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• Assessment of spur dikes performance with the effect of seepage. • Investigated the scour depth migration with time and space. • Maximum scour depth identification with the effect of downward seepage. • Bed elevation changes in the field of spur dike with and without seepage. The current work uses an experimental method to analyse a multiscale statistical assessment of scour depth surrounding the spur dikes with downward seepage. The experiments used three T-shape spur dikes arranged in three series and provided outward from the channel's bank. A no-seepage condition and two distinct seepage velocities, V S1 = 0.09 mm/s and V S2 = 0.18 mm/s, were used in the experiments. Seepage from a channel affects the morphological behaviour and stability of the channel's bed, leading to changes in its hydrodynamic characteristics. In addition, downward seepage movement shifts the flow near the channel boundary and weakens the particles' stability. Thus, the streamwise velocity and Reynolds shear stress increase near the bed. As the seepage velocity increases, the local scour formation also rises relative to the channel's bed until the channel reaches a state of equilibrium. The celerity of scour depth for both time and length scales depends on the amount of downward seepage. Initially, higher seepage leads to faster changes in the scour depth, but over time, this rate decreases until it becomes constant. The bedform elevations' tails are thicker initially, but they become thinner and closer to the Gaussian distribution curve over time. [ABSTRACT FROM AUTHOR]

Pemphigus vulgaris autoantibodies (PV IgG) promote cell detachment in epidermal cell cultures and acantholysis in the epidermis of neonatal BALB/c mice in vivo. We have studied the evolution of the immunologic and ultrastructural changes in the epidermis of BALB/c mice that receive parenteral injections of PV IgG. Neonatal BALB/c mice received a single i.p. injection of PV IgG (10 mg/g body weight) or control IgG from normal humans. The skin and serum of these animals was obtained at 0, 1, 3, 6, 12, 18 and 24 h post injection and examined by immunofluorescence (IF), electron microscopy (EM) and immunoelectron microscopy (IEM). PV IgG was detected in the mouse serum and bound to the epidermal cells as soon as 1 h after injection by IF and IEM. The intensity of the binding in the skin (by IF) increased sharply between 3 and 6 h and remained positive at 24 h. Early epidermal cell detachment was demonstrable by EM at 1 h as widening of the epidermal intercellular spaces (ICS)and by 6 h the ICS between desmosomes had detached completely. Desmosomal junctions are the last to separate, occurring at 12-18 h. At this point, complete cell detachment occurred in the suprabasilar layers of the epidermis. Basal cells remain attached to the underlying dermis (tombstone row). Coincident with cell detachment, intracellular tonofilaments retracted from the cell periphery and clustered in a perinuclear position. IEM confirmed the binding of PV antibodies to the surface of epidermal cells in early and established lesions. This study demonstrates that the early immunologic and ultrastructural changes that occur in human pemphigus vulgaris are reproduced in this mouse model of the disease. [ABSTRACT FROM AUTHOR]

The pathogenic effects of pemphigus vulgaris (PV) antibodies on epidermal cells can be demonstrated both in vitro using skin organ culture or primary epidermal cell cultures (PECC) and in vivo by passive transfer of PV antibodies into neonatal BALB/c mice. Although PV antibodies have been localized on the epidermal cell surface by several techniques, little is known about the fate of these autoantibodies subsequent to their surface binding. We have examined this, using murine PECC which express pemphigus antigen on their surface, and followed the fate of the bound antibody molecules. Forty-eight-hour PECC were incubated at 37°C with PV antibodies for 20 min and then with horseradish peroxidase-labelled antihuman IgG. This was considered time 0. The monolayers were fixed with glutaraldehyde after 0, 0.5, 1, 3, 6, 18, and 24 h incubation at 37°C and then processed for electron microscopy. At time 0 hour, PV antibodies is detected bound evenly along the surface of keratinocytes. Within 30 min, the bound PV antibodies becomes clustered, internalized into submembranous vesicles via surface pits, and eventually fused with lysosomes. Widening of the intercellular spaces was also seen in PECC treated with PV antibodies within the first 24 h. PECC treated with normal human IgG in parallel cultures showed no such surface binding, internalization, or cell-cell detachment. Treatment with cytochalasin-D and/or colchicine did not affect the internalization of the PV antibodies, but fusion with lysosomes was not seen in treated cultures. These findings suggest that PV antibodies binds a surface antigen and the complex is internalized and fused with lysosomes in a process that may have pathophysiologic relevance. [ABSTRACT FROM AUTHOR]

