Changes in localization of human discs large (hDlg) during keratinocyte differentiation are [corrected] associated with expression of alternatively spliced hDlg variants.

Alternative spliced variants of the human discs large (hDlg) tumour suppressor are characterized by combinations of insertions. Here, using insertions I2- and I3-specific antibodies, we show that I2 and I3 variants have distinct distributions in epidermal and cervical epithelia. In skin and cervix, I3 variants are found in the cytoplasm. Cytoplasmic localization of I3 variants decreases as cervical keratinocytes differentiate, concomitant with relocalization to the cell periphery. I2 variants are found at the cell periphery of differentiated epidermal and cervical keratinocytes. Nuclear localization of I2 variants was evident in both tissues, with concentration of nuclear I2 variants in basal and parabasal cervical keratinocytes. A prominent nuclear localization of hDlg in cells of hyperproliferative layers of psoriatic lesions, but not in mature differentiated keratinocytes, together with I2 redistribution in differentiating keratinocytes, suggests that nuclear hDlg functions may be pertinent to growth of undifferentiated cells. Supporting our findings in squamous tissues, a decrease of nuclear hDlg and an increase of membrane-bound and cytoplasmic hDlg upon calcium-induced keratinocyte differentiation were not concomitant processes. Furthermore, we confirm that the exit of I2 variants from the nucleus is linked to stimulation of epithelial differentiation. The dynamic redistribution of hDlg also correlated with a marked increase in the expression of I3 variants while the level of I2 variants showed only a moderate decrease. Because changes in the intracellular distribution of hDlg splice variants, and in their expression levels, correlate with changes in differentiation state we hypothesize that the different hDlg isoforms play distinct roles at various stages of epithelial differentiation.

Unraveling the molecular mechanisms of hair and nail genodermatoses.

Inherited hair and nail diseases have long been considered a group of rare and obscure disorders with a largely unknown genetic basis. In the postgenomic era, a large portion of the genes that are responsible for these genetic disorders has been identified, yielding new insights into the complex molecular pathways that regulate the development and biological function of epidermal appendages. This article reviews the recent progress accomplished in this field and discusses the novel clinical and experimental observations in several hair and nail genodermatoses.

Gene characterization of sciellin (SCEL) and protein localization in vertebrate epithelia displaying barrier properties.

Sciellin is a precursor of the cornified envelope of mammalian stratified epithelia characterized by a central core of nonidentical repeats. We characterized the genomic structure of human sciellin and showed that each homologous repeat was encoded by one exon. We also characterized mouse sciellin and showed that mouse sciellin and human sciellin (HGMW-approved symbol SCEL) share a similar gene organization and protein expression pattern. This one exon/one repeat organization is unique among other cornified envelope precursors characterized by homologous repeats. We identified an alternatively spliced isoform of human sciellin, absent in mouse, characterized by an additional repeat at the beginning of the core domain. During embryonic development, the accumulation of sciellin transcript and the accumulation of sciellin protein in the epidermis correlated with the activation of markers of terminal differentiation in epidermis. Mouse sciellin was also identified in simple epithelia with barrier properties, lending further support to its importance in epithelial function.

Dexamethasone reduces the inflammatory response to cardiopulmonary bypass in children.

