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P Verrando

Publications and source records attributed to P Verrando.

At least 19 recordsLinked to original sources
PubMed1994

The 100-kDa chain of nicein/kalinin is a laminin B2 chain variant.

We have isolated the basement membrane component nicein and performed rotary-shadow analyses using electron microscopy that showed the presence of two forms (I and II) of the protein. Molecular cloning of the cDNA that codes for the 100-kDa chain of the protein revealed that the sequence matches those independently identified for the 105-155-kDa subunit of kalinin, a recently identified basement membrane component. These data demonstrate that nicein and kalinin contain an identical chain. The length of the open reading frame in the cDNA (approximately 5200 nucleotides) and amino acid sequence obtained from the N-terminus of the 105-kDa kalinin chain showed the occurrence of a precursor polypeptide. This immature polypeptide is probably related to form I, observed by rotary shadowing, while the mature form is related to form II. It is noteworthy that nicein/kalinin subunits share discrete sequence similarities with the B2 chain of human laminin, but with a cleavage occurring within domain III that eliminates domains IV and V from the final product. The sequence of this subunit is nearly identical to that of B2t, a recently described polypeptide supposed to be related to a new laminin variant. Since nicein/kalinin expression is specifically impaired in the severe genodermatosis Herlitz junctional epidermolysis bullosa, the role and structure of this tissue-restricted laminin variant is crucial for the understanding of epidermal-dermal adhesion.

Amino Acid Sequence↗
PubMed1994

Keratinocytes from junctional epidermolysis bullosa do adhere and migrate on the basement membrane protein nicein through alpha 3 beta 1 integrin.

BACKGROUND: Junctional epidermolysis bullosa (JEB) encompasses several genodermatoses characterized by skin blistering, and possibly disturbed wound healing. Although the molecular defects underlying JEB are not known, we have demonstrated previously that nicein, an adhesive laminin-related basement membrane component, is immunologically altered in the very severe JEB of Herlitz type (H-JEB), and was expressed to a lesser extent in skin from patients with inversa JEB (I-JEB). In this study, we assessed adhesion and migration of H-JEB and I-JEB keratinocytes on exogenous nicein and laminin to get insights on the biologic function defective in JEB skin. EXPERIMENTAL DESIGN: Adhesion of cultured epidermal keratinocytes from H-JEB and I-JEB patients was assayed by quantitation of cell attachment 1 hour after seeding into microtiter wells coated with nicein or laminin. Cell migration and modulation by function-blocking antibodies to integrins was quantified by computer-assisted image analysis of the tracks left by the cells in a phagokinetic assay using gold particles coated with nicein or laminin. RESULTS: In spite of the fact that H-JEB keratinocytes do not produce normal immunoreactive nicein, they were able to adhere on exogenous nicein similarly to normal and I-JEB keratinocytes which produce nicein. Adhesion of both JEB and normal keratinocytes to laminin was weak compared with nicein. At low and high concentrations of nicein, a reduced migration response occurred with H-JEB keratinocytes whereas I-JEB cells behaved like their normal counterparts. Integrin alpha 3 beta 1 was dominantly involved in adhesion and migration of all these cells. Laminin did not support the migration of either JEB or normal keratinocytes. CONCLUSIONS: H-JEB and I-JEB keratinocytes which produce no or less nicein than normal keratinocytes are able to adhere and migrate on exogenous nicein. Integrin alpha 3 beta 1 which is specifically involved in migration and adhesion of keratinocytes on nicein does not appear altered in JEB. These data indicate that defective nicein rather than modifications of the nicein-recognizing receptor play a central role in the pathogenesis of H-JEB.

Adult↗
PubMed1993

The basement membrane protein BM-600/nicein codistributes with kalinin and the integrin alpha 6 beta 4 in human cultured keratinocytes.

