New systems for replicating DNA in vitro

In order to investigate whether soluble intercellular adhesion molecule-1 (sICAM-1) and soluble interleukin-2 receptors (sIL-2R) were present in scleroderma skin, and to compare their levels to concentrations measured in plasma and clinical parameters, we examined suction blister fluid and plasma from 13 patients with systemic sclerosis and 11 healthy volunteers. Suction blisters and biopsies were from the transition zone between normal skin and scleroderma, and uninvolved abdominal skin. The levels of sICAM-1 and sIL-2R were significantly increased in both plasma and suction blister fluid from systemic sclerosis patients compared with healthy volunteers. ICAM-1 was localized to vessels and perivascular mononuclear infiltrates by immunohistochemical methods. IL-2R was expressed by CD3-positive cells. The elevated levels of sICAM-1 and sIL-2R in suction blister fluid point towards activation of endothelial cells and T cells in both the transition zone and uninvolved skin of systemic sclerosis patients.     

Erythrodermic bullous pemphigoid is a clinical variant of bullous pemphigoid

Bullous pemphigoid (BP) is an autoimmune blistering disease of the skin. Several variants of BP have been described but until recently the relationship of these variants to generalized BP was unclear. Several studies have shown that pemphigoid nodularis, pemphigoid vegetans, localized BP and vesicular pemphigoid are true variants of BP as the circulating antibodies in these patients recognize the same 230 kDa BP antigen as found in patients with generalized BP. Erythrodermic BP is a very unusual variant characterized by an erythroderma along with blister formation. We describe the third known patient to develop erythrodermic BP and characterize the antigenic specificity of the circulating antibodies in both our newly reported patient with erythrodermic BP and in one of the two other previously reported cases of erythrodermic BP. Both patients with erythrodermic BP had circulating IgG antibodies which bound to the epidermal side of salt-split human skin in a pattern identical to two patients with immunopathologically proven generalized BP. Sera from four erythrodermic patients without blisters and from a healthy normal volunteer, as controls, failed to demonstrate detectable circulating IgF autoantibodies. Immunoprecipitation studies revealed that both patients with erythrodermic BP had circulating IgG autoantibodies which recognized, to varying degrees, the same 230 and 180 kDa BP antigens as recognized by sera from two patients with immunopathologically proven generalized BP. Sera from four erythrodermic patients without blisters and from a healthy normal volunteer, as controls, failed to recognize any specific polypeptides. These observations demonstrate that erythrodermic BP is a distinct clinical variant of BP.     

Comparison of three different experimental methods for the assessment of peripheral compartment pharmacokinetics in humans

In many cases the concentration reached in a peripheral effect compartment rather than in plasma determines the clinical outcome of therapy. Therefore, several experimental approaches have been developed for direct assessment of drug kinetics in peripheral compartments. Particularly saliva sampling, skin blister fluid sampling, and in vivo microdialysis are frequently employed for measuring peripheral drug concentrations. However, data derived from these techniques have never been directly compared. In the present study, the tissue kinetics of theophylline were measured following single dose administration simultaneously in cantharides induced skin blisters, saliva and microdialysates of subcutaneous- and skeletal muscle- tissue and compared to plasma concentrations. Theophylline was administered to 9 healthy volunteers as an i.v. infusion of 240 mg. Mean ratio (AUCsaliva/AUCplasma) was 0.63 +/- 0.05, mean ratio (AUCblister/AUCplasma) was 0.69 +/- 0.12, mean ratio (AUCmuscle/AUCplasma) was 0.41 +/- 0.10, mean ratio (AUCsubcutaneous/AUCplasma) was 0.34 +/- 0.07. The time course of the concentration(peripheral)/concentration(plasma)-ratios showed that tissue concentrations obtained by microdialysis were closely correlated to free plasma levels, whereas saliva- and cantharides blister data overestimated the corresponding free plasma concentrations. It is concluded that microdialysis represents a reliable technique for the measurement of unbound peripheral compartment concentrations and is superior to saliva- and skin blister concentration measurements.     

