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1.
Int J Mol Sci ; 22(5)2021 Mar 06.
Article in English | MEDLINE | ID: mdl-33800950

ABSTRACT

Male patients with Fabry disease (FD) are at high risk for the formation of antibodies to recombinant α-galactosidase A (AGAL), used for enzyme replacement therapy. Due to the rapid disease progression, the identification of patients at risk is highly warranted. However, currently suitable references and standardized protocols for anti-drug antibodies (ADA) determination do not exist. Here we generate a comprehensive patient-derived antibody mixture as a reference, allowing ELISA-based quantification of antibody titers from individual blood samples. Serum samples of 22 male patients with FD and ADAs against AGAL were pooled and purified by immune adsorption. ADA-affinities against agalsidase-α, agalsidase-ß and Moss-AGAL were measured by quartz crystal microbalance with dissipation monitoring (QCM-D). AGAL-specific immune adsorption generated a polyclonal ADA mixture showing a concentration-dependent binding and inhibition of AGAL. Titers in raw sera and from purified total IgGs (r2 = 0.9063 and r2 = 0.8952, both p < 0.0001) correlated with the individual inhibitory capacities of ADAs. QCM-D measurements demonstrated comparable affinities of the reference antibody for agalsidase-α, agalsidase-ß and Moss-AGAL (KD: 1.94 ± 0.11 µM, 2.46 ± 0.21 µM, and 1.33 ± 0.09 µM, respectively). The reference antibody allows the ELISA-based ADA titer determination and quantification of absolute concentrations. Furthermore, ADAs from patients with FD have comparable affinities to agalsidase-α, agalsidase-ß and Moss-AGAL.


Subject(s)
Antibodies, Neutralizing/immunology , Enzyme Replacement Therapy , Enzyme-Linked Immunosorbent Assay , Fabry Disease/immunology , alpha-Galactosidase/immunology , alpha-N-Acetylgalactosaminidase/immunology , Antibodies, Neutralizing/biosynthesis , Antibody Affinity , Dose-Response Relationship, Immunologic , Fabry Disease/blood , Fabry Disease/drug therapy , Humans , Immunoglobulin G/biosynthesis , Immunoglobulin G/immunology , Male , Recombinant Proteins/immunology , Recombinant Proteins/therapeutic use , Reference Standards , alpha-Galactosidase/blood , alpha-Galactosidase/therapeutic use , alpha-N-Acetylgalactosaminidase/blood , alpha-N-Acetylgalactosaminidase/therapeutic use
2.
Drug Alcohol Depend ; 170: 147-151, 2017 Jan 01.
Article in English | MEDLINE | ID: mdl-27907862

ABSTRACT

BACKGROUND: The present study aimed to investigate for the first time, the alteration of α-N-acetylgalactosaminidase (nagalase) concentration in alcohol-dependent individuals without liver disease, before, during and at the end of the detoxification therapy. METHODS: Forty-eight alcohol-dependent individuals without liver disease who were admitted for alcohol detoxification, and eighty-four healthy controls participated in this study. Patients' blood was obtained upon admission, two weeks later and after the completion of the detoxification period (4-5 weeks). Nagalase concentration in serum was assessed by enzyme-linked immunosorbent assay. RESULTS: Nagalase concentration was significantly elevated in the patient samples in all serum collections as compared to the normal controls, with a progressive fall from admission to discharge (p-value<0.001). Values differed significantly among the three time points, with a net shift to decrease, but remained still high, above normal control level at the end of the therapy. No significant correlations were detected among the nagalase levels and the liver enzymes values. Moreover, no significant correlation was found between the alterations of nagalase concentrations and the amount of consumed alcohol. CONCLUSIONS: The high nagalase concentrations in alcohol abuse might be associated with macrophage impairment through decreasing the endogenous macrophage-activating factor (MAF) production by Gc-protein. The possible pathogenetic association between nagalase activity and alcohol overconsumption remains a matter of further investigation. Nagalase could also serve as a marker of alcohol overconsumption for the evaluation of alcohol-dependent individuals before, as well as during the detoxification therapy.


