Normal human skin is superior to monkey oesophagus substrate for detection of circulating BP180-NC16A-specific IgG antibodies in bullous pemphigoid.

BACKGROUND: Bullous pemphigoid (BP) is the most common autoimmune subepidermal blistering skin disease. Two antigens have been identified as targets of circulating autoantibodies (autoAbs) - BP180 and BP230 - with BP180 being a critical transmembrane adhesion protein of basal keratinocytes of the epidermis. The noncollagenous domain 16A (NC16A) of BP180 is the immunodominant epitope in patients with BP, and anti-BP180-NC16A IgG antibodies (Abs) correlate to disease activity. Routine serological testing and follow-up of BP relies on indirect immunofluorescence (IIF) of serum Abs, commonly performed on monkey oesophagus (ME), and/or enzyme-linked immunosorbent assay (ELISA) testing on recombinantly produced fragments of BP180 and BP230 (BP180-NC16A, BP230-C/N). OBJECTIVES: To determine if NC16A epitopes are well represented on ME substrate. METHODS: Sera from different BP cohorts were tested by IIF on ME and normal human skin (NHS). To confirm findings, affinity-purified anti-BP180-NC16A/BP230 polyclonal Abs and recombinant anti-BP180-NC16A/BP230 monoclonal antibodies (mAbs) were used. RESULTS: For sensitive detection of BP180-NC16A-specific IgG Abs, sections of NHS are superior to the widely used ME. Confirmation comes from polyclonal affinity-purified anti-BP180-NC16A/BP230 Abs, and by mAbs cloned from a patient with active BP. CONCLUSIONS: Use of NHS is preferable over ME in routine IIF testing for BP. These results are of clinical relevance because anti-BP180-NC16A IgG titres are correlated to disease activity and detecting them reliably is important for screening, diagnosis and follow-up of patients with BP.

Reduced soluble receptor for advanced glycation end-products (sRAGE) scavenger capacity precedes pre-eclampsia in Type 1 diabetes.

OBJECTIVE: Increased advanced glycation end-products (AGEs) and their soluble receptors (sRAGE) have been implicated in the pathogenesis of pre-eclampsia (PE). However, this association has not been elucidated in pregnancies complicated by diabetes. We aimed to investigate the serum levels of these factors in pregnant women with Type 1 diabetes mellitus (T1DM), a condition associated with a four-fold increase in PE. DESIGN: Prospective study in women with T1DM at 12.2 ± 1.9, 21.6 ± 1.5 and 31.5 ± 1.7 weeks of gestation [mean ± standard deviation (SD); no overlap] before PE onset. SETTING: Antenatal clinics. POPULATION: Pregnant women with T1DM (n = 118; 26 developed PE) and healthy nondiabetic pregnant controls (n = 21). METHODS: Maternal serum levels of sRAGE (total circulating pool), N(ε)-(carboxymethyl)lysine (CML), hydroimidazolone (methylglyoxal-modified proteins) and total AGEs were measured by immunoassays. MAIN OUTCOME MEASURES: Serum sRAGE and AGEs in pregnant women with T1DM who subsequently developed PE (DM PE+) versus those who remained normotensive (DM PE-). RESULTS: In DM PE+ versus DM PE-, sRAGE was significantly lower in the first and second trimesters, prior to the clinical manifestation of PE (P < 0.05). Further, reflecting the net sRAGE scavenger capacity, sRAGE:hydroimidazolone was significantly lower in the second trimester (P < 0.05) and sRAGE:AGE and sRAGE:CML tended to be lower in the first trimester (P < 0.1) in women with T1DM who subsequently developed PE versus those who did not. These conclusions persisted after adjusting for prandial status, glycated haemoglobin (HbA1c), duration of diabetes, parity and mean arterial pressure as covariates. CONCLUSIONS: In the early stages of pregnancy, lower circulating sRAGE levels, and the ratio of sRAGE to AGEs, may be associated with the subsequent development of PE in women with T1DM.

Biological and biomechanical assessment of a long-term bioresorbable silk-derived surgical mesh in an abdominal body wall defect model.

