The distribution of blood group antigens in rodent epithelia

Summary The pattern of distribution of antigens cross-reacting with antibodies to human blood group antigens A and B and two precursor molecules was examined by immunofluorescence in the epidermis, oral mucosa and forestomach of rats and mice. Staining for blood group antigen A was negative. In all epithelia examined, blood group antigen B was present at the surface of basal and parabasal cells, and the H antigen at the surface of spinous cells. N-acetyllactosamine was present on the cell membranes in the upper spinous and granular cell layers of epidermis and forestomach epithelium and was not expressed in the oral epithelia except for a limited area in the dorsal tongue epithelium.Thus, the expression of antigen varies both regionally and, as earlier shown in human epithelium, with the stage of maturation of cells within a given epithelium. The observed sequence of expression of these antigens during maturation differs from that of human epithelia, but the present study provides a basis for further experimental studies of the role of cell surface antigens in epithelial homeostasis and maturation.     

Collagen fibril network and elastic system remodeling in a reconstructed skin transplanted on nude mice.

Wound healing of deep and extensive burns can induce hypertrophic scar formation, which is a detrimental outcome for skin functionality. These scars are characterized by an impaired collagen fibril organization with fibril bundles oriented parallel to each other, in contrast with a basket weave pattern arrangement in normal skin. We prepared a reconstructed skin made of a collagen sponge seeded with human fibroblasts and keratinocytes and grown in vitro for 20 days. We transplanted it on the back of nude mice to assess whether this reconstructed skin could prevent scar formation. After transplantation, murine blood vessels had revascularized one-third of the sponge thickness on the fifth day and were observed underneath the epidermis at day 15. The reconstructed skin extracellular matrix was mostly made of human collagen I, organized in loosely packed fibrils 5 days after transplantation, with a mean diameter of 45 nm. After 40-90 days, fibril bundles were arranged in a basket weave pattern while their mean diameter increased to 56 nm, therefore exactly matching mouse skin papillary dermis organization. Interestingly, we showed that an elastic system remodeling was started off in this model. Indeed, human elastin deposits were organized in thin fibrils oriented perpendicular to epidermis at day 90 whereas elastic system usually took years to be re-established in human scars. Our reconstructed skin model promoted in only 90 days the remodeling of an extracellular matrix nearly similar to normal dermis (i.e. collagen fibril diameter and arrangement, and the partial reconstruction of the elastic system).     

Langerhans Cell Homeostasis and Activation Is Altered in Hyperplastic Human Papillomavirus Type 16 E7 Expressing Epidermis

It has previously been shown that expression of human papillomavirus type 16 (HPV) E7 in epidermis causes hyperplasia and chronic inflammation, characteristics of pre-malignant lesions. Importantly, E7-expressing epidermis is strongly immune suppressed and is not rejected when transplanted onto immune competent mice. Professional antigen presenting cells are considered essential for initiation of the adaptive immune response that results in graft rejection. Langerhans cells (LC) are the only antigen presenting cells located in normal epidermis and altered phenotype and function of these cells may contribute to the immune suppressive microenvironment. Here, we show that LC are atypically activated as a direct result of E7 expression in the epidermis, and independent of the presence of lymphocytes. The number of LC was significantly increased and the LC are functionally impaired, both in migration and in antigen uptake. However when the LC were extracted from K14E7 skin and matured in vitro they were functionally competent to present and cross-present antigen, and to activate T cells. The ability of the LC to present and cross-present antigen following maturation supports retention of full functional capacity when removed from the hyperplastic skin microenvironment. As such, opportunities are afforded for the development of therapies to restore normal LC function in hyperplastic skin.     

New facets of keratin K77: interspecies variations of expression and different intracellular location in embryonic and adult skin of humans and mice

The differential expression of keratins is central to the formation of various epithelia and their appendages. Structurally, the type II keratin K77 is closely related to K1, the prototypical type II keratin of the suprabasal epidermis. Here, we perform a developmental study on K77 expression in human and murine skin. In both species, K77 is expressed in the suprabasal fetal epidermis. While K77 appears after K1 in the human epidermis, the opposite is true for the murine tissue. This species-specific pattern of expression is also found in conventional and organotypic cultures of human and murine keratinocytes. Ultrastructure investigation shows that, in contrast to K77 intermediate filaments of mice, those of the human ortholog are not attached to desmosomes. After birth, K77 disappears without deleterious consequences from human epidermis while it is maintained in the adult mouse epidermis, where its presence has so far gone unnoticed. After targeted Krt1 gene deletion in mice, K77 is normally expressed but fails to functionally replace K1. Besides the epidermis, both human and mouse K77 are present in luminal duct cells of eccrine sweat glands. The demonstration of a K77 ortholog in platypus but not in non-mammalian vertebrates identifies K77 as an evolutionarily ancient component of the mammalian integument that has evolved different patterns of intracellular distribution and adult tissue expression in primates.     

