Immunohistochemical differentiation between lymphangiographically verified lymphatic vessels and blood vessels

Summary In order to investigate the antigen profile in human lymphatic vessels when compared with blood vessels, postmortem retrograde lymphangiography was done via the thoracic duct on six patients. Formalin fixed, paraffin embedded tissue was stained immunohistochemically for Factor VIII-Related antigen (F VIII R:Ag), with Ulex Europaeus 1 lectin (UEA-1) and for laminin. The results show that the endothelium of blood vessels and lymphatics at all levels of the lymphatic system react positively following staining for Factor VIII-R:Ag and with UEA-1 lectin. The staining for F VIII R:Ag was generally weaker in the endothelial cells lining lymphatic vessels. Staining for the basement membrane component laminin can be used to distinguish lymphatic capillaries and smaller lymphatic collecting vessels from blood vessels.     

Branchial cleft cysts. Histologic and immunohistochemical aspects

8 cases of branchial cleft cysts and 1 case of branchiogenic carcinoma were examined immunohistochemically for detectable keratin, immunoglobulins (IgG, IgA, IgM), carcinoembryonic antigen (CEA), factor VIII related antigen (Factor VIII RGA), and lysozyme. Lectin binding patterns were also determined. Histologically, cystic lining epithelia were classified into stratified squamous epithelium without keratinization, columnar epithelium with or without cilia, or a mixture of both. Almost all of the cases indicated accompanying lymphoid structures with germinal centers. Keratin expression in epithelial cells was slightly positive, and lectin binding affinities in them were similar to those of oral squamous epithelium. CEA was found on the surface border of columnar epithelial cells, but cystic epithelia in most of the cases were devoid of lysozyme. Endothelial cells of capillary vessels showed positive binding by UEA-1 lectin and the presence of factor VIII RAG. In the lymphoid structures, there were scattered strongly positive lysozyme-staining cells as well as a few lymphocytes bearing IgG, IgA, or IgM.     

Different bindings to lectin in human submandibular gland after enzymatic digestion

Lectin binding affinities were described in human submandibular gland (SMG) in the paraffin sections following alpha-amylase, sialidase, and trypsin digestions. Lectins in the present study were used Con A (Glc, Man binding lectins), PNA, and SBA(Gal, GalNAc), RCA-1(Gal), DBA(GalNAc), WGA(GlcNAc), and UEA-1(Fuc). Lectin stainings in serous and mucous acinar cells and ductal epithelia were reported to compare enzyme treated and nontreated sections. Amylase treatment showed increasing Con A staining in connective tissue fibers and no marked changes in SMG to lectin bindings. Sialidase digestion was characteristically intense in PNA and SBA bindings in SMG cells, and also enhanced staining to UEA-1 in serous and duct cells and to WGA in mucous and duct cells were noted. Trypsin digestion indicated a slight increase to Con A binding, and was relatively strong to UEA-1 in serous and duct cells and a little strong to WGA. The results suggested that SMG serous cells contain higher amounts of Gal, GalNAc, and Fuc residues; and mucous cells were also abundant in Gal, GalNAc, and GlcNAc residues.     

Comparison of Ulex europaeus I lectin binding and factor VIII-related antigen as markers of vascular endothelium in follicular carcinoma of the thyroid

Factor VIII-related antigen (F VIII-RAg), a well established marker for endothelial cells, and the lectin Ulex europaeus agglutinin I (UEA-1), a newly described marker, were used in immunoperoxidase techniques to demonstrate endothelial cells in 30 follicular neoplasms of the thyroid and so to assist in detection of vascular invasion. Both endothelial cell markers made visible a greater number of instances of vascular invasion than were detected on routine elastic stains, but UEA-1 lectin gave more reliable staining of endothelium lining large capsular veins and of blood vessels within the tumour than did F VIII-RAg. In vessels completely occluded by tumour, examples of endothelial staining by UEA-1 in the absence of F VIII-RAg staining were found, but some such vessels appeared to lack surviving endothelial cells and in these no staining occurred. It is concluded that UEA-1 lectin is a more reliable endothelial marker in this setting than F VIII-RAg and may assist the demonstration of vascular invasion in these tumours.     

