20 research outputs found
The generation of antibodies as tools to characterise triggering receptor expressed on Myeloid Cells 2 (TREM2) and explore its role in Alzheimerās Disease
Alzheimerās disease (AD) is the most common form of dementia, accounting for 60-70% of cases. Genome-wide association and genetic meta-analysis studies have
identified several risk genes linked with late onset AD, a major one being triggering receptor expressed on myeloid cells 2 (TREM2). Variants of TREM2 have been
associated with partial loss of function, such as impaired ligand binding and reduced microglial inflammatory responses, and recent studies have demonstrated a key role
for TREM2 in microglial modulation in the healthy brain and in AD. However, conflicting results in the literature indicate that the role of TREM2 is more complex
than once thought, and a better understanding of the functions and relevance of TREM2, and its soluble counterpart, is necessary to further the knowledge of AD
pathogenesis and identify potential therapeutic targets.
There is currently a lack of reliable commercial anti-TREM2 antibodies that can detect and modulate TREM2 in human and animal systems. This thesis describes the production and characterisation of a panel of monoclonal and polyclonal antibodies raised against human and mouse TREM2. Of these antibodies, a complementary pair was selected and optimised for the establishment of an in-house ELISA for the
detection of soluble TREM2 in human samples. This assay was used to analyse a cohort of AD and mild cognitive impairment (MCI) patients alongside age-matched controls, where sTREM2 levels were found to positively correlate with age, but not
clinical status. Novel antibodies were also generated against peptides representing the two most common TREM2 variants associated with increased susceptibility to
AD; R47H and R62H.
Overall, this thesis discusses the characterisation and application of the generated anti-TREM2 antibodies and demonstrates their future potential for use in functional
assays
A novel mouse model expressing human forms for complement receptors CR1 and CR2
Background
The complement cascade is increasingly implicated in development of a variety of diseases with strong immune contributions such as Alzheimerās disease and Systemic Lupus Erythematosus. Mouse models have been used to determine function of central components of the complement cascade such as C1q and C3. However, species differences in their gene structures mean that mice do not adequately replicate human complement regulators, including CR1 and CR2. Genetic variation in CR1 and CR2 have been implicated in modifying disease states but the mechanisms are not known.
Results
To decipher the roles of human CR1 and CR2 in health and disease, we engineered C57BL/6J (B6) mice to replace endogenous murine Cr2 with human complement receptors, CR1 and CR2 (B6.CR2CR1). CR1 has an array of allotypes in human populations and using traditional recombination methods (Flp-frt and Cre-loxP) two of the most common alleles (referred to here as CR1long and CR1short) can be replicated within this mouse model, along with a CR1 knockout allele (CR1KO). Transcriptional profiling of spleens and brains identified genes and pathways differentially expressed between mice homozygous for either CR1long, CR1short or CR1KO. Gene set enrichment analysis predicts hematopoietic cell number and cell infiltration are modulated by CR1long, but not CR1short or CR1KO.
Conclusion
The B6.CR2CR1 mouse model provides a novel tool for determining the relationship between human-relevant CR1 alleles and disease
A novel mouse model expressing human forms for complement receptors CR1 and CR2.
BACKGROUND: The complement cascade is increasingly implicated in development of a variety of diseases with strong immune contributions such as Alzheimer\u27s disease and Systemic Lupus Erythematosus. Mouse models have been used to determine function of central components of the complement cascade such as C1q and C3. However, species differences in their gene structures mean that mice do not adequately replicate human complement regulators, including CR1 and CR2. Genetic variation in CR1 and CR2 have been implicated in modifying disease states but the mechanisms are not known.
