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R S Labib

Publications and source records attributed to R S Labib.

At least 37 records · Page 2Linked to original sources

Human pemphigus autoantibodies are pathogenic to squamous epithelium.

In 1957, Witbesky et al. put forward several criteria that ideally should be fulfilled in order to prove the pathogenic role of an autoantibody in a putative autoimmune disease. There can now be very little doubt of the autoimmune nature of this disease and of the primary role of autoantibodies in its pathogenesis. The evidence that supports the concept that pemphigus autoantibodies are of primary pathogenic importance in the disease is as follows: IgG class autoantibodies can be found both circulating in the serum and bound to the epithelial cell surfaces in and around lesions in patients with pemphigus. These autoantibodies, purified from the serum of pemphigus patients, can induce acantholytic lesions typical of pemphigus both in experimental animals (neonatal mice) and in human and murine epidermal cell cultures. These autoantibodies react with a specific antigen of the epidermal cell. This purified antigen has been used to immunize rabbits and the resulting antibodies are capable of inducing pemphigus-like lesions in neonatal mice.

Acantholysis

Dexamethasone inhibits plasminogen activator activity in experimental pemphigus in vivo but does not block acantholysis.

In vitro studies have suggested that autoantibody-stimulated increases in epidermal plasminogen activator (PA) may be an important pathogenetic mechanism in pemphigus vulgaris (PV). We measured PA in murine epidermis after i.p. injection of normal human IgG (NH IgG) and PV IgG, with and without exposure to dexamethasone (DEX). BALB/c neonates received i.p. injections of saline control or DEX (20 mg/kg). Twenty-four hours later, they received a second injection of saline or DEX and a single dose of NH or PV IgG (20 mg/gm body weight). After 24 hr, epidermis was obtained and was sequentially extracted in 0.14 M NaCl, pH 6.8, and 0.5% Triton X-100 in 0.1 M Tris, pH 8.1. Epidermal PA was assayed in the Triton-Tris supernatant by a two-stage colorimetric reaction and was expressed as milliPloug units per milligram of protein (mPu/A280). PA in animals injected with NH IgG was 0.21 +/- 0.11 mPu/A280 (n = 8). Epidermal PA was increased in animals with cutaneous lesions of pemphigus to 0.42 +/- 0.29 (n = 15). Treatment with DEX decreased PA levels in both animals receiving NH IgG and PV IgG by 80%, to 0.04 +/- 0.05 (n = 15) and 0.09 +/- 0.07 (n = 7), respectively. Despite the decreased PA activity, all animals in the PV IgG and the PV IgG-plus-DEX group had identical and extensive cutaneous disease, and lesions developed at the same time points. This finding shows that PV autoantibodies can stimulate increases in epidermal PA, but reduction of PA by corticosteroids does not inhibit acantholysis in vivo. There is no clear correlation between PA and disease activity in the murine model of pemphigus.

Acantholysis

Epidermal proteins. I. Differential extraction and quantitative polyacrylamide gel-electrophoretic analysis of basal spinous-cell proteins of neonatal mouse epidermis.

The study of keratinocyte proteins and their changes in different physiological, experimental, and pathological states has been facilitated and stimulated by the development of high-resolution polyacrylamide gel electrophoretic (PAGE) techniques. We describe a differential extraction system that separates the keratinocyte proteins into four major groups which are further quantitatively analyzed by PAGE: cytoplasmic-soluble proteins, nonionic-detergent-soluble proteins consisting of membrane-associated proteins, salt-dissociated proteins mainly consisting of histones, and ribosomal and keratohyaline granule proteins, and the keratins (and other intermediate-filament-associated proteins), which are further separated into disulfide-stabilized keratins and keratins that do not require reducing agents in order to dissolve in sodium dodecyl sulfate (SDS) or urea. This extraction system was applied to neonatal mouse epidermal cell preparations that consisted mainly (60%-85%) of basal cells and also of some spinous cells (10%-30%). The SDS-PAGE patterns obtained by spectrophotometric scanning were graphically compared and integrated using an IBM personal computer. The protein bands in each extract were identified by their apparent molecular weights and were quantitated as a percentage of the total in each extract and in micrograms per 15 X 10(6) cells. Some protein peaks were provisionally identified as actin, the core histones H2A, H2B, H3, and H4, ribosomal proteins, and six keratins. This study serves as the foundation for the quantitative description of molecular changes which occur during keratinocyte differentiation and for the comprehensive identification of epidermal proteins.

