Indirect Immunoperoxidase Technique Research Articles

Developmental expression of synaptophysin, synapsin I and syntaxin in the rat retina

Expression of synaptophysin, synapsin I and syntaxin was studied immunocytochemically in the developing rat retina using indirect immunoperoxidase technique. In the inner plexiform layer (IPL), syntaxin immunoreactivity appeared at postnatal day 1 (P1) whereas synaptophysin and synapsin I staining were first observed at P2. In the outer plexiform layer (OPL), synaptophysin appeared at P4, while synapsin I and syntaxin appeared at P8. In the case of synaptophysin, a punctate pattern of staining was observed from the time of its appearance (P4) in the OPL and from P12 onwards in the IPL. Synapsin I and syntaxin immunoreactivity in the OPL were of a low intensity throughout the development and in the adult stage. These findings are discussed in relation to synaptogenesis in the rat retina.

Binding of a human monoclonal antithyroglobulin antibody to cultured human thyroid cancer cells.

To develop a new method of radioimmunodetection for thyroid cancer, we tested the binding ability of a human antithyroglobulin monoclonal antibody, VB5, to primary culture of human thyroid cancer cells. VB5 was able to immunostain cytoplasmic thyroglobulin (Tg) in the acetone-fixed cancer cells when used in a labeled streptavidin-biotin method but not in a conventional indirect immunoperoxidase technique. The antibody was readily labeled with I-125 in the standard chloramin-T method, and showed specific binding to the antigen on cultured malignant thyrocytes displaceable with non-labeled VB5 or with excess Tg antigen. Although these initial results in vitro are encouraging, the observed low specific binding (about 1% at room temperature) to intact cells with a single monoclonal antibody seems insufficient to conduct any in vivo immunolocalization experiments in animals. To obtain more binding, we would need a cocktail of several monoclonal antibodies to different epitopes, and also fragmentation of antibody molecules to penetrate into cytoplasm.

ACTH/CLIP Immunoreactivity in the Cat Brain Stem

COVEÑAS, R., M. de LEÓN, J. A. NARVÁEZ, J. A. AGUIRRE, G. TRAMU AND S. GONZÁLEZ-BAŔON.ACTH/CLIP immunoreactivity in the cat brain stem.PEPTIDES 18(7) 965–970, 1997.—The distribution of adrenocorticotropin hormone/corticotropin-like intermediate lobe peptide was studied in the cat brain stem, using an indirect immunoperoxidase technique. No immunoreactive cell bodies were observed. However, a high density of immunoreactive fibers was found in the periaqueductal gray, the dorsal nucleus of the raphe, the locus coeruleus, and the marginal nucleus of the brachium conjunctivum. A moderate density was found in the central linear nucleus, the central tegmental field, the Kölliker–Fuse nucleus, the inferior central nucleus, and the postpyramidal nucleus of the raphe. A low density was found in the superior and inferior colliculi, the interpeduncular nucleus, the nucleus sagulum, the superior central nucleus, the cuneiform nucleus, the accessory dorsal tegmental nucleus, the nucleus of the solitary tract, the dorsal motor nucleus of the vagus, and the paralemniscal, magnocellular, gigantocellular, and lateral tegmental fields. Moreover, single immunoreactive fibers were observed in numerous nuclei of the cat brain stem. In comparison with previous studies carried out in the same region of the cat, as well as the rat and the human, our results point to a more widespread distribution of adrenocorticotropin hormone/corticotropin-like intermediate lobe peptide immunoreactive structures in the cat brain stem. This widespread distribution indicates that the peptide might be involved in several physiological functions of the cat brain stem.

