Common epitopes of human and murine Mallory bodies and Lewy bodies as revealed by a neurofilament antibody.

The antibody SMI 31, which is directed against a phosphorylated epitope, associated with neurofilaments and recognizes Lewy bodies in brains of patients with Parkinson's disease (Bancher C, Lassmann H, Budka H, Jellinger K, Grundgke-Iqbal I, Iqbal K, Wiche G, Seitelberger F, Wisniewski H: J Neuropathol Exp Neurol 1:81, 1989), decorated in immunofluorescence microscopy Mallory bodies (MBs) present in livers of mice chronically treated with griseofulvin and 3,5-diethoxycarbonyl-1,4-dihydrocollidine. In immunoblots it recognized very acidic MB components in a molecular weight range between 55 and 69.5 kilodaltons in addition to poorly soluble high molecular weight material. Moreover, an antibody to tau protein showed similar reactivities in immunofluorescence microscopy and immunoblotting experiments. Both antibodies also stained MBs in human liver with alcoholic hepatitis. These observations support and extend earlier findings which indicate that several intermediate filament-related cellular inclusion bodies, including MBs, share a variety of morphologic, structural and antigenic features. They also suggest the involvement of tau or tau-like proteins in MB formation.     

High molecular weight component of Mallory bodies detected by a monoclonal antibody.

To identify Mallory body (MB) constituents, monoclonal antibodies to murine MBs induced by long-term griseofulvin (GF) feeding were produced. One of these, antibody MM 120-1, specifically reacted in immunofluorescence microscopy with MBs in all developmental stages but not with other cell structures of human and mouse liver and other organs. The MM 120-1 antigen was present in murine MBs induced by griseofulvin or 3,5-diethoxycarbonyl-1,4-dihydrocollidine feeding and also in human MBs in livers of patients with alcoholic hepatitis. In immunoblots, the MM 120-1 antigen was detectable in the high molecular weight fraction of MB preparations, most of which remained in the well and at the interphase between stacking and resolving gel. No reactivity with cytokeratin polypeptides of different conformational states (i.e., isolated cytoker atin components A and D, heterotetramers A2D2, reconstituted intermediate filaments) was found. It is concluded that the antibody MM 120-1 is a highly specific and sensitive marker for murine and human MBs recognizing a high molecular weight nonkeratin component. This component could play a central role in the pathogenesis of MBs.     

Ubiquitin is present on the cytokeratin intermediate filaments and Mallory bodies of hepatocytes

To investigate the relationship of cytokeratin intermediate filaments (IFs) and Mallory bodies (MBs) to the regulatory protein ubiquitin, the griseofulvin-fed mouse was examined by double-label immunocytochemistry. In controls, immunofluorescence of hepatocytes showed that an antiserum specific to ubiquitin stained the cell border and the cytoplasm as well as the nuclear rim. In griseofulvin-fed liver cells, the MBs induced by this treatment were stained in an identical pattern by the antiserum to ubiquitin and a monoclonal antibody specific to cytokeratin (TROMA 1). Upon examination of the immunoreaction at the ultrastructural level, the ubiquitin antiserum decorated the cytokeratin filaments as well as MB filaments. Particularly striking was the coincidence of localization of TROMA 1 and ubiquitin epitopes, many IF surrounding MBs being either intensely decorated or alternatively nonimmunoreactive. These results suggest that normal cytokeratin IFs are lightly ubiquitinated, whereas MBs are heavily ubiquitinated. Immunoblot analysis of extracted cytoskeletal proteins separated by gel electrophoresis showed that extensive ubiquitination of peptides was present in the livers of the griseofulvin-fed mice. Further, the lack of ubiquitin and TROMA 1 epitopes in some liver IF suggest that loss of the TROMA 1 epitope may lead to concomitant loss of the ability to bind ubiquitin. Although the role ubiquitin plays in Mallory body formation remains to be elucidated, we suggest that its significance here may be related to its normal association with cytokeratin.     

