Tape strips in dermatology research.

Tape strips have been used widely in dermatology research as a minimally invasive method to sample the epidermis, avoiding the need for skin biopsies. Most research has focused on epidermal pathology, such as atopic eczema, but there is increasing research into the use of tape strips in other dermatoses, such as skin cancer, and the microbiome. This review summarizes the technique of tape stripping, and discusses which dermatoses have been studied by tape stripping and alternative minimally invasive sampling methods. We review the number of tape strips needed from each patient and the components of the epidermis that can be obtained by tape stripping. With a focus on protein and RNA extraction, we address the techniques used to process tape strips. There is no optimal protocol to extract protein, as this depends on the abundance of the protein studied, its level of expression in the epidermis and its solubility. Many variables can alter the amount of protein obtained from tape strips, which must be standardized to ensure consistency between samples. No study has compared different RNA extraction techniques, but our own experience is that RNA yield is optimized by using 20 tape strips and the use of a cell scraper.

Hierarchical Black Hole Mergers in Active Galactic Nuclei.

The origins of the stellar-mass black hole mergers discovered by LIGO/Virgo are still unknown. Here we show that if migration traps develop in the accretion disks of active galactic nuclei (AGNs) and promote the mergers of their captive black holes, the majority of black holes within disks will undergo hierarchical mergers-with one of the black holes being the remnant of a previous merger. 40% of AGN-assisted mergers detected by LIGO/Virgo will include a black hole with mass ≳50M_{⊙}, the mass limit from stellar core collapse. Hierarchical mergers at traps in AGNs will exhibit black hole spins (anti)aligned with the binary's orbital axis, a distinct property from other hierarchical channels. Our results suggest, although not definitively (with odds ratio of ∼1), that LIGO's heaviest merger so far, GW170729, could have originated from this channel.

Differential expression of secreted factors SOSTDC1 and ADAMTS8 cause profibrotic changes in linear morphoea fibroblasts.

BACKGROUND: Linear morphoea (LM) is a rare connective tissue disorder characterized by a line of thickened skin and subcutaneous tissue and can also affect the underlying muscle and bone. Little is known about the disease aetiology, with treatment currently limited to immune suppression, and disease recurrence post-treatment is common. OBJECTIVES: In order to uncover new therapeutic avenues, the cell-intrinsic changes in LM fibroblasts compared with site-matched controls were characterized. METHODS: We grew fibroblasts from site-matched affected and unaffected regions from five patients with LM, we subjected them to gene expression analysis and investigation of SMAD signalling. RESULTS: Fibroblasts from LM lesions showed increased migration, proliferation, altered collagen processing, and abnormally high basal levels of phosphorylated SMAD2, thereby rendering them less responsive to transforming growth factor (TGF)-ß1 and reducing the degree of myofibroblast differentiation, which is a key component of the wound-healing and scarring process in normal skin. Conditioned media from normal fibroblasts could reverse LM-affected fibroblast migration and proliferation, suggesting that the LM phenotype is driven by an altered secretome. Gene array analysis and RNA-Seq indicated upregulation of ADAMTS8 and downregulation of FRAS1 and SOSTDC1. SOSTDC1 knock-down recapitulated the reduced TGF-ß1 responsiveness and LM fibroblast migration, while overexpression of ADAMTS8 induced myofibroblast markers. CONCLUSIONS: We demonstrate that cell-intrinsic changes in the LM fibroblast secretome lead to changes observed in the disease, and that secretome modulation could be a viable therapeutic approach in the treatment of LM.

Rab3Gap1 mediates exocytosis of Claudin-1 and tight junction formation during epidermal barrier acquisition.

