Herpes gestationis. Immunopathology and characterization of the HG factor.

Five patients with herpes gestationis, a blistering disease of pregnancy, were studied immunologically. All had in vivo deposition of C3 in a linear band along the basement membrane zone of lesional and normal-appearing skin, the location of early blister formation. Immunoglobulin deposition was more variable, though four patients had evidence of in vivo bound IgG at the same site. A circulating, complement binding herpes gestationis factor was demonstrated in the sera of four of the patients, its concentration unrelated to the activity of clinical disease. Characterization of this factor by sucrose gradient ultracentrifugation, specific absorption studies, and papain digestion indicates that it is an IgG. Evidence exists for involvement of both the classical and alternate complement pathways in vivo, though in vitro studies implicate the classical pathway as the primary route of complement activation. Three offspring were studied, none with clinical involvement; one showed in vivo deposition of C3 at the basement membrane zone of normal skin and a second showed the herpes gestationis factor in cord blood.

Possible role of P-450 metabolite of arachidonic acid in vasodilator mechanism of angiotensin II type 2 receptor in the isolated microperfused rabbit afferent arteriole.

Although angiotensin II type 2 (AT2) receptor has recently been cloned, its functional role is not well understood. We tested the hypothesis that selective activation of AT2 receptor causes vasodilation in the preglomerular afferent arteriole (Af-Art), a vascular segment that accounts for most of the preglomerular resistance. We microperfused rabbit Af-Arts at 60 mmHg in vitro, and examined the effect of angiotensin II (Ang II; 10(-11)-10(-8) M) on the luminal diameter in the presence or absence of the Ang II type 1 receptor antagonist CV11974 (CV; 10(-8) M). Ang II was added to both the bath and lumen of preconstricted Af-Arts. Ang II further constricted Af-Arts without CV (by 74+/-7% over the preconstricted level at 10(-8) M; P < 0.01, n = 7). In contrast, in the presence of CV, Ang II caused dose-dependent dilation; Ang II at 10(-8) M increased the diameter by 29+/-2% (n = 7, P < 0.01). This dilation was completely abolished by pretreatment with an AT2 receptor antagonist PD123319 (10(-7) M, n = 6), suggesting that activation of AT2 receptor causes vasodilation in Af-Arts. The dilation was unaffected by inhibiting either nitric oxide synthase (n = 7) or cyclooxygenase (n = 7), however, it was abolished by either disrupting the endothelium (n = 10) or inhibiting the cytochrome P-450 pathway, particularly the synthesis of epoxyeicosatrienoic acids (EETs, n = 7). These results suggest that in the Af-Art activation of the AT2 receptor may cause endothelium-dependent vasodilation via a cytochrome P-450 pathway, possibly by EETs.

Biphasic effects of 12-O-tetradecanoylphorbol-13-acetate on the cell morphology of low calcium-grown human epidermal carcinoma cells: involvement of translocation and down regulation of protein kinase C.

The present study was performed to investigate involvement of protein kinase C in the biphasic effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on cell morphology in low calcium (0.07 mM)-grown cells of a human epidermal squamous cell carcinoma cell line. The low calcium-grown cells formed no desmosomal cell-cell contact and showed roughly circular arrangements of keratin intermediate filaments around the nucleus. Treatment with 10 ng/ml of TPA induced a rapid formation (within 15 min) of cell-cell contact and reorganization of keratin intermediate filaments from a circular organization to a radial arrangement in these low calcium-grown cells. These structural phenomena were associated with a transient increase in membrane-bound protein kinase C activity. However, the prolonged treatment longer than 24 h led to a prominent decrease in the number of cell-cell contacts, that had been once formed, and caused fibroblastic changes of cell morphology in association with a decrease in the membrane-bound protein kinase C activity. Addition of 20 microM 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, a potential inhibitor of protein kinase C, to the medium with TPA blocked the formation of cell-cell contact. Addition of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride alone to normal calcium-grown cell cultures exhibiting cell-cell contact resulted in a decrease in the number of cell-cell contacts and in the fibroblastic morphological changes after 24-h incubation. These results suggest that the effects of TPA are biphasic as follows: the initial stage, inducing cell-cell contact formation associated with the translocation of protein kinase C activity from the cytosol to the membrane; and the late stage, exhibiting a fibroblastic morphological change with a decrease in the number of cell-cell contacts associated with the down regulation of this enzyme activity by TPA.