Immune complex nephritis in alcoholic cirrhosis: detection of Mallory body antigen in complexes by means of monoclonal antibodies to Mallory bodies.

A Mallory body (alcoholic hyaline) antigen (JMB2) which is also present in intermediate filaments of epithelial origin was demonstrated immunohistochemically in renal glomeruli of three out of eleven patients with alcoholic liver damage. In two of these patients, both of whom had alcoholic cirrhosis with Mallory bodies, it was associated with mesangial deposits of IgA and C3. JMB2 was not found in glomeruli of normal controls, nor in a series of cases of glomerulonephritis in non-alcoholic patients. It is concluded that JMB2 is present in immune complexes in renal glomeruli of patients with renal disease consequent on alcoholic liver disease.     

Mapping of benzodiazepine-like immunoreactivity in the rat brain as revealed by a monoclonal antibody to benzodiazepines.

A monoclonal antibody against benzodiazepines (21-7F9) was used to study the distribution of benzodiazepine-like immunoreactivity in the rat brain. Immunodensitometry in combination with image analysis were used for quantification. The results showed a ubiquitous distribution of benzodiazepine-like immunoreactivity throughout the brain. Very high levels of benzodiazepine-like immunoreactivity were found in the Purkinje cell layer of the cerebellum, in the primary olfactory cortex, in the stratum pyramidale of the hippocampus and in the mitral cell layer of the olfactory bulb. High densities of benzodiazepine-like immunoreactivity were found in the granule cell layer of the cerebellum, the pyramidal cell layer of the olfactory tubercle, the granule layer of the dentate gyrus, the arcuate nucleus of the hypothalamus, the mammillary bodies, the interstitial nucleus of Cajal and superficial grey layer of superior colliculus. The substantia nigra pars compacta, the islands of Calleja and layers II, III, V and VI of the cerebral cortex had moderate levels of benzodiazepine-like immunoreactivity. Lower densities were found in the internal granular layer and the external plexiform layer of the olfactory bulb, in the molecular layer of the dentate gyrus, in layers I and IV of the cerebral cortex, in the nucleus caudate-putamen and most of the thalamic nuclei. The lowest density of immunoreactivity was found in the globus pallidus, and the strata radiatum, oriens and lacunosum-moleculare of the hippocampus. The distribution of endogenous benzodiazepine-like immunoreactivity was compared with the distribution of the GABA/benzodiazepine receptor by using both immunocytochemistry and receptor autoradiography. Our studies have shown a clear mismatch between the localization of the benzodiazepine-like immunoreactivity and the GABA/benzodiazepine receptors.     

Perisynaptic Schwann cells at neuromuscular junctions revealed by a novel monoclonal antibody

This study aimed to generate a probe for perisynaptic Schwann cells (PSCs) to investigate the emerging role of these synapse-associated glial cells in the formation and maintenance of the neuromuscular junction (NMJ). We have obtained a novel monoclonal antibody, 2A12, which labels the external surface of PSC membranes at the frog NMJ. The antibody reveals PSC fine processes or fingers that are interposed between nerve terminal and muscle membrane, interdigitating with bands of acetylcholine receptors. This antibody also labels PSCs at the avian neuromuscular junction and recognizes a 200 kDa protein in Torpedo electric organs. In frog muscles, axotomy induces sprouting of PSC processes beyond clusters of acetylcholine receptors and acetylcholinesterase at denervated junctional branches. PSC branches often extend across several muscle fibers. At some junctions, PSC sprouts join the tips of neighboring branches. The average length of PSC sprouts is approximately 156 µ at 3-week denervated NMJs. PSC sprouting is accompanied by a significant increase in the number of Schwann cell bodies per NMJ. Following nerve regeneration, nerve terminals reinnervate the junction along the PSC processes. In vivo observations of normal frog muscles also show PSC processes longer than nerve terminals at some junctional branches. The results suggest that nerve injury induces profuse PSC sprouting that may play a role in guiding nerve terminal regeneration at frog NMJs. In addition, antibody 2A12 reveals the fine morphology of PSCs in relation to other synaptic elements and is a useful probe in elucidating the function of these synapse-associated glial cells in vivo.     

