scholarly journals Amyloid and Tau in Neurodegeneration

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Acacia Williams ◽  
Grace Hallinan, PhD ◽  
Maria Teresa Gomez Ponce, MS ◽  
Ruben Vidal, PhD
Keyword(s):  
Cell Culture ◽  
Western Blot ◽  
Neurodegenerative Diseases ◽  
Tau Phosphorylation ◽  
Amyloid Peptide ◽  
Amyloid Angiopathy ◽  
Cell Cytotoxicity ◽  
Serum Samples ◽  
Familial Danish Dementia ◽  
C Terminus

Background and Hypothesis:Familial British dementia (FBD) and familial Danish dementia (FDD) are two autosomal dominant neurodegenerative diseases caused by mutations in the BRI2 gene. FBD and FDD are characterized by widespread cerebral amyloid angiopathy (CAA), parenchymal amyloid deposition, and neurofibrillary tangles.Previous studies have shown that accumulation of Danish amyloid peptide (ADan) induces hyperphosphorylation of tau and cell cytotoxicity; however, progress in our understanding of the interaction between ADan and tau has been hindered by the lack of antibodies against ADan. In addition, whether the British amyloid peptide (ABri) has a similar effect on tau phosphorylation remains unknown. The main goals of our project are to generate monoclonal antibodies against ADan and to test whether the ABri peptide induces hyperphosphorylation of tau and cell cytotoxicity. Experimental Design or Project Methods:A peptide homologous to the C-terminus of the ADan peptide was used to immunize 5 mice. Serum samples were tested from mice with high ELISA titers and the best 2 animals were used for cell fusion. Cell culture supernatants were screened by ELISA, western blot and immunohistochemistry. ABri peptides were used as negative controls.HEK cells expressing human tau were used to assess the interaction between ABri and tau. We performed immunocytochemistry and Western blot analysis to investigate tau phosphorylation. Results:A total of 11 clones tested positive by western blot. 3 clones tested positive for ABri and were discarded. 5 clones were tested by immunohistochemistry. 3 of the positive clones will be used for subcloning to complete clonality. Work in progress on the interaction between ABri and tau will determine whether ABri induces tau hyperphosphorylation in cell culture. Conclusion and Potential Impact:Our work will provide novel tools to study the interaction between amyloid and tau in neurodegenerative diseases and may uncover possible new targets for pharmaceutical intervention in tauopathies.

scholarly journals GSK-3 in Neurodegenerative Diseases

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Peng Lei ◽  
Scott Ayton ◽  
Ashley I. Bush ◽  
Paul A. Adlard
Keyword(s):  
Cell Culture ◽  
Neurodegenerative Diseases ◽  
Glycogen Synthase ◽  
Transgenic Animal ◽  
Tau Phosphorylation ◽  
Glycogen Synthase Kinase 3 ◽  
Cellular Processes ◽  
Amyloid A ◽  
Transgenic Animal Models

Glycogen synthase kinase-3 (GSK-3) regulates multiple cellular processes, and its dysregulation is implicated in the pathogenesis of diverse diseases. In this paper we will focus on the dysfunction of GSK-3 in Alzheimer’s disease and Parkinson’s disease. Specifically, GSK-3 is known to interact with tau,β-amyloid (Aβ), andα-synuclein, and as such may be crucially involved in both diseases. Aβproduction, for example, is regulated by GSK-3, and its toxicity is mediated by GSK-induced tau phosphorylation and degeneration.α-synuclein is a substrate for GSK-3 and GSK-3 inhibition protects against Parkinsonian toxins. Lithium, a GSK-3 inhibitor, has also been shown to affect tau, Aβ, andα-synuclein in cell culture, and transgenic animal models. Thus, understanding the role of GSK-3 in neurodegenerative diseases will enhance our understanding of the basic mechanisms underlying the pathogenesis of these disorders and also facilitate the identification of new therapeutic avenues.

scholarly journals Recombinant Dense Granular Protein (GRA5) for Detection of Human Toxoplasmosis by Western Blot

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Xiao Teng Ching ◽  
Yee Ling Lau ◽  
Mun Yik Fong ◽  
Veeranoot Nissapatorn ◽  
Hemah Andiappan
Keyword(s):  
Western Blot ◽  
Affinity Purification ◽  
Expression System ◽  
Economic Loss ◽  
Serum Samples ◽  
Serological Tests ◽  
Human Patient ◽  
Prokaryotic Expression System ◽  
Human Sera ◽  
Lysate Antigens

Toxoplasma gondiiinfects all warm-blooded animals, including humans, causing serious public health problems and great economic loss for the food industry. Commonly used serological tests require costly and hazardous preparation of wholeToxoplasmalysate antigens from tachyzoites. Here, we have evaluated an alternative method for antigen production, which involved a prokaryotic expression system. Specifically, we expressedT. gondiidense granular protein-5 (GRA5) inEscherichia coliand isolated it by affinity purification. The serodiagnostic potential of the purified recombinant GRA5 (rGRA5) was tested through Western blot analysis against 212 human patient serum samples. We found that rGRA5 protein was 100% specific for analysis of toxoplasmosis-negative human sera. Also, rGRA5 was able to detect acute and chronicT. gondiiinfections (sensitivities of 46.8% and 61.2%, resp.).

