Leukocyte analysis using monoclonal antibodies in human glomerulonephritis

The leukocyte subpopulations were analyzed within both the glomeruli and the interstitium in renal biopsies from 145 patients with various forms of glomerulonephritis. Cells were identified by monoclonal antibodies to leukocyte cell-surface antigens and immunoperoxidase labelling. Leukocytes, as defined by a monoclonal antibody to the leukocyte common antigen (PHM1), were present in normal, human renal tissue in both glomeruli (2.8 +/- 0.6 cells/glom. cross section) and interstitium (102 +/- 18 cells/mm2). Monocytes constituted the predominant infiltrating cell type in normal glomeruli (1.3 +/- 0.2) and T cells were rarely found (0.3: range 0 to 0.8), whereas both monocytes (34 +/- 10/mm2) and T lymphocytes (33 +/- 14/mm2) were found in the normal interstitium. In the non-proliferative forms of glomerulonephritis there was no significant increase in the number of glomerular inflammatory cells when compared with normal glomeruli. However, significantly increased numbers of T lymphocytes were seen in the interstitium of biopsies with minor non-specific changes (67 +/- 15/mm2), membranous nephropathy (134 +/- 30/mm2), focal glomerulosclerosis (207 +/- 53/mm2), and diabetic nephropathy (198 +/- 81/mm2). In the proliferative forms of glomerulonephritis only crescentic GN and post-infectious GN demonstrated significantly-increased glomerular monocytes and granulocytes. There was no significant increase in the number of glomerular T cells when compared with normal glomeruli. However, there was a significant increase in the number of interstitial T lymphocytes in all forms of proliferative glomerulonephritis when compared with the normal interstitial cell population.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistologic diagnosis of multiple carcinomas. The role of monoclonal antibodies against carcinoembryonic antigen

The occurrence of multiple malignant lesions in the same patient requires that management decisions be made from an accurate pathological identification of the tumours. The authors describe five patients who had colorectal adenocarcinoma preceded or followed by adenocarcinomas in other organs. Definitive identification could not be established from routine histopathologic findings. The use of immunohistochemistry helped to clarify the relationship between the various tumours; in particular, a monoclonal antibody (D14) directed against carcinoembryonic antigen (CEA) made a notable contribution. Because of the increasing number of monoclonal antibodies with well-defined tumour specificities, their use in the documentation of multiple malignant tumours has great clinical potential.     

Cellular immunity analysis using monoclonal antibodies in human glomerulonephritis

Monoclonal antibodies against class II antigens of the human major histocompatibility complex (MHC) (Edu 1), von Willebrand factor-related antigen marker of endothelial cell, T cells (Cris 1), helper/inducer T cells (T4) and cytotoxic/suppressor T cells (T8) by indirect immunofluorescence, and stain for nonspecific esterase characterizing monocytes-macrophages (Mo-Ma) were applied in 64 renal biopsies--54 glomerulonephritis (GN), 10 non-GN- and in 14 normal kidneys. Class II antigens were expressed on the endothelium of renal microvasculature in all specimens. Intraglomerular T cells and Mo-Ma were only present in GN. Mo-Ma appeared associated with endo- and extracapillary proliferation (Xc2 = 4.68; p less than 0.05), C3 (X2 = 4.21; p less than 0.05), and fibrinogen (X2 = 3.84; p less than 0.05) deposition; and those were most numerous in biopsies with intraglomerular T cells. Interstitial MHC-class II+ cells (Xc2 = 5.5; p less than 0.02), T cells (F = 3.37; p less than 0.005) and Mo-Ma (F = 2.45; p less than 0.05) were significantly higher in GN with endo- or extracapillary proliferation than in the remaining. In GN, correlations were seen between T cells and MHC-class II+ cells (r = 0.63; p less than 0.001), and Mo-Ma (r = 0.38; p less than 0.02), infiltrating the interstitium. Our results suggest that both humoral and cellular immunity contribute to macrophage glomerular infiltration in the human GN. Mononuclear cells, and no intrinsic renal cells, would be implicated in the cellular immune interactions in situ.     

