Tryptases are trypsin‐like serine proteinases found in the granules of mast cells. Although they show 40% sequence identity with trypsin and contain only 20 or 21 additional residues, tryptases display several unusual features. Unlike trypsin, the tryptases only make limited cleavages in a few proteins and are not inhibited by natural trypsin inhibitors, they form tetramers, bind heparin, and their activity on synthetic substrates is progressively inhibited as the concentration of salt increases above 0.2 M. Unique sequence features of seven tryptases were identified by comparison to other serine proteinases. The three‐dimensional structures of the tryptases were then predicted by molecular modeling based on the crystal structure of bovine trypsin. The models show two large insertions to lie on either side of the active‐site cleft, suggesting an explanation for the limited activity of tryptases on protein substrates and the lack of inhibition by natural inhibitors. A group of conserved Trp residues and a unique proline‐rich region make two surface hydrophobic patches that may account for the formation of tetramers and/or inhibition with increasing salt. Although they contain no consensus heparin‐binding sequence, the tryptases have 10–13 more His residues than trypsin, and these are positioned on the surface of the model. In addition, clustering of Arg and Lys residues may also contribute to heparin binding. Putative Asn‐linked glycosylation sites are found on the opposite side of the model from the active site. The model provides structural explanations for some to the unusual characteristics of the tryptases and a rational basis for future experiments, such as site‐directed mutagenesis.

Meta-analytic methods provide a framework around which an inquiry into MAST and SMAST score reliability was completed. Of the 470 measurement opportunities observed between 1971 and 2005, 62 (13.2%) were coupled with accurate reliability information. Weighted reliability estimates centered on.80 suggesting that the MAST and SMAST generally produce scores of similar and adequate reliability for most research purposes. However, the variability of internal consistency estimates shows that at times these tools will not produce reliable scores, particularly among female and nonclinical respondents. Multiple regression equations provide practical guidelines to improve reliability estimates for the future use of these instruments.

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Indiana University-Purdue University Indianapolis (IUPUI) / Non-alcoholic fatty liver disease, or NAFLD, is characterized by excess fat deposition in the liver. Cellular senescence is a critical hallmark of NAFLD. Cholangiocytes in the liver plays a significant role in the progression of fatty liver by contributing to senescence. p16 is the main senescent protein expressed by cholangiocytes in primary sclerosing cholangitis (PSC). Thus, we aimed to downregulate p16 by vivo-morpholino and evaluate the disease phenotypes and signaling mechanisms in a murine model of NAFLD. We found that downregulation of p16 reduced i) steatosis), ii) inflammation, iii) fibrosis, and cholangiocyte proliferation in HFD mice compared to the HFD-fed, control vivo-morpholino injected mice. Moreover, the downregulation of p16 reduced insulin-like growth factor-1 (IGF-1) in cholangiocytes, previously identified by our laboratory as a principal SASP factor secreted from cholangiocytes during NAFLD. By ingenuity pathway analysis, we found that p16 might regulates IGF-1 expression via the E2F1/FOXO1axis. Further analyses indicate that p16 downregulation reduces E2F1 mRNA transcription, inhibiting FOXO1 and subsequent IGF-1 expression in cholangiocytes. The presence of mast cells in the liver has been implicated in multiple cholangiopathies. Our lab demonstrated that mast cell stabilization by cromolyn sodium treatment reduced histamine secretion, fibrosis, and biliary proliferation in Mdr2-/- mice, a model of PSC. Thus, we aimed to determine mast cell stabilization as a therapeutic approach to managing NAFLD and its more advanced form, NASH. We found that cromolyn sodium ameliorated i) serum histamine levels, ii) intrahepatic mast cells, iii) inflammation, iv) fibrosis, v) steatosis, and cholangiocyte proliferation in methionine choline deficient diet-fed mice compared to the saline controls. Overall, we report that amelioration of senescence is a critical factor in improving the disease phenotypes in NAFLD. Biliary senescence plays a crucial role in modulating the disease progression in NAFLD, and mast cell stabilization can be used as a therapeutic approach to reduce pathological hallmarks of fatty liver. / 2024-05-22

