bluuebunny/crossref_metadata_2025_split huggingface dataset:
An online workshop integrated into Immunology course to teach pharmacogenomics to the first-year pharmacy students
Authors: Fazal Nadeem , Joly Rifka , Jassem Nuhad , Edquiban Marianne , Lakhani Baneen , Shaikh Kiran 
Published: January 2023

Abstract
Genetic basis of antibody structure is taught as a part of didactic course of Immunology to first-year pharmacy students. Therefore, we designed a three-hour workshop for P-1 students as a part of didactic Immunology course. Online workshops offer a rich multimedia learning experience, the interactive format includes guided learning videos, engaging discussion, assessment and comprehensive survey. Accreditation Council for Pharmacy Education (ACPE) Standards 2016 include pharmacogenomics, it is the responsibility of pharmacy schools to prepare pharmacists entering clinical practice to effectively use genetic data in the delivery of precision medicine. However, in a 2010 survey the state of pharmacogenomics instruction at most pharmacy schools was reported as “poor” or “not at all adequate”. Pharmacogenomics is defined as the study of how genes affects a person’s response to drugs. We proposed a new method of instruction using online learning resources to drive better learning and awareness of pharmacogenomics at a very early stage in pharmacy education. 64 students were included in this study. Two YouTube videos 45 minutes each, i.e., one describing basic genetics and the other pharmacogenomics were posted on Moodle. A case-study model was used to bring home the basic concepts. A pre- and post-workshop google forum was posted to assess student’s learning. A Moodle-based chat forum was also incorporated to take students questions during the workshop. The data showed that most of the students were unware of the importance and relevance of pharmacogenomics and the workshop improved learning this significantly. Online learning tools like workshops were found to be student-friendly and a flexible format with no scheduled meeting times.
Development of an interactive simulation and app (Pipette Master) to teach undergraduate students how to read, set and use and automatic laboratory pipette
Authors: Costabile Maurizio , Vedova Chris Della 
Published: January 2023

Abstract
An automatic pipette is a key piece of laboratory equipment. The ability to accurately and reproducibly deliver any volume is a critical skill to master for all undergraduate students in the laboratory sciences. Over many years we have noted that students struggle in learning several facets of correct pipette usage. These include, choosing the correct pipette for a given volume, correctly setting the pipette and correctly using the plunger to aspirate and dispense the liquid. Students also struggle with unit conversions between ml and μl and vice versa. Lastly, students fail to retain these skills and information in following semesters of teaching. In an attempt to help students with converting between units (μl and ml) as well as choosing the most appropriate pipette for a given volume, I developed an interactive simulation which covered all these major aspects. Using a Gilson pipette as an example, the simulation covered how to set the pipette for a number of defined volumes. An Android/iOS app of the simulation has also been developed which can be used during laboratory classes. In 2017, we polled first year undergraduate students about their level of experience, knowledge of pipettes and level of comfort in converting between units and we then explained the simulation to them. The feedback on their skills and the software will be discussed. We also tested the impact of the simulation using 2nd year students enrolled in genetics. Using a laboratory quiz, we found significant improvements in student performance when students used the simulation compared to those that did not. We suggest this interactive approach is a simple and effective way to enhance student learning of key aspects of laboratory pipettes. The simulation is freely available.
A speaking-intensive model for case studies in undergraduate immunology
Authors: Redmond Sarah , Mohamed Attia 
Published: January 2023

Abstract
Effective communication is consistently identified as a key attribute of successful graduates seeking employment or additional training. While most undergraduate programs place a focus on written communication, many interactions students have leading up to and during these new experiences require dialogue with peers, overseers, or the public. To prepare students for those interactions we use a group-presentation model in which students are responsible for three 50-minute presentations in their undergraduate immunology course. Student-led class sessions present challenges – real-time assessment, reliance on students to accurately and thoroughly convey information, and class engagement throughout the presentation. We use case studies as the basis for each presentation, with a set structure that requires presenters to address background, clinical details, scientific literature, and case-specific questions. Basic information is given to non-presenters and presenters receive additional information allowing for a preparatory meeting. Using a hybrid model for discussions, non-presenters can attempt to “solve” the case and ask questions before the class meets. In addition to the in-class assessment, students are graded on their preparation for the preparatory meeting and reflect on each other’s performance. Students were sampled at the beginning and end of the course using the CURE assessment (5 point scale), and reported a 0.74 point increase in critical thinking skills and a 0.44 point increase in speaking skills, as compared to a 0.09 point increase in other academic skills. We will share instructor, teaching assistant, and student perspectives.
Characterization of AIM2 expression in human macrophages during M. tuberculosis infection
Authors: Ontiveros Carlos Oscar , Arnett Eusondia , Schlesinger Larry S. 
Published: January 2023

