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.

Surface markers and mitogen response of cells harvested from cutaneous infiltrates in mycosis fungoides and Sézary's syndrome.

It was the purpose of this study to characterize the proliferating cells in skin lesion of Sézary's syndrome and of mycosis fungoides by means of their surface markers and their response to Phytohemagglutinine mitogen stimulation. Viable infiltrating cells were freed from skin biopsy specimens by means of a disaggregating homogenizer and the cells yielded were tested with heterologius polyvalent anti-human Ig and with anti-human T-cell globulin, as well as for spontaneous rosette formation with sheep red blood cells (SRBC) and for their response to stimulation with Phytohemagglutinine. Most of the infiltrating cells in skin lesions of mycosis fungoides and Sézary's syndrome lack receptors for anti-human Ig but form spontaneous rosettes with SRBC and have receptors for anti-T-cell globulin, indicating the T-lymphocyte nature of the infiltrating cells; however, their response to Phytohemagglutinine is weak. The results indicate the atypical, presumably neoplastic, nature of T-lymphocytes proliferating in skin lesions of mycosis fungoides and Sézary's syndrome.

Circulating Sézary cells in the diagnosis of Sézary syndrome (quantitative and morphometric analyses).

Plastic-embedded circulating Sézary cells were examined in semithin and thin sections (assisted by the nuclear contour index-NCI). Eight cases of Sézary syndrome were analyzed as well as 11 controls (3 cases of widespread eczemas and 8 cases of erythroderma), 7 cases of mycosis fungoides, and 3 healthy individuals. Discriminating criteria between Sézary syndrome and benign diseases were sought: in addition to Sézary cells (NCI greater than 6.5) intermediate lymphocytes (5.0 less than NCI less than or equal to 6.5) proved to be helpful. Cases with Sézary syndrome were clearly differentiated when the following 3 ultrastructural criteria were fulfilled: (1) Sézary cells (SC) greater than 9%; (2) intermediate lymphocytes (IL) greater than 20%; (3) the sum of SC and IL greater than 37%. A good correlation between thin and semithin sections was obtained (correlation coefficient for Sézary cells r = 0.82). Usually the values of SC were slightly higher on thin sections. The diagnosis of SS can be made on semithin sections when the ultrastructural criteria are fulfilled. In this way 8 of 12 samples of Sézary syndrome were correctly classified. Therefore, semithin sections (studied by light microscopy) are recommended as a routine method in the diagnosis of cases suspected of Sézary syndrome, whereas thin sections (studied by electron microscopy) appeared to be necessary in problem cases only.

Ultrastructural discrimination between malignant melanomas and benign nevocytic nevi using high-resolution image and multivariate analyses.

Prompted by the well-known difficulties of reliable and objective histologic differentiation between initial malignant melanoma (MM) and benign nevocytic nevi (NN), ultrastructural high-resolution image and multivariate analyses were evaluated for their diagnostic efficiency. Thirty-seven different features describing morphometry (area, circumference, and shape factor), amount of heterochromatin and euchromatin, chromatin homogeneity, and presence of smaller dark chromatin aggregations were determined by a MICROVAX 3500 computer in each of 1840 intraepidermal melanocytic nuclei of 17 MM and 20 NN. A strategy for the classification of cases based on the identification of markedly atypical melanocytic cells (MACS) was developed. MACS, selected in multivariate analysis with a linear combination of the eight most important features for cell classification, were found in 39.4% of the melanoma cells, but only in 0.3% of nevocytic nevus cells. The presence of MACS allowed a clear differentiation between MM and NN. All cases of MM had more than four MACS, whereas 17 cases of nevocytic nevi were MACS negative, and in each of the remaining three cases only one MAC was present. The percentage of MACS detected within intraepidermal parts of MM by using computerized high-resolution image analysis was found to be a highly efficient diagnostic marker. The new classification strategy has the potential of saving considerable time in subsequent studies, because preselected sampling and the calculation of only a few criteria have proven sufficient for correct classification of malignant melanomas.

Atypical cells in lymphomatoid papulosis express the Hodgkin cell-associated antigen Ki-1.

Lymphomatoid papulosis (LyP) is characterized by the presence of large multinucleated cells resembling Reed-Sternberg (RS) cells. Evidence of antigenic similarity between these two cell types has been sought by immunohistologic labeling of 10 biopsies from cases of LyP with monoclonal antibodies against Ki-1 and other RS and Hodgkin (H) cell-associated antigens. In all cases studied, a proportion of the large atypical cells expressed the Ki-1 antigen. On the contrary, in 20 biopsies of benign skin lesions or cutaneous T-cell lymphomas, Ki-1-positive cells were absent or only occasionally present. Furthermore the large atypical cells of LyP also expressed antigens (e.g., T3, T4, HLA-DR, IL-2 receptors) which we have previously demonstrated on RS cells in the majority of cases of Hodgkin's disease (HD). These findings, in conjunction with the observation that Ki-1 antigen expression can be induced on peripheral blood lymphocytes following exposure to phytohemagglutinin or HTLV I, provide evidence that the Ki-1 positive cells in LyP represent activated T cells as RS cells do in many cases of HD.

