The document analyzes regulatory regions of the human TNFAIP3 gene, which encodes the A20 protein. Three BACs containing different regulatory regions were injected into mice to create humanized mouse models. The mice were bred to be deficient in mouse A20 but express a specific human A20 transgene. Analysis found the Δ-Down BAC mice, missing enhancers downstream of TNFAIP3, had decreased human A20 expression and RA/SLE-like phenotypes. This demonstrates the importance of the downstream enhancers for inducible A20 expression and inflammation control.
Background
Influenza A viruses are medically significant pathogens responsible for higher mortality and morbidity throughout the world. Swine influenza is known to be caused by influenza A subtypes H1N1, H1N2, and H3N2, which are highly contagious, and belongs to the family Orthomyxoviridae. Efficient and accurate diagnosis of influenza A in individuals is critical for monitoring of a constantly evolving pandemic. A rapid result is important, because timely treatment can reduce disease severity and duration. Rapid antigen tests were among the first-line diagnostic tools for the detection of pandemic H1N1 (2009) virus infection during the initial outbreak. Current study focuses on the significant approach of the usage of molecular method utilizing real-time PCR for the detection of type A influenza virus (H1N1 subtype) in humans.
Methods
A total of 2000 mixed nasal/throat swab specimens collected in commercial viral transport from Apollo hospitals, Hyderabad were submitted to Institute of Preventive Medicine for molecular testing by reverse transcriptase polymerase chain reaction (RT-PCR) from 2009 to 2015 from its affiliated primary care clinics.
Results
Among the 2000 samples collected, 700 samples were positive for Human Inf A, swine Inf A, and Swine Inf H1 (fourth table in the article). One thousand two hundred samples were negative for Human Inf A, swine Inf A, and Swine Inf H1, and 100 samples were positive for Influenza A only.
Conclusion
The molecular testing of H1N1 patients helped the clinicians in timely diagnosis and treatment of these patients during the pandemic surveillance. The RT-PCR test has higher sensitivity and specificity; hence it is considered to be the best tool to use during the pandemic surveillance, as compared to the any other commercial antigen-based tests, which show a variable performance, with the sensitivities of tests from different manufacturers ranging from 9 to 77%.
Background
Influenza A viruses are medically significant pathogens responsible for higher mortality and morbidity throughout the world. Swine influenza is known to be caused by influenza A subtypes H1N1, H1N2, and H3N2, which are highly contagious, and belongs to the family Orthomyxoviridae. Efficient and accurate diagnosis of influenza A in individuals is critical for monitoring of a constantly evolving pandemic. A rapid result is important, because timely treatment can reduce disease severity and duration. Rapid antigen tests were among the first-line diagnostic tools for the detection of pandemic H1N1 (2009) virus infection during the initial outbreak. Current study focuses on the significant approach of the usage of molecular method utilizing real-time PCR for the detection of type A influenza virus (H1N1 subtype) in humans.
Methods
A total of 2000 mixed nasal/throat swab specimens collected in commercial viral transport from Apollo hospitals, Hyderabad were submitted to Institute of Preventive Medicine for molecular testing by reverse transcriptase polymerase chain reaction (RT-PCR) from 2009 to 2015 from its affiliated primary care clinics.
Results
Among the 2000 samples collected, 700 samples were positive for Human Inf A, swine Inf A, and Swine Inf H1 (fourth table in the article). One thousand two hundred samples were negative for Human Inf A, swine Inf A, and Swine Inf H1, and 100 samples were positive for Influenza A only.
Conclusion
The molecular testing of H1N1 patients helped the clinicians in timely diagnosis and treatment of these patients during the pandemic surveillance. The RT-PCR test has higher sensitivity and specificity; hence it is considered to be the best tool to use during the pandemic surveillance, as compared to the any other commercial antigen-based tests, which show a variable performance, with the sensitivities of tests from different manufacturers ranging from 9 to 77%.
Presented by Etienne de Villiers at the African Swine Fever Diagnostics, Surveillance, Epidemiology and Control Workshop, Nairobi, Kenya, 20-21 July 2011
Dr. Ben Hause - Pathogen Discovery Using Metagenomic SequencingJohn Blue
Pathogen Discovery Using Metagenomic Sequencing - Dr. Ben Hause, College of Veterinary Medicine, Kansas State University, from the 2016 Allen D. Leman Swine Conference, September 17-20, 2016, St. Paul, Minnesota, USA.
