Similarities of antisera to casein and epithelial membrane antigen

Summary Antisera raised against human milk fat globule membranes and against the casein fraction of human milk have been compared. Using an immunohistochemical stain of tissue sections it has been shown that many of the antigenic determinants detected by the different antisera are identical. A radioimmunoassay for epithelial membrane antigen (EMA) showed that casein preparations are associated with small quantities of EMA. Antisera to casein frequently contained appreciable concentrations of antibodies to EMA and this accounts for the immunohistochemical staining of non-mammary tissues.     

Study of childhood renal tumours using peroxidase conjugated lectins.

Six peroxidase conjugated lectins were used to compare their ability to bind to formalin fixed paraffin embedded tissue sections of childhood renal tumours (Wilms' tumour, mesoblastic nephroma, renal carcinoma, rhabdoid renal tumour, and bone metastasising renal tumour of childhood (BMRTC) with fetal and normal children's kidney. Lectins were found to be helpful in the differential diagnosis of renal tumours. Another important finding was that the mesenchyme of renal tumours showed differences in its reactivity among various types of kidney tumours. The results of lectin binding were not helpful in establishing the origin of kidney tumours.     

Study of childhood renal tumours using a monoclonal antibody to Tamm-Horsfall protein.

A monoclonal antibody to Tamm-Horsfall glycoprotein was used for the immuno-localization of Tamm-Horsfall protein in formalin fixed, paraffin embedded tissue sections of childhood renal tumours, normal children's kidneys, and human fetal kidneys. The procedure was a dinitrophenyl hapten sandwich staining method. The antibody, diluted 1/100,000, gave a very strong and specific staining of the loop of Henle and distal tubules of normal and fetal kidneys. No staining was seen in Wilms' tumour, mesoblastic nephroma, and bone metastasizing renal tumour of childhood. In contrast, two of seven renal carcinomas and three of four rhabdoid renal tumours were positive for Tamm-Horsfall protein.     

Antibodies to purified renal tubular epithelial antigens contain activity against laminin, fibronectin, and type IV collagen

Antibodies directed against tubular brush border antigens (RTE) are used to induce heterologous immune-complex nephritis. Among these antigens a glycoprotein with a molecular weight of 330 kilodaltons (gp330) has been shown to be of pathogenetic significance. We investigated whether antibodies other than those directed against gp330 are present in anti-RTE and whether they play a pathogenetic role. By using enzyme-linked immunosorbent assay techniques and Western blotting, we investigated polyclonal antibodies directed not only against crude RTE but also against RTEgp, a purified glycoprotein fraction of RTE, with respect to activity against glomerular basement membrane (GBM) components laminin, fibronectin, and type IV collagen. Both antibody preparations showed reactivity predominantly to the 220 kilodaltons subunit of laminin. Lower but nevertheless distinct reactivity to fibronectin and type IV collagen was also found. The antibody fraction directed against components of the GBM, which was isolated from anti-RTE IgG by affinity chromatography, showed linear binding to the GBM in indirect immunofluorescence studies. Injection of these antibodies into the renal artery also led to linear binding to the GBM with linear deposition of complement factors 3 and 9 and induced a weak and transient proteinuria. Immunoelectron microscopy revealed binding of the antibodies to glomerular epithelial and endothelial cell surfaces adjacent to the GBM. Injection of anti-RTE antibody absorbed to GBM components resulted in binding of antibodies and complement factors 3 and 9 in a fine granular pattern along the GBM, whereas injection of unabsorbed anti-RTE led to a course granular pattern. We conclude that the presence of antibodies (cross-)reacting with laminin, fibronectin, and type IV collagen in anti-RTE antibody has pathogenetic effects and could explain differences in pathogenicity between monospecific anti-gp330 antibody and polyclonal anti-RTE antibody.     

Use of antisera to epithelial membrane antigen for the cytodiagnosis of malignancy in serous effusions.

A new human antigen, designated epithelial membrane antigen (EMA), has recently been described on surface membranes of a wide variety of normal epithelium but not on connective tissue cells. The antigen is only weakly expressed on normal or reactive mesothelium. Increased expression of the antigen has been observed in most neoplasms of epithelial origin and in malignant mesothelioma. We have investigated the possibility of using this difference in the expression of the antigen to distinguish between mesothelial cells and malignant cells in cytological smears of serous effusions. This distinction cannot always be made on morphological grounds alone and problems of differential diagnosis are encountered in about 15% of all specimens of serous effusions sent for cytological examination. Using antisera to EMA we have applied an indirect immunoalkaline phosphatase technique to alcohol-fixed smears prepared from serous effusions and have found that intense staining of the antigen is confined to effusions from patients in whom there is either clinical or cytological evidence of malignancy. The technique proved to be especially useful in cytologically equivocal cases, where there were problems of differential diagnosis.     