Abstract
Tuberculosis (TB) is a leading contributor to global mortality and is a well-known threat to global health. The disease-causing bacterium, Mycobacterium tuberculosis (M.tb), has demonstrated resistance to current treatments, thus necessitating novel methods to fight the disease. The investigation of host-bacterium interactions may provide insight for development of effective host-directed therapies, thus circumventing antibiotic resistance. The body’s defense system against airborne M.tb includes alveolar macrophages (AMs) responsible for elimination of foreign agents. However, they do not effectively eliminate M.tb, leading to bacterial propagation. It was previously found that the NOD-like receptor, absent in melanoma 2 (AIM2), plays key roles in M.tb infection in murine macrophages. However, the expression of AIM2 in primary human cells during M.tb infection remains unknown. To this end, we characterized AIM2 expression in human monocyte-derived macrophages (MDMs) and AMs during M.tb infection via quantitative real-time PCR (qRT-PCR). We found that M.tb induces AIM2 transcripts in human MDMs and AMs, in a time-dependent manner. We also investigated the contribution of peroxisome proliferator-activated receptor (PPAR)γ to AIM2 expression due to its known regulation of several genes involved in the human macrophage response to M.tb. We found that PPARγ mediates AIM2 expression. In conclusion, the increased AIM2 gene expression observed in MDMs and AMs during M.tb infection necessitates further investigation into its function and its potential use as a target for M.tb host-directed therapy.
Myeloid derived suppressor cells (MDSC) from HIV-infected individuals are defective in innate immunity to Mycobacterium tuberculosis thus increasing the risk of tuberculosis
Authors: Garg Ankita J , Spector Stephen A 
Published: January 2023

Abstract
Tuberculosis (TB) during HIV-infection can occur at any disease stage, even when CD4+ T cell number are reconstituted and virus replication suppressed. We previously showed that compared to HIV-uninfected, immune suppressive CD14+ MDSC are higher in HIV-infected individuals on combined antiretroviral therapy (cART) and with virus suppression. Here we examined if MDSC isolated from HIV-infected individuals are responsible for defective immunity to M tuberculosis (Mtb). PBMCs from HIV-infected individuals on cART and with viral suppression were isolated and CD14+HLA DRhi (DRhi) and HLA DR−/lo (MDSC) cell subsets were sorted by flow cytometry (FACS) and analyzed for the expression of TLR-2, -4 and IL-27R. FACS analysis showed that MDSC and DRhi subsets had comparable expression of surface TLR-2 (p=0.21) and TLR-4 (p=0.13); expression of IL-27R was high on MDSC (p=0.002). MDSC and DRhi subsets were infected with GFP expressing Mtb Erdman strain at MOI 5 in the presence of neutralizing IL-27 or isotype match control antibody. FACS analysis at 2-hrs post infection showed a higher expression of intracellular ROS in DRhi as compared to MDSC subsets (p= 0.05). Neutralization of IL-27 increased ROS expression of DRhi (p=0.005) but not MDSC subset (p=0.07). Furthermore, MDSC as compared to DRhi subset exhibited a higher intracellular replication of Mtb at day-5 post infection (p=0.01). Lastly, gene expression of intracellular MyD88 in MDSC as compared to DRhi subset was lower in response to Mtb (p=0.05). These findings suggest that MDSC in HIV-infected individuals inhibit protective immunological response to Mtb and increase the risk of TB disease in these individuals.
Involvement of Sphingosine Kinase (SK1) during autophagosome formation plays a critical role in controlling the growth of Mycobacterium tuberculosis within Human Mesenchymal Stem Cells
Authors: Khan Arshad , Singh Vipul Kumar , Mann Lovepreet , Papanna Ramesha , Canaday David , David Sunil A , Tsetkov Andrey S , Jagannath Chinnaswamy 
Published: January 2023