Epidermal barrier acquisition during late murine gestation is accompanied by an increase in Akt kinase activity and cJun dephosphorlyation. The latter is directed by the Ppp2r2a regulatory subunit of the Pp2a phosphatase. This was accompanied by a change of Claudin-1 localisation to the cell surface and interaction between Occludin and Claudin-1 which are thought to be required for tight junction formation. The aim of this study was to determine the nature of the barrier defect caused by the loss of AKT/Ppp2r2a function. There was a paracellular barrier defect in rat epidermal keratinocytes expressing a Ppp2r2a siRNA. In Ppp2r2a knockdown cells, Claudin-1 was located to the cytoplasm and its expression was increased. Inhibiting cJun phosphorylation restored barrier function and plasma membrane localisation of Claudin-1. Expression of the Rab3 GTPase activating protein, Rab3Gap1, was restored in Ppp2r2a siRNA cells when cJun phosphorylation was inhibited. During normal mouse epidermal development, Claudin-1 plasma membrane localisation and Rab3Gap1 cell surface expression were co-incident with Akt activation in mouse epidermis, strongly suggesting a role of Rab3Gap1 in epidermal barrier acquisition. Supporting this hypothesis, siRNA knockdown of Rab3Gap1 prevented plasma membrane Claudin-1 expression and the formation of a barrier competent epithelium. Replacing Rab3Gap1 in Ppp2r2a knockdown cells was sufficient to rescue Claudin-1 transport to the cell surface. Therefore these data suggest Rab3Gap1 mediated exocytosis of Claudin-1 is an important component of epidermal barrier acquisition during epidermal development.

Circulating cell-free fetal DNA for the detection of RHD status and sex using reflex fetal identifiers.

OBJECTIVE: To determine the sensitivity and specificity of circulating cell-free fetal DNA in determining the fetal RHD status and fetal sex. METHODS: Maternal blood was collected in each trimester of pregnancy from RhD negative nonalloimmunized women. Whole blood was centrifuged, separated into plasma and buffy coat, and frozen at -80°C. DNA analysis was conducted via allele-specific primer extensions for exons 4, 5, and 7 of the RHD gene and for a 37-base pair insertion in exon 4 (RHD pseudogene; psi) three Y-chromosome sequences (SRY, DBY, and TTY2), and an extraction control (TGIFL-like X/Y). RhD serotyping on cord blood and gender assessment of the newborns were entered into a Web-based database. RESULTS: One hundred twenty women were enrolled. The median gestational age at the first venipuncture was 12.4 (range: 10.6-13.9) weeks with 120 samples drawn; 118 samples were drawn at 17.6 (16-20.9) weeks; and 113 samples at 28.7 (27.9-33.9) weeks. Overall accuracy for RHD was 99.1%, 99.1%, and 98.1% for each trimester and was 99.1%, 99.1%, and 100% for fetal sex determination. CONCLUSIONS: Fetal RHD genotyping and sex can be very accurately determined in all three trimesters using circulating cell-free fetal DNA in the maternal circulation.

Fetal blood sampling in baboons (Papio spp.): important procedural aspects and literature review.

BACKGROUND: The baboons (Papio cynocephalus) have similarities with human placentation and fetal development. Fetal blood sampling allows investigators to assess fetal condition at a specific point in gestation as well as transplacental transfer of medications. Unfortunately, assessing fetal status during gestation has been difficult and fetal instrumentation associated with high rate of pregnancy loss. Our objectives are to describe the technique of ultrasound guided cordocentesis (UGC) in baboons, report post-procedural outcomes, and review existing publications. METHODS: This is a procedural paper describing the technique of UGC in baboons. After confirming pregnancy and gestational age via ultrasound, animals participating in approved research protocols that required fetal assessment underwent UGC. RESULTS: We successfully performed UGC in four animals (five samples) using this technique. Animals were sampled in the second and third trimesters with fetal blood sampling achieved by sampling a free cord loop, placental cord insertion site or the intrahepatic umbilical vein. All procedures were without complication and these animals delivered at term. CONCLUSIONS: Ultrasound guided fetal umbilical cord venipuncture is a useful and safe technique to sample the fetal circulation with minimal risk to the fetus or mother. We believe this technique could be used for repeated fetal venous blood sampling in the baboons.

