Canine malignant histiocytosis (MH): clinical and radiographic findings.
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Animals↗
Biomedical subjects
M L Schmidt
Publications and source records attributed to M L Schmidt.
Prolonged recombinant activated factor VII (rFVIIa) treatment for severe bleeding in a factor-IX-deficient patient with an inhibitor.
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Child↗
Comparative epitope analysis of neuronal cytoskeletal proteins in Alzheimer's disease senile plaque neurites and neuropil threads.
Dystrophic neurites in senile plaque (SP) coronas and neuropil threads (NTs) massively accumulate in Alzheimer's disease (AD) cortex. Hence, they may contribute to the cognitive deficits in AD. However, the composition of these neuritic lesions is poorly understood, and it is not known if they derive from axons, dendrites or both. To gain insights into the composition and derivation of SP neurites and NTs in AD, we undertook an in situ epitope mapping study wherein we probed these lesions using 278 monoclonal antibodies specific for spatially distinct epitopes in each neurofilament (NF) subunit, or in microtubule-associated proteins, i.e., tau and microtubule-associated protein 2. The middle molecular weight NF subunit (NF-M) and tau were extensively represented in SP neurites, i.e., epitopes extending from the NH2 to COOH domains of NF-M and tau were present. In contrast, microtubule-associated protein 2 was not present in any SPs, and only epitopes in the core domain of the low (NF-L), and in the tail piece of the high molecular weight subunit were detected in SP neurites. SP cores never stained with these antibodies. NTs were similar to SP neurites in that they contained the same complement of tau epitopes, and were devoid of any microtubule-associated protein 2 immunoreactivity, but they were also distinct because they rarely contained any NF determinants. These antigenic dissimilarities between SP neurites and NTs suggest that NTs and SP neurites are distinctly separate lesions that reflect widespread disruption of the neuronal cytoskeleton in AD.
Alzheimer Disease↗
Epitope map of neurofilament protein domains in cortical and peripheral nervous system Lewy bodies.
A subset of demented elderly patients exhibit large numbers of cortical intraneuronal inclusions similar to the neurofilament (NF)-rich Lewy bodies (LB) found in pigmented subcortical neurons of patients with Parkinson's disease (PD). Because these cortical inclusions may contribute to the emergence of cognitive impairments in afflicted individuals, the authors mapped the distribution of NF epitopes in these so-called cortical LBs. This was done using ethanol-fixed tissues and a large library of monoclonal antibodies (MAbs) with well-characterized binding specificities to various regions of each NF triplet protein. Cortical LBs were examined by light, confocal, and electron microscopy, and they were compared with the subcortical LBs of PD and LBs in the peripheral nervous system (PNS). Monoclonal antibodies specific for the rod regions of each of the three NF subunits, or for phosphate-dependent and independent antigenic sites in the tail region of the high- (NF-H) and middle- (NF-M) molecular weight (Mr) NF subunits as well as other MAbs to the extreme COOH terminus of NF-L and NF-M or the head region of NF-M labeled a variable number of cortical LBs. Remarkably one of these anti-NF MAbs, RMO32, which recognized a phosphorylated epitope in the tail region of NF-M, immunolabeled nearly all cortical LBs, whereas each of the other anti-NF MAbs never labeled more than 10% of ubiquitin- or RMO32-positive cortical LBs. Further LBs in the PNS resembled those in the central nervous system (CNS) in their immunologic properties, and LBs in both sites were dominated by filamentous aggregates at the ultrastructural level. These findings suggest that NF proteins are profoundly altered during their incorporation into cortical and PNS LBs. Further the authors here identified immunologic and ultrastructural properties common to cortical LBs, PNS LBs, and classic substantia nigra LBs in PD. The accumulation of filamentous, perikaryal inclusions rich in NF proteins at diverse sites in the CNS and PNS of patients with a variety of neurodegenerative disorders suggests a widespread disruption of NF metabolism or transport.
Aged↗
Vulnerability of the neuronal cytoskeleton in aging and Alzheimer disease: widespread involvement of all three major filament systems.
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Aging↗
Defined neurofilament, tau, and beta-amyloid precursor protein epitopes distinguish Alzheimer from non-Alzheimer senile plaques.
