A Multi-Institutional Description of Processes and Outcomes of Postbaccalaureate Research Education Programs in the Mid-Atlantic Region.

ABSTRACT: Outcome data from 6 National Institutes of Health-funded Postbaccalaureate Research Education Programs (PREPs) in the Mid-Atlantic region were combined to give a multi-institutional perspective on their scholars' characteristics and progress through biomedical research training. The institutions hosting these programs were Johns Hopkins University School of Medicine, the Medical University of South Carolina, the University of Maryland School of Medicine, the University of North Carolina at Chapel Hill, Virginia Commonwealth University, and Virginia Polytechnic Institute and State University. The authors summarize the institutional pathways, demographics, undergraduate institutions, and graduate institutions for a total of 384 PREP scholars who completed the programs by June 2021. A total of 228 (59.4%) of these PREP scholars identified as Black or African American, 116 (30.2%) as Hispanic or Latinx, and 269 (70.0%) as female. The authors found that 376 of 384 scholars (97.9%) who started PREP finished their program, 319 of 376 (84.8%) who finished PREP matriculated into PhD or MD/PhD programs, and 284 of 319 (89.0%) who matriculated have obtained their PhD or are successfully making progress toward their PhD.

Lymphoedema conditions disrupt endothelial barrier function in vitro.

Lymphatic vessel contractions generate net antegrade pulsatile lymph flow. By contrast, impaired lymphatic vessels are often associated with lymphoedema and altered lymph flow. The effect of lymphoedema on the lymph flow field and endothelium is not completely known. Here, we characterized the lymphatic flow field of a platelet-specific receptor C-type lectin-like receptor 2 (CLEC2) deficient lymphoedema mouse model. In regions of lymphoedema, collecting vessels were significantly distended, vessel contractility was greatly diminished and pulsatile lymph flow was replaced by quasi-steady flow. In vitro exposure of human dermal lymphatic endothelial cells (LECs) to lymphoedema-like quasi-steady flow conditions increased intercellular gap formation and permeability in comparison to normal pulsatile lymph flow. In the absence of flow, LECs exposed to steady pressure (SP) increased intercellular gap formation in contrast with pulsatile pressure (PP). The absence of pulsatility in steady fluid flow and SP conditions without flow-induced upregulation of myosin light chain (MLCs) regulatory subunits 9 and 12B mRNA expression and phosphorylation of MLCs, in contrast with pulsatile flow and PP without flow. These studies reveal that the loss of pulsatility, which can occur with lymphoedema, causes LEC contraction and an increase in intercellular gap formation mediated by MLC phosphorylation.

PGC1α Regulates the Endothelial Response to Fluid Shear Stress via Telomerase Reverse Transcriptase Control of Heme Oxygenase-1.

OBJECTIVE: Fluid shear stress (FSS) is known to mediate multiple phenotypic changes in the endothelium. Laminar FSS (undisturbed flow) is known to promote endothelial alignment to flow, which is key to stabilizing the endothelium and rendering it resistant to atherosclerosis and thrombosis. The molecular pathways responsible for endothelial responses to FSS are only partially understood. In this study, we determine the role of PGC1α (peroxisome proliferator gamma coactivator-1α)-TERT (telomerase reverse transcriptase)-HMOX1 (heme oxygenase-1) during shear stress in vitro and in vivo. Approach and Results: Here, we have identified PGC1α as a flow-responsive gene required for endothelial flow alignment in vitro and in vivo. Compared with oscillatory FSS (disturbed flow) or static conditions, laminar FSS (undisturbed flow) showed increased PGC1α expression and its transcriptional coactivation. PGC1α was required for laminar FSS-induced expression of TERT in vitro and in vivo via its association with ERRα(estrogen-related receptor alpha) and KLF (Kruppel-like factor)-4 on the TERT promoter. We found that TERT inhibition attenuated endothelial flow alignment, elongation, and nuclear polarization in response to laminar FSS in vitro and in vivo. Among the flow-responsive genes sensitive to TERT status, HMOX1 was required for endothelial alignment to laminar FSS. CONCLUSIONS: These data suggest an important role for a PGC1α-TERT-HMOX1 axis in the endothelial stabilization response to laminar FSS.

