Clinical Evaluation of Cerebrospinal Fluid p217tau and Neurofilament Light Chain Levels in Patients with Alzheimer's Disease or Other Neurological Diseases.

BACKGROUND: The cerebrospinal fluid (CSF) levels of tau phosphorylated at threonine 217 (p217tau) or 181 (p181tau), and neurofilament light chain (NfL) are definite biomarkers of tauopathy and neurodegeneration in Alzheimer's disease (AD). OBJECTIVE: To validate their utility in excluding other neurological diseases and age-related changes in clinical settings. METHODS: We developed monoclonal antibodies against p217tau and NfL, established novel ELISAs, and analyzed 170 CSF samples from patients with AD or other neurological diseases. RESULTS: In AD, p217tau is a more specific and abundant CSF component than p181tau. However, CSF NfL levels increase age-dependently and to a greater extent in central and peripheral nervous diseases than in AD. CONCLUSIONS: CSF p217tau correlates better with AD neurodegeneration than other tau-related biomarkers and the major phosphorylated tau species. The clinical usage of NfL as a neurodegeneration biomarker in AD requires exclusion of various central and peripheral neurological diseases.

Targeting apolipoprotein E and N-terminal amyloid ß-protein precursor interaction improves cognition and reduces amyloid pathology in Alzheimer's mice.

Apolipoprotein E (apoE) interaction with amyloid ß-protein precursor (APP) has garnered attention as the therapeutic target for Alzheimer's disease (AD). Having discovered the apoE antagonist (6KApoEp) that blocks apoE binding to N-terminal APP, we tested the therapeutic potential of 6KApoEp on AD-relevant phenotypes in amyloid ß-protein precursor/presenilin 1 (APP/PS1) mice that express each human apoE isoform of apoE2, apoE3, or apoE4 (designated APP/PS1/E2, APP/PS1/E3, or APP/PS1/E4 mice). At 12 months of age, we intraperitoneally administered 6KApoEp (250 µg/kg) or vehicle once daily for 3 months. At 15 months of age, blockage of apoE and N-terminal APP interaction by 6KApoEp treatment improved cognitive impairment in most tests of learning and memory, including novel object recognition and maze tasks in APP/PS1/E2, APP/PS1/E3, and APP/PS1/E4 mice versus each vehicle-treated mouse line and did not alter behavior in nontransgenic littermates. Moreover, 6KApoEp therapy ameliorated brain parenchymal and cerebral vascular ß-amyloid deposits and decreased abundance of amyloid ß-protein (Aß) in APP/PS1/E2, APP/PS1/E3, and APP/PS1/E4 mice versus each vehicle-treated mouse group. Notably, the highest effect in Aß-lowering by 6KApoEp treatment was observed in APP/PS1/E4 mice versus APP/PS1/E2 or APP/PS1/E3 mice. These effects occured through shifting toward lessened amyloidogenic APP processing due to decreasing APP abundance at the plasma membrane, reducing APP transcription, and inhibiting p44/42 mitogen-activated protein kinase phosphorylation. Our findings provide the preclinical evidence that 6KApoEp therapy aimed at targeting apoE and N-terminal APP interaction is a promising strategy and may be suitable for patients with AD carrying the apoE4 isoform.

Structural basis of the 24B3 antibody against the toxic conformer of amyloid ß with a turn at positions 22 and 23.

Amyloid ß-protein (Aß) oligomers are involved in the early stages of Alzheimer's disease (AD) and antibodies against these toxic oligomers could be useful for accurate diagnosis of AD. We identified the toxic conformer of Aß42 with a turn at positions 22/23, which has a propensity to form toxic oligomers. The antibody 24B3, developed by immunization of a toxic conformer surrogate E22P-Aß9-35 in mice, was found to be useful for AD diagnosis using human cerebrospinal fluid (CSF). However, it is not known how 24B3 recognizes the toxic conformation of wild-type Aß in CSF. Here, we report the crystal structure of 24B3 Fab complexed with E22P-Aß11-34, whose residues 16-26 were observed in electron densities, suggesting that the residues comprising the toxic turn at positions 22/23 were recognized by 24B3. Since 24B3 bound only to Aß42 aggregates, several conformationally restricted analogs of Aß42 with an intramolecular disulfide bond to mimic the conformation of toxic Aß42 aggregates were screened by enzyme immunoassay. As a result, only F19C,A30homoC-SS-Aß42 (1) bound significantly to 24B3. These data provide a structural basis for its low affinity to the Aß42 monomer and selectivity for its aggregate form.