AKT1-mediated Lamin A/C degradation is required for nuclear degradation and normal epidermal terminal differentiation.

Nuclear degradation is a key stage in keratinocyte terminal differentiation and the formation of the cornified envelope that comprises the majority of epidermal barrier function. Parakeratosis, the retention of nuclear material in the cornified layer of the epidermis, is a common histological observation in many skin diseases, notably in atopic dermatitis and psoriasis. Keratinocyte nuclear degradation is not well characterised, and it is unclear whether the retained nuclei contribute to the altered epidermal differentiation seen in eczema and psoriasis. Loss of AKT1 function strongly correlated with parakeratosis both in eczema samples and in organotypic culture models. Although levels of DNAses, including DNase1L2, were unchanged, proteomic analysis revealed an increase in Lamin A/C. AKT phosphorylates Lamin A/C, targeting it for degradation. Consistent with this, Lamin A/C degradation was inhibited and Lamin A/C was observed in the cornified layer of AKT1 knockdown organotypic cultures, surrounding retained nuclear material. Using AKT-phosphorylation-dead Lamin A constructs we show that the retention of nuclear material is sufficient to cause profound changes in epidermal terminal differentiation, specifically a reduction in Loricrin, Keratin 1, Keratin 10, and filaggrin expression. We show that preventing nuclear degradation upregulates BMP2 expression and SMAD1 signalling. Consistent with these data, we observe both parakeratosis and evidence of increased SMAD1 signalling in atopic dermatitis. We therefore present a model that, in the absence of AKT1-mediated Lamin A/C degradation, DNA degradation processes, such as those mediated by DNAse 1L2, are prevented, leading to parakeratosis and changes in epidermal differentiation.

PA-FABP, a novel marker of human epidermal transit amplifying cells revealed by 2D protein gel electrophoresis and cDNA array hybridisation.

Human epidermal stem cells express higher levels of beta1 integrins than their more differentiated daughters, transit amplifying cells. In a search for additional stem and transit cell markers we used proteomics and differential cDNA hybridisation to compare keratinocytes fractionated on the basis of beta1 integrin expression. There were remarkably few differences between the two populations and none of the RNAs differed in abundance by more than 2-fold. Nevertheless, proteomics revealed upregulated expression of epidermal fatty acid binding protein (PA-FABP, also known as E-FABP), Annexin II and two keratin related proteins in the transit population. An unknown high molecular mass protein was upregulated in the stem cell population. The upregulation of PA-FABP was confirmed by Northern blotting and conventional and whole mount labelling of human epidermis. We conclude that PA-FABP is a novel marker of epidermal transit amplifying cells.

Bromocriptine associated with symptom exacerbation during neuroleptic treatment of schizoaffective schizophrenia.

Bromocriptine is a dopamine agonist with potential mental side effects. The authors report a case involving a patient with neuroleptic-induced remission of psychiatric symptoms in whom bromocriptine was associated with temporary exacerbation of schizophrenic symptoms. Other treatment approaches than bromocriptine are suggested for amenorrhea-galactorrhea induced by neuroleptic medication. The use of bromocriptine requires monitoring for changes in mental status.

Severe nonimmune hydrops secondary to parvovirus B-19 infection: Spontaneous reversal in utero and survival of a term infant.

We present a case of intrauterine infection with parvovirus B-19 and accompanying severe nonimmune hydrops at 26 weeks' gestation. The fetus showed progressive recovery on ultrasound. A term infant was delivered with hepatosplenomegaly as the only abnormality.

Management of pregnancies complicated by anti-Kell isoimmunization.

