Current approaches to the diagnosis and management of amyloidosis.

Amyloidosis is a collection of diseases caused by the misfolding of proteins that aggregate into insoluble amyloid fibrils and deposit in tissues. While these fibrils may aggregate to form insignificant localised deposits, they can also accumulate in multiple organs to the extent that amyloidosis can be an immediately life-threatening disease, requiring urgent treatment. Recent advances in diagnostic techniques and therapies are dramatically changing the disease landscape and patient prognosis. Delays in diagnosis and treatment remain the greatest challenge, necessitating physician awareness of the common clinical presentations that suggest amyloidosis. The most common types are transthyretin (ATTR) amyloidosis followed by immunoglobulin light-chain (AL) amyloidosis. While systemic AL amyloidosis was previously considered a death sentence with no effective therapies, significant improvement in patient survival has occurred over the past 2 decades, driven by greater understanding of the disease process, risk-adapted adoption of myeloma therapies such as proteosome inhibitors (bortezomib) and monoclonal antibodies (daratumumab) and improved supportive care. ATTR amyloidosis is an underdiagnosed cause of heart failure. Technetium scintigraphy has made noninvasive diagnosis much easier, and ATTR is now recognised as the most common type of amyloidosis because of the increased identification of age-related ATTR. There are emerging ATTR treatments that slow disease progression, decrease patient hospitalisations and improve patient quality of life and survival. This review aims to update physicians on recent developments in amyloidosis diagnosis and management and to provide a diagnostic and treatment framework to improve the management of patients with all forms of amyloidosis.

Regulation of the methylome in differentiation from adult stem cells may underpin vitamin D risk in MS.

Multiple lines of evidence indicate Multiple Sclerosis (MS) is affected by vitamin D. This effect may be mediated by methylation in immune cell progenitors. We aimed to determine (1) if haematopoietic stem cell methylation constrains methylation in daughter cells and is variable between individuals, and (2) the interaction of methylation with the vitamin D receptor binding sites. We interrogated genomic methylation levels from matching purified CD34+ haematopoietic stem cells and progeny CD14+ monocytes and CD56+ NK cells from 11 individuals using modified reduced representation bisulfite sequencing. Differential methylation of Vitamin D Receptor binding sites and MS risk genes was assessed from this and using pyrosequencing for the vitamin D regulated MS risk gene ZMIZ1. Although DNA methylation states at CpG islands and other sites are almost entirely recapitulated between progenitor and progeny immune cells, significant variation was detected at some regions between cell subsets and individuals; including around the MS risk genes HLA DRB1 and the vitamin D repressor NCOR2. Methylation of the vitamin D responsive MS risk gene ZMIZ1 was associated with risk SNP and disease. We conclude that DNA methylation settings in adult haematopoietic stem cells may contribute to individual variation in vitamin D responses in immune cells.

Amyloid Cardiomyopathy.

Amyloid cardiomyopathy is emerging as an important and under-recognised cause of heart failure and cardiac arrhythmias, especially in older adults. This disorder is characterised by extracellular deposition of amyloid fibrils that form due to misfolding of secreted light chains (AL) or transthyretin protein (ATTR). In ATTR, amyloid aggregates typically result from excessive accumulation of wild-type transthyretin (ATTRwt) or from protein structural defects caused by TTR gene variants (ATTRv). Amyloid fibril deposition may predominantly affect the heart or show multi-system involvement. Previously considered to be rare and inexorably progressive with no specific therapy, there has been enormous recent interest in ATTR cardiomyopathy due to upwardly-revised estimates of disease prevalence together with development of disease-modifying interventions. Because of this, there is a clinical imperative to have a high index of suspicion to identify potential cases and to be aware of contemporary diagnostic methods and treatment options. Genetic testing should be offered to all patients with proven ATTR to access the benefits of new therapies specific to ATTRv and allow predictive testing of family members. With heightened awareness of amyloid cardiomyopathy and expanded use of genetic testing, a substantial rise in the numbers of asymptomatic individuals who are carriers of pathogenic variants is expected, and optimal strategies for monitoring and treatment of these individuals at risk need to be determined. Pre-emptive administration of fibril-modifying therapies provides an unprecedented opportunity for disease prevention and promises to change amyloid cardiomyopathy from being a fatal to a treatable disorder.