Nephronectin influences EAE development by regulating the Th17/Treg balance via reactive oxygen species.

Blood levels of the extracellular matrix protein nephronectin (Npnt), a protein critical for kidney development, are elevated in autoimmune experimental autoimmune encephalitis (EAE) mice, which are a model for multiple sclerosis. We found here that treatment with anti-Npnt antibody directed against the α8ß1 integrin-binding site (Npnt-blocking antibody) inhibits EAE development. The selenium transporter selenoprotein P (SeP) was identified as a novel Npnt-binding partner. In EAE, Npnt induced SeP and glutathione peroxidase 1 (GPx1) expression, followed by reactive oxygen species (ROS) inhibition in CD4+ T cells; these changes were disturbed by Npnt-blocking antibody treatment, which also caused suppressed differentiation of interleukin (IL)-17-producing CD4+ T-helper cells (Th17s) and elevated differentiation of regulatory T cells (Tregs). Treatment of EAE mice with the ROS scavenger N-acetyl cysteine (NAC) blocked the Npnt-blocking antibody-induced decrease in Th17 differentiation and increase in Treg differentiation. In conclusion, the interaction between Npnt and SeP contributes to EAE development by regulating the Th17/Treg balance via the ROS level.

Characterization of a Conformation-Restricted Amyloid ß Peptide and Immunoreactivity of Its Antibody in Human AD brain.

Characterization of amyloid ß (Aß) oligomers, the transition species present prior to the formation of Aß fibrils and that have cytotoxicity, has become one of the major topics in the investigations of Alzheimer's disease (AD) pathogenesis. However, studying pathophysiological properties of Aß oligomers is challenging due to the instability of these protein complexes in vitro. Here, we report that conformation-restricted Aß42 with an intramolecular disulfide bond at positions 17 and 28 (SS-Aß42) formed stable Aß oligomers in vitro. Thioflavin T binding assays, nondenaturing gel electrophoresis, and morphological analyses revealed that SS-Aß42 maintained oligomeric structure, whereas wild-type Aß42 and the highly aggregative Aß42 mutant with E22P substitution (E22P-Aß42) formed Aß fibrils. In agreement with these observations, SS-Aß42 was more cytotoxic compared to the wild-type and E22P-Aß42 in cell cultures. Furthermore, we developed a monoclonal antibody, designated TxCo-1, using the toxic conformation of SS-Aß42 as immunogen. X-ray crystallography of the TxCo-1/SS-Aß42 complex, enzyme immunoassay, and immunohistochemical studies confirmed the recognition site and specificity of TxCo-1 to SS-Aß42. Immunohistochemistry with TxCo-1 antibody identified structures resembling senile plaques and vascular Aß in brain samples of AD subjects. However, TxCo-1 immunoreactivity did not colocalize extensively with Aß plaques identified with conventional Aß antibodies. Together, these findings indicate that Aß with a turn at positions 22 and 23, which is prone to form Aß oligomers, could show strong cytotoxicity and accumulated in brains of AD subjects. The SS-Aß42 and TxCo-1 antibody should facilitate understanding of the pathological role of Aß with toxic conformation in AD.

Gallic acid is a dual α/ß-secretase modulator that reverses cognitive impairment and remediates pathology in Alzheimer mice.

