Human Skin Specimens Research Articles

Immunohistochemical distribution of aromatase and 3B-hydroxysteroid dehydrogenase in human hair follicle and sebaceous gland.

Human hair follicles (HF) and sebaceous glands (SG) were assessed for the presence and distribution of the cytochrome P-450-aromatase (AR) and 3B-hydroxysteroid dehydrogenase (3B-HSD) enzymes. Immunohistochemical methods were used to examine both enzymes in male and female human skin specimens at various ages and different body sites. AR was found in the external root sheath of anagen, terminal HF, and in SG, whereas the 3B-HSD was found only in the SG. AR was rarely found in telogen HF. The expression of both enzymes, AR and 3B-HSD, did not vary with body site or sex. Localizing AR in the external root sheath of anagen HF suggests that AR may have a function in the HF cycle. We hypothesize that AR may be one of many enzymes or factors that play a role in the HF cycle by regulating the level of androgens formed locally, whereas 3B-HSD is localized in SG, converting weak androgen precursors to potent androgens, stimulating lipogenesis.

Histological demonstration of initial lymphatics in human skin: comparison between different extension techniques

Initial lymphatic vessels (IL) are difficult to demonstrate histologically in excised normal skin, as they are usually completely collapsed. In this report three different methods (vacuum extension, large traction extension, and small traction extension) are described by which human skin specimens can be mechanically extended. After extension specimens were Epon enbedded and the IL counted and their diameters measured. The greatest number of lymphatic vessels was found in specimens extended by vacuum, and the smallest number in small specimens extended by traction. The highest density of lymphatic vessels was found 50-300 microns below the epidermis. Our data reveal that vacuum extension is useful for investigating questions concerning the topography of IL, whereas preparations extended by traction are more suitable for obtaining information about single lymphatic vessels.

Full-Thickness Human Skin Explants for Testing the Toxicitiy of Topically Applied Chemicals

This report describes a model organ-culture system for testing the toxicity of chemical substances that are topically applied to human skin. In this system, the viable keratinocytes in the full-thickness skin explants are protected by the same keratinized layer as skin remaining on the donor, and toxicity can be assessed microscopically and/or biochemically. The human skin specimens were discards from a variety of surgical procedures. They were cut into full-thickness 1.0-cm2 explants, and briefly exposed to the military vesicant sulfur mustard (SM), which was used as a model toxicant. The explants were then organ cultured in small Petri dishes for 24 h at 36 degrees C. In the 0.03-1.0% dosage range, a straight-line dose-response relationship occurred between the concentration of SM applied and the number of paranuclear vacuoles seen histologically in the epidermis. Within the same SM dosage range, there was also a proportional decrease in 14C-leucine incorporation by the explants. Thus, the number of paranuclear vacuoles reflected decreases in protein synthesis by the injured epidermal cells. The epidermis of full-thickness untreated (control) human skin explants usually remained viable for 7 d when stored at 4 degrees C in culture medium. During storage, a relatively small number of paranuclear vacuoles developed within the epidermis, but the explants were still quite satisfactory for testing SM toxicity. Incubation (for 4 or 24 h at 36 degrees C) of such control skin explants reduced (often by 50%) the small number of paranuclear vacuoles produced during 4-7 d of storage. This reduction was probably caused by autolysis of many of the vacuolated cells. Two types of paranuclear vacuoles could be identified by both light and electron microscopy: a storage type and a toxicant type. The storage type seemed to be caused by autolysis of cell components. The toxicant type seemed to be caused by an invagination of the plasma membrane. Only toxicant-type vacuoles increased appreciably in number when skin explants were exposed to mustard, and to other toxicants.

