Treponema pallidum in leukoderma syphiliticum demonstrated by electron microscopy

Punch biopsies from syphilitic leukoderma lesions and from unaffected skin in 3 patients with secondary syphilis were studies in the transmission electron microscope. In one of the patients the pigment disorder was not preceded by any visible syphilids, and in the biopsy from the leukodermal skin in this patient Treponemata pallidium were demonstrated around vessels and inside nerve fibres in which the myelin sheaths of the axons showed evidence of degeneration. In the other 2 patients the depigmented areas appeared while macular and papular syphilitic lesions were healing. In the biopsies from the leukodermal lesions of these 2 patients and from unaffected skin of all 3 patients, no treponemes were demonstrated. The study indicates that syphilitic leukoderma is not invariably a post-inflammatory phenomenon, but the pigmented skin lesions may themselves represent stigmata of an active syphilitic infection.     

Ultrastructural observations on uninvolved skin in dermatitis herpetiformis

Clinically normal skin from patients being treated for dermatitis herpetiformis was examined with the electron microscope. Cytoplasmic processes from the basal epidermal cells penetrated the dermis through discontinuities in the basal lamina which otherwise was normal in structure and thickness. The fibrous network of the dermis consisting of collagen, small reticular fibrils, anchoring fibrils and elastin, appeared normal. In patients receiving only dapsone, membrane-bound‘vacuoles’were found just below the basal lamina. They contained a fibrillar material of low electron density and were often associated with cell processes or complete cells, the classification of which was difficult. It is suggested that these vacuoles may be implicated in the reaction between reticulin and immunological complexes. In two specimens, early blister formation was also seen in the dermal papillae. The basal lamina remained applied to the stratum basale and was only absent where basal cell processes projected into the blister space which contained fibrin deposits. Vesiculated structures observed in the region of the blisters are interpreted as abnormal sensory nerve endings. This evidence supports the view that disruption of the basal lamina and blister formation in dermatitis herpetiformis are secondary to an earlier reaction.     

Drug-induced linear IgA disease: target antigens are heterogeneous

No information is available concerning the target antigen(s) involved in drug-induced linear IgA disease. The purpose of this study was to define better drug-induced linear IgA desease by studying its immunopathological, immunoultrastructural and immunochemical characteristics in three patients. In the first patient. IgA deposits were seen in the lamina lucida. In the second and the third patients. IgA deposits were seen on each side of the lamina densa. Immunoblotting of the first patient's serum showed that IgA reacted with a protein of 230 kDa similar to the bullous pemphigoid-associated antigen 1 (BPAG 1). The second patient's serum did not react with specific bands. In the third patient IgA antibodies reacted with a protein of 97 kDa on epidermal extracts. Our findings suggest that, as in idiopathic forms, the target antigen is not unique in drug-induced linear IgA disease. In some patients, drug-induced linear IgA disease may represent an IgA class response to the BPAG 1.     

HISTOGENESIS OF HYALINOSIS CUTIS ET MUCOSAE

Light microscopically, reticulum fibers and argyrophilic materials were found by reticulum staining, and lipid droplets by Sudan III staining, around the blood vessels and in the interstices of the smooth muscle fibers of tunica dartos; toluidine blue staining of thick sections of Epon-embedded tissue revealed poorly-stained, glass-like, amorphous hyalin with configuration of collagen fibers in the same areas and hyalinized muscle fibers in tunica dartos. Electron microscopy disclosed that multiplication of the basal lamina and its transition to the fibrogranular amorphous material, probably a hyalin substance, was seen around the true smooth muscle cells of tunica dartos and also around the blood vessels. In addition, dilated rough-surfaced endoplasmic reticulums with fibrogranular content were often observed in the endothelial cells, pericytes, and smooth muscle cells, with Golgi apparatus in some parts. These findings suggest that hyalinosis cutis et mucosae is due to the accumulation and invasion of hyalin into the normal collagen fibers due to a genetic abnormality in the synthetic processes of smooth muscle cells, vascular endothelial cells, pericytes and some fibroblast-like connective tissue cells. In my speculation, the hyalin substance in this disease may be normal collagen invaded by basal lamina glycoprotein, most probably involving type III and/or type IV collagen.     

Porphyria-like cutaneous changes induced by tetracycline hydrochloride photosensitization.