Kindler syndrome. Clinical and ultrastructural findings

BACKGROUND: Kindler syndrome is a genodermatosis that combines clinical features of hereditary epidermolysis bullosa and poikiloderma congenitale. The ultrastructural level of blister formation has not been well characterized. OBSERVATIONS: Two brothers with Kindler syndrome had a history of primarily acral blistering since infancy as well as photosensitivity. Blister formation was found through the basal layer. Marked tonofilament clumping was found in intact keratinocytes adjacent to the blisters. The younger brother (aged 21 years) had actinic keratoses, which have not been previously described in Kindler syndrome. CONCLUSIONS: The findings of basal layer separation in both spontaneous and induced blisters in Kindler syndrome suggest this is the true level of blister formation. The finding of actinic keratoses in a young patient with Kindler syndrome suggests that some patients may be at increased risk for early solar-induced skin disease. The presence of clumped tonofilaments in keratinocytes adjacent to blistered areas suggests an abnormality of keratin 5 or 14 could be present and may play a role in blister formation in patients with Kindler syndrome.     

Growth and configurational stability of circular,buckling-driven film delaminations

A study is presented of delamination at the interface between a thin elastic film bonded to a substrate under conditions in which the film is subject to equi-biaxial compression. The focus is on initially circular delaminations. When the initial delamination is sufficiently large it buckles away from the substrate producing a blister which in turn induces a driving force on the interface crack tip. A two-part theoretical analysis of the coupled buckling/fracture problem is conducted: the axisymmetric growth of the circular blister, and the stability of the circular blister to nonaxisymmetric perturbations of the interface crack front. A simple criterion is identified which excludes the possibility of wide-spread delamination. Experiments are reported for a model film/substrate system (mica bonded to aluminum) chosen to allow visualization of the interface and to permit compressive stresses in the film to be generated over the full range of interest by exploiting the large thermal expansion mismatch of the system. The experiments bear out the theoretical prediction of a regime of axisymmetric growth which gives way to nonaxisymmetric blisters after a blister becomes sufficiently large. The study suggests that the wavy-circular and worm-like blister morphologies which are usually observed for delaminated films are a manifestation of the configurational instability of the interface crack front.     

Calculation of diffusion-limited kinetics for the reactions in collision coupling and receptor cross-linking

Both enzyme (e.g., G-protein) activation via a collision coupling model and the formation of cross-linked receptors by a multivalent ligand involve reactions between two molecules diffusing in the plasma membrane. The diffusion of these molecules is thought to play a critical role in these two early signal transduction events. In reduced dimensions, however, diffusion is not an effective mixing mechanism; consequently, zones in which the concentration of particular molecules (e.g., enzymes, receptors) becomes depleted or enriched may form. To examine the formation of these depletion/ accumulation zones and their effect on reaction rates and ultimately the cellular response, Monte Carlo techniques are used to simulate the reaction and diffusion of molecules in the plasma membrane. The effective reaction rate at steady state is determined in terms of the physical properties of the tissue and ligand for both enzyme activation via collision coupling and the generation of cross-linked receptors. The diffusion-limited reaction rate constant is shown to scale with the mean square displacement of a receptor-ligand complex. The rate constants determined in the simulation are compared with other theoretical predictions as well as experimental data.     

An in vitro diffusion model for the study of calcification of bovine pericardium tissue

Bovine pericardium (BP) is extensively used for the production of heart valve bioprostheses. BP has excellent mechanical properties but a limited lifespan because of intrinsic subsurface calcification in vivo. In this study, the in vitro mineralization of BP was investigated by a novel diffusion cell model. In two sets of experiments, glutaraldehyde-treated BP membranes were placed between two compartments, both of which contained calcium phosphate solutions made by equilibration of octacalcium phosphate (Exp I) or dicalcium phosphate dihydrate (Exp II) in phosphoric acid. The movement of calcium (Ca), phosphate (P), and protons through the BP membrane was followed throughout the diffusion process. Histology, scanning electron microscopy, wet chemical analysis, and energy dispersive X-ray analyses provided good evidence of subsurface mineralization of BP that resembled in vivo mineral deposition. Energy dispersive x-ray microanalyses found a Ca/P heterogeneity of the early subsurface mineral that formed in the membrane. The use of a diffusion cell to model BP calcification under well-characterized conditions has led to in vitro mineralization that more closely matches that observed in vivo. The results suggest that this in vitro diffusion model can be used to study the mechanism of pathological mineralization. This model has the potential to provide rapid, inexpensive, basic information about the mineralization process.     