Microglia in the mature and developing quail brain as revealed by a monoclonal antibody recognizing hemopoietic cells.

The monoclonal antibody QH1, which recognizes quail endothelial and hemopoietic cells, was found to label microglia in the developing and mature brain of the quail. Forms of microglia similar to those described in mammals were labelled. Ameboid microglia predominated at embryonic stages, became less numerous in late embryonic development, and disappeared completely by day 10 post-hatch (P10). Poorly ramified microglia were present as early as day 5 of incubation (E5), and were progressively replaced by mature ramified microglia from E14 onwards. From P10 onwards, ramified microglia were the only microglial form seen in the quail brain.     

27 kDa extracellular matrix protein revealed by a monoclonal antibody raised against rat testis

In search of unique components of the seminiferous tubule extracellular matrix, monoclonal antibodies were raised against an isolated seminiferous tubule extracellular matrix, and the monoclonal antibody 12G11 was cloned. By immunofluorescence microscopy in eight kinds of rat tissues (testis, lung, liver, small intestine, cardiac muscle, skeletal muscle, kidney, and brain), 12G11 antigen existed only in the testis. Immunoelectron microscopy revealed that the antigen is localized in the basement membrane of the seminiferous tubule and in the basement membranes of myoid cells. For a biochemical analysis, eight kinds of rat extracellular matrices were isolated and solubilized with 8 M urea and 2% beta-mercaptoethanol. Immunoblot analysis of these samples in 0.8% agarose gel also showed that the antigen was specific for the testis, and in a two high-molecular weight aggregates. These aggregates seemed to contain type IV collagen and laminin chains. The antigen of 12G11 antibody was shown to be 27 kDa by 10% SDS-PAGE followed by immunoblotting. From these data, the existence of a testis specific 27 kDa basement membrane protein, which associate with type IV collagen and laminin, was suggested.     

Unexplained in-vitro fertilization failure: implication of acrosomes with a small reacting region, as revealed by a monoclonal antibody.

To determine the acrosomal characteristics related to in-vitro fertilization (IVF) outcome, spermatozoa from 50 men whose wives had resorted to IVF have been studied by indirect immunofluorescence microscopy with anti-human pro-acrosin monoclonal antibody 4D4 (mAb 4D4), prior to and after incubation in a capacitating medium. The antibody labelled only the acrosomal principal region (APR), revealing its shape (i.e. normal, small or amorphous) and its status (i.e. unreacted, partially or totally reacted). The IVF outcome distinguished: (i) spermatozoa which were able to fertilize at least one oocyte in vitro (group I; n = 25) and (ii) spermatozoa which failed to fertilize any oocyte in vitro (group II; n = 25). The semen characteristics of the two sperm groups, including the acrosome morphology, were similar according to conventional analysis. The mAb 4D4 detected in both the whole and the swim-up sperm cell fractions a lower percentage of normal APR in group II (< 50% for 10 patients in group II versus one patient from group I), which was related to a higher percentage of small APR. Moreover, after 21 h incubation, group II had a lower acrosomal loss index. The spermatozoa of five patients of this infertile group II did not undergo acrosomal modification whereas spermatozoa of all group I patients underwent the acrosomal reaction. The data showed that the relationship between acrosomal anomalies and IVF failure is mainly due to an increased incidence of acrosomes with a reduced size of the region involved in the acrosome reaction. Immunodiagnosis of this acrosomal region by means of mAb 4D4 is informative for IVF outcome.     