Five patients manifested cutaneous changes indistinguishable from those noted in some porphyric disorders, consisting of fragility, denudation, and blister formation of sun-exposed skin. Microscopical examination showed subepidermal bulla formation and the desposition of PAS-positive, diastase-resistant material and IgG in or around the upper dermal blood vessel walls. There was also electron microscopical evidence of vascular basal lamina reduplication and the deposition of a fine fibrillar material in and around these vessels. However, no abnormal porphyrin formation was noted. All five patients had been receiving 250 mg of tetracycline hydrochloride twice a day for at least six months and had had extensive sun exposure prior to the onset of the condition. For four patients, discontinuing the medication led to complete remission, despite subsequent sun exposure; the fifth patient was much improved, but her skin was still somewhat fragile seven months later. We concluded that these cutaneous changes resulted from a low-grade photosensitization by tetracycline hydrochloride.     

Discrepancy between the localization of in vivo bound immunoglobulins in the skin and in vitro binding sites of circulating anti-BMZ antibodies in bullous pemphigoid: immunoelectron microscopic studies

The immunoelectron microscopic reaction products showing in vivo bound immunoglobulins or complement in the basement membrane zone (BMZ) of the lesional skin of 12 patients with bullous pemphigoid (BP) were homogeneously distributed in the lamina lucida in 3 cases, just beneath the basilar surface of the basal cells in 5 cases, and were associated with the basal lamina in 4 cases. No reaction products could be found beneath the melanocytes in any of the first 2 groups; however, positive reaction products were found on the basal lamina beneath some melanocytes, and were associated with the detached basal lamina in the dermis in the latter 4 cases. In vitro binding sites of circulating anti-BMZ antibodies in the sera from 7 patients with BP indicated that the antibodies reacted with the basilar surface of the hemidesmosomes, with a weak reaction on the basal lamina in 5 cases and with no reaction in 2 cases. No positive findings were obtained beneath the melanocytes or on the basal lamina under the melanocytes. The discrepancy between the antigenic sites and the localization of immune deposits in BP is assumed to be due to the renewal of the hemidesmosomes and constant clearance of immune deposits in vivo. The in vivo bound immunoglobulins and complement seen in the BP skin are the result of their accumulation in the lamina lucida or in association with the basal lamina.     

Ultrastructure of skin in primary systemic amyloidosis.

Amyloid masses were found in the dermis of two brothers suffering from primary amyloidosis. The masses consisted of fibrils, composed in turn of twin hollow filaments of amyloid. An amyloid filament appeared as a 3 nm thick lucent core with a 2 nm thick wall. Occasionally 4--6 filaments were packed together in one fibril. The twin filaments were slightly twisted, with a twisting angle of 2.5 degrees and a coiling pitch of 1 160 nm. Wavy shapes of amyloid fibrils were also seen in elastic fibres. Amyloid fibrils were found in elastic fibres, under the basal lamina of the epidermis, sweat gland epithelium and Schwann cells; also around perineural cells, perivascular cells and, in one of the brothers, in collagen fibril bundles. No amyloid fibrils were found under the endothelial basal lamina. It would appear that amyloid fibrils are pathological fibrils belonging to the elastic fibre-basal lamina system.     

The localization of target antigens and autoantibodies in linear IgA disease is variable: correlation of immunogold electron microscopy and immunoblotting

Linear IgA disease (LAD) of adults and children is a dapsone-responsive, autoimmune subepidermal blistering disease characterized by linear IgA deposits at the basement membrane zone (BMZ) of the skin and mucosa. Circulating IgA antibodies to BMZ components are often present. In this study we investigated the ultrastructural localization of the antigens and autoantibodies in six patients with LAD (five adults and one child). Using a direct postembedding immunogold electron microscopy (EM) technique, three different patterns of IgA antibody deposition were seen in the skin of four patients with LAD. The IgA deposits localized within the uppermost part of the lamina lucida and to the basal surface of the hemidesmosome in two patients, to the lamina lucida in one, and to the lamina densa in the fourth patient. Using an indirect immunogold EM technique and serum or purified blister fluid from two additional LAD patients, we showed that the serum autoantibodies of one patient bound to the hemidesmosome of the BMZ, while the autoantibodies in the blister fluid of the other patient bound to the lamina densa and sublamina densa including the anchoring fibrils in a labelling pattern similar to that of the monoclonal antibody (LH7.2) to collagen VII. All the autoantibodies binding to the hemidesmosome or lamina lucida recognized a protein in epidermal extracts of molecular weight 180 kDa or its breakdown product of 97 kDa, 200 kDa or 230 kDa. The antibodies binding to the lamina densa recognized proteins of 180 and 285 kDa. The antibodies that bound to the lamina densa and anchoring fibrils recognized collagen VII. In this immunogold EM study we have shown four patterns of IgA labelling in six patients with LAD, associated with five different antigens as recognized by immunoblotting. These results, together with our previous immunofluorescence and immunoblotting findings add support to the contention that LAD is a heterogeneous disease as regards both the target antigens and epitopes.     