Histochemical studies of lectin binding patterns in keratinized lesions,including malignancy

Summary Histochemical detection of lectin binding was carried out using the HRP-conjugated lectin method in hyperkeratinized lesions including leukoplakia,carcinoma in situ, Paget's disease, keratoacanthoma, and condyloma acuminatum. The lectins used for demonstrating sugar residues were: Con A (hexose), PNA and RCA-1 (Gal), DBA and SBA (GalNAc), UEA-1 (Fuc), and WGA (GlcNAc).Lectin binding in normal squamous epithelium showed regional distribution patterns of keratinized, spinous and basal layer types. Histo-chemical localization of lectin binding was generally at the cellular surface and in the intercellular substance and sometimes in the cytoplasm of normal epithelial cells. Dysplastic cells or carcinoma cell, in contrast, displayed a loss of cellular surface and intercellular staining. Paget's cells were devoid of lectin staining. In keratoacanthoma and condyloma specimens, spinous cells, which were PAS-positive, showed an intense PA/Con A-HRP staining and moderate binding by other lectins, which was somewhat decreased when compared with that in the surrounding intact epithelium. The cytochemical distribution of epithelial lectin binding might be indicative of the expression of normal stratification and keratinocytic differentiation, and the disappearance of this typical epithelial pattern may suggest severe dysplasia and malignancy.Abbreviations HRP horseradish peroxidase - PBS phosphate-buffered saline - DAB diamino-benzidine - Con A Concanavalin A - PNA peanut lectin,Arachis hypogaea - RCA-1 Caster bean,Ricinus communis - DBA Horse gram,Dolichos biflorus - SBA Soybean,Glycine max - UEA-1 Gorse seed,Ulex europeus - WGA Wheat germ,Triticum vulgaris - Gal d-galactose - GalNAc n-acetyl-d-galactosamine - Fuc l-fucose - GlcNAc n-acetyl-d-glucosamine     

Lectin binding properties of cells from serous effusion and peritoneal washing specimens.

The diagnostic value of staining cells from serous effusion and peritoneal washing specimens with a panel of four lectins was investigated and the results compared with those achieved with polyclonal anti-carcinoembryonic antigen (anti-CEA) sera. Cell blocks from 42 pleural effusions, 25 peritoneal effusions, and 14 peritoneal washing specimens were stained with Con A (D-mannosyl, D-glucosyl), WGA (n-acetyl glucosamine), UEA-1 (L-fucose), SBA (n-acetyl galactosamine) and anti-CEA. Con A and WGA were not useful in discriminating mesothelial cells from adenocarcinomas. In contrast, UEA-1 and SBA binding was present in 30 of 46 (65%) and in 31 of 46 (67%), respectively, of adenocarcinomas tested, but negative in 21 cases with reactive mesothelial cells, 10 cases with benign mesothelial cells, and one case of mesothelioma. All mesothelial cells were also negative for CEA, but 24 of 46 (52%) of adenocarcinomas were positive. All three cases of lymphoma were negative with UEA-1, SBA, and anti-CEA. UEA-1 and SBA lectins identified a higher proportion of metastatic adenocarcinomas than CEA and stained most cases of adenocarcinomas metastatic from sites which usually fail to express CEA. Combination of staining results for UEA-1, SBA, and anti-CEA produced a test with high sensitivity and specificity, identifying 40 of 46 (87%) of adenocarcinomas tested, with no false positive results. It is concluded that UEA-1 and SBA staining of effusion specimens warrants further study, and may provide a useful adjunct to CEA staining.     

Lectin binding pattern in human retinal pigment epithelium.

A comparative lectin histochemical study of human retinal pigment epithelium (RPE) was performed to investigate the lectin binding pattern of normal, reactive and proliferating RPE. Normal RPE with attached sensory retina was found to bind the lectins Con A, WGA, PNA and RCA I. Reactive and proliferating RPE in retinal detachment and in photocoagulation scars revealed the same lectin binding pattern although its cellular topography changed. RPE-macrophages showed an additional reaction with SBA. In periretinal membranes of human PVR the typical lectin binding pattern of Con A, WGA, PNA and RCA I was found in pigmented and in a subpopulation of non-pigmented cells, suggesting that these lectin-positive elements were of RPE-origin. Additionally, single pigmented cells positive for SBA were found indicating macrophage differentiation. Thus lectin histochemistry provides a tool for cytochemical identification of RPE and its morphologic variants by revealing a specific combination of sugar-binding sites.     

Prognostic value of the immunocytochemical detection of extramural venous invasion in Dukes'' C colorectal adenocarcinomas. A preliminary study.

