bluuebunny/crossref_metadata_2025 huggingface dataset:
In-class activities as formative assessments in immunology: student buy-in and utilization
Authors: Brown Deborah M , Couch Brian A. , Brazeal Kathleen R.
Published: January 2023

Immunology is a difficult discipline to teach due to complex concepts, cellular interactions, jargon, and requirement of prerequisite knowledge across biology. Formative assessment (FA) techniques have been shown to improve student learning through increased engagement with material, instructor feedback, revision of understanding and student ownership of learning; yet implementation of these student-centered learning strategies can prove challenging. An in-class activity (ICA) worksheet FA was used in two semesters of an upper level Immunology course and a survey was used to measure student perception, buy-in and utilization of the FA. The ICA was administered each week and covered a key concept of the material presented. ICA worksheets examined the biochemical signaling pathways of T cell receptor signaling using an on line video by HHMI; HLA codominance and polygeny with flow cytometry data and Mendelian inheritance pedigrees; and CD4 T cell subset generation with an on-line modeling program developed by Cell Collective (www.cellcollective.org). Overall, student perceptions about ICA were favorable with a high level of “buy-in” across multiple categories/themes. In addition, ICA for immunology had a higher level of utilization (especially for later use) compared with other Biology courses using FAs. Within the immunology course, students viewed the ICA more favorable for later use and discussion compared to a pre-class FA, just in time teaching (JiTT). In contrast, students perceived the JiTT FA as providing more helpful feedback to correct misunderstandings. Utilizing student survey responses for both ICA and JiTT FAs will help improve in-class discussion, instructor feedback, and learning outcomes in immunology.
Development of a 4-year, comprehensive curriculum model for undergraduate immunology education
Authors: Bruns Heather A. , Justement Louis B
Published: January 2023

In addition to protecting individuals against infectious diseases, it is now well appreciated that the immune system influences a wide range of diseases including autoimmunity, cancer, and inflammatory syndromes that affect every organ in the body. Because it plays a key role in maintaining health, the immune system is important for several clinical specialties beyond infectious diseases. In spite of this, the development of robust curricula and educational programs at the national level that that are focused on teaching undergraduate students about the immune system are still in the earliest stages. This is in contrast to the current state of other specialized science majors. Because immunology is a highly conceptual discipline and understanding how the multiple cellular and soluble components of the immune system work in concert requires knowledge in a number of disciplines, time is needed for students to process and apply this information in meaningful ways. Concomitantly, knowledge in the field of immunology is expanding rapidly, bolstering the need for increased time in the curriculum to facilitate the ability of educators to convey information so that it can be effectively understood and applied. To address the need for undergraduate education in immunology, and the challenges faced in a multidisciplinary major, we have developed a 4-year program at the University of Alabama at Birmingham that provides a broad-based curriculum; delving into the normal and pathological workings of the immune system. We propose that it is time to follow this model, and that of a handful of other immunology programs to create a national curriculum that can be effectively implemented at the national level to promote undergraduate education in immunology.
Building Foundations for First Year Medical Students: Integration of Immunology, Microbiology, Pharmacology, and Infectious Disease Using Active Teaching and Self-Directed Learning
Authors: Steinel Natalie C , Lee Michael
Published: January 2023

In an era of decreasing basic science curriculum at medical schools, we sought to re-imagine how to optimally deliver three core basic science disciplines (immunology, microbiology, and pharmacology) together with infectious disease in a 5-week course. This course, was developed as part of a new 1-year pre-clinical basic science curriculum at the recently established Dell Medical School (DMS) at the University of Texas at Austin, featured a fully integrated curriculum in which the majority of the sessions were team-taught. In line with the goals and missions of the DMS, material was covered using primarily self-directed and flipped classroom approaches that incorporated approaches from the educational and cognitive psychology literature. Analysis of student data of our integrated course shows a strong correlation between student performance on formative and summative assessments as well as a correlation between summative exam performance and attendance in flipped classroom sessions. This suggests that, despite the abbreviated course time, engaging with the course as designed impacts achievement and may lead to superior outcomes. This offers valuable information that can be used to advise students on how to be successful in this type of high-impact course. It also may afford us the ability to predict which students may be at academic risk prior to the final summative exam.
Development of a CURE course aimed at characterization of bacterial Toll/interleukin 1 receptor (TIR) domain-containing proteins
Authors: Snyder Michelle , Snyder Greg A , Schmidt Katrina
Published: January 2023

Course-based undergraduate research experiences (CUREs) can engage a large, diverse group of students in authentic research, promote student research skills, student content knowledge, scientific literacy, critical thinking and analysis skills, support student self-efficacy, and at the same time provide a means to advance faculty research productivity. These varied goals of a CURE course, at times, can prove to be competing. For example, courses aimed at providing advanced levels of student input in design of research questions, hypotheses and protocols can veer from faculty research goals. In an effort to balance these competing objectives, we designed a CURE course engaging students at Towson University with research ongoing at the University of Maryland, School of Medicine aimed at characterizing the function of bacterial Toll/interleukin 1 receptor (TIR) domain containing proteins. Bacterial TIR proteins were originally characterized as virulence factors that impede mammalian innate immune signaling, but recently also have been shown to exhibit enzymatic NADase activity. Students in the CURE course used bioinformatics analysis of modeled structures of bacterial TIRs to predict sites for NADase function and hypothesized the effects of mutations of these sites on TIR functions. Students then designed and created recombinantly-expressed proteins mutated at their predicted sites and tested their mutant proteins for both NADase function and potential inhibition of mammalian innate immune signaling. This course outline thus allowed student engagement in project design and hypothesis formation, while also maintaining projects aimed at advancing a specific faculty research program.
Using IF-AT strategy in the dental immunology curriculum has a beneficial impact on performance and long term retrieval
Authors: Alard Pascale
Published: January 2023

