81 research outputs found
Proinflammatory and antigen-specific CD4+ T cells in Rheumatoid Arthritis
Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease primarily
affecting peripheral joints. In this thesis work we aimed to increase the knowledge
about proinflammatory and antigen-specific CD4+ T cell subsets involved in RA
pathogenesis and I also investigated the effect of T cell directed therapy (abatacept) on
CD4+ T cell subsets.
Abatacept (CTLA4-Ig) is a biologic therapy that blocks T cell co-stimulation by
interfering with the binding of CD28 to CD80/86. Treatment of abatacept leads to
reduced TH1 and TH17 cytokine production in ACPA-positive RA patients and
diminished frequencies of several Treg subsets. This was also confirmed by in vitro
studies where abatacept was added in vitro to cell cultures.
Approximately 60% of RA patients have antibodies against citrullinated proteins
(ACPA) and their presence is associated to HLA-DRB1*04, one of the strongest genetic
risk factors associated with RA. It is believed that T cells recognizing citrullinated
epitopes presented by HLA-DRB1*04 alleles may drive the development of ACPA and
disease. We have used MHC class II tetramer technology in order to identify and
enumerate autoantigen-specific T cells in blood and synovial fluid of RA patients.
Several citrullinated epitopes of CILP, a-enolase, fibrinogen and vimentin were
identified. The frequency of citrulline-specific T cells was higher in the blood of RA
patients compared to HLA-matched healthy controls and the citrulline-specific T cells
in RA patients were of a memory TH1 phenotype. Furthermore, we enumerated and
characterized a-enolase native and citrulline-specific T cells in blood and synovial fluid
of HLA-DRB1*04:01 RA patients. Higher frequencies of citrullinated a-enolase
specific T cells were present in synovial fluid compared to blood and T cells
recognizing the citrullinated variant of a-enolase were also more often of a memory
phenotype (i.e. had encountered their cognate antigen in vivo) than those recognizing
the native a-enolase epitope. Some T cells showed cross-reactivity between the two
investigated epitopes. HLA-DRB1*04:01-IE transgenic mice were used to substantiate
our findings.
Another potentially contributing T cell subset to RA pathology is an expanded T cell
subset that lacks the co-stimulatory molecule CD28, often referred to as CD4+CD28null
T cells, which are present in approximately 1/3 of RA patients. These are
proinflammatory cells and their frequency in blood can be up to 50 % of all CD4+ T
cells, but they are infrequent in synovial fluid. CD28null T cells are different than
conventional CD4+ T cells in several aspects, but we demonstrate that even within the
CD28null subset there are differences due to their localization. We compared cells from
blood and synovial fluid. CD28null cells from synovial fluid expressed more CXCR3
and CCR6 than those form the circulation and CD28null cells from synovial fluid were
able to produce IL-17 even though they displayed a hypomethylated IFNG promoter.
During my thesis studies, several novel HLA-DRB1*04:01 restricted citrullinated T cell
epitopes have been identified. The auto-reactive T cells did not overlap with the
CD28null phenotype. We have demonstrated the proof of principle that auto-reactive T
cells can be identified by MHC class II tetramer technology in an assay not dependent
on in vitro stimulation
Increasing Tetrahydrobiopterin in Cardiomyocytes Adversely Affects Cardiac Redox State and Mitochondrial Function Independently of Changes in NO Production
Tetrahydrobiopterin (BH4) represents a potential strategy for the treatment of cardiac remodeling, fibrosis and/or diastolic dysfunction. The effects of oral treatment with BH4 (Sapropterinβ’ or Kuvanβ’) are however dose-limiting with high dose negating functional improvements. Cardiomyocyte-specific overexpression of GTP cyclohydrolase I (mGCH) increases BH4 several-fold in the heart. Using this model, we aimed to establish the cardiomyocyte-specific responses to high levels of BH4. Quantification of BH4 and BH2 in mGCH transgenic hearts showed age-based variations in BH4:BH2 ratios. Hearts of mice (\u3c6 \u3emonths) have lower BH4:BH2 ratios than hearts of older mice while both GTPCH activity and tissue ascorbate levels were higher in hearts of young than older mice. No evident changes in nitric oxide (NO) production assessed by nitrite and endogenous ironβnitrosyl complexes were detected in any of the age groups. Increased BH4 production in cardiomyocytes resulted in a significant loss of mitochondrial function. Diminished oxygen consumption and reserve capacity was verified in mitochondria isolated from hearts of 12-month old compared to 3-month old mice, even though at 12 months an improved BH4:BH2 ratio is established. Accumulation of 4-hydroxynonenal (4-HNE) and decreased glutathione levels were found in the mGCH hearts and isolated mitochondria. Taken together, our results indicate that the ratio of BH4:BH2 does not predict changes in neither NO levels nor cellular redox state in the heart. The BH4 oxidation essentially limits the capacity of cardiomyocytes to reduce oxidant stress. Cardiomyocyte with chronically high levels of BH4 show a significant decline in redox state and mitochondrial function
Daily steps and all-cause mortality: a meta-analysis of 15 international cohorts
Background Although 10000 steps per day is widely promoted to have health benefits, there is little evidence to support
this recommendation. We aimed to determine the association between number of steps per day and stepping rate
with all-cause mortality.
