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E-cadherin is a calcium-sensitive, cell-to-cell, adhesion molecule that is expressed widely in normal human epithelial tissue. Abnormal expression has been described in colorectal, breast and nasopharyngeal squamous cell carcinomas, where loss of E-cadherin is associated with an increased metastatic potential. We have examined, by standard immunohistochemical techniques using the monoclonal antibody HECD-1 (E-cadherin monoclonal antibody), the distribution of E-cadherin in normal human skin and in non-melanoma neoplastic lesions. In the normal epidermis, E-cadherin was strongly expressed on the surface of keratinocytes and specialized epithelial structures. Staining was absent from the lower pole of basal keratinocytes in contact with the basement membrane. Weak cytoplasmic staining was also noted in basal keratinocytes. No reactivity was demonstrated in dermal structures. The assessment of cutaneous tumours demonstrated an altered pattern of staining in most cases. Cell surface expression was reduced in 28 of 30 cases of basal cell carcinomas (BCC). Twenty showed an additional feature of positive staining on the dermal aspect of peripheral cells of tumour lobules. In squamous cell carcinomas (SCC) (n = 16), surface expression was attenuated in eight and absent in a further four. Strong surface expression, similar to normal skin was seen in all examples of Bowen's disease (n = 6), viral wart (n = 3), seborrhoeic keratosis (n = 3) and actinic keratosis (n = 4). This study demonstrates that, in BCC and SCC, but not in premalignant lesions, cell-surface expression of E-cadherin is reduced, consistent with the observation that the loss of E-cadherin is associated with tumour invasion.

Tumour necrosis factor alpha (TNF-alpha) is a potent immunoregulatory cytokine produced by many cutaneous cells, including keratinocytes, mast cells and Langerhans cells. To explore its potential role in inflammatory skin disease, we have studied immunohistochemically the effects of intradermal recombinant human TNF-alpha (rHuTNF-alpha) on cutaneous inflammatory cells, adhesion molecules and Langerhans cells in normal human skin. Volunteers receive rHuTNF-alpha 100 U (group A), 5000 U (group B), or 100 U daily for 5 days (group C), and biopsies were taken at 6 h (groups A and B), or 6 h after the final injection (group C). An inflammatory cell infiltrate developed in all cases: following single injections of either 100 or 5000 U rHuTNF-alpha this was predominantly neutrophilic, whereas following multiple injections of 100 U few neutrophils were seen, although many lymphocytes (CD3+, CD4+) were present. In all groups there was an increase in cells of monocyte/macrophage lineage (CD36+). TNF-alpha induced a dose- and time-dependent decrease in CD1a+ epidermal Langerhans cell numbers and an increase in dermal CD1a+ cells, suggesting migration of Langerhans cells away from the epidermis. TNF-alpha induced endothelial E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in all groups, and adhesion molecule expression by interstitial dermal dendritic cells (ICAM-1 and VCAM-1) and keratinocytes (ICAM-1) was observed. These findings indicate that TNF-alpha is a potent modulator of cutaneous immune function in vivo, and this central role in the cutaneous immune response suggests that TNF-alpha may be an attractive target for therapeutic inhibition.

Cellular adhesion molecules play a central role in leucocyte migration through peripheral blood and tissues. A crucial stage in these events in selectin-mediated adhesion involving E-selectin expressed on activated endothelium interacting with a range of carbohydrate ligands expressed by specific subpopulations of leucocytes. As such mechanisms may be relevant to bone marrow-derived dendritic epidermal Langerhans' cell (LC) migration, expression of these carbohydrate ligands was assessed immunocytochemically in whole skin biopsies and in epidermal cell suspensions obtained from adult humans. Double-labelling experiments revealed that sialyl Lewis x, recognized by the monoclonal antibody CSLEX1, was expressed on epidermal LC (n = 9). Furthermore, expression was enhanced at 24 hr following epicutaneous application of antigen and in the inflammatory disorder psoriasis (n = 10). E-selectin was concomitantly strongly expressed on dermal endothelium in psoriasis and allergic contact dermatitis. Intradermal injection of the T-cell-derived cytokine interferon-gamma (IFN-gamma) led to increased LC expression of sialyl Lewis x. In epidermal cell suspensions, in contrast to keratinocytes, CD1a+ cells expressed sialyl Lewis x, intensity of which was enhanced after 4 days in culture. CSLEX1 staining could be abolished and CD15 (non-sialated Lewis x) expression induced by saponification and treatment with neuraminidase. Expression of other selectin ligands was also examined. While the cutaneous lymphocyte antigen defined by the monoclonal antibody HECA-452 reacted with a small minority of LC, sialyl Lewis a and sulphatide were not expressed under any experimental conditions. These studies indicate that E-selectin-sialyl Lewis x interactions are potentially important in LC migration, both into and out of skin.