In postsurgical staging of colorectal adenocarcinomas, it is sometimes difficult to determine the range of possible venous spread. Distinguishing between the extramural veins (especially when the neoplastic embolus takes up the whole lumen and the endothelium cannot be identified) and the smallest extramural lymph nodes (when they are completely replaced by metastatic carcinoma, leaving the capsule alone) is also difficult. This work proposes a more precise definition of true venous invasion to improve histopathologic staging. Immunohistochemical techniques employing commercial antibodies against Factor VIII RAG, with and without enzymatic digestion, and UEA I lectin for residual endothelium detection, were applied, as well as antibodies against vimentin, desmin, and alpha sm-1 actin to detect wall components. The immunohistochemical evaluation of colorectal adenocarcinomas, in particular by anti-alpha sm-1 actin antibodies, permitted a reliable morphologic distinction of the true venous invasion. This factor proved to be relevant for survival rate prediction.     

人胚胎升结肠和降结肠粘膜凝集素受体定位的比较研究

用凝集素亲和细胞化学ABC法比较分析了63例7～38周人胚胎升结肠和降结肠粘膜凝集素受体的定位分布.发现升结肠粘膜能表达刀豆素A受体和大豆素受体,降结肠粘膜能表达刀豆素体、大豆素受体和荆豆素-1受体,且升结肠和降结肠的刀豆素A受体和大豆素受体存在量的差异,两段结肠粘膜均不表达麦胚凝集素受体.表明:在胚胎发育过程中,升结肠和降结肠粘膜的糖复合物既有一定的相似性,也存在质和量的差异.     

Differential lectin binding on walls of thoraco-cervical blood vessels and lymphatics in rats.

Lectin binding in the walls of large to medium-sized blood vessels and lymphatics in the rat thoraco-cervical region was examined histochemically. The tunica intima of the aorta and superficial cervical artery showed positive reactions with wheat germ agglutinin (WGA) and Concanavalin A (ConA) but not with Dolichus biflorus agglutinin (DBA). The tunica media of the aorta exhibited intense WGA binding, especially on the smooth muscle cells, but the tunica media of the superficial cervical artery did not react with the lectin. Neither ConA nor DBA bound to the tunica media of the aorta and superficial cervical artery. The tunica adventitia of both arteries contained sites binding the three lectins, although DBA reactivity declined as the vascular diameter decreased. The tunica intima of the superior vena cava and azygos vein exhibited positive WGA and ConA binding, whereas DBA binding was noted on only part of the tunica intima of the superior vena cava and not on that of the azygos vein. The tunica media and tunica adventitia were reactive for all three lectins. The WGA and ConA binding sites in the tunica adventitia showed loose networks, suggesting lectin binding on connective tissue elements interlacing among smooth muscle bundles. Lectin binding sites in the walls of lymphatics exhibited an arrangement similar to those in the walls of the veins. Moreover valves protruding into the lumen showed intense WGA and ConA binding and scattered DBA binding. Three other lectins (Ulex europaeus agglutinin, peanut agglutinin, Maclura pomifera) were examined, but they showed no reactions with the vessels. Thus, the differential binding of lectins on the walls of blood vessels and lymphatics of various sizes suggests the functional complexity of monosaccharide residues in the vascular walls.     

Expression of the Vascular Endothelial Growth Factor C Receptor VEGFR-3 in Lymphatic Endothelium of the Skin and in Vascular Tumors

It is difficult to identify lymph vessels in tissue sections by histochemical staining, and thus a specific marker for lymphatic endothelial cells would be more practical in histopathological diagnostics. Here we have applied a specific antigenic marker for lymphatic endothelial cells in the human skin, the vascular endothelial growth factor receptor-3 (VEGFR-3), and show that it identifies a distinct vessel population both in fetal and adult skin, which has properties of lymphatic vessels. The expression of VEGFR-3 was studied in normal human skin by in situ hybridization, iodinated ligand binding, and immunohistochemistry. A subset of developing vessels expressed the VEGFR-3 mRNA in fetal skin as shown by in situ hybridization and radioiodinated vascular endothelial growth factor (VEGF)-C bound selectively to a subset of vessels in adult skin that had morphological characteristics of lymphatic vessels. Monoclonal antibodies against the extracellular domain of VEGFR-3 stained specifically endothelial cells of dermal lymph vessels, in contrast to PAL-E antibodies, which stained only blood vessel endothelia. In addition, staining for VEGFR-3 was strongly positive in the endothelium of cutaneous lymphangiomatosis, but staining of endothelial cells in cutaneous hemangiomas was weaker. These results establish the utility of anti-VEGFR-3 antibodies in the identification of lymphovascular channels in the skin and in the differential diagnosis of skin lesions involving lymphatic or blood vascular endothelium.     

Characterization of stromal cells with myoid features in lymph nodes and spleen in normal and pathologic conditions.