Anomalous expression of costimulatory molecules B7-1, B7-2 and CD28 in primary biliary cirrhosis

Background: T lymphocytes require two important signals for efficient activation: 1) recognition of antigens bound to self major histocompatibility complex antigens, and 2) simultaneous stimulation via so-called costimulatory molecules. Interaction of the costimulatory B7 molecules on antigen presenting cells with CD28 on T lymphocytes appears to be particularly important, as it modifies secretion of cytokines, especially interleukin 2. In primary biliary cirrhosis biliary epithelial cells aberrantly express major histocompatibility complex class II antigens and may function as antigen presenting cells. Methods: We studied expression of HLA-DR, B7-1, B7-2 and CD28 on cryostat liver sections in 16 patients with primariy biliary cirrhosis, three patients each with autoimmune hepatitis and primary sclerosing cholangitis and nine patients with chronic viral hepatitis (five hepatitis B, four hepatitis C) using mouse monoclonal antibodies in an indirect immunoperoxidase technique. Results: In advanced primary biliary cirrhosis, HLA-DR was found on 57% of bile ducts, B7-2 on 5% of bile ducts, and B7-1 could not be detected on any bile duct. Neither B7-1 nor B7-2 was seen on bile ducts in the four patients with early primary biliary cirrhosis. HLA-DR+ bile ducts also lacked expression of B7 molecules in autoimmune hepatitis. In contrast, HLA-DR, B7-1 and B7-2 were expressed simultaneously on professional antigen presenting cells such as macrophages in epitheloid granulomas. Conclusion: HLA-DR+ biliary epithelial cells in primary biliary cirrhosis insufficiently co-express B7-1 or B7-2 molecules. Therefore, they must either use different costimulatory molecules, or otherwise are deficient in lymphocyte activation. Since recognition of antigen in the absence of B7-CD28 interaction may lead to anergy of lymphoyctes, this might contribute to the impaired cytokine secretion found in primary biliary cirrhosis.

Prolactin receptor in rat bile ducts in ontogeny

Expression of prolactin receptors (PR) in the nuclei and plasma membrane of the bile duct cells is demonstrated in 1-45-day-old male and female rats with the use of indirect immunoperoxidase technique. The intensity of PR staining in the bile duct cells is not sex-dependent; it increases from the 1st day till the 45th day and disappears by the 65th day of postnatal ontogeny. The PR-specific staining of hepatocytes also increases during the first 45 days of postnatal life and becomes sex-dependent by the 65th day.

Transcytosis of Protein through the Mammalian Cerebral Epithelium and Endothelium: III. Receptor-Mediated Transcytosis through the Blood–Brain Barrier of Blood-Borne Transferrin and Antibody against the Transferrin Receptor

Diferric-transferrin (Tf; 80K mol. wt.) and the OX26 antibody (150K mol. wt.) against the transferrin receptor (TfR) were evaluated in the rat at light and ultrastructural levels as potential vehicles for the blood to brain transcellular transfer (transcytosis) of native horseradish peroxidase (40K mol. wt.), which by itself does not cross the blood–brain barrier (BBB). OX26, the Fab fragment of OX26 (50K mol. wt.), and Tf complexed to two ferric ions were conjugated to HRP irreversibly in a 1:1 molar ratio. The indirect immunoperoxidase technique with OX26 as the monoclonal primary antibody applied to the surface of cryostat sections or delivered intravenously to the live rat revealed TfRs on BBB capillaries, arterioles, and venules; TfRs were absent on non-BBB vessels supplying the circumventricular organs (i.e., median eminence, choroid plexus). OX26–HRP and OX26(Fab)–HRP delivered intravenously and diferric-Tf–HRP administered into the carotid artery labeled BBB vessels throughout the CNS without discernible disruption of the BBB or extravasation of the blood-borne probes into the brain parenchyma. No reaction product for the probes was observed in sites deficient in a BBB. Each of the macromolecular conjugates was endocytosed by BBB endothelia and labeled presumptive endocytic vesicles, endosomes, and dense bodies. OX26–HRP and Tf–HRP, but not OX26(Fab)–HRP, appeared to undergo transcytosis through BBB endothelia for subsequent labeling of perivascular cells. Distinct differences in the intracellular and extracellular distributions between OX26–HRP and Tf–HRP were identified: (1) endocytosis and sequestration of blood-borne OX26–HRP within BBB endothelia were more prominent than those for diferric-Tf–HRP; (2) only OX26–HRP labeled the Golgi complex in BBB endothelia; (3) peroxidase labeling of CNS perivascular clefts and perivascular cells in rats receiving diferric-Tf–HRP was conspicuous at less than 1 h postinjection but not so in rats with blood-borne OX26–HRP at 5 min through 6 h postinjection; and (4) peroxidase-labeled CNS neurons and glial cells were identified readily in rats receiving diferric-Tf–HRP. The results suggest that the receptor-mediated, transendothelial transfer of Tf–HRP from blood to brain is more efficient and direct than that of OX26–HRP. Labeling of the Golgi complex in BBB endothelia with blood-borne OX26–HRP implies that the transendothelial transfer of OX26–HRP follows intraendothelial pathways associated with the process of adsorptive transcytosis. A diagram is provided depicting the possible intracellular and transcellular pathways within BBB endothelia available to blood-borne diferric-Tf and OX26 as vectors for delivery into the CNS of non-lipid-soluble macromolecules that otherwise are denied entry by the blood–brain fluid barriers.

