Immunolocalization of 14-3-3 isoforms in brains with Pick body disease

Immunolocalization of 14-3-3 protein isoforms in relation to Pick bodies in Pick body disease (PBD) brains was investigated. Weakly granular immunoreactivity of 14-3-3 proteins was found in neurons in control subjects and in Pick body disease brains. In addition to this granular immunoreactivity, many Pick bodies were immunopositive for 14-3-3 proteins as confirmed with double-immunofluorescence with an anti-PHF tau (AT8) and anti-14-3-3 that recognizes all its isoforms (common). When probed with isoform-specific antibodies, Pick bodies were positive for beta, gamma, epsilon, eta, tau, and zeta isoform and exhibited immunostaining pattern similar to that observed with the anti-14-3-3 proteins (common). In addition, immunoreactivity of sigma isoform, so far considered to be exclusively extraneuronal, was unexpectedly found in Pick bodies, normal hippocampal neurons and brain homogenate from age-matched controls. Although localization of 14-3-3 proteins in Pick bodies suggests their involvement in Pick body formation, their role may be variable dependent on the isoforms differently expressed in different area in the brain.     

Diagnostic value of 14-3-3beta immunoblot and T-tau/P-tau ratio in clinically suspected Creutzfeldt-Jakob disease

Creutzfeldt-Jakob disease (CJD) is a fatal prion disease characterized by rapid neurodegeneration. The clinical diagnosis is difficult and reliable diagnostic markers are highly desired. Here, we assess the sensitivity, specificity and predictive values of cerebrospinal fluid (CSF) tests for 14-3-3 protein and total tau (T-tau)/phospho-tau (P-tau) ratio among 36 patients with clinically suspected CJD. Nine of the 36 patients had CJD. These cases were all autopsy-proven. The 14-3-3 test was positive in 4 of the 9 CJD cases and falsely positive in 7 of the 27 patients with other diagnoses. The resulting figures for sensitivity, specificity, and positive and negative predictive values were 44%, 74%, 36% and 80%, respectively. The best result for distinguishing CJD from other diagnoses was obtained using a combined test for T-tau and T-tau/P-tau ratio and cut-off values of >1,400 pg/ml and >25, respectively. The resulting figures for sensitivity, specificity, and positive and negative predictive values were 78%, 93%, 78% and 93%, respectively. The achieved accuracy (89%) was significantly higher than that for the 14-3-3 test (67%; P=0.023). The high predictive values achieved by the combined tau test suggest that it could be used in conjunction with other diagnostic methods for accurate intra vitam diagnosis of CJD.     

Cysticercosis immunodiagnosis using 18- and 14-kilodalton proteins from Taenia crassiceps cysticercus antigens obtained by immunoaffinity chromatography

Monoclonal antibodies (MAb) against Taenia crassiceps and Taenia solium cysticerci were produced and showed cross-reactivity with a 14-kDa protein from T. solium and with 18- and 14-kDa proteins from T. crassiceps. These MAbs and antibodies from cerebrospinal fluid (CSF) as well as serum samples from patients with neurocysticercosis (NC) reacted with 18- and 14-kDa T. crassiceps proteins purified by immunoaffinity chromatography using a Sepharose column coupled with MAbs (anti-excretory/secretory or anti-vesicular fluid antigens). Immunoaffinity-purified 18- and 14-kDa proteins were used in the design of a diagnostic enzyme-linked immunosorbent assay (ELISA) to detect antibodies in 23 CSF and 20 serum samples from patients with NC, showing 100% sensitivity. The test specificity was determined using 42 noninflammatory CSF samples and 70 inflammatory CSF samples from patients with other neurological disorders (OND), showing 100% and 99.1% (confidence interval, 97.3% to 100%) specificity, respectively. A false-positive CSF sample result in the OND group was from a human immunodeficiency virus-positive patient with meningoencephalitis. By using serum samples from 194 healthy individuals, the specificity was 100%. Analysis of an additional 16 serum samples from individuals with other parasitic diseases (13 with intestinal parasitosis and 3 with schistosomiasis) showed negative results. Three (10%) serum samples from patients with hydatidosis were positive in our ELISA and in ELISA with T. solium cysticerci antigens. Two of them were also positive by immunoblotting. The use of 18- and 14-kDa T. crassiceps immunoaffinity-purified proteins for detection of anti-cysticercus antibodies in CSF and/or serum samples using an ELISA system showed a good performance and high specificity for serum samples, dispensing with the use of confirmatory tests, such as immunoblotting, for checking specificity.     