BACKGROUND: The patient's own repair of a body wall defect would be enhanced by a mechanically robust, physically tailorable, bioresorbable mesh scaffold that: (1) supports rapid host tissue ingrowth and development, while (2) reducing the complications associated with permanent synthetic and allogenic and xenogenic biological implants. METHODS: A full-thickness 1-cm-diameter abdominal wall defect was made in 36 Sprague-Dawley rats, which were divided according to the implant material used; SeriFascia surgical mesh (test) and Mersilene mesh (control). Histopathology, histomorphometry, tissue ingrowth, and biomechanical analysis were performed 30 and 94 days post-implantation. RESULTS: The biological response to the test and control groups were equivalent, with significantly greater tissue ingrowth observed in the test group (P < 0.05). A significant 33 and 57% reduction of test device mass was observed at 30 and 94 days, respectively. The ultimate burst load for the test group defect decreased after 30 days to the initial strength of the control mesh and remained constant for the duration of the study. CONCLUSION: SeriFascia surgical mesh initially bioresorbed at an ideal rate that supported the transfer of load-bearing responsibility to developing host repair tissue. The results indicate the development of functional native tissue that could potentially minimize any long-term complication associated with presently available mesh implants.

Anti-angiogenic factors and pre-eclampsia in type 1 diabetic women.

AIMS/HYPOTHESIS: Elevated anti-angiogenic factors such as soluble fms-like tyrosine kinase 1 (sFlt1), a soluble form of vascular endothelial growth factor receptor, and endoglin, a co-receptor for TGFbeta1, confer high risk of pre-eclampsia in healthy pregnant women. In this multicentre prospective study, we determined levels of these and related factors in pregnant women with type 1 diabetes, a condition associated with a fourfold increase in pre-eclampsia. METHODS: Maternal serum sFlt1, endoglin, placental growth factor (PlGF) and pigment epithelial derived factor were measured in 151 type 1 diabetic and 24 healthy non-diabetic women at each trimester and at term. RESULTS: Approximately 22% of the diabetic women developed pre-eclampsia, primarily after their third trimester visit. In women with pre-eclampsia (diabetic pre-eclampsia, n = 26) vs those without hypertensive complications (diabetic normotensive, n = 95), significant changes in angiogenic factors were observed, predominantly in the early third trimester and prior to clinical manifestation of pre-eclampsia. Serum sFlt1 levels were increased approximately twofold in type 1 diabetic pre-eclampsia vs type 1 diabetic normotensive women at the third trimester visit (p < 0.05) and the normal rise of PlGF during pregnancy was blunted (p < 0.05). Among type 1 diabetic women, third trimester sFlt1 and PlGF were inversely related (r(2) = 42%, p < 0.0001). Endoglin levels were increased significantly in the diabetic group as a whole vs the non-diabetic group (p < 0.0001). CONCLUSIONS/INTERPRETATION: Higher sFlt1 levels, a blunted PlGF rise and an elevated sFlt1/PlGF ratio are predictive of pre-eclampsia in pregnant women with type 1 diabetes. Elevated endoglin levels in women with type 1 diabetes may confer a predisposition to pre-eclampsia and may contribute to the high incidence of pre-eclampsia in this patient group.

Down syndrome with pure partial trisomy 21q22 due to a paternal insertion (4;21) uncovered by uncultured amniotic fluid interphase FISH.

OBJECTIVE: To emphasize the usefulness and reliability of fluorescence in situ hybridization (FISH) on uncultured amniotic fluid cells in the prenatal diagnosis of common chromosomal aneuploidies. METHODS: FISH analyses utilizing centromeric, locus-specific or whole chromosome paint DNA probes specific for chromosomes X, Y, 13, 18, 21, and 4 were performed on uncultured amniotic fluid cells or the peripheral blood specimen from the father. Routine chromosome analysis was carried out as well. RESULTS: A prenatal case with partial trisomy 21 due to a paternal cryptic insertion (4;21) was ascertained by a rapid overnight FISH on uncultured amniotic fluid cells. The fetus was delivered at term and had classical features of Down syndrome. CONCLUSION: Our results stress the importance of FISH on uncultured amniotic fluid cells to supplement routine cytogenetics, especially in cases with abnormal ultrasound findings.

Description of ciliary body anatomy and identification of sites for transscleral cyclophotocoagulation in the equine eye.