Inflammatory cell infiltrates and cell adhesion molecule expression have been examined in normal human skin after intradermal injection of sensory neuropeptides substance P (n = 6), vasoactive intestinal polypeptide (n = 6), and calcitonin gene-related peptide (n = 6) together with PBS as control (n = 4). Each neuropeptide induced rapid, time-dependent neutrophil influx into dermis, which was initially observed at 15 min and persisted for 8 h after injection. Increases in numbers of neutrophils with time after substance P, vasoactive intestinal polypeptide and calcitonin gene-related peptide were highly significant when compared with controls p < 0.005, p < 0.005, p < 0.005, respectively (analysis of variance). Substance P additionally induced marked eosinophilic accumulation at 4 and 8 h in four of six subjects. These changes paralleled rapid translocation of P-selectin from cytoplasmic Weibel-Palade granules to luminal membranes by 15 min, and significant up-regulation of E-selectin expression at 4 and 8 h. Increases in percentage of E-selectin positive vessels with respect to time after each neuropeptide were highly significant when compared with controls, p < 0.005, p < 0.005, p < 0.005 (ANOVA), respectively, and were significantly correlated with neutrophil infiltrates, r = 0.55, p < 0.001. VCAM-1 was not expressed, and constitutive ICAM-1 expression on dermal endothelium was unchanged at all time points examined (0-8 h). Induction of endothelial adhesion molecule expression by neuropeptides provides a mechanism for neutrophil accumulation in neurogenic inflammation. Substance P-induced eosinophil accumulation in the absence of VCAM-1 expression suggests that mechanisms distinct from VCAM-1/very late antigen-4 binding mediate selective tissue eosinophilia.

Background: Vascular cell adhesion molecule-1 (VCAM-1) is an endothelial protein with adhesive properties for inflammatory cells including lymphocytes. Its role in skin disease and regulation in vivo are uncertain., Objective: Our purpose was to determine expression of VCAM-1 in normal and inflamed skin and the effect on this of the T-cell-derived cytokine interferon gamma (IFN-gamma)., Methods: VCAM-1 was detected immunohistochemically in frozen-section biopsy specimens of inflammatory dermatoses and skin tumors. Volunteers received intradermal IFN-gamma and underwent biopsy 2 hours to 6 days later., Results: In normal skin, VCAM-1 was present on perivascular dendritic cells and some follicular keratinocytes. In allergic contact dermatitis, atopic dermatitis, and psoriasis, VCAM-1 was variably upregulated on dermal endothelium and dendritic cells, but was most pronounced in lichen planus. IFN-gamma led to marked upregulation of endothelial and dermal dendritic cell VCAM-1., Conclusion: VCAM-1 may be involved in the pathogenesis of various skin diseases and in vivo, IFN-gamma is a potent modulator of its expression.

Recombinant human interferon-gamma was administered intradermally (10 micrograms in 0.1 ml) to healthy adult human volunteers from day 1 to day 3, and epidermal cell proliferation was measured on whole skin biopsies at day 6. Three independent parameters were assessed, namely, a) epidermal keratin-16 expression, b) keratinocyte proliferating cell nuclear antigen expression, and c) keratinocyte silver nucleolar organizer region counts. Significantly increased scores for each parameter were observed after interferon-gamma injection (P < 0.01 in each case) compared to site-matched controls. Keratin-16 expression was confined to suprabasal epidermis, whereas proliferating cell nuclear antigen and silver nucleolar organizer region counts were particularly elevated in the basal epidermis. Taken together with previous findings, these studies indicate both proinflammatory and growth regulatory roles for interferon-gamma in human skin. These data are likely to be of particular importance to pathophysiological mechanisms of psoriasis and related cutaneous inflammatory diseases.