Identification of human spermatozoa antigens using monoclonal antibodies and the alkaline phosphatase anti-alkaline phosphatase-technique

Both cytoplasmic and surface-membrane antigens of human spermatozoa were detected by means of monoclonal antibodies (MoAbs) and of the alkaline phosphatase anti-alkaline phosphatase- (APAAP-) technique. Several advantages of this technique for the identification of sperm could be demonstrated. The labeling of cytocentrifuge preparations from 16 ejaculates proved the presence of glycosphingolipids, nuclear and mitochondrial antigens of spermatozoa. However, there were no HLA-molecules and other leukocyte antigens on sperm cells.     

Immunohistochemical localization of osteonectin in developing human and calf bone using monoclonal antibodies

Summary Osteonectin was immunolocalized in human fetal and calf neonatal developing bone using newly developed monoclonal antibodies. The protein was localized to the cytoplasm of osteoblasts and young osteocytes. In bone matrix, strong reactivity was found in newly laid down osteoid. Bone matrix immunoreactivity was enhanced by pretreatment of sections with proteases, possibly because of an unmasking of epitopes engaged in protein-protein interactions. Osteonectin immuno-reactivity was also found in preosteoblasts in all types of human fetal osteogenesis (membranous, endochondral, subperiosteal, and mantellar (Meckel's cartilage) ossification), and in some chondrocytes of metaphyseal growth plate, posibly modulating towards an osteoblastic phenotype.     

Immunoreactivity of recombinant human glandular kallikrein using monoclonal antibodies raised against prostate-specific antigen

The gene encoding human glandular kallikrein (KLK2) was expressed in Escherichia coli, and the corresponding protein (hK2) was produced by fermentation. The hK2 was characterized by Western blotting and epitope map using monoclonal antibodies (MAbs) specific for another protease, prostate-specific antigen (PSA) with high structural identity (80%). MAbs that recognized three different epitopes were bound to hK2, representing 7 out of 23 MAbs tested. One epitope was localized to the sequence region around amino acid position 78, which is believed to be glycosylated in hK2. The affinities of MAbs recognizing hK2 were similar to those for PSA, suggesting that common epitopes seem to contain very conserved structures. The result may help in designing specific diagnostic assays for the assessment of prostate cancer. Prostate 31:84–90, 1997. © 1997 Wiley-Liss, Inc.     

Cellular composition of crescents in human rapidly progressive glomerulonephritis identified using monoclonal antibodies

The relative contributions of glomerular epithelial cells, macrophages, and other cell types to the formation of cellular crescents characteristic of human rapidly progressive glomerulonephritis remain controversial. To identify and quantitate the cell types present during different stages of glomerular crescent formation, immunoperoxidase labelling of cryostat sections from renal biopsies with cellular (n = 9) or sclerosed (n = 3) crescents was performed using monoclonal antibodies to cell-specific antigens of leucocytes, epithelial cells, and other glomerular cell types. Fresh cellular crescents consisted of macrophages (34.5 +/- 7.0%; mean +/- SEM) plus lesser proportions of polymorphs (12.8 +/- 4.7%) and epithelial cells (10.4 +/- 1.5%). Sclerosed crescents contained fewer macrophages (5.1 +/- 1.0%), but similar proportions of polymorphs (11.1 +/- 2.9%) and epithelial cells (11.5 +/- 2.1%). Lymphocytes were not detected within crescents. Many of the remaining unlabelled cells morphologically resembled fibroblasts and expressed surface fibronectin, though fibroblast-specific cell markers were not available. These results show that macrophages and not epithelial cells constitute the major cell type within cellular crescents. Therapeutic manoeuvres directed against macrophages may, therefore, be of clinical value in the management of human crescentic rapidly progressive glomerulonephritis.     