Methods In this meta-analysis, we identified studies investigating the effect of daily step count on all-cause mortality
in adults (aged β₯18 years), via a previously published systematic review and expert knowledge of the field. We asked
participating study investigators to process their participant-level data following a standardised protocol. The primary
outcome was all-cause mortality collected from death certificates and country registries. We analysed the doseβ
response association of steps per day and stepping rate with all-cause mortality. We did Cox proportional hazards
regression analyses using study-specific quartiles of steps per day and calculated hazard ratios (HRs) with inversevariance weighted random effects models.
Findings We identified 15 studies, of which seven were published and eight were unpublished, with study start dates
between 1999 and 2018. The total sample included 47 471 adults, among whom there were 3013 deaths (10Β·1 per
1000 participant-years) over a median follow-up of 7Β·1 years ([IQR 4Β·3β9Β·9]; total sum of follow-up across studies was
297 837 person-years). Quartile median steps per day were 3553 for quartile 1, 5801 for quartile 2, 7842 for quartile 3,
and 10 901 for quartile 4. Compared with the lowest quartile, the adjusted HR for all-cause mortality was 0Β·60 (95% CI
0Β·51β0Β·71) for quartile 2, 0Β·55 (0Β·49β0Β·62) for quartile 3, and 0Β·47 (0Β·39β0Β·57) for quartile 4. Restricted cubic splines
showed progressively decreasing risk of mortality among adults aged 60 years and older with increasing number of
steps per day until 6000β8000 steps per day and among adults younger than 60 years until 8000β10000 steps per day.
Adjusting for number of steps per day, comparing quartile 1 with quartile 4, the association between higher stepping
rates and mortality was attenuated but remained significant for a peak of 30 min (HR 0Β·67 [95% CI 0Β·56β0Β·83]) and
a peak of 60 min (0Β·67 [0Β·50β0Β·90]), but not significant for time (min per day) spent walking at 40 steps per min or
faster (1Β·12 [0Β·96β1Β·32]) and 100 steps per min or faster (0Β·86 [0Β·58β1Β·28]).
Interpretation Taking more steps per day was associated with a progressively lower risk of all-cause mortality, up to a
level that varied by age. The findings from this meta-analysis can be used to inform step guidelines for public health
promotion of physical activity
Daily steps and all-cause mortality: a meta-analysis of 15 international cohorts
Background
Although 10β000 steps per day is widely promoted to have health benefits, there is little evidence to support this recommendation. We aimed to determine the association between number of steps per day and stepping rate with all-cause mortality.
Methods
In this meta-analysis, we identified studies investigating the effect of daily step count on all-cause mortality in adults (aged β₯18 years), via a previously published systematic review and expert knowledge of the field. We asked participating study investigators to process their participant-level data following a standardised protocol. The primary outcome was all-cause mortality collected from death certificates and country registries. We analysed the doseβresponse association of steps per day and stepping rate with all-cause mortality. We did Cox proportional hazards regression analyses using study-specific quartiles of steps per day and calculated hazard ratios (HRs) with inverse-variance weighted random effects models.