When considering health professions education, student’s ability to retrieve acquired knowledge long term is critical. The immunology program for dental students is very intensive as they learn a tremendous amount of information in a short time (2.5 weeks), including memorization/understanding of new terminologies and concepts. In this study, we tested the impact of Immediate Feedback Assessment Technique (IF-AT) strategy on performance and long term retrieval. Two IF-AT sessions were held and accounted for 5% of the grade. Students were quizzed individually using iclicker, then divided into groups of 4–6 students who discussed the possible answers before selecting an answer using the IF-AT scratch forms. Students individually retook the quiz later and the concepts were discussed. Students found the exercise helpful in keeping up with studying and achieving a deeper understanding of the concepts. Students’ scores improved when taking the quiz in groups, and students answering correctly during the individual retake of the quiz increased from 60% to 85–90%. Performance on the exam shows that 89.3% of the students correctly answered questions whose topics had been covered during the IF-AT sessions versus 83.7% for questions about uncovered topics. The average of the class on the exam was 7 points higher, and was not due to a more performant class, i.e., students did not do better in other course exams unrelated to Immunology. Finally, students took the same quiz two months later without studying for it to assess long-term retrieval, and 70%–80 % of the students answered the questions correctly. In conclusion, holding IF-AT sessions is very beneficial as it improves the performance of the students on the exam and helps with long term retrieval.
Immunology that supports STEM participation for High School students
Authors: Sika-Paotonu Dianne , Anderson Jane , Cashin Phillipa , Cockburn Alana
Published: January 2023

The global burden of cancer is growing. The impact on Pacific communities living in New Zealand (NZ) and in the Pacific Region is significant and remains of concern. Pacific Peoples in NZ and in the Region remain overrepresented in adverse health statistics and outcomes. To improve health outcomes for Pacific communities, various strategies that include increasing the Pacific Health workforce have been proposed. These career pathways require participation in STEM and health related courses at high school and higher education learning levels. Pacific students remain underrepresented amongst these groups. This work sought to use Immunology based examples for improving the interest, engagement and participation of underrepresented student groups in High School the science laboratory. A teaching session was organized to include cancer related Immunology teaching content that was associated with subsequent laboratory workshop activities and was presented by a Pacific Biomedical Scientist. A question and answer was also set-up for the students to support their STEM learning in this context. Feedback indicated students who attended the teaching session found the Immunology related content helpful for their understanding of the overall STEM related laboratory and workshop content. Enthusiastic in-class interactions and engagement was observed. This work demonstrated the value of designing an in-class/laboratory environment that supports positive interactions and participation by students, by incorporating Immunology within the laboratory focus that is appropriately delivered and contextualized.
The importance of cultural context in supporting STEM related Immunology content for High School students
Authors: Sika-Paotonu Dianne , Cockburn Alana
Published: January 2023

Students and young people of Indigenous Māori and Pacific Peoples heritage living in NZ, remain underrepresented for STEM and health related courses. Health and STEM focused careers require participation in science and health related subject matter throughout high school and at higher education learning levels. This work sought to support, promote and encourage positive learning experiences for STEM related teaching content that was Immunologically based for a group of high school female students from underrepresented population groups that included Māori and Pacific students. Rheumatic Fever related Penicillin research work was presented to an all-girls high school class, comprised of students who were of predominantly Māori and Pacific heritage to support appropriate knowledge translation of STEM related Immunology based work that was also relevant to Māori and Pacific communities. This was carried out by a female Pacific Biomedical Scientist. Student feedback highlighted the session was very well-received and enjoyed by those in attendance. Strong interest was also expressed by the students who participated in the Rheumatic Fever related Penicillin based focus to the in-class interactions. A key element for undertaking this work was the inclusion of a female Pacific Biomedical Scientist in the teaching, aligning with the role modelling concept important for the Pacific students in particular who were involved with this STEM related outreach effort. These findings highlight the importance and relevance of cultural context in fostering positive learning and teaching interactions in the classroom for female high school students when engaging with Immunologically based STEM related content.
The utility and appropriate contextualisation of Immunology based cancer related teaching examples within Medical Sciences Education
Authors: Sika-Paotonu Dianne , Baltazar Patricia , dudley Joshua , Gilespie Alana , Logan Monica , Su Terrence , Tam Victoria , Walton Michaela
Published: January 2023

