In vitro pemphigus vulgaris model using organotypic cultures of human epidermal keratinocytes.

Using the Combi-ring-dish (CRD), a new culture device, organotypic cultures of human epidermal keratinocytes were grown on bovine eye lens capsules. In these highly differentiated cultures, typical suprabasal acantholysis was induced by pemphigus vulgaris antibodies. This in vitro pemphigus vulgaris model may be used to analyse keratinocyte-derived factors causing acantholysis in experimental pemphigus.

Autoaggressive inflammation of the myenteric plexus resulting in intestinal pseudoobstruction.

After a 3-year history of severe constipation, a 16-year-old girl required surgery to be relieved of impacted stools. Histologic examination showed ganglionitis in the myenteric plexus of the large bowel and ileum, whereas the submucosal plexus was spared. At this time, antineuronal nuclear antibodies (ANNA-1, anti-Hu) were found at high titer in the serum of the patient. One and a half years earlier, a paravertebral ganglioneuroblastoma had been removed. Histologic examination had shown undifferentiated neuroblasts and morphologically mature ganglion cells with both cell types embedded in an inflammatory infiltrate morphologically similar to the lymphoplasmocytic infiltration seen in the myenteric plexus. The patient's serum was found to bind to nuclei of mouse intestinal tract neurons, thus fulfilling defining criteria for ANNA-1. The serum also reacted with antigens of defined molecular weight in a Western blot, thus fulfilling defining criteria for anti-Hu. Expression of the Huantigen could be visualized in the nuclei of the patient's tumor cells by immunohistochemistry. These tests showed that an antitumor inflammatory response was the cause of the bowel disease. This is the first report of a tumor from the neuroblastoma group that caused paraneoplastic intestinal pseudoobstruction. Ganglionitis and subsequent aganglionosis are the hallmark of the morphologic diagnosis which cannot be obtained by suction biopsy in patients with intact submucosal plexus. Instead, serum testing for autoantibodies can reveal the etiology.

Follicular tumor in the sellar region without primary cancer of the thyroid. Heterotopic carcinoma?

A case of a follicular tumor in the sellar region is described without evidence of a primary tumor within the thyroid gland or at a site known to harbor ectopic thyroid tissue. Large amounts of thyroglobulin were readily demonstrated mainly within the colloid of the follicles. The possible development from heterotopic thyroid tissue at this unusual site is discussed.

Factor VIII-related antigen in malignant hemangioendothelioma of the thyroid: additional evidence for the endothelial origin of this tumor.

Malignant hemangioendothelioma of the thyroid gland, which most often originates in a hemorrhagic nodule, is a well-known entity in European alpine regions with endemic goiter. In other parts of the world it very rarely has been diagnosed. This tumor may display considerable morphologic variation and often has been interpreted as a variant of undifferentiated carcinoma. In 13 out of 20 thyroid tumors, classified by light microscopy as malignant hemangioendothelioma, Factor VIII-related antigen, a marker for endothelial cells, was demonstrated in neoplastic cells with the help of immunohistochemical technics applied to conventional paraffin sections. In one case, in which material suitable for electron microscopy was available, Weibel-Palade bodies were found in tumor cells. These findings add strong support to the notion of an endothelial origin of this neoplasm.

Thymic lymphopoiesis: protected from, or influenced by, external stimulation?

The mechanisms regulating thymic lymphopoiesis are still a matter of debate. Intracortical proliferation and differentiation of thymocytes are thought to be controlled by locally produced humoral factors and close contact with epithelial, possibly also phagocytic, cells, and restricted by products of the major histocompatibility complex. The observation of a translocation of intraabdominally introduced PVP-coated silica particles (Percoll) via parathymic lymph vessels and through the thymic capsule into the cortical parenchyma demonstrates that the thymic cortex is accessible to materials carried with the transcapsular flux of interstitial fluid, and that this barrier is less effective than the blood-thymus barrier. The proliferative activity of cortical thymocytes following an intraabdominal injection of particulate tetanus toxoid was compared in sites adjacent to, and distant from, parathymic lymph nodes. Absolute numbers of DNA-synthesizing thymocytes were found to be much higher in cortical areas close to the lymph nodes, where lymphatic vessels are most numerous, than on the opposite sides of the thymic lobes. Taken together, these findings indicate that--in addition to intrinsic control mechanisms--cortical thymocyte production may be influenced by peripheral stimulation to some extent, and that materials from sites which are drained by parathymic lymph nodes may be important in this respect.