Chronic bullous dermatosis in childhood (linear IgA dermatosis)

A 5-year-old boy presented with disseminated, partly grouped blisters indicative of chronic bullous dermatosis of childhood (CBDC) following a gastrointestinal infection 2 weeks earlier. CBDC has long been differentiated from adult linear IgA disease. Clinical and laboratory studies revealed substantial clinical and immunological overlap between the two blistering disorders, whereas recent investigations suggest heterogeneity of the target antigen involved. Pathohistological and immunofluorescence-microscopical characteristics of a subepidermal blister and linear IgA and granular C3 deposition at the basement membrane together with the typical history and clinical signs were decisive in the differential diagnosis. The disease promptly cleared up after daily administration of 16 mg methylprednisolone-21-acetate tapering and 25 mg dapsone. Immunohistological detection of collagen IV at the base of a blister made it possible to localize the split above the lamina densa. The demonstration of collagen IV stresses the importance of immunodermatopathology in the differential diagnosis of subepidermal blistering diseases.     

Glucose content in human skin: relationship with blood glucose levels

In order to ascertain the dynamic relationship between the extracellular glucose in upper skin layers and blood glucose, skin suction blisters were raised in six Type 1 diabetic patients during a three-step glucose clamp. Blister glucose closely paralleled venous glucose (mean of r = 0.998). However, in three patients blister glucose was constantly lower than plasma glucose and this appeared to be related to their slower formation of skin blisters. A substantial difference in skin blister suction time was noted among patients and it was found that suction time was linearly correlated to glycosylated haemoglobin (HbA1C) (n = 6, r = 0.865, p = 0.026). It is concluded that a non-invasive blood glucose monitoring system could be successfully based on measurement of alterations in skin glucose contents.     

Characterization of the nuclear localization signal in the DNA helicase involved in Werner''s syndrome

We have previously shown that the 180 kDa bullous pemphigoid antigen (BPAG2) is distributed on the lateral-apical (as a pool) and ventral (as hemidesmosomes) cell membranes of monolayer cultured keratinocytes and that addition of IgG purified from bullous pemphigoid (BP) patients (BP-IgG) causes the internalization of immune complexes of BPAG2 and BP-IgG from the lateral-apical cell membrane. This internalization of BPAG2 is believed to inhibit the Ca2+ induced reformation of hemidesmosomes on the ventral cell membrane, possibly by inhibiting the supply of the antigen from the lateral-apical to the ventral membranes to form hemidesmosomes. The purpose of this paper is to examine, by using biopsy specimens from BP patients (12 cases), whether or not this internalization of BPAG2 is generated in situ. The fates of BPAG2, 230 kDa BPA (BPAG1) and bound BP-IgG were traced by immunofluorescence microscopy using monoclonal antibodies to BPAG1, BPAG2 and human IgG. In more than half of the lesional and perilesional biopsy specimens, internalization of BPAG2 into the basal cells was observed, while in normal skin BPAG2 was detected on the whole surface of the basal cells without its internalization. No internalization of BPAG1 was detected in BP and normal epidermis. These results suggest that binding of BP-IgG on the lateral-apical cell surface of basal cells causes internalization of BPAG2 in situ in the epidermis of BP patients similar to that seen in cultured cell systems, and that this internalization of immune complexes of BPAG2 and BP-IgG may play an important part in blister formation in BP.     