Coexistence of heterogeneous populations ofToxoplasma gondii parasites within parasitophorous vacuoles of murine macrophages as revealed by a bradyzoite-specific monoclonal antibody

The expression of bradyzoite-specific antigens (Bsa) ofToxoplasma gondii was studied in murine bonemarrow-derived macrophages that had previously been infected with tachyzoites. Growth conditions that allowed only restricted replication ofToxoplasma gondii resulted in heterogeneous populations; (1) Bsa-positive and Bsa-negative parasites could be observed within one parasitophorous vacuole (heterogeneous vacuole community), and (2) homogeneous Bsa-positive and homogeneous Bsa-negative vacuole communities coexisted within one macrophage host cell. These observations suggest that stage conversion does not seem to be a synchromous event for a vacuole community.Parts of this paper were presented (by U.G.) at the first BIOMED Workshop onToxoplasma gondii Research in Europe, Würzburg, January 28, 1993; similar results were described by J.F. Dubremetz at the same workshop     

Human alpha-fetoprotein epitopes as revealed by monoclonal antibodies.

The epitope specificity of 12 anti-human alpha-fetoprotein monoclonal antibodies (Mabs) was estimated in an enzyme-linked immunosorbent assay (ELISA). A combination of two different approaches: (i) Mabs binding to heterologous alpha-fetoprotein (AFP); and (ii) cooperative Mabs binding to human AFP (hAFP) when tested in pair mixtures; was used. This double-approach methodology was found to be more reliable for the definition of Mab specificities than either method alone. The anti-hAFP Mabs studied recognised eight unique non-repeated epitopes on hAFP. Two of the epitopes were specific for humans, whereas six were common to other species (mouse, rat, calf, dog, pig and cat) with a characteristic species distribution for each epitope. All epitopes were present on hAFP synthesised by hepatoma, yolk sac tumour and embryo.     

Identification of the conserved,conformation-dependent cytokeratin epitope recognized by monoclonal antibody (lu-5)

Summary The epitope recognized by the murine monoclonal antibody (mAB lu-5) recently described as a formaldehyde-resistant, pan-epithelial marker of great value in tumour diagnosis is located on the surface of cytokeratin filaments. It has been preserved during vertebrate evolution from amphibia to man. As this epitope is not reactive after SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the epitope-bearing protein has been identified by a dot-blot antibody binding assay, using purified proteins in which the epitope is reconstituted. We show that the epitope is present in most cytokeratin polypeptides of both the acidic (type I) and basic (type II) subfamily but does not occur in other cytoskeletal proteins. The location of this widespread epitope is discussed with respect to homologies of amino acid sequences of cytokeratins and their conformations.     

Extensive nuclear localization of alpha-synuclein in normal rat brain neurons revealed by a novel monoclonal antibody

Synuclein was initially named for its localization in both presynaptic nerve terminals and portions of nuclear envelope. However, subsequent studies only confirmed the presynaptic localization of this protein in the brain; its nuclear localization in the neurons remained elusive. Here, two new monoclonal antibodies against alpha-synuclein (alpha-SYN) were produced. Epitope mapping using phage peptide display showed that the epitopes of the two antibodies were localized in two distinct specific sequences of the C-terminal domain of alpha-SYN. One antibody named 3D5 recognized amino acids 115-121 of alpha-SYN and the other antibody named 2E3 identified the amino acids 134-138 of the protein. Western blot analysis demonstrated that both 2E3 and 3D5 detected a 19 kD protein from rat and human brain homogenates, which was identical to the molecular size of recombinant alpha-SYN. However, immunohistochemical staining on normal adult rat brain sections showed that the two antibodies revealed distinct patterns of subcellular localization of alpha-SYN immunoreactivity. Both 3D5 and 2E3 detected the presynaptic alpha-SYN but only 3D5 detected the nuclear alpha-SYN. The nuclear localization of alpha-SYN was further confirmed by Western blot analysis in isolated nuclear fraction where the same size of alpha-SYN was detected, and by immunoelectron microscopy using colloidal gold probes where gold particles were specifically localized in portions of peri- and intra-nucleus. The nuclear positive neurons were distributed extensively in almost all the brain regions. This is the first report well characterizing the extensive localization of alpha-SYN in the neuronal nuclei throughout the brain in normal conditions. This finding indicates an important physiological function of this molecule in the nuclei of brain neurons, which deserves further investigations.     