Increased levels of epsilon and gamma isoforms of 14-3-3 proteins in cerebrospinal fluid in patients with Creutzfeldt-Jakob disease.

We established four hybridoma cell lines producing monoclonal antibodies (MAbs) against 14-3-3 proteins. Immunoblot analysis revealed that epsilon and gamma isoforms were specifically increased in premortem cerebrospinal fluid samples from patients with sporadic Creutzfeldt-Jakob disease. Furthermore, dot immunoblot analysis showed that MAbs were more specific for native antigen than polyclonal antibodies were.     

A monoclonal antibody Hy20-54-16-3L to lambda (lambda) light chain of human immunoglobulin reacts with amyloid in Alzheimer's disease brain

We have previously described a monoclonal antibody Hy20-54-16-3L raised against crude primary (AL) amyloid fibrils that preferentially labelled amyloid deposits in the brains of patients with Alzheimer's disease (AD) (Ishii et al., Neuropathol. Appl. Neurobiol., 12 (1986) 441-445). In the present study, we analyzed the protein from an AL-amyloid laden spleen by gel filtration and SDS-PAGE; a partial amino acid sequence of the Hy20-54-16-3L-immunoreactive band excised from Western blots was determined and found to be a lambda light chain of human immunoglobulins. Additionally we have confirmed that this monoclonal antibody reacts with amyloid in AD brain by using a peroxidase-linked immunoelectron microscope technique. It is concluded that an epitope shared with a lambda light chain is included in or associated with amyloid deposits in the AD brain.     

Increased Levels of ɛ and γ Isoforms of 14-3-3 Proteins in Cerebrospinal Fluid in Patients with Creutzfeldt-Jakob Disease

We established four hybridoma cell lines producing monoclonal antibodies (MAbs) against 14-3-3 proteins. Immunoblot analysis revealed that and γ isoforms were specifically increased in premortem cerebrospinal fluid samples from patients with sporadic Creutzfeldt-Jakob disease. Furthermore, dot immunoblot analysis showed that MAbs were more specific for native antigen than polyclonal antibodies were.     

A refined method for molecular typing reveals that co-occurrence of PrP(Sc) types in Creutzfeldt-Jakob disease is not the rule

Molecular typing in Creutzfeldt-Jakob disease (CJD) relies on the detection of distinct protease-resistant prion protein (PrP(Sc)) core fragments, which differ in molecular mass or glycoform ratio. However, the definition and correct identification of CJD cases with a co-occurrence of PrP(Sc) types remains a challenge. With antibodies recognizing a linear epitope in the octapeptide repeat PrP region, supposed to distinguish between the two major PrP(Sc) isoforms (ie, types 1 and 2), it was recently shown that all type 2 cases display an associated band with a type 1 migration pattern, which led to the conclusion that multiple PrP(Sc) types regularly coexist in CJD. We studied brain samples from 53 sporadic CJD and 4 variant CJD subjects using a high-resolution electrophoresis, a wide range of proteinase K (PK) activities, the 'type 1-selective' antibody 12B2, and several unselective antibodies. We found that the type 1-like band detected by 12B2 in all CJD subtypes associated with PrP(Sc) type 2 is not a PK-resistant PrP(Sc) core but rather matches the physicochemical properties of partially cleaved fragments, which result from the several PK cleavage sites included in the N-terminal portion of PrP(Sc). Furthermore, using gels with high resolution and a relatively high PK activity, we were able to increase the detection sensitivity of either type 1 or 2, when coexisting, to amount corresponding to 3-5% of the total PrP(Sc) signal (ie, weak band of one type/total PrP(Sc)). Our results show that there are many pitfalls associated with the use of 'type 1 selective' antibodies for CJD typing studies and that co-occurrence of PrP(Sc) types in CJD is not the rule. The present results further validate the rationale basis of current CJD classification and the qualitative nature of molecular typing in CJD.     