We have examined the expression of three endothelial adhesion molecules (intercellular adhesion molecule-1 [ICAM-1], endothelial leukocyte adhesion molecule-1 [ELAM-1], and vascular cell adhesion molecule-1 [VCAM-1]) in normal human skin following intradermal injection of stratum corneum-derived interleukin-1 alpha (SCIL-1 alpha). In control skin, constitutive expression of ICAM-1 was found on endothelial cells and at low levels on dermal dendritic cells but not on keratinocytes. ELAM-1 and VCAM-1 were present in low levels on endothelium and perivascular dendritic cells, respectively. SCIL-1 alpha injection produced marked endothelial ELAM-1 upregulation. Double staining with neutrophil elastase demonstrated that many ELAM-1-positive vessels contained marginating neutrophils and that interstitial neutrophils were clustered around ELAM-1-positive vessels. An increase in dermal dendritic cell ICAM-1 expression occurred and in two of three biopsies there was keratinocyte expression of ICAM-1 in the SCIL-1 alpha-injected tissue. Also, there was upregulation of VCAM-1 on vascular endothelium and an increase in the dermal dendritic cell expression of this molecule. These results give in vivo confirmation that SCIL-1 alpha modulated endothelial and dermal dendritic cell adhesion molecule expression, and show that endothelial VCAM-1 is regulated in vivo by SCIL-1 alpha, thus providing a regulatable ICAM-1-independent means of mononuclear cell recruitment.

Objective: To describe the association between epithelial cell IgM, which has previously been associated with an increased incidence of loss of renal graft in children, with a novel cutaneous eruption and unexplained native renal disease., Design: Observational study on children with epithelial cell antibody presenting with unexplained renal or skin disease., Setting: General paediatric department and regional paediatric nephrology unit., Patients: Six children (five girls, one boy), who presented to the unit in 1989-90., Results: Three children, two of whom had a history of a hyperpigmented rash, presented with hypertension, proteinuria, and impaired renal function. Renal biopsy specimens from two of these children showed severe arteriolar endothelial cell swelling with arteriolar occlusion. These children fully recovered after treatment with antihypertensive drugs. The third child developed end stage renal failure and required dialysis. Three other children presented with an unusual cutaneous eruption but no evidence of renal disease. Histology of the skin lesions showed acute epidermal necrosis and features consistent with a viral infection., Conclusions: The aetiology and pathogenesis of the epithelial cell antibody are unknown. These cases indicate that it may have a role in native kidney disease and focal epidermal necrosis. Clinical and histological features suggest that the antibody may be associated with a viral infection.

Gamma glutamyl transpeptidase (GGT) is an enzyme expressed by some epithelial neoplasms but not normal interfollicular epidermis. In order to examine the relationship between malignant change and de-differentiation we studied histochemically the expression of GGT in human foetal skin, various inflammatory dermatoses and epidermal neoplasms. In foetal skin GGT was detectable after 7 weeks' gestation, reached a maximum at 11 weeks and was undetectable by 24 weeks. It was expressed strongly by squamous cell carcinoma and focally in Bowen's disease and actinic keratoses. There was no GGT expression in basal cell carcinoma or most benign skin tumours, but keratoacanthomas were weakly positive. Keratinocytes in the vicinity of malignant melanocytes also expressed GGT. This study suggests that GGT expression, while not a simple marker of malignancy, may represent reversion to a less differentiated or 'foetal' phenotype.

A number of double immunocytochemical labeling techniques are available that allow simultaneous identification of two antigens in one tissue section. Those most commonly used are (a) the acid elution method, which uses acid buffer to remove one set of antibodies, thus allowing a second set to be applied; (b) the use of noncross-reacting combinations of immunoglobulin subclass and subclass-specific antibodies; and (c) application of the diaminobenzidine (DAB) reaction product as an antibody-blocking reagent, preventing cross-reaction of one set of antibodies with those of a second labeling method. We assessed each technique, using a variety of immunoenzyme methods, for the ability to label antigens that are anatomically separate in one tissue section and those that are colocalized on the same cell surface. Acid elution methods increased antibody cross-reaction. The use of immunoglobulin subclass antibodies resulted in inferior staining. The DAB blocking method, using peroxidase antiperoxidase (DAB substrate) and alkaline phosphatase antialkaline phosphatase (APAAP) (fast red substrate) optimally labeled non colocalized antigens. The substitution of fast blue as the APAAP substrate proved optimal for colocalized antigens, producing brown and blue single-labeled and black double-labeled cells.