Eight antisera and one monoclonal antibody to synthetic peptides that corresponded to domains extending over the entire length of the beta-amyloid precursor protein (beta-APP), and an antiserum to the full-length 695-amino acid form of the beta-APP, were raised to probe the composition of the core and corona of senile plaques (SPs). We localized distinct beta-APP domains, including the beta-amyloid protein or A4 region, within the SPs of 13 end-stage Alzheimer disease (AD) and 13 age-matched control samples of hippocampus and entorhinal cortex. The composition of SPs also was probed with antibodies to defined epitopes in tau (tau) as well as the large and mid-size neurofilament (NF) proteins. The most important observations were that beta-APP domains outside the A4 region were largely restricted to SP coronas in the AD samples, together with tau and NF determinants, whereas the same epitopes were absent from A4-positive blood vessels and exceptionally rare in non-AD SPs. Indeed, samples from a subset of the non-AD cases contained a considerable number of A4-positive SPs totally devoid of any of the other beta-APP, tau, and NF epitopes. These observations suggest that the deposition of the A4 protein in AD SPs results from the local processing of beta-APPs in association with tau and NF protein fragments. It is unclear whether this association is fortuitous or linked by common mechanisms. However, differences between the complement of beta-APP, tau, and NF protein epitopes in AD versus non-AD brains implicate a defect involving one or more steps in the posttranslational modification, degradation, or elimination of these proteins in AD brains, and this may account for the massive numbers of SPs that characterize AD.
Alzheimer Disease↗
Relative abundance of tau and neurofilament epitopes in hippocampal neurofibrillary tangles.
Neurofibrillary tangles (NFTs) derive, in part, from normal neuronal cytoskeletal proteins, ie, large portions of tau (tau) but only restricted segments of the peripheral domains of the high- and middle-molecular weight neurofilament subunits. To learn more about the events leading to the incorporation of tau and neurofilament epitopes into NFTs, the relative abundance of tau and NF determinants in these lesions was quantitatively analyzed in hippocampi from Alzheimer disease (AD) patients and age-matched controls using monoclonal antibodies specific for tau or for NF proteins. Immunostained NFTs appeared qualitatively the same in both AD and controls, ie, every epitope found in AD NFTs occurred also in the NFTs of the control patients. However, in hippocampi with only a few tangles, tau epitopes, but no NF epitopes, were detected in NFTs. In contrast, both tau and NF epitopes were present in those tangles that were found in hippocampi with abundant NFTs. Nevertheless, the number of tau-positive NFTs generally exceeded the number of NF-positive NFTs. These findings indicate that tau epitopes are more abundant than NF epitopes in NFTs and that the formation of NFTs may be linked to a derangement in the normal metabolism of tau that is more extensive than alterations in NF protein metabolism.
Antibodies, Monoclonal↗
Distribution of phosphate-independent MAP2 epitopes revealed with monoclonal antibodies in microwave-denatured human nervous system tissues.
In contrast with results obtained in experimental animals, antibodies to microtubule associated protein-2 (MAP2) preferentially label abnormal structures in human nervous system tissue samples, but the normal sites at which MAP2 is expressed are not well-defined. To determine the distribution of MAP2 in the human central (CNS) and peripheral (PNS) nervous systems, we prepared monoclonal antibodies (MAbs) specific to MAP2, and compared the localization of this MAP in postmortem bovine and human tissues as well as in several human neural cell lines that express either neurofilament (NF) or glial filament (GF) proteins. Eight MAbs specific for phosphate-independent epitopes in bovine and human MAP2 were obtained, and those that performed well in tissues produced immunoreactivity confined to the somatodendritic domain of neurons in bovine and human CNS and PNS tissues. Other neural cells (e.g. astrocytes) did not express MAP2 immunoreactivity using these MAbs. Postmortem delays of less than 24 h prior to tissue denaturation did not affect the distribution of MAP2 immunoreactivity. However, microwave denaturation of these tissues preserved MAP2 immunoreactivity better than fixation with Bouin's solution or formalin. Microwave treatment also improved the immunoreactivity of several MAbs for NF and GF proteins. Finally, MAP2 was not detected in human neural cell lines that express NF (2) or GF (1) proteins. We conclude that microwave denaturation provides an effective means to preserve the immunoreactivity of normal human neuronal cytoskeletal proteins, and that this method of tissue denaturation allows the normal distribution of MAP2 to be defined in postmortem samples of human CNS and PNS tissues.
Acetates↗
Distribution of tau proteins in the normal human central and peripheral nervous system.