Intranuclear immunolocalization of 14-3-3 protein isoforms in brains with spinocerebellar ataxia type 1

Immunolocalization of 14-3-3 protein isoforms, one of the interacters with ataxin 1, was investigated in spinocerebellar ataxia type 1 (SCA 1) brains using isoform-specific antibodies. Samples from the pons and from the cerebellum of four SCA1 cases and three controls were studied. The intensity of the immunoreactivity (IR) and its subcellular topography were analyzed. In control subjects, granular immunoreactivity for an epitope common to all known isoforms of 14-3-3 proteins (14-3-3 COM) found in the cytoplasm of some pontine and dentate nucleus neurons was weak. It was observed in some Purkinje cells, while its intensity varied. Many nuclei of those neurons and Purkinje cells of SCA1 were intensely immunopositive for 14-3-3 COM, while it was less in their cytoplasm. Expanded polyglutamine epitope was colocalized to 14-3-3 COM epitope in some pontine neurons, sometimes accumulated in intranuclear inclusion-like structures. This findings support previous reports that 14-3-3 proteins stabilize mutant ataxin 1 in nucleus and possibly lead to neurodegeneration. However, nuclear localization of 14-3-3 proteins in SCA1 brains was dependent on its isoforms, i.e. pontine neurons intensely positive for beta, Purkinje cells for tau and dentate nucleus neurons for both, while all of those neurons were consistently positive for zeta isoform, although sigma isoform tended to be located in the cytoplasm. Nuclear accumulation and isoform- and region-dependent subcellular localizations of 14-3-3 proteins may be related to SCA1 pathology, which exhibits marked regional variability.     

3例很可能克雅脑病患者的临床表现及脑电图动态分析

目的：分析克雅脑病（CJD）患者的临床表现、病程及脑电图的动态变化。方法：回顾性分析我院3例诊断为很可能CJD的患者,行脑电图、头部MRI、CSF14-3—3蛋白检查。结果：主要临床表现为进行性痴呆、共济失调、肌阵挛、精神行为异常、锥体外系征及缄默等。3例脑电图均有弥漫性异常,均出现典型周期性三相波,1例在视频脑电图监测中发现早期周期性三相波。2例行脑脊液14—3—3蛋白检查,1例阳性。结论：对中年以上起病,快速进展的痴呆,且合并有注意力不集中、记忆力减退、性格改变、肌阵挛、共济失调、视力障碍,以及进行性精神障碍患者,应高度怀疑CJD的可能。脑电图、MRI以及脑脊液14—3—3蛋白的检测,特别是视频脑电图监测有可能更有利于发现早期的三相波,有助于对可疑病例进行早期的诊断。     

人S100蛋白的表达及其抗血清的制备和脑脊液中S100含量测定方法的建立

目的：建立定量检测脑脊液（CSF）及血清中S100蛋白的方法，探讨S100蛋白的检测在辅助诊断克－雅病（CJD）中的应用。方法：利用脑cDNA文库，经PCR获得了S100基因并克隆至原核表达载体pGEX-2T上，在大肠埃希菌中表达了谷胱甘肽－S－转移酶（GST）－S100融合蛋白；融合蛋白经亲和纯化后，免疫家兔，制备抗体；抗体经纯化后，用生物素（BNHS）标记，建立了可定量检测S100蛋白的生物素－亲和素系统ELISA方法，并初步用于临床脑脊液的检测中。结果：所表达的GST－S100蛋白相对分子质量约为35000，以其为抗原制备的S100特异性抗血清具有良好的免疫反应性。建立了定量检测脑脊液中S100蛋白的双抗体夹心ELISA方法，对3例“可能性的CJD”患者（14－3－3蛋白阳性）和15例无痴呆症状患者脑脊液进行检测，结果显示，3例CJD患者脑脊液S100含量均超过2．900μg/L,而在无痴呆症状患者组中14例患者脑脊液S100含量都低于0．180μg/L。对正常人和CJD患者血清进行检测，显示S100蛋白含量个体间差异很大。结论：所建立的方法可用于脑脊液中S100蛋白的检测，进一步扩大标本量有助于明确脑脊液中S100蛋白的检测在辅助诊断CJD中的价值。     

