Expression of Cholera Toxin B–Proinsulin Fusion Protein in Lettuce and Tobacco Chloroplasts – Oral Administration Protects Against Development of Insulitis in Non-Obese Diabetic Mice

Lettuce and tobacco chloroplast transgenic lines expressing the cholera toxin B subunit–human proinsulin (CTB-Pins) fusion protein were generated. CTB-Pins accumulated up to ~16% of total soluble protein (TSP) in tobacco and up to ~2.5% of TSP in lettuce. Eight milligrams of powdered tobacco leaf material expressing CTB-Pins or, as negative controls, CTB–green fluorescent protein (CTB-GFP) or interferon–GFP (IFN-GFP), or untransformed leaf, were administered orally, each week for 7 weeks, to 5-week-old female non-obese diabetic (NOD) mice. The pancreas of CTB-Pins-treated mice showed decreased infiltration of cells characteristic of lymphocytes (insulitis); insulin-producing β-cells in the pancreatic islets of CTB-Pins-treated mice were significantly preserved, with lower blood or urine glucose levels, by contrast with the few β-cells remaining in the pancreatic islets of the negative controls. Increased expression of immunosuppressive cytokines, such as interleukin-4 and interleukin-10 (IL-4 and IL-10), was observed in the pancreas of CTB-Pins-treated NOD mice. Serum levels of immunoglobulin G1 (IgG1), but not IgG2a, were elevated in CTB-Pins-treated mice. Taken together, T-helper 2 (Th2) lymphocyte-mediated oral tolerance is a likely mechanism for the prevention of pancreatic insulitis and the preservation of insulin-producing β-cells. This is the first report of expression of a therapeutic protein in transgenic chloroplasts of an edible crop. Transplastomic lettuce plants expressing CTB-Pins grew normally and transgenes were maternally inherited in T1 progeny. This opens up the possibility for the low-cost production and delivery of human therapeutic proteins, and a strategy for the treatment of various other autoimmune diseases

Modeling the cumulative genetic risk for multiple sclerosis from genome-wide association data

BACKGROUND: Multiple sclerosis (MS) is the most common cause of chronic neurologic disability beginning in early to middle adult life. Results from recent genome-wide association studies (GWAS) have substantially lengthened the list of disease loci and provide convincing evidence supporting a multifactorial and polygenic model of inheritance. Nevertheless, the knowledge of MS genetics remains incomplete, with many risk alleles still to be revealed. METHODS: We used a discovery GWAS dataset (8,844 samples, 2,124 cases and 6,720 controls) and a multi-step logistic regression protocol to identify novel genetic associations. The emerging genetic profile included 350 independent markers and was used to calculate and estimate the cumulative genetic risk in an independent validation dataset (3,606 samples). Analysis of covariance (ANCOVA) was implemented to compare clinical characteristics of individuals with various degrees of genetic risk. Gene ontology and pathway enrichment analysis was done using the DAVID functional annotation tool, the GO Tree Machine, and the Pathway-Express profiling tool. RESULTS: In the discovery dataset, the median cumulative genetic risk (P-Hat) was 0.903 and 0.007 in the case and control groups, respectively, together with 79.9% classification sensitivity and 95.8% specificity. The identified profile shows a significant enrichment of genes involved in the immune response, cell adhesion, cell communication/signaling, nervous system development, and neuronal signaling, including ionotropic glutamate receptors, which have been implicated in the pathological mechanism driving neurodegeneration. In the validation dataset, the median cumulative genetic risk was 0.59 and 0.32 in the case and control groups, respectively, with classification sensitivity 62.3% and specificity 75.9%. No differences in disease progression or T2-lesion volumes were observed among four levels of predicted genetic risk groups (high, medium, low, misclassified). On the other hand, a significant difference (F = 2.75, P = 0.04) was detected for age of disease onset between the affected misclassified as controls (mean = 36 years) and the other three groups (high, 33.5 years; medium, 33.4 years; low, 33.1 years). CONCLUSIONS: The results are consistent with the polygenic model of inheritance. The cumulative genetic risk established using currently available genome-wide association data provides important insights into disease heterogeneity and completeness of current knowledge in MS genetics

Author Correction: A systems biology approach uncovers cell-specific gene regulatory effects of genetic associations in multiple sclerosis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

A systems biology approach uncovers cell-specific gene regulatory effects of genetic associations in multiple sclerosis.