Endothelial leucocyte adhesion molecule-1 (ELAM-1) is a recently described endothelial surface glycoprotein which is inducible by interleukin 1 (IL-1), tumour necrosis factor-alpha (TNF-alpha) or bacterial lipopolysaccharide (LPS). Using an immunohistochemical technique and a monoclonal antibody (1.2B6) specific for ELAM-1 we have found marked vascular endothelial expression of ELAM-1 in many cutaneous inflammatory disorders, including allergic contact dermatitis, atopic dermatitis and psoriasis, and in dermal infiltrates associated with benign, premalignant and malignant keratinocyte proliferation. In normal skin, minimal levels of ELAM-1 expression were detected. In psoriasis, double-immunoenzyme staining studies revealed a close spatial relationship between ELAM-1 expression and neutrophil margination, suggesting a functional link. Recombinant human interferon-gamma (30 micrograms) injected intradermally in normal adult human volunteers did not substantially upregulate ELAM-1 in contrast to its marked effect on intercellular adhesion molecule-1 (ICAM-1) expression, indicating that this cytokine is probably not involved in ELAM-1 induction in vivo. These results indicate that ELAM-1 is widely induced in cutaneous inflammation with a time course of expression that is longer than that observed in vitro. As ELAM-1 acts as an adhesion ligand for neutrophils, and perhaps monocytes, the expression of this molecule in cutaneous lesions is likely to be an indication of the ability of vascular endothelium to recruit these cells from the circulation. Furthermore, the cytokine inducibility of ELAM-1 is indirect evidence for functional interactions between perivascular mononuclear cells, other resident cells and the blood vessel wall.

A 59-year-old Caucasian housewife presented with a 2-year history of marked loss of tissue substance from the finger and toe pulps and the heel pads. There was no clinical evidence or history of urticaria or other inflammatory change. Investigations demonstrated a raised plasma cortisol secondary to a left adrenal adenoma. Skin biopsies showed abnormalities of dermal collagen, but no evidence of elastin destruction. This case presents an unusual variant of the cutaneous atrophy associated with Cushing's syndrome.

The clinical and histopathologic responses to intradermal platelet-activating factor (PAF-acether) in atopic subjects, without evidence of atopic dermatitis are documented. An immediate acute wheal and flare reaction was observed in all volunteers. Histopathologically, the reaction was characterized by a predominantly neutrophilic response, which was seen at 30 minutes and was maximal at 4 hours. Eosinophils were observed in the infiltrate as early as 30 minutes after injection, and were maximal by 12 hours. The specific PAF-acether antagonist BN52063 antagonized the acute flare response to intradermal PAF-acether but had little effect on cellular recruitment at the site of injection.

In a study of the direct effects of interferon-gamma (IFN-gamma) on normal human skin, healthy adult male volunteers received either 3 micrograms (n = 4) or 30 micrograms (n = 9) of recombinant IFN-gamma administered intradermally over 3 days. Biopsies were taken on day 6 and histopathological examination of fixed paraffin-embedded sections from sites which had received 30 micrograms IFN-gamma revealed a moderate perivascular lymphohistiocytic dermal infiltrate with mast cells. Immunophenotyping of 5 microns cryostat sections demonstrated that 3 micrograms IFN-gamma induced keratinocyte HLA-DR expression in the absence of any significant infiltrate. More intense keratinocyte HLA-DR expression was produced by 30 micrograms IFN-gamma in all specimens, with HLA-DP concurrently expressed in three biopsies. The ratio of CD4:CD8 cells within the infiltrate was approximately 3:1. CD1 + cells within the epidermis were markedly depleted by 30 micrograms IFN-gamma, while CD1-labelled cells were observed in the dermal perivascular infiltrate. Intradermal IFN-gamma induces similar immunopathological changes to those observed in many of the inflammatory dermatoses.