Structure of basement membranes in malignant melanoma and nevocytic nevi

Basement membranes found around tumor cells in nevocytic nevi, Spitz's nevi, and malignant melanomas were analyzed by electron microscopy and antibody staining for several basement membrane proteins. Nevocytic nevi and Spitz's nevi showed a distinct, occasionally discontinuous lamina densa regardless of whether they were located in junctional zones of the epidermis or within the dermis. All basement membranes around nests of aggregated nevus cells, however, lacked anchoring fibrils. This correlated with the absence of type VII collagen. In contrast, type IV collagen, laminin, and nidogen were present at the periphery of the nevus cell clusters in agreement with the presence of an intact lamina densa. Aggregated tumor cells in malignant melanomas were bordered by a lamina densa when located in a junctional position and lacked this structure when they had migrated into the dermis. This process was accompanied by a drastically reduced staining for collagen type IV and nidogen, whereas laminin was still detectable. Anchoring fibrils and their molecular correlate, type VII collagen, were consistently absent. These observations demonstrate major alterations in the composition of basement membranes around malignant melanomas, which can be an important factor for the invasive growth and formation of metastases of these tumors.     

Basal lamina of the dermal capillary: concentric multilamination following regeneration processes

Concentric multilamination of the basal lamina around the subepidermal capillaries was observed after reaction to dinitrochlorobenzene. It consisted of luminal obstruction, necrosis of endothelial cells and pericytes, regeneration of these cells, and new deposits of basal lamina. Beneath the two basal laminae, delicate collagenous fibrils were present. These findings help explain the similar but more pronounced lamination of the vascular basal lamina in patients with diabetes, protoporphyria, etc. and the endothelial production of fibrillar collagen under certain condition.     

Neurothekeoma of Gallager and Helwig (dermal nerve sheath myxoma variant): report of a case with electron microscopic and immunohistochemical studies

A patient presented with a frontal nodule of the scalp. Histopathological examination revealed a myxomatous multilobulate tumor composed of epithelioid cells with variable pleomorphism. Perineurium-like structures were seen but only around isolated lobules located at the tumor periphery. Electron microscopy revealed polygonal cells and cells with elongated cytoplasmic processes. Many cells had myelinoid figures. A basement membrane-like lamina was noted around some cells. Some of the tumor cells were immunoreactive for myelin basic protein. This finding suggests that the tumor cells are of schwannian type. Neurothekeoma of Gallager and Helwig is a rare, probably benign tumor with fairly distinctive histopathologic characteristics. It appears to be a variant of dermal nerve-sheath myxoma.     

Hyaluronan and CD44 in psoriatic skin. Intense staining for hyaluronan on dermal capillary loops and reduced expression of CD44 and hyaluronan in keratinocyte-leukocyte interfaces

The distributions of hyaluronan (HA) and its presumptive receptor, CD44, were studied in skin samples from 13 psoriasis vulgaris patients, using an HA-specific probe (HABC), and monoclonal antibodies, respectively. The general distribution of HA and CD44 in psoriatic lesional epidermis resembled that in normal epidermis. However, areas of epidermis invaded by leukocytes showed a local depletion of HA and CD44, particularly at the contact areas of keratinocytes to lymphocytes and neutrophils. Removal by cellular uptake or extracellular degradation of CD44 and HA may be required for tight adherence between a keratinocyte and a leukocyte. On the dermal side, the tips of the prolonged dermal papillae in psoriatic lesions were intensively stained with HABC. The dilated capillaries and the space below the tip basal lamina, in particular, were heavily covered with HA. Occasionally, a similar intense staining was seen around an enlarged capillary in uninvolved psoriatic skin. CD44-positive leukocytes were found around the affected capillaries. The accumulation of HA in the dermal papillae may support the growth of psoriatic lesions, since HA stimulates the growth of capillaries as well as attracting inflammatory cells.     