Distribution of laminin, fibronectin, and interstitial collagen type III in soft tissue tumours

The distributions of laminin, fibronectin, and interstitial collagen type III have been investigated in a series of 60 soft tissue tumours by immunochemistry. Positive laminin staining was seen in sites predicted by the distribution of ultrastructurally visible basal lamina. Pericellular laminin was present in all benign tumours of Schwann cell and smooth muscle origin examined, in the two malignant Schwannomas examined, and in six of 13 leiomyosarcomas. It was also evident around nests of cells in an alveolar soft part sarcoma and around malignant endothelial cells in an angiosarcoma. In fibroblastic and fibrohistiocytic tumours it was found only in blood vessel walls. The results of laminin staining led to revision of the original histopathological diagnosis in seven of the 60 cases studied. Fibronectin was abundant in the stroma of most neoplasms, both benign and malignant. It was also found in a distribution parallel to that of laminin. In some tumours this was clearly distinguishable from the distribution of interstitial collagen. Intracellular fibronectin was shown consistently only in mast cell granules. Its demonstration in synovial cells, fibroblasts, and histiocytes was more variable. Interstitial collagen type II had the most irregular distribution of the three proteins. It was as plentiful in tumours of smooth muscle origin as in tumours of fibroblastic origin, but was scanty in fibrous histiocytomas. Its distribution appeared similar to that of laminin and fibronectin in leiomyomas, but differed from these two proteins in Schwann cell tumours and other neoplasms. In one leiomyosarcoma fibronectin, laminin, and type III collagen appeared to be lost concomitantly from tumour cell peripheries.     

The distribution of epithelial membrane antigen in the kidney and its tumours

The distribution of epithelial membrane antigen (EMA) in the kidney and its tumours has been studied using a polyclonal anti-EMA antiserum and the immunoperoxidase-antiperoxidase technique (PAP). Twelve fetal and 10 adult kidneys examined showed EMA to be confined to the distal tubular or collecting duct epithelium or their embryological precursors. The examination of 55 primary renal tumours showed EMA to be present on the epithelial tumours, on tubular epithelium in nephroblastoma, but not on mesenchymal tumour cells nor on undifferentiated areas of nephroblastoma.     

Chordoma. Antibodies to epithelial membrane antigen and carcinoembryonic antigen in differential diagnosis

Seven chordomas, chondrosarcomas, and mucinous colorectal carcinomas, five liposarcomas, and five ependymomas were evaluated for the presence of epithelial membrane antigen (EMA) and carcinoembryonic antigen (CEA) by using an immunoperoxidase technique. All of the chordomas and mucinous carcinomas were cytoplasmic EMA positive, while the chondrosarcomas, liposarcomas, and ependymomas were negative. Among the tumors that were studied, only the mucinous carcinomas were positive for CEA. The EMA positivity of a chordoma reflects its epithelial nature and is a valuable aid in making the differential diagnosis between a chordoma and nonepithelial tumors, while the absence of CEA in a chordoma aids in making the distinction between a chordoma and a mucinous carcinoma.     

Determination of factor VIII-related antigen using commercial antisera.

Commercially available antiserum to factor VIII was used in several tests to determine whether it might serve as a reference between research laboratories involved in investigation of the factor VIII complex and whether the antiserum might be useful in the screening of large populations of patients with hereditary disorders of factor VIII. In Ouchterlony plates, the antiserum gave a single line of identity with concentrated factor VIII, cryoprecipitate, and human plasma. The antiserum was capable of inhibiting the ristocetin response of normal platelets. Testing antigenic factor VIII by the Laurell technic with the commercial antiserum on plasmas from normal and stressed normal controls, patients with von Willebrand's disease, patients with hemophilia A, and obligate carriers of hemophilia A gave diagnostic and reproducible results.     