BACKGROUND: A randomized, prospective, double-blind study of 29 children was performed to evaluate the hypothesis that dexamethasone administration prior to cardiopulmonary bypass would decrease the inflammatory mediator release and improve the postoperative clinical course. METHODS: Fifteen children received dexamethasone (1 mg/kg intravenously) and 14 (controls) received saline solution 1 hour prior to CPB. Serial blood analyses for interleukin-6, tumor necrosis factor-alpha, complement component C3a, and absolute neutrophil count were performed. Postoperative variables evaluated included temperature, supplemental fluids, alveolar-arterial oxygen gradient, and days of mechanical ventilation. RESULTS: Dexamethasone caused an eightfold decrease in interleukin-6 levels and a greater than threefold decrease in tumor necrosis factor-alpha levels after CPB (p < 0.05). Complement component C3a and absolute neutrophil count were not affected by dexamethasone. The mean rectal temperature for the first 24 hours postoperatively was significantly lower in the group given dexamethasone than in the controls (37.2 degrees +/- 0.4 degrees C versus 37.7 degrees +/- 4 degrees C; p = 0.007). Dexamethasone-treated patients required less supplemental fluid during the first 48 hours (22 +/- 28 mL/kg versus 47 +/- 34 mL/kg; p = 0.04). Compared with controls, dexamethasone-treated children had significantly lower alveolar-arterial oxygen gradients during the first 24 hours (144 +/- 108 mm Hg versus 214 +/- 118 mm Hg; p = 0.02) and required less mechanical ventilation (median duration, 3 days versus 5 days; p = 0.02). CONCLUSIONS: Dexamethasone administration prior to CPB in children leads to a reduction in the postbypass inflammatory response as assessed by cytokine levels and clinical course.

Fatal hypermagnesemia in a child treated with megavitamin/megamineral therapy.

We report a case of fatal hypermagnesemia resulting from the unsupervised use of high doses of magnesium oxide administered as part of a regimen of megavitamin and megamineral therapy to a child with mental retardation, spastic quadriplegia, and seizures. The treatment regimen was given at the recommendation of a dietician working as a private nutritional consultant without the involvement or notification of the child's pediatrician. Hypermagnesemia is an uncommon but serious side effect of the use of magnesium containing compounds. These compounds are widely used as laxatives and dietary supplements, and serious side effects are uncommon when used in appropriate dosages and with adequate supervision. The use of alternative medical therapies, including megavitamin/megamineral therapy, is widespread. Many patients use alternative medicine or seek care from alternative medicine practitioners without the recommendation or knowledge of their primary physicians. Despite unproved benefit, many alternative therapies may be safe. However, unsupervised use of generally safe treatments can result in serious side effects. This case report serves to illustrate the characteristic pathophysiologic changes of severe hypermagnesemia, an entity rarely seen in pediatric practice, and more importantly, it alerts primary care and subspecialty pediatricians to be aware of and monitor the use of alternative medical therapies in their patients.

Hot spot mutations in keratin 2e suggest a correlation between genotype and phenotype in patients with ichthyosis bullosa of Siemens.

Ichthyosis bullosa of Siemens (IBS) is a rare disorder of cornification characterized by blister formation in the upper suprabasal layers of the epidermis. Molecular analysis of IBS has identified mutations in the keratin 2e (K2e) gene, which is located in the type II keratin gene cluster on chromosome 12q. We have studied two IBS families and have identified heterozygous point mutations in codon 493 of the K2e gene in both families. Whereas a non-conservative amino acid substitution at position 117 of the 2B region of K2e (E117K) was associated with a severe phenotype in family 1, family 2 showed mild clinical features as a result of a conservative substitution (E117D). These data suggest a phenotype-genotype correlation in these families.

Myocardial injury in children with respiratory syncytial virus infection.

OBJECTIVE: Respiratory syncytial virus (RSV) infection is associated with a number of extrapulmonary manifestations, including a sepsis-like syndrome characterized by any combination of hypothermia, fever, apnea, hypovolemia, and myocardial dysfunction. We hypothesized that RSV can have a direct injurious effect on the myocardium of infants and children that can be detected by the presence of cardiac troponin I (cTnI), a highly sensitive and specific marker of myocardial injury, in the blood of patients infected with the virus. DESIGN: Serial cTnI measurements were obtained from patients admitted with documented RSV infection to the pediatric intensive care unit (PICU). PARTICIPANTS: Data were collected and analyzed from 22 RSV infected patients and 11 control patients. RESULTS: Elevated levels of cTnI were detected in 54.5% (12/22) of the study population during their PICU admission. The average cTnI level was significantly higher in the RSV infected group than in controls. There was a significant association between the presence of a positive troponin assay and the occurrence of a cardiovascular event, the need for inotropic support, and the requirement of mechanical ventilation. Patients who required inotropic support had a significantly higher cTnI level than the rest of the study population. CONCLUSION: A large percentage of children admitted to the PICU with RSV infection have myocardial damage as detected by the use of commercially available troponin assays. Additionally, in a portion of these patients, this damage is clinically significant, leading to cardiovascular instability and the need for inotropic support.