Several plant-derived compounds have demonstrated efficacy in pre-clinical Alzheimer's disease (AD) rodent models. Each of these compounds share a gallic acid (GA) moiety, and initial assays on this isolated molecule indicated that it might be responsible for the therapeutic benefits observed. To test this hypothesis in a more physiologically relevant setting, we investigated the effect of GA in the mutant human amyloid ß-protein precursor/presenilin 1 (APP/PS1) transgenic AD mouse model. Beginning at 12 months, we orally administered GA (20 mg/kg) or vehicle once daily for 6 months to APP/PS1 mice that have accelerated Alzheimer-like pathology. At 18 months of age, GA therapy reversed impaired learning and memory as compared with vehicle, and did not alter behavior in nontransgenic littermates. GA-treated APP/PS1 mice had mitigated cerebral amyloidosis, including brain parenchymal and cerebral vascular ß-amyloid deposits, and decreased cerebral amyloid ß-proteins. Beneficial effects co-occurred with reduced amyloidogenic and elevated nonamyloidogenic APP processing. Furthermore, brain inflammation, gliosis, and oxidative stress were alleviated. We show that GA simultaneously elevates α- and reduces ß-secretase activity, inhibits neuroinflammation, and stabilizes brain oxidative stress in a pre-clinical mouse model of AD. We further demonstrate that GA increases abundance of a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10, Adam10) proprotein convertase furin and activates ADAM10, directly inhibits ß-site APP cleaving enzyme 1 (BACE1, Bace1) activity but does not alter Adam10 or Bace1 transcription. Thus, our data reveal novel post-translational mechanisms for GA. We suggest further examination of GA supplementation in humans will shed light on the exciting therapeutic potential of this molecule.

Antibodies against nephronectin ameliorate anti-type II collagen-induced arthritis in mice.

The extracellular matrix protein nephronectin (Npnt) is known to be critical for kidney development, but its function in inflammatory diseases is unknown. Here, we developed a new enzyme-linked immunosorbent assay system to detect Npnt in various autoimmune diseases, which revealed that plasma Npnt levels are increased in various mouse autoimmune models. We also report that antibodies against the α8ß1 integrin-binding region of Npnt protect mice from anti-type II collagen-induced arthritis, suggesting that Npnt may be a potential therapeutic target molecule for the prevention of autoimmune arthritis.

Combined treatment with the phenolics (-)-epigallocatechin-3-gallate and ferulic acid improves cognition and reduces Alzheimer-like pathology in mice.

"Nutraceuticals" are well-tolerated natural dietary compounds with drug-like properties that make them attractive as Alzheimer's disease (AD) therapeutics. Combination therapy for AD has garnered attention following a recent National Institute on Aging mandate, but this approach has not yet been fully validated. In this report, we combined the two most promising nutraceuticals with complementary anti-amyloidogenic properties: the plant-derived phenolics (-)-epigallocatechin-3-gallate (EGCG, an α-secretase activator) and ferulic acid (FA, a ß-secretase modulator). We used transgenic mice expressing mutant human amyloid ß-protein precursor and presenilin 1 (APP/PS1) to model cerebral amyloidosis. At 12 months of age, we orally administered EGCG and/or FA (30 mg/kg each) or vehicle once daily for 3 months. At 15 months, combined EGCG-FA treatment reversed cognitive impairment in most tests of learning and memory, including novel object recognition and maze tasks. Moreover, EGCG- and FA-treated APP/PS1 mice exhibited amelioration of brain parenchymal and cerebral vascular ß-amyloid deposits and decreased abundance of amyloid ß-proteins compared with either EGCG or FA single treatment. Combined treatment elevated nonamyloidogenic soluble APP-α and α-secretase candidate and down-regulated amyloidogenic soluble APP-ß, ß-C-terminal APP fragment, and ß-secretase protein expression, providing evidence for a shift toward nonamyloidogenic APP processing. Additional beneficial co-treatment effects included amelioration of neuroinflammation, oxidative stress, and synaptotoxicity. Our findings offer preclinical evidence that combined treatment with EGCG and FA is a promising AD therapeutic approach.

Combination therapy with octyl gallate and ferulic acid improves cognition and neurodegeneration in a transgenic mouse model of Alzheimer's disease.

