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1.  CCR2 Regulates Monocyte Recruitment As Well As CD4+ Th1 Allorecognition After Lung Transplantation 
Graft rejection remains a formidable problem contributing to poor outcomes after lung transplantation. Blocking chemokine pathways have yielded promising results in some organ transplant systems. Previous clinical studies have demonstrated upregulation of CCR2 ligands following lung transplantation. Moreover, lung injury is attenuated in CCR2-deficient mice in several inflammatory models. In this study, we examined the role of CCR2 in monocyte recruitment and alloimmune responses in a mouse model of vascularized orthotopic lung transplantation. The CCR2 ligand MCP-1 is upregulated in serum and allografts following lung transplantation. CCR2 is critical for the mobilization of monocytes from the bone marrow into the bloodstream and for the accumulation of CD11c+ cells within lung allografts. A portion of graft-infiltrating recipient CD11c+ cells expresses both recipient and donor MHC molecules. Two-photon imaging demonstrates that recipient CD11c+ cells are associated with recipient T cells within the graft. While recipient CCR2 deficiency does not prevent acute lung rejection and is associated with increased graft infiltration by T cells, it significantly reduces CD4+ Th1 indirect and direct allorecognition. Thus, CCR2 may be a potential target to attenuate alloimmune responses after lung transplantation.
PMCID: PMC3746750  PMID: 20420631
Allorecognition; CD4+ T cells; chemokine receptors; lung transplantation; monocytes
2.  Epigenetics and lupus 
Arthritis Research & Therapy  2012;14(Suppl 3):A1.
PMCID: PMC3467478
3.  Strategies for Incorporating Novel Post-Translational Modification Enrichment Methods in a Quantitative Proteomics Environment 
Low-stoichiometry post-translational modifications (PTMs) such as phosphorylation, acetylation, s-nitrosylation (SNO), and s-acylation are integral to intracellular signaling and are thought to capture the most immediate response of a cell upon external stimulus. These modifications, among others, have been the subject of intense focus in the mass spectrometry world because of the ability of the mass spectrometer to identify the peptide and many times the site of modification with a much higher degree of specificity than antibody-based approaches. However, PTMs present a distinct analytical challenge because they exist at low levels in biological samples and therefore must be enriched in order to be measured with the mass spectrometer in a high-throughput manner from a complex system. Three categorical methods exist to enrich PTMs, including chemical enrichment, antibody enrichment, and PTM-switch methods.
In a biomedical research environment, one of the most challenging aspects to implementing any enrichment strategy is to properly qualify a technique so that it can be performed with high specificity and reproducibility. Each of the three categories of enrichments has unique analytical concerns and challenges. Recently, our laboratory has sought to extend the study of PTMs from primarily a qualitative environment to a quantitative one, in order to study selected PTMs in systems where isotopic labeling is not possible, such as animal models or clinical studies.[1-4] This presentation will focus on the key steps in the development and deployment of label-free quantitation using three of our recently-published PTM-switch enrichment approaches, S-nitrosothiol resin-assisted capture (SNO-RAC), S-acylation resin-assisted capture (Acyl-RAC), and the acetyl biotin-switch technique (Acetyl-BST).
PMCID: PMC3630577
4.  Genetically Determined Amerindian Ancestry Correlates with Increased Frequency of Risk Alleles for Systemic Lupus Erythematosus 
Arthritis and rheumatism  2010;62(12):3722-3729.
To analyze if genetically determined Amerindian ancestry predicts the increased presence of risk alleles of known susceptibility genes for systemic lupus erythematosus.
Single nucleotide polymorphisms within 16 confirmed genetic susceptibility loci for SLE were genotyped in a set of 804 Mestizo lupus patients and 667 Mestizo normal healthy controls. In addition, 347 admixture informative markers were genotyped. Individual ancestry proportions were determined using STRUCTURE. Association analysis was performed using PLINK, and correlation of the presence of risk alleles with ancestry was done using linear regression.
A meta-analysis of the genetic association of the 16 SNPs across populations showed that TNFSF4, STAT4, PDCD1, ITGAM, and IRF5 were associated with lupus in a Hispanic-Mestizo cohort enriched for European and Amerindian ancestry. In addition, two SNPs within the MHC region, previously associated in a genome-wide association study in Europeans, were also associated in Mestizos. Using linear regression we predict an average increase of 2.34 risk alleles when comparing a lupus patient with 100% Amerindian ancestry to an SLE patient with 0% American Indian Ancestry (p<0.0001). SLE patients with 43% more Amerindian ancestry are predicted to carry one additional risk allele.