Identification of a 160-kD Molecule as a Component of the Basement Membrane Zone and as a Minor Bullous Pemphigoid Antigen

The antigens in normal human skin defined by antibodies in patients with bullous pemphigoid (BP) were studied by Western immunoblots. Eighteen (90%) of 20 BP sera reacted to a 230-kD antigen. Seven (35%) of the sera reacted to a 160-kD antigen. Two of these reacted only to the 160-kD antigen and five also reacted to the 230-kD antigen. Antibodies to the 160-kD antigen were not present in 25 control sera obtained from normal individuals or patients with other bullous diseases. The 160-kD antigen was present in epidermal extracts of four different specimens of normal human skin but not in dermal extracts or extracts of control cells including melanoma, fibroblasts, lung carcinoma, and colon carcinoma. Monospecific sera with antibodies to either the 230-kD or to the 160-kD antigen reacted solely to their respective target antigens, but not to both, in extracts of epidermis that contained both antigens. The 160-kD antigen broke down to a 140-kD fragment, while the 230-kD antigen was unchanged in the absence of protease inhibitors. Western blot affinity purified antibody to the 160-kD antigen bound only to the basement membrane zone on the epidermal side of 1M NaCl split skin. These results indicate that a 160-kD antigen is a normal component of the basement membrane zone of human skin. The antigen is located on the epidermal side of skin split with 1M NaCl. It is a minor BP antigen, antibodies to which are present in some patients with BP.

End-stage renal disease and diabetes catalyze the formation of a pentose-derived crosslink from aging human collagen.

Structure elucidation of a specific fluorophore from the aging extracellular matrix revealed the presence of a protein crosslink formed through nonenzymatic glycosylation of lysine and arginine residues. The unexpected finding that a pentose instead of a hexose is involved in the crosslinking process suggested that the crosslink, named pentosidine, might provide insight into abnormalities of pentose metabolism in aging and disease. This hypothesis was investigated by quantitating pentosidine in hydrolysates of 103 human skin specimens obtained randomly at autopsy. Pentosidine level was found to increase exponentially from 5 to 75 pmol/mg collagen over lifespan (r = 0.86, P less than 0.001). A three- to tenfold increase was noted in insulin-dependent diabetic and nondiabetic subjects with severe end-stage renal disease requiring hemodialysis (P less than 0.001). Moderately elevated levels were also noted in some very old subjects, some subjects with non-insulin dependent diabetes, and two subjects with cystic fibrosis and diabetes. The cause of the abnormal pentose metabolism in these conditions is unknown but may relate to hemolysis, impaired pentose excretion, cellular stress, and accelerated breakdown of ribonucleotides. Thus, pentosidine emerges as a useful tool for assessment of previously unrecognized disorders of pentose metabolism in aging and disease. Its presence in red blood cells and plasma proteins suggests that it might be used as a measure of integrated pentosemia in analogy to glycohemoglobin for the assessment of cumulative glycemia.

Laser light transmission and laser Doppler blood flow measurements on human, rat and guinea pig cochlea

In order to test the applicability of laser-Doppler flowmetry in monitoring cochlear blood flow clinically, the thickness and the helium-neon laser light transmission of specimens of human, rat and guinea pig promontory bone and human skin were determined. Furthermore, comparative laser-Doppler measurements were taken from the promontory in patients, rats and guinea pigs. Due to the different thicknesses of the promontory bone in different species, the light transmission was found to be considerably higher for the animal cochlea (rat, 15%; guinea pig, 6.6%) than the human cochlea (1.7%). However, a clearly higher laser-Doppler signal was recorded from both the human and the rat cochleas as compared with the guinea pig. The relative laser light attenuation by the human skin specimens corresponded to that of the human promontory bone. The findings are discussed with regard to the suitability of the laser-Doppler method for blood flow measurements in the human cochlea.

Influence of absorbing and scattering media on the propagation of ultrasound

The preservation of the sound field produced by a single element ultrasound transducer was investigated for the assessment of the applicability of diffraction correction strategies. A variety of purely absorbing and absorbing plus scattering phantoms was employed. The acoustic parameters (velocity of sound, frequency-dependent attenuation coefficient, and differential backscattering coefficient) were estimated for these phantoms and compared to the parameters of human liver and skin specimens. The focal beam properties were estimated with a small diamond reflector positioned beyond the investigated media. The changes of the beam characteristics in the phantoms could be explained completely by the overall attenuation, i.e., either by absorption exclusively or by absorption plus scattering. In liver tissue, the averaged beam broadening could be explained by the attenuation, but great and irregular distortions of the sound beam were observed. The distortions by skin, subcutaneous fat, and muscle layers were even more severe. An important conclusion from the present study is, therefore, that diffraction correction strategies should be considered as a first-order approximation when applied to in vivo diagnostic procedures.

