Remarkable increases of α1-antichymotrypsin in brain tissues of rodents during prion infection.

α1-Antichymotrypsin (α1-ACT) belongs to a kind of acute-phase inflammatory protein. Recently, such protein has been proved exist in the amyloid deposits which is the hallmark of Alzheimer's disease, but limitedly reported in prion disease. To estimate the change of α1-ACT during prion infection, the levels of α1-ACT in the brain tissues of scrapie agents 263K-, 139A- and ME7-infected rodents were analyzed, respectively. Results shown that α1-ACT levels were significantly increased in the brain tissues of the three kinds of scrapie-infected rodents, displaying a time-dependent manner during prion infection. Immunohistochemistry assays revealed the increased α1-ACT mainly accumulated in some cerebral regions of rodents infected with prion, such as cortex, thalamus and cerebellum. Immunofluorescent assays illustrated ubiquitously localization of α1-ACT with GFAP positive astrocytes, Iba1-positive microglia and NeuN-positive neurons. Moreover, double-stained immunofluorescent assays and immunohistochemistry assays using series of brain slices demonstrated close morphological colocalization of α1-ACT signals with that of PrP and PrPSc in the brain slices of 263K-infected hamster. However, co-immunoprecipitation does not identify any detectable molecular interaction between the endogenous α1-ACT and PrP either in the brain homogenates of 263K-infected hamsters or in the lysates of prion-infected cultured cells. Our data here imply that brain α1-ACT is increased abnormally in various scrapie-infected rodent models. Direct molecular interaction between α1-ACT and PrP seems not to be essential for the morphological colocalization of those two proteins in the brain tissues of prion infection.

Circulating inflammatory biomarkers in relation to brain structural measurements in a non-demented elderly population.

The aim of this investigation was to determine whether circulating inflammatory biomarkers c-reactive protein (CRP), interleukin-6 (IL6), and alpha 1-antichymotrypsin (ACT) were related to structural brain measures assessed by magnetic resonance imaging (MRI). High-resolution structural MRI was collected on 680 non-demented elderly (mean age 80.1years) participants of a community-based, multiethnic cohort. Approximately three quarters of these participants also had peripheral inflammatory biomarkers (CRP, IL6, and ACT) measured using ELISA. Structural measures including brain volumes and cortical thickness (with both global and regional measures) were derived from MRI scans, and repeated MRI measures were obtained after 4.5years. Mean fractional anisotropy was used as the indicator of white matter integrity assessed with diffusion tensor imaging. We examined the association of inflammatory biomarkers with brain volume, cortical thickness, and white matter integrity using regression models adjusted for age, gender, ethnicity, education, APOE genotype, and intracranial volume. A doubling in CRP (b=-2.48, p=0.002) was associated with a smaller total gray matter volume, equivalent to approximately 1.5years of aging. A doubling in IL6 was associated with smaller total brain volume (b=-14.96, p<0.0001), equivalent to approximately 9years of aging. Higher IL6 was also associated with smaller gray matter (b=-6.52, p=0.002) and white matter volumes (b=-7.47, p=0.004). The volumes of most cortical regions including frontal, occipital, parietal, temporal, as well as subcortical regions including pallidum and thalamus were associated with IL6. In a model additionally adjusted for depression, vascular factors, BMI, and smoking status, the association between IL6 and brain volumes remained, and a doubling in ACT was marginally associated with 0.054 (p=0.001) millimeter thinner mean cortical thickness, equivalent to that of approximately 2.7years of aging. None of the biomarkers was associated with mean fractional anisotropy or longitudinal change of brain volumes and thickness. Among older adults, increased circulating inflammatory biomarkers were associated with smaller brain volume and cortical thickness but not the white matter tract integrity. Our preliminary findings suggest that peripheral inflammatory processes may be involved in the brain atrophy in the elderly.

Pancreatic-type Acinar Cell Carcinoma of the Stomach Included in Multiple Primary Carcinomas.