The observation is reported that in low-passage human keratinocyte colonies cultured under conditions that allow full epidermal differentiation (i) the basement membrane protein BM-600/nicein, identified by the mAb GB3, is codistributed with laminin and collagen type IV as well as with the bullous pemphigoid antigen in footprints deposited by growing and migrating colonies; (ii) the integrin heterodimer alpha 6 beta 4 is codistributed with the same molecules suggesting its spatial association with basement membrane components; (iii) the distribution pattern of alpha 6 beta 4 and BM-600/nicein underneath individual cells is identical and is characterized by a typical "leopard skin" pattern complementary to the distribution of submembraneous F-actin microfilament network; (iv) a rabbit polyclonal antiserum to kalinin (R4012) used in double-label immunofluorescence staining with mAb GB3 shows that this protein has the same distribution as BM-600/nicein and this suggests that they are identically located; and (v) immunoprecipitation with mAb GB3 to BM-600/nicein and BM165 to kalinin shows identical bands suggesting that nicein and kalinin represent the same molecular entity. We suggest that alpha 6 beta 4 displays not only an adhesive role for keratinocytes in view of its reported association to hemidesmosomes but may also be involved in organizing the molecules of the epithelial extracellular matrix, including those forming the basement membrane zone and hemidesmosomes, a function proposed for other integrins in other cellular systems.

Actins↗
PubMed1993

Labeling of fractured human skin with antibodies to BM 600/nicein, epiligrin, kalinin and other matrix components.

A variety of methods were used to fracture the dermal-epidermal junction (DEJ) of human skin. These included warm and hot phosphate buffered saline, trypsin, cold 1 M salt, potassium bromide and proteolytic digestion with dispase. The localization and sensitivity of basement membrane components (bullous pemphigoid antigen, BM 600/nicein, epiligrin, kalinin, laminin, collagens IV and VII (EBA antigen) and linkin) were determined after the DEJ was fractured by each method. We found that the basement membrane zone proteins, BM 600/nicein, epiligrin and kalinin remained with the dermal side of the DEJ fractured through the lamina lucida by cold salt, phosphate buffered saline and potassium bromide. BM 600/nicein, epiligrin and kalinin were not detected after treatment with trypsin. In contrast, laminin, another glycoprotein in the lamina lucida, was insensitive to all of the procedures, but co-localized to the dermal side of DEJ-fractured skin. We also found that separation of the DEJ with brief exposure of skin to 56 degrees C provided a useful substrate for testing the autoantibodies in the sera of patients with epidermolysis bullosa acquisita (EBA). Heat-separated skin can be prepared in a significantly shorter period of time than salt-separated skin.

Antibodies↗
PubMed1993

Nicein (BM-600) in junctional epidermolysis bullosa: polyclonal antibodies provide new clues for pathogenic role.

We have raised polyclonal antibodies against each of three subunits of the new basement membrane component nicein (formerly BM-600), the antigen recognized by the monoclonal antibody GB3 (Biochem Biophys Acta 942:45-56, 1988). Preparation of such antibodies was achieved from gel electrophoresis purification of nicein isolated by immuno-affinity chromatography. These antibodies were reactive with each transblotted denatured nicein subunit and recognized the native protein both in cultured keratinocytes and in all normal human basement membranes where the GB3 antigen is located. A reciprocal immuno-cross-reactivity was detected with the antibodies directed against the 100-kD and 150-kD (sometimes resolved as a 146-150-kD doublet) subunits of nicein, showing that they share some identical epitopes. In tissues and keratinocyte cultures from patients with the Herlitz form of junctional epidermolysis bullosa (H-JEB), GB3 is unable to recognize nicein, and the question arises whether this is due to an absence of synthesis or a structural abnormality of the protein. We report here that the polyclonal antibody directed against the 150-kD subunit of nicein binds its antigen in H-JEB patients (although usually less intensely than in control skin), whereas the other two antibodies either do not recognize or recognize only weakly their respective antigen subunits. These data suggest that nicein is present but structurally altered in basement membranes from H-JEB tissues. Furthermore, in non-Herlitz junctional and dystrophic types of epidermolysis bullosa, all three polyclonal antibodies recognize their antigens normally. Consequently, such antibodies should serve as potentially useful molecular tools for studying the expression of nicein in H-JEB.

Antibodies↗
PubMed1993

Basement membrane proteins kalinin and nicein are structurally and immunologically identical.