An ultrastructural study of lesional skin from 4 subjects with hyperkeratosis lenticularis perstans (Flegel's disease) demonstrated variable numbers of lamellar bodies of normal size and architecture within cells of the stratum granulosum and the upper stratum spinosum. Their characteristic lamellate inclusions were frequently observed within the intercellular spaces. These observations suggest that the lamellar bodies are qualitatively normal in Flegel's disease.

Localized persistent pigmented macules and plaques due to a chronic form of pigmented purpuric dermatitis are described in forty-two patients. The designation 'lichen aureus' for this eruption can be justified because of the striking yellowish or bronze-like colour assumed by many of the lesions. The lower legs were the commonest sites but other body regions can be affected also. Histologically lichen aureus differs from other pigmented purpuric dermatoses in the density of the lichenoid tissue reaction and the marked accumulation of pigment-containing macrophages. Recently developed endothelial cell markers have been studied in selected cases.

Immunohistological studies have been performed on tissues from various dermatological conditions using two monoclonal antibodies, RFD1 and NA1/34. These reagents were used to determine whether antigen expression restricted to interdigitating cells (RFD1+) and Langerhans' cells (NA1/34+) in normal tissues might occur together on dendritic cells involved in cutaneous inflammatory reactions. The results presented demonstrate that in psoriasis, allergic contact dermatitis and atopic dermatitis a proportion of the inflammatory dendritic cells express both antigens.

An immunoelectronomicroscopic method has been employed to demonstrate in situ the T lymphocyte nature of the dermal and epidermal infiltrates to mycosis fungoides. A specific antiserum to the human T lymphocyte surface antigen (HTLA) was used in an indirect reaction with a peroxidase labeled anti-immunoglobulin. After histochemically revealing the peroxidase activity, T cells were easily identified by the deposition of electron dense material on the cytoplasmic membrane. Counterstaining with uranyl acetate and lead citrate enabled us morphologically to observe the maturity of infiltrating T lymphocytes and to identify Sézary cells and other cells such as eosinophils in the infiltrate. Our results confirm the T cell nature of the dermal infiltrate in mycosis fungoides and show that the epidermal infiltrate of cells forming Pautrier micro-abscesses are predominantly T lymphocytes.

The Dowling-Meara variant of epidermolysis bullosa simplex (EBS) is characterized microscopically by clumping of the keratin tonofilaments within areas of incipient blistering, thereby raising the possibility that an abnormality of the keratin cytoskeleton might underlie blister formation in this condition. In order to investigate keratin expression in Dowling-Meara EBS, the staining profile of a panel of antikeratin monoclonal antibodies was examined in perilesional skin from 5 affected subjects, using a standard immunoperoxidase technique. Normal labelling characteristics were demonstrated by antibodies identifying keratins of the basal and suprabasal compartments of normal interfollicular epidermis and of simple epithelia (keratin 19). In addition, a keratin expressed in hyperproliferative epidermal states was shown to be absent. The results suggest that the profile of keratin synthesis is normal in Dowling-Meara EBS and that the tonofilament clumping may be, therefore, the result of a post synthetic modification of keratin molecules.

The influence of the sequential stages of conventional formaldehyde fixation and paraffin embedding of cutaneous tissue on monoclonal antibody labeling of cell surface antigens is described. The effects of variation in fixation time, dehydration, clearing, wax embedding, and enzyme treatment of cutaneous sections were examined. By curtailing fixation time, using cold ethanol dehydration, and limited cold clearing with xylene, immunoreactivity of several important monoclonal antibodies was retained. Wax embedding could be achieved at 58 degrees C for 1 h or by using low-melting-point wax at 42 degrees C for 3 h. Thus was derived an optimal processing procedure which afforded good tissue morphology and allowed reliable reproducible labeling by monoclonal antibodies to cell surface antigens.

A detailed examination of material from 10 cases of cutaneous endometriosis revealed a range of histopathologic features similar to those found within the uterine endometrium at each of the main phases of the menstrual cycle. However, there appeared to be a poor correlation between histologic appearance and menstrual stage. Recognition of these cyclic variations is important for the accurate diagnosis of cutaneous endometriomas.