To date, there is no effective Alzheimer's disease (AD)-modifying therapy. Nonetheless, combination therapy holds promise, and nutraceuticals (natural dietary compounds with therapeutic properties) and their synthetic derivatives are well-tolerated candidates. We tested whether combination therapy with octyl gallate (OG) and ferulic acid (FA) improves cognition and mitigates AD-like pathology in the presenilin-amyloid ß-protein precursor (PSAPP) transgenic mouse model of cerebral amyloidosis. One-year-old mice with established ß-amyloid plaques received daily doses of OG and FA alone or in combination for 3 months. PSAPP mice receiving combination therapy had statistically significant improved cognitive function versus OG or FA single treatment on some (but not all) measures. We also observed additional statistically significant reductions in brain parenchymal and cerebral vascular ß-amyloid deposits as well as brain amyloid ß-protein abundance in OG- plus FA-treated versus singly-treated PSAPP mice. These effects coincided with enhanced nonamyloidogenic amyloid ß-protein precursor (APP) cleavage, increased α-secretase activity, and ß-secretase inhibition. We detected elevated expression of nonamyloidogenic soluble APP-α and the α-secretase candidate, a disintegrin and metalloproteinase domain-containing protein 10. Correspondingly, amyloidogenic ß-carboxyl-terminal APP fragment and ß-site APP-cleaving enzyme 1 expression levels were reduced. In parallel, the ratio of ß- to α-carboxyl-terminal APP fragment was decreased. OG and FA combination therapy strikingly attenuated neuroinflammation, oxidative stress, and synaptotoxicity. Co-treatment afforded additional statistically significant benefits on some, but not all, of these outcome measures. Taken together, these data provide preclinical proof-of-concept for AD combination therapy.

Reactivity of anti-HNK-1 antibodies to branched O-mannose glycans associated with demyelination.

Human natural killer-1 (HNK-1) epitope, a highly-expressed glycan in the nervous system, is critical for normal synaptic plasticity and spatial learning. HNK-1 epitope modifies N-glycans on several neural glycoproteins, and also modifies O-mannosyl glycans. A branching enzyme for O-mannosyl glycans (GnT-IX, Core M2 synthase) exhibits brain-specific expression, and the product core M2 glycans are also limited to the brain. In a previous study, we showed that cuprizone-induced demyelination increased HNK-1-capped core M2 glycan expression, while GnT-IX deficiency ameliorated demyelination, suggesting that these glycans could be useful diagnostic markers for demyelination status and act as therapeutic targets. Nevertheless, a lack of appropriate detection tools hampered further analysis of HNK-1-capped O-mannosyl glycans. In the present study, we chemoenzymatically synthesized HNK-1-capped core M2 glycans for antibody production, and confirmed that the resulting immune sera reacted with HNK-1-capped core M2 glycans. We then examined several HNK-1-related antibodies, including the Cat-315 antibody, for reactions with HNK-1-capped core M2 glycans. Finally, we confirmed the increased HNK-1 epitope expression in demyelinated brains of cuprizone-fed mice.

Methylene blue modulates ß-secretase, reverses cerebral amyloidosis, and improves cognition in transgenic mice.

Amyloid precursor protein (APP) proteolysis is required for production of amyloid-ß (Aß) peptides that comprise ß-amyloid plaques in the brains of patients with Alzheimer disease (AD). Here, we tested whether the experimental agent methylene blue (MB), used for treatment of methemoglobinemia, might improve AD-like pathology and behavioral deficits. We orally administered MB to the aged transgenic PSAPP mouse model of cerebral amyloidosis and evaluated cognitive function and cerebral amyloid pathology. Beginning at 15 months of age, animals were gavaged with MB (3 mg/kg) or vehicle once daily for 3 months. MB treatment significantly prevented transgene-associated behavioral impairment, including hyperactivity, decreased object recognition, and defective spatial working and reference memory, but it did not alter nontransgenic mouse behavior. Moreover, brain parenchymal and cerebral vascular ß-amyloid deposits as well as levels of various Aß species, including oligomers, were mitigated in MB-treated PSAPP mice. These effects occurred with inhibition of amyloidogenic APP proteolysis. Specifically, ß-carboxyl-terminal APP fragment and ß-site APP cleaving enzyme 1 protein expression and activity were attenuated. Additionally, treatment of Chinese hamster ovary cells overexpressing human wild-type APP with MB significantly decreased Aß production and amyloidogenic APP proteolysis. These results underscore the potential for oral MB treatment against AD-related cerebral amyloidosis by modulating the amyloidogenic pathway.