Tumor necrosis factor alpha induces invasiveness of human skin fibroblasts in vitro.

The chemotactic response of fibroblasts plays an important role during wound healing and fibrosis. Several substances have been found to mediate fibroblast migration in vitro. In the tissue, however, fibroblasts have also the potential to pass through connective tissue barriers following a chemotactic stimulus. Since tumor necrosis factor alpha (TNF alpha) is a cytokine released by mononuclear cells during wound healing, we have been interested in studying its effect on the regulation of fibroblast chemotaxis and invasive migration. TNF alpha did not attract skin fibroblasts nor did it alter their chemotactic response towards other chemoattractants like fibroblast conditioned medium or fibronectin. However, whereas normal skin fibroblasts did not invade a collagen I gel, preincubation of the cells with TNF alpha markedly induced their invasive migration into the gel. This seems to be associated with a specific degradation of type I collagen, because TNF alpha did not promote the invasion of skin fibroblasts through a reconstituted basement membrane (Matrigel).

Differentiation between lymphomas and pseudolymphomas of the skin by computerized DNA-image cytometry.

The histologic and immunohistologic differential diagnosis between pseudolymphomas (PL) and malignant lymphomas (ML) of the skin can be difficult. Since DNA cytometry has been found to be of both diagnostic and prognostic value in various neoplasms, its ability to discriminate between ML and PL in Feulgen-stained imprints of 17 PL and 49 ML skin biopsies was examined by high-resolution image analysis. The reliability of the following algorithms of DNA distribution was evaluated: 1) 2cDI (2c-deviation index), which reflects the variation of the nuclear DNA values around the diploid DNA peak; 2) percentage of cells having a DNA value greater than or equal to 5c (5cER; 5c-exceeding rate); 3) percentage of cells presenting with a DNA value greater than or equal to 4c (4cER). A 2cDI of 0.1 was found to be the most reliable marker for the differentiation between PL and ML. On the basis of this feature, 16 of 17 cases of PL and 46 of 49 cases of ML were correctly classified. The sensitivity, specificity, and efficiency of this feature were 94%. A 5cER greater than or equal to 1% had a specificity of 100%, but the sensitivity was only 43%. For the 4cER, a sensitivity of 61% and a specificity of 94% were found. In conclusion, the calculation of the 2cDI and the 5cER based on high-resolution image analysis provided additional helpful diagnostic features, and therefore should be included as a diagnostic tool. If the 5cER is at least 1%, the diagnosis of a ML can be confirmed with a specificity of 100%.

Occurrence of IgE-bearing epidermal Langerhans cells in atopic eczema: a study of the time course of the lesions and with regard to the IgE serum level.

Uninvolved and lesional skin of untreated and treated patients with atopic eczema has been investigated immunohistochemically to determine the conditions in which IgE-bearing CD1a+ Langerhans cells/indeterminate cells (LC/IC) occur in this disease. IgE-bearing epidermal dendritic cells were present in patients with elevated IgE serum level (greater than 300 UI/ml) and the staining pattern was stronger in lesional skin. On double immunostaining, a subpopulation of CD1a+ LC/IC was found not to bear IgE molecules as determined by the ratio IgE+/CD1a+ cells on serial sections as well. The ratio IgE+/CD1a+ cells decreased in patients who underwent a local therapy with glucocorticosteroids. These results suggest that the expression of IgE receptors and/or binding of IgE molecules on epidermal LC/IC in atopic eczema may be controlled by a complex network of mediators from the epidermis or the inflammatory infiltrate, or both, and that this phenomenon could be down regulated by glucocorticosteroids.

Interferon gamma in survivors of the Chernobyl power plant accident: new therapeutic option for radiation-induced fibrosis.