A cross-institutional analysis of the effects of broadening trainee professional development on research productivity.

PhD-trained scientists are essential contributors to the workforce in diverse employment sectors that include academia, industry, government, and nonprofit organizations. Hence, best practices for training the future biomedical workforce are of national concern. Complementing coursework and laboratory research training, many institutions now offer professional training that enables career exploration and develops a broad set of skills critical to various career paths. The National Institutes of Health (NIH) funded academic institutions to design innovative programming to enable this professional development through a mechanism known as Broadening Experiences in Scientific Training (BEST). Programming at the NIH BEST awardee institutions included career panels, skill-building workshops, job search workshops, site visits, and internships. Because doctoral training is lengthy and requires focused attention on dissertation research, an initial concern was that students participating in additional complementary training activities might exhibit an increased time to degree or diminished research productivity. Metrics were analyzed from 10 NIH BEST awardee institutions to address this concern, using time to degree and publication records as measures of efficiency and productivity. Comparing doctoral students who participated to those who did not, results revealed that across these diverse academic institutions, there were no differences in time to degree or manuscript output. Our findings support the policy that doctoral students should participate in career and professional development opportunities that are intended to prepare them for a variety of diverse and important careers in the workforce.

Lymphatic Valves Separate Lymph Flow Into a Central Stream and a Slow-Moving Peri-Valvular Milieu.

The lymphatic system plays a pivotal role in the transport of fats, waste, and immune cells, while also serving as a metastatic route for select cancers. Using live imaging and particle tracking, we experimentally characterized the lymph flow field distal from the inguinal lymph node in the vicinity of normal bileaflet and malformed unileaflet intraluminal valves. Particle tracking experiments demonstrated that intraluminal lymphatic valves concentrate higher velocity lymph flow in the center of the vessel, while generating adjacent perivalvular recirculation zones. The recirculation zones are characterized by extended particle residence times and low wall shear stress (WSS) magnitudes in comparison to the rest of the lymphangion. A malformed unileaflet valve skewed lymph flow toward the endothelium on the vessel wall, generating a stagnation point and a much larger recirculation zone on the opposite wall. These studies define physical consequences of bileaflet and unileaflet intraluminal lymphatic valves that affect lymph transport and the generation of a heterogeneous flow field that affects the lymphatic endothelium nonuniformly. The characterized flow fields were recreated in vitro connecting different flow environments present in the lymphangion to a lymphatic endothelial cell (LEC) pro-inflammatory phenotype. Unique and detailed insight into lymphatic flow is provided, with potential applications to a variety of diseases that affect lymph transport and drug delivery.

Career planning courses increase career readiness of graduate and postdoctoral trainees.

Background: Given national calls for intentional career development during graduate and post-graduate scientific training, this study assessed career readiness development within the context of academic career courses. The current study evaluated the effects of academic career courses offered at two institutions that were specifically designed to increase career awareness, interest, and career-related confidence among doctoral students and postdoctoral fellows. Methods: Participants enrolled in a career course at trainees' respective academic institutions and responded to pre- and post-course surveys (n=32, n=148). The paper offers a thematic analysis of each of the two courses using an individualized learning plan career development framework and describes the results of their respective pretest-posttest evaluations which indicated increases in career readiness. Results: Though the format and content provided in each course varied, participation was associated with increases in career readiness. Participants reported increased career-awareness including a greater familiarity with different types of careers overall. Furthermore, interest in tenure track faculty careers increased in both samples, which may assuage fears that exposure to diverse career pathways could reduce interest in academic careers. Transferrable skills, including career planning and awareness also significantly increased. Course participants reported an increase in the number and type of mentors they interacted with beyond their principal faculty mentor (other faculty, professional PhDs, peers, and administrative staff). Conclusions: Findings provide supporting evidence for the benefits of implementing structured career development efforts during PhD training; even with varying content, delivery methods, and instructor type, both academic career courses led to significant gains in career awareness and readiness. Successful development and delivery of academic career courses, with a focus on career planning skills, suggest that institutions can utilize these and are an effective way to prepare PhDs for their transition from training positions into careers.