To support effective learning for students undertaking undergraduate Medical Sciences Education, multiple elements are required. These include clinically relevant Immunological course components and related research teaching examples with appropriate contextualisation. This learning and teaching effort involved incorporation of Immunologically based clinical research examples within undergraduate Medical Science Education classes relevant to current cancer related health issues associated with Māori and Pacific population groups living in New Zealand (NZ) and the Pacific Region. The overall aim was to support effective learning for students undertaking undergraduate Medical Sciences Education classes using clinically relevant Immunological based research examples that were contextualised and relevant to current health priorities associated with Māori and Pacific population groups. Immunologically based Cancer research work and examples of relevance to Māori and Pacific population groups were presented to 4th Year Medical students undertaking Medical Sciences Education classes. Feedback indicated these in-class examples helped raise awareness of current health issues and priorities facing Māori and Pacific population groups. Verbal and written student feedback also indicated in-depth understanding of the subject matter presented. Some students pursued efforts to secure research placements focused on Pacific related cancer-based research work. This work demonstrated the importance and utility of clinically relevant Immunological teaching examples that are contextualised and support learning and application of scientific research content for undergraduate Medical Science Education.
Teaching Undergraduate Immunology Using Journal Club Discussions and Case Studies
Authors: Dinkel Brittney A
Published: January 2023

With immunology being a rapidly progressing field, it is important to not only teach the basic immunology concepts but also teach current methods and navigation of primary literature. Based off our curriculum, students entering the 400-level immunology course already have had a brief introduction to immunology through the microbiology course. This allows us to dive more deeply into the material using primary literature and regular journal club discussions. Approximately half of the class each week provides background information and key concepts. The second half of the class each week is focused around either a recent publication focused around the concepts discussed during lecture or a case study that works to incorporate the understanding of multiple concepts into a disease or related condition. The journal clubs not only increase the depth and level of understanding around the topics but also works to expose students at small liberal arts colleges to techniques and data analysis that cannot be done on campus due to various limitations. Assessment is based off paper discussion forms, in-class discussion participation, and ability to analyze data and apply the knowledge during an exam.
Germinal center expansion but not plasmablast differentiation is directly proportional to peptide-MHCII density via CD40-CD40L signaling strength
Authors: Jing Zhixin , McCarron Mark J , Dustin Michael L , Fooksman David R
Published: January 2023

Although cognate peptide-MHCII (pMHCII) is critical for B cell selection in the germinal center (GC), it is unclear how cell intrinsic differences in peptide levels contribute to selection and cell fate decisions. Here, we applied the a-DEC205-OVA (dec) model system, to deliver different levels of OVA peptide to GC B cells in situ in order to interrogate how intermediate and high levels of pMHCII affect selection and cell fate on a per cell basis. We used Ly75+/− (DEC-het) B cells, which expressed ~50% of surface DEC205 protein compared to WT B cells, and presented proportional amount of OVA peptide after dec treatment. Using competitive co-transfers, we found that WT B cells expanded two-fold more than DEC-het B cells. This 2-fold difference in expansion was maintained at a wide range of dec administration. To study T cell recognition in situ, we developed a novel caged ovalbumin peptide, which is readily detected by OT-II T cells upon photo-uncaging, and revealed that initial T cell recognition in the GC leads to increased B-T serial contacts. Differential CD40 signaling, was both necessary and sufficient to mediate 2-fold differences in GC B cell expansion and also promoted GC-like B cell morphology, suggesting CD40 signaling is a rheostat of pMHCII dose. Surprisingly, we found that while plasmablast numbers were increased upon dec stimulation, both WT and DEC-het GC B cells were equally capable of differentiating into plasma cells, suggesting that pMHCII density does not directly control the output or quality of plasma cells generated. Thus, these results delineate distinct roles pMHCII play in expansion vs. differentiation in the GC.

Supported by grants from NIH (R01 HL141491-05)
Influenza infection reduces disease in a B-cell dependent EAE model of multiple sclerosis
Authors: Allushi Bujana , Chlebicz Magdalena , Labombarde Jocelyn , Turner Sean , Kovats Susan , Axtell Robert C
Published: January 2023

Multiple sclerosis (MS) is an autoimmune disease characterized by demyelination of neurons and inflammation in the central nervous system. MS afflicts approximately 2.5 million people worldwide. Understanding how viral infection affects MS disease is of great importance because the immune-suppressive effects of some MS therapies can make patients more vulnerable to viral infections. The goal of our project is to elucidate the effect of respiratory viral infection in a B cell-driven experimental autoimmune encephalomyelitis (EAE), which models the immune pathology of MS. Here, we describe a comorbid animal model in which mice were immunized with hMOG1–125 to induce EAE and subsequently infected prior to disease onset with a sublethal dose of the H1N1 Influenza A mouse-adapted A/Puerto Rico/8/1934 virus (IAV). Our data show that comorbid mice have a less severe EAE paralysis phenotype than mice with EAE alone. Flow cytometry analyses show that the lungs of comorbid mice had increased neutrophil numbers and a reciprocal reduction in MHCII+ CD86+ inflammatory monocytes. Comorbid mice also had fewer germinal center B cells in the lungs and reduced levels of IAV-specific IgM and IgG antibodies compared to IAV-infected controls. Secondary IAV challenge of comorbid mice revealed deficits in immunological B cell memory. Overall, our data show that EAE disrupts the antiviral immune response, including by decreasing the IAV-specific B cell response. Paradoxically, viral infection decreased EAE severity, indicating that the antiviral immune response attenuates B cell-dependent neuroinflammation. Future studies will determine mechanisms by which inflammation induced by viral infection modulates neuroinflammatory diseases.