Epidermal cell-to-cell or basal cell-to-substrate adhesion in vitro, involves as yet, unknown mechanisms. The rate of attachment of newly dissociated neonatal mouse epidermal cells and the rate of keratinocyte detachment from preformed epidermal monolayers was investigated under a variety of experimental conditions. In the present investigation, we obtained the following results: (1) variables such as temperature, nature of substrate, and presence of cations in the growth medium were important in the initial cell-to-substrate attachment; (2) removal of Ca++ and Mg++ from the growth medium was associated with a very low attachment rate (<5% at 24 hr and 48 hr); (3) the initial cell-to-substrate attachment of epidermal cells decreased about 50% when maintained in medium deficient in either Mg++ or Ca++ indicating that both cations are important in the cell-to-substrate attachment; (4) keratinocyte-detachment from preformed monolayers increased in medium deficient in Ca++, Mg++ as well as in medium only deficient in Ca++. However, detachment in cultures maintained in Mg++ deficient medium was similar to controls. In conclusion, Ca++ is one factor that may regulate epidermal cell-to-cell and cell-to-substrate interactions in vitro. Mg++ appears to influence also in the initial attachment process of epidermal basal cells. [ABSTRACT FROM AUTHOR]

It is known that during trypsinization of the skin, the epidermis is first separated from the dermis and individual keratinocytes are dissociated by disruption of the epidermal intercellular spaces. The desmosomal unit is separated at the level of the intercellular space and the split desmosomes are internalized in plasma membrane- limited vesicles; however the fate of the hemidesmosome under such conditions has not been studied. We have recently shown (Mutasim et al: J Invest Dermatol 84:47-53, 1985) that autoantibodies from the sera of patients with bullous pemphigoid bind in vitro to hemidesmosomes but not to desmosomes providing a highly specific marker for these organelles. Utilizing these autoantibodies, we studied the fate of the hemidesmosome during trypsin dissociation of epidermal basal cells derived from the skin of neonatal BALB/c mice. During trypsinization, portions of the dermal face of the plasma membrane which include hemidesmosomes formed pits which pinched off to produce vesicles that moved toward the nucleus. This was accompanied by retraction of the tonofilaments away from the cell periphery. The mechanism of this internalization process is not yet known, but may involve contractile elements of the cytoskeleton. The highly specific binding of bullous pemphigoid autoantibodies to the hemidesmosome may prove helpful in future biochemical and immunocytochemical studies of this organelle. [ABSTRACT FROM AUTHOR]

The sera of patients with pemphigus vulgaris (PV) and bullous pemphigoid (BP) contain autoantibodies which react with antigens present in a variety of mammalian squamous epithelia. The biologic role of these epithelial antigens is unknown; however, they do appear to be markers of epithelial differentiation. We have examined many human tissues that may be used as sources of large amounts of BP and PV antigens, including the human amnion, which is composed of the amnion reflectum and placentum (epithelial monolayers) and amnion cord (stratified epithelium). Human amnion was obtained from normal term deliveries and specimens of each area of the amnion were processed for light and electron microscopy. Samples of amnion were snap-frozen in liquid nitrogen and 4-μm sections were used as a substrate for BP and PV antibodies by indirect immunofluorescence (IF) techniques. Well-characterized BP serum (indirect IF titer 1:2560), PV serum (indirect IF titer 1:160), and normal human serum (negative indirect IF) were utilized as sources of BP and PV autoantibodies and a negative control, respectively. Linear staining of the BMZ was produced by BP antibodies in 8/8 specimens of amnion reflectum, 8/8 specimens of amnion placentum, and 3/3 specimens of amnion cord. Staining of the epithelial intercellular spaces was produced by PV antibodies of 2/3 specimens of amnion cord and none of the amnion reflectum (0/8) or amnion placentum (0/8) tested. Normal human serum produced no specific staining of the amnion. The light and the ultrastructural features of human amnion basement membrane zone and intercellular spaces resembles closely their epidermal counterparts. The restricted distribution of BP antigen in amnion epithelial basal cells (amnion reflectum, placentum, and cord) and PV antigen in stratified amnion epithelium (amnion cord) reinforces their relationship with epithelial differentiation. [ABSTRACT FROM AUTHOR]

IgG isolated from sera of patients with pemphigus vulgaris (PV) has been shown to induce cell detachment when added to primary epidermal cell cultures (PEOC). We studied the specificity of this phenomenon. IgG fractions were purified from the sera of five patients with PV and control IgG fractions from the sera of normal donors and patients with bullous pemphigoid (BP), systemic lupus erythematosus (SLE), and anti-AB blood group sera (anti-AB). IgG fractious were added to PECC either at initial plating (0 hours), at media change (48 hours), or sequentially at both times, and cell detachment was quantitated at 72 and 96 hours, Significant cell detachment occurred only when PV IgG was added to the growth media sequentially at 0 and 48 hours (p = 0.001), and this effect was dose-dependent for either dose. Substitution of an unrelated IgG (BP, SLE, or anti- AB) at either time points reduced cell detachment to near control values. Furthermore, cell detachment was inhibited by the addition of the proteinase inhibitors α2 macroglobulin (70% inhibition of detachment), aprotinin (63% inhibition), soybean and lima bean trypsin inhibitor (62 and 64%, respectively), and pepstatin (49%), but not by the inhibitors chymostatin, leupeptin, or antipain. These data confirm that PV IgG induces increased cell detachment in PECC and shows that tins effect is specific for PV IgG, is dose-dependent, and may be inhibited by certain proteinase inhibitors. [ABSTRACT FROM AUTHOR]

