data suggest that decreased in vitro expression is correlated with decreased levels of translatable collagenase mRNA but that other factors, such as the collagenase inhibitor and altered specific activity of the enzyme, may be important in modulating collagenase activity.

We investigated kinetic and pharmacologic properties of the uptake of serotonin and dopamine by blood platelets from patients with Huntington disease and from persons at high genetic risk of developing Huntington disease. Uptake proved normal.