BACKGROUND: One of the remarkable clinical consequences of the Chernobyl accident was skin involvement, leading to extensive cutaneous fibrosis. Apart from surgery, no established treatment is available. METHODS: A group of survivors, working in or present at the accident site on April 26, 1986, and a few days thereafter, were examined, treated, and followed-up in 6-month intervals from September 1991 to November 1995. Eight individuals were identified as suffering from excessive cutaneous fibrosis. Skin thickness was measured with high-frequency (20 MHz) ultrasound in a clinically well-defined target skin lesion, in addition to histologic confirmation of radiation fibrosis. Interferon gamma was scheduled for all patients on a low-dose regimen (3 x 50 microg/week s.c.). In 2 patients, interferon was discontinued after the first injection, due to withdrawal of consent. In 6 patients, interferon was continued for 30 months, with 1 injection weekly for a further 6 months. Treatment was discontinued in November 1994. Four patients in the treated group and 1 of the 2 patients treated only once ("untreated patients") were reexamined 1 year later. RESULTS: In all individuals treated for 36 months, a significant (p < 0.005) reduction of radiation fibrosis could be determined, in contrast to a significant (p < 0.005) increase in the 2 untreated patients. Follow-up 1 year after discontinuation of the interferon treatment demonstrated significant (p < 0.005) recurrence of fibrosis. CONCLUSION: Low-dose interferon appears to be a safe and effective treatment of cutaneous radiation fibrosis following accidental exposure to high doses of ionizing radiation. Long-term supportive therapy may be required.

Effect of retinoids on collagen production by chondrocytes in culture.

The effect of vitamin A and of some of its derivatives on chondrocytes in culture has been studied. In the presence of retinoids the proliferation of the cells decreased and they lost their characteristic polygonal shape and assumed a fibroblast-like morphology. All retinoids also caused dedifferentiation of chondrocytes as indicated by the induction of types I and III collagen. 13-cis retinoic acid (= isotretinoin) was the most active derivative in this aspect. Since appropriate control of the synthesis of extracellular matrix proteins is a prerequisite for their normal physiological function, alterations such as those observed here may be involved in the pathogenesis of side effects which are observed during the treatment of dermatological disorders with retinoic acid derivatives.

Lung involvement in scleroderma.

Lung involvement (LI) was studied by lung function (LF) in 101 scleroderma patients (circumscribed scleroderma, n = 17; progressive systemic scleroderma [PSS], n = 84; with the subtypes I, acroscleroderma [n = 19]; 2, proximal ascending scleroderma [n = 61]; 3, trunk scleroderma [n = 4]). Eighteen percent of morphea, 32 percent of type 1, 56 percent of type 2, and 75 percent of type 3 patients had impaired LF. The LI was more frequent (57 percent vs 45 percent) and more severe (20 percent vs 3 percent) in PSS with systemic inflammation (form A) compared to those without (form B). Elevated lymphocytes/neutrophils in bronchoalveolar lavage (BAL) were found associated with form A and severe LI. The LF of patients showing an inflammatory cell pattern in initial BAL (n = 3) worsened, whereas those with normal BAL findings (n = 4) did not. Collagenase activity in BAL was significantly elevated in those with elevated lymphocytes/neutrophils in lavage. Patients with type 2 or 3 of PSS, especially form A, carry a higher risk of developing severe LI than circumscribed scleroderma, type 1, or form B patients. Differential cell count and collagenase activity in BAL is correlated with active disease and provides prognostic information.

Effect of iron limitation on protein composition and ultrastructure of Haemophilus ducreyi.

Protein profiles of whole cells of Haemophilus ducreyi grown in the presence or absence of the iron chelator desferal, were compared by polyacrylamide gel electrophoresis. Each of four strains produced novel proteins in the range 43-160 kDa when cultured under conditions of reduced iron availability. At some sub-inhibitory concentrations, desferal produced enhanced growth, possibly due to it functioning as an exogenous siderophore. Organisms grown under conditions of reduced iron availability ultrastructurally showed also large periplasmic spaces between cytoplasm and outer membrane.

Prognostic index in malignant melanoma.

A retrospective study was undertaken of 146 surgically treated subjects with primary cutaneous melanoma of which 73 were disease-free for five to nine years and 73 developed later metastases. A prognostic factor was south to determine patients with poor prognoses. The best overall method was shown to be the evaluation of the prognostic index defined as the product of tumor thickness and the number of mitoses per square millimeter. However, for establishing a group of patients with no incidence of metastases, the mitotic rate proved to be as good a factor as the prognostic index and better than tumor thickness or levels of invasion. The application of this prognostic index seems therefore to be useful in selecting for further treatment stage I melanoma patients with poor prognoses, eg, prophylatic lymph node dissection and immunochemotherapy.

Late irradiation damage to the skin caused by soft X-ray radiation therapy of cutaneous tumors.

