Serum Proteins In Patients Research Articles

Multiplex Microscopy Assay for Assessment of Therapeutic and Serum Antibodies against Emerging Pathogens.

The emergence of novel pathogens, exemplified recently by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlights the need for rapidly deployable and adaptable diagnostic assays to assess their impact on human health and guide public health responses in future pandemics. In this study, we developed an automated multiplex microscopy assay coupled with machine learning-based analysis for antibody detection. To achieve multiplexing and simultaneous detection of multiple viral antigens, we devised a barcoding strategy utilizing a panel of HeLa-based cell lines. Each cell line expressed a distinct viral antigen, along with a fluorescent protein exhibiting a unique subcellular localization pattern for cell classification. Our robust, cell segmentation and classification algorithm, combined with automated image acquisition, ensured compatibility with a high-throughput approach. As a proof of concept, we successfully applied this approach for quantitation of immunoreactivity against different variants of SARS-CoV-2 spike and nucleocapsid proteins in sera of patients or vaccinees, as well as for the study of selective reactivity of monoclonal antibodies. Importantly, our system can be rapidly adapted to accommodate other SARS-CoV-2 variants as well as any antigen of a newly emerging pathogen, thereby representing an important resource in the context of pandemic preparedness.

NCAM1 and GDF15 are biomarkers of Charcot-Marie-Tooth disease in patients and mice.

Molecular markers scalable for clinical use are critical for the development of effective treatments and the design of clinical trials. Here, we identify proteins in sera of patients and mouse models with Charcot-Marie-Tooth disease (CMT) with characteristics that make them suitable as biomarkers in clinical practice and therapeutic trials. We collected serum from mouse models of CMT1A (C61 het), CMT2D (GarsC201R, GarsP278KY), CMT1X (Gjb1-null), CMT2L (Hspb8K141N) and from CMT patients with genotypes including CMT1A (PMP22d), CMT2D (GARS), CMT2N (AARS) and other rare genetic forms of CMT. The severity of neuropathy in the patients was assessed by the CMT Neuropathy Examination Score (CMTES). We performed multitargeted proteomics on both sample sets to identify proteins elevated across multiple mouse models and CMT patients. Selected proteins and additional potential biomarkers, such as growth differentiation factor 15 (GDF15) and cell free mitochondrial DNA, were validated by ELISA and quantitative PCR, respectively. We propose that neural cell adhesion molecule 1 (NCAM1) is a candidate biomarker for CMT, as it was elevated in Gjb1-null, Hspb8K141N, GarsC201R and GarsP278KY mice as well as in patients with both demyelinating (CMT1A) and axonal (CMT2D, CMT2N) forms of CMT. We show that NCAM1 may reflect disease severity, demonstrated by a progressive increase in mouse models with time and a significant positive correlation with CMTES neuropathy severity in patients. The increase in NCAM1 may reflect muscle regeneration triggered by denervation, which could potentially track disease progression or the effect of treatments. We found that member proteins of the complement system were elevated in Gjb1-null and Hspb8K141N mouse models as well as in patients with both demyelinating and axonal CMT, indicating possible complement activation at the impaired nerve terminals. However, complement proteins did not correlate with the severity of neuropathy measured on the CMTES scale. Although the complement system does not seem to be a prognostic biomarker, we do show complement elevation to be a common disease feature of CMT, which may be of interest as a therapeutic target. We also identify serum GDF15 as a highly sensitive diagnostic biomarker, which was elevated in all CMT genotypes as well as in Hspb8K141N, Gjb1-null, GarsC201R and GarsP278KY mouse models. Although we cannot fully explain its origin, it may reflect increased stress response or metabolic disturbances in CMT. Further large and longitudinal patient studies should be performed to establish the value of these proteins as diagnostic and prognostic molecular biomarkers for CMT.