Ectopic expression of the nude gene induces hyperproliferation and defects in differentiation: implications for the self-renewal of cutaneous epithelia.

Nude mice are characterized by the absence of visible hair, epidermal defects, and the failure to develop a thymus. This phenotype results from loss-of-function mutations in Whn (Hfh11), a winged-helix transcription factor. In murine epidermis and hair follicles, endogenous whn expression is induced as epithelial cells initiate terminal differentiation. Using the promoter for the differentiation marker involucrin, transgenic mice that ectopically express whn in stratified squamous epithelia, hair follicles, and the transitional epithelium of the urinary tract were generated. Transgenic epidermis and hair follicles displayed impaired terminal differentiation and a subset of hair defects, such as delayed growth, a waved coat, and curly whiskers, correlated with decreased transforming growth factor (TGF)-alpha expression. The exogenous Whn protein also stimulated epithelial cell multiplication. In the epidermis, basal keratinocytes exhibited hyperproliferation, though transgene expression was restricted to suprabasal, postmitotic cells. Hair follicles failed to enter telogen (a resting period) and remained continuously in an abnormal anagen (the growth phase of the hair cycle). Ureter epithelium developed severe hyperplasia, leading to the obstruction of urine outflow and death from hydronephrosis. Though an immune infiltrate was present occasionally in transgenic skin, the infiltrate was not the primary cause of the epithelial hyperproliferation, as the immune reaction was not observed in all affected transgenics, and the transgene induced identical skin and urinary tract abnormalities in immunodeficient Rag1-null mice. Given the effects of the transgene on cell proliferation and TGFalpha expression, the results suggest that Whn modulates growth factor production by differentiating epithelial cells, thereby regulating the balance between proliferative and postmitotic populations in self-renewing epithelia.

Identification of sporadic mutations in the helix initiation motif of keratin 6 in two pachyonychia congenita patients: further evidence for a mutational hot spot.

Pachyonychia congenita (PC) is a rare, autosomal dominant, ectodermal dysplasia characterized most distinctly by the presence of symmetric nail hypertrophy. In the Jadassohn-Lewandowsky form, or PC-1, additional cutaneous manifestations may include palmoplantar hyperkeratosis, hyperhidrosis, follicular keratoses, and oral leukokeratosis. Mutations have previously been identified in the 1A helix initiation motif of either keratin 6 or keratin 16 in patients with PC-1. In the current study, we have identified 2 sporadic, heterozygous mutations in the 1A helix region of the K6 isoform (K6a). The first mutation identified was a 3 base pair deletion (K6adelta N171). The second mutation was a C-to-A transversion resulting in an amino acid substitution (K6a N171K). These data, in combination with previous reports, provide further evidence that this location is a mutational hot spot.

Complete repair of Tetralogy of Fallot with absent pulmonary valve including the role of airway stenting.