Lav/HTLV-III Neutralizing Antibodies in the Sera of Patients with Aids, Lymphadenopathy Syndrome and Asymptomatic Seropositive Individuals

1986 ◽  
Vol 72 (3) ◽  
pp. 219-224 ◽  
Author(s):  
Georgine P. Faulkner-Valle ◽  
Anita De Rossi ◽  
Oriella Dalla Gassa ◽  
Luigi Chieco-Bianchi
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Neutralizing Antibodies ◽  
Elisa Test ◽  
Antibody Specificity ◽  
Serum Samples ◽  
Neutralizing Activity ◽  
The Mean ◽  
Asymptomatic Individuals

Serum samples which had previously been found positive for LAV/HTLV-III antibodies by the ELISA test and then confirmed by radioimmunoassay (Western blot) were tested for the presence of neutralizing antibodies. No neutralizing activity was found in the sera of a group of patients with the clinical diagnosis of AIDS. However in patients with LAS and other related pathologic conditions the percentage of sera positive for neutralizing antibodies was 27 % and 55 % respectively. At least 50 % of the sera from seropositive asymptomatic individuals had neutralizing activity but with the exception of the haemophiliac group the mean titre was much lower than that of LAS patients. No relationship was found between the neutralizing titre and the antibody specificity detected by Western blot analysis for p41 and p120.

SOX Antibodies in Small-Cell Lung Cancer and Lambert-Eaton Myasthenic Syndrome: Frequency and Relation With Survival

2009 ◽  
Vol 27 (26) ◽  
pp. 4260-4267 ◽  
Author(s):  
Maarten J. Titulaer ◽  
Rinse Klooster ◽  
Marko Potman ◽  
Lidia Sabater ◽  
Francesc Graus ◽  
...  
Keyword(s):  
Western Blot ◽  
High Throughput Screening ◽  
Small Cell Lung Carcinoma ◽  
Diagnostic Value ◽  
Small Cell ◽  
Enzyme Linked Immunosorbent Assay ◽  
Small Cell Lung ◽  
Serum Samples ◽  
Cell Lung Carcinoma ◽  
Myasthenic Syndrome

Purpose SOX1 antibodies are common in small-cell lung carcinoma (SCLC) with and without paraneoplastic syndrome (PNS) and can serve as serological tumor marker. Addition of other antibodies might improve its diagnostic power. We validated an enzyme-linked immunosorbent assay (ELISA) to assess the diagnostic value of serum antibodies in SCLC and Lambert-Eaton myasthenic syndrome (LEMS). Clinical outcome with respect to SOX antibodies was evaluated, as the SOX-related antitumor immune response might help to control the tumor growth. Patients and Methods We used recombinant SOX1, SOX2, SOX3, SOX21, HuC, HuD, or HelN1 proteins in an ELISA to titrate serum samples and validated the assay by western blot. We tested 136 consecutive SCLC patients, 86 LEMS patients (43 with SCLC), 14 patients with SCLC and PNS (paraneoplastic cerebellar degeneration or Hu syndrome), 62 polyneuropathy patients, and 18 healthy controls. Results Our ELISA was equally reliable as western blot. Forty-three percent of SCLC patients and 67% of SCLC-LEMS patients had antibodies to one of the SOX or Hu proteins. SOX antibodies had a sensitivity of 67% and a specificity of 95% to discriminate between LEMS with SCLC and nontumor LEMS. No difference in survival was observed between SOX positive and SOX negative SCLC patients. Conclusion SOX antibodies are specific serological markers for SCLC. Our assay is suitable for high throughput screening, detecting 43% of SCLC. SOX antibodies have diagnostic value in discriminating SCLC-LEMS from nontumor LEMS, but have no relation to survival in patients with SCLC.

scholarly journals Detecting HIV-1 Tat in Cell Culture Supernatants by ELISA or Western Blot

2016 ◽  
pp. 329-342 ◽  
Author(s):  
Fabienne Rayne ◽  
Solène Debaisieux ◽  
Annie Tu ◽  
Christophe Chopard ◽  
Petra Tryoen-Toth ◽  
...  
Keyword(s):  
Cell Culture ◽  
Western Blot ◽  
Culture Supernatants ◽  
Hiv 1

scholarly journals Phage Display Detection of Mimotopes that Are Shared Epitopes of Clinically and Epidemiologically Relevant Enterobacteria