We have identified a novel IgG anitikeratin autoantibody in the serum of a Brazilian pemphigus foliaceus patient (Cascas-42). This antibody is specific for the 59 kD acidic murine keratin and its 56.5 kD human counterpart (Moll's catalogue #10), and is distinct from the pemphigus antibody system. Antikeratin autoantibodies present in the Cascas-42 serum were purified by affinity chromatography with a 59 kD murine keratin-agarose column (IAP-Cascas-42 antibodies). The specificity of the IAP-Cascas-42 antibodies was tested by indirect immunofluorescence and immunoelectron microscopy against epidermal cryosections, trypsin- dissociated keratinocytes, and epidermal cell cultures. The serum was also tested with extracts from unlabeled and surface 125I-labeled keratinocytes (lodo-Gen method) by immunoblot analysis of one- and two-dimensional poly- acrylamide gel electrophoresis. The IAP-Cascas-42 antibodies bind the intercellular spaces of murine epidermis, and the cell surfaces of viable, dissociated murine keratinocytes, as well as murine epidermal cells in culture by immunofluorescence and immunoelectron microscopy. These autoantibodies did not stain cytoplasmic keratins and did not react with parallel human epidermal substrates. The Cascas-42 serum identified the 59 kD murine acidic keratin and its 56.5 kD human coun- terpart in epidermal extracts by two-dimensional poly- acrylamide gel electrophoresis and immunoblot analysis. In addition, surface radioiodination of viable murine ke- ratinocytes selectively labeled the 59 kD keratin suggesting that a domain of this molecule is exposed on the cell surface. The 125I-labeled 59 kD keratin was also recognized by the Cascas-42 serum by immunoblotting and autoradiography. These studies suggest that in murine epidermis, the 59 kD keratin is a transmembrane protein with an extra-cellular domain recognized by the IAP-Cascas--42 antibodies. [ABSTRACT FROM AUTHOR]

Brazilian pemphigus foliaceus (fogo selvagem) is a cutaneous blistering disease endemic to certain areas of South America that has distinctive epidemiologic features suggestive of an infectious disease transmitted by an insect vector. Patients with the disease have anti-epithelial autoantibodies, both circulating in the serum and bound to lesional epidermis. In order to examine the possible pathogenic role of these autoantibodies, IgG from the sera of these patients was purified and injected into the peritoneum of neonatal BALB/c mica. Thirty-four of 46 mice (74%) receiving parenteral IgG fractions from these patients developed cutaneous lesions that were identical to the human disease by clinical, histologic, immunologic, and ultrastructural criteria. High-titer Brazilian pemphigus foliaceus sera produced lesions more consistently and rapidly than low-titer sera. When injections were discontinued, new lesions ceased to appear and old lesions resolved. The extent of disease correlated with the titer of human antiepithelial antibodies detected in the mouse serum (z < 0.01). Similar concentrations of IgG fractions obtained from sera of unaffected Brazilians living in endemic areas and from American donors did not induce disease when injected into littermates. These results establish that the antiepithelial autoantibodies play an important role in the pathogenesis of the cutaneous lesions in Brazilian pemphigus foliaceus. [ABSTRACT FROM AUTHOR]

Bullous pemphigoid (BP) antibodies are known to react with an antigen of the basement membrane zone (BMZ) of squamous epithelia and produce, by the indirect immunofluorescence technique, linear fluorescence at the BMZ. Direct and indirect immunoelectron microscopy (IEM) have demonstrated BP antigen to be within the lamina lucida, in close association with the basal cell membrane. Trypsin-dissociated epidermal basal cells bind BP antibodies in a polar distribution, presumably because the BP antigen is restricted to the dermal pole of the basal cell membrane. In this study we have utilized newborn BALB/c mouse skin to obtain both dissociated basal cells (by trypsinization) and epiderinal sheets (by dithiothreitol treatment). We show that viable basal cells, which are impermeable to IgG molecules, do not react with HP antibodies. When the basal cell plasma membrane is disrupted by cytospin centrifugation, air drying, freezing and thawing, or hypotonic lysis, or permeated by non-ionic detergents (saponin), cells become reactive with BP antibodies. Basal cell cytoskeletons, prepared by sequential treatment with Triton X-100, deoxyribonuclease and 2 M NaCl continue to react with BP antibodies. Similarly, viable epidermal sheets fail to bind BP antibodies. When epidermal sheets are treated with non-ionic detergents, water, or freezing and thawing prior to incubation with BP antibodies, linear BMZ fluorescence is observed. IEM study of saponin-treated basal cells shows the immunoreactants to be localized on intracytoplasmic vacuoles which represent internalized hemidesmosomes. IEM of permeated epidermal sheets shows the immunoreactants as aggregates on the inner surface of the dermal pole of the basal cell membrane. These observations suggest that the BP antigen is intracellular and is in close association with the basal cell cytoskeleton and hemidesmosomes. [ABSTRACT FROM AUTHOR]