Findings
We identified 15 studies, of which seven were published and eight were unpublished, with study start dates between 1999 and 2018. The total sample included 47β471 adults, among whom there were 3013 deaths (10Β·1 per 1000 participant-years) over a median follow-up of 7Β·1 years ([IQR 4Β·3β9Β·9]; total sum of follow-up across studies was 297β837 person-years). Quartile median steps per day were 3553 for quartile 1, 5801 for quartile 2, 7842 for quartile 3, and 10β901 for quartile 4. Compared with the lowest quartile, the adjusted HR for all-cause mortality was 0Β·60 (95% CI 0Β·51β0Β·71) for quartile 2, 0Β·55 (0Β·49β0Β·62) for quartile 3, and 0Β·47 (0Β·39β0Β·57) for quartile 4. Restricted cubic splines showed progressively decreasing risk of mortality among adults aged 60 years and older with increasing number of steps per day until 6000β8000 steps per day and among adults younger than 60 years until 8000β10β000 steps per day. Adjusting for number of steps per day, comparing quartile 1 with quartile 4, the association between higher stepping rates and mortality was attenuated but remained significant for a peak of 30 min (HR 0Β·67 [95% CI 0Β·56β0Β·83]) and a peak of 60 min (0Β·67 [0Β·50β0Β·90]), but not significant for time (min per day) spent walking at 40 steps per min or faster (1Β·12 [0Β·96β1Β·32]) and 100 steps per min or faster (0Β·86 [0Β·58β1Β·28]).
Interpretation
Taking more steps per day was associated with a progressively lower risk of all-cause mortality, up to a level that varied by age. The findings from this meta-analysis can be used to inform step guidelines for public health promotion of physical activity.
Funding
US Centers for Disease Control and Prevention
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Epidermal Growth Factor Receptor Activation in Glioblastoma through Novel Missense Mutations in the Extracellular Domain
Background:
Protein tyrosine kinases are important regulators of cellular homeostasis with tightly
controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory
constraints on kinase activity, can promote malignant transformation, and appear to be a major
determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase
domain, for example, have recently been identified in patients who showed clinical responses
to EGFR kinase inhibitor therapy.
Methods and Findings:
Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR)
kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR
coding sequence in glioma tumor samples and cell lines. We identified novel missense
mutations in the extracellular domain of EGFR in 13.6% (18/132) of glioblastomas and 12.5% (1/
8) of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene
dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells.
Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR
kinase inhibitors.
Conclusions:
Our results suggest extracellular missense mutations as a novel mechanism for oncogenic
EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for
treatment of glioblastoma
Target selection and annotation for the structural genomics of the amidohydrolase and enolase superfamilies
To study the substrate specificity of enzymes, we use the amidohydrolase and enolase superfamilies as model systems; members of these superfamilies share a common TIM barrel fold and catalyze a wide range of chemical reactions. Here, we describe a collaboration between the Enzyme Specificity Consortium (ENSPEC) and the New York SGX Research Center for Structural Genomics (NYSGXRC) that aims to maximize the structural coverage of the amidohydrolase and enolase superfamilies. Using sequence- and structure-based protein comparisons, we first selected 535 target proteins from a variety of genomes for high-throughput structure determination by X-ray crystallography; 63 of these targets were not previously annotated as superfamily members. To date, 20 unique amidohydrolase and 41 unique enolase structures have been determined, increasing the fraction of sequences in the two superfamilies that can be modeled based on at least 30% sequence identity from 45% to 73%. We present case studies of proteins related to uronate isomerase (an amidohydrolase superfamily member) and mandelate racemase (an enolase superfamily member), to illustrate how this structure-focused approach can be used to generate hypotheses about sequenceβstructureβfunction relationships
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SCIPP: An Expanded Community of Practice - Community Publishing