Radiosensitization of human lung cancer cells by the novel purine-scaffold Hsp90 inhibitor, PU-H71.

The molecular chaperone heat shock protein 90 (Hsp90) is involved in the maturation and stabilization of a wide range of oncogenic client proteins for oncogenesis and malignant cell proliferation, which renders this protein a promising target in the development of cancer therapeutics. PU-H71 is a purine-scaffold Hsp90 inhibitor with less toxicity in normal cells than in cancer cells. In this study, we examined the in vitro radiosensitizing activity and molecular mechanisms of action of PU-H71 in human lung cancer cell lines. PU-H71 enhanced the sensitivity of the SQ-5 and A549 cancer cells to radiation. When the cancer cells were pre-treated with PU-H71, the repair of DNA double-strand breaks (DSBs) was markedly inhibited after irradiation compared with the cells that were not pre-treated with PU-H71, as evaluated by counting the foci of phosphorylated histone H2AX (γ-H2AX). We further demonstrated that post-irradiation, PU-H71 inhibited Rad51 foci formation, a critical protein for the homologous recombination pathway of DNA DSB repair. These data indicate that targeting Hsp90 with PU-H71 may be novel therapeutic strategy for radioresistant carcinomas.

Novel sandwich ELISA for detecting the human soluble (pro)renin receptor.

The (pro)renin receptor ((P)RR) plays a key role in the activation of the local renin-angiotensin system via interaction with renin and prorenin. A truncated form that is cleaved by furin, referred to as soluble (pro)renin receptor (s(P)RR), is secreted into the extracellular space. An accurate measurement of s(P)RR levels in vivo is an important issue in elucidating the roles of (P)RR in physiology and pathophysiology. To address this, we developed a sandwich ELISA that is applicable to human subjects. The standard curve of this ELISA showed a high linearity (125-8,000 pg/ml) with a correlation coefficient of >0.99. The recovery rate was approximately 90% in human blood and urine samples. The s(P)RR levels in plasma of healthy volunteers was in the range from 15.2 to 35.1 ng/ml (n = 20). Intra- and inter-assay coefficient of variations were less than 5.5% and 7.5%, respectively. It thus appears that this ELISA is a reliable tool for measuring s(P)RR levels in human subjects.

[Experience using the isocenter verification device in proton therapy equipment].

In this study, we developed an isocenter verification device for use in proton therapy. Radiation and mechanical isocenters were verified for treatment equipment including room lasers, a digital radiography system and the beam axis of a rotational gantry. The special feature of this device is its ability to correlate the position of the three isocenters in one measurement and thus improve accuracy compared to the conventional method using three separate devices. The reproducibility of the method and the fluctuation of the position of the beam axis isocenter were both investigated using this device for almost a year. Monthly measurements of the isocenter position were acquired for two gantries and it was found that the fluctuation was +/- 0.10mm for the up-to-down direction and +/- 0.16mm for the right-to-left direction in Gantry 1 and was +/-0.14mm for the up-to-down direction and +/-0.18mm for the right-to-left direction in Gantry 2. We could be measured with a repeatability of +/-0.18 mm or less by using developed device for the relative positional relationship between each isocenters. Because we can confirm results in approximately 30 minutes, we can perform a quality control after a clinical practice.

Leucine-rich α-2-glycoprotein is a marker for idiopathic normal pressure hydrocephalus.