Immunoelectron microscopic studies in IgA linear dermatosis

Summary The authors investigated three cases of IgA linear dermatosis by immunoelectron microscopy. In two of the cases there were additionally some IgG deposits in the basement membrane zone.The arrangement of the IgA deposits was found to vary. In two cases it was of the dermal and the lamina lucida type at the same time, and in one case it was so close to basal cell membranes as to leave an electronlucent space on the side facing the basal lamina. In the third case, the arrangement (only IgG and complement were studied) was exclusively of the dermal type.In one of the cases the deposits had first, i.e., 11/2 years earlier, been confined to the lamina lucida, whereas now they were also seen below the basal lamina, although the clinical condition of the patient had remained unchanged. It would seem that the localization of IgA deposits in the basement membrane zone may vary, depending on the evolution of the disease.     

The ultrastructural changes in the skin in dermatitis herpetiformis after withdrawal of dapsone

The development of skin lesions in patients with dermatitis herpetiformis after the withdrawal of their dapsone therapy was studied with the electron microscope. In control biopsies from patients prior to cessation of treatment, membrane-bound vacuoles were found beneath the basal lamina of the epidermis as previously described. After dapsone withdrawal, there was an apparent increase in the number of vacuoles and occasionally several vacuoles appeared to have coalesced forming an early blister. At this stage, the basal lamina and associated hemidesmosomes were normal although in places there were small discontinuities in the basal lamina. Where the reaction was more intense, vacuoles and cells, mainly eosinophils, were embedded in fibrin de posits. Above this, the basal lamina was usually disrupted with involvement of the basal epidermal cells. These results suggest that the vacuoles do play a part in the formation of the pathological lesion in dermatitis herpetiformis. In addition, the basal lamina is shown to be only secondarily involved. The nature of the vacuoles has still to be elucidated.     

Extracellular binding sites of IgA anti-jejunal antibodies on normal small bowel detected by indirect immunoelectronmicroscopy

Patients with dermatitis herpetiformis (DH) have IgA deposition in the papillary dermis and in the lamina propria of the small bowel. In addition, most of DH patients' sera contain IgA class anti-reticulin antibodies, anti-endomysium antibodies (EMA), and anti-jejunal antibodies (JAB) during times of gluten intake. In previous studies, JAB and EMA seemed to be identical and related to the group of anti-reticulin antibodies. In the present study, pre-embedding en bloc immunoelectronmicroscopic methods were applied for analysis of the ultrastructural binding sites of JAB on monkey and rabbit small bowels. These substrates were incubated with sera from DH patients strongly positive for JAB. Simultaneous investigations with the PAP technique and with 5 nm gold-labeled protein A or second antibodies visualized the bound IgA identically: it was associated with collagen fibrils underlying the epithelial and cryptal basement membranes and with collagen fibrils around capillaries. Staining was also detected along the endomysial collagen fibrils of smooth muscle layers, around elastica and smooth muscle cells of blood vessel walls, and along collagen fibrils near smooth muscle cells in the lamina propria. Neither the peroxidase product nor gold deposition was detected directly on the fibers, but was associated with amorphous material surrounding collagen fibers of different diameters. The distribution of JAB-stained structures corresponded to the localization of reticulin network of the small bowel. Our data indicate that JAB recognize an antigen or antigens associated with an amorphous component of the reticulin-collagen structure of jejunum and may have identical binding sites, as anti-reticulin antibodies and EMA.     

Expression of a pemphigoid gestationis-related antigen by human placenta

We studied the antigen binding of sera from 25 patients with pemphigoid gestationis (PG) using skin and amnion as substrates in an indirect immunofluorescence technique. In addition, skin was studied after prior incubation with I M NaCl which allows BMZ cleavage through the lamina lucida. Linear BMZ staining was obtained with sera from patients with PG using human amnion as substrate. This was seen with both full-term and second trimester placentae, but not with first trimester placentae. In NaCl incubated skin all sera bound to the epidermal aspect of the separated tissue. The pattern of staining in skin, chemically split skin and amnion was identical to that previously seen with the sera of patients with bullous pemphigoid suggesting that the antigen against which circulating anti-BMZ antigens are directed may be the same in the two conditions.     

Scanning electron microscopy of the epidermal lamina densa in normal human skin.