SERUM LEVELS AND IN SITU EXPRESSION OF TNF-α AND TNF-α BINDING PROTEINS IN INFLAMMATORY LIVER DISEASES

Cells respond to tumour necrosis factor-α (TNF-α) via binding to 75-kDa (type A) and 55-kDa (type B) receptors which have different intracellular signalling pathways and can also circulate as soluble molecules. Both receptors are co-expressed in many tissues, but their relative contributions to cellular TNF responses is for most situations unknown. In patients with viral and non-viral inflammatory liver diseases serum TNF-α was determined by an immunoenzymetic assay and soluble type A and B TNF receptors (TNF-αr) by enzyme linked immunological and biological assays (ELIBA). In addition, cellular expression of TNF and its binding proteins were studied in liver biopsies by an indirect immunoperoxidase technique. Secretion of TNF-α and upregulation of TNF-αr-A were particularly prominent in viral hepatitis. Strong TNF-α-in-situ production by mononuclear cells could be demonstrated in liver biopsies from patients with acute viral hepatitis. However, TNF-αr-A was detected only on hepatocytes. Serum TNF-αr-A was elevated two-fold in relative abundance over TNF-αr-B and was correlated to serum TNF-α ( r= 0.6464, P< 0.0001). Soluble TNF-αr levels normalized, when the viral hepatitis was cleared, and successful therapy of hepatitis B was associated with a temporary rise of TNF-αr-A during the initial flare of aminotransferases. Patients with alcoholic hepatitis had also evidence of TNF-α activation but clearly differed from patients with viral induced liver diseases: Soluble TNF-αr-A and TNF-αr-B were highly elevated in equal proportions. In situ analysis in liver biopsies revealed a distinctive pattern of TNF-αr expression with strong cytoplasmic staining for both TNF-αr-A and B on scattered hepatocytes in addition to infiltrating mononuclear cells. The data propose that TNF release during antiviral immune responses is predominantly associated with TNF-αr-A upregulation and shedding, whereas upregulation and shedding of TNF-αr-B is more prominent in alcoholic hepatitis. As cytotoxicity and apoptosis by TNF are mediated mainly via TNF-αr-B, our results are consistent with more severe TNF-α induced liver damage in alcoholic hepatitis as compared to viral hepatitis.

The mast cells of the multiple sclerosis brain

Traditional staining methods, plus indirect immunoperoxidase techniques for IgE and mast cell tryptase (MC Tr) were used to study the mast cells (MCs) of multiple sclerosis (MS) and normal brains. The MCs varied in number in MS amongst perivascular inflammatory cells as well as free in the parenchyma, especially inside and around ‘chronic active’ plaques. Since MCs do not migrate, and rarely divide in maturity, they must have developed locally. Staining for IgE was moderately strong on and within MCs, and weak within some plasma cells. MC Tr reacted strongly both within CNS and outside it. Being a strong neutral proteinase, MC Tr, plus IgE, could conceivably have played some role in the pathogenesis of the MS plaques.

Immunohistochemical characterization of hepatic lymphocytes in acute hepatitis A, B, and C.

