36 research outputs found
Co-Delivery of Immunomodulators and Autoantigen as Antigen-Specific Immunotherapy for the Treatment of a Murine Model of Multiple Sclerosis
Multiple Sclerosis (MS) is an autoimmune disease characterized by the breakdown of immune tolerance towards autoantigen in the myelin sheath surrounding the neurons. Current therapies for MS and other autoimmune diseases focus on treating the symptoms and not the cause of the disease. A major setback in improving current therapeutics for autoimmunity is the lack of antigen specificity. Successful antigen-specific immunotherapy (ASIT) would allow for improved treatment of autoimmune diseases. This thesis investigates the creation of ASIT for autoimmunity through the co-delivery of an immunomodulator and autoantigen. First, immunomodulatory peptides targeting the B7 costimulatory pathway and cell adhesion were tested for their ability to suppress a murine model of MS, experimental autoimmune encephalomyelitis (EAE), utilizing a co-delivery vehicle developed in our lab, soluble antigen arrays. Peptides targeting different surface markers were all found to suppress EAE when co-delivered with autoantigen, demonstrating the ability of different immunomodulators to create effective ASIT. Expanding upon the idea of combinational ASIT, eleven different small molecules immunomodulators were screened for properties indicative of autoimmune suppression in an antigen-specific splenocyte system. This screen revealed that several compounds, most notably dexamethasone, had the ability to skew the antigen-specific immune response towards autoimmune suppression. The ability of dexamethasone to act as an effective immunomodulator in ASIT for autoimmunity was confirmed in vivo in the treatment of EAE. Co-delivery of dexamethasone and autoantigen in an oil-in-water emulsion, incomplete Freund’s adjuvant, was found to suppress EAE and shift the immune response. Overall, the results presented dissertation provide evidence for the successful creation of ASIT for autoimmunity by combining immunomodulator and autoantigen
Targeted Genomic Sequencing of TSC1 and TSC2 Reveals Causal Variants in Individuals for Whom Previous Genetic Testing for Tuberous Sclerosis Complex Was Normal
Tuberous sclerosis complex (TSC) is caused by inactivating variants in TSC1 and TSC2. Somatic mosaicism, as well as the size and complexity of the TSC1 and TSC2 loci, makes variant identification challenging. Indeed, in some individuals with a clinical diagnosis of TSC, diagnostic testing fails to identify an inactivating variant. To improve TSC1 and TSC2 variant detection, we screened the TSC1 and TSC2 genomic regions using targeted HaloPlex custom capture and next-generation sequencing (NGS) in genomic DNA isolated from peripheral blood of individuals with definite, possible or suspected TSC in whom no disease-associated variant had been identified by previous diagnostic genetic testing. We obtained >95% target region coverage at a read depth of 20 and >50% coverage at a read depth of 300 and identified inactivating TSC1 or TSC2 variants in 83/155 individuals (54%); 65/113 (58%) with clinically definite TSC and 18/42 (43%) with possible or suspected TSC. These included 19 individuals with deep intronic variants and 54 likely cases of mosaicism (variant allele frequency 1-28%; median 7%). In 13 cases (8%), we identified a variant of uncertain significance (VUS). Targeted genomic NGS of TSC1 and TSC2 increases the yield of inactivating variants found in individuals with suspected TSC.</p
Single-Step Grafting of Aminooxy-Peptides to Hyaluronan: A Simple Approach to Multifunctional Therapeutics for Experimental Autoimmune Encephalomyelitis
The immune response to antigens is directed in part by the presence or absence of costimulatory signals. The ability to coincidently present both antigen and, for example, a peptide that inhibits or activates the costimulatory pathway, would be a valuable tool for tolerization or immunization, respectively. A simple reaction scheme utilizing oxime chemistry was identified as a means to efficiently conjugate different peptide species to hyaluronan. Peptides synthesized with an aminooxy N-terminus reacted directly to hyaluronan under slightly acidic aqueous conditions without the need for a catalyst. The resulting oxime bond was found to rapidly hydrolyze at pH 2 releasing peptide, but was stable at higher pH values (5.5 and 7). Two different peptide species, a multiple sclerosis antigen (PLP) and an ICAM-1 ligand (LABL) known to block immune cell stimulation, were functionalized with the aminooxy end group. These peptides showed similar reactivity to hyaluronan and were conjugated in an equimolar ratio. The resulting hyaluronan with grafted PLP and LABL significantly inhibited disease in mice with experimental autoimmune encephalomyelitis, a model of multiple sclerosis. Aminooxy-peptides facilitate simple synthesis of multifunctional hyaluronan graft polymers, thus enabling novel approaches to antigen-specific immune modulation
