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Biomedical subjects

David J Rawlings

Publications and source records attributed to David J Rawlings.

At least 19 recordsLinked to original sources
PubMed2007

Wiskott-Aldrich syndrome protein is required for regulatory T cell homeostasis.

Wiskott-Aldrich syndrome protein (WASp) is essential for optimal T cell activation. Patients with WAS exhibit both immunodeficiency and a marked susceptibility to systemic autoimmunity. We investigated whether alterations in Treg function might explain these paradoxical observations. While WASp-deficient (WASp(-/-)) mice exhibited normal thymic Treg generation, the competitive fitness of peripheral Tregs was severely compromised. The total percentage of forkhead box P3-positive (Foxp3(+)) Tregs among CD4(+) T cells was reduced, and WASp(-/-) Tregs were rapidly outcompeted by WASp(+) Tregs in vivo. These findings correlated with reduced expression of markers associated with self-antigen-driven peripheral Treg activation and homing to inflamed tissue. Consistent with these findings, WASp(-/-) Tregs showed a reduced ability to control aberrant T cell activation and autoimmune pathology in Foxp3(-/-)Scurfy (sf) mice. Finally, WASp(+) Tregs exhibited a marked selective advantage in vivo in a WAS patient with a spontaneous revertant mutation, indicating that altered Treg fitness likely explains the autoimmune features in human WAS.

Adoptive Transfer↗
PubMed2006

Mutifactorial analysis of risk factors for reduced bone mineral density in patients with Crohn's disease.

AIM: To determine the prevalence of osteoporosis in a cohort of patients with Crohn's disease (CD) and to identify the relative significance of risk factors for osteoporosis. METHODS: Two hundred and fifty-eight unselected patients (92 M, 166 F) with CD were studied. Bone mineral density (BMD) was measured at the lumbar spine and hip by dual X-ray absorptiometry. Bone formation was assessed by measuring bone specific alkaline phosphatase (BSAP) and bone resorption by measuring urinary excretion of deoxypyridinoline (DPD) and N-telopeptide (NTX). RESULTS: Between 11.6%-13.6% patients were osteoporotic (T score < -2.5) at the lumbar spine and/or hip. NTX levels were significantly higher in the patients with osteoporosis (P < 0.05) but BSAP and DPD levels were not significantly different. Independent risk factors for osteoporosis at either the lumbar spine or hip were a low body mass index (P < 0.001), increasing corticosteroid use (P < 0.005), and male sex (P < 0.01). These factors combined accounted for 23% and 37% of the reduction in BMD at the lumbar spine and hip respectively. CONCLUSION: Our results confirm that osteoporosis is common in patients with CD and suggest that increased bone resorption is the mechanism responsible for the bone loss. However, less than half of the reduction in BMD can be attributed to risk factors such as corticosteroid use and low BMI and therefore remains unexplained.

Adrenal Cortex Hormones↗
PubMed2006

Immunomodulation of transgene responses following naked DNA transfer of human factor VIII into hemophilia A mice.

A robust humoral immune response against human factor VIII (hFVIII) following naked DNA transfer into immunocompetent hemophilia A mice completely inhibits circulating FVIII activity despite initial high-level hFVIII gene expression. To prevent this undesirable response, we compared transient immunomodulation strategies. Eight groups of mice (n = 4-9 per group) were treated with naked DNA transfer of pBS-HCRHPI-hFVIIIA simultaneously with immunosuppressive reagents that included cyclosporine A (CSA), rapamycin (RAP), mycophenylate mofetil (MMF), a combination of CSA and MMF, a combination of RAP and MMF, a monoclonal antibody against murine CD40 ligand (MR1), recombinant murine Ctla4Ig, and a combination of MR1 and Ctla4Ig. All animals except those receiving only CSA exhibited delayed or absent immune responses against hFVIII. The most effective immunosuppressive regimen, the combination of Ctla4Ig and MR1, prevented inhibitor formation in 8 of 9 animals; the ninth had transient low-titer antibodies. All 9 mice of this group produced persistent, therapeutic levels of hFVIII for more than 6 months. When challenged with the T-dependent antigen bacteriophage Phix174, tolerized mice exhibited normal primary and secondary antibody responses, suggesting that transient immunomodulation to disrupt B/T-cell interaction at the time of plasmid injection effectively promoted long-term immune tolerance specific for hFVIII.