In human brain, antibodies to tau proteins primarily label abnormal rather than normal structures. This might reflect altered immunoreactivity owing to post-mortem proteolysis, disease, or species differences. We addressed this issue by comparing the distribution of tau in bovine and human post-mortem nervous system tissues and in human neural cell lines, using new monoclonal antibodies (MAb) specific for phosphate-independent epitopes in bovine and human tau. In neocortex, hippocampus, and cerebellum, immunoreactive tau was widely expressed but segregated into the axon-neuropil domain of neurons. In spinal cord and peripheral nervous system, tau immunoreactivity was similarly segregated but less abundant. No immunoreactive tau was detected with our MAb in glial cells or in human neural cell lines that express neurofilament or glial filament proteins. Post-mortem delays in tissue denaturation of less than 24 hr did not affect the distribution of tau, but the method used to denature tissues did, i.e., microwave treatment preserved tau immunoreactivity more effectively than chemical fixatives such as Bouin's solution, and formalin-fixed tissue samples reacted poorly with our anti-tau MAb. We conclude that the distribution of tau proteins in human nervous system is similar to that described in perfusion-fixed experimental animals, and that visualization of normal immunoreactive tau in human tissues is critically dependent on the procedures used to denature post-mortem tissue samples. Furthermore, microenvironmental factors in different neuroanatomical sites may affect the regional expression of tau.
Animals↗
Selective expression of epitopes in multiphosphorylation repeats of the high and middle molecular weight neurofilament proteins in Alzheimer neurofibrillary tangles.
Here we review our recent "epitope analyses" of a few of the fibrous intraneuronal inclusions that are distinctive hallmarks of human neurodenerative conditions using a large library of monoclonal antibodies (MAbs) raised to normal neuronal cytoskeletal proteins. Analyses of the low (NF-L), middle (NF-M), and high (NF-M), and high (NF-H) molecular weight neurofilament (NF) proteins with greater than 500 MAbs enumerated epitopes shared by NF proteins and the intraneuronal neurofibrillary tangles (NFTs) that occur in the hippocampus and brainstem of Alzheimer's disease (AD) subjects. We identified the NF-H multi-phosphorylation repeat domain, i.e. repeats of Lys-Ser-Pro-X (where X is a small uncharged amino acid and Ser acts as a phosphate acceptor), as the determinant recognized by 15/16 MAbs that detected NFTs in sections of AD hippocampus, and 11 of the same 16 MAbs recognised NF-M multi-phosphorylation repeats. Further, the antigen binding regions of these MAbs were shown to comprise 13 separate classes based on their differential binding to 12 synthetic peptides derived from the NF-H and NF-M multi-phosphorylation sites, NF subunits of 10 diverse mammalian and sub-mammalian species, and normal human tau (tau). None of these anti-NF MAbs recognized NFTs in the brainstem of subjects with progressive supranuclear palsy (PSP), but NFTs in AD brainstem sections were reactive with five of these MAbs. Both PSP and AD brainstem NFTs were recognized by MAbs specific for tau and paired helical filament antigens.(ABSTRACT TRUNCATED AT 400 WORDS)
Alzheimer Disease↗
Analysis of epitopes shared by Hirano bodies and neurofilament proteins in normal and Alzheimer's disease hippocampus.
Hirano bodies (HBs) are intraneuronal inclusions that are abundant in Alzheimer's disease (AD) and share epitopes with normal cytoskeletal proteins, i.e., actin, actin-associated proteins, and microtubule-associated proteins. To evaluate immunologic similarities between neurofilament (NF) proteins and HBs, we probed hippocampal HBs with anti-NF monoclonal antibodies (Mabs) and characterized the epitopes shared by NFs and HBs. Hippocampal HBs were stained by only 4 of greater than 500 MAbs raised to NF proteins, and were confined to stratum lacunosum and stratum pyramidale of hippocampus. Normal blood vessels, neuronal structures (e.g. axons), glia, and portions of senile plaques and extraneuronal tangles also were stained by these MAbs. Two (RM061 and RM0129) of the 4 MAbs were exclusively specific for the middle molecular weight human NF subunit (NF-M). The other 2 (RM054 and RM0298) recognized human NF-M and actin. Further, RM054 cross-reacted with human vinculin, and RM0298 recognized the low molecular weight human NF subunit (NF-L). Stratum lacunosum HBs stained with these MAbs accumulated with age until the 2nd to 3rd decades of life, and declined in number thereafter. In contrast, immunoreactive HBs in stratum pyramidale accumulated more gradually (peak values occurred in the 8th to 9th decades), and were significantly more numerous in AD subjects compared with controls. We conclude that: (a) Hippocampal HBs contain NF-M- and NF-L-like epitopes; (b) Stratum lacunosum HBs wax and then wane in number during normal aging; (c) Statistical analysis of the number of HBs in stratum pyramidale versus stratum lacunosum revealed a significant correlation between these data and the presence of AD.