Biografia, mobilidade e cultura atlântica: a micro-escala do tráfico de escravos em Benguela, séculos XVIII-XIX
Determination of Genes Involved in Hygienic Behavior in the Honey Bee, Apis mellifera
Honeybees are an integral part of the agricultural industry, especially in the Midwestern United States. Honeybees serve as pollinators of crops, an invaluable job of honeybees, as well as producers of honey. Diseases that kill members of the colony threaten honeybee colonies, and there are few options available to treat these diseases. Some honeybees exhibit a behavior—termed hygienic behavior—in which the bees clean out diseased brood from the hive, thus conferring a natural resistance to three diseases of bees: American foulbrood, chalkbrood, and varroa mite. However, it is not found in all colonies and only a certain number of honeybees contain this behavior in a given colony. This desirable behavior can be traced to genetics—meaning hygienic honeybees exhibit this behavior partly due to information they carry in their DNA (genetic material). We have undertaken a large genetic screen in cooperation with Dr. Marla Spivak of the Entomology department at the University of Minnesota. We have identified differences between genes in brains from hygienic honeybees vs. non-hygienic honeybees using a technique called microarray, and are now validating these differences using a secondary biochemical technique. Identifying specific genes that allow these bees to protect their colonies is a critical step towards implementing this behavior in unprotected honeybee colonies
Co-delivery of autoantigen and B7 pathway modulators suppresses experimental autoimmune encephalomyelitis
Autoimmune diseases such as multiple sclerosis (MS) are characterized by the breakdown of immune tolerance to autoantigens. Targeting surface receptors on immune cells offers a unique strategy for reprogramming immune responses in autoimmune diseases. The B7 signaling pathway was targeted using adaptations of soluble antigen array (SAgA) technology achieved by covalently linking B7-binding peptides and disease causing autoantigen (proteolipid peptide (PLP)) to hyaluronic acid (HA). We hypothesized that co-delivery of a B7-binding peptide and autoantigen would suppress experimental autoimmune encephalomyelitis (EAE), a murine model of MS. Three independent B7-targeted SAgAs were created containing peptides to either inhibit or potentially stimulate the B7 signaling pathway. Surprisingly, all SAgAs were found to suppress EAE disease symptoms. Altered cytokine expression was observed in primary splenocytes isolated from SAgA-treated mice, indicating that SAgAs with different B7-binding peptides may suppress EAE through different immunological mechanisms. This antigen-specific immunotherapy using SAgAs can successfully suppress EAE through co-delivery of autoantigen and peptides targeting with the B7 signaling pathway
Arginase deficiency masked by cerebral palsy and coagulopathy—Three varied presentations of Latin American origin
Abstract Arginase deficiency (ARG1‐D) is an autosomal recessive inborn error of metabolism that is often misdiagnosed. Classic presentation of ARG1‐D includes progressive symptoms of spasticity, delayed development, cognitive impairment, protein avoidance, and seizures. Patients who present atypically may evade diagnosis and require a thoughtful diagnostic workup. Here, we discuss three females of Latin American origin with differing clinical presentations, but who all have the same intronic pathogenic variant in ARG1. Importantly, we found that each case included elevated coagulopathy on laboratory testing and discussed one case in particular with manifestation of bleeding. When diagnosed early, treatment is favorable and can prevent progressive decline. While many states have added ARG1‐D to their expanded newborn screening panels, still many states and countries do not screen for ARG1‐D, and it can be missed in a healthy newborn. We aim to bring awareness to not only the classic presentation as a necessary consideration for otherwise unexplained spastic diplegia but also to the varied presentations of ARG1‐D