Animals

Experimentally induced pemphigus vulgaris in neonatal BALB/c mice: a time-course study of clinical, immunologic, ultrastructural, and cytochemical changes.

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.

Acantholysis

A pool of bullous pemphigoid antigen(s) is intracellular and associated with the basal cell cytoskeleton-hemidesmosome complex.

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 epidermal sheets (by dithiothreitol treatment). We show that viable basal cells, which are impermeable to IgG molecules, do not react with BP antibodies. When the basal cell plasma membrane is disrupted by cytospin centrifugation, air drying, freezing and thawing, or hypotonic lysis, or permeated by nonionic 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 nonionic 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.

Animals

The use of human pemphigoid autoantibodies to study the fate of epidermal basal cell hemidesmosomes after trypsin dissociation.

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.

Animals

Brazilian pemphigus foliaceus autoantibodies are pathogenic to BALB/c mice by passive transfer.

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 antiepithelial 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 mice. 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 less than 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.

Animals

Pemphigus and pemphigoid antigens are expressed in human amnion epithelium.

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-micron 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. The abundance and availability of these tissues facilitate extraction and characterization of BP and PV antigens.

Amnion

Demonstration of pemphigus antibodies on the cell surface of murine epidermal cell monolayers and their internalization.

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 degrees 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 degrees 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.

Animals

Specificity and inhibition of the epidermal cell detachment induced by pemphigus IgG in vitro.

IgG isolated from sera of patients with pemphigus vulgaris (PV) has been shown to induce cell detachment when added to primary epidermal cell cultures (PECC). 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 fractions 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 alpha 2 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 this effect is specific for PV IgG, is dose-dependent, and may be inhibited by certain proteinase inhibitors.

Animals

Induction of pemphigus in neonatal mice by passive transfer of IgG from patients with the disease.

We examined the role of circulating autoantibodies in the pathogenesis of pemphigus vulgaris by passively transferring IgG fractions from five patients with pemphigus vulgaris into neonatal Balb/c mice, in doses of 1.5 to 16 mg per gram of body weight per day. Cutaneous blisters and erosions with the histologic, ultrastructural, and immunofluorescence features of pemphigus occurred in 39 to 55 mice given intraperitoneal injections of IgG from patients with pemphigus and in none of 58 control mice given normal human IgG. IgG fractions with high titers of pemphigus antibodies were most effective in inducing disease, and this effect was dose dependent. Titers of circulating IgG in mouse serum closely correlated with the extent of disease induced (P less than 0.002). This study strongly supports the proposed role of pemphigus autoantibodies in the pathogenesis of pemphigus vulgaris in human beings and demonstrates that pemphigus can be passively transferred to laboratory animals.

Animals

Enzymatic oxidation of polyamines. Relationship to immunosuppressive properties.

A method for the detection of spermine oxidase activity was developed using the amino acid analyzer to follow the changes in composition of incubation mixture of spermine or spermidine. Spermine oxidase activity could be detected in fetal calf serum (FCS), and six major spermine oxidation products were revealed in the incubation mixture. Incubation of spermine or spermidine with FCS produced oxidation products which suppressed lipopolysaccharide-induced mitogenesis and the murine mixed lymphocyte reaction. Oxidation was inhibited by the spermine oxidase inhibitors hydroxylamine and isonicotinic acid hydrazide, and inhibition of the immune response was no longer evident suggesting that the inhibitory material was a product of the action of the enzyme on spermic and spermidine. When FCS was used as the source of the spermine oxidase, some suppression via a cytotoxic effect could not be excluded. Mouse amniotic fluid was found to contain high levels of a spermine oxidase having a somewhat different specificity than that in FCS, and this enzyme produced a noncytotoxic immune inhibitor from cadaverine, spermidine and spermine. These data raise questions concerning the role polyamine oxidation products may play in the immunosuppression associated with pregnancy and in the generation of nonantigen-specific suppressors by cells cultured in FCS or other media containing polyamine oxidase activity.