BACKGROUND/AIM: Pancreatic-type acinar cell carcinoma (ACC) in the stomach is extraordinarily rare. We pathologically examined two cases with multiple primary carcinomas, including gastric tumors. PATIENTS AND METHODS: Gastric cancer specimens were examined by immunostaining and electron microscopy. RESULTS: Both cases had cancer cells with acinar patterns, resembling pancreatic ACC. The cancer cells in the first case were positive for exocrine markers, including chymotrypsin, lipase and alpha-1 antichymotrypsin (ACT), as well as neuroendocrine markers, including chromogranin A and synaptophysin. The cancer cells in the second case were positive for chymotrypsin and alpha-1 ACT, while being slightly positive for chromogranin A and synaptophysin. Ultrastructurally, cancer cells contained zymogen granules in both cases. The final diagnosis was pancreatic mixed acinar-neuroendocrine carcinoma and pure pancreatic ACC, respectively. CONCLUSION: We confirmed two cases with gastric pancreatic-type ACC included in multiple primary carcinomas. This type of double cancer has not been reported previously.

Immunohistochemical comparison of p53, Ki-67, CD68, vimentin, α-smooth muscle actin and alpha-1-antichymotry-psin in oral peripheral and central giant cell granuloma.

INTRODUCTION: Giant cell lesions are characterised histologically by multinucleated giant cells in a background of ovoid to spindle-shaped mesenchymal cells. There is a major debate whether these lesions are separate entities or variants of the same disease. Our aim was to study the nature of multinucleated and mononuclear cells from peripheral giant cell granuloma (PGCG), and central giant cell granuloma (CGCG) and giant cell tumor (GCT) of long bones using immunohistochemistry evaluation and to determine whether there is a correlation between recurrence and the markers used. MATERIALS AND METHODS: Ki-67, p53, Vimentin, smooth muscle specific actin, CD68 and alpha-1-antichymotrypsin were used to study 60 giant cell lesions. These included 26 CGCG, 28 PGCG, and 6 GCT cases using an avidin-biotin-complex immunohistochemistry standard method. RESULTS: All studied cases showed the same results except the percentage of Ki-67 positive mononuclear cells in PGCG was significantly higher than that of both CGCG and GCT (p<0.05). Interestingly, no statistical correlation between recurrence and the markers used was found. CONCLUSION: Our results may suggest that these lesions have the same histogenesis. The mononuclear stromal cells, both histiocytic and myofibroblastic, are thought to be responsible for the behavior of these lesions whereas the multinucleated cells are considered as reactive. This might support the argument that PGCG, CGCG and GCT are different variants for the same disease. Further studies using molecular techniques are required to elucidate why some of these lesions behave aggressively than others.

Pancreatoblastoma in an adult.

Pancreatoblastoma is a malignant pancreatic tumor that rarely occurs in adults. We report a case of an adult female with pancreatoblastoma. A mass was detected in the pancreatic head using computed tomography and ultrasonography. The clinical diagnosis was a solid-pseudopapillary neoplasm of the pancreas. However, after the operation, the final diagnosis was pancreatoblastoma, which showed two lines of differentiation: Acinar differentiation and squamoid corpuscles. The patient is currently in good condition.

Proteomic identification of proteinase inhibitors in the porcine enamel matrix derivative, EMD(®).