Abstract
Mycobacterium tuberculosis (Mtb) evades host defense mechanisms by infecting and persisting in Mesenchymal Stem Cells (MSCs). Thus, MSCs contain M. tuberculosis DNA and harbor viable bacilli, and our recent study showed that autophagy plays a critical role in restricting the growth of Mtb in human MSCs. Activation of human MSCs using rapamycin enhanced the bactericidal function of MSCs. Here, we examined the autophagy pathway associated with the control of Mtb growth within MSCs. When compared to human macrophages, MSCs had increased expression of Sphingosine Kinase 1 (SK1) during Mtb infection, which was also found to be associated with overall increase in autophagic flux and LC3B positive puncta. SK1 also more often co-localized with gfpMtb containing autophagosomes within MSCs when compared to human macrophages. Percentage of SK1 and LC3B double positive M. tuberculosis containing autophagosomes were also significantly higher in MSCs compared to macrophages suggesting that Mtb induces SK1 mediated autophagy in MSCs during infection. To validate the role of SK1 mediated autophagy in inhibition of Mtb growth within MSCs, we knocked down SK1 using siRNA and scrambled control and examined the survival of M. tuberculosis. SK1-siRNA treated MSCs showed an enhanced survival of Mtb, and replication compared to control siRNA treated cells over 21 days of incubation. These results together indicate that SK1 mediates autophagy, autophagosome formation and autophagy dependent inhibition of Mtb growth within MSCs.
Deletion of MCP-1 enhances susceptibility to carbapenem-resistant Klebsiella pneumoniae via modulating neutrophil and macrophage function
Authors: Jin Liliang , Ghimire Laxman , Paudel Sagar , Cai Shanshan , Jeyaseelan Samithamby 
Published: January 2023

Abstract
Monocyte chemoattractant protein-1 (MCP-1) is important for monocyte recruitment to the lungs in response to bacterial infection. MCP-1 is also essential for neutrophil-dependent host defense during Escherichia coli infection. Carbapenem-Resistant Klebsiella pneumoniae (CRKP) infection has been rapidly growing as a life-threatening nosocomial disease in the U.S. and world. However, the role of chemokines and cellular responses in protective immunity to pulmonary infection with CRKP is largely unknown. Here, we investigated the role of MCP-1 in pulmonary host defense against CRKP infection using MCP-1 knockout (KO) mice. We determined survival, bacterial burden, and neutrophil influx, cytokine/chemokine expression as well as cellular functions, such as formation of neutrophil extracellular traps (NETs) and macrophage extracellular traps (METs). MCP-1 is essential for survival and restricting bacterial outgrowth organs. Compared to the C57Bl/6 (control) mice, KO mice showed impaired influx of neutrophils in the airways as assessed by nuclear cell morphology in BALF and by myeloperoxidase activity (MPO) in lung parenchyma. Moreover, the NETs and METs induced by CRKP were reduced in KO mice in both in vitro or in vivo. Intriguingly, subsequent to CRKP infection, KO mice demonstrated increased pyroptosis through enhanced activation of caspase-1, higher expression of IL-1β and cleaved gasdermin-D in the lungs. These findings enhance our understanding of the critical role of MCP-1 in modulating neutrophil function, such as NETosis, METosis and pyroptosis and suggest that therapies targeting modulation of NETs, METs and pyroptosis during CRKP infection can be explored through clinical trials.
Type III interferons are expressed in tuberculosis granulomas and may influence signaling in epithelioid macrophages
Authors: Talukdar Priyanka , Mattila Joshua T. 
Published: January 2023