Subject(s)
Alcoholism/blood , alpha-N-Acetylgalactosaminidase/blood , Adult , Alcoholism/drug therapy , Diazepam/therapeutic use , Enzyme-Linked Immunosorbent Assay , Female , GABA Modulators/therapeutic use , Humans , Macrophage-Activating Factors/blood , Male , Middle Aged , Therapeutics
3.
Cancer Lett ; 283(2): 222-9, 2009 Oct 08.
Article in English | MEDLINE | ID: mdl-19394758

ABSTRACT

Currently clinical outcome in melanoma is not predictable by known serum biomarkers. The only reliable tool for the diagnosis of this tumor is the histopathological assay after surgical removing. We used a proteomic approach in order to identify novel non-invasive serum biomarkers of melanoma. Serum proteomic maps showed different patterns in relation to the presence and progression of the tumor in five regions of the map. Differently expressed spots were identified by MALDI-TOF mass spectrometry. Significant increases of expression were found for transthyretin (TTR) and angiotensinogen (AGT) while vitamin D binding protein (DBP) expression was decreased in presence of melanoma. Interestingly, protein expression came back to control values in stages I and II of the disease after 1 month since surgical removal of suspected melanoma. We related the decrease of DBP spot to the impaired immune response of cancer patients. In fact cancer cells release the alpha-N-acetylgalactosaminidase that can deglycosylate DBP thus interfering with the immune cascade response in which DBP is involved, leading to immunosuppression in melanoma patients. Specific enzymatic activity of serum alpha-N-acetylgalactosaminidase was significantly increased in stage III melanoma patients, but not in early stages. This enzymatic assay may provide a non-invasive way of evaluation of melanoma severity.


Subject(s)
Biomarkers, Tumor/blood , Melanoma/blood , Skin Neoplasms/blood , alpha-N-Acetylgalactosaminidase/blood , Disease Progression , Electrophoresis, Gel, Two-Dimensional , Humans , Melanoma/enzymology , Melanoma/pathology , Neoplasm Staging , Proteomics , Skin Neoplasms/enzymology , Skin Neoplasms/pathology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
4.
J Med Virol ; 81(1): 16-26, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19031451

ABSTRACT

Serum Gc protein (known as vitamin D3-binding protein) is the precursor for the principal macrophage activating factor (MAF). The MAF precursor activity of serum Gc protein of HIV-infected patients was lost or reduced because Gc protein is deglycosylated by alpha-N-acetylgalactosaminidase (Nagalase) secreted from HIV-infected cells. Therefore, macrophages of HIV-infected patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Since Nagalase is the intrinsic component of the envelope protein gp120, serum Nagalase activity is the sum of enzyme activities carried by both HIV virions and envelope proteins. These Nagalase carriers were already complexed with anti-HIV immunoglobulin G (IgG) but retained Nagalase activity that is required for infectivity. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent macrophage activating factor (termed GcMAF), which produces no side effects in humans. Macrophages activated by administration of 100 ng GcMAF develop a large amount of Fc-receptors as well as an enormous variation of receptors that recognize IgG-bound and unbound HIV virions. Since latently HIV-infected cells are unstable and constantly release HIV virions, the activated macrophages rapidly intercept the released HIV virions to prevent reinfection resulting in exhaustion of infected cells. After less than 18 weekly administrations of 100 ng GcMAF for nonanemic patients, they exhibited low serum Nagalase activities equivalent to healthy controls, indicating eradication of HIV-infection, which was also confirmed by no infectious center formation by provirus inducing agent-treated patient PBMCs. No recurrence occurred and their healthy CD + cell counts were maintained for 7 years.


Subject(s)
HIV Infections/drug therapy , Immunotherapy/methods , Macrophage-Activating Factors/therapeutic use , Vitamin D-Binding Protein/therapeutic use , CD4 Lymphocyte Count , CD4-Positive T-Lymphocytes/virology , Humans , Macrophage-Activating Factors/administration & dosage , Plasma/virology , Viral Load , Vitamin D-Binding Protein/administration & dosage , alpha-N-Acetylgalactosaminidase/blood
5.
Cancer Immunol Immunother ; 57(7): 1007-16, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18058096

