Influenza vaccine format mediates distinct cellular and antibody responses in human immune organoids

Highly effective vaccines elicit specific, robust, and durable adaptive immune responses. To advance informed vaccine design, it is critical that we understand the cellular dynamics underlying responses to different antigen formats. Here, we sought to understand how antigen-specific B and T cells were activated and participated in adaptive immune responses within the mucosal site. Using a human tonsil organoid model, we tracked the differentiation and kinetics of the adaptive immune response to influenza vaccine and virus modalities. Each antigen format elicited distinct B and T cell responses, including differences in their magnitude, diversity, phenotype, function, and breadth. These differences culminated in substantial changes in the corresponding antibody response. A major source of antigen format-related variability was the ability to recruit naive vs. memory B and T cells to the response. These findings have important implications for vaccine design and the generation of protective immune responses in the upper respiratory tract

QuantiMus: A Machine Learning-Based Approach for High Precision Analysis of Skeletal Muscle Morphology.

Skeletal muscle injury provokes a regenerative response, characterized by the de novo generation of myofibers that are distinguished by central nucleation and re-expression of developmentally restricted genes. In addition to these characteristics, myofiber cross-sectional area (CSA) is widely used to evaluate muscle hypertrophic and regenerative responses. Here, we introduce QuantiMus, a free software program that uses machine learning algorithms to quantify muscle morphology and molecular features with high precision and quick processing-time. The ability of QuantiMus to define and measure myofibers was compared to manual measurement or other automated software programs. QuantiMus rapidly and accurately defined total myofibers and measured CSA with comparable performance but quantified the CSA of centrally-nucleated fibers (CNFs) with greater precision compared to other software. It additionally quantified the fluorescence intensity of individual myofibers of human and mouse muscle, which was used to assess the distribution of myofiber type, based on the myosin heavy chain isoform that was expressed. Furthermore, analysis of entire quadriceps cross-sections of healthy and mdx mice showed that dystrophic muscle had an increased frequency of Evans blue dye+ injured myofibers. QuantiMus also revealed that the proportion of centrally nucleated, regenerating myofibers that express embryonic myosin heavy chain (eMyHC) or neural cell adhesion molecule (NCAM) were increased in dystrophic mice. Our findings reveal that QuantiMus has several advantages over existing software. The unique self-learning capacity of the machine learning algorithms provides superior accuracy and the ability to rapidly interrogate the complete muscle section. These qualities increase rigor and reproducibility by avoiding methods that rely on the sampling of representative areas of a section. This is of particular importance for the analysis of dystrophic muscle given the "patchy" distribution of muscle pathology. QuantiMus is an open source tool, allowing customization to meet investigator-specific needs and provides novel analytical approaches for quantifying muscle morphology

QuantiMus: A Machine Learning-Based Approach for High Precision Analysis of Skeletal Muscle Morphology

Skeletal muscle injury provokes a regenerative response, characterized by the de novo generation of myofibers that are distinguished by central nucleation and re-expression of developmentally restricted genes. In addition to these characteristics, myofiber crosssectional area (CSA) is widely used to evaluate muscle hypertrophic and regenerative responses. Here, we introduce QuantiMus, a free software program that uses machine learning algorithms to quantify muscle morphology and molecular features with high precision and quick processing-time. The ability of QuantiMus to define and measure myofibers was compared to manual measurement or other automated software programs. QuantiMus rapidly and accurately defined total myofibers and measured CSA with comparable performance but quantified the CSA of centrally-nucleated fibers (CNFs) with greater precision compared to other software. It additionally quantified the fluorescence intensity of individual myofibers of human and mouse muscle, which was used to assess the distribution of myofiber type, based on the myosin heavy chain isoform that was expressed. Furthermore, analysis of entire quadriceps cross-sections of healthy and mdx mice showed that dystrophic muscle had an increased frequency of Evans blue dye+ injured myofibers. QuantiMus also revealed that the proportion of centrally nucleated, regenerating myofibers that express embryonic myosin heavy chain (eMyHC) or neural cell adhesion molecule (NCAM) were increased in dystrophic mice. Our findings reveal that QuantiMus has several advantages over existing software. The unique self-learning capacity of the machine learning algorithms provides superior accuracy and the ability to rapidly interrogate the complete muscle section. These qualities increase rigor and reproducibility by avoiding methods that rely on the sampling of representative areas of a section. This is of particular importance for the analysis of dystrophic muscle given the “patchy” distribution of muscle pathology. QuantiMus is an open source tool, allowing customization to meet investigatorspecific needs and provides novel analytical approaches for quantifying muscle morphology