Tumor-associated antigens recognized by human monoclonal antibodies

Background: Nonhuman monoclonal antibodies (MoAbs) of desired specificities have been studied in cancer treatment and tumor targeting with minimal success. Attempts of using humanized chimeric antibodies have not improved significantly their clinical applications. We have engaged in the development of human MoAbs by incorporating the in vitro immunization protocols to the nodal lymphocytes of cancer patients. Three human MoAbs thus generated were found to be strongly reactive with various human malignancies. The antigens recognized by the three antibodies were selected for immunochemical and biochemical characterizations. Methods: The antigens investigated were AgSK1, PA 1-2 and PA 3-1. The patterns of each antigen expression in various human cancer cell lines were studied by the immunocytochemical staining technique. The expression of AgSK1 in association with cellular proliferation was examined by the flow cytometry analysis. In studying the biochemical natures of these antigens, their sensitivies toward various chemical and physical treatments were determined. The antigens that were shown to be proteins were subjected to SDS-PAGE and Western blot for estimations of molecular weights. Results: The AgSK1 was detected in 10 human carcinoma cell lines but in none of the melanoma cell lines. This suggests that SK1 may be an epithelial or carcinoma marker. The phenotypic expressions of AgSK1 were shown to be associated with proliferation of carcinoma cells. Biochemically AgSK1 was a sialophycoprotein with an estimated molecular weight of 42–44 kilodaltons (kDa). HuMAb PA1-2 demonstrated a unique staining pattern at both the cytoplasmic and intercellular interface. The stained filamentlike structures extending from cell to cell indicated that Ag PA1-2 might play a role in cellular interactions. Biochemically, Ag PA1-2 appeared to be an asialocarbohydrate. The Ag PA3-1 was a cytoplasmic glycoprotein expressed by all 13 cell lines. The estimated molecular weights of PA3-1 were 164, 104, and 40 kDa. Conclusions: Tumor-associated antigens recognized by the human MoAbs may be more relevant clinically than those recognized by the mouse immune system. Carcinoma-specific human MoAbs are desirable for cancer treatment and tumor localization.     

Identification of rectal ganglion cells using monoclonal antibodies

A monoclonal antibody has been developed that reacts specifically with ganglion cells of the myenteric and submucous plexuses of the gastrointestinal tract. This antibody also recognizes an axonal antigen that is distributed throughout the circular muscle of normal colon and rectum. Aganglionic segments of colon and rectum from patients with Hirschsprung's disease showed no specific antibody binding using a fluorescent labelled assay. The use of monoclonal antibodies should provide further insights into the pathophysiology of Hirschsprung's disease and allied disorders.     

Generation of human monoclonal antibodies against colon cancer

Lymphocytes from regional lymph nodes of patients with colon cancer were fused with a human lymphoblastoid cell line with or without in vitro immunization. The efficacy of these two protocols for the generation of human monoclonal antibodies against colon cancer was investigated. The hyperplastic lymph nodes adjacent to the tumor were the best source of B lymphocytes. Fusion frequency and the number of tumor-reactive clones were markedly increased when the in vitro immunization protocol was applied prior to fusion. As a stimulant in in vitro immunization, the supernatant of pokeweed mitogen-stimulated T lymphocytes was superior to the supernatant of mixed lymphocytes culture. Carcinoembryonic antigen at 20 micrograms/L seemed to be the optimal dose for in vitro immunization. The reactivities of human monoclonal antibodies thus generated were measured by enzyme-linked immunosorbent assay and confirmed by immunoperoxidase staining. Combining in vitro immunization with lymphocytes of cancer patients may lead to the successful production of clinically useful human monoclonal antibodies.     