Comparative effects of aspirin, a synthetic thrombin inhibitor and a monoclonal antiplatelet glycoprotein IIb/IIIa antibody on coronary artery reperfusion, reocclusion and bleeding with recombinant tissue-type plasminogen activator in a canine preparation.

The comparative effects of intravenous aspirin, the synthetic thrombin inhibitor (2R,4R)-4-methyl-1-[N2-(3-methyl-1,2,3,4-tetrahydro-8- quinolinesulfonyl)-L-arginyl]-2-piperidinecarboxylic acid monohydrate (Argatroban) and F(ab')2 fragments of monoclonal antibody 7E3 against platelet glycoprotein IIb/IIIa (7E3-F[ab']2) on thrombolysis, reocclusion and bleeding associated with 0.45 mg/kg body weight bolus injections of recombinant tissue-type plasminogen activator (rt-PA) were studied in a canine coronary artery thrombosis model. Coronary patency was monitored for 2 h both by flow probe and by coronary angiography. Four groups were studied: Group I = pretreated with 17 mg/kg intravenous aspirin (n = 6), Group II = pretreated with 200 micrograms/kg per min intravenous Argatroban for 60 min (n = 5), Group III = pretreated with aspirin and Argatroban (n = 5) and Group IV = pretreated with 0.8 mg/kg intravenous 7E3-F(ab')2 (n = 5). In Group I, reflow occurred in four of six dogs, but did not persist; reflow was induced in Group II in four of five dogs, persisting in one; in Group III, reflow occurred in all five dogs, persisting in four; in Group IV reflow was achieved in four of five dogs, persisting in two. The frequency of persistent reflow in Group III was significantly higher than in the combined Groups I and II (p = 0.012), whereas the time to reflow was significantly shorter in the groups receiving Argatroban than in the aspirin group (median 25 versus 55 min, p = 0.04). There were no significant differences between Groups III and IV.(ABSTRACT TRUNCATED AT 250 WORDS)

An inhibitory factor against monocyte spreading in the sera of patients with systemic lupus erythematosus.

The effect of the sera of patients with systemic lupus erythematosus (SLE) on monocyte function was studied using cell spreading as an indicator. Monocyte spreading induced by exogenous stimuli was shown to be inhibited by SLE sera. Gel filtration of SLE sera on Sephadex G-200 revealed that the factor responsible for this inhibition had a molecular weight of about 50,000. Pretreatment of monocytes with the inhibitory factor led to suppression of cell spreading induced by subsequent stimulation, but this hyporeactivity was reversible. Spreading of monocytes was rapidly aborted by the addition of this inhibitory factor. Thus, the inhibitory factor appeared to affect monocyte itself, but its effect seemed to be transient.

Apoptosis in relevant clinical situations: contribution of apoptosis in myocardial infarction.

Myocardial infarction is associated with increased TUNEL-positivity in cardiac resident and infiltrated cells. Apoptosis of proliferated interstitial myofibroblasts and infiltrated inflammatory cells may have a role in terminating tissue repair processes after infarction. Lateral and endocardial border zones of infarction within the risk area have frequent appearance of TUNEL-positive cardiomyocytes. Although the typical ultrastructural morphology of apoptosis has rarely been detected in ischaemic cardiomyocytes, there are many reports in which the TUNEL method was used for assessment of cardiomyocyte apoptosis. It has become evident that TUNEL-positivity reflects a wide range of cellular conditions; viable cells undergoing DNA repair, apoptosis, and necrosis. Therefore, it is controversial whether TUNEL-positive cardiomyocytes in infarcted myocardium are all apoptotic. Methods which will be more specific for identifying apoptosis are required for future study. TUNEL-positivity can be attenuated by anti-apoptotic interventions such as inhibition of caspases, mitochondrial protection, free radical scavenging, and some conventional pharmacotherapies. However, it remains to be determined whether anti-apoptotic interventions result in satisfactory reduction of infarct size. The injurious impact of myocardial ischaemia comes from a mixture of pro-apoptotic and necrosis-promoting signals, and the target of both signals is mitochondria. Through a common pathway they may cause permeability transition. Interventions which act only at the post-mitochondrial stage of apoptosis may fail to reduce infarct size, whereas those acting at pre-mitochondrial and mitochondrial stages may reduce infarct size. Progress in investigating the basic mechanisms of apoptosis and recognition of the modes of cardiomyocytes death will contribute to advances in cardioprotective therapy in myocardial infarction.