Amniotic Fluid

Pathogenic effects of bullous pemphigoid autoantibodies on rabbit corneal epithelium.

Bullous pemphigoid (BP) is associated with circulating autoantibodies reactive with an antigen(s) of the basement membrane zone (BMZ) of skin and mucosae. The pathogenicity of these autoantibodies, although suspected, is unconfirmed. We have investigated the effects of BP autoantibodies on a closely related tissue, the corneal epithelium of the rabbit. IgG fractions from the sera of seven patients with BP were purified by (a) ammonium sulfate precipitation, (b) ion exchange chromatography, or (c) gel filtration. Control IgG was prepared by ion exchange chromatography of pooled normal human gamma globulins. 32 rabbits received corneal intrastromal injections of BP IgG fractions (50 microliter, 0.95-2.05 mg total dose) in one eye, and control IgG (50 microliter, 1.8 mg) in the contralateral cornea. 28 of 32 BP IgG injections produced corneal inflammatory lesions, 10 of which developed visible blisters. Histologically, lesions showed polymorphonuclear cells clustering along the BMZ, and subepithelial blister formation. Immunofluorescence showed in vivo bound IgG and C3 at the BMZ. The intensity of inflammation was dose dependent and correlated often with in vitro complement fixation titers of the fractions. None of 32 corneas injected with control IgG became inflamed. BP IgG fractions injected intradermally into the ear skin of rabbits failed to produce inflammation. This may be due to slow clearance of IgG in the cornea, and optimal binding by the corneal epithelium. The intracorneal injections of BP IgG reproduce the clinical, histological, and immunological features of BP. This study provides evidence that BP autoantibodies are pathogenic.

Animals

Studies on extracellular proteases of Streptococcus sanguis. Purification and characterization of a human IgA1 specific protease.

Extracellular caseinolytic activity was found in the culture fluid of Streptococcus sanguis ATCC 10556 grown in a dialyzed culture medium. This activity was due to multiple proteases that differed in their elution from hydroxyapatite, sensitivity to enzyme inhibitors, specificity and optimum pH. IgA protease, which splits human immunoglobulin A1 into intact Fc and Fab could be effectively separated from these relatively non-specific proteases and purified to apparent homogeneity in 20% yield by a five-step procedure. Although the bulk of the dextran sucrase activity was separated from the IgA protease, a small amount of sucrase activity remained with the final IgA protease preparation. In polyacrylamide gel electrophoresis at pH 9.5 both activities were located in the single protein band detected in this preparation. A quantitative method for the assay of IgA protease was developed, based on radial immunodiffusion to quantitate the Fab produced. This was used to follow the specific activity and yield during purification, and to characterize some of the catalytic properties of the enzyme. At an enzyme/substrate ratio of 1: 400 (w/w) the protease could effect 50% proteolysis of IgA in overnight incubation at 37 degrees C. The optimum activity was at pH 8.0, and 50% inhibition was achieved at 4 . 10(-4) M o-phenanthroline or 8 . 10(-4) M ethylene diamine tetraacetate. Concentrations of diisopropyl phosphofluoridate, phenylmethyl-sulfonyl fluoride, iodoacetate and p-chloromercuribenzoate up to 10(-2) M were without effect on the IgA protease activity. Full reactivation of the chelator inhibited enzyme could be achieved by the addition of Mg2+, Mn2+ or Ca2+.

Edetic Acid