OBJECTIVE: To determine the most appropriate anatomical location for diode laser probe placement to accurately photoablate the equine ciliary body using a contact, transscleral approach. DESIGN: Original research. PROCEDURES: Forty-two freshly enucleated adult equine eyes were evaluated. The horizontal, medial vertical, central vertical, and lateral vertical lengths of the cornea were measured from limbus to limbus. Needles were inserted perpendicular to the sclera at specific distances posterior to the external limbus at the 1, 2, 4, 5, 6, 7, 8, 10, 11 and 12 o'clock positions. The per cent frequency that needles penetrated internal anatomical regions (lens, lens zonules, pars plicata, anterior, middle, and posterior pars plana, ora ciliaris retinae, or retina), when inserted at 2, 4, 6, and 8 mm posterior to the limbus, were calculated for all eyes combined, for right vs. left, for males vs. females, and for three age groups. The internal distance from the limbus to the anterior pars plana, and from the anterior pars plana to the ora ciliaris retinae were also measured in six of the horses (12 eyes). Mean distances and standard deviations were computed for all parameters. RESULTS: Average corneal sizes and standard deviations were: 30.24 mm +/- 1.53 (horizontal); 24.69 mm +/- 1.52 (central vertical); 22.79 mm +/- 1.49 (medial vertical); and 19.79 mm +/- 1.55 (lateral vertical). Internal distances of the pars plicatas ranged from 5.33 mm +/- 0.49 to 10.67 mm +/- 1.15. Internal distances of the pars planas ranged from 0.33 mm +/- 0.49 to 3.17 mm +/- 0.39. High probabilities of penetrating the pars plicata correspond to positions 4 mm posterior to the external limbus dorsotemporal 10, 11 o'clock (OD), and 1, 2 o'clock (OS), dorsonasal 1 o'clock (OD) and 11 o'clock (OS), and ventrotemporal 5-7 o'clock (OU). Low probabilities of penetrating the pars plicata correspond to positions ventronasal 4 o'clock (OD), 8 o'clock (OS) and dorsonasal 2 o'clock (OD), 10 o'clock (OS) at 4 and 6 mm posterior to the external limbus as well as ventrotemporal 4 o'clock (OS), 8 o'clock (OD) at 6 mm posterior to the external limbus. CONCLUSIONS: Transscleral cyclophotocoagulation may be a viable alternative to medical therapy for control of intraocular pressure in horses with glaucoma. Overall, the most accurate anatomical position on the sclera for cyclophotocoagulation of the equine eye is 4-6 mm posterior to the limbus, avoiding the nasal quadrants. Accurate transscleral cyclophotocoagulation should optimize the therapeutic outcome and minimize potential side-effects such as retinal detachment and cataract formation.

Central role of the plakoglobin-binding domain for desmoglein 3 incorporation into desmosomes.

The carboxy-termini of classical cadherins and desmocollins have been shown to play an important role in initiating desmosome assembly. In this study we wanted to determine whether the carboxy- terminal cytoplasmic domains of desmoglein 3 are important for targeting it to the desmosome. By generating stably transfected A431 cell lines with chimeric constructs encoding for the extracellular domain of E-cadherin and the transmembrane and intracellular region of human desmoglein 3, we could show that the cytoplasmic tail is sufficient to target the protein to the desmosome. By generating truncations of the carboxy-terminus we investigated the importance of the various intracellular subdomains. Whereas the construct encoding the intracellular cadherin-type segment domain still allowed its incorporation into the desmosome, further truncation, leaving only the intracellular anchor domain, did not. Deletion of the 87 amino acid long plakoglobin-binding site within the intracellular cadherin-type segment domain demonstrated that this region is essential for targeting desmoglein 3 to the desmosome. Absent the plakoglobin-binding site the chimeric molecule colocalizes with beta-catenin rather than desmoplakin. We conclude that binding of plakoglobin to desmoglein 3 is an important step in desmosome assembly and leads to the incorporation of desmoglein 3 into the desmosome.

Endoplasmic reticulum and cis-Golgi localization of human T-lymphotropic virus type 1 p12(I): association with calreticulin and calnexin.

Human T-lymphotropic virus type 1 (HTLV-1) is a complex retrovirus encoding regulatory and accessory genes in four open reading frames (ORF I to IV) of the pX region. We have demonstrated an important role of pX ORF I expression, which encodes p12(I), in establishment of HTLV-1 infection in a rabbit model and for optimal viral infectivity in quiescent primary lymphocytes. These data indicated that p12(I) may enhance lymphocyte activation and thereby promote virus infection. To further define the role of p12(I) in cell activation, we characterized the subcellular localization of p12(I) in transfected 293T cells and HeLa-Tat cells by multiple methods, including immunofluorescence confocal microscopy, electron microscopy, and subcellular fractionation. Herein, we demonstrate that p12(I) accumulates in the endoplasmic reticulum (ER) and cis-Golgi apparatus. The location of p12(I) was unchanged following treatments with both cycloheximide (blocking de novo protein synthesis) and brefeldin A (disrupting ER-to-Golgi protein transport), indicating that the protein is retained in the ER and cis-Golgi. Moreover, using coimmunoprecipitation assays, we identify the direct binding of p12(I) with both calreticulin and calnexin, resident ER proteins which regulate calcium storage. Our results indicate that p12(I) directly binds key regulatory proteins involved in calcium-mediated cell signaling and suggest a role of p12(I) in the establishment of HTLV-1 infection by activation of host cells.

