scholarly journals Identification of the minimal active soluble TREM2 sequence for modulating microglial phenotypes and amyloid pathology

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Xuan Sheng ◽  
Yunling Yao ◽  
Ruizhi Huang ◽  
Ying Xu ◽  
Yifei Zhu ◽  
...  
Keyword(s):  
Cell Viability ◽  
Inflammatory Responses ◽  
Solid Phase ◽  
Amyloid Plaque ◽  
Dot Blot ◽  
Soluble Trem2 ◽  
Plaque Load ◽  
Dot Blot Assay ◽  
Csf Levels ◽  
5Xfad Mice

Abstract Background TREM2 is a microglial receptor genetically linked to the risk for Alzheimer’s disease (AD). The cerebrospinal fluid (CSF) levels of soluble TREM2 (sTREM2) have emerged as a valuable biomarker for the disease progression in AD and higher CSF levels of sTREM2 are linked to slower cognitive decline. Increasing sTREM2 in mouse models of amyloidosis reduces amyloid-related pathology through modulating microglial functions, suggesting a beneficial role of sTREM2 in microglia biology and AD pathology. Methods In the current study, we performed serial C- and N-terminal truncations of sTREM2 protein to define the minimal sequence requirement for sTREM2 function. We initially assessed the impacts of sTREM2 mutants on microglial functions by measuring cell viability and inflammatory responses. The binding of the sTREM2 mutants to oligomeric Aβ was determined by solid-phase protein binding assay and dot blot assay. We further evaluated the impacts of sTREM2 mutants on amyloid-related pathology by direct stereotaxic injection of sTREM2 proteins into the brain of 5xFAD mice. Results We found that both sTREM2 fragments 41–81 and 51–81 enhance cell viability and inflammatory responses in primary microglia. However, the fragment 51–81 exhibited impaired affinity to oligomeric Aβ. When administrated to the 5xFAD mice brain, the sTREM2 fragment 41–81, but not 51–81, increased the number of plaque-associated microglia and reduced the plaque deposition. Interestingly, the fragment 41–81 was more efficient than the physiological form of sTREM2 in ameliorating Aβ-related pathology. Conclusions Our results indicate that the interaction of sTREM2 truncated variants with Aβ is essential for enhancing microglial recruitment to the vicinity of an amyloid plaque and reducing the plaque load. Importantly, we identified a 41-amino acid sequence of sTREM2 that is sufficient for modulating microglial functions and more potent than the full-length sTREM2 in reducing the plaque load and the plaque-associated neurotoxicity. Taken together, our data provide more insights into the mechanisms underlying sTREM2 function and the minimal active sTREM2 sequence represents a promising candidate for AD therapy.

Interleukin-1 excretion in urine specimens of renal transplant recipients with acute rejection using matrix dot-blot assay

1996 ◽  
Vol 63 (4) ◽  
pp. 421-423
Author(s):  
A. Mukhtar
Keyword(s):  
Renal Transplant ◽  
Acute Rejection ◽  
Inflammatory Responses ◽  
Mesangial Cells ◽  
Interleukin 1 ◽  
Renal Transplant Recipients ◽  
Transplant Recipients ◽  
Dot Blot ◽  
Dot Blot Assay ◽  
Urine Specimens

Interleukin-1 is a polypeptide mediator involved in the regulation of inflammatory responses. Mesangial cells IL-1 may act as an autocrine growth factor (Lovett et al., 1983). The local release of such growth factors may be important in the development of mesangial proliferative lesions characteristic of many forms of immune-mediated glomerulonephritis (Lovett et al., 1986). Secretion of IL-1 in urine specimens of renal transplant recipients with acute rejection was studied using matrix APAAP dot-blot assay, to determine any alteration of IL-1 changes that could reveal any pathological correlation. Twelve urine specimens from 6 persons with acute rejection revealed IL-1 staining in the dot-blot assay while there was no staining for IL-1 in the urine specimens of healthy persons. The immunological changes correlated with acute renal rejection could lead to autocrine and apocrine secretion of IL-1 its secretion in urine.

scholarly journals Rapid Screening of the Epidermal Growth Factor Receptor Phosphosignaling Pathway via Microplate-Based Dot Blot Assays

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Amedeo Cappione ◽  
Janet Smith ◽  
Masaharu Mabuchi ◽  
Timothy Nadler
Keyword(s):  
Large Scale ◽  
Solid Phase ◽  
Growth Factor Receptor ◽  
Cost Effective ◽  
Protein Microarrays ◽  
Rapid Screening ◽  
Dot Blot ◽  
A431 Cells ◽  
Dot Blot Assay ◽  
Multiple Samples