Abatacept↗
PubMed2006

HS1 functions as an essential actin-regulatory adaptor protein at the immune synapse.

HS1, the leukocyte-specific homolog of cortactin, regulates F-actin in vitro and is phosphorylated in response to TCR ligation, but its role in lymphocyte activation has not been addressed. We demonstrate that HS1-deficient T cells fail to accumulate F-actin at the immune synapse (IS) and, upon TCR ligation, form actin-rich structures that are disordered and unstable. Early TCR activation events are intact in these cells, but Ca2+ influx and IL-2 gene transcription are defective. Importantly, HS1 tyrosine phosphorylation is required for its targeting to the IS and for its function in regulating actin dynamics and IL-2 promoter activity. Phosphorylation also links HS1 to multiple signaling proteins, including Lck, PLCgamma1, and Vav1, and is essential for the stable recruitment of Vav1 to the IS. Taken together, our studies show that HS1 is indispensable for signaling events leading to actin assembly and IL-2 production during T cell activation.

Actins↗
PubMed2006

The CARMA1 signalosome links the signalling machinery of adaptive and innate immunity in lymphocytes.

The recognition of antigen by B- or T-cell receptors initiates an intracellular signalling cascade that results in the nuclear translocation and activation of the transcription factor nuclear factor-kappaB (NF-kappaB). NF-kappaB is an important regulator of lymphocyte development and function, and its dysregulation is associated with many immune disorders. Defining the mechanisms that transmit signals from the antigen receptor to NF-kappaB is therefore an important goal for immunologists. In this Review, we merge information gleaned from research of the innate immune system with what we know about antigen-receptor signals in the adaptive immune system, to propose a cohesive model of how antigen receptors activate NF-kappaB.

Animals↗
PubMed2006

Low bone mineral density measurements in care home residents--a treatable cause of fractures.

PURPOSE: to assess predictors of fracture risk and treatment for osteoporosis among elderly care home residents. SUBJECTS AND METHODS DESIGN: cross-sectional survey; SETTING: residents of care homes in Newcastle upon Tyne, UK; PARTICIPANTS: representative sample from residential care (87), nursing homes (105) and specialist homes for elderly people with dementia [elderly mentally infirm (EMI)]: residential (124) and nursing (76); MAIN OUTCOME MEASURES: dual-energy X-ray absorptiometry bone mineral density (BMD) at calcaneum; functional assessments, including cognition, using Mini-Mental State Examination (MMSE), Clifton Assessment Procedure for the Elderly-Behaviour Rating Score (CAPE-BRS) and Functional Assessment Staging Test (FAST) scores; current drug prescription. RESULTS: MMSE, CAPE, FAST (all ANOVA P < 0.001) and weight (ANOVA P < 0.02) were lower in EMI homes. Drugs with sedative effects (chi-square, P < 0.0001) were more likely and calcium and vitamin D (CaD) supplementation (chi-square, P < 0.02) less likely in EMI care. For residential care, the odds ratio (OR) for sedative drugs in EMI was 2.13 (95% CI 1.11-4.06) with no significant difference between nursing homes. For CaD supplementation, the OR for EMI nursing homes was 0.19 (95% CI 0.05-0.72) and for EMI residential homes 0.38 (NS to 95% CI 0.12-1.27). BMD was low: mean T-score was -2.29 (95% CI -2-48 to -2.09) and Z-score -0.96 (95% CI -1.16 to -0.76) with a prevalence of osteoporosis (T-score < -1.6) of 69.2%. MMSE and FAST scores did not predict BMD. In EMI residential care, a decrease of CAPE score by 5 points was associated with a decrease in T-score by 0.6 (95% CI 0.15-1.1). CONCLUSIONS: of the tools used to assess function, only CAPE predicted low BMD in EMI residential care. Rates of CaD supplementation are particularly low in EMI care, where risk factors for fracture were the greatest. We conclude that fracture risk is neglected in these homes, and targeted education and treatment are warranted.

Absorptiometry, Photon↗
PubMed2005

CD38 signaling regulates B lymphocyte activation via a phospholipase C (PLC)-gamma 2-independent, protein kinase C, phosphatidylcholine-PLC, and phospholipase D-dependent signaling cascade.