OBJECTIVE: To assess the efficacy of managing pregnancies complicated by anti-Kell isoimmunization using the methods developed for evaluating anti-Rh-D isoimmunization. METHODS: We reviewed 156 anti-Kell-positive pregnancies seen from 1959 to 1995, which were managed with serial maternal titers, amniotic fluid deltaOD450 determination, and funipuncture. Data on maternal titers, paternal phenotypes, invasive fetal testing and therapies, and neonatal outcomes were collected and analyzed to determine whether severely affected pregnancies were identified in time for successful fetal and neonatal therapy. RESULTS: Twenty-one fetuses were affected, eight with severe disease, and two fetuses in this group died. All of the severely affected fetuses were associated with maternal serum titers of at least 1:32. A critical titer of 1:32 was found to be 100% sensitive for identifying the affected pregnancies. The affected group had significantly higher amniotic fluid deltaOD450 values over the range of gestational ages than did the unaffected group (P < .001). The upper Liley curve was a specific discriminator for the diagnosis of affected fetuses, and the lower curve was specific for the diagnosis of unaffected or mild cases. CONCLUSION: Fetal anemia due to anti-Kell isoimmunization might be due in part to erythropoietic suppression, but it is still largely a hemolytic process. The methods based on a hemolytic process, including use of a critical maternal serum titer of 1:32, serial amniotic fluid analyses when the titer was exceeded, and liberal use of funipuncture, were successful in identifying severely affected fetuses.

Articulated needle guide: report on the first 30 cases.

This report represents our initial experience with a new type of needle guide for amniocentesis, transabdominal chorionic villus biopsies, and funipuncture developed at the Ohio State University and tested at the Harris Birthright Centre for Fetal Medicine. This articulated needle guide is designed to combine the advantages of the existing needle guides and the freehand technique. The needle guide offers improved maneuverability compared with standard needle guides and maintains easy visualization of the needle.

Anti-M isoimmunization: management and outcome at the Ohio State University from 1969 to 1995.

OBJECTIVE: To review the management strategies and outcome in gravidas with anti-M isoimmunization over the past 26 years at The Ohio State University. METHODS: Data collected from 115 pregnancies found to have anti-M antibody at The Ohio State University from September 1969 through February 1996 were reviewed retrospectively. We analyzed indirect antiglobulin tests, amniotic fluid with spectrophotometric examination, direct antiglobulin tests, M antigen status, antepartum course, and perinatal outcome. RESULTS: Anti-M antibody was found in 90 women who had 115 pregnancies over 26 years. Among those with positive indirect antiglobulin tests, 104 pregnancies had titers at or below 1:4. Only one patient with an initial low titer experienced more than a three-fold increase to 1:64. Two women underwent a total of eight amniocenteses when titers were at or above 1:128. Forty-two (60%) of the 70 infants tested were positive for M antigen. Nine infants required phototherapy. Eight of these infants were delivered preterm. There was an increase in the number of women seen with anti-M antibody in pregnancy at our institution, with nearly 10% of all gravidas with a positive antibody screen having anti-M alloantibodies. There were no cases of hemolytic disease of the newborn, mild or severe. CONCLUSION: The prevalence of anti-M isoimmunization may be increasing. The incidence of severe hemolytic disease of the newborn due to anti-M is extremely low. We found no cases in our review of 115 pregnancies, although there have been several cases of severe hemolytic disease of the newborn reported. If anti-M is detected in pregnancy, the titer is low (no more than 1:4), and there is no history of prior pregnancy complications suggesting a hemolytic disease process, we recommend no further testing other than an indirect antiglobulin test at 28 weeks to look for the emergence of other alloantibodies. However, if the initial titer is elevated or there is a concerning obstetric history, serial titers should be performed and amniocenteses reserved for rising titers.

Does extra-amniotic infection cause preterm labor? Gas-liquid chromatography studies of amniotic fluid in amnionitis, preterm labor, and normal controls.

Gas-liquid chromatography (GLC) was used to identify short-chain organic acid byproducts of bacterial metabolism in amniotic fluid from seven normal control patients, six women with overt amnionitis, and six preterm labor patients. Microbiologic culture for aerobic and anaerobic bacteria was also carried out. Positive GLC findings were generally associated with positive cultures, except in five of the preterm labor patients whose GLCs were positive despite negative cultures. The origin of the short-chain organic acids found in these women is unclear; extra-amniotic bacterial growth may explain this finding.