Expression profiling on a large scale, as is the case in drug discovery, is often accomplished through use of sophisticated solid-phase protein microarrays or multiplex bead technologies. While offering both high-throughput and high-content analysis, these platforms are often too cost prohibitive or technically challenging for many research settings. Capitalizing on the favorable attributes of the standard ELISA and slot blotting techniques, we developed a modified dot blot assay that provides a simple cost-effective alternative for semiquantitative expression analysis of multiple proteins across multiple samples. Similar in protocol to an ELISA, but based in a membrane bound 96-well microplate, the assay takes advantage of vacuum filtration to expedite the tedious process of washing in between binding steps. We report on the optimization of the assay and demonstrate its use in profiling temporal changes in phosphorylation events in the well-characterized EGF-induced signaling cascade of A431 cells.

scholarly journals Specific Ligand Binding Attributable to Individual Epitopes of Gonococcal Transferrin Binding Protein A

2002 ◽  
Vol 70 (2) ◽  
pp. 732-740 ◽  
Author(s):  
Heather P. Masri ◽  
Cynthia Nau Cornelissen
Keyword(s):  
Ligand Binding ◽  
Fusion Proteins ◽  
Iron Uptake ◽  
Solid Phase ◽  
Protein A ◽  
Dot Blot ◽  
Affinity Tags ◽  
Amino Terminal ◽  
Dot Blot Assay ◽  
Specific Ligand

ABSTRACT The gonococcal transferrin receptor complex comprises two iron-regulated proteins, TbpA and TbpB. TbpA is essential for transferrin-iron uptake and is a TonB-dependent integral outer membrane protein. TbpB is thought to increase the efficiency of iron uptake from transferrin and is lipid modified and surface exposed. To evaluate the structure-function relationships in one of the components of the receptor, TbpA, we created constructs that fused individual putative loops of TbpA with amino-terminal affinity tags. The recombinant proteins were then overexpressed in Escherichia coli, and the fusions were recovered predominately from inclusion bodies. Inclusion body proteins were solubilized, and the epitope fusions were renatured by slow dialysis. To assess transferrin binding capabilities, the constructs were tested in a solid-phase dot blot assay followed by confirmatory quantitative chemiluminescent enzyme-linked immunosorbent assays. The constructs with only loop 5 and with loops 4 and 5 demonstrated dose-dependent specific ligand binding in spite of being out of the context of the intact receptor. The immunogenicities of individual TbpA-specific epitopes were investigated by generating rabbit polyclonal antisera against the fusion proteins. Most of the fusion proteins were immunogenic under these conditions, and the resulting sera recognized full-length TbpA in immunoblots. These results suggest that individual epitopes of TbpA are both immunogenic and functional with respect to ligand binding capabilities, and the vaccine implications of these findings are discussed.

scholarly journals CYP46A1 Activation by Efavirenz Leads to Behavioral Improvement without Significant Changes in Amyloid Plaque Load in the Brain of 5XFAD Mice

2019 ◽  
Vol 16 (3) ◽  
pp. 710-724 ◽  
Author(s):  
Alexey M. Petrov ◽  
Morrie Lam ◽  
Natalia Mast ◽  
Jean Moon ◽  
Yong Li ◽  
...  
Keyword(s):  
Amyloid Plaque ◽  
Plaque Load ◽  
5Xfad Mice ◽  
Behavioral Improvement ◽  
The Brain

APPLICATION OF REAL-TIME PRC TECHNIQUE AND REVERSE DOT-BLOT FOR DETECTION THE HIGH RISK TYPES OF HUMAN PAPILLOMAVIRUS CAUSING CERVICAL CANCER

2017 ◽  
pp. 99-103
Author(s):  
Van Bao Thang Phan ◽  
Hoang Bach Nguyen ◽  
Van Thanh Nguyen ◽  
Thi Nhu Hoa Tran ◽  
Viet Quynh Tram Ngo
Keyword(s):  
Cervical Cancer ◽  
High Risk ◽  
Real Time ◽  
Real Time Pcr ◽  
Dot Blot ◽  
The Real ◽  
Dot Blot Assay ◽  
Hpv Types ◽  
High Risk Hpv ◽  
Reverse Dot Blot