Hemodynamic regulation of perivalvular endothelial gene expression prevents deep venous thrombosis.

Deep venous thrombosis (DVT) and secondary pulmonary embolism cause approximately 100,000 deaths per year in the United States. Physical immobility is the most significant risk factor for DVT, but a molecular and cellular basis for this link has not been defined. We found that the endothelial cells surrounding the venous valve, where DVTs originate, express high levels of FOXC2 and PROX1, transcription factors known to be activated by oscillatory shear stress. The perivalvular venous endothelial cells exhibited a powerful antithrombotic phenotype characterized by low levels of the prothrombotic proteins vWF, P-selectin, and ICAM1 and high levels of the antithrombotic proteins thrombomodulin (THBD), endothelial protein C receptor (EPCR), and tissue factor pathway inhibitor (TFPI). The perivalvular antithrombotic phenotype was lost following genetic deletion of FOXC2 or femoral artery ligation to reduce venous flow in mice, and at the site of origin of human DVT associated with fatal pulmonary embolism. Oscillatory blood flow was detected at perivalvular sites in human veins following muscular activity, but not in the immobile state or after activation of an intermittent compression device designed to prevent DVT. These findings support a mechanism of DVT pathogenesis in which loss of muscular activity results in loss of oscillatory shear-dependent transcriptional and antithrombotic phenotypes in perivalvular venous endothelial cells, and suggest that prevention of DVT and pulmonary embolism may be improved by mechanical devices specifically designed to restore perivalvular oscillatory flow.

Investigating Effects of Fluid Shear Stress on Lymphatic Endothelial Cells.

Recent studies using in vivo models have characterized lymph flow and demonstrated that lymph flow plays a key role in the later stages of lymphatic vascular development, including vascular remodeling, to create a hierarchical collecting vessel network and lymphatic valves (Sweet et al., J Clin Invest 125, 2995-3007, 2015). However, mechanistic insights into the response of lymphatic endothelial cells to fluid flow are difficult to obtain from in vivo studies because of the small size of lymphatic vessels and the technical challenge of lymphatic endothelial cell isolation. On the other hand, in vitro experiments can be tailored to isolate and test specific mechanotransduction pathways more cleanly than conditions in vivo. To measure in vitro the cellular response to flow, cultured primary lymphatic endothelial cells can be exposed to highly specific fluid forces like those believed to exist in vivo. Such in vitro studies have recently helped identify FOXC2 and GATA2 as important transcriptional regulators of lymphatic function during valve formation that are regulated by lymph flow dynamics. This chapter discusses the methods used to expose primary lymphatic endothelial cells (LECs) to lymph fluid dynamics and the relationship of these in vitro studies to in vivo lymphatic biology.

An evidence-based evaluation of transferrable skills and job satisfaction for science PhDs.