Profiling and classification tree applied to renal epithelial tumours

Aims:  Selection of the relevant combination from a growing list of candidate immunohistochemical biomarkers constitutes a real challenge. The aim was to establish the minimal subset of antibodies to achieve classification on the basis of 12 antibodies and 309 renal tumours.
Methods and results:  Seventy-nine clear cell (CC), 88 papillary (PAP) and 50 chromophobe (CHRO) renal cell carcinomas, and 92 oncocytomas (ONCO) were immunostained for renal cell carcinoma antigen, vimentin, cytokeratin (CK) AE1–AE3, CK7, CD10, epithelial membrane antigen, α-methylacyl-CoA racemase (AMACR), c-kit, E-cadherin, Bcl-1, aquaporin 1 and mucin-1 and analysed by tissue microarrays. First, unsupervised hierarchical clustering performed with immunohistochemical profiles identified four main clusters—cluster 1 (CC 67%), 2 (PAP 98%), 3 (CHRO 67%) and 4 (ONCO 100%)—demonstrating the intrinsic classifying potential of immunohistochemistry. A series of classification trees was then automatically generated using Classification And Regression Tree software. The most powerful of these classification trees sequentially used AMACR, CK7 and CD10 (with 86% CC, 87% PAP, 79% CHRO and 78% ONCO correctly classified in a leave-one-out cross-validation test). The classifier was also helpful in 22/30 additional cases with equivocal features.
Conclusion:  The classification tree method using immunohistochemical profiles can be applied successfully to construct a renal tumour classifier.     

层粘蛋白及其受体、增殖细胞核抗原与肝细胞癌肿瘤生物学特性的关系

目的:探讨层粘蛋白(LN)、层粘蛋白受体(LN-R)及增殖细胞核抗原(PCNA)与原发性肝细胞癌(HCC)恶性程度的关系.方法:采用SABC法分别检测42例肝细胞癌及癌旁组织中LN、LN-R及PCNA的表达情况.结果:LN、LN-R及PCNA在肝细胞癌中的表达均显著高于癌旁组织(P＜0.05),且与肝细胞癌Edmondson分级呈正相关;浸润型(IHCC)生长、有转移、肿瘤直径＞3cm者LN、LN-R及PCNA的表达高,而膨胀型(EHCC)生长、无转移、肿瘤直径≤3cm者多表达较低;在高增殖指数组LN、LN-R表达高于低增殖指数组(P＜0.05).结论:细胞内LN、LN-R及PCNA可作为评判肝细胞癌恶性程度及临床预后的良好指标.     

Peripheral nerve regeneration using silicone rubber chambers filled with collagen, laminin and fibronectin

A 10 mm gap of rat sciatic nerve was created between the proximal and distal nerve stumps, which were sutured into silicone rubber tubes filled with an extracellular gel containing collagen, laminin and fibronectin. Empty silicone rubber tubes were used as controls. Six weeks after implantation, all extracellular elements were completely degraded and absorbed, and 90% of the animals from the extracellular gel group exhibited regeneration across the nerve gaps, whereas only 60% in the control group. Both qualitative and quantitative histology of the regenerated nerves revealed a more mature ultrastructural organization with 28% larger cross-sectional area and 28% higher number of myelinated axons in the extracellular gel group than the controls. These results showed that the gel mixture of collagen, laminin and fibronectin could offer a suitable growth medium for the regeneration of axons.     

Immunocytochemical investigation of intermediate filament proteins and epithelial membrane antigen in spindle cell tumours of the breast

Seven consecutive cases of primary spindle celled tumours of the breast have been studied immunohistologically using antisera to the intermediate filament proteins (IFP) vimentin, cytokeratin, and desmin, and with an antibody to epithelial membrane antigen. Representative paraffin sections were examined using a peroxidase-antiperoxidase method. In three cases, very occasional foci of epithelial differentiation were apparent by conventional microscopy, and in one case, adjacent ductal carcinoma in situ was present. The remaining three cases were composed of spindle cell elements entirely, with no evidence of epithelial differentiation morphologically. Immunoreactivity of spindle cell elements for vimentin was found in all seven cases, and for cytokeratin in six cases. One case showed immunoreactivity for vimentin, cytokeratin, and desmin, and one case only for vimentin. Epithelial membrane antigen was not identified in the spindle cell elements of any tumour, but was present in the invasive epithelial component of three cases and the in situ component of one case. We conclude that many spindle cell tumours of breast show immunohistological evidence of epithelial differentiation and can be regarded as spindle cell carcinomas. However, in some cases IFP expression may be complex and histogenesis cannot be determined. This technique can aid histological diagnosis in some cases.     