BACKGROUND: The purpose of this investigation was 2-fold: (a) to compare two recently described proteins, the anchoring filament protein, kalinin and the hemidesmosome-associated protein, nicein (formerly called BM-600) which are both absent in junctional epidermolysis bullosa (JEB) Herlitz's disease; (b) to further define the structural defect in JEB Herlitz's disease. EXPERIMENTAL DESIGN: Cultured keratinocytes were analyzed with monoclonal antibodies (mAbs) against kalinin and nicein by indirect immunofluorescence. These mAbs were also used to immunoprecipitate radiolabeled proteins from keratinocyte cultures and to immunoaffinity purify proteins from keratinocyte conditioned culture medium. The precipitated or purified products were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, partial V8 protease digestion, and rotary shadowing. RESULTS: Kalinin and nicein mAbs show identical immunofluorescent staining patterns on cultured keratinocytes. Kalinin and nicein mAbs immunoprecipitate peptides from radiolabeled normal human keratinocyte cell and medium fractions that are electrophoretically identical. Partial V8 protease digestion patterns of the reduced 140 kilodalton peptides precipitated by nicein and kalinin mAbs are identical. Kalinin (like nicein) is absent from JEB Herlitz keratinocyte conditioned medium although K-laminin, another anchoring filament component, is present in these cultures. Kalinin, purified from conditioned keratinocyte medium by antibody affinity chromatography with K140-Sepharose (mAb against kalinin) and nicein purified from conditioned keratinocyte medium with GB3-Sepharose (mAb against nicein) are electrophoretically identical by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunologically identical by immunoblotting using kalinin and nicein mAbs. Rotary shadowed images of kalinin and nicein molecules are identical. CONCLUSIONS: We demonstrate that kalinin and nicein are identical by biochemical and immunologic analysis. We also verify that kalinin, like nicein, is absent in the conditioned medium of cultured JEB Herlitz keratinocytes, although another anchoring filament protein, K-laminin, is secreted by these cultures. These results correlate with previous immunofluorescent findings that show that while kalinin or nicein is absent in basement membranes of individuals with JEB Herlitz's disease, K-laminin appears to be present.

Antibodies, Monoclonal↗
PubMed1992

The 6/2 (AA3) polyclonal antibody identifying a 37 kD keratinocyte protein reacts also with BM-600/nicein, the basement membrane component bound by the monoclonal antibody GB3.

An unexpected finding concerning our previously reported polyclonal antibody raised against an extract from human amnion (pAb 6/2, also termed AA3), and which recognizes an epidermal keratinocyte protein, is presented in this study. Using the immunoblot technique, pAb 6/2 binds to a 37 kD intracellular protein antigen. We have subsequently found that, by radioimmunoprecipitation performed after metabolic labelling with 35S-methionine of cultured keratinocytes, pAb 6/2 recognizes the 600 kD epidermal basement membrane component (termed BM-600/nicein) which was reported to be bound by the monoclonal antibody mAb GB3. Specifically, pAb 6/2 reacts with immunoaffinity chromatography-isolated BM-600/nicein blotted onto nitrocellulose. The data suggest the existence of two immunological reactivities borne by pAb 6/2, each of them being directed against, respectively, the 37 kD (seen in immunoblots) and the 600 kD protein (seen in immunoprecipitations). The data further suggest possible independent expression of these two proteins in cell culture. In comparison with the staining pattern of normal skin, immunofluorescence was previously noted to be impaired (pAb 6/2) or absent (mAb GB3) in lethal junctional epidermolysis bullosa. Thus, we conclude that mAb GB3, rather than pAb 6/2, is a more appropriate probe for the comprehensive biochemical study of this genodermatosis.

Antibodies↗
PubMed1991

Monoclonal antibody GB3 defines a widespread defect of several basement membranes and a keratinocyte dysfunction in patients with lethal junctional epidermolysis bullosa.

An antigen expressed at the dermal-epidermal junction as well as in some other human basement membranes (BM) has been detected by the use of a monoclonal antibody termed GB3. This antigen, synthesized by cultured normal human keratinocytes, has been identified as a 600-kilodalton glycoprotein different from other known components of BM. Using indirect immunofluorescence, GB3 was found to be not reactive with the epidermal BM in patients with lethal junctional epidermolysis bullosa. The present study demonstrates (by indirect immunofluorescence) that GB3 defines a widespread defect of several BM in these patients. Furthermore, it gives evidences for an intrinsic biologic defect of lethal junctional epidermolysis bullosa epidermal keratinocytes using in vitro culture of these cells. Whether the lack of GB3 reactivity is the consequence of a true absence of the antigen or an alteration of its molecular structure is not yet known. Nevertheless, GB3 is a useful probe for both rapid and prenatal diagnosis of lethal junctional epidermolysis bullosa, which will give new insights into the molecular comprehension of this disorder.