BACKGROUND: Although radiotherapy of skin tumors has lost its former preeminence, there is still need for this modality. The aim of this study was, therefore, to determine the frequency of radiogenic ulcers and tumors following soft x-ray therapy of skin lesions. STUDY DESIGN: A total of 612 radiation sites in 522 patients were retrospectively analyzed by means of medical records. All patients received at least a total dose of more than 12 Gy and had a minimum follow-up time of 10 years. The average radiation dose was about 80 Gy, ranging from 56 to 184 Gy. Determined was occurrence of radiogenic tumors after more than 10 years and of ulcers during the entire follow-up period. The frequency of radiogenic tumors and ulcers was related to the total dose applied and the patient's age at the time of irradiation. RESULTS: In the 612 radiation fields used, 58 ulcers (9.4%), 12 basal cell carcinomas (2%), and nine squamous cell carcinomas (1.5%) were observed. There was no relationship between the total dose of radiation and the frequency of tumors; in contrast, radiogenic ulcers increased with a higher total dose. Radiogenic ulcers occurred more often in patients who were of a younger age at the time of irradiation. CONCLUSIONS: The risk of developing radiogenic ulcers and tumors after soft x-ray therapy is not very high. Since most of the ulcers can be prevented by modern dose recommendation (time dose fractionation factor), soft x-ray therapy can be considered as a safe and effective means of therapy, especially in older patients.

A probable case of rotavirus exanthem.

A generalized maculopapular exanthem and signs of hepatitis developed in a 28-year-old man one week after his two sons had suffered from rotavirus gastroenteritis. The patient's serum contained rotavirus antibody at titers of 1:256 and 1:512. Other known causes of exanthemata were excluded by clinical and laboratory investigations. The epidemiologic evidence and the results of serological tests suggested that the rotavirus caused the patient's exanthem.

Monocytic leukemia. Clinically appearing as 'malignant reticulosis of the skin'.

Enzyme cytochemical (hydrolytic enzymes for cell differentiation), immunocytologic (B and T lymphocyte differentiation), and electron microscopic studies of skin infiltrates facilitate the proper diagnosis of myelomonocytic and lymphoreticular proliferations. With use of these methods, the original clinical diagnosis of malignant reticulosis of the skin was corrected to monocytic leukemia in a 65-year-old woman. Because primary involvement of the skin preceded monocytosis of the blood, it was concluded that the cutaneous infiltrates in our patient resulted from proliferation of tumor cells in the skin rather than from homing of the cells to, or settling of the cells in, the skin.

Acquired epidermolysis bullosa with the clinical feature of Brunsting-Perry cicatricial bullous pemphigoid.

A 56-year-old woman with the typical clinical feature of cicatricial bullous pemphigoid of the Brunsting-Perry type was studied. Histologic examination of a lesion skin biopsy specimen demonstrated a subepidermal blister. Direct immunofluorescence microscopy revealed linear deposits of IgG, IgM, and C3 located on both the roof and the floor of the blister. Immunofluorescence antigen mapping using cryostat sections of a spontaneous blister and antisera against defined basement membrane components localized the bullous pemphigoid antigen and type IV collagen in the roof of the blister. This dermal type of blister formation was confirmed by electron microscopy, which showed the cleavage level below the lamina densa. In direct immunoelectron microscopy, granular deposits of C3 and IgG were found attached to and just beneath the lamina densa in a pattern identical to the distribution of anchoring fibrils. These findings are diagnostic of acquired epidermolysis bullosa, a blistering disease that has much more clinical heterogeneity than previously suggested.

Erythema induratum of Bazin. Evidence of T-lymphocyte hyperresponsiveness to purified protein derivative of tuberculin: report of two cases and treatment.

BACKGROUND AND DESIGN: Erythema induratum of Bazin, a chronic form of nodular vasculitis, may be associated with chronic infections by Mycobacterium tuberculosis. However, the true origin of the disease is a subject of speculation and remains elusive. Two female patients (58 years old and 33 years old) with a minimum 10-year history of chronic tender ulcerating nodules on the lower aspects of the legs were studied both clinically and in the response of their peripheral T cells to purified protein derivative of tuberculin. RESULTS: Both patients with no previous history of tuberculosis had strongly positive skin test results at a Mantoux 10(-4) dilution (1 unit of purified protein derivative). In response to full-course triple-agent (isoniazid, rifampicin, and ethambutol) chemotherapy, a complete remission of clinical symptoms was seen in both cases and no relapse occurred after discontinuation of therapy. A marked increase in peripheral T-lymphocyte response to purified protein derivative was found before onset of and during successful therapy. CONCLUSIONS: The present clinical observations together with the pronounced cellular response to purified protein derivative suggest a tuberculous origin of erythema induratum of Bazin.

Hemorrhagic bullous amyloidosis. A histologic, immunocytochemical, and ultrastructural study of two patients.

In patients with bullous hemorrhagic amyloidosis of the skin, the skin lesions were the first manifestations of a plasma cell dyscrasia. Both cases were characterized by similar clinical, histologic, and ultrastructural findings showing an intradermal blister within deposits of amyloid substances. Immunohistologic investigations with a panel of antibodies directed against amyloid fibril proteins showed reactivity of the amyloid deposits with an anti-A lambda serum in both patients.