A solid-phase anti-C3 assay for detection of immune complexes in six distinguished forms

A solid-phase anti-C3 assay detecting immune complexes associated with C3 fragments is described. Two monoclonal antibodies against C3 fragments are used; one recognizes an epitope expressed on C3b, C3c and C3i, the other recognizes an epitope expressed on C3dg and iC3b. Combining these monoclonal anti-C3s with antibodies against class-specific immunoglobulins in enzyme-linked immunosorbent assay (ELISA), immune complexes (ICs) are detected in six distinguished forms; C3b-IgG-IC, iC3b/C3dg-IgG-IC, C3b-IgA-IC, iC3b/C3dg-IgA-IC, C3b-IgM-IC and iC3b/C3dg-IgM-IC. About three-fourths of the plasma samples from patients with active SLE or IgA-nephritis were found positive in one or more types of ICs. IgA-type ICs were found very frequently in patients with autoimmune or renal diseases.     

14-3-3 proteins interact with the β-thymosin repeat protein Csp24

Conditioned stimulus pathway protein 24 (Csp24) is a β-thymosin-like protein that is homologous to other members of the family of β-thymosin repeat proteins that contain multiple actin binding domains. Actin co-precipitates with Csp24 and co-localizes with it in the cytosol of type-B photoreceptor cell bodies. Several signal transduction pathways have been shown to regulate the phosphorylation of Csp24 and contribute to cellular plasticity. Here, we report the identification of the adapter protein 14-3-3 in lysates of the Hermissenda circumesophageal nervous system and its interaction with Csp24. Immunoprecipitation experiments using an antibody that is broadly reactive with several isoforms of the 14-3-3 family of proteins showed that Csp24 co-precipitates with 14-3-3 protein, and nervous systems stimulated with 5-HT exhibited a significant increase in co-precipitated Csp24 probed with a phosphospecific antibody as compared with controls. These results indicate that post-translational modifications of Csp24 regulate its interaction with 14-3-3 protein, and suggest that this mechanism may contribute to the control of intrinsic enhanced excitability.     

A common idiotope on human rheumatoid factors identified by a hybridoma antibody

Human monoclonal and polyclonal anti-IgG autoantibodies [rheumatoid factors (RFs)] are composed primarily of kappa light chains, and may display cross-reactive idiotypes. However, the nature of the shared idiotope(s) has remained unclear. We have prepared a murine hybridoma antibody (17-109) that recognizes an idiotope present on 30% (3/10) of human IgM-RF paraproteins, and absent on immunoglobulins without RF activity. The idiotope was measurable on isolated, intact kappa light chains, but not on light-chain tryptic peptides, nor on isolated heavy chains. A comparison of the binding to 17-109 of five IgM-RF paraproteins, with known kappa chain amino acid sequences, suggested a relationship between the idiotope recognized by the hybridoma and the complementarity-determining regions. The serum of patients with rheumatoid arthritis contained idiotope positive material that bound specifically to a 17-109 immunoadsorbent column. Moreover, the 17-109 anti-idiotope antibody partially inhibited the binding to IgG of IgM-RF and IgA-RF in serum, but did not effect the binding to antigen of IgM and IgA anti-tetanus toxoid antibodies. These results suggest that a significant proportion of IgM-RF paraproteins share an idiotope located at or near the complementarity-determining regions of the kappa light chain. Human serum RFs include a kappa light chain family that is idiotopically related to the kappa chains on IgM-RF paraproteins.     

Human cerebral malaria: characterization of malarial antibodies in cerebrospinal fluid.

Anti-malarial antibodies were quantified in cerebrospinal fluid (CSF) of 17 cases of cerebral malaria, 16 presumptive cases (no demonstrable parasitaemia in peripheral blood but responding to i.v. quinine therapy) of cerebral malaria, and 15 controls. A schizont-enriched Plasmodium knowlesi antigen was used in an ELISA. Anti-malarial antibodies of IgA and IgM isotypes were not detectable in most of the CSF samples analysed, although serum antibody titres were high. However, 88% of CSF from cerebral malaria and 56% of presumptive cerebral malaria cases had significant levels of IgG anti-malarial antibodies in comparison to control CSF. The antibody levels did not correlate with the severity of coma but correlated well with the duration of coma. The CSF malarial antibody titres were independent of degree of parasitaemia. The possible role of CSF anti-malarial antibodies in cerebral malaria in the light of recent demonstrations of intrathecal synthesis of immunoglobulins and deposition of immune complex in cerebral tissues is discussed.     

