Remembering Our Past: Teaching the History of Anatomy at Indiana University

Most students pursuing careers in anatomy or related disciplines have a limited understanding of how, over the centuries, the intricate structure of the human body came to be known. To provide students with the relevant historical perspective, we developed a team-taught survey course in the history of anatomical sciences—including gross anatomy, histology, neuroanatomy, and embryology—from antiquity to the present. Taught entirely via Zoom during the Spring semester of 2021, History of Anatomy (2 semester hours credit) met once per week for approximately 2 hours. Enrollment consisted of 5 undergraduate students majoring in Biology (2), Human Biology (2), or Anthropology (1), as well as 3 graduate students pursuing either a master’s degree in Clinical Anatomy (1) or a Ph.D. in Anatomy Education (2). Three of the students had no prior coursework in anatomy. Through assigned readings, lectures, and discussions, the class explored the work of the great anatomists and their discoveries. A particular emphasis was placed on the evolution of anatomy as a discipline and the cultural influences, scientific controversies, and ethical dilemmas facing its practitioners. Syllabus topics included critical appraisals of the role of gender, race, and ethnicity in anatomical discovery. A key feature of the course was the opportunity for students to engage in robust discussions about such controversial issues as: Eurocentric biases in our understanding of human anatomy and the untold story of Muslim contributions to anatomical knowledge well before Vesalius; Competing claims of priority for who “discovered” the pulmonary circulation; The underappreciated role of women in the history of anatomy and medicine; The ethical quandary of teaching anatomy from archival fetal specimens obtained before the era of informed consent; Accusations that famed anatomist William Hunter used the bodies of murdered pregnant women to create his anatomical atlas of the gravid uterus; Complicity of Eduard Pernkopf and other Nazi-era anatomists in the unethical use of executed victims to obtain images for a renowned anatomical atlas. All students were assessed through weekly homework (written responses to study questions), a mid-term writing assignment, and a term paper about an historical topic of the student’s choosing. Graduate students had the additional requirement of a class presentation about their term paper topic. The end-of-course evaluation suggested that the course was well-received by the students (mean Likert score = 4.63 on a 5-point scale; n = 6). Based on this positive reception, we plan to offer History of Anatomy again on a recurring basis. We believe that by knowing our history, both the good and the bad, future practitioners of anatomy and related disciplines will be less likely to perpetuate the biases and ethical transgressions of earlier eras.American Association for Anatomy Spring Meetin

Neutrophils Are Resistant to Yersinia YopJ/P-Induced Apoptosis and Are Protected from ROS-Mediated Cell Death by the Type III Secretion System

The human innate immune system relies on the coordinated activity of macrophages and polymorphonuclear leukocytes (neutrophils or PMNs) for defense against bacterial pathogens. Yersinia spp. subvert the innate immune response to cause disease in humans. In particular, the Yersinia outer protein YopJ (Y. pestis and Y. pseudotuberculosis) and YopP (Y. enterocolitica) rapidly induce apoptosis in murine macrophages and dendritic cells. However, the effects of Yersinia Yop J/P on neutrophil fate are not clearly defined.In this study, we utilized wild-type and mutant strains of Yersinia to test the contribution of YopJ and YopP on induction of apoptosis in human monocyte-derived macrophages (HMDM) and neutrophils. Whereas YopJ and YopP similarly induced apoptosis in HMDMs, interaction of human neutrophils with virulence plasmid-containing Yersinia did not result in PMN caspase activation, release of LDH, or loss of membrane integrity greater than PMN controls. In contrast, interaction of human PMNs with the virulence plasmid-deficient Y. pestis strain KIM6 resulted in increased surface exposure of phosphatidylserine (PS) and cell death. PMN reactive oxygen species (ROS) production was inhibited in a virulence plasmid-dependent but YopJ/YopP-independent manner. Following phagocytic interaction with Y. pestis strain KIM6, inhibition of PMN ROS production with diphenyleneiodonium chloride resulted in a reduction of PMN cell death similar to that induced by the virulence plasmid-containing strain Y. pestis KIM5.Our findings showed that Yersinia YopJ and/or YopP did not induce pronounced apoptosis in human neutrophils. Furthermore, robust PMN ROS production in response to virulence plasmid-deficient Yersinia was associated with increased PMN cell death, suggesting that Yersinia inhibition of PMN ROS production plays a role in evasion of the human innate immune response in part by limiting PMN apoptosis