Changes in the expression of blood-group carbohydrates during oral mucosal development in human fetuses

Abstract. The distribution of blood group carbohydrate chains with antigen A, B, H type 2 chain (A and B precursor), and N-acetyllactosamine (H type 2 precursor) specificity was studied in human oral epithelium from different anatomical regions. These represented various epithelial differentiation patterns such as non-keratinized, parakeratinized, and orthokeratinized stratified squamous epithelium. The material included buccal and palatal epithelium from 20 persons with blood group A or O, gingival, and alveolar epithelium from 10 persons with blood group A or B, and buccal metaplastically keratinized epithelium from nine blood group A, two blood group B, and nine blood group O individuals. The blood group carbohydrate chains were examined in tissue sections by immunofluorescence microscopy. The A and B blood group antigens were detected by human blood group sera, and antigen H type 2 chains and N-acetyllactosamine by murine monoclonal antibodies. Each antigen showed a similar staining pattern in buccal and alveolar epithelium (non-keratinized) which differed considerably from that seen in palatal and gingival epithelium (ortho- and parakeratinized). The expression of blood group antigens A or B and the precursor antigen H type 2 chains in metaplastically keratinized buccal epithelium was found to differ significantly from that seen in normal non-keratinized buccal epithelium. The regional variations demonstrated in cell surface carbohydrates are suggested to reflect differences in tissue differentiation.     

Regional variations of cell surface carbohydrates in human oral stratified epithelium

The distribution of blood group carbohydrate chains with antigen A, B, H type 2 chain (A and B precursor), and N-acetyllactosamine (H type 2 precursor) specificity was studied in human oral epithelium from different anatomical regions. These represented various epithelial differentiation patterns such as non-keratinized, parakeratinized, and orthokeratinized stratified squamous epithelium. The material included buccal and palatal epithelium from 20 persons with blood group A or O, gingival, and alveolar epithelium from 10 persons with blood group A or B, and buccal metaplastically keratinized epithelium from nine blood group A, two blood group B, and nine blood group O individuals. The blood group carbohydrate chains were examined in tissue sections by immunofluorescence microscopy. The A and B blood group antigens were detected by human blood group sera, and antigen H type 2 chains and N-acetyllactosamine by murine monoclonal antibodies. Each antigen showed a similar staining pattern in buccal and alveolar epithelium (non-keratinized) which differed considerably from that seen in palatal and gingival epithelium (ortho- and parakeratinized). The expression of blood group antigens A or B and the precursor antigen H type 2 chains in metaplastically keratinized buccal epithelium was found to differ significantly from that seen in normal non-keratinized buccal epithelium. The regional variations demonstrated in cell surface carbohydrates are suggested to reflect differences in tissue differentiation.     

The serine protease marapsin is expressed in stratified squamous epithelia and is up-regulated in the hyperproliferative epidermis of psoriasis and regenerating wounds

The trypsin-like serine protease marapsin is a member of the large protease gene cluster at human chromosome 16p13.3, which also contains the structurally related proteases testisin, tryptase epsilon, tryptase gamma, and EOS. To gain insight into the biological functions of marapsin, we undertook a detailed gene expression analysis. It showed that marapsin expression was restricted to tissues containing stratified squamous epithelia and was absent or only weakly expressed in all other tissues, including the pancreas. Marapsin was constitutively expressed in nonkeratinizing stratified squamous epithelia of human esophagus, tonsil, cervix, larynx, and cornea. In the keratinizing stratified squamous epidermis of skin, however, its expression was induced only during epidermal hyperproliferation, such as in psoriasis and in murine wound healing. In fact, marapsin was the second most strongly up-regulated protease in psoriatic lesions, where expression was localized to the upper region of the hyperplastic epidermis. Similarly, in the hyperproliferative epithelium of regenerating murine skin wounds, marapsin localized to the suprabasal layers, where keratinocytes undergo squamous differentiation. The transient up-regulation of marapsin, which closely correlated with re-epithelialization, was virtually absent in a genetic mouse model of delayed wound closure. These results suggested a function during the process of re-epithelialization. Furthermore, in reconstituted human epidermis, a model system of epidermal differentiation, members of the IL-20 subfamily of cytokines, such as IL-22, induced marapsin expression. Consistent with a physiologic role in marapsin regulation, IL-22 was also strongly expressed in re-epithelializing skin wounds. Marapsin's restricted expression, localization, and cytokine-inducible expression suggest a role in the terminal differentiation of keratinocytes in hyperproliferating squamous epithelia.     