管线管氢致开裂试样检测及分析

对管线管氢致开裂(HIC)试验后试样进行超声波探伤和金相分析。结果表明:HIC试验后未发现氢致裂纹缺陷,但超声波探伤可检测到大型B类夹杂物及表面氢鼓泡。本试验所取试样较大B类夹杂物出现在管体壁厚中心与外壁之间,可以大致推断较大B类夹杂位于原始铸坯1/2半径到表层细晶区之间,其成因与铸坯芯部成分偏析无关。氢鼓泡中有大颗粒状氧化镁及硫化钙夹杂,其余区域为铝酸钙夹杂,故夹杂物是形成氢鼓泡的诱因之一。     

Linear immunoglobulin A (IgA) bullous dermatosis of childhood: identification of the target antigens and study of the cellular sources

A 4-year-old girl developed numerous tense blisters on the body. The blisters healed without scarring. Histopathological and immunofluorescence studies showed findings consistent with linear immunoglobulin A (IgA) bullous dermatosis of childhood. Immunoelectron microscopy revealed deposition of IgA in the lamina lucida of the basement membrane zone of the dermal-epidermal junction. Circulating IgA autoantibody was positive at the titre of 1 : 128 and recognized the antigens located on epidermal sites of 1 mol/l NaCl-split skin. Immunofluorescence staining of cultured normal human fibroblasts and cultured DJM-1 cells (derived from human squamous cell carcinoma of skin) with the patient's sera demonstrated that both of these cells synthesize the antigens in vitro, although fibroblasts produce the antigens more abundantly. When DJM-1 cells were injected intracutaneously into nude mice, the antigens recognized by the sera were present mainly around the tumour cell islands in a linear pattern, while the dermal-epidermal junction of mouse skin was negative, suggesting that epidermal cells may contribute directly to synthesis and deposition of the antigens at the basement membrane. By immunoprecipitation using cultured normal human fibroblasts, the patient's sera could precipitate at least two specific molecules at 100-kDa and 145-kDa molecular weight.     

Protein concentration of subcutaneous interstitial fluid in the human leg. A comparison between the wick technique and the blister suction technique

The wick technique and the blister suction technique are the most common methods for sampling of subcutaneous interstitial tissue fluid in man. The blister suction technique has the advantage of being less invasive than the wick technique, but the reliability of this method is still controversial. The aim of this study was to evaluate whether the simpler blister suction technique using large (8 mm) blisters could replace the wick technique in the investigation of patients with postreconstructive leg edema. Fifteen patients with ipsilateral leg edema following infrainguinal bypass surgery for lower limb atherosclerosis were investigated. The two different fluid sampling techniques were applied simultaneously on both legs. The concentration of total protein and albumin as well as colloid osmotic pressure of the subcutaneous interstitial tissue fluid in the leg were measured in all fluid samples. Agreement analysis was applied to compare the two methods, while the correspondence between the methods was estimated with linear regression analysis. The agreement index was found to be positive for all variables from the operated as well as from the contralateral control limb. Furthermore, all values were within the agreement limit. The best agreement between the two methods was found for colloid osmotic pressure on the operated side. According to the equation of linear regression, there was a slight overestimation of the wick values compared to the observed blister values. In conclusion, there was a good methodological agreement between the blister suction technique and the wick technique. The less invasive blister suction technique should be regarded as the method of choice for the investigation of subcutaneous interstitial tissue fluid in patients with postreconstructive leg edema.     

Peritoneal protein loss in children with nephrotic syndrome during peritoneal dialysis