The distinctive histologic findings in acute hepatitis A, B, and C suggest that different immunological mechanisms are involved in the pathogenesis of these diseases. This study was undertaken to define the immune response in each type of acute hepatitis by identification of the intrahepatic lymphocyte subsets. Thirty paraffin-embedded liver biopsy specimens from 10 patients with acute hepatitis A, 10 patients with acute hepatitis B, and 10 patients with acute hepatitis C were evaluated. Immunohistochemical staining was performed by the indirect immunoperoxidase technique using the following monoclonal or polyclonal antibodies: CD45RO, CD20-cy, CD57, and Mac387. Inflammatory infiltrates varied from specimen to specimen. However, CD45RO+ memory T cells were the predominant infiltrating mononuclear cells in all specimens. In the portal areas, CD45RO+ memory T cells were the most prominent in AHC, followed by AHA and AHB, and the difference between AHC and AHB was statistically significant. CD20-cy+ B cells were seen mainly in the portal areas, and were significantly less common in AHB than in AHA and AHC. In addition, the ratio of CD20-cy+ B cells to CD45RO+ memory was significantly lower in AHB than in the other types of acute hepatitis. The necrotic areas in all specimens contained mainly CD45RO+ memory cells in association with a few CD57+ NK and T cells or CD20-cy+ B cells. Our study revealed differences of the intrahepatic lymphocyte subsets among the various types of acute hepatitis, but the meaning of these differences is presently unknown. Therefore, further studies are required to fully elucidate the mechanism of the immune response in acute hepatitis.

An immunocytochemical mapping of ACTH/CLIP in the cat diencephalon

Using an indirect immunoperoxidase technique, the location of cell bodies and fibres containing adrenocorticotropin hormone/corticotropin-like intermediate lobe peptide (ACTH/CLIP) was studied in the cat diencephalon. Immunoreactivity was observed in several diencephalic nuclei of the cat in which no immunoreactivity has been previously reported. In this sense, a low density of immunoreactive cell bodies was found in the nucleus ventromedialis hypothalami; a high density of immunoreactive fibres was found in the medial preoptic area; a moderate density in the lateral preoptic area and in the nuclei centralis thalami (pars medialis), interventralis thalami, interanteromedialis thalami, parafascicularis and praemamillaris (pars ventralis and pars dorsalis); a low density in the nuclei habenularis lateralis and reuniens thalami, and single fibres were found in the nuclei lateralis thalami (pars anterior), habenularis medialis, parataenialis, corpus geniculatum mediale, ventralis thalami (pars medialis) and in the fornix. Our results point to a more widespread distribution of ACTH/CLIP immunoreactive structures in the cat diencephalon in comparison with previous studies carried out in the same region of this feline.

Autoimmunity to the GM2-1 Islet Ganglioside Before and at the Onset of Type I Diabetes

Recently, the GM2-1 pancreatic islet ganglioside, proposed as a potential autoantigen in type I diabetes autoimmunity, has been biochemically characterized and found to be a novel ganglioside structure. In the present study, we aimed to determine whether an autoimmune response toward this novel islet molecule is 1) present in type I diabetes and is specifically directed against this molecule and not to gangliosides in general and 2) predictive of disease in high-risk subjects. To this end, the following patients have been studied: 1) 24 newly diagnosed type I diabetic subjects, 20 islet cell autoantibody (ICA)-negative first-degree relatives of type I diabetic subjects, and 25 age-matched normal control individuals; and 2) 31 prospectively evaluated ICA+ first-degree relatives of type I diabetic subjects who were followed for up to 10 years, during which 14 of them developed type I diabetes. A direct assay for autoantibodies to GM2-1 and to other pancreatic gangliosides (GM3, GD3, GD1a) was developed using an indirect immunoperoxidase technique performed directly on thin layer chromatography plates. Anti-GM2-1 autoantibodies (all belonging to the IgG class) were expressed in a high percentage of newly diagnosed type I diabetic subjects (71%), while no significant difference was found in the expression of antibodies directed against other pancreatic gangliosides (GM3, GD3, GD1a) among the different groups studied. Anti-GM2-1 autoantibodies were also present in ICA+ relatives (64%) (P < 0.001 vs. control subjects and ICA-relatives): in this group, life table analysis of progression to diabetes showed that anti-GM2-1 autoantibodies were significantly (P < 0.001) associated with disease, occurring in all relatives developing type I diabetes within 5 years and thus identifying a cohort of ICA+ subjects with markedly increased diabetes risk.

Mouse fetal antigen 1 (mFA1), the circulating gene product of mdlk, pref-1 and SCP-1: isolation, characterization and biology.