More presentations at http://www.swinecast.com/2016-leman-swine-conference-material
Recent advances in African swine fever vaccine development at the Internation...ILRI
Presentation by Lucilla Steinaa at a Global African Swine Fever Research Alliance (GARA)/International Alliance for Biological Standardization (IABS) webinar on current efforts in African swine fever vaccines, 6 May 2021
Improving methodologies for rapid diagnosis of coinfection in plants (updated...Benjamin Schwessinger
This is a talk by Gamran Green an undergrad summer student from University of New South Wales at ANU 2016/17. Nice example of how MinION, Python, and Blast can be used to introduce students to novel technologies. Gamran didn't have any experience in plants, plant pathology, genomics, or scripting before the start of the project. Two months later this. Well done!
Presented by Etienne de Villiers at the African Swine Fever Diagnostics, Surveillance, Epidemiology and Control Workshop, Nairobi, Kenya, 20-21 July 2011
Dr. Ben Hause - Pathogen Discovery Using Metagenomic SequencingJohn Blue
Pathogen Discovery Using Metagenomic Sequencing - Dr. Ben Hause, College of Veterinary Medicine, Kansas State University, from the 2016 Allen D. Leman Swine Conference, September 17-20, 2016, St. Paul, Minnesota, USA.
More presentations at http://www.swinecast.com/2016-leman-swine-conference-material
Recent advances in African swine fever vaccine development at the Internation...ILRI
Presentation by Lucilla Steinaa at a Global African Swine Fever Research Alliance (GARA)/International Alliance for Biological Standardization (IABS) webinar on current efforts in African swine fever vaccines, 6 May 2021
Improving methodologies for rapid diagnosis of coinfection in plants (updated...Benjamin Schwessinger
This is a talk by Gamran Green an undergrad summer student from University of New South Wales at ANU 2016/17. Nice example of how MinION, Python, and Blast can be used to introduce students to novel technologies. Gamran didn't have any experience in plants, plant pathology, genomics, or scripting before the start of the project. Two months later this. Well done!
Dr. Laura Miller - Comparative analysis of signature genes in PRRSV-infected ...John Blue
Comparative analysis of signature genes in PRRSV-infected porcine monocyte-derived dendritic cells at differential activation statuses - Dr. Laura Miller, Virus and Prion Diseases of Livestock Research Unit, National Animal Disease Center, USDA-ARS, from the 2015 North American PRRS Symposium, December 4 - 5, 2015, Chicago, IL, USA.
More presentations at http://www.swinecast.com/2015-north-american-prrs-symposium
understanding of the human immune system, and thereby cancer immunology.
αβT-cells are the primary constituents of human cell-mediated adaptive immunity.
The antigen specificity of each αβT-cell is encoded in the 500-600 bp transcript
encompassing the variable portion of the rearranged TCRα and TCRβ subunits,
which can be read via NGS in a process termed repertoire sequencing. Until now,
the main challenge the field faces is the lack of a technology that can provide a
contiguous read of 600 bp to minimize the complexity of designing bias-prone
primers and informatics challenges of stitching short reads. Here we leverage the
long read capability of Ion 530™ chip to comprehensively sequence all three CDR
domains of the TCRβ chain. The Ion 530™ chip offers greater than 15 M productive
reads, allowing a multiplex of 2-4 samples with sufficient coverage for most repertoire
profiling studies. Initial testing with leukocyte total RNA demonstrates that this
multiplex PCR assay produced repertoires that were much more similar to data
derived from 5’-RACE protocol than the commonly used BIOMED-2 primer set. This
result suggested that the use of long reads minimizes bias by allowing targeting of
less variable regions. To further assess the performance of the assay, we designed a
model system of 30 plasmid controls containing common human T-cell CDR3
sequences. Each plasmid was amplified individually and sequenced to confirm the
detection of a single clonal population. Analytical sensitivity of the assay and
accuracy of the accompanied analysis solution were further evaluated by spiking in
plasmid concentrations from 10 pg to 0.0001 pg (5 million to 50 copies) in a
background of 100 ng cDNA reverse transcribed from leukocyte total RNA. Results
showed the assay offers linearity over 5 orders of magnitude of decreasing input
concentration. In summary, we have demonstrated a NGS workflow for TCRβ
sequencing that offers multiplex flexibility on Ion S5 with sample to answer in less
than 48 hours.
Genomic gene expression changes resulting from Trypanosomiasis: a horizontal study Examining expression changes elucidated by micro arrays in seminal tissues associated with the pathophysiology of Trypanosomiasis during disease progression
Analyses of Regulatory Regions of Human TNFAIP3 Gene
1. Analyses of Regulatory Regions of
Human TNFAIP3 Gene with
Humanized Mouse Models
Mark Liber
Hospital for Special Surgery
2. Some Background on A20
• A20’s function is cell-type dependent
• A20 exerts its activities via multiple mechanisms,
including ubiquitin editing, primarily to negatively
regulate proinflammatory signaling
• A20 expression and activity is regulated
transcriptionally
Catrysse,L, et al.A20 in inflammation and autoimmunity. Trends in Immunology, January 2014, Vol. 35, No. 1
3. Some Background on A20
• Human genetic studies (GWAS) have identified A20
as a disease susceptibility gene
– Various SNPs have been associated with
autoimmune disease
• Gene targeting studies confirm the important role of
A20 in controlling tissue homeostasis
Catrysse,L, et al.A20 in inflammation and autoimmunity. Trends in Immunology, January 2014, Vol. 35, No. 1
4. Biological Consequences of A20 Deficiency
• A20-deficient mice die prematurely as a result of
cachexia and severe inflammation and tissue
damage in multiple organs.