Amerindian ancestry increased the number of risk alleles for lupus.
PMCID: PMC3078084  PMID: 20848568
5.  Methylation of the OP-1 promoter: potential role in the age-related decline in OP-1 expression in cartilage 
An age-related decline in chondrocyte production of OP-1 (BMP-7) may contribute to cartilage loss in osteoarthritis. This study was designed to determine if increased methylation of the OP-1 promoter might serve as a mechanism for the age-related decline in OP-1 expression.
Human articular chondrocytes were isolated from cartilage obtained after death from tissue donors (ages 19-86 years) without a known history of arthritis. DNA was obtained from isolated chondrocytes in primary culture and analyzed for OP-1 promoter methylation by PCR after bisulfite treatment. Cultured cells were treated with the DNA methyltransferase inhibitor 5-azacytidine and OP-1 production was measured in the media by ELISA. RNA was isolated to measure expression of IGF-1, the IGF-1 receptor, aggrecan, and OP-1 by real-time PCR.
Methylation of the OP-1 promoter was detected in chondrocytes isolated from tissue obtained from older adults and there was a positive correlation between age and OP-1 methylation status (n=22, R2=0.277, p=0.014). Inhibition of methylation in cultured cells with 5-azacytidine increased chondrocyte production of OP-1 protein and increased the expression of the IGF-1, the IGF-1 receptor, aggrecan, and OP-1 genes but not GAPDH.
Age-related methylation of the OP-1 promoter may contribute to a decrease in OP-1 production in cartilage and a decrease in expression of OP-1 responsive genes such as IGF-1, the IGF-1 receptor, and aggrecan.
PMCID: PMC2692619  PMID: 18829350
6.  Defective T-cell ERK signaling induces interferon-regulated gene expression and overexpression of methylation-sensitive genes similar to lupus patients 
Genes and immunity  2008;9(4):368-378.
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies against a host of nuclear antigens. The pathogenesis of lupus is incompletely understood. Environmental factors may play a role via altering DNA methylation, a mechanism regulating gene expression. In lupus, genes including CD11a and CD70 are overexpressed in T cells as a result of promoter hypomethylation. T-cell DNA methyltransferase expression is regulated in part by the extracellular signal-regulated kinase (ERK) signaling pathway. In this study, we investigate the effects of decreased ERK pathway signaling in T cells using transgenic animals. We generated a transgenic mouse that inducibly expresses a dominant-negative MEK in T cells in the presence of doxycycline. We show that decreased ERK pathway signaling in T cells results in decreased expression of DNA methyltransferase 1 and overexpression of the methylation-sensitive genes CD11a and CD70, similar to T cells in human lupus. Our transgenic animal model also develops anti-dsDNA antibodies. Interestingly, microarray expression assays revealed overexpression of several interferon-regulated genes in the spleen similar to peripheral blood cells of lupus patients. This model supports the contention that ERK pathway signaling defects in T cells contribute to the development of autoimmunity.
PMCID: PMC2825163  PMID: 18523434
epigenetics; T cell; autoimmunity; interferon; methylation; lupus
9.  Randomised controlled trial and cost consequences study comparing initial physiotherapy assessment and management with routine practice for selected patients in an accident and emergency department of an acute hospital 
Objective: The Department of Health is reviewing the effectiveness of accident and emergency (A&E) departments. This study aimed to compare health and economic effects of physiotherapy initial assessment and management with routine practice in an A&E department.
Methods: Randomised controlled trial and cost and consequences study. Patients presenting at A&E were eligible if suspected at triage to have soft tissue injury without fracture. The efficacy end point was "days to return to usual activities". Secondary end points included patient satisfaction with their care and further health outcomes and cost data.
Results: 766 of 844 (915) patients were randomised. The median days before return to usual activities (available for 73% of those randomised) was greater in the physiotherapist group (41 days compared with 28.5 days; hazard ratio 0.85 p = 0.071). The physiotherapy group expressed greater satisfaction with their A&E care (on a scale of 1 to 5, median was 4.2 compared with 4.0, p<0.001), were more likely to be given advice and reassurance, and more likely to be provided with aids and appliances. Costs were the same between the two arms.
Conclusion: There is evidence that physiotherapy leads to a prolonged time before patients return to usual activities. This study shows no clear danger from physiotherapy intervention and long term outcomes may be different but given these findings, a best estimate is that introducing physiotherapist assessment will increase costs to the health service and society. Routine care should continue be provided unless there is some reason why it is not feasible to do so and an alternative must be found.