The interaction of Multiple Sclerosis risk loci with Epstein-Barr virus phenotypes implicates the virus in pathogenesis.

Translating the findings of genome wide association studies (GWAS) to new therapies requires identification of the relevant immunological contexts to interrogate for genetic effects. In one of the largest GWAS, more than 200 risk loci have been identified for Multiple Sclerosis (MS) susceptibility. Infection with Epstein-Barr virus (EBV) appears to be necessary for the development of Multiple Sclerosis (MS). Many MS risk loci are associated with altered gene expression in EBV infected B cells (LCLs). We have interrogated this immunological context to identify interaction between MS risk loci and EBV DNA copy number, intrinsic growth rate and EBV encoded miRNA expression. The EBV DNA copy number was associated with significantly more risk alleles for MS than for other diseases or traits. EBV miRNAs BART4-3p and BART3-5p were highly associated with EBV DNA copy number and MS risk loci. The poliovirus receptor (PVR) risk SNP was associated with EBV DNA copy number, PVR and miRNA expression. Targeting EBV miRNAs BART4-3p and BART3-5p, and the gene PVR, may provide therapeutic benefit in MS. This study also indicates how immunological context and risk loci interactions can be exploited to validate and develop novel therapeutic approaches.

Transcribed B lymphocyte genes and multiple sclerosis risk genes are underrepresented in Epstein-Barr Virus hypomethylated regions.

Epstein-Barr Virus (EBV) infection appears to be necessary for the development of Multiple Sclerosis (MS), although the specific mechanisms are unknown. More than 200 single-nucleotide polymorphisms (SNPs) are known to be associated with the risk of developing MS. About a quarter of these are also highly associated with proximal gene expression in B cells infected with EBV (lymphoblastoid cell lines-LCLs). The DNA of LCLs is hypomethylated compared with both uninfected and activated B cells. Since methylation can affect gene expression, and so cell differentiation and immune evasion, we hypothesised that EBV-driven hypomethylation may affect the interaction between EBV infection and MS. We interrogated an existing dataset comprising three individuals with whole-genome bisulfite sequencing data from EBV transformed B cells and CD40L-activated B cells. DNA methylation surrounding MS risk SNPs associated with gene expression in LCLs (LCLeQTL) was less likely to be hypomethylated than randomly selected chromosomal regions. Differential methylation was independent of genomic features such as promoter regions, but genes preferentially expressed in EBV-infected B cells, including the LCLeQTL genes, were underrepresented in the hypomethylated regions. Our data does not indicate MS genetic risk is affected by EBV hypomethylation.

Evidence from genome wide association studies implicates reduced control of Epstein-Barr virus infection in multiple sclerosis susceptibility.

BACKGROUND: Genome wide association studies have identified > 200 susceptibility loci accounting for much of the heritability of multiple sclerosis (MS). Epstein-Barr virus (EBV), a memory B cell tropic virus, has been identified as necessary but not sufficient for development of MS. The molecular and immunological basis for this has not been established. Infected B cell proliferation is driven by signalling through the EBV produced cell surface protein LMP1, a homologue of the MS risk gene CD40. METHODS: We have investigated transcriptomes of B cells and EBV-infected B cells at Latency III (LCLs) and identified MS risk genes with altered expression on infection and with expression levels associated with the MS risk genotype (LCLeQTLs). The association of LCLeQTL genomic burden with EBV phenotypes in vitro and in vivo was examined. The risk genotype effect on LCL proliferation with CD40 stimulation was assessed. RESULTS: These LCLeQTL MS risk SNP:gene pairs (47 identified) were over-represented in genes dysregulated between B and LCLs (p < 1.53 × 10-4), and as target loci of the EBV transcription factor EBNA2 (p < 3.17 × 10-16). Overall genetic burden of LCLeQTLs was associated with some EBV phenotypes but not others. Stimulation of the CD40 pathway by CD40L reduced LCL proliferation (p < 0.001), dependent on CD40 and TRAF3 MS risk genotypes. Both CD40 and TRAF3 risk SNPs are in binding sites for the EBV transcription factor EBNA2, with expression of each correlated with EBNA2 expression dependent on genotype. CONCLUSIONS: These data indicate targeting EBV may be of therapeutic benefit in MS.

Novel approaches to detect serum biomarkers for clinical response to interferon-beta treatment in multiple sclerosis.