OBJECTIVE: Cerebrospinal fluid (CSF) shunting can improve symptoms of elderly patients' idiopathic normal pressure hydrocephalus (iNPH). However, adjunctive means for confirming the diagnosis remain unavailable. We have previously reported the specific increase of leucine-rich alpha-2-glycoprotein (LRG) in iNPH CSF, and the present study investigates its potential clinical applications. METHODS: We performed CSF tap test (TT) on 90 patients (mean age 73.4 years) and shunting in 52 patients (mean age 73.5 years), evaluating symptom improvement and higher cerebral functions-mini-mental state examination (MMSE) and Frontal Assessment Battery (FAB) before and 12 months after shunting. LRG and tau protein concentrations in TT CSF were simultaneously measured using enzyme-linked immunosorbent assay. We then compared the predictive value of these concentrations with TT results regarding successful shunting outcomes. RESULTS: Positive combinations of TT and LRG concentrations of 67 ng/ml or higher, gave 81.6% sensitivity and 78.6% specificity. Therefore we used LRG (67 ng/ml) and tau (200 pg/ml) cut-off values, dividing patients into four groups. In group A (LRG ≥ 67 ng/ml and tau < 200 pg/ml) 31 of 34 patients (91.2%) had a positive TT and all operated 22 patients were shunt responders. Dementia MMSE and FAB scores in them increased from a baseline of 22.05(SE ± 0.96) to 25.65 (±0.85) and 11.38 (±0.68) to 13.08 (±0.57) respectively. In group B, (LRG ≥ 67 ng/ml and tau ≥ 200 pg/ml), the mean MMSE score increased from 17.62 (±2.03) to 21.62 (±1.96), and the FAB decreased slightly from 9.25 (±1.15) to 10.5 (±1.59), without improvement beyond the range of dementia. In group C, (LRG < 67 ng/ml, tau < 200 pg/ml), the mean MMSE score improved from 22.06 (±1.25) to 24.29 (±1.23) and the FAB score improved slightly from 12.0 (±0.72) to 12.87 (±0.72). Finally, in group D, (LRG < 67 ng/ml, tau ≥ 200 pg/ml), there was almost no improvement in MMSE score CONCLUSIONS: A combination of positive TT and biomarkers quantification such as LRG and tau protein, can reliably predict shunting outcome in iNPH patients.

Thrombin-cleaved osteopontin levels in synovial fluid correlate with disease severity of knee osteoarthritis.

OBJECTIVE: although osteoarthritis (OA) is generally assessed using standard radiographic images in clinical practice, biochemical markers can be used to detect the disease and determine its severity. Osteopontin (OPN) is an extracellular matrix glycoprotein that is a potential inflammatory cytokine. Presence of the thrombin-cleaved form of OPN is well correlated with various inflammatory diseases. We examined whether thrombin-cleaved OPN in synovial fluid (SF) and synovium could be associated with the severity of knee OA. METHODS: SF samples were obtained from 139 knees with OA. Thrombin-cleaved OPN product was determined using Western blotting. Levels of thrombin-cleaved and full-length OPN in SF were determined by ELISA. Synovium samples were analyzed by immunohistochemistry using an antibody specific to the thrombin-cleaved form. RESULTS: western blotting showed the presence of thrombin-cleaved OPN in SF from patients with advanced OA. Concentrations of OPN full-length in OA knees were not statistically different from those in controls (p = 0.134). In contrast, levels of OPN N-half were significantly higher in OA knees than in controls (p = 0.042). Statistically significant correlation was found between thrombin-cleaved OPN and disease severity by Kellgren-Lawrence grade 1, 2, 3, and 4 (R = 0.274, p < 0.001). Immunohistochemistry of the synovium showed stronger reactivity in samples from subjects with advanced OA. CONCLUSION: local generation of thrombin-cleaved OPN was increased with greater OA severity.

Antitumor activity of anti-C-ERC/mesothelin monoclonal antibody in vivo.