• A20-deficient mouse embryonic fibroblasts (MEFs)
and myeloid cells fail to terminate TNF-and LPS-
induced NF-κB activation and show high levels of
proinflammatory cytokines
Lee, E.G. et al. (2000) Failure to regulate TNF-induced NF-kappaB
and cell death responses in A20-deficient mice. Science 289, 2350–
2354
5. A20 acts through various pathways within the cell…
Catrysse,L, et al.A20 in inflammation and autoimmunity. Trends in Immunology, January
2014, Vol. 35, No. 1
6. Catrysse,L, et al.A20 in inflammation and autoimmunity. Trends
in Immunology, January 2014, Vol. 35, No. 1
…which leads to tissue abormalities throughout
the body and autoimmune disease
7. Our Project
• Three different BACs containing different
regulatory regions of the human TNFAIP3
gene were injected into pronuclei of fertilized
mouse eggs of C57BL/6 background
• These 3 groups of humanized mice were born,
raised in our facility, and bred
– Mice were bred to be deficient in mouse A20, but
positive for their specific human A20 (hA20)
transgene (TG+/- A20-/-)
8. GWAS SNPs
1. RP11-10J5
2. CTD-2657E11
3. RP11-953L22
CD14+
Unstim.
TNF 24h
TTtoA
H3K27ac
Our BACs & the their location with respect to the
A20 (TNFAIP3) locus
ΔDOWN-BAC
ΔUP-BAC
WT-BAC
WT-BAC : 5’: 76 kb bp; 3’: 83 kb
D-DOWN-BAC1: 5’: 174 kb ; 3’: 36.7 kb
D-UP-BAC: 5’: 25.6 kb ; 3’: 190 kb
TT>A: 41578 bp downstream of TSS
9. Simplified figure of Our BACs
3’
TNFAIP3
Δ-
DOWN
BAC
Δ-UP
BAC
1. Δ-Down BAC: missing regulatory elements downstream of the TNFAPI3 gene
2. WT-BAC: is centered around the TNFAIP3 gene
3. Δ-Up BAC: missing regulatory elements upstream of the TNFAIP3 gene
WT-BAC
10. Aims
• To identify any phenotypic differences
between the mice of various human BACs and
determine which regulatory regions around
the TNFAIP3 gene are most important for A20
expression and regulation of inflammation
11. Comparing the BACs involved…
• Harvesting and studying primary cells from these
mice
– Measured hA20 RNA and protein expression in bone
marrow-derived macrophages, splenic T & B
lymphocytes, and fibroblast-like synoviocytes (FLS)
• Analyzing of serum autoimmune markers
• Challenging mice with antigens and comparing
their ability to tolerate infection and curtail
inflammation
13. The decrease in mRNA and protein levels in Δ-Down BAC transgenic line
demonstrates that the enhancers located in the region between 36.7-83
kb downstream of TNFAIP3 TSS are required for inducible human A20
expression, and their absence may be at least partially responsible for
RA and SLE-like phenotypes observed
1. RP11-10J5
3. RP11-953L22
ΔUP-BAC
WT-BAC
A20 gene
Conclusion
2. CTD-2657E11
ΔDOWN-BAC
14. Clinical Implications
• Further study of the TNFAIP3 regulatory
elements downstream of the gene may
provide insight into the genetic causes of
phenotypes related to RA and SLE, and
overexpression of these regions may provide
therapeutically beneficial down the road
15. Skills Gained and Utilized
• Animal breeding
• Immunohistochemistry
• Fluorescence microscopy
• Data Analysis
• Scientific presentation
• Creation of mouse bone marrow chimeras
• Mouse intraperitoneal and intraorbital injections
• Utilization of the Quantstudio 3D Digital PCR system (Copy number determination)
• Cell culture with THP1 and IMR90 cell lines
• Primary cell culture (mouse and human monocytes, macrophages, B lymphocytes, T lymphocytes, and fibroblast-
like synoviocytes)
• PBMC purification from human blood
• Quantitative real-time PCR
• Southern blotting
• Western blotting
• Flow cytometry and cell sorting
• ELISA
• ChIP-sequencing
• Genomic DNA purification and amplification
• Restriction enzyme digest
• Mini-and maxipreparations of plasmid DNA for BAC construction