The lamina densa of normal human epidermis was exposed by treatment with 1 M sodium chloride and was examined by high-power scanning electron microscopy before and after trypsinization. Localization of type IV collagen in the lamina densa was also studied by transmission and scanning immunoelectron microscopy. Before trypsinization, the surface of the lamina densa consisted of microridges and microvalleys. The microridges varied in height and were connected with each other. They were arranged in a concentric fashion around the tips of the dermal microprojections. At a higher magnification, the surface of the lamina densa was composed of densely packed cobblestone-like structures approximately 7-15 nm in size, between which were interspaces 4-11 nm wide. These structures expressed type IV collagen. After trypsinization, the lamina densa was found to be composed of microfilaments approximately 10 nm thick showing beaded appearances. These microfilaments exhibited the same cobblestone-like structures as the lamina densa surface. Observation of the torn lamina densa demonstrated anchoring fibrils and oxytalan fibers that were attached to the lamina densa itself. Another kind of filament about 7 nm thick linked the anchoring fibrils and the oxytalan fibers. Beneath the lamina densa was a network of fibers about 40-50 nm thick, which was composed of collagen fibers and possibly also elaunin fibers. In conclusion, this study revealed the detailed surface ultrastructure of the epidermal lamina densa and its underlying filamentous elements.     

Immunoelectron microscopy in subepidermal autoimmune bullous diseases: a prospective study of IgG and C3 bound in vivo in 32 patients

Thirty-two patients suffering from subepidermal autoimmune bullous disease were studied prospectively by clinical examination and immunoelectron microscopy. Clinically, 1 patient had herpes gestationnis, 14 typical bullous pemphigoid (BP), 3 epidermolysis acquisita (EBA), 3 cicatricial pemphigoid (CP), and 11 patients overlapping clinical diseases. These 11 patients shared clinical features of BP, EBA, or CP and a clinical diagnosis could not be done safely. Immunoelectron microscopy revealed diaminobenzidine deposits in 20 patients on the epidermal side of dermo-epidermal junction in the lamina lucida as in BP. In 5 patients, deposits located mostly under the anchoring fibril zone, in the floor of a sublamina densa dermoepidermal separation for 2 of them, were consistent with a diagnosis of EBA. In 6 patients, deposits were located mostly in the lamina densa, in the floor of a dermoepidermal separation occurring in the lamina lucida for 3 of them. This suggests that some of these 6 patients had neither EBA or BP, but another autoimmune bullous disease again, an uncharacterized component of dermoepidermal junction located in the lamina densa. Finally, a correlation exists between the sites of IgG and/or C3 components on epidermal or dermal side of dermoepidermal junction and the presence or absence of characteristic clinical features such as scar, milia formation, or mucosal involvement.     

Herpes gestationis. Ultrastructure and ultrastructural localization of in vivo-bound complement.

Ultrastructural localization of C3 deposition in the skin of two patients with herpes gestationis was determined by using a peroxidase-antiperoxidase multistep technique. The tissue preparations can be stored for long periods of time and identical sections may be used for light and electron microscopic examination. The reaction products were seen throughout the entire lamina lucida and the basal cell plasma membrane appeared to be accentuated. The most remarkable ultrastructural changes in normal-appearing skin were the destruction of the basal cell membranes on the dermal side, localized cytoplasmic dissolution, and intracellular edema unaccompanied by inflammatory cells. Early, nonvesicular lesions showed basal cell degeneration and dermal inflammatory cells. Necrosis and loss of basal cells occurred in the next stage which resulted in microvesicles in which collagen or a well-preserved basal lamina formed the vesicle base. In the later blister stage, the basal lamina was usually lost. It is suggested that damage of basal cell membranes on their dermal side leads to the destruction of basal cells with the subsequent protrusion of epidermal and junctional substances into the dermis. This may result in inflammatory cell infiltration and blister formation.     

ULTRASTRUCTURAL ASPECTS OF DERMATITIS HERPETIFORMIS (DUHRING''S DISEASE)

Summary.— Biopsies of clinically normal skin and erythematous and recent vesicular lesions of 3 patients with dermatitis herpetiformis (DH) were studied by electron microscopy.
The early events at the dermo–epidermal junctional zone were the presence of glycogen and lipid particles in the basal cells, rarefaction of half-desmosomes, and small duplications of the basal lamina.
In some areas of the dermo-epidermal junction of the erythematous skin separation between epithelial basal cells and basal lamina was observed. In the vesicles fibrin, with its typical cross-banding pattern, was seen.
The early changes observed in the epithelial germinative cells were interpreted as evidence of disturbance in dermo-epidermal interaction, possibly caused by immunoglobulin deposition at this level. The abnormal synthesis of half-desmosomes by the basal cells could be the cause of supra-laminar dis-junction.