Antibodies, Monoclonal↗
PubMed1990

GB3 monoclonal antibody for the diagnosis of junctional epidermolysis bullosa: results of a multicenter study.

GB3 monoclonal antibody detects a normal basement membrane component (GB3 antigen) that is variably expressed in junctional epidermolysis bullosa. To assess the accuracy of GB3 in the diagnosis of junctional epidermolysis bullosa, we have reviewed its use in 250 cases of the major types of epidermolysis bullosa. In the majority of cases of the simplex and dystrophic forms of epidermolysis bullosa, GB3 antigen is normally expressed. In the Herlitz variant of junctional epidermolysis bullosa, GB3 antigen expression is consistently abnormal, but in the non-Herlitz and indeterminate forms of junctional epidermolysis bullosa, 40% of cases express GB3 antigen normally. We propose that GB3 monoclonal antibody is useful in the accurate identification of patients with Herlitz junctional epidermolysis bullosa and may prove equal to electron microscopy for the diagnosis of this disease. For the non-Herlitz variants, it should not be used as an alternative to electron microscopy but may be of special value in the determination of prognosis.

Antibodies, Monoclonal↗
PubMed1990

Cultured epithelia from junctional epidermolysis bullosa letalis keratinocytes express the main phenotypic characteristics of the disease.

Keratinocytes from a 1-week-old male infant with junctional epidermolysis bullosa letalis (JEBL) were grown in vitro and then grafted as multi-layered epithelia onto nude mice, to investigate whether the defect in the dermo-epidermal cohesiveness in the disease is of epidermal and not mesodermal origin. In culture, there was a birefringent ring of cells at the edges of the keratinocyte colonies and in places some cells looked as though they had been ejected from the periphery of the colony. At confluence, the multi-layered epithelia were easily detached from the culture flasks using only mechanical agitation. On microscopy the fully-differentiated epithelium on days 21, 30 and 40 after grafting sometimes showed blistering at the dermal-epidermal junction. No labelling was noted using a GB3 monoclonal antibody, that reacts with normal human keratinocytes in culture and with the dermo-epidermal basement membrane zone in normal skin. This indicates that the defect of JEBL may be reproduced in culture and also after grafting the cultured epithelial onto a wound without an epidermis. This suggests a possible role for the junctional structure recognized by GB3 in dermo-epidermal cohesiveness.

Animals↗
PubMed1988

The new basement membrane antigen recognized by the monoclonal antibody GB3 is a large size glycoprotein: modulation of its expression by retinoic acid.

Further biochemical investigations on the hemidesmosome-associated epidermal basement membrane component recognized by the monoclonal antibody GB3 are presented in this study. We previously found that the expression of this constituent is impaired in a severe genodermatosis termed lethal junctional epidermolysis bullosa. We demonstrate now that this factor is a very large glycoprotein (apparent molecular weight, 600 kDa) made up of polypeptides in the range of 93.5 to 150 kDa, and containing N-linked oligosaccharide chains. Both endo-beta-N-acetylglucosaminidases and neuraminidase hydrolysis, as well as concanavalin A binding experiments were performed on the GB3 radioimmunoprecipitated polypeptides from cultured human keratinocytes. They showed that the antigen subunits probably bear both 'high-mannose' and 'complex' type glycosidic chains. The chronic exposure of cultured human keratinocytes to retinoic acid (10(-8) to 10(-6) M) resulted in no apparent changes in the overall bulk of these glycosidic chains, but a dose-dependent increase of synthesis and secretion of the antigen was observed. A relative induction factor of 4 was obtained in cultures treated with 10(-6) M retinoic acid. This induction was also observed morphologically by indirect immunofluorescence at the basement membrane zone from cultured human keratinocytes grown on dead de-epidermized dermis. These results further emphasize the influence of glycoproteins in cell-cell and cell-substratum attachment. Furthermore, the ability to modulate this antigen may be relevant for the understanding of the molecular defect involved in lethal junctional epidermolysis bullosa.