Characterization of human skin-derived CD1a-positive lymph cells.

The phenotype and function of CD1a+ lymph cells is of considerable interest. By means of microsurgical lymph cannulation human lymph derived from normal skin was sampled. Cells were isolated and processed for immunocytochemistry, electron microscopy, flow cytometry and functional assays. The majority of the cells, (62%), were T cells. The other cells comprised CD1a+ cells (7%), monocytes/macrophages (8%), and B cells (1%); the remainder were erythrocytes or uncharacterized cells. The CD1a+ cells reacted with antibodies against protein S-100, HLA-DR, the Lag antigen, CD4, CD11a, CD11b, CD18, CD25, CD40, CD54, CD80 and CD86. Interestingly, a small prolow portion the of CD1a+ cells (about 5%) reacted with an antibody to CD14. The CD1a+ cells did not react with an antibody against human follicular dendritic cells nor were they CD19-, CD23-, E-cadherin- or factor XIIIa-positive. Both allogenic and antigen-specific T cell proliferation stimulated by antigen-presenting lymph cells were strongly inhibited by adding anti-CD80 and anti-CD86 antibodies. By electron microscopy Birbeck granules were detected in only 22% of the CD1a+ lymph cells and these cells exhibited an extensive ruffling of the surface. These findings demonstrate that CD1a+ lymph cells, which do not express the dermal dendritic cell marker factor XIIIa, resemble dendritic cells formerly designated as 'veiled' as well as lymphoid dendritic cells, suggesting that after migration to the regional lymphoid organs, Langerhans cells form a more differentiated population of dendritic cells specialized in sensitizing T lymphocytes. Our results add further support to the view that resident Langerhans cells may be precursors of lymphoid dendritic cells acquiring the final phenotype in the microenvironment of the lymph node.

High doses of antigen-nonspecific IgG do not inhibit pemphigus acantholysis in skin organ cultures.

A patient suffering from severe pemphigus vulgaris was treated using large-volume plasma exchange in combination with an immunosuppressive regimen. As some recent reports have shown evidence that polyclonal, polyspecific human IgG in high doses through the i.v. route (IGIV) protect target platelets in idiopathic thrombocytopenic purpura from attack by antiplatelet autoantibodies and/or immune complexes, we also administered IGIV to this pemphigus-vulgaris patient. In order to test the hypothesis that IGIV might protect in vitro-cultured human skin from acantholysis induced by pemphigus antibodies, studies with skin organ cultures were carried out using plasma from another pemphigus-vulgaris patient who had undergone plasma exchange. The preincubation of either the skin explants or the pemphigus plasma with various concentrations of IGIV (ranging from 0.15 to 15 mg/ml in the culture medium) did not prevent acantholysis induced by the pemphigus plasma nor did it inhibit the binding of the specific antibodies visualized by direct immunofluorescence. Thus, the assumption that IGIV may coat the pemphigus antigens on epidermal cells making them inaccessible to pathogenic autoantibodies was not substantiated by our tests in vitro; likewise, the hypothesis of functionally blocking autoantibody activity by means of anti-idiotype effects of IGIV cannot be supported.

Hormonotherapy of meningiomas with medroxyprogesterone acetate. Immunohistochemical demonstration of the effect of medroxyprogesterone acetate on growth fractions of meningioma cells using the monoclonal antibody Ki-67.