Mesothelioma is an aggressive cancer often caused by chronic asbestos exposure, and its prognosis is very poor despite the therapies currently used. Due to the long latency period between asbestos exposure and tumor development, the worldwide incidence will increase substantially in the next decades. Thus, novel effective therapies are warranted to improve the prognosis. The ERC/mesothelin gene (MSLN) is expressed in wide variety of human cancers, including mesotheliomas, and encodes a precursor protein cleaved by proteases to generate C-ERC/mesothelin and N-ERC/mesothelin. In this study, we investigated the antitumor activity of C-ERC/mesothelin-specific mouse monoclonal antibody, 22A31, against tumors derived from a human mesothelioma cell line, ACC-MESO-4, in a xenograft experimental model using female BALB/c athymic nude mice. Treatment with 22A31 did not inhibit cell proliferation of ACC-MESO-4 in vitro; however, therapeutic treatment with 22A31 drastically inhibited tumor growth in vivo. 22A31 induced antibody-dependent cell-mediated cytotoxicity by natural killer (NK) cells, but not macrophages, in vitro. Consistently, the F(ab')(2) fragment of 22A31 did not inhibit tumor growth in vivo, nor did it induce antibody-dependent cell mediated cytotoxicity (ADCC) in vitro. Moreover, NK cell depletion diminished the antitumor effect of 22A31. Thus, 22A31 induced NK cell-mediated ADCC and exerted antitumor activity in vivo. 22A31 could have potential as a therapeutic tool to treat C-ERC/mesothelin-expressing cancers including mesothelioma.

Establishment of novel mAb to human ERC/mesothelin useful for study and diagnosis of ERC/mesothelin-expressing cancers.

Malignant mesothelioma is a highly aggressive tumor of the serosal cavity that arises from the mesothelial cells of the pleura, peritoneum, or pericardium. The immunohistochemical diagnosis of epithelioid mesothelioma from biopsy or surgically resected specimens has been actively pursued, using markers such as mesothelin. Several markers have indeed been helpful for confirming the diagnosis of mesothelioma and distinguishing between mesothelioma and adenocarcinoma. The authors have developed a novel mAb to human C-ERC/mesothelin, which performed well when used in western blotting, fluorescence-activated cell sorting, immunocytochemistry and immunohistochemistry, and which therefore will be useful in studying the molecular biology of mesothelin, in addition to improving the diagnosis and therapy of mesothelin-expressing cancers.

Thrombin-cleaved osteopontin in synovial fluid of subjects with rheumatoid arthritis.

OBJECTIVE: Osteopontin (OPN) is an extracellular matrix glycoprotein that has been recognized as a potential inflammatory cytokine. The function of OPN is modulated by protease digestion, and a thrombin-cleaved form of OPN is involved in the pathogenesis of various inflammatory disorders. We examined thrombin-cleaved OPN products in synovial fluid from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: Synovial fluid samples were obtained from knees of 20 patients with RA and 111 patients with OA. Thrombin-cleaved OPN product was determined using Western blotting. Levels of thrombin- cleaved and full-length OPN in synovial fluid were determined by ELISA. Synovia were analyzed by immunohistochemistry using an antibody specific to the thrombin-cleaved form. RESULTS: Immunoblotting showed the presence of thrombin-cleaved OPN in synovial fluid from patients with RA and OA. ELISA results showed no difference between concentrations of full-length OPN in the synovial fluid of RA and OA patients; however, thrombin-cleaved OPN concentrations in RA synovial fluid samples were roughly 30-fold higher compared with OA samples (p < 0.001). Synovial fluid concentrations of thrombin-cleaved OPN in RA did not correlate with C-reactive protein levels. Immunohistochemistry of the synovium showed stronger reactivity in RA than in OA samples. CONCLUSION: Local generation of thrombin-cleaved OPN was increased in RA joints. Thrombin-cleaved OPN may be a useful biochemical marker of RA.

Cytoplasmic and serum galectin-3 in diagnosis of thyroid malignancies.

In order to address whether galectin-3 in the sera and fine needle aspirates serve as a diagnostic marker distinguishing between benign and malignant thyroid nodules, we developed an enzyme-linked immunosorbent assay. We quantified galectin-3 in fine needle aspirates from a series of 118 patients with thyroid nodules and serum galectin-3 from another series of 46 patients, which were compared with final histology after thyroidectomy. Relative galectin-3 value (ng/mg), defined as galectin-3 concentration (ng/ml) divided by total protein concentration (mg/ml) in fine needle aspirates, was significantly higher in papillary carcinoma than in the other thyroid entities. There was no significant difference in serum galectin-3 level among patients with thyroid nodules and healthy individuals. Accordingly, relative galectin-3 value allows a definitive diagnosis of papillary carcinoma independent of cellular morphology, whereas serum galectin-3 does not serve as a marker for papillary carcinoma.