PhD recipients acquire discipline-specific knowledge and a range of relevant skills during their training in the life sciences, physical sciences, computational sciences, social sciences, and engineering. Empirically testing the applicability of these skills to various careers held by graduates will help assess the value of current training models. This report details results of an Internet survey of science PhDs (n = 8099) who provided ratings for fifteen transferrable skills. Indeed, analyses indicated that doctoral training develops these transferrable skills, crucial to success in a wide range of careers including research-intensive (RI) and non-research-intensive (NRI) careers. Notably, the vast majority of skills were transferrable across both RI and NRI careers, with the exception of three skills that favored RI careers (creativity/innovative thinking, career planning and awareness skills, and ability to work with people outside the organization) and three skills that favored NRI careers (time management, ability to learn quickly, ability to manage a project). High overall rankings suggested that graduate training imparted transferrable skills broadly. Nonetheless, we identified gaps between career skills needed and skills developed in PhD training that suggest potential areas for improvement in graduate training. Therefore, we suggest that a two-pronged approach is crucial to maximizing existing career opportunities for PhDs and developing a career-conscious training model: 1) encouraging trainees to recognize their existing individual skill sets, and 2) increasing resources and programmatic interventions at the institutional level to address skill gaps. Lastly, comparison of job satisfaction ratings between PhD-trained employees in both career categories indicated that those in NRI career paths were just as satisfied in their work as their RI counterparts. We conclude that PhD training prepares graduates for a broad range of satisfying careers, potentially more than trainees and program leaders currently appreciate.

Predictors of Student Productivity in Biomedical Graduate School Applications.

Many US biomedical PhD programs receive more applications for admissions than they can accept each year, necessitating a selective admissions process. Typical selection criteria include standardized test scores, undergraduate grade point average, letters of recommendation, a resume and/or personal statement highlighting relevant research or professional experience, and feedback from interviews with training faculty. Admissions decisions are often founded on assumptions that these application components correlate with research success in graduate school, but these assumptions have not been rigorously tested. We sought to determine if any application components were predictive of student productivity measured by first-author student publications and time to degree completion. We collected productivity metrics for graduate students who entered the umbrella first-year biomedical PhD program at the University of North Carolina at Chapel Hill from 2008-2010 and analyzed components of their admissions applications. We found no correlations of test scores, grades, amount of previous research experience, or faculty interview ratings with high or low productivity among those applicants who were admitted and chose to matriculate at UNC. In contrast, ratings from recommendation letter writers were significantly stronger for students who published multiple first-author papers in graduate school than for those who published no first-author papers during the same timeframe. We conclude that the most commonly used standardized test (the general GRE) is a particularly ineffective predictive tool, but that qualitative assessments by previous mentors are more likely to identify students who will succeed in biomedical graduate research. Based on these results, we conclude that admissions committees should avoid over-reliance on any single component of the application and de-emphasize metrics that are minimally predictive of student productivity. We recommend continual tracking of desired training outcomes combined with retrospective analysis of admissions practices to guide both application requirements and holistic application review.

Diversity Exiting the Academy: Influential Factors for the Career Choice of Well-Represented and Underrepresented Minority Scientists.

A national sample of PhD-trained scientists completed training, accepted subsequent employment in academic and nonacademic positions, and were queried about their previous graduate training and current employment. Respondents indicated factors contributing to their employment decision (e.g., working conditions, salary, job security). The data indicate the relative importance of deciding factors influencing career choice, controlling for gender, initial interest in faculty careers, and number of postgraduate publications. Among both well-represented (WR; n = 3444) and underrepresented minority (URM; n = 225) respondents, faculty career choice was positively associated with desire for autonomy and partner opportunity and negatively associated with desire for leadership opportunity. Differences between groups in reasons endorsed included: variety, prestige, salary, family influence, and faculty advisor influence. Furthermore, endorsement of faculty advisor or other mentor influence and family or peer influence were surprisingly rare across groups, suggesting that formal and informal support networks could provide a missed opportunity to provide support for trainees who want to stay in faculty career paths. Reasons requiring alteration of misperceptions (e.g., limited leadership opportunity for faculty) must be distinguished from reasons requiring removal of actual barriers. Further investigation into factors that affect PhDs' career decisions can help elucidate why URM candidates are disproportionately exiting the academy.

Preparing Postbaccalaureates for Entry and Success in Biomedical PhD Programs.