Mild TSC phenotype and non-penetrance associated with a frameshift variant in TSC2 prompts caution in evaluating pathogenicity of frameshift variants
Introduction: Technological advances in genetic testing, particularly the adoption of noninvasive prenatal screening (NIPS) for single gene disorders such as tuberous sclerosis complex (TSC, OMIM# 613254), mean that putative/possible pathogenetic DNA variants can be identified prior to the appearance of a disease phenotype. Without a phenotype, accurate prediction of variant pathogenicity is crucial. Here, we report a TSC2 frameshift variant, NM_000548.5(TSC2):c.4255_4256delCA, predicted to result in nonsense-mediated mRNA decay (NMD) and cessation of TSC2 protein production and thus pathogenic according to ACMG criteria, identified by NIPS and subsequently detected in family members with few or no symptoms of TSC. Due to the lack of TSC-associated features in the family, we hypothesized that the deletion created a non-canonical 5′ donor site resulting in cryptic splicing and a transcript encoding active TSC2 protein. Verifying the predicted effect of the variant was key to designating pathogenicity in this case and should be considered for other frameshift variants in other genetic disorders. Methods: Phenotypic information on the family members was collected via review of the medical records and patient reports. RNA studies were performed using proband mRNA isolated from blood lymphocytes for RT-PCR and Sanger sequencing. Functional studies were performed by transient expression of the TSC2 variant proteins in cultured cells, followed by immunoblotting. Results:No family members harboring the variant met any major clinical diagnostic criteria for TSC, though a few minor features non-specific to TSC were present. RNA studies supported the hypothesis that the variant caused cryptic splicing, resulting in an mRNA transcript with an in-frame deletion of 93 base pairs r.[4255_4256del, 4251_4343del], p.[(Gln1419Valfs*104), (Gln1419_Ser1449del)]. Expression studies demonstrated that the canonical function of the resulting truncated TSC2 p.Gln1419_Ser1449del protein product was maintained and similar to wildtype.Conclusion: Although most frameshift variants are likely to result in NMD, the NM_000548.5(TSC2):c.4255_4256delCA variant creates a cryptic 5′ splice donor site, resulting in an in-frame deletion that retains TSC2 function, explaining why carriers of the variant do not have typical features of TSC. The information is important for this family and others with the same variant. Equally important is the lesson that predictions can be inaccurate, and that caution should be used when designating frameshift variants as pathogenic, especially when phenotypic information to corroborate testing results is unavailable. Our work demonstrates that functional RNA- and protein-based confirmation of the effects of DNA variants improves molecular genetic diagnostics.</p
Codelivery of antigen and an immune cell adhesion inhibitor is necessary for efficacy of soluble antigen arrays in experimental autoimmune encephalomyelitis
Autoimmune diseases such as multiple sclerosis (MS) are typified by the misrecognition of self-antigen and the clonal expansion of autoreactive T cells. Antigen-specific immunotherapies (antigen-SITs) have long been explored as a means to desensitize patients to offending self-antigen(s) with the potential to retolerize the immune response. Soluble antigen arrays (SAgAs) are composed of hyaluronic acid (HA) cografted with disease-specific autoantigen (proteolipid protein peptide) and an ICAM-1 inhibitor peptide (LABL). SAgAs were designed as an antigen-SIT that codeliver peptides to suppress experimental autoimmune encephalomyelitis (EAE), a murine model of MS. Codelivery of antigen and cell adhesion inhibitor (LABL) conjugated to HA was essential for SAgA treatment of EAE. Individual SAgA components or mixtures thereof reduced proinflammatory cytokines in cultured splenocytes from EAE mice; however, these treatments showed minimal to no in vivo therapeutic effect in EAE mice. Thus, carriers that codeliver antigen and a secondary “context” signal (e.g., LABL) in vivo may be an important design criteria to consider when designing antigen-SIT for autoimmune therapy