Different altered stage correlative expression of high abundance acute-phase proteins in sera of patients with epithelial ovarian carcinoma

Different altered stage correlative expression of high abundance acute-phase proteins in sera of patients with epithelial ovarian carcinoma

Detection of differentially expressed proteins in sera of patients with hepatocellular carcinoma by sodium dodecyl sulphate poly-acrylamide gel electrophoresis (SDS-PAGE)

Hepatocellular carcinoma (HCC) is considered as one of the major liver cancers prevailing in the world. The survival rate for the patients diagnosed with HCC is very low as average survival time is only one year. Due to the lack of early diagnosis and poor knowledge of pathophysiological mechanisms, screening of HCC remains possible only at advanced stages. Furthermore, due to non-effective treatment and poor diagnostic techniques led to high fatality rate. Previously, number of HCC markers has been identified but have very less specificity. This study was designed to look into and find out certain HCC specific serological proteins which may be used as biomarker for the early diagnosis of the said disease. In this study all collected samples were analyzed by SDS-PAGE. The results revealed that four different proteins were up-regulated in HCC diagnosed patients compared to the control sample. These proteins may be used as potential biomarkers for the early diagnosis of HCC. The further investigations of the relevant proteins may reveal important clues for the early diagnosis of HCC. Keywords: Biomarker proteins; HCC; SDS-PAGE; Early diagnosis; Liver Cancer http://dx.doi.org/10.19045/bspab.2017.60006

Evaluation of Serum Total Sialic Acid and Lipid Associated Sialic Acid as a Tumour Marker

The present study was done to assess the levels of total sialic acid & lipid associated sialicAcid. Lactate dehydrogenase and total proteins in sera of patients with various malignant diseases and compared with sera of non malignant diseases, benign tumors & controls. Serum total sialic acid, LASA & LDH values are significantly increased in malignancies when compared to the controls (P<0.001), indicating altered metabolism of tumour cell surface glycoprotein & sialoglycolipids and there is decrease of total proteins in malignancies especially Gastro intestinal tract malignancies (P<0.001). We had followed up 23 patients suffering from different malignancies and were received treatment. All these patients showed clinical improvement gradually at an end of 20 days followed up period. The TSA, LASA, & LDH significantly decreased (P<0.001, P<0.01, P<0.05) respectively. So this study suggests sialic acid as a tumour marker which shows good diagnostic potential.

Citrullination: the loss of tolerance and development of autoimmunity in rheumatoid arthritis

Rheumatoid arthritis is a chronic inflammatory disease characterized by synovial inflammation and pannus formation leading to destruction of cartilage and bone. Several self proteins have been suggested to be disease-driving autoantigens. Proteins are encoded by a limited number of genes in our genome. Post-translational modifications such as citrullination of the arginine residues, can increase the morphological and the functional diversity of the proteome. The positivity of anti-citrullinated peptides autoantibodies occurs then at an early stage of the disease development. Several factors, among which the synovial tissue inflammatory and the nitric oxide reaction, are involved in the regulation of the citrullination reaction. All of them have to be analysed and considered to understand the loss of tolerance and the development of autoimmunity leading to the disease.

Different altered stage correlative expression of high abundance acute-phase proteins in sera of patients with epithelial ovarian carcinoma

BackgroundThe general enhanced expression of α1-antichymotrypsin (ACT), clusterin (CLU), α1-antitrypsin (AAT), haptoglobin β-chain (HAP), and leucine rich glycoprotein (LRG) in the sera of patients with epithelial ovarian carcinoma (EOCa) was recently reported. In the present study, we compared the expression of the serum acute-phase proteins (APPs) in the patients according to their stages of cancer.ResultsDifferent altered stage correlative expression of the high abundance serum APPs was demonstrated in sera of the patients studied. While the expression of ACT, HAP and AAT appeared to demonstrate positive correlation with the three initial stages of the cancer, inverse correlation was apparently detected in the expression of LRG and CLU. For patients who were diagnosed with stage IV of the cancer, expression of the serum APPs did not conform to the altered progression changes.ConclusionOur results highlight the potential prognostic significance of selective high abundance serum APPs in patients with EOCa.

Disease association of antibodies to human and mycobacterial hsp70 and hsp60 stress proteins.