Interferon beta (IFNbeta) is the most common immunomodulatory treatment for relapsing-remitting multiple sclerosis (RRMS). However, some patients fail to respond to treatment. In this study, we identified putative clinical response markers in the serum and plasma of people with multiple sclerosis (MS) treated with IFNbeta. In a discovery-driven approach, we use 2D-difference gel electrophoresis (DIGE) to identify putative clinical response markers and apply power calculations to identify the sample size required to further validate those markers. In the process we have optimized a DIGE protocol for plasma to obtain cost effective and high resolution gels for effective spot comparison. APOA1, A2M, and FIBB were identified as putative clinical response markers. Power calculations showed that the current DIGE experiment requires a minimum of 10 samples from each group to be confident of 1.5 fold difference at the p<0.05 significance level. In a complementary targeted approach, Cytometric Beadarray (CBA) analysis showed no significant difference in the serum concentration of IL-6, IL-8, MIG, Eotaxin, IP-10, MCP-1, and MIP-1alpha, between clinical responders and non-responders, despite the association of these proteins with IFNbeta treatment in MS.

The multiple sclerosis whole blood mRNA transcriptome and genetic associations indicate dysregulation of specific T cell pathways in pathogenesis.

Multiple sclerosis (MS) is an autoimmune disease with a genetic component, caused at least in part by aberrant lymphocyte activity. The whole blood mRNA transcriptome was measured for 99 untreated MS patients: 43 primary progressive MS, 20 secondary progressive MS, 36 relapsing remitting MS and 45 age-matched healthy controls. The ANZgene Multiple Sclerosis Genetics Consortium genotyped more than 300 000 SNPs for 115 of these samples. Transcription from genes on translational regulation, oxidative phosphorylation, immune synapse and antigen presentation pathways was markedly increased in all forms of MS. Expression of genes tagging T cells was also upregulated (P < 10(-12)) in MS. A T cell gene signature predicts disease state with a concordance index of 0.79 with age and gender as co-variables, but the signature is not associated with clinical course or disability. The ANZgene genome wide association screen identified two novel regions with genome wide significance: one encoding the T cell co-stimulatory molecule, CD40; the other a region on chromosome 12q13-14. The CD40 haplotype associated with increased MS susceptibility has decreased gene expression in MS (P < 0.0007). The second MS susceptibility region includes 17 genes on 12q13-14 in tight linkage disequilibrium. Of these, only 13 are expressed in leukocytes, and of these the expression of one, FAM119B, is much lower in the susceptibility haplotype (P < 10(-14)). Overall, these data indicate dysregulation of T cells can be detected in the whole blood of untreated MS patients, and supports targeting of activated T cells in therapy for all forms of MS.

Extreme lymphoproliferative disease and fatal autoimmune thrombocytopenia in FasL and TRAIL double-deficient mice.

To delineate the relative roles of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas ligand in lymphocyte biology and lymphoproliferative disease, we generated mice defective in both molecules. B6.GT mice develop severe polyclonal lymphoproliferative disease because of accumulating CD3(+)CD4(-)CD8(-)B220(+) T cells, CD4(+) and CD8(+) T cells, and follicular B cells, and mice die prematurely from extreme lymphocytosis, thrombocytopenia, and hemorrhage. Accumulating lymphocytes resembled antigen-experienced lymphocytes, consistent with the maximal resistance of B6.GT CD4(+) and CD8(+) T cell to activation-induced cell death. More specifically, we show that TRAIL contributes to Fas ligand-mediated activation-induced cell death and controls lymphocyte apoptosis in the presence of interferon-gamma once antigen stimulation is removed. Furthermore, dysregulated lymphocyte homeostasis results in the production of anti-DNA and rheumatoid factor autoantibodies, as well as antiplatelet IgM and IgG causing thrombocytopenia. Thus, B6.GT mice reveal new roles for TRAIL in lymphocyte homeostasis and autoimmune lymphoproliferative syndromes and are a model of spontaneous idiopathic thrombocytopenia purpura secondary to lymphoproliferative disease.

Tolerance induction in a large-animal model using total lymphoid irradiation and intrathymic bone marrow.