RESULTS: To decipher the roles of human CR1 and CR2 in health and disease, we engineered C57BL/6J (B6) mice to replace endogenous murine Cr2 with human complement receptors, CR1 and CR2 (B6.CR2CR1). CR1 has an array of allotypes in human populations and using traditional recombination methods (Flp-frt and Cre-loxP) two of the most common alleles (referred to here as CR1
CONCLUSION: The B6.CR2CR1 mouse model provides a novel tool for determining the relationship between human-relevant CR1 alleles and disease
Novel monoclonal antibodies against mouse C1q: characterisation and development of a quantitative ELISA for mouse C1q
Recent studies have identified roles for complement in synaptic pruning, both physiological during development and pathological in Alzheimerās disease (AD). These reports suggest that C1q initiates complement activation on synapses and C3 fragments then tag them for removal by microglia. There is an urgent need to characterise these processes in rodent AD models; this requires the development of reagents and methods for detection and quantification of rodent C1q in fluids and pathological tissues. These will enable better evaluation of the role of C1q in disease and its value as disease biomarker. We describe the generation in C1q-deficient mice of novel monoclonal antibodies against mouse and rat C1q that enabled development of a sensitive, specific, and quantitative ELISA for mouse and rat C1q capable of measuring C1q in biological fluids and tissue extracts. Serum C1q levels were measured in wild-type (WT), C1q knockout (KO), C3 KO, C7 KO, Crry KO, and 3xTg and APPNL-G-F AD model mice through ageing. C1q levels significantly decreased in WT, APPNL-G-F, and C7 KO mice with ageing. C1q levels were reduced in APPNL-G-F compared to WT at all ages and in 3xTg at 12 months; C3 KO and C7 KO, but not Crry KO mice, also demonstrated significantly lower C1q levels compared to matched WT. In brain homogenates, C1q levels increased with age in both WT and APPNL-G-F mice. This robust and adaptable assay for quantification of mouse and rat C1q provides a vital tool for investigating the expression of C1q in rodent models of AD and other complement-driven pathologies
Plasma biomarkers and genetics in the diagnosis and prediction of Alzheimer's disease
Plasma biomarkers for Alzheimer's disease-related pathologies have undergone rapid developments during the past few years, and there are now well-validated blood tests for amyloid and tau pathology, as well as neurodegeneration and astrocytic activation. To define Alzheimer's disease with biomarkers rather than clinical assessment, we assessed prediction of research-diagnosed disease status using these biomarkers and tested genetic variants associated with the biomarkers that may reflect more accurately the risk of biochemically defined Alzheimer's disease instead of the risk of dementia. In a cohort of Alzheimer's disease cases (N=1439, mean age 68 years [SD=8.2]) and screened controls (N=508, mean age 82 years [SD=6.8]), we measured plasma concentrations of the 40 and 42 amino acid-long amyloid Ī² fragments (AĪ²40 and AĪ²42, respectively), tau phosphorylated at amino acid 181 (P-tau181), neurofilament light (NfL), and glial fibrillary acidic protein (GFAP) using state-of-the-art Single molecule array (Simoa) technology. We tested the relationships between the biomarkers and Alzheimer's disease genetic risk, age at onset, and disease duration. We also conducted a genome-wide association study for association of disease risk genes with these biomarkers. The prediction accuracy of Alzheimer's disease clinical diagnosis by the combination of all biomarkers, APOE and polygenic risk score reached AUC=0.81, with the most significant contributors being Īµ4, AĪ²40 or AĪ²42, GFAP and NfL. All biomarkers were significantly associated with age in cases and controls (p<4.3x10-5). Concentrations of the AĪ²-related biomarkers in plasma were significantly lower in cases compared with controls, whereas other biomarker levels were significantly higher in cases. In the case-control genome-wide analyses, APOE-Īµ4 was associated with all biomarkers (p=0.011- 4.78x10-8), except NfL. No novel genome-wide significant SNPs were found in the case-control design; however, in a case-only analysis, we found two independent genome-wide significant associations between the AĪ²42/AĪ²40 ratio and WWOX and COPG2 genes. Disease prediction modelling by the combination of all biomarkers indicates that the variance attributed to P-tau181 is mostly captured by APOE-Īµ4, whereas AĪ²40, AĪ²42, GFAP and NfL biomarkers explain additional variation over and above APOE. We identified novel plausible genome wide-significant genes associated with AĪ²42/AĪ²40 ratio in a sample which is fifty times smaller than current genome-wide association studies in Alzheimer's disease