The Haemophilus influenzae Hap autotransporter binds to fibronectin,laminin, and collagen IV

Nontypeable Haemophilus influenzae (NTHI) initiates infection by colonizing the upper respiratory tract mucosa. NTHI disease frequently occurs in the context of respiratory tract inflammation, where organisms encounter damaged epithelium and exposed basement membrane. In this study, we examined interactions between the H. influenzae Hap adhesin and selected extracellular matrix proteins. Hap is an autotransporter protein that undergoes autoproteolytic cleavage, with release of the adhesive passenger domain, Hap(s), from the bacterial cell surface. We found that Hap promotes bacterial adherence to purified fibronectin, laminin, and collagen IV and that Hap-mediated adherence is enhanced by inhibition of autoproteolysis. Adherence is inhibited by pretreatment of bacteria with a polyclonal antiserum recognizing Hap(s). Purified Hap(s) binds with high affinity to fibronectin, laminin, and collagen IV but not to collagen II. Binding of Hap(s) to fibronectin involves interaction with the 45-kDa gelatin-binding domain but not the 30-kDa heparin-binding domain of fibronectin. Taken together, these observations suggest that interactions between Hap and extracellular matrix proteins may play an important role in NTHI colonization of the respiratory tract.     

Differential expansion of human endothelial monolayers on basement membrane and interstitial collagens, laminin and fibronectin in vitro

In this study the ability of a human endothelial cell monolayer to expand over specific components of the basement membrane and extracellular matrix was investigated over a 5-day period. The method was intended as a model to study the mechanisms of endothelial regeneration. All components were coated onto sterile coverslips at a concentration of 10 micrograms/ml. The highest expansion was obtained on fibronectin, laminin and collagen type III, all three being statistically significantly greater than on the uncoated control surface (0.002 greater than p greater than 0.0001). Collagens types I and IV and a high molecular weight fragment mixture of type IV (IV-F, consisting of 75, 120 and 140 kD fragments) elicited approximately similar expansion rates, significantly higher than the control (0.02 greater than p greater than 0.003), although significantly lower (approximately 15%) than collagen type III, fibronectin and laminin (p less than 0.001). The high monolayer expansion on collagen type III is surprising, as it is a relatively minor biosynthetic product of the endothelial cell. It could, however, be of significance in wound healing, in which endothelial cells come into contact with this interstitial collagen. In addition, the similar results obtained with collagens IV and IV-F indicate that expansion of the endothelial monolayer is not dependent on the integrity of the tetrameric structure of type-IV collagen.     

Glomerular basement membrane expansion in passive Heymann nephritis. Absence of increased synthesis of type IV collagen, laminin, or fibronectin.

The distribution and synthetic rate of glomerular basement membrane components was examined in the Passive Heymann Nephritis model of experimental membranous nephropathy. The extensive tissue injury that developed included subepithelial electron-dense deposits, podocyte foot process effacement, and expansion of the glomerular basement membrane. Levels of mRNA for type IV collagen, laminin, and fibronectin from isolated glomeruli was quantitated by slot-blot analysis and showed no change in experimental animals as compared to controls at either 1 week, 3 weeks, or 3 months after disease induction. Immunoelectron microscopy with gold-labeled anti-laminin IgG revealed no difference in the number of particles bound to the glomerular basement membrane of experimental animals and controls. Immunofluorescence with both type IV collagen antisera and anti-laminin antibody showed no difference in the intensity or pattern of staining. Despite extensive glomerular damage and glomerular basement membrane thickening, no evidence was found for either an increase in the synthetic rate of type IV collagen, laminin, or fibronectin or for an accumulation of basement membrane laminin within the damaged glomeruli. Alternate processes, such as diminished density of matrix components or accumulation of other unmeasured matrix constituents, presumably account for the expansion of the glomerular basement membrane seen in experimental membranous nephropathy.     

A study of the Ca antigen in epithelial tumours of the ovary.

The expression of Ca antigen in ovarian epithelial tumours has been studied by an immunohistochemical technique using the Ca 1 monoclonal antibody. The antigen was found to be present in some examples of each of the histological subtypes of ovarian epithelial neoplasms and was detected in benign, borderline and malignant tumours. It is concluded that the presence of Ca antigen is of no discriminatory value in the assessment of malignancy in ovarian epithelial neoplasms.     

A rapid factor VIII-related antigen electroimmunoassay. A micromethod using commercial antisera.

A simple, rapid micromethod for determining Factor VIII-related antigen is outlined. The simplicity of the method, using commercial antisera, prepackaged buffer, plastic plates that readily accept the gel without precoating with agarose, and standard equipment makes the procedure adaptable to the routine laboratory. The results give a straight-line graph to the 0.50 units/ml range, and values as low as 0.03125 U/ml can be determined using log-log graph paper. A second range of standards gives values from 0.10 to 0.750 U/ml when plotted on semilog graph paper. A permanent record can be made after a first rapid visualization process, which permits reading the plates within 15 min of the two-and-a-half-hour electrophoresis time.