ABSTRACT

Serum vitamin D binding protein (Gc protein) is the precursor for the principal macrophage-activating factor (MAF). The MAF precursor activity of serum Gc protein of colorectal cancer patients was lost or reduced because Gc protein is deglycosylated by serum alpha-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Deglycosylated Gc protein cannot be converted to MAF, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent macrophage-activating factor (GcMAF) ever discovered, but it produces no side effect in humans. Macrophages treated with GcMAF (100 microg/ml) develop an enormous variation of receptors and are highly tumoricidal to a variety of cancers indiscriminately. Administration of 100 nanogram (ng)/ human maximally activates systemic macrophages that can kill cancerous cells. Since the half-life of the activated macrophages is approximately 6 days, 100 ng GcMAF was administered weekly to eight nonanemic colorectal cancer patients who had previously received tumor-resection but still carried significant amounts of metastatic tumor cells. As GcMAF therapy progressed, the MAF precursor activities of all patients increased and conversely their serum Nagalase activities decreased. Since serum Nagalase is proportional to tumor burden, serum Nagalase activity was used as a prognostic index for time course analysis of GcMAF therapy. After 32-50 weekly administrations of 100 ng GcMAF, all colorectal cancer patients exhibited healthy control levels of the serum Nagalase activity, indicating eradication of metastatic tumor cells. During 7 years after the completion of GcMAF therapy, their serum Nagalase activity did not increase, indicating no recurrence of cancer, which was also supported by the annual CT scans of these patients.


Subject(s)
Colorectal Neoplasms/therapy , Macrophage-Activating Factors/therapeutic use , Vitamin D-Binding Protein/therapeutic use , Aged , Aged, 80 and over , Antigens, Neoplasm/immunology , Biomarkers, Tumor/blood , Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/immunology , Female , Glycosylation , Humans , Immunotherapy , Macrophage Activation , Macrophage-Activating Factors/metabolism , Macrophages/immunology , Male , Middle Aged , Neuraminidase/metabolism , Vitamin D-Binding Protein/metabolism , alpha-N-Acetylgalactosaminidase/blood
6.
AIDS Res Hum Retroviruses ; 22(3): 262-71, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16545013

ABSTRACT

Serum vitamin D3-binding protein (Gc protein) is the precursor for the principal macrophage-activating factor (MAF). The precursor activity of serum Gc protein was lost or reduced in HIV-infected patients. These patient sera contained alpha-N-acetylgalactosaminidase (Nagalase), which deglycosylates serum Gc protein. Deglycosylated Gc protein cannot be converted to MAF and thus loses MAF precursor activity, leading to immunosuppression. Nagalase in the blood stream of HIV-infected patients was complexed with patient immunoglobulin G, suggesting that this enzyme is immunogenic, seemingly a viral gene product. In fact, Nagalase was inducible by treatment of cultures of HIV-infected patient peripheral blood mononuclear cells with a provirus-inducing agent. This enzyme was immunoprecipitable with polyclonal anti-HIV but not with anticellular constitutive enzyme or with antitumor Nagalase. The kinetic parameters (km value of 1.27 mM and pH optimum of 6.1), of the patient serum Nagalase were distinct from those of constitutive enzyme (km value of 4.83 mM and pH optimum of 4.3). This glycosidase should reside on an envelope protein capable of interacting with cellular membranous O-glycans. Although cloned gp160 exhibited no Nagalase activity, treatment of gp160 with trypsin expressed Nagalase activity, suggesting that proteolytic cleavage of gp160 to generate gp120 and gp41 is required for Nagalase activity. Cloned gp120 exhibited Nagalase activity while cloned gp41 showed no Nagalase activity. Since proteolytic cleavage of protein gp160 is required for expression of both fusion capacity and Nagalase activity, Nagalase seems to be an enzymatic basis for fusion in the infectious process. Therefore, Nagalase appears to play dual roles in viral infectivity and immunosuppression.


Subject(s)
HIV Envelope Protein gp160/metabolism , HIV-1/pathogenicity , alpha-N-Acetylgalactosaminidase/biosynthesis , alpha-N-Acetylgalactosaminidase/metabolism , Baculoviridae/genetics , Cells, Cultured , Cloning, Molecular , Enzyme Induction , HIV Envelope Protein gp160/genetics , HIV-1/enzymology , HIV-1/genetics , Humans , Hydrogen-Ion Concentration , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin G/metabolism , Kinetics , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/enzymology , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/virology , Mitomycin/pharmacology , Models, Biological , Precipitin Tests , Trypsin/pharmacology , alpha-N-Acetylgalactosaminidase/blood
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