The mouse homologue to human fetal antigen 1 (hFA1) was purified from mouse amniotic fluid by cation exchange chromatography and immunospecific affinity chromatography. Mouse FA1 (mFA1) is a single chain glycoprotein with an M(r) of 42-50 kDa (SDS-PAGE). The N-terminal amino acid sequence (39 residues) revealed 74% identity to hFA1 and 100% identity to the translated cDNAs referred to as mouse dlk, pref-1 and SCP-1. mFA1 is the secreted processed molecule encoded by the mRNA defined by these identical mouse cDNAs. Monospecific rabbit anti-mFA1 antibodies, purified by ammonium sulfate precipitation and immunospecific affinity chromatography, were used for immunohistochemical and quantitative ELISA techniques. The indirect immunoperoxidase technique demonstrated mFA1 within the endocrine structures of adult mouse pancreas, whereas the exocrine tissue remained unstained. FA1-positive staining was also seen in the pituitary gland and the mouse adrenal gland. The concentration of mFA1 in a pool of amniotic fluid was assessed at 25 micrograms ml-1 and the serum concentration in normal nonpregnant adult mice (n = 28) was 11.3 +/- 5.0 ng ml-1 (2 SD). During pregnancy the concentration of mFA1 in maternal serum increased above the nonpregnant reference value at midpregnancy, reaching a maximum concentration (> 0.35 microgram ml-1) 2 days prepartum. The maternal serum concentration was positively correlated with the number of fetuses. After delivery the rate of disappearance of mFA1 in maternal serum was very fast with a t1/2 < 1 h. The concentration of mFA1 in newborn mice was about 15 micrograms ml-1 and did not reach normal adult values until the age of > 50 days.

An immunocytochemical mapping of β-endorphin (1–27) in the cat diencephalon

The distribution of β-endorphin (1–27) immunoreactive cell bodies and fibres was studied in the diencephalon of the cat using an indirect immunoperoxidase technique. In the thalamus, almost all the immunoreactive fibres were found in the midline region and in nuclei located near the midline, whereas in the hypothalamus fibres containing β-endorphin (1–27) were visualized extending by the whole structure. The hypothalamus showed a higher density of β-endorphin (1–27) immunoreactive fibres than the thalamus, as well as immunoreactive cell bodies, since in the thalamus no β-endorphin (1–27) immunoreactive neuron was located. The densest network of immunoreactive fibres was observed in the epithalamus (nucleus periventricularis anterior) and in the hypothalamic nuclei arcuatus, hypothalami ventromedialis, suprachiasmaticus, periventricularis hypothalami, hypothalamus dorsomedialis, area hypothalamica dorsalis, hypothalamus anterior, filiformis, hypothalamus posterior and regio praeoptica. In the hypothalamus, a high density of perikarya containing β-endorphin (1–27) was observed in the nucleus arcuatus and a low density in the nucleus hypothalami ventromedialis. The distribution of β-endorphin (1–27) immunoreactive fibres and perikarya is compared with the location of other neuropeptides in the cat diencephalon. Our findings reveal that b-endorphin (1–27) immunoreactive structures are widely distributed in the cat diencephalon, suggesting that the peptide might be involved in several physiological functions.

Distribution of von Willebrand factor in capillary endothelial cells of rat lungs with pulmonary fibrosis

To determine the value of von Willebrand factor (vWF) antigen as a marker of endothelial injury in radiation-induced fibrosis of rat lungs, we studied endothelial immunoreactivity to antibodies against vWF using the indirect immunoperoxidase technique combined with morphometric analysis. Using immunoelectron microscopy of LR White embedded lung samples to detect vWF, immunogold-labelled Weibel Palade bodies were found in endothelial cells of capillary endothelium. The irradiated lungs showed a statistically significant elevation of vWF expression, ie, vWF positive endothelia per unit area, and a significant increase of vWF expression per unit of parenchyma as well. The results suggest that vWF antigen expression and the number of vWF positive structures is modulated under condition of injury in radiation-induced fibrosis.

Immunotyping of blasts in human bone marrow.