Supported by grants from NIH (R01AI137047 and R01EY027346) A grant awarded to Dr. Kovats and Dr. Axtell from the Presbyterian Health Foundation
CD4+ T cells targeting a hybrid insulin:chromogranin A self-antigen are necessary and sufficient for autoimmune diabetes initiation
Authors: Dwyer Alexander J , Mitchell Jason S , Spanier Justin A , Silva Nubia , Alkhatib Mohannad , Tucker Christopher G , Morales Milagros Silva , Fife Brian T
Published: January 2023

Autoimmune diabetes induction requires CD4+ T cell responses directed against pancreatic islet self-antigens, and recent evidence implicates insulin C-chain:chromogranin A (InsC-ChgA) hybrid peptides as critical targets. Despite suggested contributions of InsC-ChgA-specific CD4+ T cells to diabetes pathology, the mechanisms underlying antigen encounter and pathogenicity are not understood. Here, we demonstrate these cells are absent in pancreata of adult diabetes-resistant mice, yet undergo rapid activation and expansion in neonatal non-obese diabetic (NOD) mice. InsC-ChgA-specific cells were clonally restricted to a public TRBV15 CDR3 sequence paired with shared α-chains in multiple mice, and the expansion of these cells was dependent upon early interactions with XCR1+ type 1 conventional dendritic cells. We also demonstrate pathogenicity upon transfer of InsC-ChgA-specific cells into NOD.Rag1−/− mice and disease abrogation with therapeutic administration of an anti-InsC-ChgA:I-Ag7 monoclonal antibody. Most importantly, this antibody prevents spontaneous diabetes. Together, these data establish the pathogenicity of InsC-ChgA-specific cells, demonstrating sufficiency and necessity for autoimmune diabetes.

Supported by grants from NIH (R01 AI156276, P01 AI35296, 2T32 AI007313)
Age-related susceptibility to grey matter demyelination and neurodegeneration is associated with meningeal neutrophil accumulation in an animal model of MS
Authors: Le Zuo Michelle Xiao , Fettig Naomi M , Osborne Lisa C , Gommerman Jennifer L , Ramaglia Valeria
Published: January 2023

People living with multiple sclerosis (MS) experience episodic central nervous system (CNS) white matter lesions instigated by autoreactive T cells. With age, MS patients show evidence of grey matter demyelination and experience devastating non-remitting symptomology. What drives progression is unclear and has been hampered by the lack of suitable animal models. Here we show that passive experimental autoimmune encephalomyelitis (EAE) induced by an adoptive transfer of young Th17 cells induces a non-remitting clinical phenotype that is associated with persistent leptomeningeal inflammation and cortical pathology in old, but not young SJL/J mice. While the quantity and quality of T cells did not differ in the brains of old vs young EAE mice, an increase in neutrophils and a decrease in B cells was observed in the brains of old mice. Neutrophils were also found in the leptomeninges of a subset of progressive MS patient brains that showed evidence of leptomeningeal inflammation and subpial cortical demyelination. Taken together, our data show that while Th17 cells initiate CNS inflammation, subsequent clinical symptoms and grey matter pathology are dictated by age and associated with other immune cells such as neutrophils.
Dendritic cells regulate innate cell trafficking into lymph nodes during inflammation
Authors: Gerner Michael Y , Wu Yixuan , Huang Jessica Y
Published: January 2023

Dendritic cells (DCs) have well-established roles in antigen presentation, and we recently showed that within several hours of immunization, lymph node (LN) resident DCs undergo activation and reposition into the T cell zone (TZ) to drive early T cell activation. Concurrently, monocytes are also recruited into LNs via high endothelial venules (HEVs) and cooperate with DCs to enhance the generation of adaptive responses. However, the mechanisms driving monocyte recruitment are unknown. Here, we used mouse models of immunization, quantitative imaging, and RNA sequencing to study early innate responses in draining LNs. We find that during inflammation, monocytes as well as neutrophils rapidly infiltrated the LN parenchyma and the TZ, with the timing of cell influx being concordant with that of DC activation and repositioning. As the DCs migrated towards the TZ, they passed through regions highly enriched in HEVs. DCs also expressed multiple inflammatory chemokines involved in monocyte and neutrophil trafficking, as well as high levels of the integrin ligand, ICAM-1, thus generating chemokine- and adhesion molecule-rich perivascular microenvironments. Depletion of DCs or ablation of ICAM-1 in DCs significantly decreased innate cell trafficking into LNs, with the recruited cells being largely trapped near HEVs and less able to infiltrate the TZ. Disruption of DC repositioning with conditional CCR7 ablation also resulted in the mislocalization of neutrophils and monocytes within the LNs. Thus, in addition to their classical roles as antigen presenting cells, during inflammation, DCs facilitate the recruitment of several innate cell types into LNs and guide them into the TZ to promote the generation of functional immune responses.