Platelet activating factor (PAF-acether) is released from a variety of inflammatory cell types and has properties appropriate to a mediator of allergy and inflammation. Here, we have examined the interaction between PAF-acether and the prostaglandins, PGE2 and ZK 36374 (a stable analogue of prostacyclin, PGI2) in the skin of guinea-pigs and human volunteers. PGE2 and ZK 36374 significantly potentiated increased plasma protein extravasation induced by PAF-acether in guinea-pigs, assessed by extravasation of I-125-HSA. In addition, PGE2 significantly potentiated the ability of PAF-acether to elicit acute wheal (volume) and flare responses in human skin. The inflammatory properties of PAF-acether should not be considered in isolation since this phospholipid interacts synergistically with prostaglandins which are recognised as modulators of inflammation.

Lymphocyte function associated antigen 1 (LFA-1) and its ligand intercellular adhesion molecule 1 (ICAM-1) are cell surface adhesion molecules important in many lymphocyte-mediated responses. Recent in vitro studies have demonstrated that the cytokine interferon-gamma (IFN-gamma) can induce ICAM-1 expression by keratinocytes, and that lymphocytes adhere to IFN-gamma treated keratinocytes. In view of the importance of keratinocyte/lymphocyte interactions in the pathogenesis of cutaneous disease, we have examined the effects of in vivo IFN-gamma on cutaneous expression of LFA-1 and ICAM-1. Fourteen volunteers received intradermal IFN-gamma (dose: 1 or 10 micrograms) daily for 3 d. Biopsy was obtained on day 6. Cryostat sections were stained by the peroxidase antiperoxidase technique employing murine monoclonal antibodies to CD11, CD18, and ICAM-1. IFN-gamma intensified ICAM-1 expression by dermal endothelial cells and induced keratinocyte expression of ICAM-1. Furthermore, after administration of 10 micrograms of IFN-gamma LFA-1 positive (LFA + ve) lymphocytes were observed along the basement membrane zone closely related to ICAM-1 + ve basal keratinocytes and also surrounding dermal endothelium. Exposure to IFN-gamma induced expression of both CD11a and CD18 antigens on epidermal Langerhans cells. These studies suggest that the distribution of adherence molecules expression within cutaneous tissue in vivo is modulated by IFN-gamma, and that these alterations may be important in interactions involving cutaneous immunocompetent cells.

The effect of synthetic Paf-acether has been studied in guinea-pig skin, following intradermal injection, and in guinea-pig lung, following intravenous administration. Histopathological responses to Paf-acether were assessed by both light microscopy and electron microscopy. In addition, plasma protein extravasation and platelet accumulation were quantitatively assessed using radiolabelling techniques. Intradermal injection of Paf-acether, but not lyso-Paf, elicited acute increased vascular permeability, accompanied by intravascular accumulation of platelets and neutrophils. There was evidence, 2-8 h after intradermal injection of Paf-acether, of perivascular infiltration with neutrophils. At 24 h there was a mixed cellular infiltrate comprising mononuclear cells in addition to neutrophils. Following systemic administration of Paf-acether, aggregates of platelets in close association with neutrophils were evident within the pulmonary vasculature. Intravenous injection of Paf-acether, but not lyso-Paf, caused intrathoracic accumulation of radiolabelled platelets. These results suggest that Paf-acether has properties consistent with those of a mediator of inflammation.

In this paper we look at the regional variation of Langerhans cell (LC) numbers in tissue sections between different sites in the same individual and identical sites in different individuals. We also looked at the reproducibility of identification of LC in tissue sections by their OKT6 reactivity. In this way we could assess the validity of comparative enumeration in random tissue sections. Stepped sections were obtained and stained by the OKT6 method. The slides were projected, the cells counted, and an image analysis system used to measure the length of basement membrane. In this way we could calculate the number of LC per millimeter of basement membrane. Our subjects were six age- and sex-matched volunteers. We found that the number of LC on the trunk was significantly lower than on any of the limbs. However, each individual had significant site variation of LC that was different for each one. This makes direct comparison of individual sections invalid and suggests that the most suitable control, when counting LC in lesional skin, is adjacent normal skin.