BACKGROUND: The passage of proteins across the glomerular filtration barrier is mainly determined by the size of the protein. In nephrotic syndrome (NS) the glomerular permselectivity is affected, causing proteinuria. Some authors suggest the existence of a generalized basement membrane defect. The permeability characteristics of the peritoneal basement membrane in children with NS are not known. METHODS: The transperitoneal transport of proteins with a different molecular weight (beta2-microglobulin MW 11800 D, albumin MW 69000 D, IgG MW 160000 D, and alpha2-macroglobulin MW 820000 D) was studied in a study group (group A) consisting of six stable nephrotic children (three with glomerulosclerosis and three with congenital nephrotic syndrome, one of them with mesangial sclerosis) and compared to a control group (group B) consisting of eight stable children on peritoneal dialysis. After a dwell of 6 h with Dianeal 1.36% dialysate and serum samples were collected. For each patient the dialysate to plasma (D/P) ratios of the four proteins were calculated. The D/P ratios of the nephrotic patients in group A were compared to the D/P ratios of the patients in the control group B. Data were expressed as mean +/- SD. RESULTS: The values for the D/P ratios (in percentage) of beta2-microglobulin, albumin, IgG and alpha2-macroglobulin in group A were 19.6+/-9.9, 2.7+/-1.7, 1.6+/-0.9, and 0.5+/-0.4, compared to 24.9+/-10.2, 4.0+/-2.3, 2.2 +/- 1.2, and 0.7 +/- 0.3 in the control group B. The ratios were plotted against MW on a double logarithmic scale. In all patients a linear relationship between molecular weight and D/P ratio of the proteins was obtained. The D/P ratios of the study group did not differ significantly from the control group. CONCLUSION: We conclude that the size selectivity of the capillary permeability is not affected in the peritoneal membrane in children with NS due to glomerulosclerosis and congenital nephrotic syndrome.     

Gelatinase B-deficient mice are resistant to experimental bullous pemphigoid

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease characterized by deposition of autoantibodies at the basement membrane zone. In an experimental BP model in mice, the subepidermal blistering is mediated by antibodies directed against the hemidesmosomal protein BP180 (collagen XVII, BPAG2), and depends on complement activation and neutrophil infiltration. Gelatinase B is present in BP blister fluid and can cleave BP180. In this study we investigated the role of gelatinase B in the immunopathogenesis of experimental BP using mice containing targeted disruption of the gelatinase B (MMP-9, 92 kD gelatinase) gene. Gelatinase B-deficient mice were resistant to the blistering effect of intracutaneous anti-mBP180 antibodies, although these mice showed deposition of autoantibodies at the basement membrane zone and neutrophil recruitment to the skin comparable to that observed in the control mice. Interleukin 8 given intradermally concomitantly with pathogenic anti-mBP180 elicited a significant neutrophil recruitment into the skin in gelatinase B-deficient mice, but blistering did not occur. However, gelatinase B-deficient mice reconstituted with neutrophils from normal mice developed blistering in response to anti-mBP180 antibodies. These results implicate neutrophil-derived gelatinase B in the pathogenesis of experimental BP and might lead to novel therapeutic strategies for BP.     

A case of nonscarring inflammatory epidermolysis bullosa acquisita: characterization of IgG autoantibodies by immunofluorescence, immunoblotting and immunogold electron microscopy

We report a case of nonscarring inflammatory epidermolysis bullosa acquisita in a 59-year-old Japanese woman. She developed blisters and erosions on her lip, trunk and extremities. Sodium aurothiomalate was effective for the skin lesions. The patient had been free from bullous skin lesions for the last 13 years and had shown no scarring. Indirect immunofluorescence (IF) study on 1 M NaCl-split skin revealed IgG autoantibodies against the dermal side of the split skin. Immunoblotting using normal human dermal extracts disclosed IgG autoantibodies reactive with the 290 and 145 kD antigens. Circulating IgG autoantibodies were deposited on the lamina densa by immunoelectron microscopy. IF mapping using several antibodies for the components of the basement membrane zone revealed blister formation at the lamina densa. These results suggest that the cleavage at the lamina lucida does not necessarily exclude the diagnosis of EBA and that the definite diagnosis of EBA should be confirmed by immunoblotting or immunoelectron microscopic study.     