An evaluation of indirubin analogues as phosphorylase kinase inhibitors

Phosphorylase kinase (PhK) has been linked with a number of conditions such as glycogen storage diseases, psoriasis, type 2 diabetes and more recently, cancer (Camus S. et al., Oncogene 2012, 31, 4333). However, with few reported structural studies on PhK inhibitors, this hinders a structure based drug design approach. In this study, the inhibitory potential of 38 indirubin analogues have been investigated. 11 of these ligands had IC50 values in the range 0.170 – 0.360 µM, with indirubin-3’-acetoxime (1c) the most potent. 7-bromoindirubin-3’-oxime (13b), an antitumor compound which induces caspase-independent cell-death (Ribas J. et al., Oncogene, 2006, 25, 6304) is revealed as a specific inhibitor of PhK (IC50 = 1.8 µM). Binding assay experiments performed using both PhK-holo and PhK-γtrnc confirmed the inhibitory effects to arise from binding at the kinase domain (γ subunit). High level computations using QM/MM-PBSA binding free energy calculations were in good agreement with experimental binding data, as determined using statistical analysis, and support binding at the ATP-binding site. The value of a QM description for the binding of halogenated ligands exhibiting -hole effects is highlighted. A new statistical metric, the ‘sum of the modified logarithm of ranks’ (SMLR), has been defined which measures performance of a model for both the “early recognition” (ranking earlier/higher) of active compounds and their relative ordering by potency. Through a detailed structure activity relationship analysis considering other kinases (CDK2, CDK5 and GSK-3α/β), 6’(Z) and 7(L) indirubin substitutions have been identified to achieve selective PhK inhibition. The key PhK binding site residues involved can also be targeted using other ligand scaffolds in future work

A Protein Aggregation Based Test for Screening of the Agents Affecting Thermostability of Proteins

To search for agents affecting thermal stability of proteins, a test based on the registration of protein aggregation in the regime of heating with a constant rate was used. The initial parts of the dependences of the light scattering intensity (I) on temperature (T) were analyzed using the following empiric equation: I = Kagg(T−T0)2, where Kagg is the parameter characterizing the initial rate of aggregation and T0 is a temperature at which the initial increase in the light scattering intensity is registered. The aggregation data are interpreted in the frame of the model assuming the formation of the start aggregates at the initial stages of the aggregation process. Parameter T0 corresponds to the moment of the origination of the start aggregates. The applicability of the proposed approach was demonstrated on the examples of thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscles and bovine liver glutamate dehydrogenase studied in the presence of agents of different chemical nature. The elaborated approach to the study of protein aggregation may be used for rapid identification of small molecules that interact with protein targets

Purification of Isoform Specific Actin Capping Protein Antibodies and Immunofluorescent Studies

Actin is a protein that is vital to muscle contraction and cell motility. Actin is synthesized as a monomer and polymerizes into a filament with two very distinct ends; a pointed end and a barbed end. Actin assembly is regulated by a variety of proteins including Actin Capping Protein (CP) that binds the barbed end. CP is composed of an a and a p subunit. In vertebrates, the a subunit has three isoforms: al, a2 and a3 and three beta isoforms: p 1, p2 and p3. The a and p isoforms sequences are very similar in many species, suggesting that the isoforms have specific functions. The al and a2 specific fusion proteins were prepared and expressed. The proteins were purified and used to produce isoform specific antibodies in a rabbit and a chicken. The antibodies were purified from the serum in the blood using affinity chromatography. I will evaluate the specificity of the antibodies will be evaluated using Western Blot analysis. Protein constructs were prepared, expressed, and purified the proteins. The proteins were used to generate polyclonal antibodies in chicken and rabbit. I propose to use the purified isoform specific antibodies to determine the localization of the a isoforms in murine tissues using immunofluorescence. Tissues from heart, kidney, skeletal muscle, spleen, liver, and lung, will be probed with the antibodies, and tagged with a fluorescent marker

