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  Creation of a hyperplane device for horizontal cellular migration assays

  Nicholas M. Bittner, Nelofar Nargis, Ghoulem Ifrene, Brent Jeffrey Voels, and Colin K. Combs

  Cell culture studies routinely seek to monitor cell migration in response to chemoattractant stimuli. Common assays of cell migration employ well inserts and vertical cell migration assessment. This approach does not allow real-time monitoring of cell behavior. To address this need, we sought to develop a horizontal culture platform conducive to time course cell assessment changes in migration, morphology, phenotype etc. Modification of a commercial chamber slide allowed us to quantify cell migration in response to a 20% serum gradient. Based upon this finding, we designed and fabricated a prototype chamber slide for high replicate, real time assessment of cell migration in the serum gradient. The novel chamber slide design was effective for quantifying not only cell migration differences but visualizing cell movement. Optimization of the fabricated design will provide a novel tool for cell biology research.

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  Continued food-allergen consumption exacerbates beta-amyloid accumulation in allergen-sensitized AppNL-G-Fmice.

  Afrina Brishti

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  Dietary whey protein increases brain leukocytes in mice regardless of their hypersensitivity status

  Dilini Ekanayake

  Cow’s milk allergy (CMA) often manifests as milder reactions and may be linked to neurological problems. Previously, we demonstrated that C57BL/6J mice sensitized to a bovine whey allergen, β-lactoglobulin (BLG, Bos d 5), moderately increased BLG-specific IgE levels and exhibited behavioral changes without severe allergic reactions. When these non-anaphylactic CMA mice were placed on a whey-protein (WP)-containing diet for 2 weeks to simulate continuous dairy consumption, we found neuropathology indicative of neuroinflammation and cortical demyelination. Since immune cells migrate to the central nervous system (CNS) and promote neuroinflammation in demyelinating conditions such as multiple sclerosis, we hypothesized that the number of leukocytes would increase in BLG-sensitized mouse brains to orchestrate neuropathology. To test this hypothesis, we used flow cytometry to determine the number and phenotypes of leukocytes in the brains of naïve, sham, and BLG-sensitized mice after the 2 weeks of the WP diet. The frequencies of cells expressing common leukocyte marker CD45, pan T cell marker CD3, cytotoxic T cell marker CD8, integrin CD11b, myeloid cell marker CD14, and co-stimulatory marker CD86 significantly increased, regardless of the sensitization status. The percentages of these cells were low in mice that never received WP. This result indicated that WP diet consumption alone increased CNS leukocyte populations. Additional immunophenotyping is needed to determine whether the identified cells can be differentiated among the experimental groups. Detailed characterization of CNS leukocyte phenotypes and dynamics will help elucidate the mechanism of CMA-induced neuroinflammation and cortical demyelination.

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  Leukocytes as mediators of gut-brain communication

  Dilini Ekanayake

  Food allergies

  ▪ Reactions range from mild/delayed to severe/rapid. ▪ People with mild allergic reactions have increased re-exposure risks. ▪ Cow’s milk allergy tends to manifest with milder allergic reactions.

  Cow’s milk allergy (CMA)

  ▪ CMA has been associated with behavioral and neurological disorders. ▪ How allergic inflammatory signals from the gut reach the brain is unclear

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  Milk allergen increases intestinal immune cells in association with neuroinflammation and behavioral changes

  Dilini Ekanayake

  Cow’s milk allergy (CMA)

   CMA h as been associated with neurological disorders.

   How allergic inflammatory signals from the gut reach the brain is unclear.

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  Cortical demyelination and depression-like behavior are associated with histaminergic dysregulation in a mouse model of peripheral inflammation

  Danielle Germundson-Hermanson

  ▪ Demyelinating diseases of the central nervous system are increasing in prevalence world-wide1,2 and manifest as motor, behavioral, and/or cognitive defects3 . The etiology and pathophysiology of demyelinating diseases remain unclear.

  ▪ We have previously observed cortical demyelination in our mouse model of non-anaphylactic cow’s milk allergy4 . The demyelination was associated with depression-like behaviors and region-specific increases in brain histamine and H3 receptor (H3R) levels4,5 .

  ❖ The brain’s central histaminergic system is tightly controlled and regulates many behaviors. Additionally, signaling through H3R plays a crucial role in oligodendrocyte differentiation, and thus, demyelination and remyelination6 .

  ▪ We hypothesized that excess histamine produced during the hypersensitivity response would influence behavior through dysregulation of the central histaminergic system, resulting in neuroinflammation and demyelination.

  ▪ To test our hypothesis, we treated our food allergy mouse model with thioperamide, an H3R antagonist, and examined whether blocking histaminergic signaling would ameliorate the aberrant behaviors and demyelination.

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  Histamine H3 receptor antagonism mitigates food-hypersensitivity-associated depressive behavior and neuropathology in a mouse model of cow’s milk allergy

  Danielle Germundson-Hermanson

  Background Histamine is best known for causing allergy symptoms, but it also regulates blood-brain barrier permeability1 and oligodendrocyte differentiation2. Thus, brain histamine levels are tightly controlled.