Structural homology between microbial and human stress proteins has been postulated to be a basis for autoimmunization in chronic inflammatory diseases. Therefore, we estimated by ELISA titration the antibody levels to mycobacterial (M) and human (H) recombinant hsp70 and M-hsp65 heat-shock proteins in sera of patients with Crohn's disease (n = 29), ulcerative colitis (n = 20) and nontuberculous mycobacterial disease of the lungs (n = 20). Antibodies to H-hsp60, separated by two-dimensional gel electrophoresis, were tested in six sera of each group of patients. In Crohn's disease, antibody titres to the M-hsp65 antigen without detectable H-hsp60 binding were significantly elevated in 52% of the patients. In contrast titres to both M-hsp70 and H-hsp70 were demonstrable and correlated, but increased over control values only in four (14%) patients. The antibody pattern in ulcerative colitis was found to be quite different: anti-H-hsp60 binding was demonstrable in most patients, although anti-M-hsp65 titres were not elevated. Furthermore, 25% of patients had significantly elevated titres to M-hsp70, but not to H-hsp70. In non-tuberculous mycobacterial pulmonary disease, about 50% of patients had elevated titres to both hsp65 and hsp71 mycobacterial antigens but not to the corresponding human proteins; patients with Mycobacterium xenopi infection had the highest titres in this group. These results demonstrate the existence of distinct disease-associated patterns in the human antibody response to stress protein antigens. However, these data are not sufficient to imply sensitization with mycobacteria in patients with inflammatory bowel diseases, since certain epitopes of heat-shock proteins are shared by several bacterial genera.

Distribution of retroviral p15E-related proteins in neoplastic and non-neoplastic human tissues, and their role in the regulation of the immune response.

In patients with head and neck carcinomas and in patients with chronic purulent upper airway infections, low molecular weight retroviral p15E-like factors are found. These factors are responsible for partial defects in the cellular immune response. We studied the distribution of these p15E-related proteins in neoplastic, inflamed and normal human tissues and related these findings with the presence of p15E-like factors in patients' sera. Demonstration of p15E-like proteins in sera of patients with upper airway infections and of patients with head and neck carcinomas correlated exclusively with the presence of p15E in normal and pathologic epithelium of the upper respiratory tract. p15E was not demonstrated in epithelia of other localizations. Our results suggest that chronic stimulation or neoplastic transformation of the epithelia of the upper respiratory tract stimulates the production of p15E-like proteins leading to their reported immunosuppressive actions.

Detection,identification and diagnostic value of differential proteins in sera of patients with HBVrelated primary hepatic carcinoma

Objective:To detect and identify differentially expressed proteins in the sera of patients with HBV-related primary hepatic carcinoma and discuss their possible role in early diagnosis of primary hepatic carcinoma.Methods:The surface-enhanced laser desorption/ionization time-of-flight mass spectroscopy(SELDI-TOF-MS)was used to screen for the differentially expressed serum proteins in patients with HBV-related primary hepatic carcinoma,liver cirrhosis,chronic hepatitis,and healthy adults.Then the most differential protein sample was isolated and purified by ionic exchange chromatography and was subjected to mass spectrometry analysis.The screening results were used to establish a diagnosis model for hepatic carcinoma and the accuracy and sensitivity of the model were assessed.Results:CM10(weak cation exchange)chip found 44 differentially expressed protein peaks(P0.05)in hepatic carcinoma group compared with those in the healthy adult group,with 21 up-regulated and 23 down-regulated.Fifty-one differential peaks were identified in hepatic carcinoma group compared with those in the liver cirrhosis group(P0.05),with 47 up-regulated and 4 down-regulated.The expression of protein with a mass-to-charge ratio(m/z)of 5 805 gradually increased in order in the healthy adult,chronic hepatitis,liver cirrhosis and hepatic carcinoma patients;and the expression in hepatic carcinoma group was significantly higher than that in the healthy adult group(P0.01);peptide mass fingerprint(PMF)after enzyme hydrolysis showed that 2 of the peptides were partially identical to chondroitin sulfate synthase 2.A diagnosis model of hepatic carcinoma was successfully established based on the differentially expressed proteins,with an accuracy of 94.82%(23+32/58),a sensitivity of 88.46%(23/26),and a specificity of 100%(32/32).Conclusion:We have successfully screened out the differentially expressed proteins in the sera of patients with HBV-related primary hepatic carcinoma;the most differentially expressed protein(5 805)has 2 peptides partially identical to chondroitin sulfate synthase 2 in sequence.The established model may help to diagnose HBV-related primary hepatic carcinoma.