Abstract
Granulomas are the pathologic hallmark of Mycobacterium tuberculosis (Mtb) infection. Balanced pro- and anti-inflammatory cytokines regulate inflammation in tuberculosis (TB) and determine outcomes at the lesional and organismal level. Understanding what constitutes protective and detrimental inflammation in TB is important for the improvement of current TB therapeutics. The type III interferon family is composed of four isoforms and includes IL-29, IL-28A, IL-28B, and the recently classified IFNλ4. These cytokines may be important regulators of inflammation in TB yet little is known about their relationship with TB or whether they have different properties in granulomas than type I interferons (IFNα/β), a family of cytokines associated with severe TB. To better understand the relationship between IFNλ, IFNα, and TB we characterized IFNλ expression in formalin-fixed paraffin-embedded granulomas from cynomolgus macaques and investigated cytokine expression and STAT phosphorylation by macaque peripheral blood, lung, and lymph node cells. We found that IL-29 and IL-28B are expressed by epithelioid macrophages and neutrophils in granulomas and IFNλ4 had a unique expression in the transcriptionally active region of epithelioid macrophage nuclei suggesting a possible regulatory role for IFNλ4 in these cells. Experiments were also performed to compare IFNα and IFNλ’s effect on antimycobacterial responses in monocyte-derived macrophages, and ability to induce STAT phosphorylation and pro- and anti-inflammatory cytokine expression by T cells, macrophages, monocytes, and neutrophils. Our findings suggest that IFNλ may have unappreciated roles in regulating inflammation at the site of disease in TB.
Phenotypic analysis of alveolar macrophage populations in old mice and their response to Mycobacterium tuberculosis infection
Authors: Lafuse William P , Rajaram Murugesan VS , Wu Qian , Moliva Juan Ignacio , Torrelles Jordi B , Turner Joanne , Schlesinger Larry S. 
Published: January 2023

Abstract
The elderly are more susceptible to pulmonary infections including tuberculosis. Here, we examined the impact of aging on the phenotype and response of mouse alveolar macrophages (AMs) to Mycobacterium tuberculosis (M.tb). AMs were isolated from bronchoalveolar lavage fluid (BALF) of young (3 month) and old (18 month) C57BL/6 mice. AMs from old mice expressed higher mRNA levels of CCL2, IFN-β, IL-10, IL-12p40, TNFα and MIF than young mice and old mice contained higher levels of CCL2, IFN-β, IL-1β and MIF in their BALF. Flow cytometry analysis of AMs showed two distinct AM populations, a major CD11c+ CD11b− and a minor CD11c+CD11b+ population, the latter was significantly increased in old mice. Expression of CD206, TLR2, CD16/CD32, MHC class II and CD86 was higher in the CD11c+CD11b+ AMs and these AMs expressed monocytic markers Ly6C, C3CR1, and CD115, suggesting they are of monocytic origin. Sorted CD11c+CD11b+ AMs expressed higher mRNA levels of CCL2, IL-1β, and IL-6, whereas CD11c+ CD11b− AMs expressed higher mRNA levels of immune-regulatory cytokines IFN-β and IL-10. CD11c+CD11b+ AMs phagocytosed more M.tb, and M.tb in these AMs expressed higher RNA levels of genes required its survival. Our studies identify two distinct AM populations in old mice, specifically a novel CD11c+CD11b+ AM population which is hyper-inflammatory and phagocytoses M.tb more efficiently, yet is more permissive for M.tb survival than the CD11c+CD11b− population which is more immuno-regulatory in nature. Our results also indicate that the lung cytokine environment of old mice contains an unusual array of both pro-inflammatory and immune-regulatory cytokines.
Alveolar macrophage responses to Mycobacterium tuberculosis reveal human-to-human variation in important immunobiology pathways gleaned from functional genomics study
Authors: Azad Abul , Papp Audrey , Zhou Xiaofei , Pietrzak Maciej , Wang Min , Arnett Eusondia , Montoya Maria , Handelman Samuel , Rempala Grzegorz , Lin Shili , Sadee Wolfgang , Schlesinger Larry 
Published: January 2023