BACKGROUND AND OBJECTIVE: The porcine enamel matrix derivative, EMD(®), which is the active component of Emdogain(®), is used widely in periodontics because of its ability to promote the regeneration of soft and hard tissues and to reduce inflammation. Previous studies have used indirect methods to explain its angiogenic and proliferative effects on cells associated with wound healing. In this study we used proteomic techniques to identify proteins in EMD other than amelogenins. MATERIAL AND METHODS: Proteins in EMD were separated by two-dimensional gel electrophoresis and were identified using mass spectrometry. Proteomic results were validated by western blot analysis of Emdogain. RESULTS: Fourteen proteins of porcine origin were identified and included the serine and cysteine proteinase inhibitors alpha1-antichymotrypsin and fetuin A, respectively. Alpha1-antichymotrypsin is an acute-phase factor that has been reported to indirectly down-regulate the expression of the gelatinase MMP-9. Fetuin A, a major glycoprotein component of bone and teeth, is a potent inhibitor of ectopic calcification of vascular and soft tissues and has been implicated in both osteogenesis and bone resorption. It also facilitates plasma membrane repair in damaged fibroblasts. CONCLUSION: EMD contains a number of high-molecular-weight compounds which include the proteinase inhibitors, fetuin A and alpha1-antichymotrypsin.

Proteolytic activation of matrix metalloproteinase-9 in skin wound healing is inhibited by alpha-1-antichymotrypsin.

An excessive amount of matrix metalloproteinase-9 (MMP-9) has been well documented in inflammatory diseases, including chronic wounds and cancers. Secreted as a zymogen, proMMP-9 can be irreversibly converted to a mature form through cleavage of the N-terminal propeptide domain. Although the converting enzyme for proMMP-9 in human tissues is unknown, we previously found that tumor necrosis factor-alpha (TNF-alpha) promotes activation of proMMP-9 in human skin, and characterized the converting activities as tissue-associated chymotrypsin-like proteinases. On the other hand, the pathophysiologic inhibitor to prevent proMMP-9 maturation also remains elusive. In this regard, we observed the presence of the inhibitory property in burn blister fluid that abrogates the skin extract-mediated activation of proMMP-9. Then we determined that alpha-1-antichymotrypsin (alpha-ACT), an acute-phase factor abundantly present in the blister, effectively inhibited proMMP-9 activation in human and rodent skin. In contrast, the aminophenylmercuric acetate-induced "cysteine switch" and activation of proMMP-9 were not affected by alpha-ACT. TNF-alpha-induced activation of proMMP-9 by the explants of human skin was inhibited by alpha-ACT but not by related alpha-1-antitrypsin. alpha-ACT specifically attenuated maturation of proMMP-9 but not proMMP-2 or proMMP-13. Furthermore, short peptides that mimic the reactive center loop (RCL) of alpha-ACT were sufficient to inhibit the conversion. Mutation analysis demonstrated that a conserved leucine within the RCL was critical for alpha-ACT-exerted inhibition. In chronic wounds, a large amount of mature MMP-9 was associated with fragmentation and inactivation of alpha-ACT. Taken together, these results demonstrate that, to the best of our knowledge, alpha-ACT is a previously unreported pathophysiologic inhibitor that controls proMMP-9 activation in skin tissue.

Preparation of highly stable oligo(ethylene glycol) derivatives-functionalized gold nanoparticles and their application in LSPR-based detection of PSA/ACT complex.

A sandwich immunoassay for PSA/ACT complex detection based on gold nanoparticle aggregation using two probes was developed. The functionalized colloidal gold nanoparticles (AuNPs) showed highly stable not only in the presence of high ionic strength but also in a wide pH range. The functionalized AuNPs were tagged with PSA/ACT complex monoclonal antibody and goat PSA polyclonal antibody and served as the probes to induce aggregation of the colloidal particles. As a result, PSA/ACT complex was detected at concentrations as low as 1 ng/ml. This is the first time that a new aggregation sandwich-immunoassay technique using two gold probes has been used, and the results are generally applicable to other LSPR-based immunoassays.

Double-enhancement strategy: A practical approach to a femto-molar level detection of prostate specific antigen-alpha1-antichymotrypsin (PSA/ACT complex) for SPR immunosensing.