Effect of anti-LFA1 (CD11a) monoclonal antibodies in acute rejection in human kidney transplantation

A murine IgG1 monoclonal antibody, 25-3 (Immunotech, France), directed against the alpha chain (CD11a) of the human LFA1 molecule was used in the treatment of 7 histologically documented first acute rejection in first kidney transplantations under cyclosporine. Four patients (group I) received 20 mg/day for 2 days and 10 mg/day for 8 days of 25-3 MoAb. One developed Quincke's edema after the first injection of 25-3 and was immediately withdrawn from the study. In 2 patients, whose serum creatinine continued to increase, 25-3 MoAb was replaced by steroids, followed by ALG after 3 and 4 days of treatment, respectively. In the last case, rejection was reversed by 25-3 MoAb alone. As the clinical response of rejection to 25-3 was poor, another group of 3 patients (group II) was treated with 25-3 at a dose of 40 mg/day for 2 days, 20 mg/day for 2 days, and 10 mg/day for 6 days, but 25-3 was still unsuccessful in reversing acute rejection, and rescue treatment was initiated between days 5 and 8 in all cases. MoAb tolerance was excellent in 3 patients. With the exception of the one case of Quincke's edema, only minor side effects were noted in the last 3 recipients. 25-3 MoAb serum trough levels peaked between 1.5-3.5 micrograms/ml at day 3 in group I and between 2-9 micrograms/L at day 2 in group II. Surprisingly, only one patient, in group I, exhibited a borderline IgG immune response against 25-3. These findings suggest that the 25-3 anti-CD11a MoAb is ineffective in controlling the course of acute rejection in kidney transplantation. However as already reported for another anti-LFA1 or with an anti-CD4 MoAb in mouse, 25-3 would be the first example in humans of a MoAb that does not elicit a strong immune response against its own determinants. This property might have important applications if 25-3 can prevent rejection in a prophylactic protocol or block the immune response against other MoAbs.     

The specificity of nephritogenic antibodies. IV. Binding of monoclonal antithymocyte antibodies to rat kidney

Polyclonal rabbit antirat thymocyte serum (ATS) has been shown to form in situ glomerular immune aggregates following perfusion into normal rat kidney ex vivo. This may be due to the presence of T-cell-like epitopes in the rat kidney, or it may be a result of contaminating anti-connective-tissue antibodies in ATS. To exclude the latter possibility we investigated binding to the rat kidney of three different (mouse) monoclonal antirat thymocyte antibodies (anti-T-cell MoAbs), directed to Thy 1.1 antigen, as well as (control) anti-B-cell MoAbs. The MoAbs were incubated in vitro with kidney sections or perfused into the blood-free rat kidney ex vivo. It was shown using immunofluorescence (IF) and immunoelectron microscopy (IEM) (peroxidase technique) that the anti-T-cell MoAbs used, in contrast to anti-B-cell MoAbs, are able to bind with glomerular capillary walls, and with mesangial structures after incubation in vitro or perfusion ex vivo. Although staining patterns are not completely identical, the reaction product is clearly demonstrated throughout the glomerular basement membrane (GBM) and along the plasma membranes of endothelial and epithelial cells, after contact with either of the three anti-T-cell MoAbs used. It is concluded that the presence of T-cell-like epitopes in the rat kidney may lead to immune complex formation following contact with anti-T-cell MoAbs. The nephritogenicity of rabbit ATS, as well as of some batches of clinically used ATS, may also be explained by this mechanism rather than by the usually assumed presence of contaminating antibodies in these polyclonal antisera.     

Radioimmunodetection of colorectal cancer, using anti-CEA monoclonal antibodies

Aiming at radioimmunodetection of colorectal cancer, anti-CEA monoclonal antibodies (CEA102) were produced by immunization with purified CEA. CEA102 showed high specificity with colorectal cancer by mixed hemadsorption assay and immunoperoxidase technique. The antigen detected by CEA102 was confirmed to be carcinoembryonic antigen (CEA) and its molecular weight was estimated to be ca. 180,000 by biochemical analysis. The in vivo study using nude mice grafted a human colorectal cancer or a human malignant melanoma showed greater accumulation of 125I-labeled CEA102 in CEA-positive colorectal cancer than in nude mouse tissues and CEA-negative malignant melanoma. Moreover we successfully obtained scans with good localization of the grafted colorectal cancer on FCR (Fuji Computed Radiography). Using 131I-labeled CEA102 liver metastasis in the patient with colorectal cancer was successfully detected by external scanning with gamma-camera. These results suggest that radiolabeled CEA102 is useful for the detection of colorectal cancer.     