Abstract
Human alveolar macrophages (AMs) are the primary target of Mycobacterium tuberculosis (M.tb) in the lung, where M.tb establishes intracellular residence and growth. This study characterizes M.tb-AM interactions and processes driving inter-subject variability. AMs were obtained by bronchoalveolar lavage from 28 healthy donors and cultured in the presence and absence of virulent M.tb strain H37Rv engineered to produce bioluminescence. Luminescence intensity (RLU) was measured at 2, 24, 48, and 72h post infection. At 2, 24, and 72h, we also measured 27 secreted proteins and AM RNA transcriptomes using AmpliSeq, targeting 20,804 genes, including 2,228 non-coding RNAs. RLUs reveal large differences in M.tb uptake at 2h, varying rates of intracellular adaptation between 2 and 24h, and a range of growth rates from 48 to 72h, resulting in discrete donor groups for each type of interaction. We identify 5039 significant differentially expressed RNAs (DE genes), revealing immune-related pathways, gene networks, and hub genes. A sub-group of three AMs with rapid growth rates (72/48h RLU ratios) displays the most significant DE RNA profiles, including TREM1 networks, IL8 and IL1B, CXCL10, and most prominently IDO1 at 72h, recently identified as a key protein characteristic of active TB, in a cluster of 73 tightly co-expressed genes. Large inter-individual differences in secreted proteins are mostly consistent with the RNA results. Our results identify known and novel key genes in the early phase of M.tb infection in a subset of HAMs, potentially relevant to in vivo infection and TB risk. These key genes can guide a search for genetic variants that contribute to TB risk, and potential targets for diagnostics, treatment, and prevention strategies.
Non-canonical activation of Caspase 8 mediates pyroptosis
Authors: Poltorak Alexander 
Published: January 2023

Abstract
Cell death and inflammation are intimately linked during homeostasis, host defense, and tumorigenesis. Pathogenic Yersinia inhibits the MAP kinase TGFβ-activated kinase 1 (TAK1) via the effector YopJ, thereby silencing cytokine expression while activating caspase-8–mediated cell death. Here, using Yersinia pseudotuberculosis in corroboration with costimulation of lipopolysaccharide and (5Z)-7-Oxozeaenol, a small-molecule inhibitor of TAK1, we show that caspase-8 activation during TAK1 inhibition results in cleavage of both gasdermin D (GSDMD) and gasdermin E (GSDME) in murine macrophages, resulting in pyroptosis. The ensuing cell death is rapid and morphologically is similar to pyroptosis. Loss of GsdmD delays membrane rupture, reverting the cell-death morphology to apoptosis. The IL1 response arises from asynchrony of macrophage death during bulk infections in which two cellular populations provide signal 1 for transcriptional upregulation and signal 2 for the inflammasome assembly. Furthermore, we found that human macrophages are resistant to YopJ-mediated pyroptosis, with dampened IL-1β production. Our results uncover a form of caspase-8–mediated pyroptosis and suggest a hypothesis for the increased sensitivity of humans to Yersinia infection compared with the rodent reservoir. Altogether, this knowledge will be fundamental for our understanding of the mechanism of regulation of cell death and the discovery of new drug targets that can span areas from infections to tissue injury both in and ex vivo. This work was supported by the NIH grant AI056234 and Russian Science Foundation Grant 15-15-00100 (to A.P.)
Calpain drives pyroptotic vimentin cleavage, intermediate filament loss, and cell rupture that mediates immunostimulation
Authors: Davis Michael Alan , Fairgrieve Marian R. , den Hartigh Andreas , Yakovenko Olga , Duvvuri Bhargavi , Lood Christian , Fink susan , Thomas Wendy E. , Gale Michael J 
Published: January 2023

Abstract
Pyroptosis is an inflammatory form of programmed cell death following cellular damage or infection. It is a lytic process driven by gasdermin D-mediated cellular permeabilization and presumed osmotic forces thought to induce swelling and rupture. We found that pyroptotic cells do not spontaneously rupture in culture but lose mechanical resilience. As a result cells were susceptible to rupture by extrinsic forces such as shear stress or compression. Cell analyses revealed that all major cytoskeleton components were disrupted during pyroptosis and that sensitivity to rupture was calpain-dependent and linked with cleavage of vimentin and loss of intermediate filaments. Moreover, while release of LDH, HMGB1, and IL-1β occurred without rupture, rupture was required for release of large inflammatory stimuli—ASC-specks, mitochondria, nuclei, and bacteria. Importantly, supernatants from ruptured cells were more immunostimulatory than those from non-ruptured cells. These observations reveal undiscovered cellular events occurring during pyroptosis, define the mechanisms driving pyroptotic rupture, and highlight the immunologic importance of this event.