Indiana University-Purdue University Indianapolis (IUPUI) / Neurofibromatosis type I (NF1) is a common genetic disease that affects over 200,000 patients in North America, Europe, and Japan. Individuals with NF1 display a wide variety of pathologies; importantly, 15-40% of NF1 patients are affected by plexiform neurofibromas. Neurofibromas are complex tumors consisting of tumorgenic Schwann cells surrounded by endothelial cells, fibroblasts, and inflammatory mast cells. These peripheral nerve sheath tumors contribute significantly to the morbidity and mortality associated with NF1. Currently, no medical therapies exist for treating neurofibromas. Recent evidence indicates that the hematopoietic tumor microenvironment carries out a crucial function in the formation of plexiform neurofibromas. Neurofibromatosis is the result of mutations at the NF1 locus, which encodes the GTPase activating protein neurofibromin. Neurofibromin is a negative regulator of the proto-oncogene Ras. Ras hyperactivation is the molecular basis of NF1 associated phenotypes, and it has been demonstrated that restoration of Ras signaling to wild type levels can correct NF1 associated phenotypes in vitro and in vivo. In keeping with the long term goal of detecting potential molecular targets for medical therapies to treat human plexiform neurofibromas, we have identified the kinase Pak1 as a possible downstream intermediary of Ras signaling in NF1 deficient cells. Studies described here utilized murine genetic models to study the effects of genetic inactivation of Pak1 on molecular signaling and cellular functions related to neurofibromas. We demonstrate that inactivation of Pak1 leads to correction of SCF mediated gain-in-function phenotypes seen in Nf1 haploinsufficient mast cells, in vivo and in vitro. However, by using a conditional Nf1 knockout mouse that is a reliable model of plexiform neurofibroma formation, we shown that loss of Pak1 alone in the hematopoeitic compartement is not sufficient to prevent neurofibroma formation. Additionally, we describe a key role for Pak1 in regulating PDGF and TGF-β mediated fibroblast functions, in vitro and in vivo. These studies provide insight into the causes of debilitating tumors related to a common genetic disease, and this research could potentially lead to the development of medical therapies for these tumors, increasing the quality of life for tens of thousands of affected individuals each year.

Indiana University-Purdue University Indianapolis (IUPUI) / Stem cell factor (SCF) mediated c-Kit signaling, and downstream activation of Phosphatidylinositol-3 Kinase (PI-3K) is critical for multiple biological effects mediated by mast cells. Mast cells express multiple regulatory subunits of PI-3Kinase, including p85α, p85β, p50α and p55α. In the present study, we have examined the relationship between p85α and p85β subunit in mast cell development and show that loss of p85α in mast cell progenitors impairs their growth, maturation and survival whereas loss of p85β enhances this process. To further delineate the mechanism (s) by which p85α provides specificity to mast cell biology, we compared the amino acid sequences between p85α and p85β subunits. The two isoforms share significant structural homology in the two SH2 domains, but show significant differences in the N-terminal SH3 domain as well as the BCR homology domain. To determine whether the c-Kit induced reduction in growth of mast cells is contributed via the N-terminal SH3 or the BCR homology domain, we cloned and expressed the shorter splice variant p50α, and various truncated mutant versions of p85α in p85α deficient mast cells. We demonstrate both invitro and invivo that while the SH3 and the BH domains of p85 are dispensable for mast cell maturation; they are essential for normal growth and survival. In contrary to existing dogma on redundant functional role of PI-3K regulatory subunits, this study proves that p85α and p85β regulatory subunits of PI-3K have unique roles in mast cell development. We prove that p85α deficiency impairs the expression of multiple growth, survival and maturation related genes whereas p85β deficiency inhibits c-Kit receptor internalization and degradation. This novel finding on negative role of p85β in mast cell development has significant clinical implication, as this knowledge could be used to develop treatments for mast-cell-associated leukemia and mastocytosis.

Background and Research Aim: Stress affects the immune system, which influences host physiology. Mast cells have been associated with several stress-induced changes in gut physiology. Mast cells also have the potential to influence the brain and behaviour. We investigated how mast cells influenced the body, brain, and behaviour during stress. Methodology: We investigated the behaviour of mast cell deficient animals and deficient animals that received whole bone marrow (WBM) transplants. We also studied the effects of mast cell stabilization during stress on changes in gut motility, via ex vivo. recordings of intestinal segments, and brain, via behavioural measurements and flow cytometry analysis of proinflammatory monocyte trafficking to the brain. Results: Mast cell deficiency leads to several behavioural changes related to activity level, exploration, and sociability. Furthermore, deficient animals that received WBM transplants demonstrated social and anxiety-like behaviour that differed from their deficient counterparts. Mast cell stabilization during stress prevented many of the stress-induced changes in gut motility commonly observed in the intestine. Mast cell stabilization during stress also prevented proinflammatory monocyte trafficking to the brain and was associated with reduced anxiety-like behaviour. Conclusion: Our findings support the role of mast cells in baseline behaviour, suggesting the presence of mast cells is needed for normal social and anxiety-related functioning. We also found that mast cell activation contributes to stress-induced intestinal dysmotility, suggesting that mast cells should be a target for interventions of stress-related gut motility disorders, such as irritable bowel syndrome. Lastly, our findings on the role of mast cells in monocyte trafficking and anxiety adds to our knowledge of neuroimmune interactions during stress and supports a potential role for mast cells in anxiety-related mood disorders, where stabilization of mast cells during stressful events may be of benefit. / Thesis / Master of Science (MSc) / Stress affects the immune system, which influences the body. Mast cells of the immune system are involved in several stress-induced changes in the body. They can also influence the brain and behaviour. We investigated how mast cells influence the changes that occur in the gut, brain, and behaviour during stress. Using mouse models, we prevented mast cells from activating during stress and looked at this effect on gut movement, changes in brain chemistry, and behaviour. We also compared the behaviour of mast cell deficient mice and deficient mice that gained mast cells. We found that by preventing mast cells from activating during stress, we can prevent several stress-associated changes in gut movement, brain chemistry, and anxiety behaviour. We also found that mast cells affect anxiety and social behaviour. These results suggest that mast cells impact the body, brain chemistry, and behaviour in stress and non-stress conditions.