Expression of high‐abundance proteins in sera of patients with endometrial and cervical cancers: Analysis using 2‐DE with silver staining and lectin detection methods

The expression of high-abundance serum proteins in newly diagnosed patients with endometrial adenocarcinoma (EACa), squamous cell cervical carcinoma (SCCa) and cervical adenocarcinoma (ACCa), relative to control female subjects, was analyzed by subjecting serum samples to 2-DE followed by image analysis of the silver-stained protein profiles. The three cohorts of cancer patients demonstrated different altered expression of serum high-abundance proteins compared to negative control women. The expression of alpha1-antitrypsin, alpha1-B glycoprotein, cleaved high-molecular-weight kininogen (light chain) and antithrombin III were consistently altered in all the patients. However, clusterin was upregulated only in the patients with EACa, while those with SCCa and ACCa were typically characterized by the upregulated expression of zinc alpha-2-glycoprotein. The aberrant expression of selective serum proteins in the various cohorts of cancer patients was validated by competitive ELISA as well as by lectin detection. Analysis by using the champedak galactose binding lectin further highlighted an unidentified protein that may be differently glycosylated in the sera of the EACa patients that were studied.

Proteomics-based Identification of Human Acute Leukemia Antigens That Induce Humoral Immune Response

The identification of panels of tumor antigens that elicit an antibody response may have utility in cancer screening, diagnosis, and establishing prognosis. Until now, autoimmunity in cancer has been mainly revealed in solid tumors. The aim of this study was to apply the proteomic approach to the identification of proteins that commonly elicit a humoral response in acute leukemia (AL). Sera from 21 newly diagnosed patients with AL, 20 patients with solid tumors, and 22 noncancer controls were analyzed for antibody-based reactivity against AL proteins resolved by two-dimensional electrophoresis. As a result, autoantibody against a protein identified by mass spectrometry as Rho GDP dissociation inhibitor 2 was detected in sera from 15 of 21 patients with AL (71%). By contrast, such antibody was detected in sera from one of 20 patients with solid tumors (5%) and one of 22 noncancer controls (4.5%). Five other protein autoantibodies were also found in AL patients with a high frequency and constituted the major target antigens of the AL autoimmune response. The findings of autoantibodies against Rho GDP dissociation inhibitor 2 and other proteins in sera of patients with AL suggest that the proteomic approach we have implemented may have utility for the development of a serum-based assay for AL screening and diagnosis.

Serological investigation of latex allergy in Argentina.

Atopy to latex has been reported in different high-risk groups of subjects in whom it is mainly caused by proteins from natural latex that are responsible for eliciting a variety of clinical symptoms, some of them systemic. Thus, identification of subjects sensitized to latex proteins is of great health importance, because it would become possible to advise them to avoid contact with latex-containing products. Protein extracts from ammoniated latex were prepared by incubation with PBS buffer either with or without detergents, followed by ultracentrifugation. Three immunoenzymic methods were developed (EAST, ELISA, and immunoblotting) to detect the presence of specific IgE antibodies against latex proteins in sera of patients from different groups at risk. The protein content of the latex extracts ranged from 5.3 to 8.8 mg/mL. The prevalence of specific IgE against latex proteins was 9/28 (32.1%) in children with multiple surgeries, 17/98 (17.3%) in health care workers, and 23/123 (18.6%) in outpatients assisted at the Allergy Department. None of the sera belonging to the healthy control group showed the presence of specific IgE. Therefore, this protein extract from latex could be used to detect specific IgE antibodies in serum by immunoenzymic methods. Serologic results for latex-specific IgE found are in accordance with those reported in the literature of other countries.

The native form and maturation process of hepatitis C virus core protein.