Introduction: Infection with HPV is the main cause of cervical cancer. Determining HPV infection and the types of HPV plays an important role in diagnosis, treatment and prognosis of cervicitis/cervical cancer. Aims: Determining proportion of high-risk HPV types and the occurrence of coinfection with multiple HPV types. Methods: 177 women with cervicitis or abnormal Pap smear result were enrolled in the study. Performing the real-time PCR for detecting HPV and the reverse DOT-BLOT assay for determining type of HPV in cases of positive PCR. Results: 7 types of high-risk HPV was dectected, the majority of these types were HPV type 18 (74.6%) and HPV type 16 (37.6%); the proportion of infection with only one type of HPV was 30.4% and coinfection with multiple HPV types was higher (69.6%), the coinfected cases with 2 and 3 types were dominated (32.2% and 20.3%, respectively) and the coinfected cases with 4 and 5 types were rare. Conclusion: Use of the real-time PCR and reverse DOT-BLOT assay can determine the high-risk HPV types and the occurrence of coinfection with multiple HPV types. Key words: HPV type, Reverse DOT-BLOT, real-time PCR,PCR, cervical cancer

Use of monoclonal and polyclonal antibodies to detect prostatic acid phosphatase by immunoblotting.

1986 ◽  
Vol 32 (10) ◽  
pp. 1832-1835 ◽  
Author(s):  
P C Patel ◽  
L Aubin ◽  
J Côte
Keyword(s):  
Monoclonal Antibodies ◽  
Acid Phosphatase ◽  
Western Blot ◽  
Polyclonal Antibodies ◽  
Prostatic Acid Phosphatase ◽  
The Other ◽  
Dot Blot ◽  
Western Blots ◽  
Dot Blot Assay ◽  
Polyclonal Antisera

Abstract We investigated two techniques of immunoblotting--the Western blot and the dot blot--for use in detecting prostatic acid phosphatase (PAP, EC 3.1.3.2). We used polyclonal antisera to human PAP, produced in rabbits by hyperimmunization with purified PAP, and PAP-specific monoclonal antibodies in the immunoenzymatic protocols. We conclude that PAP can be readily detected by Western blots with use of polyclonal antisera, but not with monoclonal antibodies. On the other hand, using a dot blot assay, we could easily detect PAP with both polyclonal and monoclonal antibodies.

scholarly journals Bacterial sepsis increases hippocampal fibrillar amyloid plaque load and neuroinflammation in a mouse model of Alzheimer's disease

2021 ◽  
Vol 152 ◽  
pp. 105292
Author(s):  
Jacob M. Basak ◽  
Aura Ferreiro ◽  
Lucy S. Cohen ◽  
Patrick W. Sheehan ◽  
Collin J. Nadarajah ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Mouse Model ◽  
Amyloid Plaque ◽  
Bacterial Sepsis ◽  
Model Of Alzheimer’S Disease ◽  
Plaque Load

scholarly journals Impact of Gut Microbiome Manipulation in 5xFAD Mice on Alzheimer’s Disease-Like Pathology

2021 ◽  
Vol 9 (4) ◽  
pp. 815
Author(s):  
Malena dos Santos Guilherme ◽  
Vu Thu Thuy Nguyen ◽  
Christoph Reinhardt ◽  
Kristina Endres
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Gut Microbiome ◽  
Microbial Composition ◽  
Aβ Peptides ◽  
Plaque Deposition ◽  
Glycation End Products ◽  
Plaque Load ◽  
5Xfad Mice ◽  
Building Capability

The gut brain axis seems to modulate various psychiatric and neurological disorders such as Alzheimer’s disease (AD). Growing evidence has led to the assumption that the gut microbiome might contribute to or even present the nucleus of origin for these diseases. In this regard, modifiers of the microbial composition might provide attractive new therapeutics. Aim of our study was to elucidate the effect of a rigorously changed gut microbiome on pathological hallmarks of AD. 5xFAD model mice were treated by antibiotics or probiotics (L. acidophilus and L. rhamnosus) for 14 weeks. Pathogenesis was measured by nest building capability and plaque deposition. The gut microbiome was affected as expected: antibiotics significantly reduced viable commensals, while probiotics transiently increased Lactobacillaceae. Nesting score, however, was only improved in antibiotics-treated mice. These animals additionally displayed reduced plaque load in the hippocampus. While various physiological parameters were not affected, blood sugar was reduced and serum glucagon level significantly elevated in the antibiotics-treated animals together with a reduction in the receptor for advanced glycation end products RAGE—the inward transporter of Aβ peptides of the brain. Assumedly, the beneficial effect of the antibiotics was based on their anti-diabetic potential.

Quantitating Apoptosis by a Nonradioactive DNA Dot Blot Assay

1994 ◽  
Vol 221 (2) ◽  
pp. 431-433 ◽  
Author(s):  
A.O. Hueber ◽  
M. Pierres ◽  
H.T. He
Keyword(s):  
Dot Blot ◽  
Dot Blot Assay
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