Interleukin 1 alpha and beta in psoriatic skin: enzymoimmunoassay, immunoblot studies and effect of systemic retinoids.

Interleukin 1 alpha (IL-1 alpha) and interleukin 1 beta (IL-1 beta) proteins were studied by enzymoimmunoassay (EIA) and Immunoblot analysis in the 10,000 g supernatant of normal and psoriatic (lesional and nonlesional) human skin specimens. By EIA IL-1 alpha was the principal form detected in all the specimens, which contrasts with the predominance of IL-1 beta in human blood monocytes. In psoriatic plaques relatively less IL-1 alpha and more IL-1 beta were detected. On Immunoblot analysis the mature form (17 kD) was not detected in normal skin, which showed only 52-kD immunoreactive forms. In contrast the 17-kD form was found in psoriatic skin. This indicates either a distinct processing of IL-1 molecules or a contribution of inflammatory cells infiltration to the IL-1 pool in psoriatic plaques. During systemic retinoids therapy the amount of both IL-1 species decreased in lesional and nonlesional psoriatic skin.

Demonstration of antigen-specific T cells and histopathological alterations in mice experimentally inoculated with Borrelia burgdorferi

Antigen-specific T-cell responses and histopathological changes were studied in mice experimentally inoculated with Borrelia burgdorferi B31. Inbred mice with different H-2 haplotypes and/or different genetic backgrounds were inoculated with B. burgdorferi organisms and tested for antigen-specific T-cell responses in vivo (delayed-type hypersensitivity [DTH]) and in vitro (T-cell proliferation). Comparable DTH responses were found after inoculation with either inactivated (in the presence of adjuvants) or viable microorganisms in all mouse strains, except BALB/c, irrespective of the H-2 haplotype (b, d, k, or s) tested and the sex of the animals. Moreover, in mice presensitized to B. burgdorferi, DTH responses could be induced only with antigen preparations derived from the corresponding strain but not with those obtained from either related spirochetes such as Treponema phagedenis and Leptospira interrogans or unrelated bacteria such as Mycobacterium tuberculosis. T cells isolated from lymph nodes or spleens of mice previously sensitized to B. burgdorferi but not those from naïve mice could be induced for antigen-specific proliferation in vitro, as revealed by [3H]thymidine incorporation. Histopathological examination of mice inoculated with viable B. burgdorferi organisms revealed significant perivascular infiltrates consisting mainly of mononuclear and a few polymorphonuclear leukocytes in different organs (brain, heart, lungs, liver, and kidneys) and the appearance of giant multinucleated cells within the spleen similar to those found in human skin specimens of patients suffering from cutaneous manifestations of Lyme disease. Our findings suggest that mice are a suitable animal model with which to study the immune response to B. burgdorferi and the pathogenesis of Lyme disease.

Bullous pemphigoid antigen concentration in normal human skin in relation to body area and age.

The amount of bullous pemphigoid antigen (BPA) in normal human skin on different areas of the body and in bodies of different ages was determined by making endpoint titer estimations using the indirect immunofluorescence technique. Two sera with bullous pemphigoid antibodies were tested with 36 specimens of normal human skin from six cadavers. The greatest expression of BPA was found in plantar sites, whereas the lowest endpoint titers were seen in flexural arm biopsy specimens. Age-dependent differences in BPA expression were detected as well. The oldest individuals tested produced the highest endpoint titers in contrast to the lowest endpoint titers in the youngest. This study suggests that the amount of BPA depends not only on the body region where the biopsy specimen is taken but also on the age of the donor. This is important to know in connection with indirect immunofluorescence assays for BP antibodies on human skin.