Supported by NIH grants R01AI134713, R21AI142667, T32GM007270 (J.H.), and F31AI161316 (J.H.).
The tetraspanin CD53 as a novel regulator for B cell trafficking
Authors: Greenberg Zev Joshua , Li Weikai , Schuettpelz Laura Graves
Published: January 2023

Migration and homing of B cells involves a myriad of adhesion molecules and chemokines that enable the movement of B cells from one part of the body to another. This tightly regulated process is important to facilitate maturation and optimize B cell function. Accordingly, loss of proper adhesive interactions leads to reduced immunoglobulin production and impaired immune function. As a family, tetraspanins regulate cellular growth, survival, and migration. Specifically, the tetraspanin CD53 has been implicated in early B cell development and immunoglobulin production; yet a role for CD53 in B cell trafficking has not been studied. Using a Cd53−/− mouse, we find that splenic B cells have significantly reduced transmigration when placed on a monolayer of human dermal microvascular endothelial cells, compared to WT B cells (1.7% vs 4.2% transmigration, p<0.0001). We next profiled chemotaxis in response to CXCL12 using a transwell assay and found significantly less migration of Cd53−/− bone marrow-derived pre-B cells (3.2% vs 5.0% of input, p<0.01) and Cd53−/− splenic B cells (14% vs 20% of input, p<0.001), compared to WT littermates. Splenic B cells from Cd53−/− mice also had significantly impaired in vivo homing to both the bone marrow (2.8% vs 7.7%, p<0.01) and spleen (2.6% vs 7.3%, p<0.001) compared to WT splenic B cells. Upon ex vivo stimulation of splenic B cells with CXCL12, loss of CD53 resulted in significantly less phosphorylation of the signaling mediators Akt (7.8% vs 12.2%, p<0.001) and Erk (12% vs 23%, p<0.0001), measured by flow cytometry. Together, these data suggest that the CXCL12 receptor, CXCR4, is a candidate binding partner for CD53 and that loss of CD53 abrogates normal B cell migration and homing.

Supported by institutional funding from Washington University in St. Louis
Delineating the mechanistic pathway and therapeutic potential of Siglec-6 in Chronic Lymphocytic Leukemia
Authors: Nunes Jessica Judith , Ventura Ann , Zapolnik Kevan , Mo Xiaokui , Zhang Liwen , Chen Min , Byrd John , Rader Christoph , Muthusamy Natarajan
Published: January 2023

Sialic acid-binding immunoglobulin-like lectins (Siglec) regulate immune function via glycan recognition. Here, we show for the first time the functional relevance of Siglec-6 and its ligand sialyl Tn (sTn) in cell adhesion and migration in chronic lymphocytic leukemia (CLL). Siglec-6+ primary B-CLL cells have significantly enhanced adhesion and migration towards sTn+ CLL− bone marrow stromal cells. A Siglec-6 targeted antibody inhibited homing of Siglec-6+ MEC1 cells and B-CLL cells to the spleen and bone marrow in NSG mice. Mass spectrometry analysis in MEC1 cells revealed interaction of Siglec-6 with DOCK8, a guanine nucleotide exchange factor. Stimulation of Siglec-6+ MEC1 cells with sTn resulted in Cdc42 activation, WASP recruitment and enhanced actin polymerization, which were compromised in Siglec-6 or DOCK8 KO MEC1 cells. Additionally, cell fractionation experiments revealed that Siglec-6+ MEC1 cells and B-CLL cells had higher levels of DOCK8 at the cell membrane when compared to MEC1 Siglec-6 KO cells and normal donor B cells respectively, indicating that Siglec-6 may be responsible for tethering DOCK8 to the cell membrane and is important for promoting actin polymerization at the leading cell edge. To study the therapeutic potential of Siglec-6, we generated a Siglec-6+ CLL mouse model (Siglec-6 x TCL1). Ex vivo experiments with a Siglec-6/CD3 targeted bispecific antibody showed specific killing of Siglec-6+ leukemic B cells. In summary, our studies have identified a novel role for Siglec-6 in promoting DOCK8 dependent CLL cell migration and opens up new therapeutic avenues to eliminate Siglec-6+ malignant B cells. Ongoing studies are focused on evaluating in vivo efficacy of Siglec-6/CD3 targeted bispecific antibody.

Natarajan Muthusamy: NIH-R21 Grant and Pelotonia Idea grant Jessica Nunes: Pelotonia Graduate Fellowship
Effects of Mycoplasma pneumoniae CARDS Toxin on mitochondrial homeostasis
Authors: Liendo Nathaniel Steven , Benavides Andrea Milenia , Segovia Jesus
Published: January 2023

Mycoplasma pneumoniae is an atypical bacterial pathogen known to cause mild respiratory infections and pneumonia. M. pneumoniae produces Community-Acquired Respiratory Distress Syndrome toxin (CARDS toxin), a virulence factor that plays a primary role in pathogenesis. CARDS toxin is known to have dramatic vacuolating and inflammatory properties, however these properties have not been fully characterized.

Recent evidence from our lab has supported that the toxin localizes on the mitochondria, affecting homeostasis and triggering inflammation. Our current study is focused on characterizing the effects of CARDS toxin on mitochondrial stress and production of mitochondrial reactive oxygen species (ROS). A549 lung epithelial cells and IMR-90 lung fibroblasts were treated with recombinant CARDS toxin exogenously, and mitochondrial ROS was measured. Additionally, we sought to determine whether ADP-ribosylation or vacuolization were required for CARDS toxin-induced mitochondrial stress by expressing CARDS toxin truncations on a mammalian expression plasmid. Endogenously expressed CARDS toxin truncations were fused to either EGFP or mCherry2. CARDS toxin was detected by western blot and IFA was used to track CARDS toxin localization in the cell.