Stromal cells with myoid features were identified in rat or human lymph nodes and spleen in normal and pathologic conditions, using antibodies to desmin, alpha-smooth muscle actin, and smooth muscle myosin. In normal lymph nodes, myoid cells (MCs) were present in the superficial and deep paracortex as well as in the medulla, and absent in lymphoid follicles. In the spleen, they were numerous in the red pulp, less abundant in periarteriolar lymphocyte sheaths of the white pulp, and absent in lymphoid follicles. On double immunostaining, alpha-smooth muscle actin and smooth muscle myosin were coexpressed with desmin only in the deep paracortex and parafollicular areas of the lymph nodes, as well as in the MCs of the periarteriolar lymphocyte sheaths and marginal zone of the spleen; the remaining MCs expressed only desmin. When examined by means of electron microscopy, MCs showed a dendritic shape and cytoplasmic bundles of microfilaments with dense bodies scattered between them. When compared with normal conditions, MCs showed changes of distribution and number in several pathologic situations. Additional findings were 1) staining of pericytes surrounding high endothelium venules of lymph nodes with alpha-smooth muscle actin antibodies in man and rat and with desmin antibodies in rats; 2) staining of endothelial cells in these venules with desmin antibodies in rats. It is concluded that a subset of reticular cells in lymph nodes and spleen, as well as pericytes and endothelial cells in high endothelium venules display cytoskeletal features suggesting a myoid differentiation and function.     

Extracutaneous infantile haemangioma is also Glut1 positive

AIM: To investigate whether extracutaneous infantile haemangioma-like tumours are immunohistochemically similar to cutaneous infantile haemangiomas. METHODS: Mammary, salivary gland, liver (one each), and placental (two cases) capillary haemangiomas and typical examples of cutaneous (eight cases) infantile haemangioma were investigated immunohistochemically for alpha smooth muscle actin and Glut1, a proposed marker for the skin localised lesion. Positive internal controls included red blood cells, perineurium, trophoblast, and endothelial cells of the placental capillaries. Extralesional vessel endothelium acted as a negative control (except in the placenta). The liver haemangioma and both chorioangiomas presented in patients with Beckwith-Wiedemann syndrome. RESULTS: The endothelial cells of all the vascular lesions were Glut1 positive. These were consistently surrounded by a rim of alpha smooth muscle actin positive pericytic cells. Controls reacted appropriately. CONCLUSIONS: All infantile haemangiomas were immunohistochemically positive for Glut1: expression of this molecule was not limited to infantile haemangiomas of the skin. These tumours comprise proliferations of both endothelial and pericytic cells. The association with Beckwith-Wiedemann syndrome may provide a clue to the molecular genetics of infantile haemangioma.     

Lectin histochemistry of experimental murine rhabdomyosarcomas

Methylcholanthrene-induced murine rhabdomyosarcomas and skeletal muscle of 10 and 18 d old murine embryos were investigated by lectin histochemistry (WGA, RCA-I, LCA, Con-A, PSA, UEA-I, PNA) and by immunohistochemistry (vimentin, desmin, myoglobulin). In rhabdomyosarcomas as well as in the developing skeletal muscle a clear trend was visible. A decrease of vimentin positivity and an increase of desmin positivity were associated with a diminution of binding sites for WGA, RCA, and LCA. No binding moieties for these lectin could be demonstrated in myoglobin positive normal and neoplastic rhabdomyomatous cells at all. The homologous expression or absence of markers reflected the cellular variability in rhabdomyosarcomas and may be explained as a phenomenon of different tumor cell maturation. The results show that rhabdomyosarcomatous cells are imitating the normal skeletal muscle development.     

Evidence for the origin of Kaposi''s sarcoma from lymphatic endothelium.

Previous studies utilizing enzyme histochemistry, electron microscopy, and immunohistochemistry have failed to establish the cell of origin in Kaposi's sarcoma. The authors have rigorously tested the prevailing hypothesis that the lesion defined as Kaposi's sarcoma is derived from vascular endothelial cells. They use seven markers to characterize endothelial cells: three antigens (Factor VIII-related antigen, HLA-DR/Ia, macrophage/endothelial antigens), three enzymes (5'-nucleotidase, ATPase, alkaline phosphatase), and lectin binding (Ulex europaeus I). They applied the markers first to normal skin and lymph node, and then to biopsy specimens from 40 patients with Kaposi's sarcoma. Normal blood vessel endothelium was positive for all seven markers, but normal lymphatic endothelium was negative for all of the markers except 5'-nucleotidase and Ulex europaeus lectin. The neoplastic cells in 40 cases of Kaposi's sarcoma closely resembled those of normal lymphatic endothelium but not those of blood vessel endothelium. This suggests that Kaposi's sarcoma may originate in lymphatic endothelium.     