Evaluating the role of group 2 innate lymphoid cells in muscular dystrophy

Duchenne muscular dystrophy (DMD) is a lethal, x-linked genetic neuromuscular disorder caused by mutations in the dystrophin gene. The resulting non-functional dystrophin protein renders muscle myofibers susceptible to contraction-induced injury, and normal ambulation leads to muscle degeneration and eventual death. Further contributing to disease is the activation of chronic immune responses caused by asynchronous and continuous bouts of injury. This distributes the homeostasis-promoting balance of immune responses, further causing exacerbated muscle degeneration and impaired regeneration. Importantly, different facets of the immune system, such as type I or type II immunity, are implicated as being injury-promoting or pro-regenerative, respectively. In acute injury settings, type II immune responses such as the activation of M2-like macrophages and eosinophils are critical for efficient muscle regeneration. It is suspected that a similar type II immune response is activated in dystrophic muscle, but its role in promoting regeneration is disrupted by competing pro-inflammatory responses. However, the regulation of type II immunity in dystrophic muscle is largely unknown. Intriguingly, a recently identified subset of immune cells, group 2 innate lymphoid cells (ILC2), have been shown to be potent regulators of type II immunity and promote repair in other diseases. The focus of this dissertation is aimed at understanding the role of ILC2s in regulating skeletal muscle type II immune responses during chronic muscle disorders such as DMD (Chapter 3). We found that mdx muscle ILC2s were increased in number and expressed higher levels of type II cytokines interleukin-5 and interleukin-13 compared to wildtype controls, indicating that muscle degeneration activates ILC2s. We also sought to identify how muscle ILC2s are activated and found that fibro/adipogenic progenitors were the primary source of IL-33, an alarmin known to activate ILC2s. We found that muscle ILC2s are activated by exogenous cytokines, including IL-2/anti-IL-2 complex (IL-2c) and IL-33, which we also used to induce the expansion of ILC2s in vivo. Using additional mouse models in which ILC2s are genetically depleted, we found that ILC2s are potent regulators of skeletal muscle eosinophilia. As ILC2s have also been shown to activate M2-like macrophages in other tissues, we also evaluated this interaction in dystrophic skeletal muscle. To our surprise, ILC2s did not regulate macrophage numbers or phenotype in mdx muscle during this study.In addition to investigating the role of ILC2s in muscle dystrophy, another aim of this work was focused on the development of software that evaluates the morphological features of skeletal muscle (Chapter 2). Muscle function is commonly assessed by evaluating and quantifying histological features of muscle cross-sections. This includes evaluating myofiber size, the expression of markers for injury and regeneration, as well as measuring centrally-located myofiber nuclei. Historically, the time-consuming and tedious nature of manually quantifying muscle samples hindered accurate evaluation, as many analyses were limited to the sampling of parts of the entire cross-section. Furthermore, morphological features are much more complex in diseased muscle compared to healthy, which further inhibited the ability to use software to automatically evaluate the tissue in a high-throughput manner. To address these issues in the field, and provide a tool that we could use to evaluate how perturbations to the immune system effect muscle pathology, we developed QuantiMus. QuantiMus is a machine learning-based software that allows the accurate detection of muscle myofibers as well as the location of nuclei (i.e., centrally-located) and intra-myofiber presence of investigator-chosen proteins stained by immunofluorescence in entire muscle cross-sections. To date, we have successfully used QuantiMus to measure myofiber regeneration, myofiber injury, centrally-nucleated fibers, and to determine the myofiber type distribution of entire muscle cross-sections. The ability to measure full cross-sections is an advancement in the field that allows investigators to avoid random sampling of tissue sections and prevents bias in the data. All together, this study defines a role for ILC2s in regulating skeletal muscle eosinophilia during muscular dystrophy. We also developed software that allows us to evaluate muscle morphology in a high-throughput manner. Future directions will be aimed at understating what, in addition to regulating muscle eosinophilia, role ILC2s play in regulating DMD pathogenesis. In part, these future studies will encompass the power of QuantiMus to histologically evaluate how perturbations to ILC2s and eosinophils regulate pathology

Generation of Rabbit and Chicken Polyclonal Antibodies

Actin, a cytoskeletal component of all eukaryotic cells, plays an important role in diverse cell functions, including maintaining cell shape and contributing to cell motility. Actin filament length and stability is regulated by a variety of accessory proteins including actin capping protein (CP). In vertebrates, three alpha isoforms (α1, α2, α3) and three beta isoforms (β1, β2, β3) have been identified. We hypothesize that the alpha isoforms have distinct functions in tissues and cells which suggests that the proteins have different localization patterns. To evaluate the expression of the alpha proteins, we are generating alpha isoforms\u27 specific antibodies which will be used in future localization studies. Because alpha isoform antibodies do not exist, I am preparing two new polyclonal anti-mouse CP sera, one generated in chicken and one generated in rabbit, which will allow for double localization studies. The immunogens were peptides for mouse CP α1and CP α2, one specific for mouse al and one specific for mouse α2. Fusion protein constructs were prepared in pGEX-6, a glutathione S transferase vector. The protein were expressed In E. coli and purified by affinity chromatography. The protein concentration was determined by Bradford analysis and used as an immunogen in both chicken and rabbit. We have determined the titer of the production antibodies using Western Blot analysis. The α1 antibodies, generated in chicken, have a reactive titer of 10-6. α2 antibody production in rabbit is underway