Cerebrospinal fluid complement system biomarkers in demyelinating disease
Background: Multiple sclerosis (MS) can be difficult to differentiate from other demyelinating diseases, notably neuromyelitis optica spectrum disorder (NMOSD). We previously showed that NMOSD is distinguished from MS by plasma complement biomarkers. Objective: Here, we measure cerebrospinal fluid (CSF) complement proteins in MS, NMOSD and clinically isolated syndrome (CIS), a neurological episode that may presage MS, to test whether these distinguish NMOSD from MS and CIS. Materials and methods: CSF (53 MS, 17 CIS, 11 NMOSD, 35 controls) was obtained; complement proteins (C4, C3, C5, C9, C1, C1q, Factor B (FB)), regulators (Factor I (FI), Factor H (FH), FH-Related Proteins 1, 2 and 5 (FHR125), C1 Inhibitor (C1INH), Properdin) and activation products (terminal complement complex (TCC), iC3b) were quantified by ELISA and results expressed relative to CSF total protein (Ī¼g/mg). Results: Compared to control CSF, (1) levels of C4, C1INH and Properdin were elevated in MS; (2) TCC, iC3b, FI and FHR125 were increased in CIS; and (3) all complement biomarkers except TCC, FHR125, Properdin and C5 were higher in NMOSD CSF. A statistical model comprising six analytes (C3, C9, FB, C1q, FI, Properdin) plus age/gender optimally differentiated MS from NMOSD
Tissueāresident macrophages actively suppress ILā1beta release via a reactive prostanoid/ILā10 pathway
The alarm cytokine interleukinā1Ī² (ILā1Ī²) is a potent activator of the inflammatory cascade following pathogen recognition. ILā1Ī² production typically requires two signals: first, priming by recognition of pathogenāassociated molecular patterns leads to the production of immature proāILā1Ī²; subsequently, inflammasome activation by a secondary signal allows cleavage and maturation of ILā1Ī² from its proāform. However, despite the important role of ILā1Ī² in controlling local and systemic inflammation, its overall regulation is still not fully understood. Here we demonstrate that peritoneal tissueāresident macrophages use an active inhibitory pathway, to suppress ILā1Ī² processing, which can otherwise occur in the absence of a second signal. Programming by the transcription factor Gata6 controls the expression of prostacyclin synthase, which is required for prostacyclin production after lipopolysaccharide stimulation and optimal induction of ILā10. In the absence of secondary signal, ILā10 potently inhibits ILā1Ī² processing, providing a previously unrecognized control of ILā1Ī² in tissueāresident macrophages
Plasma biomarkers and genetics in the diagnosis and prediction of Alzheimer's disease
Plasma biomarkers for Alzheimerās disease-related pathologies have undergone rapid developments during the past few years, and there are now well-validated blood tests for amyloid and tau pathology, as well as neurodegeneration and astrocytic activation. To define Alzheimerās disease with biomarkers rather than clinical assessment, we assessed prediction of research-diagnosed disease status using these biomarkers and tested genetic variants associated with the biomarkers that may reflect more accurately the risk of biochemically defined Alzheimerās disease instead of the risk of dementia.
In a cohort of Alzheimerās disease cases (N=1439, mean age 68 years [SD=8.2]) and screened controls (N=508, mean age 82 years [SD=6.8]), we measured plasma concentrations of the 40 and 42 amino acid-long amyloid Ī² fragments (AĪ²40 and AĪ²42, respectively), tau phosphorylated at amino acid 181 (P-tau181), neurofilament light (NfL), and glial fibrillary acidic protein (GFAP) using state-of-the-art Single molecule array (Simoa) technology. We tested the relationships between the biomarkers and Alzheimerās disease genetic risk, age at onset, and disease duration. We also conducted a genome-wide association study for association of disease risk genes with these biomarkers.