Our findings support our hypothesis that CARDS toxin disrupts mitochondrial homeostasis and triggers mitochondrial ROS production. Our results provide valuable insight into the effects of CARDS toxin on mitochondrial function, which will help us better characterize the role of CARDS toxin during M. pneumoniae pathogenesis.

Supported by NIH T34GM008073
TLR-9 agonist CpG activates macrophages and modulates expression of microRNAs involved in cancer drug resistance and various signaling pathways
Authors: Kulangara Varsha Kontham , Zhong Yin , Yang Xiaoming , Nagarkatti Prakash S , Nagarkatti Mitzi
Published: January 2023

TLR-9 agonist, CpG induces pro-inflammatory immune response. We studied the immune response elicited in C57Bl/6 female mice by repeated stimulation of TLR-9 through multiple doses of CpG administration. Complete blood count analysis after CpG treatment revealed peripheral pancytopenia and the spleen showed splenomegaly. Flow cytometry analyses of spleen and liver mononuclear cells disclosed macrophage activation, evidenced by substantial increase in F4/80+ cells in the treatment group compared to control. Analysis of serum by ELISA demonstrated enhanced levels of pro-inflammatory cytokines, IL12/23, IL6 and TNFα in the CpG-treated group whereas no significant change was observed in the amount of anti-inflammatory cytokine, IL10. The spleen cells were further subjected to microarray analysis in order to examine the modulation of microRNA expression pattern following CpG treatment. Investigation of miRNA-mRNA relationships using the ingenuity pathway analysis (IPA) revealed differential expression of miRNAs associated with cancer drug resistance, HOTAIR and BAG signaling pathways along with microRNAs involved in immunomodulation. The major microRNAs altered with CpG treatment included mir27a, mir181, mir130a, mir487a, mir29b1, mir130a, mir3473b, mir21a, mir142 and mir144 targeting genes involved in drug resistance, apoptosis, cell cycle, angiogenesis and major histocompatibility complex-related proteins. Together, our studies demonstrate that chronic macrophage activation may lead to uncontrolled activation and proliferation of immune cells. (Supported by NIH grants P01AT003961, P20GM103641, R01AI129788, R01ES030144, R01AI160896 and R01AI123947).

Supported by NIH grants P01AT003961, P20GM103641, R01AI129788, R01ES030144, R01AI160896 and R01AI123947
CXCR4 promotes the stop signal and degranulation of cytotoxic T cells infiltrating influenza-infected lungs
Authors: Mrass Paulus , Byrum Janie , Torres David , Cannon Judy L
Published: January 2023

Cytotoxic T cells can promote protective immunity or exacerbate lung damage during influenza infections. Fine-tuning the functional activity of cytotoxic T cells in situ within influenza-infected lungs is a potential strategy to balance immunity and immunopathology. Using a murine model of influenza, we found that CXCR4 expression strongly correlated with cytotoxic T cell degranulation and that inhibition of CXCR4 reduced the degranulation of cytotoxic T cells in vitro and in vivo. Live tissue imaging revealed that influenza-specific T cells had prolonged dwell times when they were in regions with high levels of influenza antigen. Inhibition of CXCR4 led to increased T cell speed and decreased stop times in influenza-high areas. Moreover, inhibition of CXCR4 expedited the recovery of flu-infected mice. These data identify CXCR4 as a positive regulator of the stop-signal in lung-infiltrating CD8+ T cells and suggest that targeting this pathway could enhance recovery from influenza-induced weight loss.
Type I Diabetes induced by OT-I T-Cells requires ITK-mediated TCR signaling.
Authors: Perrenoud Loni Jean , Conley James , Berg Leslie
Published: January 2023

Activation of CD8+ T-cells induces changes in T cell trafficking, promoting effector T cell migration into tissues. ITK, a tyrosine kinase activated by TCR signaling, has previously been shown to be required for CD8+ T cell migration into the gastrointestinal tract during viral infection. This finding prompted us to investigate whether ITK signaling was important for effector T cell trafficking into non-infected tissue, as might occur in autoimmune disease. Using the RIP-mOVA mouse model, in which the OVA protein is expressed in the β-islet cells of the pancreas, we examined the ability of WT versus ITK-deficient OT-I T cells to infiltrate the pancreas inducing Type I diabetes. To test this, WT or ITK-deficient OT-I cells were adoptively transferred into RIP-mOVA recipients. Mice were immunized with OVA peptide plus LPS, and monitored for urine glucose. We also examined OT-I cell numbers in the pancreatic draining lymph node and in the pancreas 72hr post-immunization. We found that in the absence of ITK, OT-I cells were impaired in their ability to induce diabetes, with only ~1/3 of the recipients progressing to disease; in contrast 100% of recipients receiving WT OT-I cells developed Type I diabetes. In addition, the numbers of infiltrating ITK-deficient OT-I cells in the pancreas was significantly reduced compared to the numbers of WT OT-I cells detected. These findings indicate that ITK has a key role in generating pathogenic tissue-infiltrating CD8+ T cells capable of migration into β-islets of the mouse pancreas. While exploration of the mechanism underlying this impaired migration needs further investigation, these data provide support for therapeutic potential of an ITK inhibitor in patients with tissue-specific autoimmune diseases.