BACKGROUND: Immunological unresponsiveness of T cells to alloantigen can be induced by intrathymic injection of donor-specific antigen in small-animal models. Intrathymic tolerance to vascularized grafts in large animals has not previously been reported. METHODS: Thirty-two dogs were allocated into dog leukocyte antigen DP locus allele (class II)-matched donor-recipient pairs. Female recipients were paired with male donors. Tissue typing was based on restriction fragment length polymorphism. Recipients were given 18 Gy total lymphoid irradiation in 16 fractions (1.125 Gy each) over 4 weeks. Thoracotomy after the 6th fraction permitted perithymic (n=4) or intrathymic (n=4) injection of donor bone marrow (BM) or intrathymic injection of saline (n=5). Another group received intravenous peripheral BM infusion (n=3). Fifty days postthoracotomy recipients underwent bilateral nephrectomy and donor-specific kidney transplantation. Acute rejection, suspected when serum creatinine was more than 600 mumol/L or urea was more than 40 mmol/L, was confirmed histologically. Full-thickness skin grafts followed more than 100 days posttransplantation. Tissue samples were taken for Y-chromosome polymerase chain reaction. RESULTS: One intrathymic (25%) and three perithymic (75%) BM recipients developed tolerance to renal allografts. Three intrathymic BM recipients rejected after 27, 32, and 54 days and one perithymic BM recipient rejected after 42 days. All recipients given peripheral BM or saline had rejected by 29 and 38 days, respectively. All recipients surviving more than 100 days posttransplantation, accepted donor specific and rejected dog leukocyte antigen-DP locus allele (class II) identical third-party skin grafts. Polymerase chain reaction detected intrathymic but not hematopoietic chimerism in sex-mismatched pairs. CONCLUSIONS: Fractionated total lymphoid irradiation and perithymic or intrathymic donor-specific BM induced tolerance to renal and skin allografts without inducing hematopoietic chimerism.

BAFF is a biological response marker to IFN-beta treatment in multiple sclerosis.

Multiple sclerosis (MS) is a complex autoimmune disease characterized by the destruction of the myelin sheath of neurons. Interferon beta (IFN-beta) is currently the major drug used to treat MS. Some patients fail to respond to this treatment, in some cases due to the development of neutralizing antibodies (NAb) to IFN-beta. We used microarray analysis and RT-PCR to measure gene expression in whole blood, 9-15 h postinjection, in patients with and without NAbs to IFN-beta. The canonical marker of biological response to IFN-beta, myxovirus resistance protein A, was upregulated in all NAb- patients while remaining unchanged in NAb+ patients. Genes functioning in immune response pathways were dominant in the set of differentially expressed genes: 73 immune response genes were identified as upregulated and 29 genes were identified as downregulated. B-cell activating factor (BAFF) is a strong candidate marker for biological and clinical response as well as for predisposition to NAb development. We demonstrate that it is responsive to IFN-beta in vitro and in vivo, and that its soluble form is elevated in serum from NAb- but not NAb+ patients. We conclude BAFF is a good biomarker for IFN-beta response, and requires further studies to determine its value as a marker for clinical response and NAb predisposition.

Association of common T cell activation gene polymorphisms with multiple sclerosis in Australian patients.

Susceptibility to multiple sclerosis (MS) may be influenced by the interaction of several genes within a biological pathway. T cell activation and costimulation may be potentially important in MS pathogenesis. We have therefore investigated associations between MS and polymorphisms in the CD152 (CTLA-4), CD28, CD80 and CD86 genes in Australian patients. We found no significant MS association with CTLA-4 exon 1 +49 alleles, and meta-analysis showed no significant association across nine comparable datasets (OR=1.04, p=0.54), nor with primary progressive MS across seven datasets (OR=1.19, p=0.21). Haplotype analysis showed a trend towards a decrease of the CTLA-4-1722C, -1577G, +49G haplotype in +49 G positive MS patients compared with controls (p=0.06). Screening of CD28, CD80 and CD86 genes identified novel polymorphisms in the putative promoter regions of CD28 (-372 G/A) and CD86 (exon 2 -359 deletionAAG). There was a significant increase of the CD28 -372 G allele frequency in MS patients vs. controls (p=0.045) and a trend towards a significant interaction between this allele and the CTLA-4 +49 G allele (OR=4.00, p=0.058). Our results suggest that the CTLA-4 +49 alone is not associated with overall susceptibility to MS, but may be important in clinical subsets of patients and/or may interact epistatically with other gene polymorphisms.