Cardiac myxoma. A retrospective immunohistochemical study

Seven cardiac myxomas were studied by immunoperoxidase and immunofluorescence in formalin fixed and paraffin embedded tissues. Specific antisera to factor VIII related antigens, vimentin, myosin of smooth muscle, actin, desmin, alpha-1-antitrypsin, muramidase, fibrin and prekeratin antigens were used. All myxoma cells reacted positively with antibodies to vimentin and showed no staining reaction with antibodies to alpha-1-antitrypsin, muramidase, myosin, or prekeratin. Factor VIII related antigen was found only in endothelial cells and not in myxoma cells proper. Fibrin was found in patchy areas within the stroma. Antisera to actin and desmin failed to react with formalin fixed tissue. Our results suggest that the main cellular component of cardiac myxoma is a primitive mesenchymal cell without immunohistochemical evidence of more specific differentiation.     

Altered pericyte-endothelial relations in the rat retina during aging: implications for vessel stability

Mural cells (smooth muscle cells and pericytes) regulate blood flow and contribute to vessel stability. We examined whether mural cell changes accompany age-related alterations in the microvasculature of the central nervous system. The retinas of young adult and aged Wistar rats were subjected to immunohistofluorescence analysis of -smooth muscle actin (SMA), caldesmon, calponin, desmin, and NG2 to identify mural cells. The vasculature was visualized by lectin histochemistry or perfusion of horse-radish peroxidase, and vessel walls were examined by electron microscopy. The early stage of aging was characterized by changes in peripheral retinal capillaries, including vessel broadening, thickening of the basement membrane, an altered length and orientation of desmin filaments in pericytes, a more widespread SMA distribution and changes in a subset of pre-arteriolar sphincters. In the later stages of aging, loss of capillary patency, aneurysms, distorted vessels, and foci of angiogenesis were apparent, especially in the peripheral deep vascular plexus. The capillary changes are consistent with impaired vascular autoregulation and may result in reduced pericyte–endothelial cell contact, destabilizing the capillaries and rendering them susceptible to angiogenic stimuli and endothelial cell loss as well as impairing the exchange of metabolites required for optimal neuronal function. This metabolic uncoupling leads to reactivation of “physiological hypoxia” and angiogenesis in CNS aging.     

Ulex europaeus I lectin as a marker for vascular endothelium in human tissues

Ulex europaeus I agglutinin, a lectin specific for some alpha-L-fucose-containing glycocompounds, was used in fluorescence microscopy to stain cryostat sections of human tissues. The endothelium of vessels of all sizes was stained ubiquitously in all tissues studied as judged by double staining with a known endothelial marker, antibodies against human clotting factor VIII. Cultured human umbilical vein endothelial cells, but not fibroblasts, also bound Ulex lectin. The staining was not affected by the blood group type of the tissue donor. In some tissues Ulex lectin presented additional binding to epithelial structures. Also, this was independent on the blood group or the ability of the tissue donor to secrete soluble blood group substances. Lotus tetragonolobus agglutinin, another lectin specific for some alpha-L-fucose-containing moieties failed to react with endothelial cells. Our results suggest that Ulex europaeus I agglutinin is a good histologic marker for endothelium in human tissues.     

Lectin-binding patterns in the embryonic human paraxial mesenchyme

The paraxial mesenchyme in seven human embryos aged between Carnegie stages 12 and 17 was studied by lectin histochemistry with the lectins AIA, Con A, GSA II, LFA, LTA, PNA, RCA I, SBA, SNA, WGA. The paraxial mesenchyme was found to be segmented into sclerotomes by intersegmental vessels and from late stage 12 by intrasclerotomal clefts dividing each sclerotome into a cranial and caudal half. The lectins Con A, GSA II, LFA, LTA, SBA and SNA did not react at all in the paraxial mesenchyme. Staining for AIA, PNA, RCA I and WGA was found in the developing sclerotomes. However, no differences in the staining pattern between the two sclerotomal halves could be seen. It was striking that in contrast to the chick embryo no differences in binding for PNA between the cranial and caudal sclerotomal parts was observed. These findings reveal that PNA-binding sites do not play the same functional role in segmented axonal outgrowth and neural crest immigration into cranial sclerotomal halves in the human embryo, as found in chick embryonic development. Beginning with the stage 16-embryo, the already condensed caudal sclerotomal halves express Con A-, RCA- and PNA-binding sites. The staining for PNA in particular marked the differentiation of chondrogenous structures developing in this half. From the late stage 12 or stage 13, the walls of intersegmental and other vessels showed binding sites for AIA, PNA, RCA I, SNA and WGA.