Adolescent↗
Intraneuronal and extracellular neurofibrillary tangles exhibit mutually exclusive cytoskeletal antigens.
We examined the possibility that neurofibrillary tangles (NFTs) were heterogeneous in postmortem hippocampus from 22 patients with or without senile dementia of the Alzheimer type. Intraneuronal NFTs and extracellular, or "ghost," NFTs were recognized in situ by only one or the other of two monoclonal antibodies. The first monoclonal antibody, RMO87, stained only intraneuronal NFTs and is specific for phosphate-dependent epitopes in tau and the two high molecular weight neurofilament proteins. The second monoclonal antibody, 2.2B10, is specific for glial fibrillary acidic protein, and it stained only the RMO87-negative extracellular NFTs. Treatment of sections with alkaline phosphatase or sodium dodecyl sulfate, and the isolation of NFTs from hippocampus, did not expose RMO87 binding sites in extracellular NFTs. These observations indicate that neurofilament-like and tau-like epitopes can be lost from NFTs in situ, and that at least two populations of morphologically and immunochemically distinct NFTs exist.
Aged↗
Alzheimer disease tangles share immunological similarities with multiphosphorylation repeats in the two large neurofilament proteins.
Immunological and structural analyses of neurofilament (NF) proteins with greater than 500 anti-NF monoclonal antibodies (mAbs) enumerated epitopes shared by NF proteins and Alzheimer neurofibrillary tangles. We identified the multiphosphorylation domain of the rat heaviest NF subunit--tandem repeats of Lys-Ser-Pro-Xaa (where Xaa is a small uncharged amino acid and serine is phosphorylated)--as the determinant recognized by 15 of the 16 mAbs from this collection of greater than 500 mAbs that detected neurofibrillary tangles. Most (11) of these 16 mAbs also recognized the previously characterized multiphosphorylation repeat in the human middle sized NF subunit. However, although these mAbs shared the ability to recognize NFTs, the antigen-binding domains of these 16 mAbs represented 13 separate classes based on their differential recognition of 12 synthetic peptides derived from the rat heaviest NF subunit and the human middle-sized NF subunit multiphosphorylation sites, NF subunits of 10 diverse species, and normal human tau protein. We conclude that NFTs share highly specific immunological and structural properties with specific rat heaviest NF subunit and human middle-sized NF subunit multiphosphorylation repeats.
Alzheimer Disease↗
Properties of antigenic determinants that distinguish neurofibrillary tangles in progressive supranuclear palsy and Alzheimer's disease.
Neurofibrillary tangles (NFT) in Alzheimer's disease (AD) and in progressive supranuclear palsy (PSP) differ in their location and morphology, but both appear to express the same or similar epitopes. These immunologic data may signify that both types of NFTs contain the same components and arise as a result of the same mechanisms. To explore this hypothesis, we probed PSP and AD samples of brain stem, where both PSP and AD NFTs occur, using a large panel of monoclonal antibodies (MAbs). The epitopes expressed in brain stem PSP and AD NFTs were compared with those in the NFTs of AD hippocampus. NFTs in PSP hippocampus were too infrequent for comparative analysis. The MAbs were raised to neurofilament and tau proteins, or to AD paired helical filaments. All MAbs raised to tau (three) and paired helical filaments (two) recognized brain stem PSP NFTs and AD NFTs in brain stem and hippocampus. However, 12 anti-NF MAbs specific for multiphosphorylation repeat domains or other phosphate-dependent and independent epitopes did not bind PSP NFTs, but they did detect AD NFTs in hippocampus, and 5 of these MAbs also recognized brain stem AD NFTs. We conclude that some populations of AD NFTs contain antigenic determinants that are not found in PSP NFTs. This may reflect the effect of different pathologic events specific to PSP and AD, or the selective formation of NFTs in different groups of neurons in each of these disorders.
Aged↗
Phosphate dependent and independent neurofilament epitopes in the axonal swellings of patients with motor neuron disease and controls.
