Objectives

To analyze if genetically determined Amerindian ancestry predicts the increased presence of risk alleles of known susceptibility genes for systemic lupus erythematosus.

Methods

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.

Results

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.

Conclusion

Amerindian ancestry increased the number of risk alleles for lupus.

Summary

Objective

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.

Methods

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.

Results

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.

Conclusion

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.

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.

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.

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.

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.

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.

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.

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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.

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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.

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.

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.

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.

Groups of South African Bantu boys and girls of 14 years, compared with local Caucasian children, have lower cortical thickness, cortical score, and crosssection and volume scores for second metacarpal. Yet, when these parameters on groups of aged Bantu men and women, 70-79 years, are compared with published values on corresponding Caucasians, there are no significant differences. Further, aged Bantu have lower prevalences of hip fracture and severe collapse of vertebral bodies. Since a low calcium intake, and in aged Bantu women numerous pregnancies and long lactations, are compatible with satisfactory bone data, it is questioned whether the present insistence on a high calcium intake is justified.