The diagnostic potential of immunocytochemical investigation of human bone marrow has not been fully realized due to difficulties in morphological identifying of immunostained cells. We used an indirect immunoperoxidase technique after May-Grünwald-Giemsa staining for simultaneous morphological and immunocytochemical analysis of blasts in human bone marrow. Six healthy bone marrow donors were investigated. Most blasts I expressed CD34, CD38 and HLA-DR. Expression of c-kit (CD117) was observed on 42 +/- 9% of blasts I. Granulocytomonocytopoietic character was demonstrated by expression of CD13 (33 +/- 15%) and CD45RA (23 +/- 10%) and erythropoietic character was demonstrated by expression of CD36 (22 +/- 8%) and CD45RO (30 +/- 11%). A very low proportion of blasts I were Thy-1 and CD61 positive; 34 +/- 6% of blasts I expressed CD22, representing B lymphoid committed progenitors. CD3, CD15, and glycophorin A expression was not detected. Blasts II and III and proerythroblasts did not show CD34 positivity. We conclude that blasts I are morphologically identifiable cells with a high percentage of CD34, CD38, and HLA-DR positivity. They are a pool of committed progenitor cells for erythropoiesis, granulocytomonocytopoiesis, megakaryocytopoiesis, and B cell development. Blast II and proerythroblast represent the first morphologically identifiable cells of granulocytopoiesis and erythropoiesis.

Adenocarcinoma of duodenum and ampulla of Vater: clinicopathology study and expression of p53, c-neu, TGF-alpha, CEA, and EMA.

Oncogenes, tumor suppressor genes, and growth factors are being explored as to their role in the initiation and progression of most neoplasms, but little information exists on the expression of oncoproteins or growth factors in adenocarcinoma of the duodenum or ampulla of Vater. This report covers expressions of p53, c-neu, TGF-alpha, CEA, and EMA in duodenal adenocarcinoma and ampullary adenocarcinoma, as well as correlations between expressions and tumor stage, histological grade and patient survival. The expression of p53, c-neu, TGF-alpha, CEA, and EMA has been studied in 15 duodenal adenocarcinomas and in eight ampullary adenocarcinomas by avidin-biotin-peroxidase complex indirect immunoperoxidase technique. The positive reaction for p53, c-neu, TGF-alpha, CEA, and EMA in duodenal adenocarcinoma was 20%, 60%, 60%, 73%, and 100%, respectively, and in ampullary adenocarcinoma, 13%, 100%, 50%, 63%, and 100%. Among the duodenal tumors, C-neu and p53 expression was noted more frequently in groups with high histological grades. Patients with c-neu positive duodenal adenocarcinoma had a shorter survival than the patients with c-neu negative duodenal adenocarcinoma (P < 0.01). C-neu product may serve as an unfavorable prognostic indicator in duodenal adenocarcinoma. No statistically significant correlation was found between the expressions of CEA, EMA, p53, and TGF-alpha and patient survival, tumor stage, or histological grade in either duodenal or ampullary adenocarcinomas.

Mapping of α-melanocyte-stimulating hormone-like immunoreactivity in the cat diencephalon

Using an indirect immunoperoxidase technique, we studied the location of α-melanocyte-stimulating hormone-like fibers and cell bodies in the cat diencephalon. In the thalamus, almost all the immunoreactive fibers were found in the midline region, whereas in the hypothalamus immunoreactive fibers were observed in the whole structure. The hypothalamus showed a higher density of both immunoreactive fibers and cell bodies; no immunoreactive neurons were found in the thalamus. The densest network of immunoreactive fibers was observed in the epithalamus (nucleus periventricularis anterior) and in the hypothalamic nuclei filiformis, hypothalami ventromedialis, arcuatus, periventricularis hypothalami, area hypothalamica dorsalis, and hypothalamus posterior. A high density of immunoreactive neurons was found in the nucleus arcuatus, in the hypothalamus lateralis, and in the area hypothalamica dorsalis; a low density was found in the nucleus hypothalami ventromedialis and in the hypothalamus dorsomedialis. By comparison with the studies of previous researchers, these data showed a more widespread distribution of α-melanocyte-stimulating hormone-like immunoreactive fibers and perikarya in the feline hypothalamus. Moreover, our findings indicate that the peptide is widely distributed in the cat diencephalon, suggesting that α-melanocyte-stimulating hormone might be involved in several physiological functions.