The effect of medroxyprogesterone acetate (MPA) on growth fractions of ex vivo meningiomas is demonstrated in using the Ki-67 monoclonal antibody in three cases of meningiomas operated on in two stages and in meningioma specimens from a group of eight patients operated on in one single stage after MPA therapy. Growth fractions in samples from five meningioma patients not treated with MPA were determined for comparison. In the three cases of two-stage operation of the tumors, the percentage of Ki-67-positive cells in meningioma tissue was lower by a factor of 6, 5, and 3, respectively, after MPA therapy. In meningioma specimens from patients receiving no MPA therapy, Ki-67-positive cells were present in 1.02 +/- 0.48%; in samples from MPA-treated tumors the percentage of Ki-67-positive cells was 0.41 +/- 0.40 (different at p less than 0.02 [Wilcoxon's test]). In comparison to our previously published data on untreated meningiomas analyzed for progesterone receptors (PR), MPA significantly reduced the PR activity. There was no obvious correlation between PR activity and potential suppression of the tumor growth fraction. It is concluded that MPA is attractive because it reduces the growth fractions of most meningiomas and might be suitable for adjuvant hormonotherapy.

Autoradiographic study of the localization of 2,2', 4,4', 5,5'-hexachlorobiphenyl in liver and skin tissue after in vitro uptake.

Cellular distribution of the lipophilic environmental pollutant, 2,2', 4,4', 5,5'-hexachlorobiphenyl (6-CB), was determined by autoradiography after in vitro uptake of the 14C-labeled compound into liver and skin tissue preparation. Light microscopic data of cryostat sections showed that 6-CB is homogeneously distributed in liver tissue. In skin the distribution pattern depended on the conditions of incubation. If skin slices were incubated with 6-CB prior to preparation of cryostat thin sections, the epidermis was practically free of 6-CB and the radioactivity was found mainly in the stratum reticulare of the dermis. If, however, cryostat sections of skin were directly incubated, 6-CB was more homogeneously distributed with an accumulation in the epidermis. Liver and skin sections delipidated with acetone or 95% ethanol prior to incubation with 6-CB took up very little of the compound. Delipidation of sections preincubated with 6-CB resulted in total extraction of 6-CB. The results suggest that human stratum corneum is a barrier to the penetration of 6-CB and that this compound is located in lipid structures of liver and skin.

Fibronectin in pemphigus.

In the skin organ culture model of pemphigus, fibronectin concentrations of 300 and 500 micrograms/ml inhibited pemphigus plasma-induced acantholysis and intraepidermal binding of the pemphigus antibodies examined by direct immunofluorescence. A direct interaction of fibronectin with pemphigus antibodies could not be demonstrated by chromatography of pemphigus plasma on immobilized fibronectin or by incubation with fibronectin and subsequent precipitation with antifibronectin. We then measured fibronectin concentrations in the plasma of 15 patients suffering from various types of pemphigus and presenting different activities of their disease. Immunoreactive fibronectin levels in untreated patients with active disease were generally in the low normal or even clearly in the subnormal range. They had the tendency to decrease when the disease subsided during therapy with high doses of glucocorticoids, sometimes in combination with azathioprine. The fibronectin concentration in the blister fluid of a patient with acute, untreated pemphigus vulgaris was similar to that in a plasma sample taken at the same time. Skin biopsies of pemphigus patients exhibited an essentially normal fibronectin pattern in direct immunofluorescence. We discuss a possible protective role of fibronectin in pemphigus e.g., by reducing the permeability for pemphigus antibodies in the dermal-epidermal junction zone.

The role of gut-associated lymphoid tissues in the generation of immunoglobulin-bearing lymphocytes in sheep.