The Antibody MY4 Recognizes CD14 on Porcine Monocytes and Macrophages

Several monoclonal antibodies directed against the human CD14 antigen have been established. We now report that the antibody My4, but not LeuM3, reacts with porcine monocytes. Among porcine peripheral blood mononuclear cells (PBMC), 14.6% of the cells stain with the CD14 antibody My4, which is similar to the percentage obtained with the antiporcine monocyte antibody 74-22-15. Two-colour immunofluorescence reveals that My4 and 74-22-15 antigens are coexpressed on the same cells, and cell sorter-purified My4⁺ cells exhibit the morphology of monocytes. Whole blood analysis (which also shows staining of granulocytes) reveals that the average percentage of My4⁺ monocytes amongst all leucocytes is 5.8% with 580 cells/μl. Furthermore, porcine peritoneal macrophages (PM) and alveolar macrophages (AM), both stain for My4, with a four-fold lower level on AM. Treatment of cells with phosphatidylinositol-specific phospholipase C decreases My4 staining, but does not affect staining with antibody 74-22-15. Immunoprecipitation with the My4 antibody from surface labelled pig mono-nuclear cells demonstrates a 54 kDa band similar to human CD14, and Western blotting with pig serum demonstrates two bands similar to the alpha and beta forms of human soluble CD14. Finally, the My4 antibody is capable of blocking lipopolysaccharide- (LPS)-induced interleukin-6 production in isolated PBMC. These data show that the My4 antibody recognizes genuine CD14 on porcine monocytes and macrophages.     

Quantitation of IgG kappa and IgG lambda in the cerebrospinal fluid by sandwich ELISA method

IgG kappa and IgG lambda concentrations were quantified in 96 paired CSF and sera using Hevylite? antibodies in an in-house developed sandwich ELISA method. In 56 of these samples, the results were compared with a qualitative isoelectric focusing/affinity-mediated immunoblotting assay for oligoclonal IgG kappa and IgG lambda. Normal IgG kappa/lambda ratio in the CSF was the same as in serum. In 19/33 patients with intrathecal oligoclonal IgG synthesis, skewed IgG kappa/lambda ratio was observed (increased in 16 and decreased in 3 cases). The analysis of light chain composition of intrathecally synthesised immunoglobulins could contribute to our understanding of intrathecal humoral immune response, although its diagnostic utility is limited.     

Expression of the p75 neurotrophin receptor by striatal cholinergic neurons following global ischemia in rats is associated with neuronal degeneration

A protein capture assay was used to measure 14-3-3 (-isoform) in the cerebrospinal fluid (CSF) of patients with either variant or sporadic Creutzfeldt-Jakob disease (CJD). The results were compared with those obtained using Western blotting. Elevated levels of 14-3-3 were found in 58% of variant CJD (vCJD) patients and 82% of sporadic CJD (spCJD) patients using the protein capture assay. Using a Western blotting technique, the presence of CSF 14-3-3 was detected in 58% of vCJD patients and in 89% of spCJD patients. When the results from the protein capture assay and the Western blot were combined, 14-3-3 was detected in 77% of vCJD patients and in 91% of spCJD patients. These results suggest that although analysis of CSF 14-3-3 is not as useful in vCJD as it is in the sporadic form of the disease, a combination of these two techniques results in increased sensitivity of 14-3-3 for the diagnosis of vCJD.     

Immunolocalisation of 14-3-3 isoforms in normal and scrapie-infected murine brain.