Tetralogy of Fallot (TOF) with absent pulmonary valve (APV) represents an extreme form of tetralogy where pulmonary insufficiency and mild annular stenosis often results in massive pulmonary arterial (PA) dilatation. The aneurysmal left and right PAs often compress the adjacent trachea and bronchi, leading to airway obstruction and respiratory failure in infancy. Between 1991 and 1997, 11 patients underwent a single stage repair of TOF and APV using a valved (10 patients) or nonvalved (1 patient) homograft conduit and PA reduction arterioplasty. There was one (1/11 [9.1%) perioperative and one (9.1%) late death. Both deaths were related to airway complications. Morbidity associated with postoperative respiratory complications and ventilator-dependency due to underlying tracheobronchomalacia is an important problem. Intermediate follow-up shows a high incidence of reintervention for conduit stenosis and/or insufficiency and tracheobronchial compression. These infants also required multiple hospitalizations for recurrent respiratory infections secondary to their tracheobronchomalacia. Stenting of the right and left main bronchi with balloon expandable metallic stents is a new experimental therapy that has been useful in two recent patients with respiratory failure despite satisfactory intracardiac repair. It may provide an attractive alternative therapy to prolonged mechanical ventilation with positive end expiratory pressure in patients with severe tracheobronchomalacia. Complete repair with a valved homograft conduit and reduction pulmonary arterioplasty in infancy at the time of diagnosis is the procedure of choice for infants with TOF with APV. With this approach the patient outcome is essentially determined by their airway status and airway management.

cDNA cloning and characterization of sciellin, a LIM domain protein of the keratinocyte cornified envelope.

Sciellin is a precursor of the cornified envelopes of mammalian keratinizing tissues. We have cloned the cDNA encoding sciellin by screening a human keratinocyte expression library with a sciellin-specific monoclonal antibody. The composite cDNA of 2.35 kilobase pairs encodes a protein of 75.3 kDa with a pI of 10.09. The translated sequence has a central domain containing 16 repeats of 20 amino acids each that is rich in Gln and Lys residues, which are potential transglutaminase substrates, and a carboxyl domain, which contains a single LIM motif. Sciellin cDNA probes hybridize to bands of 3.4 and 4.4 kilobase pairs on Northern blots of cultured human keratinocyte RNA. The gene was mapped to human chromosome band 13q22 by fluorescence in situ hybridization. Radiation hybrid mapping demonstrated that sciellin is linked to the sequence tagged site marker WI-457 with a logarithm of the odds score of 7.77. In situ hybridization of human foreskin tissue sections demonstrated that sciellin is expressed in the stratum granulosum. Immunofluorescent staining with a polyclonal rabbit antibody made to a recombinant sciellin protein showed peripheral cytoplasmic localization in the upper cell layers of epidermis and in stratified squamous epithelia such as the oral cavity, esophagus, and vagina. Simple and columnar epithelia, with the exception of the amnion, showed no reaction.

Hair growth cycle affects hair follicle destruction by ruby laser pulses.

It has been shown that normal mode ruby laser pulses (694 nm) are effective in selectively destroying brown or black pigmented hair follicles in adult Caucasians. This study investigated how the various stages of the hair follicle growth cycle influence follicle destruction by ruby laser treatment, using a model of predictable synchronous hair growth cycles in the infantile and adolescent mice. A range of ruby laser pulse fluences was delivered during different stages of the hair growth cycle, followed by histologic and gross observations of the injury and regrowth of hair. Actively growing and pigmented anagen stage hair follicles were sensitive to hair removal by normal mode ruby laser exposure, whereas catagen and telogen stage hair follicles were resistant to laser irradiation. Selective thermal injury to follicles was observed histologically, and hair regrowth was fluence dependent. In animals exposed during anagen, intermediate fluences induced nonscarring alopecia, whereas high fluences induced scarring alopecia. The findings of this study suggest treatment strategies for optimal laser hair removal.

Localization and characterization of the RNA binding protein TLS in skin and stratified mucosa.