Experiments were designed to examine the relative contributions of Peyer's patches and mesenteric lymph nodes to the population of circulating immunoglobulin-bearing lymphocytes in sheep. The ileum, with more than 90% of the total Peyer's patches, the mesenteric lymph nodes, or both, were removed from lambs at different stages of development and the composition of the cell populations in lymph from different sources and in the blood was examined. Lambs which had had the ileum removed before or within a few days of birth were deficient in small lymphocytes bearing membrane immunoglobulin. This deficit remained for at least the first year of the animals' lives. Neither the removal of mesenteric lymph nodes nor removal of the ileum had any statistically significant effect on the total output of cells or on the population of IgA-producing cells in lymph draining from the gut.

Production and characterization of monoclonal antibodies against human neuroblastoma.

MAb were derived from mice immunized with cells of the human neuroblastoma line IMR-32. Five hybridomas were selected according to their selective binding to human cell lines, tumors and normal tissues. One of them, CE7, reacted with all sympatho-adrenomedullary cells (neuroblastoma, ganglioneuroblastoma, ganglioneuroma, pheochromocytoma, adrenal medulla, sympathetic ganglion cells). Weak cross-reactivities were observed with melanocytes and with some human melanoma and glioma cell lines. The antigen recognized by CE7 was markedly expressed on neuroblastoma tumors of all histological grades, independently of the adrenergic or cholinergic nature of these cells. MAb derived from clones AD2, BC1, BC4 and CB10 bound variably to some, but not to all, neuroblastoma cells. By using these MAb, 3 phenotypes of neuroblastoma lines could be distinguished. The binding profiles of these types, however, showed no correlation with origin of the cell lines or stage of the disease.

[Immunohistochemical findings in otosclerotic lesions]. / Immunohistochemische Befunde in otosklerotischen Läsionen.

Despite numerous scientific efforts, the etiology of otosclerosis still remains unknown. Pathogenically, there are several signs of a chronic inflammatory process of the bony otic capsule. In this study, we tried to characterize the components of chronic inflammation by immunohistochemical techniques. Within otosclerotic lesions a mixed cellular infiltrate can be observed, consisting of lymphocytes, macrophages and plasma cells. Macrophages which are capable of presenting antigen in association with major histocompatibility antigens (MHC) class I and class II to CD8(+)-, and CD4(+)-T cells, respectively, were found in otosclerotic lesions based on their expression of the MAC387 antigen. Furthermore, HLA-DR positive cells and complement C3 have been found in resorption lacunae of otosclerotic lesions. Several osteoblasts and chondrocytes in active otosclerotic lesions reveal a strong surface expression of beta-2-microglobulin, indicating an increased MHC class I antigen expression in active otosclerotic lesions. In agreement with recently published data we found that a large fraction of the lymphoid cells are antigen-primed T-cells expressing an alpha/beta T-cell receptor in association with CD3 molecules on their surfaces. CD4+ lymphocytes which functionally represent lymphokine-secreting cells are activated through the specific recognition of antigen, presented in context with MHC class II molecules such as HLA-DR. Therefore, the presence of MHC class II positive cells are crucial for the initiation of a local immune response. Thus, our observation of HLA-DR positive cells in otosclerotic lesions is of particular interest.(ABSTRACT TRUNCATED AT 250 WORDS)

Overexpression of the c-erbB-2 protein in human breast tumor cell lines.

The c-erbB-2 proto-oncogene is amplified in a high percentage of primary human breast tumors, suggesting that the overexpression of this gene may be involved in the development of human breast cancer. We have investigated five human breast tumor cell lines and have detected amplified c-erbB-2 gene copies in two of them. This amplification leads to overexpression of the c-erbB-2 protein. In addition, two other cell lines have elevated protein levels without gene amplification, suggesting that other mechanisms can lead to overexpression of the c-erbB-2 protein. These results are similar to those that we obtained during a study of primary breast tumors (Berger et al.: Cancer Res 48:1238-1243, 1988). These breast tumor cell lines should be useful for an analysis of c-erbB-2 expression and of the mechanisms that in some cases lead to overexpression.