Alteration in the arrangement of the keratin-type intermediate filaments during mitosis in cultured human keratinocytes.

The behavior of the keratin-type intermediate filaments (KIFs) during mitosis was characterized in cultured human keratinocytes by immunofluorescence microscopy using polyclonal antibodies to keratin. The structural relationship of KIFs with microtubules (MTs) was also studied at the same time using a monoclonal antibody to alpha-tubulin. The KIFs and MTs showed similar but different cytoskeletal networks and underwent structural rearrangements independently during the cell cycle. KIFs in keratinocytes formed two different arrangements during meta- and anaphase: a global aggregation of filaments around the spindle and a fibrous array radiating from the central, global aggregation of filaments to the cell periphery where they were connected with those of the adjacent cells at desmosomal sites. These radiating fibrous portions of KIFs appeared to play a role in retaining the cell in its correct relationship to the surrounding cells during mitosis. This behavior of KIFs in normal keratinocytes was different from the KIF-alterations which had been previously described in SV40-transformed keratinocytes and other cells which expressed two different IFs (keratin and vimentin).

Identification of IgA binding structures in skin of patients with dermatitis herpetiformis.

Immunoelectronmicroscopic and ultrastructural identification of the structures which bind IgA in skin of patients with dermatitis herpetiformis (DH) reveals them to be, in part, a complex of fibrillar components which are covered with amorphous substances. One of the fibers has a diameter of 80--130 A and appears to be tubular, resembling dermal microfibrillar bundles or microtubular elements of elastic fibers. The structures, at times, are associated with the dermal microfibrillar bundles and are found within an elastic fiber system which is in close proximity to the dermal-epidermal junction. Taken together, these findings suggest that the structures which bind IgA are unique to DH and might be a part of an abnormal dermal microfibrillar bundle-elastic fiber system.

Effects of pemphigus antibody on the regeneration of cell-cell contact in keratinocyte cultures grown in low to normal Ca++ concentration.

Pemphigus is an autoimmune blistering disease of epidermal cells in which autoantibodies to the surface develop. The present study was performed to determine whether the binding of pemphigus antibodies to the surface of keratinocytes can inhibit the regeneration of cell-cell contact induced by altering from low to normal Ca++ concentration medium. Human keratinocytes (a cell line of squamous cell carcinoma, DJM-1 cell) were grown in low Ca++ medium for 4 days, then the cells were incubated in normal Ca++ medium containing 10% pemphigus (4 patients with pemphigus vulgaris and 4 patients with pemphigus foliaceus) or normal serum (treated at 56 degrees C, for 30 min) for various incubation periods (2, 6, 12, 24 h). The cells were fixed and stained with antikeratin antibody by the indirect immunofluorescence method so that the detachment of cell-cell contact was able to be clearly visualized by observing the cytoskeletal arrays of keratin filaments. The cells grown in normal Ca++ medium showed detachments of cell-cell contact 24-36 h after addition of any one of the pemphigus sera used in this study. The cells grown in low Ca++ medium formed no cell-cell contacts and expressed no pemphigus antigens. However, re-formation of cell-cell contacts and reexpression of the antigens were confirmed by immunofluorescence microscopy 30 min after the addition of Ca++ to the medium. The addition of any pemphigus vulgaris and foliaceus sera with Ca++ did not inhibit the regeneration of cell-cell contact and exerted no effects on the contact during the subsequent 12 h. However, after 24 h, these cells again lost the contact. These results indicate that pemphigus antibody and antigen reaction on the cell surface did not directly inhibit the Ca++-induced re-formation of cell-cell contact.