Translocated in liposarcoma (TLS), a member of the Ewing's sarcoma family of RNA binding proteins, is targeted to the product of RNA POL II and functions in nuclear events as well as in nuclear-cytoplasmic transport of mRNA. It has been most extensively studied in cell lines, but was identified in several rat tissues by northern blot analysis, with adipose tissue showing the highest expression followed by whole skin. This paper describes a protein with amino acid sequence homology to TLS that was isolated from bovine tongue epithelium using an affinity column made with an antibody to the cornified envelope precursor sciellin. Using reverse transcriptase polymerase chain reaction technology and total RNA isolated from bovine tongue epithelium, a cDNA was obtained whose nucleotide sequence coded for a protein homologous to human TLS. Nuclear staining in all layers of human epidermis and bovine stratified epithelium was observed with an antibody to TLS, whereas peripheral staining of the upper layers of these tissues was observed with the antibody to sciellin. Cultured cells gave similar results; however, adult tissue required boiling in citrate buffer to unmask antigenic sites before reacting with the TLS antibody. Western blots of extracts of human and bovine keratinocytes using TLS and sciellin antibodies showed that the two proteins shared at least one epitope, but that they were different. TLS was lost from the nucleus following inhibition of RNA POL II activity and the protein was identified in CNBr extracts of purified keratinocytes cornified envelopes by western blot. These results clearly indicate that TLS functions as an RNA binding protein in keratinocytes in vivo and in vitro. Furthermore the sequestration of TLS to the cell envelope may play a role in regulating its nuclear-cytoplasmic transport and effect its role in transcription.

Primary mouse keratinocyte cultures contain hair follicle progenitor cells with multiple differentiation potential.

The interfollicular epidermis contains a single type of terminally differentiated keratinocytes, whereas hair follicles are composed of a minimum of six or seven distinct types. Whether or not these various populations of terminally differentiated keratinocytes originate from one or more progenitor cells has not been established. A related and important question is whether keratinocyte progenitor cells with a pluripotent potential, able to form not only epidermis but also hair follicles, can be maintained in vitro for any period of time. We have addressed these questions using skin reconstitution assays with admixed populations of genetically labeled, cultured keratinocytes. Examination of reconstituted epidermis and hair follicles showed that neither was composed of a random mixture of differently labeled keratinocytes, as would be predicted if they originated from a random reassociation of cells. Instead, the reconstituted interfollicular epidermis contained distinct columnar units, comprising all the overlying layers and most likely derived from a single progenitor cell. In contrast, hair follicles were found to be composed of cells of multiple origin, with each population showing a striking localization to a separate concentric region. The vast majority of reconstituted follicles appeared to derive from a minimum of two or, in a significant fraction of cases, three progenitor cells, one for the generation of the shaft (cuticle, cortex, and medulla), one for the inner root sheath, and the third for the outer root sheath. The general implications of these findings for epidermis and hair follicle formation and for keratinocyte stem cell cultivation are discussed.

A novel nonepidermolytic palmoplantar keratoderma: a clinical and histopathologic study of six cases.

BACKGROUND: Some hereditary palmoplantar keratodermas (PPK) have been defined at the molecular level. OBJECTIVE: Our purpose was to establish the cause of a hereditary PPK with unique histopathologic findings in the epidermis. METHODS: Investigative studies included light and electron microscopy and determination of genomic DNA sequence. RESULTS: Six patients with PPK were found to have unique changes in the epidermis characterized by orthokeratosis, parakeratosis, perinuclear vacuolization, and keratohyalin granules that varied in size and shape and were located in the cell periphery. Electron microscopy showed the perinuclear region contained many ribosomes and vacuoles and was surrounded by a tonofibril shell. Family involvement suggested a dominant disorder. However, no mutation of keratin genes 1, 6a, 9, or 16 was found. CONCLUSION: The histopathologic features of this unique PPK most closely resemble Curth-Macklin ichthyosis for which the genetic basis has not been established. Further genetic studies are needed.

Severe paroxysmal sinus bradycardia associated with high-frequency oscillatory ventilation.