Supported by NAID TRC SIGNLG
Mechanosensory channel Piezo1 contributes to T lymphocyte migration through direct interaction with integrins
Authors: Laverty Miranda S , Benson Bryan L , Aguilar Alicia , Kingsley Daniel , Myers Jay T , Choi Sung-Hee , Huang Alex Y
Published: January 2023

Response to mechanical signals is an important mechanism by which T lymphocytes drive inflammation. A potential contributor in this process is Piezo1, a mechanosensitive cation channel which is abundantly expressed in T lymphocytes. Previously, our lab has shown that Piezo1 colocalizes with high affinity LFA-1 on activated human T lymphocytes during chemokine-activated crawling, suggesting a role for Piezo1 in the adhesion cascade. However, the mechanism by which Piezo1 contributes to the regulation of the adhesion cascade is unknown. We hypothesize that Piezo1 mediates T lymphocyte migration through direct contact with integrin to regulate its function. We investigate the role of Piezo1 in the expression, binding affinity, and turnover of LFA-1 and VLA-4. Co-immunoprecipitation revealed a preferential association of Piezo1 with the alpha subunit of integrins, specifically LFA-1, MAC-1, and VLA-4 in the active forms. Moreover, microfluidic devices were used to study the contribution of Piezo1 on T lymphocytes under laminar flow-induced shear stress. We found that activated T lymphocytes with CRISPR/Cas9-mediated Piezo1 ablation resulted in a reduced chemokine-induced crawling speed that correlates to the efficiency of Piezo1 gene ablation. Finally, in studies of lymph node homing where fluorescently labeled T lymphocytes were tracked in vivo, Piezo1 deficient cells exhibit increased ingress and decreased egress through the lymph node, suggesting an intricate role of Piezo1 on T lymphocyte migration. Taken together, our data support the hypothesis that Piezo1 modulates inflammatory processes through complex integrin interactions and mediates T lymphocyte migration from the lymph node to the site of inflammation.

Case Postbaccalaureate Research Education Program grant NIH/NIGMS 5R25GM075207-15; St. Baldrick’s Foundation; Hyundai Hope-on-Wheels Program; The I’m Not Done Yet Foundation; and Pediatric Cancer Research Foundation
The CD6-ALCAM pathway selectively modulates pathogenic T cell migration
Authors: Marrocco Valeria , Ampudia Jeanette , Connelly Stephen , Ng Cherie
Published: January 2023

CD6 is a costimulatory receptor on T cells that, through binding to its ligand ALCAM, modulates both the activity and trafficking of effector T cells (Teff). ALCAM has been seen to be overexpressed on various tissues during inflammation and is associated with increased infiltration of pathogenic CD6high CD4+ T cells in patients with autoimmune and inflammatory diseases. However, the role of the CD6-ALCAM pathway in the chemotaxis of pathogenic T cells into inflamed tissues is not yet clear.

Here, we investigate the contribution of CD6 in mediating chemokine-induced migration of Teff cells through the endothelial barrier by using itolizumab, a humanized anti-CD6 monoclonal antibody that inhibits the CD6-ALCAM interaction and reduces CD6 levels on T cells in the presence of monocytes. The migration of Teff cells from PBMCs or in vitro polarized TH17 cells expressing high levels of CD6, was tested under static conditions using a Boyden chamber assay incorporating human umbilical vein endothelial cells (HUVEC) which express high levels of ALCAM.

Analysis of migrating CD4+ T cells from total PBMCs showed that cells expressing higher levels of CD6 preferentially migrated through the HUVEC layer in response to CXCL12. Blocking the CD6-ALCAM pathway reduces the migration of CD4+ CCR7− CD45RA− TEM cells by ~60%. Furthermore, itolizumab treatment reduced the migration of pathogenic TH17 when co-cultured with monocytes.

This data demonstrates that the CD6-ALCAM pathway contributes to the chemokine-driven migration of Teff cells into inflamed organs through the endothelial layers. Consequently, blockade of this pathway will not only inhibit the activity of Teff but may also selectively affect their infiltration into inflamed organs.
Improving the Effectiveness of Combining Voice-Over PowerPoint Presentations and Review Games for Delivering Immunology Content.
Authors: Cusick John K , Goel Pranshul , Tang Justin Shiann , Abram Alyssa , Shyu Yennie , Gerriets Valerie
Published: January 2023

This study aims to improve the combined delivery of voice-over PowerPoint lectures and review games to increase student engagement and learning. Medical students were assigned immunology voice-over lectures prior to in-class or online learning sessions, in which students participated in teams to entertain sample exam questions in review games. Students provided quantitative feedback using a seven-point Likert scale and provided suggestions on how to improve the delivery of this pedagogy. The participating students were divided into groups that had differing access to either the voice-overs and/or review games before taking a formative quiz on the subject material. We are currently unable to conclude whether voice-overs or review games are more effective at increasing student performance on formative quizzes, yet future studies are planned to address this. More importantly, substantial feedback received from students through this ongoing study has been incorporated to improve the delivery of combining pre-assigned voice-overs with in-class gaming. Students overwhelmingly favored practice questions embedded within voice-overs and preferred multiple voice-overs of shorter length over long voice-overs for a given subject. Additionally, students greatly appreciated a brief question-and-answer review of the main points of the voice-over before beginning the in-class review game. Collectively, the students preferred this active learning pedagogy over traditional lectures. This study has provided valuable feedback to enhance the delivery of an active learning pedagogy that is relevant to both undergraduate and graduate-level immunology courses and is readily adaptable for either in-class or online learning sessions.