Desmoglein isoform distribution affects stratum corneum structure and function.

Desmogleins are desmosomal cadherins that mediate cell-cell adhesion. In stratified squamous epithelia there are two major isoforms of desmoglein, 1 and 3, with different distributions in epidermis and mucous membrane. Since either desmoglein isoform alone can mediate adhesion, the reason for their differential distribution is not known. To address this issue, we engineered transgenic mice with desmoglein 3 under the control of the involucrin promoter. These mice expressed desmoglein 3 with the same distribution in epidermis as found in normal oral mucous membranes, while expression of other major differentiation molecules was unchanged. Although the nucleated epidermis appeared normal, the epidermal stratum corneum was abnormal with gross scaling, and a lamellar histology resembling that of normal mucous membrane. The mice died shortly after birth with severe dehydration, suggesting excessive transepidermal water loss, which was confirmed by in vitro and in vivo measurement. Ultrastructure of the stratum corneum showed premature loss of cohesion of corneocytes. This dysadhesion of corneocytes and its contribution to increased transepidermal water loss was confirmed by tape stripping. These data demonstrate that differential expression of desmoglein isoforms affects the major function of epidermis, the permeability barrier, by altering the structure of the stratum corneum.

Early postpartum discharge at a university hospital. Outcome analysis.

OBJECTIVE: To determine whether implementation of an early hospital discharge policy was effective in safely reducing hospital stay and acceptable to patients. STUDY DESIGN: The study was a prospective, observational trial. Early discharge was defined as discharge from the hospital either on the first day after vaginal delivery or on the second day after delivery by cesarean section. A "stayover mom" policy was subsequently implemented to allow a mother the option to remain in the hospital overnight after discharge. This prospective study was divided into three consecutive three-month periods: (1) routine care (n = 576), (2) early discharge (n = 622), and (3) early discharge with stay-over mom (n = 574). Primary outcome measurements were length of hospital stay and patient willingness. RESULTS: With proper review of written instructions, all eligible patients were willing to be discharged early. As compared with the routine care group, the length of hospital stay was shorter by 0.8 days (95% CI, 0.4-1.2 days) in the early discharge group (P < .01) and by 1.1 days (95% CI, 0.5-1.7 days) in the early discharge with stayover mom group (P < .005). The stayover mom policy was more desirable because of limitations in obtaining timely transportation and concern about the infant warranting continued observation. The need for maternal readmission was rare (15 cases, 0.8% of total), with endometritis equally common (n = 3 in each group). CONCLUSION: Early postpartum discharge, especially including a stayover mom policy, was acceptable to our predominantly Medicaid population. The reduced hospital stay was not associated with increased maternal morbidity.

Toxin in bullous impetigo and staphylococcal scalded-skin syndrome targets desmoglein 1.

Exfoliative toxin A, produced by Staphylococcus aureus, causes blisters in bullous impetigo and its more generalized form, staphylococcal scalded-skin syndrome. The toxin shows exquisite specificity in causing loss of cell adhesion only in the superficial epidermis. Although exfoliative toxin A has the structure of a serine protease, a target protein has not been identified. Desmoglein (Dsg) 1, a desmosomal cadherin that mediates cell-cell adhesion, may be the target of exfoliative toxin A, because it is the target of autoantibodies in pemphigus foliaceus, in which blisters form with identical tissue specificity and histology. We show here that exfoliative toxin A cleaved mouse and human Dsg1, but not closely related cadherins such as Dsg3. We demonstrate this specific cleavage in cell culture, in neonatal mouse skin and with recombinant Dsg1, and conclude that Dsg1 is the specific receptor for exfoliative toxin A cleavage. This unique proteolytic attack on the desmosome causes a blister just below the stratum corneum, which forms the epidermal barrier, presumably allowing the bacteria in bullous impetigo to proliferate and spread beneath this barrier.