PMCID: PMC1726666  PMID: 15662054
11.  Investigation of female survival benefit in metastatic melanoma 
British Journal of Cancer  1999;80(12):2025-2033.
Epidemiological studies show female survival benefit in advanced metastatic melanoma. In investigating a possible mechanism for this female survival benefit, we have previously reported that the female steroid 17β-oestradiol significantly reduces invasion of a human melanoma cell line (A375-SM cells) and ocular melanoma cells through fibronectin. Neither cell type was found to possess oestrogen receptor-α. The aim of the current study was to obtain further information on the extent to which progression of cutaneous melanoma might be sex steroid sensitive by (a) examining the relationship between circulating sex steroids, sex hormone binding globulin and disease progression; (b) examining the relationship between sex steroid structure and the ability of steroids to reduce invasion of a melanoma cell line in vitro; and (c) examining the effects of sex steroids on proliferation of these cells in vitro. We report a significant reduction in circulating oestrone with disease progression in male but not female patients. Examining steroids for their ability to inhibit invasion of A375-SM cells through fibronectin in vitro, oestrogenic compounds (17β-oestradiol and oestrone) were found to inhibit invasion; in this respect, oestrone was approximately 50 times more potent than 17β-oestradiol; steroids lacking the benzene ring structure did not inhibit invasion, indeed dehydroepiandrosterone (DHEA) which acts as a precursor to androgenic steroids significantly enhanced invasion. Proliferation of A375-SM cells was unaffected by 17β-oestradiol, oestrone or dihydrotestosterone when cells were cultured on plastic; in contrast, all three steroids induced modest proliferation of cells when grown on fibronectin with dihydrotestosterone the most mitogenic of the three steroids. These data are consistent with sex steroids playing a role in melanoma progression. © 1999 Cancer Research Campaign
PMCID: PMC2363135  PMID: 10471056
melanoma; invasion; oestrogen; testosterone; oestrogen receptor-β
13.  Environmentally induced autoimmune diseases: potential mechanisms. 
Environmental Health Perspectives  1999;107(Suppl 5):737-742.
Environmental and other xenobiotic agents can cause autoimmunity. Examples include drug-induced lupus, toxic oil syndrome, and contaminated l-tryptophan ingestion. Numerous mechanisms, based on (italic)in vitro(/italic) evidence and animal models, have been proposed to explain how xenobiotics induce or accelerate autoimmunity. The majority of these can be divided into three general categories. The first is those inhibiting the processes involved in establishing tolerance by deletion. Inhibiting deletion can result in the release of newly generated autoreactive cells into the periphery. The second mechanism is the modification of gene expression in the cells participating in the immune response, permitting lymphocytes to respond to signals normally insufficient to initiate a response or allowing the antigen-presenting cells to abnormally stimulate a response. Abnormal gene expression can thus disrupt tolerance maintained by suppression or anergy, permitting activation of autoreactive cells. The third is the modification of self-molecules such that they are recognized by the immune system as foreign. Examples illustrating these concepts are presented, and related mechanisms that have the potential to similarly affect the immune system are noted. Some mechanisms appear to be common to a variety of agents, and different mechanisms appear to produce similar diseases. However, evidence that any of these mechanisms are actually responsible for xenobiotic-induced human autoimmune disease is still largely lacking, and the potential for numerous and as yet unidentified mechanisms also exists.
PMCID: PMC1566247  PMID: 10502539
14.  Mechanisms of drug-induced lupus. II. T cells overexpressing lymphocyte function-associated antigen 1 become autoreactive and cause a lupuslike disease in syngeneic mice. 
Journal of Clinical Investigation  1996;97(12):2866-2871.
Current theories propose that systemic lupus erythematosus develops when genetically predisposed individuals are exposed to certain environmental agents, although how these agents trigger lupus is uncertain. Some of these agents, such as procainamide, hydralazine, and UV-light inhibit T cell DNA methylation, increase lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18) expression, and induce autoreactivity in vitro, and adoptive transfer of T cells that are made autoreactive by this mechanism causes a lupuslike disease. The mechanism by which these cells cause autoimmunity is unknown. In this report, we present evidence that LFA-1 overexpression is sufficient to induce autoimmunity. LFA-1 overexpression was induced on cloned murine Th2 cells by transfection, resulting in autoreactivity. Adoptive transfer of the transfected, autoreactive cells into syngeneic recipients caused a lupuslike disease with anti-DNA antibodies, an immune complex glomerulonephritis and pulmonary alveolitis, similar to that caused by cells treated with procainamide. These results indicate that agents or events which modify T cell DNA methylation may induce autoimmunity by causing T cell LFA-1 overexpression. Since T cells from patients with active lupus have hypomethylated DNA and overexpressed LFA-1, this mechanism could be important in the development of human autoimmunity.