Short-term skin preservation at 4 degrees C: skin storage configuration and tissue-to-volume medium ratio.

This study was designed to examine the effect of the storage configuration of skin and the ratio of tissue-to-storage medium on the viability of skin stored under refrigeration. Human skin specimens were stored in four physical configurations in RPMI 1640 tissue culture media at 4 degrees C. Skin was transferred to surgically created defects on nude mice after specific storage intervals. Grafts were examined grossly and microscopically after ten days. In the rolled configuration, on storage day 15, the viability of the outside of the roll was significantly better than the inside (P less than 0.01). The graft viability of the outside of the skin rolls was similar for both tissue-to-media ratios as well as for both free-floating configurations (P = 0.27). These findings suggest the optimum cold storage configuration is free floating, and 300 cm2/100 mL is an appropriate skin surface area to volume media ratio. This proportion of tissue to media is in agreement with the minimum ratio currently recommended by the Skin Council of the American Association of Tissue Banks.

Filaggrin expression in normal and pathological skin. A marker of keratinocyte differentiation.

Filaggrin is a basic, histidine-rich protein synthesized by cells of keratinizing epithelia, that subserves major physiological functions in maturating epidermis. Its precursor, profilaggrin, constitutes a major component of kertohyalin granules. In this work the expression of profilaggrin/filaggrin was studied in 140 specimens of normal human adult and fetal skin, cultured epithelia before and after allo- and xeno-grafting, and several cases of keratinization disorders and tumours. An avidin-biotin-alkaline phosphatase technique was applied on deparaffinized tissue sections, by using a specific monoclonal antibody (AKH1) to profilaggrin/filaggrin. The results show that the expression of profilaggrin/filaggrin is variously altered in keratinization disorders, whereas in epithelial proliferations it seems to decrease with an increasing degree of dedifferentiation of the tumour. Therefore the expression of these antigens can be considered to be a sensitive marker of maturation of normal epidermis and provides a new tool for the study of differentiation of the surface epithelium in skin diseases.

Heterogeneity of Pemphigus Vulgaris Antigens

The immunologic specificity of pemphigus vulgaris (PV) antigens was studied by serologic analysis. Serum samples of 19 patients with PV antibodies were tested for reactivity with seven specimens of normal human skin by indirect immunofluorescence. Some PV antibodies reacted to antigens expressed in all specimens of skin, while others reacted to antigens expressed in only some specimens of skin. This was not due to differences in the titer of PV antibodies among serum samples or to regional variation or individual-to-individual fluctuations in the expression of a single PV antigen. The patterns of cross-reactivity suggest that PV antigens are heterogeneous. Two different groups can be defined serologically: (1) a "major" (PV) antigen expressed in all individuals, antibodies to which are the most common in PV; (2) a family of "minor" PV antigens whose expression varies from individual to individual. Several immunologically distinct PV antigens are expressed concurrently in the same specimen of skin.

A simple in vitro technique for studies on induction of class II transplantation antigens on keratinocytes

A new technique is described for evaluation of the induction and kinetics of class II transplantation antigens on keratinocytes. By culturing small rat or human skin specimens for 2-5 days in media containing gamma-interferon, class II antigens were detected on keratinocytes by immunohistochemistry. This technique is rapid and technically simple compared to keratinocyte cultures and raises the possibility of studying other cells in the skin.