Axonal accumulations of neurofilaments (NFs) may result from abnormalities intrinsic to NF subunits (e.g., proteolytic cleavage, altered phosphorylation), or from abnormalities extrinsic to NFs that retard the transport of these neuron-specific intermediate filaments. To evaluate this hypothesis, we probed the NF-rich axonal swellings seen in normal spinal cords, "globules", and the larger ones, "spheroids", seen in the spinal cords of patients with motor neuron disease by using a library of monoclonal antibodies that recognize human NF proteins. These monoclonal antibodies discriminate different phosphorylation states of individual NF subunits. The NF protein determinants of globules and spheroids were similar to each other and to the determinants that predominate in normal spinal cord axons. NF protein-positive inclusion bodies were only seen in the anterior horn cells of one patient with motor neuron disease, and they contained NF protein determinants similar to those normally expressed in perikarya. Thus, the NFs in globules and spheroids appear to be derived from axonal NF proteins, and both kinds of axonal swellings may arise by similar mechanisms. Although our data do not exclude a structural defect in NF proteins to account for the accumulation of NFs in these axonal swellings, the hypotheses being advanced to explain the formation of NF-rich globules and spheroids based on intrinsic alterations of NF proteins must consider that the immunological integrity of disparate NF protein epitopes in different states of phosphorylation is retained in both globules and spheroids.
Adolescent↗
Enzymatic detection of native and derivatized horseradish peroxidase in sodium dodecyl sulfate polyacrylamide gels.
We developed procedures for the restoration of peroxidatic activity in native horseradish peroxidase (HRP) and HRP conjugated to wheat germ agglutinin (WGA-HRP) following electrophoresis in SDS-polyacrylamide gels (SDS-PAGE). After extraction of SDS with isopropanol from gels containing HRP and WGA-HRP, the peroxidatic activity in these probes could be demonstrated by tetramethylbenzidine (TMB) chemistry. This procedure also showed HRP enzyme activity in electrophoresed tissue homogenates containing HRP. Both free HRP as well as WGA-HRP preparations contain several molecular weight species that display peroxidatic activity. These findings are important for cell biological studies utilizing these substances as molecular probes. The procedures described here should be useful for the analysis of the enzymatically active molecular forms of these frequently used markers in vitro and in vivo.
Animals↗
Immunochemical detection of diverse molecular species of horseradish peroxidase recovered from rat superior cervical ganglion following retrograde transport.
The sympathetic innervation of the submandibular gland (SMG) by neurons of the superior cervical ganglion (SCG) was used as a model system to characterize the different molecular species of retrogradely transported horseradish peroxidase (HRP). Following injections of 3 mg of HRP into the SMG, enzymatically active HRP was demonstrated by histochemistry in SCG neurons for up to 14 days. Parallel immunochemical studies were conducted on electroblots of SCG homogenates from other rats subjected to similar SMG injections of HRP. This technique allowed the detection of nanogram amounts of HRP. These experiments demonstrated that an immunochemically detectable 40 Kd species of HRP was retrogradely transported and retained in SCG neurons for up to 6 days. Beyond 8 days, only lower molecular weight (15-30 Kd) immunoreactive breakdown products of HRP were observed in SCG homogenates. Similar spontaneous HRP breakdown products displayed enzymatic activity in non-denaturing gels. These studies suggest that retrogradely transported HRP is degraded by neurons to lower molecular weight species which retain enzymatic activity. We conclude that morphological studies of retrogradely transported HRP visualized by histochemical methods reflect the response of neurons to a variety of enzymatically active molecular forms of HRP.
Animals↗
Unimpaired function of human phagocytes in the presence of phagocytosis-resistant group A streptococci.
The resistance to phagocytosis of the group A streptococci has been attributed mainly to the presence of the surface antigen, M protein. In the present study, we addressed the question of whether the phagocytosis resistance of the group A streptococci is due to their ability to impair the function of the phagocytic cells. The results of these studies demonstrate that the presence of a large excess of a phagocytosis-resistant strain of streptococci does not significantly interfere with either the antibody-independent or the antibody-dependent phagocytosis of streptococci. Apparently, a phagocytosis-resistant strain of streptococci does not bring about a generalized deactivation of the phagocytic plasma membrane. This suggests that if the resistance of the group A streptococci is due to any deactivating influence at all on the phagocytic plasma membrane, it is likely to be confined to the contact area of the cocci with the phagocyte.
Antibodies↗