A central role for alpha beta T cells in the pathogenesis of murine lupus.

We have previously shown that female transgenic mice expressing IFN-gamma in the epidermis, under the control of the involucrin promoter, develop inflammatory skin disease and a form of murine lupus. To investigate the pathogenesis of this syndrome, we generated female IFN-gamma transgenic mice congenitally deficient in either alpha beta or gamma delta T cells. TCR delta-/- transgenics continued to produce antinuclear autoantibodies and to develop severe kidney lesions. In contrast, TCR beta-/- IFN-gamma transgenic mice failed to produce antinucleosome, anti-dsDNA, or antihistone autoantibodies, and kidney disease was abolished. Both alpha beta- and gamma delta-deficient transgenics continued to develop IFN-gamma-associated skin disease, lymphadenopathy, and splenomegaly. The data show that the autoantibody-mediated pathology of murine lupus in IFN-gamma transgenic mice is completely alpha beta T cell dependent and that gamma delta T cells cannot drive autoantibody production. These results imply that production of antinuclear autoantibodies in IFN-gamma transgenic animals is Ag driven, and we identified clusters of apoptotic cells in the epidermis of the mice as a possible source of self Ags. Our findings emphasize the relevance of this murine lupus model to the human disease.     

A sequential double immunoenzymic staining procedure to obtain cell kinetic information in normal and hyperproliferative epidermis

Summary A sequential double immunoenzymic staining procedure was developed using the monoclonal antibody anti-BrdUrd and Ki67 in order to determine whether hyperproliferative skin disorders, such as psoriasis, are characterized by an increased growth fraction rather than a much shorter cell cycle time of all germinative cells. Ki67 binds to a proliferation-associated nuclear antigen in a variety of human cell types, and anti-BrdUrd can be used to identify DNA-synthesizing cells. Although in hyperproliferative epidermis the absolute numbers of BrdUrd-positive cells as well as Ki67-positive cells were grossly increased, the ratio of these values was not changed compared to the ratio found in the epidermis of the clinically uninvolved skin of psoriatic patients and in normal epidermis. This suggests an increased growth fraction in hyperproliferative epidermis.Our data show that immunohistochemical double-staining techniques can be a valuable tool in the study of cell cycle kinetics in epithelial tissues.     

Immunological demonstration of a calcium-unresponsive protein kinase C of the delta-type in different species and murine tissues. Predominance in epidermis

An antiserum raised against a delta-protein kinase C (delta-PKC)-specific peptide recognized the purified calcium-unresponsive 76-kDa protein kinase of porcine spleen in the native and the denatured form. This antiserum was used to demonstrate the delta-PKC-like enzyme in spleen of different species, in various cell types and in murine tissues by immunoblotting of the respective extracts. Due to species differences, delta-PKC-like kinases with slightly different molecular weights were observed. The enzyme was found to be present in primary murine keratinocytes, primary bovine endothelial cells, and many cell lines originating from human, rat, and murine tissues. It was present also in all murine tissues tested, predominantly in epidermis, uterus, placenta, lung, brain, spleen, and kidney. In contrast to the conventional alpha, beta, gamma-PKC, it was located almost exclusively in the particulate fraction. The delta-like PKC could be demonstrated in the epidermis and brain of newborn mice, and in both tissues its concentration increased dramatically between day 7 and 14 after birth. The delta-PKC-like kinase of mouse epidermis (p82-kinase) was down-regulated after topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA) to mouse skin. The amount of the enzyme decreased to less than 20% of the controls within 16 h and recovered almost completely within 72 h after TPA. The existence of the delta-PKC-like kinase in mouse skin, papillomas, and carcinomas could also be demonstrated by immunocytochemical staining of the respective sections. The enzyme was observed predominantly in epithelial layers. A remarkable immunostaining of nuclei in skin sections disappeared after TPA treatment of the animals.     