The prediction accuracy of Alzheimerās disease clinical diagnosis by the combination of all biomarkers, APOE and polygenic risk score reached AUC=0.81, with the most significant contributors being Īµ4, AĪ²40 or AĪ²42, GFAP and NfL. All biomarkers were significantly associated with age in cases and controls (p<4.3x10-5). Concentrations of the AĪ²-related biomarkers in plasma were significantly lower in cases compared with controls, whereas other biomarker levels were significantly higher in cases.
In the case-control genome-wide analyses, APOE-Īµ4 was associated with all biomarkers (p=0.011- 4.78x10-8), except NfL. No novel genome-wide significant SNPs were found in the case-control design; however, in a case-only analysis, we found two independent genome-wide significant associations between the AĪ²42/AĪ²40 ratio and WWOX and COPG2 genes.
Disease prediction modelling by the combination of all biomarkers indicates that the variance attributed to P-tau181 is mostly captured by APOE-Īµ4, whereas AĪ²40, AĪ²42, GFAP and NfL biomarkers explain additional variation over and above APOE. We identified novel plausible genome wide-significant genes associated with AĪ²42/AĪ²40 ratio in a sample which is fifty times smaller than current genome-wide association studies in Alzheimerās disease
Increased circulating levels of Factor H-Related Protein 4 are strongly associated with age-related macular degeneration.
Funder: V.C. was primarily funded by the Department of Healthās NIHR Biomedical Research Centre for Ophthalmology at Moorfields Eye Hospital and UCL Institute of Ophthalmology, and an MRC research grant (MR/P025838/1)Age-related macular degeneration (AMD) is a leading cause of blindness. Genetic variants at the chromosome 1q31.3 encompassing the complement factor H (CFH, FH) and CFH related genes (CFHR1-5) are major determinants of AMD susceptibility, but their molecular consequences remain unclear. Here we demonstrate that FHR-4 plays a prominent role in AMD pathogenesis. We show that systemic FHR-4 levels are elevated in AMD (P-valueā=ā7.1āĆā10-6), whereas no difference is seen for FH. Furthermore, FHR-4 accumulates in the choriocapillaris, Bruch's membrane and drusen, and can compete with FH/FHL-1 for C3b binding, preventing FI-mediated C3b cleavage. Critically, the protective allele of the strongest AMD-associated CFH locus variant rs10922109 has the highest association with reduced FHR-4 levels (P-valueā=ā2.2āĆā10-56), independently of the AMD-protective CFHR1-3 deletion, and even in those individuals that carry the high-risk allele of rs1061170 (Y402H). Our findings identify FHR-4 as a key molecular player contributing to complement dysregulation in AMD
Liposomal and Ethosomal Gels for the Topical Delivery of Anthralin: Preparation, Comparative Evaluation and Clinical Assessment in Psoriatic Patients
To enhance anthralin efficacy against psoriasis and reduce its notorious side effects, it was loaded into various liposomal and ethosomal preparations. The nanocarriers were characterized for drug encapsulation efficiency, size, morphology and compatibility between various components. Optimum formulations were dispersed in various gel bases and drug release kinetics were studied. Clinical efficacy and safety of liposomal and ethosomal Pluronic®F-127 gels were evaluated in patients having psoriasis (clinicaltrials.gov identifier is NCT03348462). Safety was assessed by recording various adverse events. Drug encapsulation efficiency ≥97.2% and ≥77% were obtained for liposomes and ethosomes, respectively. Particle sizes of 116 to 199 nm and 146 to 381 nm were observed for liposomes and ethosomes, respectively. Fourier-Transform infrared (FT-IR) spectroscopy and differential scanning calorimetry (DSC) studies confirmed the absence of interaction between anthralin and various nanocarrier components. Tested gel bases showed excellent ability to sustain drug release. At baseline, the patients had a median Psoriasis Area and Severity Index (PASI) of 3.4 for liposomes and 3.6 for ethosomes without significant difference. After treatment, mean PASI change was −68.66% and −81.84% for liposomes and ethosomes, respectively with a significant difference in favor of ethosomes. No adverse effects were detected in both groups. Anthralin ethosomes could be considered as a potential treatment of psoriasis