On the blister formation in copper alloys due to the helium ion implantation

A new approach to the blister-formation phenomenon is discussed by means of the mathematical solution on a uniformly loaded circular plate with clamped edges (circular diaphragm). In the present investigation, it was found that blister formation depends on the mechanical properties of the alloys and the near-surface concentration of the implanted gas, which itself is contingent on the crystallographic orientation by means of the stopping power of the implanted atoms. The reported model is based on the fact that at certain depths from the surface, the pressure in the cavities approaches the yield stress of the metal and blistering starts. The thickness of this thin film depends on the mechanical properties of the specific metal. Once a blister cavity is formed, the deformation of the thin film to form a blister cap depends on the buildup of pressure in the cavity contingent on the implanted dose. For the present model, it is sufficient to say that the thickness of the blister’s cap cannot be correlated with the projected range of the implantation, as assumed by other authors. The implanted helium concentration needed to buildup enough gas pressure to create a blister at a depth which is close to the projected range is higher by 50 times than the gas helium concentration in the cavity. Experimental results, such as the fact that the blisters have burst at the edge of the circular skin, where the maximum stresses are developed, and the fact that at high implantation energy (large projected range), the bursting of the blisters occurs by multilayer caps, support the present model.     

车轮钢中的白点(一)

研究了车轮钢中白点形成的机理以及白点对断口形貌的影响.结果表明,氢和空位聚集导致鼓泡形核和长大,随着鼓泡内氢气压力的升高,裂纹从鼓泡核壁形核、扩展导致白点形成.白点的断口为准解理,和氢致滞后开裂断口相同,含白点试样的断口形貌依赖于断裂方式和试样厚度.钢中的白点除了产生二次裂纹和局部准解理断面外,对各种断口形貌均没有明显的影响.     

Determination of fracture initiation in hydride blisters using acoustic emission

An experimental study based on acoustic emission techniques was carried out to determine the conditions that lead to the initiation and growth of cracks in and from zirconium hydride blisters. The stress to initiate, at room temperature, a crack in a blister previously grown on a tensile speci-men could be accurately determined using an acoustic emission (AE) method based on linear event-location techniques. It was found that the applied stress to first form a crack in an unbroken blister decreases with blister depth but also that this value is statistically distributed. This is likely due to a size distribution of microcracks or incipient flaws in the blister. The propagation, by the delayed hy-dride cracking (DHC) mechanism, of a crack from a pregrown blister at 516 K seems to require both a critical applied stress, which decreases with blister depth, and approach of the testing temperature from above. However, DHC initiation was possible at 563 K (approached from below) for blisters grown under stress, provided the applied stress was sufficiently high. The stress intensity factor,K _IB , to initiate DHC ranged from 10.7 to 15.4 MPa √m. This is above the range forK _IH the thresholdK ₁ for DHC obtained in other experiments. The characteristics of AE generated by crack propagation from a blister due to DHC always follows the same pattern. It has a low event rate both at the beginning and at the end of DHC and a maximum value in between. The DHC initiation stage has a high proportion of high amplitude events. The peak in the distribution of events with peak am-plitude shifts to lower peak amplitudes as DHC progresses. An explanation for this trend is sought in terms of the maximum hydride size required near the crack tip to propagate the crack as a func-tion ofK ₁ .     

Autoantigens of cicatricial pemphigoid and their pathogenetic significance

Cicatrical pemphigoid (CP) comprises a group of patients with a chronic subepidermal blistering disease which primarily involves mucous membranes; lesions characteristically heal with scarring. Immunofluorescence investigations typically demonstrate deposits of tissue bound and circulating immunoreactants of the IgG and less frequently of the IgA class in a linear pattern along the basement membrane zone. These autoantibodies are thought to play an important role in the blister formation of CP. Most patients show binding to the bullous pemphigoid antigen 2 (BPag2), collagen type XVII, with a molecular-weight of 180 kD. A smaller group of patients with CP have autoantibodies to laminin 5. Animal models confirm that autoantibodies binding to these two adhesion molecules (BPag2 and laminin 5) are important in blister formation. There are other autoantigens described in CP; however, they are only found in small groups of CP patients and most of them are not further characterised. The described molecules are part of the hemidesmosomal adhesion complex. Impaired function of any component of this complex may lead to a separation of the epidermis from the dermis; better knowledge about the single molecules and the exact localisation of epitopes within these molecules may lead to further understanding of the clinical picture.