Five experiments were conducted comparing differential intestinal immune responses to two isolates of Eimeria acervulina (EA), EA1 and EA2. In three experiments, broiler chicks were divided into control (non-challenged), EA1, or EA2 challenged (14 days of age) groups. On day 6 post-challenge (PC), changes in body weight were determined, intestinal lesions were scored, and duodenal tissue was evaluated for morphometric alterations and mucosal mast cell responses. EA1 produced duodenal lesions and reduced villus height to crypt depth ratios when compared to controls; however, no differences were found in mast cell counts. EA2 produced differing results, and observed data were suggestive of an intestinal secretory response when compared to EA1 or controls. In Experiment 4, tissues were analyzed from day 2 through day 6 PC. Villus atrophy and crypt hyperplasia were heightened on day 5 PC in both challenged groups. Mast cell counts were significantly greater on days 3 and 4 PC in EA1 birds. In Experiment 5, EA2 oocysts were cleaned with 5.25% sodium hypochlorite to evaluate the possibility of a bacterial contaminant contributing to the pathogenesis of intestinal alterations. Weight gains were decreased by challenge and villus heights and crypt depths were significantly altered in challenged birds, resulting in lower villus to crypt ratios, however, there were no differences in mast cell number. These data are indicative of differential host response and immunovariability between different isolates of the same Eimeria species and are suggestive of mast cell involvement in coccidial immunity in broiler chickens. / Master of Science

Pre-pubertal bovine mammary development involves ductal and stromal tissue changes. In mice, this process is impacted by presence of innate immune cells. Whether or not such immune cells are present or involved in bovine mammary development is unknown. We studied the presence, location and changes in numbers of eosinophils, mast cells and macrophages in pre-pubertal bovine mammary tissue. Chemical stains and immunofluorescence were used to identify the cells in formalin fixed, paraffin embedded mammary tissue. The first set (ONT) included samples (n=4/week) from birth to 6 weeks of age. Another set (OVX) determined the influence of ovaries, 19 animals were intact or ovariectomized 30 days before sampling. They were 90, 120 or 150 days old at examination. The third set (EST) allowed examination of the potential influence of exogenous estrogen on innate immune cells in the mammary gland. Samples were from calves given estrogen implants (n=6) or placebo (n=4) at 56 days old, and sampled at 70 days old. We examined 20 images each of NEAR and FAR stroma from every animal. More eosinophils were observed in NEAR versus FAR in the ONT and OVX , more mast cells observed in NEAR versus FAR in ONT. More macrophages were observed in NEAR versus FAR in ONT and EST. We show, for the first time, that innate immune cells are present in prepubertal bovine mammary tissue and that abundance is related to the epithelial structure. We suggest a possible role for these cells in control of bovine mammary development. / Master of Science

Reach truck masts are subjected to oscillations, which have significant impacts on the dynamics of the entire vehicle. Mast oscillations can cause undesirable outcomes in extreme situations and therefore it is desirable to be able to predict these outcomes before they occur. A forklift manufacturer in Mjölby initiated a thesis with the intention to obtain a model that can simulate mast sway for situations where oscillations occur. The objective of the thesis was to create a model of Triplex masts and find dependencies between model parameters and variables such as fork height and load. The thesis was conducted modelling the mast with a rigid multibody approach where torsion springs and dampers were used between mast parts to simulate mast elasticity. Clearance at the connections were considered and included in the model. The obtained model constitutes of 8 parameters that could be tuned to attain different oscillation characteristics. Parametric optimisation was carried out to find optimal sets of parameters for compliance with sway measurement tests with different load and fork height cases.  The thesis has resulted in a model that is able to simulate mast sway with different oscillation characteristics depending on model parameters. Performed parametric optimisation resulted in parameters that reveal useful information about how model parameters depend on load and fork height. The method used for obtaining optimal parameters can likewise be applied to other mast models in order to gain insight into model parameters as functions of load and fork height.