2020 ◽  
Vol 8 (5) ◽  
pp. 780
Author(s):  
Armando Navarro ◽  
Delia Licona-Moreno ◽  
Alejandro Monsalvo-Reyes ◽  
Ulises Hernández-Chiñas ◽  
Carlos A. Eslava-Campos
Keyword(s):  
Amino Acid ◽  
Western Blot ◽  
Phage Display ◽  
Synthetic Peptides ◽  
Amino Acid Sequences ◽  
Serum Samples ◽  
E Coli ◽  
Peptide Mimotopes ◽  
Human Sera ◽  
Shared Epitopes

Background: Escherichia coli and Salmonella are etiologic agents of intestinal infections. A previous study showed the presence of shared epitopes between lipopolysaccharides (LPSs) of E. coli O157 and Salmonella. Aim: Using phage display, the aim of this study is to identify mimotopes of shared epitopes in different enterobacterial LPSs. Methods: We use anti-LPS IgG from E. coli O157 and Salmonella to select peptide mimotopes of the M13 phage. The amino acid sequence of the mimotopes is used to synthesize peptides, which are in turn used to immunize rabbits. The antibody response of the resulting sera against the LPSs and synthetic peptides (SPs) is analyzed by ELISA and by Western blot assays, indicating that LPS sites are recognized by the same antibody. In a complementary test, the reactions of human serum samples obtained from the general population against the SPs and LPSs are also analyzed. Results: From the last biopanning phase, sixty phagotopes are selected. The analysis of the peptide mimotope amino acid sequences shows that in 4 of them the S/N/A/PF motif is a common sequence. Antibodies from the sera of immunized rabbits with SP287/3, SP459/1, SP308/3, and SP073/14 react against both their own peptide and the different LPSs. The Western blot test shows a sera reaction against both the lateral chains and the cores of the LPSs. The analysis of the human sera shows a response against the SPs and LPSs. Conclusion: The designed synthetic peptides are mimotopes of LPS epitopes of Salmonella and E. coli that possess immunogenic capacity. These mimotopes could be considered for use in the design of vaccines against both enterobacteria.

Evaluation of reactivity to Echinococcus spp. among rural inhabitants in Poland

2015 ◽  
Vol 60 (3) ◽  
Author(s):  
Ewa Cisak ◽  
Jacek Sroka ◽  
Angelina Wójcik-Fatla ◽  
Violetta Zając ◽  
Jacek Dutkiewicz
Keyword(s):  
Western Blot ◽  
Echinococcus Granulosus ◽  
Cross Reactivity ◽  
Farm Animals ◽  
Control Group ◽  
Laboratory Diagnostics ◽  
Group 14 ◽  
Serum Samples ◽  
The City ◽  
Echinococcus Spp

AbstractA group of 172 rural inhabitants from eastern Poland (68 males and 104 females, mean age 49.0 ± 12.0 years) was examined for the presence of antibodies against Echinococcus granulosus and Echinococcus multilocularis. A population of 38 healthy urban dwellers from the city of Lublin (17 males and 21 females, mean age 36.2 ± 9.6 years) were examined as a control group. Sera of 22 rural inhabitants (12.8%) reacted positively to Echinococcus granulosus hydatid fluid antigen in the screening test. A cross-reactivity was observed with two serum samples that tested positive in ELISA for E. granulosus. Three serum samples were tested positive for E. multilocularis using the Em2plus ELISA assay and also positive for Western blot. None of the members of control group showed the presence of a seropositive reaction to Echinococcus spp. The reactivity to Echinococcus spp. among rural inhabitants decreased with age and this correlation was statistically significant (R = -0.197151, p = 0.009535). The percentage of positive findings was the highest (50.0%) in the youngest age group (14-20). No significant correlations were found between responses to interview questions (possession of domestic and farm animals, contact with wild animals, eating unwashed berries, drinking unboiled water) and the presence of seropositive reactions to Echinococcus spp. The presented results seem to indicate that echinococcosis is still a current problem in Poland that should not be neglected and, moreover, indicates the need for improvement in the routine laboratory diagnostics of Echinococcus spp. by standardizing the ELISA and Western blot tests.