PMCID: PMC507381  PMID: 8675699
15.  Ligand recognition by murine anti-DNA autoantibodies. II. Genetic analysis and pathogenicity. 
Journal of Clinical Investigation  1996;97(7):1748-1760.
Although anti-DNA autoantibodies are an important hallmark of lupus, the relationships among anti-DNA structure, reactivity, and pathogenicity have not been fully elucidated. To further investigate these relationships, we compare the variable genes and primary structure of eight anti-DNA mAbs previously obtained from an MRL/MpJ-lpr/lpr mouse along with the ability of three representative mAbs to induce nephritis in nonautoimmune mice using established adoptive transfer protocols. One monospecific anti-single-stranded (ss) DNA (11F8) induces severe diffuse proliferative glomerulonephritis in nonautoimmune mice whereas another anti-ssDNA with apparently similar in vitro binding properties (9F11) and an anti-double-stranded DNA (4B2) are essentially benign. These results establish a murine model of anti-DNA-induced glomerular injury resembling the severe nephritis seen in lupus patients and provide direct evidence that anti-ssDNA can be more pathogenic than anti-double-stranded DNA. In vitro binding experiments using both protein-DNA complexes and naive kidney tissue indicate that glomerular localization of 11F8 may occur by recognition of a planted antigen in vivo. Binding to this antigen is DNase sensitive which suggests that DNA or a DNA-containing molecule is being recognized.
PMCID: PMC507240  PMID: 8601641
17.  Treating activated CD4+ T cells with either of two distinct DNA methyltransferase inhibitors, 5-azacytidine or procainamide, is sufficient to cause a lupus-like disease in syngeneic mice. 
Human antigen-specific CD4+ T cells become autoreactive after treatment with various DNA methylation inhibitors, including 5-azacytidine, procainamide, and hydralazine. This suggests a mechanism that could contribute to the development of some forms of autoimmunity. In this report we have asked whether T cells treated with DNA methylation inhibitors can induce autoimmunity. Murine CD4+ T cells were treated with 5-azacytidine or procainamide and were shown to respond to syngeneic antigen-presenting cells, similar to CD4+ human T cell clones treated with these drugs. Functional characterization demonstrated that cells treated with either drug spontaneously lysed syngeneic macrophages and secreted IL-4, IL-6, and IFN-gamma. Adoptive transfer of 5-azacytidine- or procainamide-treated cells into unirradiated syngeneic recipients induced an immune complex glomerulonephritis and IgG anti-DNA and antihistone antibodies. These experiments demonstrate that T cells treated with either of two distinct DNA methyltransferase inhibitors are sufficient to induce a lupus-like disease. It is possible that the lysis of macrophages, together with the release of cytokines promoting B cell differentiation, contributes to the autoantibody production and immune complex deposition. These results suggest that environmental agents that inhibit DNA methylation could interact with T cells in vivo to produce a lupus-like illness, a mechanism that could have relevance to drug-induced and idiopathic lupus.
PMCID: PMC293525  PMID: 7686923
18.  Identification of two related markers for common acute lymphoblastic leukemia as heat shock proteins. 
Journal of Clinical Investigation  1990;85(1):200-207.
By direct analysis of the polypeptide constituents of leukemic cells, we have previously detected several polypeptides that are restricted in their expression to acute lymphoblastic leukemia (ALL). In this study, we provide evidence that two polypeptides designated L2 and L4 are structurally related and represent novel markers for common ALL. Partial amino acid sequence analysis did not uncover differences between L2 and L4. The sequences obtained correspond to a previously cloned human gene designated hsp 27 that is expressed, following heat shock treatment, in a variety of cells. 32Pi incorporation studies indicate that L4 is an unphosphorylated form and L2 is a phosphorylated form of hsp27. The two forms were inducible by heat shock in leukemic and nonleukemic lymphoid cells. Thus, in acute leukemia, the common ALL subtype is uniquely characterized by the constitutive expression of a polypeptide that represents a major cellular phosphoprotein.
PMCID: PMC296406  PMID: 2295696
19.  Birthweights of babies born at home in a black rural community of Bophuthatswana, southern Africa. 
Archives of Disease in Childhood  1983;58(3):176-179.