  Previous Findings We demonstrated that histamine and histamine H3 receptor (H3R) levels were elevated in a mouse model of cow’s milk allergy (CMA) in association with intracranial mast cell activation, depression-like behaviors, and cortical demyelination3,4.

  Gaps in Knowledge In humans, food allergies are often associated with neuropsychiatric disorders5-7, but the involvement of allergy induced histamine in triggering behavioral changes is unclear.

  Hypothesis Repeated allergen consumption can lead to central histaminergic dysfunction through H3R, ultimately resulting in cortical demyelination and aberrant behaviors.

  Study Objectives We aimed to elucidate if antagonizing H3R could improve the behavioral and neuropathological outcomes in our mouse model after induction of CMA with repeated dietary allergen exposure.

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  Repeated allergen exposure induces histaminergic dysregulation and depression-like behaviors in a non-anaphylactic food allergy mouse model

  Danielle Germundson-Hermanson

  ▪ Food allergy is often comorbid with neuropsychiatric disorders such as anxiety1,2, depression1,2, OCD3, ADD/ADHD4, and autism5. ➢ The mechanism of how food allergy may impact brain function and behavior in certain allergic individuals has yet to be elucidated.

  ▪ Mast cells (MCs) are not only the effector cell of the hypersensitivity response but are also important in maintaining brain homeostasis6. ➢ MCs have been implicated in neuropsychiatric and neurodegenerative disorders such as multiple sclerosis7,8.

  ▪ We have previously found in a mouse model of cow’s milk allergy (CMA) that intracranial MC numbers9 and histamine 3 receptor (H3R)10 increased after acute allergic challenge in association with neuroinflammation and behavioral changes.

  ▪ We hypothesized that upon exposure to an allergen, MC-derived histamine would cause dysregulation of the brain’s histaminergic system, increasing neuroinflammation and altering behavior.

  ▪ To test our hypothesis, we preformed two studies using our mouse model of non-anaphylactic CMA to 1) observe changes in behavior and brain pathophysiology and 2) determine the role of excess histamine in our mouse model in the development of aberrant behaviors.

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  Continuous consumption of whey protein affects motor and cognitive functions in a mouse model of cow’s milk hypersensitivity

  Geetika Verma

  Hypersensitization to a food allergen can occur in the absence of immediate allergic reactions, a condition referred to as asymptomatic sensitization. Using mice asymptomatically sensitized to a bovine whey allergen, β-lactoglobulin (BLG, Bos d 5), we have previously shown that chronic exposure to the allergen results in behavior changes with increased neuroinflammation and cortical demyelination. However, whether allergen avoidance can resolve these pathologies is unknown. Thus, we aimed to compare immunological and behavioral changes in sham- or BLG-sensitized mice with or without the removal of dietary whey after the mice had consumed either a control diet (CTL) or a whey-protein-containing diet (WP) for 2 weeks. As previously reported, BLG-sensitized mice did not display overt physical reactions during the 2-week allergen exposure period, while their plasma levels of BLG-specific IgE and IgG1 were elevated compared to the respective sham groups. Following an additional 2 weeks on either the CTL or WP diet, these antibodies remained elevated in some of the sensitized mice in both diet groups. The gripstrength test indicated that sham mice that switched to the CTL diet after the 2-week WP diet increased limb strengths. In contrast, BLG-sensitized mice on either diet, as well as sham mice that stayed on the WP diet, showed a decline in limb strengths, suggesting that WP consumption, whether during sensitization or as part of the daily diet, affected their motor function. Furthermore, removing dietary WP improved spatial memory performance with the cross-maze test in both sham and BLG-sensitized mice. These results suggested that continuous exposure to a dietary allergen may influence motor and cognitive functions

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  Continuous consumption of whey protein maintains neuroinflammation in a mouse model of asymptomatic cow’s milk allergy

  Geetika Verma

  Using a mouse model of cow’s milk allergy (CMA), we previously showed that C57BL/6J mice sensitized to a bovine whey allergen, β-lactoglobulin (BLG), exhibited anxiety/depression-like behavior and neuroinflammation in the absence of overt anaphylaxis upon allergen exposure

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  Investigating the potential role of Akkermansia muciniphila supplementation in neuroinflammation: A progress report

  Geetika Verma

  Gut bacteria are important for proper development and function of the host immune system (1). Recent studies have provided association of altered microbiome in both food allergy and neuropsychiatric disorders (2, 3) indicating potential role of microbiome in regulating Gut-Brain-Axis. Akkermansia muciniphila belongs to the phylum Verrucomicrobia, is a commensal mucin degrading bacterial species (4). A. muciniphila protects the gut barrier by facilitating host mucus production. Since patients with food allergy have increased gut permeability, protection of intestinal barrier by increased mucus production may be beneficial in preventing allergen and pathogen infiltrations, hence, minimizing inflammation. A. muciniphila is well known in preventing obesity and diabetes but its role in food allergy and neuropsychiat ric disorders has yet be ascertained.