Low Immunogenicity to Romiplostim in Clinical Studies with ITP Subjects.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3425-3425 ◽  
Author(s):  
Vibha Jawa ◽  
Martha Hokom ◽  
Jenny Hu ◽  
Yao Zhuang ◽  
Dietmar Berger ◽  
...  
Keyword(s):  
Clinical Studies ◽  
Neutralizing Antibodies ◽  
Single Chain ◽  
Serum Samples ◽  
Binding Domains ◽  
C Terminus ◽  
Before And After ◽  
Immunogenicity Assessment ◽  
Covalently Linked

Abstract Romiplostim, a member of the thrombopoietin (TPO) mimetic class, is an Fc-peptide fusion protein (peptibody) that activates intracellular transcriptional pathways leading to increased platelet production via the TPO receptor (also known as cMpl). The peptibody molecule contains two identical single-chain subunits each consisting of human immunoglobulin IgG1 Fc domain, covalently linked at the C-terminus to a peptide containing two TPO receptor-binding domains. Due to the general concern regarding the immunogenic potential for all therapeutic proteins and the specific concern for monitoring antibodies capable of neutralizing thrombopoietin (TPO), an extensive immunogenicity assessment program was developed to support romiplostim. Romiplostim has been engineered to have no amino acid sequence homology to endogenous TPO. A low theoretical risk of developing conformational antibodies that cross-react against TPO exists. This risk was addressed by using an immunogenicity assessment strategy that relied upon a surface plasmon resonance based biosensor immunoassay using the Biacore 3000 capable of simultaneously monitoring antibodies that bind to romiplostim, TPO, or the active peptide portion of romiplostim (TMP). Samples that tested positive for binding antibodies in the Biacore immunoassay were then tested in the definitive functional biological assay to identify any antibodies capable of neutralizing the biological effect of romiplostim or TPO. Serum samples from 236 actively treated subjects were obtained both before and after exposure to romiplostim and were tested for romiplostim and TPO antibodies. In baseline samples, seventeen subjects (7.1%) tested romiplostim antibody positive and 12 subjects (5.1%) tested TPO antibody positive for pre-existing binding antibodies. After romiplostim exposure, twenty-five out of 236 (10.5%) subjects with ITP developed binding antibodies against romiplostim (inclusive of antibodies to both peptide and the whole molecule) and 12 out of 236 (5.1%) subjects with ITP developed binding antibodies against TPO. The antibodies that developed against romiplostim did not cross react with TPO and the antibodies that developed against TPO did not cross react with romiplostim. The incidence of anti-romiplostim neutralizing antibodies among 236 subjects with ITP who were treated with romiplostim across 10 clinical studies was 0.4% (1 out of 236). No cases of anti-TPO neutralizing antibodies were detected in romiplostim treated samples. In conclusion, after thorough immunogenicity assessment of all subjects treated with romiplostim using sensitive methods to detect binding and neutralizing antibodies, only one subject was found positive for the presence of antibodies capable of neutralizing romiplostim that was negative at the time of follow up 4 months later. As expected, none of the subjects treated were positive for antibodies capable of neutralizing TPO. No clinical sequelae were observed in association to the presence of antibodies.

scholarly journals Comparison of serological and clinical findings in Turkish patients with cystic echinococcosis

2006 ◽  
Vol 43 (4) ◽  
pp. 220-225 ◽  
Author(s):  
A. Yolasigmaz ◽  
K. Reiterová ◽  
M. Turk ◽  
E. Reyhan ◽  
A. Bozdag ◽  
...  
Keyword(s):  
Western Blot ◽  
Rural Areas ◽  
Cystic Echinococcosis ◽  
Correct Diagnosis ◽  
Clinical Findings ◽  
Serum Samples ◽  
First Case ◽  
Long Time ◽  
Back Ache ◽  
The Individual

AbstractCystic echinococcosis (CE), caused by the cestode Echinococcus granulosus, is potentially dangerous for humans. The aim of this study was to examine serological and clinical findings regarding cysts localisation and individual responses in 54 patients with CE. The majority of patients in this study were females (63 %) and the average age was 46.3 years. Most of the patients lived in rural areas or kept a dog (46 %) for a long time. The most frequent symptoms were hypochondrial pain (48.9 %), epigastrial discomfort (27.7 %), vomiting (21.3 %), minor cough (12.8 %), urticaria (6.3 %), weakness (4.3 %), fever (2.1 %), side-or back-ache (4.3 %). However, 17 % of the patients showed no symptoms. In every case, the ultrasound (USG) and/or computer tomography (CT) investigations were positive. In most cases (53.2 % of the patients) a single cyst was found but 46.8 % of the patients had multiple cyst formations (from 2 to 9 cysts) located in the liver. Sporadic lung, splenetic, mesenterial, tibial and cerebral localisations were also found. The patients were individually treated with albendazol (10–15 mg/kg) five days prior and six months after the surgical treatment. Serum samples were investigated by the serological techniques: IHAT, ELISA and Western blot using hydatid fluid antigen. In the patient sera, the specific antibody levels were mostly increased after surgery. Different results were obtained only in two patients. In the first case, seroconversion was delayed. In the other case all ELISA results were negative, however, the Western blot analysis and surgery proved the presence of CE. The results suggest that the different antibody response of patients depends on the individual immune response. Multiple localization and various stages of CE cysts demonstrate the necessity of a complex approach for the confirmation of a correct diagnosis.

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