The CD38 cell surface receptor is a potent activator for splenic, B lymphocytes. The molecular mechanisms regulating this response, however, remain incompletely characterized. Activation of the nonreceptor tyrosine kinase, Btk, is essential for CD38 downstream signaling function. The major Btk-dependent substrate in B cells, phospholipase C-gamma2 (PLC-gamma2), functions to generate the key secondary messengers, inositol-1,4,5 trisphosphate and diacylglycerol. Surprisingly, CD38 ligation results in no detectable increase in phosphoinositide metabolism and only a minimal increase in cytosolic calcium. We hypothesized that Btk functioned independently of PLC-gamma2 in the CD38 signaling pathway. Accordingly, we demonstrate that CD38 cross-linking does not result in the functional phosphorylation of PLC-gamma2 nor an increase in inositol-1,4,5 trisphosphate production. Furthermore, splenic B cells exhibit a normal CD38-mediated, proliferative response in the presence of the phosphoinositide-PLC inhibitor, U73122. Conversely, protein kinase C (PKC) beta-deficient mice, or PKC inhibitors, indicated the requirement for diacylglycerol-dependent PKC isoforms in this pathway. Loss of PKC activity blocked CD38-dependent, B cell proliferation, NF-kappaB activation, and subsequent expression of cyclin-D2. These results suggested that an alternate diacylglycerol-producing phospholipase must participate in CD38 signaling. Consistent with this idea, CD38 increased the enzymatic activity of the phosphatidylcholine (PC)-metabolizing enzymes, PC-PLC and phospholipase D. The PC-PLC inhibitor, D609, completely blocked CD38-dependent B cell proliferation, IkappaB-alpha degradation, and cyclin-D2 expression. Analysis of Btk mutant B cells demonstrated a partial requirement for Btk in the activation of both enzymes. Taken together, these data demonstrate that CD38 initiates a novel signaling cascade leading to Btk-, PC-PLC-, and phospholipase D-dependent, PLC-gamma2-independent, B lymphocyte activation.

ADP-ribosyl Cyclase↗
PubMed2005

Phosphorylation of the CARMA1 linker controls NF-kappaB activation.

PKC isoforms and CARMA1 play crucial roles in immunoreceptor-dependent NF-kappaB activation. We tested whether PKC-dependent phosphorylation of CARMA1 directly regulates this signaling cascade. B cell antigen receptor (BCR) engagement led to the progressive recruitment of CARMA1 into lipid rafts and to the association of CARMA1 with, and phosphorylation by, PKCbeta. Furthermore, PKCbeta interacted with the serine-rich CARMA1 linker, and both PKCbeta and PKCtheta phosphorylated identical serine residues (S564, S649, and S657) within this linker. Mutation of two of these sites ablated the functional activity of CARMA1. In contrast, deletion of the linker resulted in constitutive, receptor- and PKC-independent NF-kappaB activation. Together, our data support a model whereby CARMA1 phosphorylation controls NF-kappaB activation by triggering a shift from an inactive to an active CARMA1 conformer. This PKC-dependent switch regulates accessibility of the CARD and CC domains and controls assembly and full activation of the membrane-associated IkappaB kinase (IKK) signalosome.

Animals↗
PubMed2005

Bruton's tyrosine kinase regulates immunoglobulin promoter activation in association with the transcription factor Bright.

Bright (B-cell regulator of immunoglobulin heavy chain transcription) binding to immunoglobulin heavy chain loci after B-cell activation is associated with increased heavy chain transcription. Our earlier reports demonstrated that Bright coimmunoprecipitates with Bruton's tyrosine kinase (Btk) and that these proteins associate in a DNA-binding complex in primary B cells. B cells from immunodeficient mice with a mutation in Btk failed to produce stable Bright DNA-binding complexes. In order to determine if Btk is important for Bright function, a transcription activation assay was established and analyzed using real-time PCR technology. Cells lacking both Bright and Btk were transfected with Bright and/or Btk along with an immunoglobulin heavy chain reporter construct. Immunoglobulin gene transcription was enhanced when Bright and Btk were coexpressed. In contrast, neither Bright nor Btk alone led to activation of heavy chain transcription. Furthermore, Bright function required both Btk kinase activity and sequences within the pleckstrin homology domain of Btk. Bright was not appreciably phosphorylated by Btk; however, a third tyrosine-phosphorylated protein coprecipitated with Bright. Thus, the ability of Bright to enhance immunoglobulin transcription critically requires functional Btk.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2005

Lifecourse study of bone health at age 49-51 years: the Newcastle thousand families cohort study.