Differential expression of HLA-A and B-alleles on uveal melanoma as determined by immuno-histology

HLA molecules play an important role in the presentation of antigens to the immune system, including tumor-specific antigens. Uveal melanomas vary in the level of expression of monomorphic HLA molecules. However, since the HLA system is polymorphic and since antigen-presentation may be linked to the expression of specific HLA alleles, the authors wondered whether allelic differences in expression existed on uveal melanomas. In order to test this, tissue sections from 23 uveal melanomas were stained in an indirect immunoperoxidase technique with monoclonal antibodies against monomorphic and polymorphic determinants of HLA molecules. All uveal melanomas showed a high level of expression of the monomorphic determinants of HLA-Class I. The polymorphic HLA-Class I molecules A2, A3, Bw4 and Bw6 varied in expression, with a higher expression of HLA-A than of HLA-B. A low level of expression of both ß2-microglobulin and HLA-B locus products was associated with a large tumor diameter. Expression of HLA-Class II molecules was low (0 to 35%). The observation that expression of the HLA-A allelic products was higher than of the HLA-B subtypes may have implications for the search of tumorspecific peptides for immunotherapeutic use: it may be worthwile to select peptides that specifically bind to HLA-A and not to HLA-B.

T-lymphocyte immunophenotyping in polymyositis and dermatomyositis.

The objective was to elucidate the immunological abnormalities underlying polymyositis (PM) and dermatomyositis (DM). The phenotype of peripheral blood mononuclear cell (PBMNC) subsets and cell surface expression of activation (CD25 and HLA-DR) and adhesion (LFA-1) molecules was studied in 12 patients with PM and in 10 patients with DM. PBMNC subsets and expression of T-cell activation molecules were evaluated by cytofluorography. Double immunofluorescence and indirect immunoperoxidase techniques were applied to muscle biopsies to define T-cell phenotype and LFA-1/ICAM-1 expression. In PM, the absolute number of circulating cytotoxic (CD8+CD28+) T cells was selectively reduced. T cells showed increased expression of activation molecules, CD25 and HLA-DR, and increased adhesion capacity as the absolute numbers of CD3+CD25+, CD8+HLA-DR+, CD3+LFA-1+('bright') and CD8+ CD8+LFA-1+('bright') cells were higher than in healthy donors and DM patients. In PM muscle biopsies, T cells were mainly CD3+CD8+ and LFA-1+; additionally, endothelial cells and myofibres surrounded by T cells showed positive staining for ICAM-1. In DM, there was a general lymphopenia that led to a decreased absolute number of all T-lymphocyte subsets. It is proposed that in PM, in contrast to DM, LFA-1/ICAM-1 interactions enable activated CD8+ T cells to migrate selectively into the inflamed muscle and to adhere to myofibres, leading to tissue injury.

DIFFERENTIAL EXPRESSION OF ADHESION MOLECULES DURING RAT SMALL BOWEL ALLOGRAFT REJECTION

The adhesion molecules intercellular adhesion molecule 1 (ICAM-1), lymphocyte function-associated antigen-1 (LFA-1) (alpha and beta chains), and very late activation antigen-4 (VLA-4) have an essential role in cell-cell interactions and the initiation of immune responses. This study used an indirect immunoperoxidase technique to investigate the expression of these molecules in the lamina propria of allografts and isografts after heterotopic rat small bowel transplantation. Normal untransplanted small bowel served as additional controls. Overall, ICAM-1 and LFA-1 alpha expression was significantly higher in allografts, although there was variable expression of these molecules in isografted animals. There were temporal differences in the expression of ICAM-1 and LFA-1 alpha in that increased ICAM-1 expression was more pronounced in the the early posttransplant period, whereas there was a progressive increase in LFA-1 alpha as rejection developed. In contrast, there was no difference between allograft and isograft expression of LFA-1 beta and VLA-4. This study has demonstrated a preferential increase in adhesion molecule expression with developing rat small bowel allograft rejection and suggests that adhesion molecules are involved in the development and progression of allograft rejection. Although the observed differences in antigen expression are not as marked as those previously reported in other organ transplants, appropriate adhesion molecules may present suitable targets for immunotherapeutic protocols after small bowel transplantation.