Cytokeratin 8 protects from hepatotoxicity, and its ratio to cytokeratin 18 determines the ability of hepatocytes to form Mallory bodies

In alcoholic hepatitis, a severe form of alcohol-induced toxic liver injury, as well as in experimental intoxication of mice with the porphyrinogenic drugs griseofulvin and 3,5-diethoxycarbonyl-1, 4-dihydrocollidine, hepatocytes form cytoplasmic protein aggregates (Mallory bodies; MBs) containing cytokeratins (CKs) and non-CK components. Here we report that mice lacking the CK8 gene and hence CK intermediate filaments in hepatocytes, but still expressing the type I partner, ie, the CK18 gene, do not form MBs but suffer from extensive porphyria and progressive toxic liver damage, leading to the death of a considerable number of animals (7 of 12 during 12 weeks of intoxication). Our observations show that 1) in the absence of CK8 as well as in the situation of a relative excess of CK18 over CK8 no MBs are formed; 2) the loss of CK8 is not compensated by other type II CKs; and 3) porphyria and toxic liver damage are drastically enhanced in the absence of CK8. Our results point to a protective role of CKs in certain types of toxic liver injury and suggest that MBs by themselves are not harmful to hepatocytes but may be considered as a product of a novel defense mechanism in hepatocytes.     

Cholinergic innervation of the main and the accessory olfactory bulbs of the rat as revealed by a monoclonal antibody against choline acetyltransferase

Summary The main and accessory olfactory bulbs (MOB and AOB) of the rat were immunohistochemically stained with a monoclonal antibody against choline acetyltransferase (ChAT) in order to know the difference in the distribution patterns of cholinergic fibers between these two structures. A few ChAT-immunoreactive cell bodies were found in the superficial and middle parts of the external plexiform layer (EPL) of the MOB, in the granule cell layer (GCL) of the MOB, and in the GCL of the AOB. The frequency in appearance of these cells was 0.9 cells/section in the MOB and 0.3 cells/section in the AOB. While the glomerular layer (GL) and the superficial part of the EPL were most densely innervated in the MOB, the internal plexiform layer received the richest innervation in the AOB. There were no immunoreactive structures in the olfactory nerve layer of the MOB and in the vomeronasal nerve layer and glomerular layer of the AOB. In addition to a relatively homogenous distribution of cholinergic fibers in the MOB and AOB, there were several foci of very dense network of immunoreactive fibers at the posterior level of the OB. These foci formed a part of the modified glomerular complex that was recently identified using 2-deoxyglucose method and was presumed to be related to suckling behaviour in the neonatal rat.     

Cytokeratin expression during neoplastic progression of human transitional cell carcinomas as detected by a monoclonal and a polyclonal antibody

Antibodies to intermediate filament proteins were used to study different cell layers in normal human transitional epithelium, 16 human transitional cell carcinomas, and two cell lines derived from human bladder carcinomas. Conventional rabbit antisera to human skin keratins stained all layers of the transitional epithelium from bladder, ureter, and kidney. A slightly higher staining intensity was found in the basal and superficial layers as compared with the intermediate cell layers. A monoclonal antibody to cytokeratin 18 (RGE 53), however, stained only the superficial cell layer of transitional epithelium, the so-called umbrella cells. In well-differentiated (grade I) transitional cell carcinomas, RGE 53 stained only the superficial cells of papillary structures. In higher grade papillary tumors, RGE 53 also stained cells within the basal and intermediate layers, whereas in high-grade, invasive tumors almost all tumor cells were RGE 53 positive. These results show that monoclonal antibodies to cytokeratins can provide both an indication of processes involved in neoplastic progression of bladder tumors and a means of studying the molecular relationship of the tumor cells to normal cells.