In Africa, data on incidence of babies' low birthweight are based virtually exclusively on information derived from deliveries in hospitals. The incidence of low birthweight is about double that prevailing in developed countries. However, in a rural region in southern Africa, after painstakingly overcoming local strongly entrenched custom, it was found possible to organize the measurement of newborn babies born at home. It was found that the incidence of low birthweight was about half of that among babies born in hospitals--that is the proportion was similar to that of developed populations.
PMCID: PMC1627813  PMID: 6838247
21.  Purification and Some Properties of Diplococcin from Streptococcus cremoris 346 
Eleven of 150 Streptococcus cremoris strains examined produced the bacteriocin diplococcin. The diplococcin activity spectrum was restricted to S. cremoris and Streptococcus lactis strains, and none of a wide range of other gram-positive or gram-negative strains were inhibited. The diplococcin produced by S. cremoris 346 was purified by ammonium sulfate precipitation and column chromatography. Purified diplococcin was very unstable at room temperature and lost 75% of its activity after heating at 100°C for 1 min. The proteolytic enzymes trypsin, pronase, and α-chymotrypsin completely inactivated diplococcin. The amino acid composition showed a high content of acidic and neutral acids and a correspondingly low content of basic amino acids, including one residue of ornithine per mole. From the amino acid analysis a molecular weight of 5,300 was estimated. Diplococcin was readily distinguished from the S. lactis bacteriocin nisin by its restricted activity spectrum, its biological properties, and by cross-reaction experiments.
PMCID: PMC243643  PMID: 16345704
22.  Effects of surface-active agents on neutrophil receptors. 
Infection and Immunity  1978;21(1):28-33.
An easily performed assay to identify the C3b and Fc receptors on human neutrophils was developed. Salmonella typhimurium were treated with fluorescein and then incubated in nonimmune fresh human serum, which led to C3b fixation via activation of the alternative pathway. Similarly, type II pneumococci were treated with fluorescein and opsonized with type-specific rabbit antiserum. Neutrophils bearing C3b and Fc receptors formed rosettes with the respective bacteria, which were easily readable because of their bright fluorescence. Incubation of neutrophils at 37 degrees C with C3-coated bacteria generated 54 +/ 4% C3b rosettes, whereas neutrophils incubated with immunoglobulin G-coated bacteria yielded 75 +/ 7% rosettes. Incubation at 4 degrees C inhibited the formation of C3b rosettes but not Fc rosettes. Heat inactivation of the fresh human serum at 56 degrees C for 30 min completely inhibited the formation of the C3b rosettes, and addition of heat-aggregated immunoglobulin G to the polymorphonuclear leukocyte blocked the ability of the polymorphonuclear leukocyte to bind immunoglobulin G-coated bacteria. Addition of 1.0 mM N-ethylmaleimide, 0.1 mg of trypsin per ml, 10 mM H2O2, O2- generated by xanthine-xanthine oxidase, and 8 times 10(-4) M hydrocortisone inhibited the C3b receptor, but did not inhibit the Fc receptor. In neutrophils, the selective effect of the various inhibitors suggests that the Fc and C3b receptors are distinct entities.
PMCID: PMC421952  PMID: 30696
23.  Acute sacroiliitis due to Salmonella okatie. 
British Medical Journal  1977;1(6074):1449-1450.
PMCID: PMC1607665  PMID: 861685
24.  Cardio-thoracic ratio in Negroes in Southern Africa 
Postgraduate Medical Journal  1972;48(564):584-589.
Negro groups in West, Central and Southern Africa, also in Jamaica, have mean cardio-thoracic ratios significantly greater than those in corresponding age-sex groups of Caucasians.
To throw more light on the situation, studies on young and elderly Negroes have been made in certain groups in Southern Africa, also on local Caucasian groups. Only slight differences in ratio were found between local Negro groups, and Caucasian groups in South Africa, also in Wales and Tecumseh (U.S.A.). Yet ratios in Negro males from Malawi and Mozambique, resident in South Africa, were significantly greater than values in local Negroes. High ratios are not therefore invariable for Negroes. The difference observed relates to heart, not thoracic diameter. Since the phenomenon, which concerns heart position, is apparent in the young, almost certainly it is of ethnic origin. Investigations on cardiomegaly in African populations must take this factor into reckoning. Its presence or absence locally may readily be assessed by determining ratios in relatively small numbers of young people of both sexes.
PMCID: PMC2495413  PMID: 4263410
25.  Personal View 
British Medical Journal  1975;2(5973):745.
PMCID: PMC1673909

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