The maturation and subcellular localization of hepatitis C virus (HCV) core protein were investigated with both a vaccinia virus expression system and CHO cell lines stably transformed with HCV cDNA. Two HCV core proteins, with molecular sizes of 21 kDa (p21) and 23 kDa (p23), were identified. The C-terminal end of p23 is amino acid 191 of the HCV polyprotein, and p21 is produced as a result of processing between amino acids 174 and 191. The subcellular localization of the HCV core protein was examined by confocal laser scanning microscopy. Although HCV core protein resided predominantly in the cytoplasm, it was also found in the nucleus and had the same molecular size as p21 in both locations, as determined by subcellular fractionation. The HCV core proteins had different immunoreactivities to a panel of monoclonal antibodies. Antibody 5E3 stained core protein in both the cytoplasm and the nucleus, C7-50 stained core protein only in the cytoplasm, and 499S stained core protein only in the nucleus. These results clearly indicate that the p23 form of HCV core protein is processed to p21 in the cytoplasm and that the core protein in the nucleus has a higher-order structure different from that of p21 in the cytoplasm. HCV core protein in sera of patients with HCV infection was analyzed in order to determine the molecular size of genuinely processed HCV core protein. HCV core protein in sera was found to have exactly the same molecular weight as the p21 protein. These results suggest that p21 core protein is a component of native viral particles.

Detection of MAGE-4 protein in sera of lung cancer patients.

We investigated the level of MAGE‐4 protein in sera of patients with primary lung cancer to understand better the biological roles of the MAGE proteins. MAGE‐4 protein was detected as a non‐degraded form in botb the supernatant of a MAGE‐4+ tumor cell line and in a patient's serum. Serum level of the MAGE‐4 protein in lung cancer patients (n= 100, mean = 1.17 ng/ml) was significantly higher than that in either patients with benign pulmonary diseases (n= 80, 0.33 ng/ml) or healthy donors (n=68, 0.32 ng/ml). It was higher than the cutoff level (1.15 ng/ml) in 34 of 100 cancer patients, but not in anyone in the other groups.

Detection of MAGE-4 protein in sera of patients with head-and-neck squamous-cell carcinoma.

The MAGE-4 gene, a member of the MAGE gene family, is expressed in various cancers, including head-and-neck squamous-cell carcinomas (HN-SCC), but is not expressed in any normal tissues except for the testis and placenta. The aim of this study was to determine whether serum MAGE-4 protein is a useful tumor marker for detection of HN-SCC. An enzyme-linked immunosorbent assay was used to measure serum level of MAGE-4 protein. The serum level of MAGE-4 in pre-operative HN-SCC patients was significantly higher than that in patients with non-malignant diseases (NMD) of the head and neck, volunteers undergoing cancer screening (VOL), or healthy donors (HD). When the cut-off level was determined at 1.15 ng/ml (mean plus 3 SD of HD), sera from 28 of 96 patients with HN-SCC (p < 0.0001 vs. the other groups), 7 of 82 patients with NMD, 2 of 92 with VOL, and 0 of 68 HD were positive for MAGE-4. Serum levels of MAGE-4 protein in all 7 HN-SCC patients whose sera were positive for MAGE-4 before operation decreased after operation, and, in one patient, a renewed rise in serum level was followed by recurrence. These results indicate that MAGE-4 protein is detectable in sera of a significant number of HN-SCC patients, and that serum MAGE-4 protein might be a useful tumor marker to monitor the recurrence of MAGE-4-positive HN-SCC.

Detection of Antiendometrial Antibodies in Sera of Patients With Endometriosis by Dual‐Colored, Double‐Labeling Immunohistochemical Method and Western Blot

This study was undertaken to determine whether specific binding activities against endometrial proteins in sera of patients with endometriosis are detectable and, if so, to identify endometrial antigens involved in autoimmunity in endometriosis. Sera from 33 patients with endometriosis and 20 cord sera (controls) were tested against endometria of patients and their protein extracts by dual-colored, double-labeling immunohistochemical method, and Western blotting. Antiendometrial binding activities were detected in sera of 2 (10.0%) control patients and 13 (48.2%) patients with endometriosis by the immunohistochemical method. Endogenous immunoglobulin G (IgG) binding to endometrial proteins had molecular weights (MW) of 26, 28, 54, 85, 107 and 116 kDa. Most sera of both control and patients showed reactivity against endometrial proteins with MW of 34, 36, 56 and 77 kDa. However, there were specific IgG autoantibodies reactive against the endometrial proteins of 71, 92, and 103 kDa in sera of 55.2% (16/29) of patients but not in the control sera. Over 80% (10/12) of patients' sera with binding activities detectable by the immunohistochemical method also tested positive by Western blot analysis. These data show that specific IgG antibodies reactive against endometrial antigens are detectable in sera from some patients with endometriosis.