OBJECTIVE: To quantify the direct and indirect effects of fetal (position in family, weight, and social class at birth), childhood (breast feeding, growth, infections, and social class in childhood, age at menarche), and adult life (social class, alcohol consumption, smoking, diet, reproductive history, exercise, hormone replacement therapy use), and adult size (height, weight) on bone health at age 49-51 years, as measured by bone mineral density, total scanned bone area of the hip and lumbar spine, and femoral neck shaft angle. DESIGN: Follow up study of the Newcastle thousand families birth cohort established in 1947. PARTICIPANTS: 171 men and 218 women who attended for dual energy x ray absorptiometry scanning. MAIN RESULTS: Fetal life explained around 6% of variation in adult bone mineral density for men, but accounted for less than 1% for women. Adult lifestyle, including effects mediated through adult weight accounted for over 10% of variation in density for men and around 6% for women. Almost half of variation in bone area for men was explained by early life. However, most of this was mediated through achieved adult height and weight. In women, less than 5% of variation in bone area was accounted for by early life, after adjusting for adult size. Most of the variation in each of the indicators for both sexes was contributed either directly or indirectly by adult lifestyle and achieved adult height and weight. CONCLUSIONS: The effect of fetal life on bone health in adulthood seems to be mediated through achieved adult height.

Absorptiometry, Photon↗
PubMed2004

Integration with the human genome of peptide sequences obtained by high-throughput mass spectrometry.

A crucial aim upon the completion of the human genome is the verification and functional annotation of all predicted genes and their protein products. Here we describe the mapping of peptides derived from accurate interpretations of protein tandem mass spectrometry (MS) data to eukaryotic genomes and the generation of an expandable resource for integration of data from many diverse proteomics experiments. Furthermore, we demonstrate that peptide identifications obtained from high-throughput proteomics can be integrated on a large scale with the human genome. This resource could serve as an expandable repository for MS-derived proteome information.

Amino Acid Sequence↗
PubMed2004

ATM protein purified from vaccinia virus expression system: DNA binding requirements for kinase activation.

The ataxia-telangiectasia mutated (ATM) gene product plays a role in responding to double stand DNA breaks. Some biochemical studies of ATM function have been hampered by lack of an efficient expression system and abundant purified ATM protein. We report the construction of a vaccinia virus expressing ATM, vWR-ATM, which was used to produce large amounts of functional FLAG-tagged ATM protein (FLAG-ATM) in HeLa cells. Kinase activity of the purified FLAG-ATM was dependent on manganese and inhibited with wortmannin. Using the FLAG-ATM recombinant protein, GST-p53 serine 15 phosphorylation increased in the presence of damaged DNA. PHAS-1 phosphorylation was found to be DNA independent. Purified FLAG-ATM was recovered in the autophosphorylated form, as demonstrated by phosphorylation of ATM serine 1981. As shown by atomic force microscopy, FLAG-ATM bound to linear DNA both at broken ends and in mid-strands. Vaccinia virus is the most efficient ATM expression system described to date.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2004

Protein kinase C betaII regulates Akt phosphorylation on Ser-473 in a cell type- and stimulus-specific fashion.

Akt (= protein kinase B), a subfamily of the AGC serine/threonine kinases, plays critical roles in survival, proliferation, glucose metabolism, and other cellular functions. Akt activation requires the recruitment of the enzyme to the plasma membrane by interacting with membrane-bound lipid products of phosphatidylinositol 3-kinase. Membrane-bound Akt is then phosphorylated at two sites for its full activation; Thr-308 in the activation loop of the kinase domain is phosphorylated by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser-473 in the C-terminal hydrophobic motif by a putative kinase PDK2. The identity of PDK2 has been elusive. Here we present evidence that conventional isoforms of protein kinase C (PKC), particularly PKCbetaII, can regulate Akt activity by directly phosphorylating Ser-473 in vitro and in IgE/antigen-stimulated mast cells. By contrast, PKCbeta is not required for Ser-473 phosphorylation in mast cells stimulated with stem cell factor or interleukin-3, in serum-stimulated fibroblasts, or in antigen receptor-stimulated T or B lymphocytes. Therefore, PKCbetaII appears to work as a cell type- and stimulus-specific PDK2.