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Yersinia pestis Two-Component Gene Regulatory Systems Promote Survival in Human Neutrophils▿

Human polymorphonuclear leukocytes (PMNs, or neutrophils) are the most abundant innate immune cell and kill most invading bacteria through combined activities of reactive oxygen species (ROS) and antimicrobial granule constituents. Pathogens such as Yersinia pestis resist destruction by the innate immune system and are able to survive in macrophages and neutrophils. The specific molecular mechanisms used by Y. pestis to survive following phagocytosis by human PMNs are incompletely defined. To gain insight into factors that govern Y. pestis intracellular survival in neutrophils, we inactivated 25 two-component gene regulatory systems (TCSs) with known or inferred function and assessed susceptibility of these mutant strains to human PMN granule extracts. Y. pestis strains deficient for PhoPQ, KdpED, CheY, CvgSY, and CpxRA TCSs were selected for further analysis, and all five strains were altered for survival following interaction with PMNs. Of these five strains, only Y. pestis ΔphoPQ demonstrated global sensitivity to a panel of seven individual neutrophil antimicrobial peptides and serine proteases. Notably, Y. pestis ΔphoPQ was deficient for intracellular survival in PMNs. Iterative analysis with Y. pestis strains lacking the PhoP-regulated genes ugd and pmrK indicated that the mechanism most likely responsible for increased resistance to killing is 4-amino-4-deoxy-l-arabinose modification of lipid A. Together, the data provide new information about Y. pestis evasion of the innate immune system

The fibronectin-binding motif within FlpA facilitates Campylobacter jejuni adherence to host cell and activation of host cell signaling

Campylobacter jejuni is a gram-negative, curved and rod-shaped bacterium that causes human gastroenteritis. Acute disease is associated with C. jejuni invasion of the intestinal epithelium. Epithelial cells infected with C. jejuni strains containing mutations in the FlpA and CadF fibronectin (Fn)-binding proteins exhibit reduced invasion of host cells and a C. jejuni CadF FlpA double mutant is impaired in the activation of epidermal growth factor receptor (EGFR) and Rho GTPase Rac1. Although these observations establish a role for Fn-binding proteins during C. jejuni invasion, their mechanistic contributions to invasion-associated signaling are unclear. We examined FlpA, a C. jejuni Fn-binding protein composed of three FNIII-like repeats D1, D2 and D3, to identify the interactions required for cellular adherence on pathogen-induced host cell signaling. We report that FlpA binds the Fn gelatin-binding domain via a motif within the D2 repeat. Epithelial cells infected with a flpA mutant exhibited decreased Rac1 activation and reduced membrane ruffling that coincided with impaired delivery of the secreted Cia proteins and reduced cell association. Phosphorylation of the Erk1/2 kinase, a downstream effector of EGFR signaling, was specifically associated with FlpA-mediated activation of β1-integrin and EGFR signaling. In vivo experiments revealed that FlpA is necessary for C. jejuni disease based on bacterial dissemination to the spleen of IL-10(-/-) germ-free mice. Thus, a novel Fn-binding motif within FlpA potentiates activation of Erk1/2 signaling via β1-integrin during C. jejuni infection

Invasion of epithelial cells by Campylobacter jejuni is independent of caveolae

Caveolae are 25-100 nm flask-like membrane structures enriched in cholesterol and glycosphingolipids. Researchers have proposed that Campylobacter jejuni require caveolae for cell invasion based on the finding that treatment of cells with the cholesterol-depleting compounds filipin III or methyl-β-cyclodextrin (MβCD) block bacterial internalization in a dose-dependent manner. The purpose of this study was to determine the role of caveolae and caveolin-1, a principal component of caveolae, in C. jejuni internalization. Consistent with previous work, we found that the treatment of HeLa cells with MβCD inhibited C. jejuni internalization. However, we also found that the treatment of HeLa cells with caveolin-1 siRNA, which resulted in greater than a 90% knockdown in caveolin-1 protein levels, had no effect on C. jejuni internalization. Based on this observation we performed a series of experiments that demonstrate that MβCD acts broadly, disrupting host cell lipid rafts and C. jejuni-induced cell signaling. More specifically, we found that MβCD inhibits the cellular events necessary for C. jejuni internalization, including membrane ruffling and Rac1 GTPase activation. We also demonstrate that MβCD disrupted the association of the β1 integrin and EGF receptor, which are required for the maximal invasion of epithelial cells. In agreement with these findings, C. jejuni were able to invade human Caco-2 cells, which are devoid of caveolae, at a level equal to that of HeLa cells. Taken together, the results of our study demonstrate that C. jejuni internalization occurs in a caveolae-independent manner