Autoimmunity and filariasis. Autoantibodies against cytoplasmic cellular proteins in sera of patients with onchocerciasis.

Onchocerciasis or "river blindness" is a vector-borne tropical disease resulting from infection with the filarial nematode Onchocerca volvulus. Disease manifestations include dermatitis, rheumatic complaints, and blindness. Recent findings have suggested an autoimmune etiology for the occurrence of chorioretinopathy, a disease of the eye which together with sclerosing keratoconjunctivitis is responsible for approximately 400,000 onchocerciasis-related cases of blindness. The identification of onchocerciasis as an important cause of tropical rheumatism prompted us to evaluate serologically the presence of systemic autoimmune disease in onchocerciasis patients and local controls from a hyperendemic area in Sierra Leone. In both groups there was a marked autoimmune response against cytoplasmic non-RNA-associated proteins consisting of autoantibodies against five major Ag with respective m.w. of 35, 51, 64, 83, and 110 kDa. These five proteins are novel autoantigens that could be distinguished from calreticulin, the human homologue of the onchocercal Ag RAL-1, and known autoantigens such as the 50-kDa La/SS-B or 52- and 60-kDa Ro/SS-A proteins by immunoblotting and ELISA assays. Furthermore, autoantibody reactivity against calreticulin was significantly higher in O. volvulus-infected individuals than in endemic controls. Autoantibody reactivity against the five major autoantigens, anti-calreticulin reactivity, and antibody reactivity against the 65-kDa arthritis-associated mycobacterial heat shock protein were intercorrelated as parts of an onchocerciasis-associated autoimmune response. The implication of autoimmunity in the disease pathogenesis of onchocerciasis could have important consequences for future research on therapeutical regimens, pathogenetic mechanisms, and serological diagnosis of onchocerciasis.

Antibodies to cytoskeletal proteins in sera of patients with angiographically assessed coronary artery disease

Circulating autoantibodies to various components of the arterial wall have been reported in atherosclerosis. To examine the occurrence of autoantibodies to cytoskeletal proteins in coronary artery disease (CAD) we studied 56 patients with angiographically demonstrable CAD and compared them with 37 controls without CAD. Enzyme-linked immunosorbent assay (ELISA) was used to analyze the serum samples. In coronary patients, antibody absorbance values at least two standard deviations above the mean for the controls were considered positive. The following numbers of positive antibody absorbances were found in the group of 56 patients: actin IgG, 6 (10.7%); cytokeratin-18 IgG, 3 (5.4%), IgA, 2 (3.6%); myosin IgA, 11 (19.6%); desmin IgG, 13 (23.2%), IgM, 3 (5.4%); vimentin IgG, 2 (3.6%), IgM, 7 (12.5%), IgA, 6 (10.7%). The specificity of desmin IgG was tested with Western blotting against extracts of human internal mammary artery. The positive antibody absorbances to one or several cytoskeletal proteins in the patients were not found to correlate with the clinical symptoms of CAD. Our results suggest an association between autoantibodies to cytoskeletal proteins, particularly to those for desmin, with angiographically assessable CAD.

Detection by western blotting of an antibody to the hepatitis C virus E1 envelope protein in sera of patients with chronic liver disease.

We detected an antibody to HCV envelope protein (E1) in sera of patients with HCV-related chronic liver diseases (20 patients with chronic hepatitis and 5 patients with liver cirrhosis) by Western blotting using the fusion protein of E1 envelope protein and beta-galactosidase as an antigen. The antibody to HCV E1 (anti-HCV E1) was detected in 8 (42%) of 19 patients positive for HCV-RNA (16 were positive and 3 were negative for antibody to C100-3) and in 1 (17%) of 6 patients negative for HCV-RNA but positive for antibody to C100-3. HCV-RNA was detected in 8 (89%) of 9 anti-HCV E1 positive sera. The value of alanine aminotransferase was significantly higher in patients positive for anti-HCV E1 than in patients negative for the antibody. Although an antibody to the envelope protein of HCV is suspected to be one of the candidates of virus-neutralizing antibodies, our results suggest this hypothesis appears to be unlikely.