The presence of T lymphocytes in the situ in cutaneous infiltrates of contact dermatitis and lichen planus has been demonstrated by an immunoelectronmicroscope technique. A specific anti-human T cell antiserum labelled with peroxidase-conjugated anti-immunoglobulin serum was used to reveal the specific human T lymphocyte antigen (HTLA) which was visualized ultrastructurally. This technique shows that labelling is limited to the surface membrane and allows study of the in situ interrelationship of T cells.

T cell antigens were studied in cutaneous sections from five patients with mycosis fungoides (MF). The method allowed cell counting to be undertaken for each monoclonal antiserum. OKT3 (pan T cell) antiserum confirmed the predominantly T lymphocytic nature of the infiltrate, labelling the majority of infiltrating cells. OKT4 (helper/inducer) antiserum positively labelled 90% of the lymphocytes identified as OKT3 +. OKT8 (suppressor) antiserum marked only single or small groups of dermal lymphocytes, which comprised 24% of the cells identified as T lymphocytes. OKT6 (anti-Langerhans) showed positive labelling of dendritic cells in the epidermis and dermis. Fewer positively labelled epidermal dendritic cells were observed in sections from patients receiving PUVA, but no difference was found in the number of OKT6 positive dermal cells. The ratio of helper to suppressor cells in the dermal infiltrate significantly exceeded the normal circulating ratio.

Sequential skin biopsies of nicotinate-treated skin from nine normal subjects, three aspirin-pretreated normal subjects and six atopic eczema patients were examined. An erythematous skin reaction was seen in the nine normal subjects and to a lesser degree in one atopic eczema patient, but not in the aspirin-pretreated subjects nor in five remaining atopics. Accumulation of a mononuclear cell perivascular infiltrate was seen from 15 min onwards in the normal subjects. Neutrophils became the predominant cell invading thickened vessel walls and in the perivascular space beginning at 2 h and persisting up to 48 h. Leucocytoclasis was observed at 24 h. Immunofluorescence studies showed only non-specific fibrinogen deposits in papillary capillaries in the three groups of subjects. The chloroacetate esterase reaction and immunohistochemical labelling with OKM I confirmed a marked neutrophilia at 2 h and 24 h. Neutrophils were seen in one atopic eczema patient, but were not observed in the remainder, nor in the skin of the aspirin-pretreated normal subjects. Topical application of nicotinate causes non-allergenic, leucocytoclastic vascular damage in normal skin which can be inhibited by aspirin and which is reduced or absent in atopic eczema.

Tissue from the acute, non-infected eruption of sixteen atopic eczema subjects was subjected to an indirect immunoperoxidase technique using monoclonal antibodies recognizing T-lymphocyte subsets, Langerhans cells and natural killer cells. Over half the cells infiltrating the dermis were T lymphocytes, including a large majority of helper T cells and relatively few suppressor T cells. Langerhans cells were present in significant proportions in the dermis and probably reflected increased antigen presentation within the affected skin. There was no evidence of increased natural killer cell presence. This study suggests that type IV hypersensitivity may be implicated in the aetiology of atopic eczema.

Both type I and type IV hypersensitivity reactions have been implicated in the pathogenesis of atopic dermatitis. Using monoclonal antibodies we have identified IgE on the surface of cutaneous dendritic cells in both lesional and nonlesional skin. Double immunofluorescence labeling demonstrates these cells to be antigen-presenting cells. Immunoglobulin E (IgE) was not identified on such cells either in atopic individuals with no history of dermatitis or in patients with a range of other dermatoses. Further studies are consistent with IgE being bound to the cell surface via an Fc-IgE receptor. We conclude that this finding is specific for atopic dermatitis and thus may provide a link between the two types of hypersensitivity reactions frequently observed.

Immunogold-silver staining (IGSS) was evaluated for the localization of cutaneous antigens in frozen and paraffin-embedded tissue, employing antibodies to a variety of intracellular, cell-surface and extracellular epitopes in an indirect immunogold reaction, followed by silver enhancement. The principal advantages of IGSS are the avoidance of toxic reagents and the production of a silver precipitate that is permanent, clearly visible and of sufficient contrast with the reaction products of alternative immunolocalizing techniques to be of value in double-labelling procedures. It seems to localize antigenic determinants well, but does not appear to be particularly sensitive, especially for the demonstration of antibodies to extracellular constituents. Limitations of IGSS include the propensity to non-specific staining, and the need to vary the duration of the development stage during silver enhancement.