Genome-wide association studies (GWAS) have identified more than 50,000 unique associations with common human traits. While this represents a substantial step forward, establishing the biology underlying these associations has proven extremely difficult. Even determining which cell types and which particular gene(s) are relevant continues to be a challenge. Here, we conduct a cell-specific pathway analysis of the latest GWAS in multiple sclerosis (MS), which had analyzed a total of 47,351 cases and 68,284 healthy controls and found more than 200 non-MHC genome-wide associations. Our analysis identifies pan immune cell as well as cell-specific susceptibility genes in T cells, B cells and monocytes. Finally, genotype-level data from 2,370 patients and 412 controls is used to compute intra-individual and cell-specific susceptibility pathways that offer a biological interpretation of the individual genetic risk to MS. This approach could be adopted in any other complex trait for which genome-wide data is available

A systems biology approach uncovers cell-specific gene regulatory effects of genetic associations in multiple sclerosis

Genome-wide association studies (GWAS) have identified more than 50,000 unique associations with common human traits. While this represents a substantial step forward, establishing the biology underlying these associations has proven extremely difficult. Even determining which cell types and which particular gene(s) are relevant continues to be a challenge. Here, we conduct a cell-specific pathway analysis of the latest GWAS in multiple sclerosis (MS), which had analyzed a total of 47,351 cases and 68,284 healthy controls and found more than 200 non-MHC genome-wide associations. Our analysis identifies pan immune cell as well as cell-specific susceptibility genes in T cells, B cells and monocytes. Finally, genotype-level data from 2,370 patients and 412 controls is used to compute intra-individual and cell-specific susceptibility pathways that offer a biological interpretation of the individual genetic risk to MS. This approach could be adopted in any other complex trait for which genome-wide data is available

Minimum Information about T Regulatory Cells: A Step toward Reproducibility and Standardization

Nephrolog

TLR-mediated STAT3 and ERK activation controls IL-10 secretion by human B cells

Pathophysiology and treatment of rheumatic disease

Expansion of autoreactive T cells in multiple sclerosis is independent of exogenous B7 costimulation

Multiple sclerosis (MS) is an inflammatory disease of the myelinated central nervous system that is postulated to be induced by myelin-reactive CD4 T cells. T cell activation requires an antigen-specific signal through the TCR and a costimulatory signal, which can be mediated by B7-1 or B7-2 engagement of CD28. To directly examine the activation state of myelin-reactive T cells in MS, the costimulation requirements necessary to activate myelin basic protein (MBP) or tetanus toxoid (TT)-reactive CD4 T cells were compared between normal controls and MS patients. Peripheral blood T cells were stimulated with Chinese hamster ovary (CHO) cells transfected either with DRB1*1501/DRA0101 chains (t-DR2) alone, or in combination with, B7-1 or B7-2. In the absence of costimulation, T cells from normal subjects stimulated with the recall antigen TT p830-843 were induced to expand and proliferate, but stimulation with MBP p85-99 did not have this effect. In marked contrast, T cells from patients with MS stimulated with MBP p85-99 in the absence of B7-1 or B7-2 signals expanded and proliferated. Thus, MBP-reactive CD4 T cells in patients with MS are costimulation independent and have been previously activated in vivo. These experiments provide further direct evidence for a role of activated MBP-specific CD4 T cells in the pathogenesis of MS

Activation of human T cell lymphotropic virus type I-infected T cells is independent of B7 costimulation

Two distinct signals are required to activate T cells: an Ag-specific signal and a costimulatory signal mediated primarily by B7-1 (CD80) and B7-2 (CD86) through interactions with CD28. Costimulation appears to be critical in regulating autoreactive T cell responses. Here, we demonstrate that, in contrast to the parental uninfected T cell clone, a T cell clone infected by human T cell lymphotropic virus type I (HTLV-I) displays a remarkably enhanced response to Ag in the absence of B7 costimulation. Chinese hamster ovary cells either transfected with DRB1*1501 (t-DR2) alone or cotransfected with DR2 and either B7-1 or B7-2 were fixed, pulsed with myelin basic protein peptide 84-102 (MBPp84-102), and used as APCs. The MBPp84-102-reactive T cell clone Ob1A12.8 required costimulation with either B7-1 or B7-2 molecules, as the response to Ag was reduced by 90% in the absence of B7 costimulation. However, this requirement for B7 costimulation was abrogated after productive infection by HTLV-I. Stimulation of HTLV-I-infected T cells by MBPp84-102/t-DR2 induced the secretion of IL-5 and IFN-gamma, which approached the level induced in the presence of B7 costimulation, whereas IL-4 was induced to one third of its maximal level. Consistently, the secretion of IL-5 and IFN-gamma was not significantly inhibited by anti-B7-1 and B7-2 Abs, whereas IL-4 was inhibited by approximately 50%. In contrast, uninfected T cells required either B7-1 or B7-2 costimulation for significant cytokine secretion, and this response was inhibited by anti-B7-1 and B7-2 Abs. These findings suggest that HTLV-I-infected autoreactive T cells have the potential to induce an autoimmune response in the absence of B7 expression in the target organ. This may be of particular interest in the elucidation of HTLV-I pathogenicity given the association of HTLV-I infection with autoimmune-like diseases