Prostate specific antigen-alpha1-antichymotrypsin was detected by a double-enhancement strategy involving the exploitation of both colloidal gold nanoparticles (AuNPs) and precipitation of an insoluble product formed by HRP-biocatalyzed oxidation. The AuNPs were synthesized and conjugated with horse-radish peroxidase-PSA polyclonal antibody by physisorption. Using the protein-colloid for SPR-based detection of the PSA/ACT complex showed their enhancement as being consistent with other previous studies with regard to AuNPs enhancement, while the enzyme precipitation using DAB substrate was applied for the first time and greatly amplified the signal. The limit of detection was found at as low as 0.027 ng/ml of the PSA/ACT complex (or 300 fM), which is much higher than that of previous reports. This study indicates another way to enhance SPR measurement, and it is generally applicable to other SPR-based immunoassays.

Analysis of potential diagnostic biomarkers in cerebrospinal fluid of idiopathic normal pressure hydrocephalus by proteomics.

BACKGROUND: The pathogenesis of idiopathic normal pressure hydrocephalus (INPH) is unknown, and the syndrome of INPH remains a diagnostic and therapeutic challenge. The present study investigated the disease-specific proteins that aid in the diagnosis and treatment of INPH and thus to study their role in the disease process. METHODS: A comparative proteomic analysis was used for clinical screening of cerebrospinal fluid (CSF) proteins in 15 patients with INPH and compared with 12 normal subjects. Furthermore, enzyme linked immunosorbent assay (ELISA) was performed for comparison with CSF proteins between individual INPH patients and controls. RESULTS: Seven proteins and their isoforms, including leucine-rich alpha-2-glycoprotein (LRG), alpha1-antichymotrypsin, apolipoprotein D, apolipoprotein J, haptoglobin alpha1, serum albumin, and alpha-1-microglobulin/bikunin precursor showed significant changes in CSF of INPH patients compared with controls by proteomic analysis. And significant higher CSF levels of LRG in INPH patients compared with controls were found by ELISA. CONCLUSIONS: These results indicate that there are significant differences in the expression of certain proteins in the CSF of patients with INPH and normal subjects. In particular, the CSF level assay of LRG suggests that LRG is a specific biomarker for INPH and has potential use in the diagnosis and indication for CSF shunting.

A strategy for sensitivity and specificity enhancements in prostate specific antigen-alpha1-antichymotrypsin detection based on surface plasmon resonance.

A biochip based on surface plasmon resonance was fabricated to detect prostate specific antigen-alpha(1)-antichymotrypsin (PSA-ACT complex) in both HBS buffer and human serum. To reduce non-specific binding and steric hindrance effect, the chemical surface of the sensor chips was constructed by using various oligo(ethylene glycol) mixtures of different molar ratios of HS(CH2)11(OCH2CH2)6OCH2COOH and HS(CH2)11(OCH2CH2)3OH. The self-assembled monolayers were biotinylated to facilitate the immobilization of streptavidin. Using the chip surfaces, PSA-ACT complex in HBS buffer and human serum was detected at 20.7 and 47.5 ng/ml by primary immunoresponse, respectively. However, the limit of detection could be simply enhanced by a sandwich strategy to improve the sensitivity and specificity of the immunoassay. An intact PSA polyclonal antibody was used as an amplifying agent in the strategy. As a result, PSA-ACT complex concentrations as low as 10.2 and 18.1 ng/ml were found in the HBS buffer and human serum sample, respectively. The result indicates that this approach could satisfy our goal without modifying the secondary interactant.

Mast cell tryptase and chymase in chronic leg ulcers: chymase is potentially destructive to epithelium and is controlled by proteinase inhibitors.