OBJECTIVE: To determine the incidence of and risk factors for unexplained paroxysmal bradycardia in children treated with high-frequency oscillatory ventilation (HFOV). DESIGN: A nested case-control study. SETTING: A university-affiliated children's hospital. SUBJECTS: All children treated with HFOV for at least 3 days during a 2-year period and a randomly chosen comparison group of 50 children treated with only conventional mechanical ventilation (CMV) for at least 3 days during the same time period. INTERVENTIONS: None. MEASUREMENTS AND RESULTS: Unexplained paroxysmal sinus bradycardia occurred in six children (12%) receiving HFOV, and was significantly more common than in children treated with CMV (0%). The bradycardic events occurred after the lung disease started to improve, and the mean airway pressure (mPaw) at the time of the bradycardias was significantly decreased from the child's maximal mPaw. The bradycardic events were effectively treated acutely with manual ventilation or atropine sulfate, and resolved completely after the patient was changed to a regimen of CMV. CONCLUSION: Unexplained paroxysmal bradycardia associated with HFOV in children is not uncommon. It completely resolves with conversion to CMV and may be related to overdistention of alveoli as compliance improves.

High-frequency oscillatory ventilation with partial liquid ventilation in a model of acute respiratory failure.

OBJECTIVE: To determine whether there is an improvement in oxygenation when partial liquid ventilation and high-frequency oscillatory ventilation are combined in the treatment of acute lung injury, compared with high-frequency oscillatory ventilation alone. DESIGN: Controlled animal trial. SETTING: Research laboratory in a university setting. SUBJECTS: Ten 3-kg piglets. INTERVENTIONS: Anesthetized piglets underwent high-frequency oscillatory ventilation, with mean airway pressure of 20 cm H2O, before induction of acute lung injury with repeated saline lavage. When PaO2 values were < 100 torr (< 13.3 kPa), five animals were randomized to receive escalating doses (3, 15, and 30 mL/kg) of perflubron at 60-min intervals. The other five animals remained on high-frequency oscillatory ventilation only. Sham dosing was performed at 60-min intervals in these animals. Arterial blood gases were obtained in both groups at baseline, after injury, and after perflubron and sham doses. MEASUREMENTS AND MAIN RESULTS: Statistically significant improvements in oxygenation were demonstrated in animals that received 3 mL/kg of perflubron with high-frequency oscillatory ventilation compared with animals receiving high-frequency oscillatory ventilation alone (253 +/- 161 vs. 90 +/- 30 torr [33.65 +/- 21.46 vs. 12.0 +/- 4.0 kPa], p < .05). Improvements in oxygenation with additional administration of perflubron were not greater than the improvements seen in the high-frequency oscillatory ventilation-only group. PaCO2 and pH were similar in both groups at all times. No hemodynamic compromise occurred in either group of animals. CONCLUSIONS: The combination of low-dose perflubron with high-frequency oscillatory ventilation leads to more rapid improvement in arterial oxygenation than high-frequency oscillatory ventilation alone, in a piglet model of acute lung injury. Although the group receiving high-frequency oscillatory ventilation alone eventually achieved PaO2 values that were equivalent to the group receiving high-frequency ventilation and perflubron, the combination of perflubron with high-frequency oscillatory ventilation may permit effective oxygenation and ventilation at lower mean airway pressures by facilitating alveolar expansion and decreasing intrapulmonary shunt.

Loose anagen syndrome and loose anagen hair.

Loose anagen syndrome, or loose anagen hair, is a recently described condition of unknown etiology that may be under-recognized. The typical patient is a child with sparse fine hair that can easily be pulled out. The diagnosis is confirmed by microscopic examination of firmly pulled hairs, many of which are in the anagen phase but lacking an inner and outer root sheath and demonstrating a ruffled cuticle. Some presentations of alopecia areata may be confused with this condition, but the pull test analysis serves to differentiate them. A variety of theories have been postulated to explain the pathophysiology of loose anagen syndrome. In some cases, there is an autosomal dominant pattern of inheritance. In most cases, this condition spontaneously improves with age.