Antibodies, Monoclonal↗
PubMed1988

Normal Merkel cells express a synaptophysin-like immunoreactivity.

Synaptophysin (SY), a specific component of the membrane of presynaptic vesicles, has been reported as a novel marker for neurons, certain neuroendocrine cells and their neoplasms including neuroendocrine carcinomas of the skin. The origin of the Merkel cells (MC) being far from clear, this study was performed to establish if normal MC express SY. It is demonstrated by immunofluorescence and immunoelectron microscopy using a monoclonal antibody SY38 to this glycoprotein that normal MC in man, rabbit and pigs express an SY-like reactivity. Although immunoblotting identification of the immunoreactive material gave negative results, it is likely that normal MC contain SY. By immunoelectron microscopy, the staining was located at the surface of cytoplasmic vesicles. In view of the possible involvement of SY in the Ca2+-dependent neurotransmitter release, the observation of an SY-like immunoreactivity in MC supports the view that they are epithelial neuroendocrine cells and that they may possess a neurosecretory function.

Animals↗
PubMed1987

Lamellar cells of sensory receptors and perineural cells of nerve endings of pig skin contain cytokeratins.

The lamellar cells of the sensory corpuscles of the pig dermis must be considered to be epithelial cells as they contain cytokeratins. The cytokeratins detected are similar to those found in simple epithelia. Moreover, lamellar cells are embedded in an extracellular matrix reminiscent of the basement membrane of epithelium since it contains laminin and collagen IV. The perineural cells surrounding the nerves of pig dermis present the same features. These results suggest that lamellar cells and perineural cells have the same origin. The nature of the lamellar and perineural cells of the rabbit or human dermis is not as clear since cytokeratins were not detected in those cells. These results, together with recent observations on Merkel cells, may indicate that epithelio-neuronal junctions are a general feature of cutaneous sensory receptors.

Animals↗
PubMed1987

Monoclonal antibody GB3, a new probe for the study of human basement membranes and hemidesmosomes.

A monoclonal antibody, GB3, has been raised against human amnion. Not only does GB3 bind to amniotic basement membrane, but it also recognizes an antigenic structure expressed by epidermal as well as by some other human basement membranes. This antigen is synthesized (and excreted) by cultured normal human epidermal keratinocytes. It is expressed to a lesser extent by the A431 epidermoid carcinoma cell line, but is not expressed by the SV40 virus-transformed SVK14 keratinocyte cell line. In ultrastructural studies, this antigen was located in the epidermal basement membrane, both in the lamina densa and in the lamina lucida, associated with hemidesmosomes. It was identified as a protein by in vitro proteolytic cleavage studies. The radio-immunoprecipitates from cultured human keratinocytes, analysed by SDS-PAGE, showed that GB3 recognized five polypeptides of 93.5, 125, 130, 146 and 150 kD under reducing conditions. They were probably linked by disulfide bonds. The tissue distribution of the antigen and the molecular weights (MWs) of its constitutive polypeptides suggest that it is different from other known components of basement membranes. It may provide a biochemical marker for hemidesmosomes. Furthermore, GB3 represents an interesting and original clinical probe, since the antigenic structure recognized by GB3 is lacking in Junctional Epidermolysis Bullosa, a lethal genodermatosis in which a dermo-epidermal splitting occurs at the level of lamina lucida.

Amnion↗
PubMed1987

Herpes gestationis factor reacts with the amniotic epithelial basement membrane.

Sera from five patients with clinically and immunopathologically proven herpes gestationis were studied by complement fixing immunofluorescence and complement fixing immuno-electron microscopy using specimens of skin, amniochorion and placenta. The results demonstrated that the complement fixation antibody (herpes gestationis factor) could bind to the basement membrane zone of skin, amnion and chorion laeve but not to that of the placental syncytiotrophoblast. These data suggest that the herpes gestationis factor may be induced by the basement membrane zone antigens of extra-villous cytotrophoblasts.

Amnion↗