BACKGROUND: Numerous mast cells are present in chronic leg ulcers. Tryptase and chymase are the major mediators of mast cells, but their significance is mostly dependent on their activity. In addition, the proteinases may affect ulcer epithelialization. OBJECTIVES: To study levels and activity of tryptase and chymase in wash samples and biopsies from chronic leg ulcers and the possible effect of these proteinases on keratinocyte growth and adherence. METHODS: Wash samples were taken from 16 patients and a superficial shave biopsy was taken in eight of these patients; a second biopsy series was obtained from the edge of chronic venous leg ulcers (n = 6). RESULTS: Significant levels of soluble tryptase activity and histamine, but low levels of chymase activity, were measured in wash samples from chronic ulcers. No tryptase-inhibiting activity, but clear chymase-inhibiting activity, was detected in the wash samples. In superficial wound bed biopsies, relatively marked levels of chymase activity together with histamine and tryptase activity were detected. In the second biopsy series, about 80% of the mast cells belonged to the MC(TC) type (tryptase- and chymase-immunopositive). However, about 55-61% of the chymase-immunopositive cells displayed chymase activity and 64 +/- 17% of the tryptase-positive cells revealed immunoreactivity of alpha(1)-antichymotrypsin. As the activity of chymase and tryptase was detected in the ulcer base in a ratio of 1:8, a preparation containing both chymase and tryptase was partially purified from human skin yielding a similar activity ratio of 1:11-13. Treatment of fibronectin-coated plastic surfaces with this preparation decreased the adherence of cultured human keratinocytes, this effect being attributable mainly to chymase. In 2-day cultures using growth factor/serum-deficient low- or high-calcium medium, the tryptase-chymase preparation inhibited the slow growth and at higher concentrations it even induced detachment of keratinocytes. This effect was attributed to chymase, and it was partially regulated by heparin and histamine. CONCLUSIONS: Even though chymase is partially inactivated in chronic leg ulcers, accumulated mast cells in the close proximity of the epithelium edge and their chymase may impair keratinocyte adherence and migration.

[Study of alpha1-antichymotrypsin (ACT) in prostatic carcinoma].

10 normal prostates and 100 prostates with tumour were studied immunohistochemically. ACT was found mainly in the cells lining the ducts. Synthesis of ACT is significantly increased in carcinoma due to mainly two parallel processes: ACT production by carcinoma cells and intensification of its production by normal cells mainly at the tumour periphery. Hyperplastic structures showed not very high ACT content, adenomatous structures revealed a higher response. On the whole, there is a parallelism between the content of ACT and prostatic specific antigen (PSA) in both normal and carcinomatous prostate, however PSA is being found in a much more wider spectrum of cells. Content of ACT in seminal fluid may be used as a parameter for diagnosis and monitoring of prostate cancer.

Benign fibrous histiocytoma of the mandible.

A very rare case of benign fibrous histiocytoma of the mandible is presented. A 49-year-old woman was admitted because of left buccal swelling and pain. Panoramic radiograph showed well-demarcated soap-bubble appearance without sclerotic rim in the left mandibular bone. A yellowish-white and partly brown solid tumor was noted in the excised mandibular bone specimen. The tumor histologically consisted of spindle cells, in which areas showing a storiform pattern and other areas composed of histiocytic cells with erythrophagocytosis and foam cells were mixed. Immunohistochemically, the tumor cells were positive for vimentin, CD68, alpha-1-antichymotrypsin and alpha-1-antitrypsin. From these findings the tumor was diagnosed as a primary BFH of the mandible. No recurrence has been noted 2 years and 11 months after surgery.

Evaluation of an automated chemiluminescent immunoassay for complexed PSA on the Bayer ACS:180 system.