An elevated level of autoantibodies against 48- to 50-kd keratins in the serum of patients with psoriasis.

Studies on the anti-keratin intermediate filament autoantibodies (anti-KIF-Abs) in sera from psoriasis (Pso) patients were performed by immunoblotting and enzyme-linked immunosorbent assay (ELISA). According to their reactivities against keratin subunits (50, 56.5, 58, and 63-68 kd), sera were divided into four groups. However, no significant differences in these reactive patterns were found between healthy volunteers and Pso patients. In the second experiment, anti-KIF-Abs in sera from Pso patients, pustulosis palmaris et plantaris (PPP) patients, atopic dermatitis (AD) patients, systemic lupus erythematosus (SLE) patients, and healthy volunteers were determined by ELISA, using as substrate keratins purified from normal human stratum corneum and 48- and 50-kd keratins purified from psoriatic stratum corneum. The serum titers of anti-KIF-Abs against 48- and 50-kd keratins in Pso patients were significantly higher than those in PPP patients, AD patients, SLE patients, or healthy volunteers. The elevated titers of anti-KIF-Abs against the 48- and 50-kd keratins in sera of Pso patients showed a significant decrease with improvement of psoriatic lesions. The above results suggest that anti-KIF-Abs against 48- and 50-kd keratins in sera of Pso patients have some relevance to the severity of the disease and can be used as a marker for the evaluation of the disease activity of psoriasis.

Reorganization of keratin intermediate filaments by the drug-induced disruption of microfilaments in cultured human keratinocytes.

It has been shown to date that a combined treatment with microtubule and microfilament inhibitors alters the cytoskeletal organization of keratin intermediate filaments in cultured HeLa, fetal mouse epidermal, and epithelial PtK2 cells, although neither of these inhibitors alone is able to do so. In the present study, we found that disruption of microfilaments with cytochalasin B induced a remarkable reorganization of keratin filaments in cultured human keratinocytes, while disruption of microtubules with colchicine did not affect keratin filaments. Keratin filament organization in the presence of cytochalasin B demonstrated a network of connecting star-like knots or foci. These foci coincided with actin aggregates that were formed by depolymerization of actin filaments as studied by double immunofluorescence using antiactin and antikeratin antibodies. Under these conditions, no change in microtubule arrangement was observed. Our observations suggest that the stability and architecture of keratin filament organization may be supported with the microfilament rather than the microtubule cytoskeleton in cultured human keratinocytes.

Characterization of bullous pemphigoid antigen synthesized by cultured human squamous cell carcinoma cells.

This report concerns the characterization of bullous pemphigoid antigen (BPA) in cultured cells derived from a human squamous cell carcinoma (SCC cells) as identified by sera of 9 patients with bullous pemphigoid (BP). Immunoglobulin G from 5 of 9 BP sera bound the cell surfaces of SCC cells with a punctate staining pattern by immunofluorescence. The other four BP sera and all 12 controls sera showed no specific staining. To characterize the antigen for pemphigoid antibody, we immunoprecipitated NP-40 extracts of cells labeled with [14C] amino acids using nine BP sera and 12 controls sera. These immunoprecipitates were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by fluorography. The five BP sera that gave positive immunofluorescence results also precipitated a protein with molecular weight (mol wt) of 110 kD, whereas the other four BP sera negative by immunofluorescence and all 12 controls sera did not precipitate this protein. These results indicate that the sera of patients with BP contain antibodies against the protein with mol wt 110 kD in the SCC cells used in this study. With use of four BP sera, two of which were positive to this mol wt 110 kD BPA and two of which were negative, immunoprecipitation was carried out in Pam cells. One 110 kD-positive serum and two 110 kD-negative sera, precipitated a BPA of mol wt 220 kD from Pam cells labeled with [35S] methionine. These results suggest that BP sera may recognize more than one antigen, in addition to possible BPA heterogeneity in different individuals.

Detachment of cultured normal human keratinocytes by contact with TNF alpha-stimulated neutrophils in the presence of platelet-activating factor.