Seed grant from California Northstate University Institute of Teaching and Learning Excellence
The zombie pirate invasion: an introduction to immunology concepts.
Authors: Csencsits-Smith Keri , Robson Rachel
Published: January 2023

Introduction

The terminology that is used to describe the parts of the immune system can be very intimidating to student learners. We have noticed that some students disengage from the learning process early on in the semester because they are overwhelmed by “jargon” (e.g. CD markers, cytokines). The classroom exercise described here serves to introduce students to the concept of immunology as the body’s defense system as well as to provide a simplified framework for students to refer to as they learn more advanced concepts.

Methods

First-year medical students at Saba University School of Medicine, located on the small Dutch Caribbean island of Saba, are engaged in this group-based exercise at the beginning of the immunology content block. We present them with the following scenario: “Our island is being invaded by zombie pirates! How can we defend against this attack?” Each learning group is assigned a specific task, such as figuring out how to communicate across the island, determining how to differentiate zombies from residents, or planning how to train the populace in defense. Students then share their answers with the class. Instructors facilitate the linking of student answers with applicable concepts in immunology, e.g. communication = cytokine signaling, zombie identification = MHC presentation, training centers = lymph nodes.

Results/Conclusion

We have found that students remember this discussion in the following weeks, and that we can use the ideas they presented as a framework for reference as we introduce immunology concepts. This activity could be adapted to a number of different educational settings, contingent on the unique geographic or structural features of the campus on which other students study.
Female minority students-plight of staying alive in education during an unending pandemic!
Authors: Fazal Nadeem , Nguyen Minh-Thu , Simmons Jazmyne Durrah , Olk Sandra , Jhatu Prabjyot
Published: January 2023

COVID-19 pandemic laid bare deep inequalities and inequities. Disruptions brought to light by the ongoing pandemic are exacerbating risks and vulnerabilities both within education careers and personal lives. Widespread economic, sociological, and psychological damage has challenged female minority students in education more than their male counterparts. We surveyed female minority students as compared to male students at Chicago State University and found mixed ideas and opinions. The concerns on top of the list were fear of contracting COVID-19, existential epiphanies, and poor study conditions. More women, conditioned to design their lives around education are not enrolling for educational opportunities. One reason is that they can’t find childcare; another is they struggle with education/life balance. It’s not one cause but the pandemic has made it complex and multi-faceted. For other students, the pandemic offered many new opportunities that were practically never heard off. Some of the most common perks were, i.e., the ability to study remotely from anywhere, cutting out big city commutes, being able to give peak time to family, and greater flexibility in setting daily chores. A common theme for most of the surveyed students was that changes forced by the current pandemic are playing out a new social justice awakening on college campuses especially minority institutions like Chicago State University. Overarching suggestion was that right decisions made today in the context of a pandemic will have long-term consequences for the future of education. Overwhelming minority students proposed a faculty mentor and a peer-mentor to address the academic advising as well as personal guide to facilitate upheavals in pandemic situations.
Is it not over yet? – pandemic-induced ongoing burn out continues for students in Azerbaijan.
Authors: Fazal Nadeem , Cebiyev Nihat , Alakbarova Gulgaz , Huseynbayli Ayshan , Garajayev Azer , Safarova Arzu Safarova
Published: January 2023

In a nagging pandemic, while there will be some regression when the dust from pandemic settles, online, remote and hybrid education seems to stay for good and is expected to maintain a substantial portion of education. Students chronic burnout continues with more questions raised every day than the answers and promises made. It is a known psychological fact that when a younger generation of students encounters life-threatening events, they tend to reflect on death and uncertainty and take stock of their lives with defining what to make of their lives and how to achieve that. We surveyed mental health, social life and its impact on academics of medical and engineering students in Azerbaijan and reported the following data. Out of 274 respondents were 51% female and 48.2% male. 86.5% were from the age group (18–25) and 12.5% from (25–35) years. Majority of the students found themselves juggling with too many balls at the same time, not everyone could keep the focus, skill set and training to succeed. Female students delayed their cultural-ordained marriageable age in pursuit of a well-settled career partner, while male students found themselves looking for an alternative career path. Majority of professional students burn-out by a loss of at least two years of their professional education. This seemed to play out more for medical students as they were not able to find experiential training and hospital residencies. This included medical and engineering students who were ambitious to seek western education but lockdowns and visa regulations held them hostage at their home countries. Azerbaijan students however, were resilient enough to withstand the dual trauma of war with their neighboring country while fighting a global war against COVID-19 pandemic.