Induction of caspase activity is YopJ/YopP dependent but delayed in human MDMs compared to J774A.1 cells.

<p>HMDMs and J774A.1 cells were incubated alone or with <i>Y. pestis</i> KIM5, KIM5 YopJ-YopP, KIM5Δ<i>yopJ</i>, KIM6 and <i>Y. enterocolitica</i> 8081v grown at 37°C. Caspase-3, -8, -9, and -2 activity was measured after 6 h of incubation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009279#s2" target="_blank"><i>Materials and Methods</i></a> and is expressed in relative fluorescence units (RFLU). Results are expressed as the mean ± SEM of three experiments. *, represents difference from HMDM and J774A.1 controls (<i>P</i><0.05).</p

Role of YopJ and YopP in J774A.1 macrophage<i>-</i>like and human MDM cell death.

<p><i>Y. pestis</i> strains with the virulence plasmid expressing YopJ (KIM5), YopP (KIM5 YopJ-YopP), or lacking YopJ (KIM5Δ<i>yopJ</i>), along with <i>Y. enterocolitica</i> 8081v and the virulence plasmid–deficient strain (KIM6) were grown at 37°C to induce expression of the TTSS and Yops. <i>Yersinia</i> strains were combined with J774A.1 and HMDM cells and compared to detergent lysed controls as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009279#s2" target="_blank">Materials and Methods</a>. The percentage of J774A.1 cell death following incubation alone or with <i>Yersinia</i> strains at the indicated time was determined by (<b>A</b>) LDH release into the supernatant and by (<b>B</b>) EthD-1 staining of J774A.1 cells. HMDM cell death was determined by measuring (<b>C</b>) LDH release and (<b>D</b>) EthD-1 uptake into HMDMs. The results are expressed as the mean ± SEM of at least three experiments. *, represents difference from J774A.1 or HMDM controls (<i>P</i><0.05).</p

The Intestinal Microbiota Influences Campylobacter jejuni Colonization and Extraintestinal Dissemination in Mice

Campylobacter jejuni is a leading cause of human foodborne gastroenteritis worldwide. The interactions between this pathogen and the intestinal microbiome within a host are of interest as endogenous intestinal microbiota mediates a form of resistance to the pathogen. This resistance, termed colonization resistance, is the ability of commensal microbiota to prevent colonization by exogenous pathogens or opportunistic commensals. Although mice normally demonstrate colonization resistance to C. jejuni, we found that mice treated with ampicillin are colonized by C. jejuni, with recovery of Campylobacter from the colon, mesenteric lymph nodes, and spleen. Furthermore, there was a significant reduction in recovery of C. jejuni from ampicillin-treated mice inoculated with a C. jejuni virulence mutant (ΔflgL strain) compared to recovery of mice inoculated with the C. jejuni wild-type strain or the C. jejuni complemented isolate (ΔflgL/flgL). Comparative analysis of the microbiota from nontreated and ampicillin-treated CBA/J mice led to the identification of a lactic acid-fermenting isolate of Enterococcus faecalis that prevented C. jejuni growth in vitro and limited C. jejuni colonization of mice. Next-generation sequencing of DNA from fecal pellets that were collected from ampicillin-treated CBA/J mice revealed a significant decrease in diversity of operational taxonomic units (OTUs) compared to that in control (nontreated) mice. Taken together, we have demonstrated that treatment of mice with ampicillin alters the intestinal microbiota and permits C. jejuni colonization. These findings provide valuable insights for researchers using mice to investigate C. jejuni colonization factors, virulence determinants, or the mechanistic basis of probiotics