SCIPP redefines and expands the existing notions about what makes for a vibrant and robust community of practice by partnering CSUSB students and professors with K-12 students, parents, and educators, along with committed community partners. SCIPP encourages curiosity in ways that leads to critical thinking, exploration, risk taking , confidence building, open-mindedness, and other personal traits that equip them with the softskills to be active, critical, and creative contributors to our communities. SCIPP pedagogy embraces our students\u27 collective wisdom and focuses on relational building where multi-directional communication is promoted and students are viewed as equal stakeholders in their own educations. SCIPP puts collaboration into action which in turn fosters community-based lifelong learning. SCIPP provides the open intellectual space for future university students (our K-12 students) to engage with existing university students in meaningful ways so as to sustain interconnected partnerships facilitating community engagement. It supports parents as experts in the education of their children and acknowledges parents as the first conduits to spark their childrenβs imagination while they actively participate in education enriching activities and programs. Everyone involved is committed to creating a secure and open atmosphere for dreaming, sharing, and learning. Together we explore the aspects of community publishing through collaborative learning in formal and informal settings relating to digital and printed medias
Epidermal Growth Factor Receptor Activation in Glioblastoma through Novel Missense Mutations in the Extracellular Domain
BACKGROUND: Protein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy. METHODS AND FINDINGS: Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR) kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132) of glioblastomas and 12.5% (1/8) of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors. CONCLUSIONS: Our results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma
Mapping genetic variations to three- dimensional protein structures to enhance variant interpretation: a proposed framework
The translation of personal genomics to precision medicine depends on the accurate interpretation of the multitude of genetic variants observed for each individual. However, even when genetic variants are predicted to modify a protein, their functional implications may be unclear. Many diseases are caused by genetic variants affecting important protein features, such as enzyme active sites or interaction interfaces. The scientific community has catalogued millions of genetic variants in genomic databases and thousands of protein structures in the Protein Data Bank. Mapping mutations onto three-dimensional (3D) structures enables atomic-level analyses of protein positions that may be important for the stability or formation of interactions; these may explain the effect of mutations and in some cases even open a path for targeted drug development. To accelerate progress in the integration of these data types, we held a two-day Gene Variation to 3D (GVto3D) workshop to report on the latest advances and to discuss unmet needs. The overarching goal of the workshop was to address the question: what can be done together as a community to advance the integration of genetic variants and 3D protein structures that could not be done by a single investigator or laboratory? Here we describe the workshop outcomes, review the state of the field, and propose the development of a framework with which to promote progress in this arena. The framework will include a set of standard formats, common ontologies, a common application programming interface to enable interoperation of the resources, and a Tool Registry to make it easy to find and apply the tools to specific analysis problems. Interoperability will enable integration of diverse data sources and tools and collaborative development of variant effect prediction methods
Nck adapter proteins: functional versatility in T cells
Nck is a ubiquitously expressed adapter protein that is almost exclusively built of one SH2 domain and three SH3 domains. The two isoproteins of Nck are functionally redundant in many aspects and differ in only few amino acids that are mostly located in the linker regions between the interaction modules. Nck proteins connect receptor and non-receptor tyrosine kinases to the machinery of actin reorganisation. Thereby, Nck regulates activation-dependent processes during cell polarisation and migration and plays a crucial role in the signal transduction of a variety of receptors including for instance PDGF-, HGF-, VEGF- and Ephrin receptors. In most cases, the SH2 domain mediates binding to the phosphorylated receptor or associated phosphoproteins, while SH3 domain interactions lead to the formation of larger protein complexes. In T lymphocytes, Nck plays a pivotal role in the T cell receptor (TCR)-induced reorganisation of the actin cytoskeleton and the formation of the immunological synapse. However, in this context, two different mechanisms and adapter complexes are discussed. In the first scenario, dependent on an activation-induced conformational change in the CD3Ξ΅ subunits, a direct binding of Nck to components of the TCR/CD3 complex was shown. In the second scenario, Nck is recruited to the TCR complex via phosphorylated Slp76, another central constituent of the membrane proximal activation complex. Over the past years, a large number of putative Nck interactors have been identified in different cellular systems that point to diverse additional functions of the adapter protein, e.g. in the control of gene expression and proliferation
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