Formalin sensitivity and differential staining of mast cells in human dermis

The characteristics of the human dermal mast cell population with respect to formalin fixation sensitivity, toluidine blue staining and alcian blue/safranin staining were studied. Thirty-seven specimens of normal human skin were bisected. One half was fixed in 10% neutral buffered formalin and the other in Carnoy's fixative. Sections were cut and stained with either toluidine blue or alcian blue/safranin. Significantly more mast cells were visualized with alcian blue/safranin than with toluidine blue. With both stains, only approximately 50% of the mast cells observed in the Carnoy's fixed tissue could be visualized in the formalin-fixed tissue. Alcian blue/safranin staining revealed three patterns of mast cell granule staining: mast cells containing only alcian blue-positive granules, mast cells containing only safranin-positive granules, and mast cells containing a mixture of alcian blue-positive granules and safranin-positive granules. Mast cells containing only alcian blue-positive granules constituted the majority of the dermal mast cell population and 73% of these mast cells were formalin-sensitive. Mast cells containing only safranin-positive granules and those containing a mixture of alcian blue-positive granules and safranin-positive granules showed no evidence of formalin sensitivity. The human dermal mast cell population, therefore, displays heterogeneity with respect to formalin fixation sensitivity and alcian blue/safranin staining. Dermal mast cells were visualized in significantly greater numbers in skin from the head compared with that from the body or limbs.

Apoptotic Keratin Bodies as Autoantigen Causing the Production of IgM-Anti-Keratin Intermediate Filament Autoantibodies

The presence of numerous keratin bodies in the upper dermis is a characteristic finding in skin lesions of patients with various dermatoses such as cutaneous graft-versus-host disease, lichen planus, or chronic discoid lupus erythematosus. These keratin bodies are generated by apoptotic keratinocyte death, consist largely of keratin intermediate filaments (KIF), and are constantly covered with immunoglobulins, mainly IgM. Apoptosis is also thought to occur under physiologic conditions in the skin as it does in other organs, but keratin bodies are not frequently reported as being found in nonlesional skin. In order to assess the frequency of keratin bodies in normal skin, we examined serial sections of 10 normal human skin specimens and 5 dermal sheets prepared from normal human skin for the presence of keratin bodies. They were visualized by direct immunofluorescence using a fluorescein isothiocyanate (FITC) rabbit antihuman IgM conjugate. In addition the KIF origin of keratin bodies was demonstrated by a double-staining immunofluorescence procedure using a FITC-conjugated rabbit antihuman IgM followed by a mouse monoclonal antibody against keratin and a sheep antimouse immunoglobulin conjugated with Texas Red. One specimen was also examined for keratin bodies at the ultrastructural level. In serial sections, all 10 normal human skin specimens had numerous keratin bodies as assessed by visualization of globular IgM deposits. Evaluated on dermal sheets, the number of keratin bodies ranged from 39-262 per mm2. Nearly all keratin bodies also stained with the antikeratin antibodies. Ultrastructurally the remarkable number of keratin bodies, which consist of filaments measuring approximately 10 nm in diameter or of more granular material, in normal human skin was confirmed. In order to investigate the capacity of KIF material in keratin bodies to function as autoantigen, we examined the sera of the 10 skin donors and, in addition, of 30 normal healthy individuals and 10 patients with rheumatoid arthritis for the occurrence and specificity of IgM-anti-KIF autoantibodies by an enzyme-linked immunosorbent assay and by immunoblot. IgM-anti-KIF autoantibodies were found in all 50 test sera. In the majority of the sera the specificity of these autoantibodies included the 51 kD and the 58 kD KIF protein, which are constituents of KIF in keratin bodies and basal keratinocytes. Quantitatively, the antibody activity of the IgM-anti-KIF autoantibodies varied from serum to serum, being highest in the sera of patients with rheumatoid arthritis.(ABSTRACT TRUNCATED AT 400 WORDS)

Loss of allogeneic T-cell activating ability and Langerhans cell markers in human epidermal cell cultures

Human Langerhans cells are the only epidermal cells that express the T6 and HLA-DR antigens and are responsible for the in vitro allogeneic T-cell proliferative responses in the mixed skin cell lymphocyte reaction (MSLR). To investigate the presence of Langerhans cells in normal human epidermal cell cultures, epidermal cell suspensions obtained from normal human skin specimens and from the subsequent epidermal cell cultures were analyzed by indirect immunofluorescence for the presence of T6 and HLA-DR determinants. In parallel, MSLRs were conducted with suspensions of cultured epidermal cells as stimulatory cells. These studies present evidence that when human epidermal cells are grown in culture, they loose both the ability to stimulate the proliferation of allogeneic T lymphocytes in vitro and their expression of HLA-DR and T6 antigens. The T6 antigens were lost during the first 2 weeks of culture, while HLA-DR determinants were still expressed by a small number of cells and were progressively lost through duration of cultures. The loss of HLA-DR antigens closely paralleled the progressive inability of human epidermal cells in culture to stimulate allogeneic T cells in MSLR.