Papilloma formation in human foreskin xenografts after inoculation of human papillomavirus type 16 DNA.

A mouse model of high-risk human papillomavirus infection was developed in which human papillomavirus (HPV) type 16 DNA was inoculated into human foreskin grafted to the skin of severe combined immunodeficient (scid) mice. Grafted skin contained human epidermis and dermis and, like normal human skin, expressed involucrin in differentiating keratinocytes. HPV type 16 DNA, attached to gold particles, was delivered directly into human epidermal cells and induced exophytic papilloma with histologic features of papillomavirus infection, including koilocytosis and expression of papillomavirus capsid antigen. This model should be useful for determining in vivo the functions of viral genes and for developing strategies to prevent and treat HPV-associated disease. It may also be of value in developing animal models of other human skin diseases.     

Expression of the WE3 antigen in newt epithelium originating from subcutaneous grafts of skin

mAb WE3 recognizes an antigen that is developmentally regulated in the wound epithelium of regenerating newt limbs. The antigen is precociously expressed when pieces of WE3-negative wound epithelium are grafted subcutaneously (Tassava et al.: Recent Trends in Regeneration Research. New York: Plenum Publishing Co., pp. 37-49, 1989). In the present study, we investigated whether the WE3 antigen is expressed in epidermis of subcutaneous grafts of skin. Small pieces of limb skin were grafted into small tunnels in the lower jaw, limb, and tail, oriented either the same as (epidermis facing out) or opposite to (epidermis facing in) the orientation of the host skin. In most cases, the epithelium migrated from the graft along the wounded surface of the tunnel, closed onto itself, and formed a multilayered "emigrant" epithelium. Infrequently, the migrating epithelium combined with the wound epithelium of the insertion wound. In no case did the epithelium migrate over the cut edge of the grafted dermis. Reactivity to mAb WE3 was first seen at 4 days after grafting, when the migrating epithelium had almost closed over onto itself. By 6 days and thereafter, the entire emigrant epithelium was reactive to mAb WE3. While initially restricted to the emigrant epithelium, at 10 days after grafting and thereafter, reactivity was also seen in the epidermis that remained in contact with the dermis. Expression of the WE3 antigen was not influenced by the orientation of the graft nor by the graft site. The results show that, compared to amputated limbs, the epithelium originating from these grafts precociously expresses the WE3 antigen. Also, epidermis of grafted skin is capable of expressing the WE3 antigen.     

Indirect immunohistochemistry on skin biopsy for the detection of persistently infected cattle with bovine viral diarrhoea virus in Italian dairy herds

Indirect immunohistochemistry (IHC) on skin biopsies for identification of persistently infected (PI) animals has been used as a parallel test to antigen and antibody ELISAs in a bovine viral diarrhoea (BVD) voluntary control program. The aim was to evaluate the reliability and feasibility of IHC on ear skin tissues to detect PI animals in field conditions, including both adult and calves under 6 months of age. In animals over 6 months of age skin biopsy and blood sample were collected at the same time, whereas in young calves blood sampling was performed when animals reached 6 months of age. One hundred and sixty-five animals were tested and immunohistochemical results were compared with those of antigen ELISA. In case of inconclusive results virus isolation and virus neutralization assays were performed. Agreement K value was 0.96. Immunohistochemical staining in positive animals was clearly detectable in the keratinocytes of the epidermis and adnexa.     

Reconstructed human epidermis for skin absorption testing: results of the German prevalidation study

Exposure to chemicals absorbed by the skin can threaten human health. In order to standardise the predictive testing of percutaneous absorption for regulatory purposes, the OECD adopted guideline 428, which describes methods for assessing absorption by using human and animal skin. In this study, a protocol based on the OECD principles was developed and prevalidated by using reconstructed human epidermis (RHE). The permeation of the OECD standard compounds, caffeine and testosterone, through commercially available RHE models was compared to that of human epidermis and animal skin. In comparison to human epidermis, the permeation of the chemicals was overestimated when using RHE. The following ranking of the permeation coefficients for testosterone was obtained: SkinEthic > EpiDerm, EPISKIN > human epidermis, bovine udder skin, pig skin. The ranking for caffeine was: SkinEthic, EPISKIN > bovine udder skin, EpiDerm, pig skin, human epidermis. The inter-laboratory and intra-laboratory reproducibility was good. Long and variable lag times, which are a matter of concern when using human and pig skin, did not occur with RHE. Due to the successful transfer of the protocol, it is now in the validation process.     