Biopsies from lesional skin of patients with histologically confirmed mycosis fungoides were embedded in resin and semi-thin ( I micrometer) sections were submitted to a variety of staining procedures, including Toluidine blue, Richardson's Huber, Giemsa, Paragon and Tribasic stains. The stained sections were examined with the light microscope and the quality of cellular morphological detail afforded by the different stains was assessed. The Toluidine blue, Richardson's and Paragon stains were found to be technically easy to perform and gave the best representation of nuclear and cytoplasmic detail Of these, the authors favoured Richardson's stain. The technique of processing the tissue, and staining a mounting procedures are detailed.

Routinely used formal saline fixation reduces the number of demonstrable mast cells in human skin by up to 30% compared with paired specimens fixed in Carnoys medium. Using metachromatic (toluidene blue), orthochromatic (alcian blue/safranin), enzymatic (chloroacetate esterase reaction) and immunofluorescence (berberine and fluorescein conjugated avidin) staining techniques, mast cells were demonstrated and quantified. Alcian blue/safranin and fluorescein-conjugated avidin were both superior to the other staining methods used. We recommend the use of Carnoys medium fixed tissue stained with either alcian blue/safranin or conjugated avidin for optimal visualization and assessment of mast cells in human skin.

The ether-linked phospholipid, Paf-acether (AGEPC) is released from a variety of inflammatory cell types and has properties consistent with those of a mediator of inflammation. We have examined the effects of locally administered Paf-acether on cellular accumulation in the skin of experimental animals and man by histological evaluation of sequential skin biopsies and quantification of accumulation of radiolabelled blood elements. In guinea-pig skin, immediate extravasation of plasma protein and intravascular accumulation of platelets and neutrophils was succeeded by a persistent mixed cellular infiltrate predominantly of neutrophils but also containing lymphocytes and histiocytes. Radiolabelling studies were consistent with these observations. Intradermal Paf-acether elicited persistent clinical and histopathological responses in human skin. The finding that Paf-acether is able to initiate cutaneous cellular accumulation may be important in the pathogenesis of inflammatory dermatoses.

The phospholipid PAF-acether is released from a range of inflammatory cell types and, in experimental animals, has properties appropriate to a mediator of inflammation. We have studied the inflammatory characteristics of intradermally injected PAF-acether in human skin. An early weal and flare response was succeeded, in a proportion of subjects, by an area of late-onset erythema at the site of the resolved weal, reminiscent of the dual response to allergen in sensitized individuals. The time-course and dose-response relationship of the early response has been determined and a synergistic interaction between PAF-acether and prostaglandin E2 established. PAF-acether should therefore be considered as a potential mediator of both acute and persisting inflammation in man.

The ultrastructure of the human mucocutaneous end organ is described. The corpuscle is divided into sublobular units comprising axon terminals surrounded by generally concentric lamellar processes which are derived from laminar cells whose nuclei are situated towards the periphery of the sublobules. Interlamellar substance which contains elastic tissue, collagen and coarse periodicity crossbanded structures intervenes between lamellar processes. Specialized zones of contact resembling desmosomes are found at intervals seemingly connecting adjacent lamellar processes and axons with lamellar processes. The ultrastructural features of this end organ are similar to that of the Meissner corpuscle despite minor differences of the light microscopical appearance, which supports the view that differences in sensory end organs are merely variations on a common organizational basis.

The effects of the sequential stages of paraffin wax embedding on immunolabelling of cutaneous cell membrane antigens by monoclonal antibodies are described. Modifications in incubation times for fixation, dehydration, clearing and wax embedding of sections were examined. Labelling of the cell surface antigens deteriorates only after formal saline fixation for a period longer than 4 hr. Dehydration in ethanol had a minor adverse influence but clearing in xylene diminished immunoreactivity markedly. Both deleterious effects could be circumvented by conducting the procedures at 4 degrees C. The wax embedding procedure at 58 degrees C adversely affected labelling after 1 hr.