The appearance of 14-3-3 proteins in the cerebrospinal fluid is characteristic of some neurodegenerative conditions which include sporadic Creutzfeldt-Jakob disease. Although 14-3-3 proteins are physiochemically well characterised and are known to be present in neuronal cells little is known of the neuroanatomical localisation of the individual isoforms. Using 14-3-3 isoform specific antibodies we have examined the distribution of the isoforms in normal murine brain and the changes observed during neurodegeneration as a result of ME7 scrapie infection. In normal brain there are two major patterns of immunolabelling. The beta, gamma, eta and zeta isoforms which exhibit a similar distribution pattern showing labelling of neuronal cell bodies often in particular anatomical nuclei. However the individual isoforms exhibit variation revealing subtle differences in location. The tau isoform was found only in the hippocampus and medulla, and the epsilon isoform was found throughout grey matter of the CNS. In the scrapie-infected murine brain, where severe pathological changes occur during the course of the disease, significant differences in the 14-3-3 isoform distribution were observed in the hippocampus and in the thalamus. Importantly, both the 14-3-3 eta isoform and prion protein were seen in the same neurones in both the cerebellar roof nuclei and in the lateral hypothalamic nuclei.Our study of 14-3-3 isoform distribution in adult murine brain clearly demonstrates a heterogeneous pattern of neurolocation for specific 14-3-3 isoforms. The fact that isoform labelling in terminal scrapie CNS is lost in some brain areas, but increases in others, suggests that the processing of these proteins during neurodegeneration may be much more complex than previously recognised.     

Radioimmunoassay of herpes-simplex and measles virus antibodies in serum and cerebrospinal fluid of patients without infectious or demyelinating diseases of the central nervous system.

A solid-phase radioimmunoassay was used to detect IgG antibodies against herpes-simplex virus antigens (capsid, envelope and excreted) and against measles virus antigen in serum and cerebrospinal fluid (CSF) specimens of 61 patients with no evidence of infectious or demyelinating disease of the central nervous system. Quantitative determinations of IgG and albumin in serum and CSF were also performed. Of the 61 serum and 61 CSF samples tested, 57 and 56 respectively contained antibodies against subunit antigens of herpes simplex virus. Antibody against measles virus was found in 59 serum and 47 CSF specimens. A positive correlation (P less than 0-001) was found between each of the four serum to CSF antibody ratios and the serum to CSF total IgG ratios. This indicated that the distribution of antiviral IgG antibodies in serum and CSF normally follows the distribution of total IgG. The ratios between viral antibody in serum and CSF were also correlated with albumin ratios (P less than 0-05). An inverse relation (P less than 0-001) was found between the age of the patients and their serum to CSF albumin ratios, but not their IgG ratios, suggesting that the albumin ratio is a useful indicator of a blood brain barrier lesion and that the IgG ratio should be used in evaluating disturbed antibody ratios.     

Identification of Bombyx mori 14-3-3 orthologs and the interactor Hsp60

The 14-3-3 protein family consists of evolutionarily conserved, acidic 30 kDa proteins composed of seven isoforms named beta, gamma, epsilon, zeta, eta, theta, and sigma in mammalian cells. The dimeric complex of 14-3-3 isoforms, acting as a molecular adaptor, plays a central role in regulation of neuronal function. Since aberrant expression of 14-3-3 is identified in the brains of Alzheimer disease and Parkinson disease, a convenient insect model, if it is available, is highly valuable for studying a pathological role of 14-3-3 in neurodegeneration. Here, we identified the silkworm Bombyx mori 14-3-3 orthologs, zeta and epsilon isoforms highly homologous in amino acid sequences to the human 14-3-3zeta and 14-3-3epsilon. By Western blot, the expression of zeta and epsilon isoforms was identified at substantial levels in the first instar larva, markedly upregulated in the second instar larva, and the highest levels were maintained in the late stage of larva, the pupa, and the adult. Furthermore, by protein overlay and immunoprecipitation, we identified Hsp60 as a 14-3-3-binding partner. The 14-3-3 proteins interacted with the N-terminal fragment of Hsp60. The 14-3-3zeta and epsilon isoforms, along with Hsp60, were expressed widely with overlapping distribution in larval and adult tissues, including brain, fat body, silk gland, Malpighian tube, midgut, ovary, testis, antenna, and pheromone gland. These observations suggest that a molecular adaptor 14-3-3 and a molecular chaperone Hsp60 cooperate to achieve a wide range of cellular functions in B. mori.