Functional interactions of human and murine lymphoid cells. I. Analysis of primary and secondary responses

In this study human T-cell responses against murine alloantigens were analyzed. The results show that optimal primary responses are obtained from peripheral blood mononuclear cells only when murine splenic adherent cells (SAC) were used as antigen. Further analysis revealed that human T cells were able to respond directly to murine cells without the need for antigen reprocessing; however, human interleukin 1 (IL-1) was required for optimal stimulation. In contrast, secondary proliferative responses to murine cellular antigens could be induced from primed T cells even in the absence of SAC and/or IL-1. These proliferative responses, and in addition, cytotoxic T-cell responses, were specific for the priming antigen. Long-term human T-cell lines specific for murine alloantigens were found to replace the need for murine T cells in antigen-specific murine B-cell responses to sheep red blood cells. The mechanism of help delivered by the human T cells appeared to be by the release of nonspecific helper-T-cell factors. The evidence presented for this is the inability of these cells to stimulate cells from mice that express the X chromosome B-cell defect xid.     

Comparative evaluation of CD8+CTL responses following gene gun immunization targeting the skin with intracutaneous injection of antigen-transduced dendritic cells

The skin is an attractive target for antigen-specific vaccination. Particle bombardment of the epidermis with plasmid DNA using the gene gun results in antigen expression in keratinocytes of the epidermis leading to antigen presentation in the draining lymph nodes by migratory dendritic cells (DC). In order to better understand the role of the skin in stimulating antigen-specific CD8+cytotoxic T cells (CTL), we compared gene gun immunization with intracutaneous injections of antigen-transduced DC. A single intracutaneous injection of antigen-transduced DC was able to induce in vivo expansion of CD8+CTL specific for the model antigen chicken ovalbumin while four simultaneous shots with the gene gun were not effective. Antigen-transduced DC were much more efficient than particle bombardment of the epidermis in stimulating adoptively transferred TCR-transgenic CD8+CTL in the draining lymph nodes. Employing the novel technique of in vivo bioluminescence imaging, we demonstrated efficient gene transfer to the skin following gene gun bombardment and confirmed that a similar amount of antigen reached the lymph node when compared with injection of antigen-transduced DC. Our results suggest that direct transfection of the skin does not optimally reach and activate appropriate antigen-presenting DC. We believe that this reflects the immunological function of the epidermis which must balance immunity and tolerance to foreign antigens. Further investigations will have to address the role of Langerhans cells for the activation of cellular immunity in the skin.     

Effects of fibroblasts of different origin on long term maintenance of xenotransplanted human epidermal kerationocytes in immunodeficient mice

We examined effects of fibroblasts of different origin on long-term maintenance of xenotransplanted human epidermal keratinocytes. A suspension of cultured epidermal cells, originating from adult human trunk skin, was injected into double mutant immunodeficient (BALB/c nu/scid) mice subcutaneously, with or without cultured fibroblastic cells of different origin. At one week after transplantation, the epidermal cells generated epidermoid cysts consisting of human epidermis-like tissue. When the epidermal cells were injected alone or together with fibroblastic cells derived from human bone marrow, muscle fascia, or murine dermis, organized epidermoid cysts regressed within 6 weeks. In contrast, when the epidermal cells were injected together with human dermal fibroblasts, generated epidermoid cysts were maintained in vivo for more than 24 weeks. Histological examination showed that the reorganized epidermis, after injection of both epidermal keratinocytes and dermal fibroblasts, retained normal structures of the original epidermis during 6 to 24 weeks after transplantation. The results indicate that human dermal fibroblasts facilitate the long-term maintenance of the reorganized epidermis after xenotransplantation of cultured human epidermal keratinocytes by supporting self renewal of the human epidermal tissue in vivo.     

Fluorescence study on the conformation of a cyclic enkephalin analog in aqueous solution

Certain patients with ovarian germ cell tumors develop a specific antibody reacting with glycoprotein-bound large carbohydrates of murine teratocarcinoma cells. The antigenic determinant was found to involve an alpha-galactosyl residue, since alpha-galactosidase from coffee bean, but not other glycosidases abolished the antigenic activity of the large glycan isolated from F9 and OTT6050 cells. Several evidences excluded the possibility that the antigen is blood group B or P1 antigen. These results indicate tumor-associated expression of an unusual alpha-galactosyl residue in human ovarian germ cell tumors.