Distribution of ATPase-positive Langerhans cells in normal adult human skin

The distribution of ATPase-positive Langerhans cells (LC) was investigated in 117 specimens of normal adult human skin and mucosa taken from different areas of the body. Although there were significant variations in the numbers of LC in each area examined, skin from the face and neck contained the highest density of cells (976 +/- 30.93/mm2). The densities of LC in trunk skin (740 +/- 28.97/mm2), scalp (693 +/- 69.56/mm2) and arm or leg skin (640 +/- 40.95/mm2) were similar. Buccal mucosa had significantly fewer LC (567 +/- 42.94/mm2) than trunk skin, and sacrococcyx skin and palm and sole skin displayed the smallest number of these cells (267 +/- 56.14/mm2 and, 189 +/- 19.15/mm2 respectively). No ATPase-positive LC were detected in the centre of two corneal specimens.

Qualitative and Quantitative Analysis of Cytosol Retinoid Binding Proteins in Human Skin

The distribution of the cytosol retinol and retinoic acid binding proteins are known to vary greatly within the different layers of the eye, a retinoid target organ. We have analyzed the cytosol retinoid binding from adult human skin, another retinoid target organ, and examined the relative contribution of the epidermis and dermis to the total retinoid binding. The mean specific activity of [3H]retinol (0.52 +/- 0.06 pmol/mg protein) and [3H]retinoic acid (3.20 +/- 0.45 pmol/mg protein) binding to cytosol preparations from different specimens of adult human skin was determined. On the average these skins bound 7-fold more retinoic acid than retinol. When skin was treated with EDTA and separated into epidermal and dermal fractions, [3H]retinol and [3H]retinoic acid binding was found in the cytosol derived from epidermis (0.36 +/- 0.03 pmol/mg protein, 3.69 +/- 0.13 pmol/mg protein, respectively) but not from dermis. To confirm that the absence of dermal binding was not due to loss during the EDTA separation, we assayed skin keratomed at 0.1, 0.2, and 0.3 mm. The skin obtained at 0.1 mm was upper epidermis and exhibited binding for both retinol and retinoid acid. The 0.2 mm skin, which added lower epidermis but little dermal contamination, had higher specific activities for both retinol and retinoic acid binding. The 0.3 mm skin which added primarily dermis, had lower specific activities for binding both retinoids. This is consistent with the concept that the epidermis is responsible for the majority of retinoid binding in adult human skin obtained from the lower limb.

Immunohistological demonstration of S-100 protein in the cutaneous nervous system.

S-100 protein, isolated from mammalian brain, has widely been used for immunohistochemical marker of the glia cells and the cells derived from the neural crest. In the present study, we made anti S-100 protein antibody and studied the immunoreactive distribution of S-100 protein in the cutaneous nervous system. Albino rabbits were immunized with S-100 protein and complete Freund adjuvant, and antiserum was purified by ion-exchange chromatography. Formalin-fixed normal human skin and sciatic nerve of rat were examined by the PAP method. S-100 protein was detected in Schwann cells of sciatic nerve of rat and cutaneous nerve bundles of human skin specimens. Meissner corpuscles and inner core cells of Pacinian corpuscles of human skin were S-100 protein positive. These findings suggest that the staining of S-100 protein with PAP method is a simple and reliable method to demonstrate the cutaneous nervous system. Also, lamellar cells of Meissner corpuscles and inner core cells of Pacinian corpuscles are indicated to be Schwann cell origin.