Animals↗
PubMed2004

Tec kinases mediate sustained calcium influx via site-specific tyrosine phosphorylation of the phospholipase Cgamma Src homology 2-Src homology 3 linker.

Tyrosine phosphorylation of phospholipase Cgamma2 (PLCgamma2) is a crucial activation switch that initiates and maintains intracellular calcium mobilization in response to B cell antigen receptor (BCR) engagement. Although members from three distinct families of non-receptor tyrosine kinases can phosphorylate PLCgamma in vitro, the specific kinase(s) controlling BCR-dependent PLCgamma activation in vivo remains unknown. Bruton's tyrosine kinase (Btk)-deficient human B cells exhibit diminished inositol 1,4,5-trisphosphate production and calcium signaling despite a normal inducible level of total PLCgamma2 tyrosine phosphorylation. This suggested that Btk might modify a critical subset of residues essential for PLCgamma2 activity. To evaluate this hypothesis, we generated site-specific phosphotyrosine antibodies recognizing four putative regulatory residues within PLCgamma2. Whereas all four sites were rapidly modified in response to BCR engagement in normal B cells, Btk-deficient B cells exhibited a marked reduction in phosphorylation of the Src homology 2 (SH2)-SH3 linker region sites, Tyr(753) and Tyr(759). Phosphorylation of both sites was restored by expression of Tec, but not Syk, family kinases. In contrast, phosphorylation of the PLCgamma2 carboxyl-terminal sites, Tyr(1197) and Tyr(1217), was unaffected by the absence of functional Btk. Together, these data support a model whereby Btk/Tec kinases control sustained calcium signaling via site-specific phosphorylation of key residues within the PLCgamma2 SH2-SH3 linker.

Amino Acid Sequence↗
PubMed2004

Sustained correction of B-cell development and function in a murine model of X-linked agammaglobulinemia (XLA) using retroviral-mediated gene transfer.

X-linked agammaglobulinemia (XLA) is a human immunodeficiency caused by mutations in Bruton tyrosine kinase (Btk) and characterized by an arrest in early B-cell development, near absence of serum immunoglobulin, and recurrent bacterial infections. Using Btk- and Tec-deficient mice (BtkTec(-/-)) as a model for XLA, we determined if Btk gene therapy could correct this disorder. Bone marrow (BM) from 5-fluorouracil (5FU)-treated BtkTec(-/-) mice was transduced with a retroviral vector expressing human Btk and transplanted into BtkTec(-/-) recipients. Mice engrafted with transduced hematopoietic cells exhibited rescue of both primary and peripheral B-lineage development, recovery of peritoneal B1 B cells, and correction of serum immunoglobulin M (IgM) and IgG(3) levels. Gene transfer also restored T-independent type II immune responses, and B-cell antigen receptor (BCR) proliferative responses. B-cell progenitors derived from Btk-transduced stem cells exhibited higher levels of Btk expression than non-B cells; and marking studies demonstrated a selective advantage for Btk-transduced B-lineage cells. BM derived from primary recipients also rescued Btk-dependent function in secondary hosts that had received a transplant. Together, these data demonstrate that gene transfer into hematopoietic stem cells can reconstitute Btk-dependent B-cell development and function in vivo, and strongly support the feasibility of pursuing Btk gene transfer for XLA.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2004

Protein kinase C family functions in B-cell activation.

Members of the protein kinase C (PKC) family play important but distinct roles in B-cell activation, as demonstrated by emerging genetic and biochemical studies. PKCbeta is indispensable for B-cell antigen receptor (BCR)-induced NF-kappaB activation and B-cell survival. Recent evidence indicates that PKCbeta might regulate inhibitor of kappaB kinase (IKK) and NF-kappaB activation through interaction with the CARMA1/Bcl10/MALT signaling complex in BCR microdomains. By contrast, the novel PKC isoform PKCdelta is specifically required to maintain the tolerance of self-reactive B cells.

Animals↗