Prostate-specific antigen (PSA), the most important tumor marker for the detection of prostate cancer, exists in serum in a free, uncomplexed form (free PSA [fPSA]), and as bound to protease inhibitors (mainly alpha1-antichymotrypsin [ACT]). The measurement of complexed PSA (cPSA) concentration in serum has been shown to have better sensitivity and specificity than serum total PSA concentration. A new chemiluminescent immunoassay for cPSA for use on the Bayer ACS:180 fully automated system (Bayer Corp, Tarrytown, NY) has been developed and evaluated. The precision of the new assay was <3.9% (within-run coefficient of variation [CV]) and <5.0% (total CV). The analytical sensitivity (95% upper limit of noise at zero calibrator) was <0.03 ng/mL. A comparison of the ACS:180 cPSA results with the cPSA concentrations calculated from the ACCESS (Beckman-Coulter) PSA and fPSA assays yielded the following regression equation: ACS:180 cPSA=0.93* (calculated ACCESS cPSA)+0.43, R=0.993, n=95. The mean dilution and spike recovery for five samples were both 98%. No interference was observed from hemoglobin, triglyceride, or bilirubin (NCCLS protocol). These results indicate that the ACS:180 cPSA assay is precise, and compares well with the calculated cPSA from ACCESS total and free-PSA results.

[Phenotype analysis of alpha1-antichymotrypsin in the Guangzhou Han population].

OBJECTIVE: To understand the phenotype distribution profile of alpha1-antichymotrypsin (alpha1-ACT) in the Han population in Guangzhou municipality so as to decide whether geographical factors affect the distribution of alpha1-ACT phenotypes. METHODS: Polyacrylamide gel isoelectric focusing followed by immunofixation techniques was employed for examining the alpha1-ACT phenotypes in 200 unrelated individuals randomly selected from the Guangzhou Han residents. RESULTS: Three phenotypes of alpha1-ACT were identified in this subject cohort, which were identical to those identified in Chongqing but with different distributions of the phenotype frequencies. CONCLUSION: Geographical factors may affect the distribution of the phenotype frequencies of alpha1-ACT, which is a promising genetic marker for the pursuit of human genetics.

Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry.

Quantitative proteome profiling using stable isotope protein tagging and automated tandem mass spectrometry (MS/MS) is an emerging technology with great potential for the functional analysis of biological systems and for the detection of clinical diagnostic or prognostic marker proteins. Owing to the enormous complexity of proteomes, their comprehensive analysis is an as-yet-unresolved technical challenge. However, biologically or clinically important information can be obtained if specific, information-rich protein classes, or sub-proteomes, are isolated and analyzed. Glycosylation is the most common post-translational modification. Here we describe a method for the selective isolation, identification and quantification of peptides that contain N-linked carbohydrates. It is based on the conjugation of glycoproteins to a solid support using hydrazide chemistry, stable isotope labeling of glycopeptides and the specific release of formerly N-linked glycosylated peptides via peptide- N-glycosidase F (PNGase F). The recovered peptides are then identified and quantified by MS/MS. We applied the approach to the analysis of plasma membrane proteins and proteins contained in human blood serum.

Three immunoassays based on monoclonal antibodies specific for prostate specific antigen (PSA), alpha-1-antichymotrypsin (ACT), and the PSA-ACT complex.

BACKGROUND: Prostate specific antigen (PSA) has been widely used as a biomarker for the screening and diagnosis of prostate cancer. PSA in serum predominantly exists as a complex with alpha-1-antichymotrypsin (ACT), and measurement of free PSA and the PSA-ACT complex may improve the utility of the serum PSA assay for differential diagnosis of prostate cancer and non-malignant prostate diseases, such as benign prostatic hyperplasia (BPH). METHODS: Monoclonal antibodies (MAbs) against PSA, ACT, and the PSA-ACT complex were produced by immunizing mice with an incubated mixture of PSA and ACT, and characterized by Western blot analyses and several enzyme-linked immunosorbant assay (ELISA) methods. RESULTS: The MAbs produced in this study are capable of distinguishing the PSA-ACT complex from free PSA and ACT. Four MAbs have been selected and utilized to construct three ELISA systems for the separate measurements of free PSA, the PSA-ACT complex, and total PSA. CONCLUSIONS: The three PSA assay systems developed in this study can specifically measure free PSA, total PSA, and the PSA-ACT complex with equal molar sensitivity. It is expected that these PSA assay systems could be useful in the diagnosis of prostate cancer.