We have found previously that the cultured human squamous cell carcinoma cell line DJM-1 detached from the substratum after 48 h contact with human neutrophils. Neutrophils appeared to become activated by contact with DJM-1 and were found to secrete a proteinase that caused the detachment; the proteinase was inhibitable by alpha 1-proteinase inhibitor. In this study we tested whether normal human keratinocytes were also detached from the substratum by contact with human neutrophils, because keratinocyte detachment (epidermal separation) occurs in several skin diseases with neutrophil infiltration beneath the epidermis. Neutrophils with or without tumor necrosis factor (TNF) alpha pretreatment were plated on keratinocytes at confluency in 24-well culture plates, co-cultured in serum-free media for 16-24 h in the presence or absence of platelet-activating factor (PAF), and assessed for rate of detachment by counting the undetached keratinocytes and by fluorescent dye labeling. Keratinocytes were found to detach only when TNF alpha-pretreated neutrophils were plated together with 10(-5) M PAF. Inhibiting direct contact between neutrophils and keratinocytes by means of a membrane filter, however, decreased the detachment markedly. alpha 1-proteinase inhibitor and ONO-5046, a synthetic elastase specific inhibitor, inhibited the detachment significantly, and alpha 1-antichymotrypsin inhibited it slightly. The mediator responsible for detachment appeared to be elastase. Monoclonal anti-CD18 inhibited the detachment only partially. In conclusion, TNF alpha-pretreated neutrophils appeared to be activated by contact with keratinocytes in the presence of 10(-5) M PAF and caused substantial detachment of keratinocytes, possibly by secreting elastase. The precise role of PAF in detachment remains to be clarified.

Immunoelectronmicroscopic localization of IgA in skin of patients with dermatitis herpetiformis.

Ultrastructural localization of in vivo-bound IgA in the skin of patients with dermatitis herpetiformis was determined by using a modified peroxidase-antiperoxidase multistep method. Three types of reaction product deposition are seen. The most common type of reaction product deposition, that which is identified by direct immunofluorescence as the speckled type of IgA deposit, shows up as clumps and yarnlike fibrils. The second type of IgA deposition, which is a linear band by direct immunofluorescence, appears to be associated with anchoring fibrils. The third of IgA deposition, which is also linear by immunofluorescence, is confined to the lamina lucida.

Circulating IgA anti-basement membrane zone antibodies in dermatitis herpetiformis.

Criculating anti-basement membrane zone antibodies of the IgA class were detected in the sera of 2 or 6 dermatitis herpetiformis (DH) patients who had linear in-vivo-bound IgA deposits and in 1 of 42 DH patients who had granular vivo-bound IgA deposits. In the former 2 patients the circulating antibodies were localized ultrastructurally to the identical site where the in vivo-bound antibodies were localized and were bound by antigens in either the lamina lucida or the subbasal lamina anchoring fibril area of the basement membrane zone of normal human skin.

Basement membrane proteoglycans are of epithelial origin in rodent skin.

Basement membrane proteoglycans in mammalian skin comprise at least one chondroitin sulfate proteoglycan and heparan sulfate proteoglycans, including perlecan. In this study, the origins of basement membrane chondroitin sulfate proteoglycan and perlecan were investigated both in vivo and in vitro. For in vivo experiments, pieces of newborn rat epidermis obtained by dispase treatment were grafted onto athymic nude mice. Three and six weeks after grafting, immunofluorescence analysis of the grafted skin was carried out, using monoclonal antibodies specific for rat basement membrane chondroitin sulfate proteoglycan and rat and mouse perlecan. While the isolated rat epidermis was shown to completely lack rat basement membrane chondroitin sulfate proteoglycan and rat basement membrane heparan sulfate proteoglycans, including perlecan, immunofluorescence staining of tissue sections from the grafted sites on mice demonstrated the presence of rat basement membrane chondroitin sulfate proteoglycan and rat perlecan on interfollicular and follicular basement membranes including that separating dermal papillae from adjacent hair follicle epithelium. In contrast, the basement membranes of all dermal capillaries were positive for mouse perlecan, but negative for rat basement membrane chondroitin sulfate proteoglycan and rat perlecan, including the basement membranes of papillary dermal capillaries beneath the rat epidermis. These data suggest that basement membrane proteoglycans of the dermal-epidermal junction and hair follicle epithelium are of epidermal (epithelial) origin in vivo. Stratified rat keratinocytes cultured on a collagen matrix at the air-liquid interface showed the synthesis of perlecan, laminin 1, and type IV collagen in basement membranes, but not clearly detectable basement membrane chondroitin sulfate proteoglycan.