Recognition of human insulin and proinsulin by monoclonal antibodies

High-affinity monoclonal antibodies (MAB) were obtained from lymph node cell fusions. Affinities ranging from 0.8 X 10(9) L/M to 5.2 X 10(9) L/M were calculated from binding studies with monoiodinated human, bovine, and porcine insulins and human proinsulin. Two monoclonal antibodies were specific for human insulin, recognizing an epitope involving the amino acid B-30 (Thr). Another two monoclonal antibodies were bound to the C-terminal end of the B-chain near B-30. The B-chain-specific monoclonal antibodies did not bind human proinsulin. One monoclonal antibody recognized the A-chain loop in the positions A-8 to A-10. This antibody bound also to human proinsulin. It was concluded that the A-chain loop is exposed on the surface of proinsulin, while the C-terminal B-chain is not available for binding. The study shows that monoclonal antibodies can be used to characterize structures of insulin and proinsulin. In contrast to x-ray studies, the molecules can be used at low concentrations in soluble form. It is suggested to use monoclonal antibodies for the screening of atypical insulins in the serum of diabetic patients and for the further refinement of insulin and proinsulin measurements.     

Intraoperative localization of colorectal cancers using radiolabelled monoclonal antibodies

Radiation detectors may allow the intraoperative localization of small cancer deposits following administration of radiolabelled tumour-associated antibodies. This technique was evaluated in 16 patients with colorectal tumours (14 cancers, one adenoma, one lipoma) with the 111In-labelled monoclonal antibody (MAb) ICR2 which recognizes the tumour-associated epithelial membrane antigen (EMA). At operation counting was carried out (3 x 20 s per site) using a hand-held radiation probe over the primary lesions and any palpable lymph nodes in the mesocolon. The tumour to normal colon (T/NC) ratio of counts recorded at operation was more than 1.5:1 in eight of the 14 patients with cancer (mean(s.d.), 1.54(0.41):1) and 0.91:1 and 1.06:1 respectively in the two patients with benign tumours. Node to normal colon ratios were higher in lymph nodes containing metastases. The uptake of radiolabelled antibody (T/NC ratio) was higher in EMA-expressing cancers than in those not expressing the target antigen (mean(s.d.), 2.45(0.65):1 versus 1.40(0.20):1, P = 0.019). An abdominal tumour model was also developed. Radioactively filled containers of 0.5-10 ml representing tumour deposits were suspended in a tank of 111In solution representing the background activity found in normal tissues. The ratio of radioactivity in the 'tumour' to that of background varied from 2:1 to 8:1. The 'tumour' was considered to be detectable if the mean counts recorded over the 'tumour' exceeded the mean of counts recorded over background by three standard deviations. At a ratio of 2:1 only 'tumours' greater than 5 ml could be detected with a sodium iodide probe and those over 10 ml could be detected with a cadmium telluride (CdTe) probe. At a ratio of 8:1, 'tumours' of 0.5 ml could be detected with either probe. At all ratios and counting periods the NaI probe was more sensitive than the CdTe.     

Characterization of monoclonal antibodies specific for human Tamm-Horsfall protein

Fifteen monoclonal antibodies have been produced to human Tamm-Horsfall protein (THP), identifying at least seven distinct epitopes. The antibodies have been used to isolate from serum an immunoreactive protein which comigrates with urinary THP. In addition, the antibodies may prove useful to set up an immunoenzymoassay for urinary THP as well as for immunoaffinity purification.