Localization of the collagenous component in skin basement membrane.

Antibodies to type IV collagen were produced by immunizing rabbits with a basement membrane collagen obtained from a transplantable mouse tumor. Using specifically purified antibodies, type IV collagen was localized ultrastructurally to the basal lamina part of the basement membrane zone.

Polymorphonuclear leukocyte-induced detachment of cultured epidermal carcinoma cells from the substratum.

In an in vitro study, intended to develop tumoricidal therapy with the use of human leukocyte, an interesting phenomenon was found. Normal human polymorphonuclear leukocytes (PMN) plated on a cell line established from malignant trichilemmal cyst (DJM-1), a kind of squamous cell carcinoma, in a serum-free media and incubated for 48 h induced the detachment of DJM-1. The detachment was more extensive as the number of PMN increased. The detachment rate was 97.0% when the number of PMN and DJM-1 in a well was in a ratio 2.4:1 and the viability of detached DJM-1 was 96.5%. Two kinds of proteinase inhibitors, especially the inhibitor of neutrophil elastase, fetal bovine serum, and monoclonal anti-laminin antibody inhibited the detachment significantly. Furthermore, when PMN were seeded in a chamber with a filter membrane bottom to prevent direct contact with DJM-1, DJM-1 detachment decreased to 14.2%. In view of these results, the following mechanism was postulated. Activated by their adhesion to DJM-1, especially between laminin receptor on PMN and laminin on DJM-1, PMN secreted proteinases, resulting in DJM-1 detachment. This phenomenon might be an expression of cytotoxicity of PMN to cancer cells, because cultured cancer cells of epithelium origin such as DJM-1 can grow only after they are firmly attached to the substratum. This phenomenon, in turn, may explain the final step in the induction of epidermal-dermal separation in subepidermal bullous diseases with PMN infiltration such as bullous pemphigoid and dermatitis herpetiformis if we could regard DJM-1 as normal keratinocyte.

Dermatitis herpetiformis: immunoelectronmicroscopic and ultrastructural studies of a patient with linear deposition of IgA.

Using immunoelectronmicroscopic techniques, we have demonstrated three distincet patterns of IgA deposition in the skin of patients with dermatitis herpetiforms. The least common of these patterns is the localization of reaction products to the lamina lucida. As this is the location of the immunoreactants in bullous pemphigoid and herpes gestationis and because the ultrastructural findings in our patient's early lesional skin differ from those usually seen in patients with dermatitis herpetiformis, we herein detail this patient's clinical, histologic, immunologic, and ultrastructural findings. The most prominent findings are (1) IgA deposition in the lamina lucida, (2) vesicle formation between basal lamina and the basal cells, and (3) fibrin-like material in the epidermis with showering into the dermis.

Herpes gestationis. Ultrastructure and ultrastructural localization of in vivo-bound complement.

Ultrastructural localization of C3 deposition in the skin of two patients with herpes gestationis was determined by using a peroxidase-antiperoxidase multistep technique. The tissue preparations can be stored for long periods of time and identical sections may be used for light and electron microscopic examination. The reaction products were seen throughout the entire lamina lucida and the basal cell plasma membrane appeared to be accentuated. The most remarkable ultrastructural changes in normal-appearing skin were the destruction of the basal cell membranes on the dermal side, localized cytoplasmic dissolution, and intracellular edema unaccompanied by inflammatory cells. Early, nonvesicular lesions showed basal cell degeneration and dermal inflammatory cells. Necrosis and loss of basal cells occurred in the next stage which resulted in microvesicles in which collagen or a well-preserved basal lamina formed the vesicle base. In the later blister stage, the basal lamina was usually lost. It is suggested that damage of basal cell membranes on their dermal side leads to the destruction of basal cells with the subsequent protrusion of epidermal and junctional substances into the dermis. This may result in inflammatory cell infiltration and blister formation.