Certain racial and ethnic groups, individuals with disabilities, and those from low socioeconomic backgrounds remain underrepresented (UR) in the biomedical sciences. This underrepresentation becomes more extreme at each higher education stage. To support UR scholars during the critical transition from baccalaureate to PhD, we established an intensive, 1-yr postbaccalaureate training program. We hypothesized that this intervention would strengthen each participant's competitiveness for leading PhD programs and build a foundation of skills and self-efficacy important for success during and after graduate school. Scholar critical analysis skills, lab technique knowledge, and Graduate Record Examination scores all improved significantly during the program. Scholars reported significant confidence growth in 21 of 24 categories related to success in research careers. In 5 yr, 91% (41/45) of scholars transitioned directly into PhD programs. Importantly, 40% (18/45) of participating postbaccalaureate scholars had previously been declined acceptance into graduate school; however, 17/18 of these scholars directly entered competitive PhD programs following our training program. Alumni reported they were "extremely well" prepared for graduate school, and 95% (39/41) are currently making progress to graduation with a PhD. In conclusion, we report a model for postbaccalaureate training that could be replicated to increase participation and success among UR scholars in the biomedical sciences.

A phenotype at last: essential role for the Yersinia enterocolitica Ysa type III secretion system in a Drosophila melanogaster S2 cell model.

The highly pathogenic Yersinia enterocolitica strains have a chromosomally encoded type III secretion system (T3SS) that is expressed and functional in vitro only when the bacteria are cultured at 26 °C. Mutations that render this system nonfunctional are slightly attenuated in the mouse model of infection only following an oral inoculation and only at early time points postinfection. The discrepancy between the temperature required for the Ysa gene expression and the physiological temperature required for mammalian model systems has made defining the role of this T3SS challenging. Therefore, we explored the use of Drosophila S2 cells as a model system for studying Ysa function. We show here that Y. enterocolitica is capable of infecting S2 cells and replicating intracellularly to high levels, an unusual feature of this pathogen. Importantly, we show that the Ysa T3SS is required for robust intracellular replication. A secretion-deficient mutant lacking the secretin gene, ysaC, is defective in replication within S2 cells, marking the first demonstration of a pronounced Ysa-dependent virulence phenotype. Establishment of S2 cells as a model for Y. enterocolitica infection provides a versatile tool to elucidate the role of the Ysa T3SS in the life cycle of this gastrointestinal pathogen.

Staphylococcus aureus α-hemolysin mediates virulence in a murine model of severe pneumonia through activation of the NLRP3 inflammasome.

Staphylococcus aureus is a dangerous pathogen that can cause necrotizing infections characterized by massive inflammatory responses and tissue destruction. Staphylococcal α-hemolysin is an essential virulence factor in severe S. aureus pneumonia. It activates the nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 (NLRP3) inflammasome to induce production of interleukin-1ß and programmed necrotic cell death. We sought to determine the role of α-hemolysin-mediated activation of NLRP3 in the pathogenesis of S. aureus pneumonia. We show that α-hemolysin activates the NLRP3 inflammasome during S. aureus pneumonia, inducing necrotic pulmonary injury. Moreover, Nlrp3(-/-) mice have less-severe pneumonia. Pulmonary injury induced by isolated α-hemolysin or live S. aureus is independent of interleukin-1ß signaling, implicating NLRP3-induced necrosis in the pathogenesis of severe infection. This work demonstrates the exploitation of host inflammatory signaling by S. aureus and suggests the NLRP3 inflammasome as a potential target for pharmacologic interventions in severe S. aureus infections.

Protective effects of the carotenoid zeaxanthin in experimental nonalcoholic steatohepatitis.

Fat infiltration and inflammation cause liver injury and fibrosis and may progress to nonalcoholic steatohepatitis (NASH) and end-stage liver disease. Currently, there are no effective treatments for NASH. Zeaxanthin is a carotenoid which has been shown to be preferentially accumulated in the adipose tissue and liver. We hypothesized that treatment with zeaxanthin may decrease oxidative stress in the liver and, possibly, halt the inflammation and fibrosis associated with NASH. Here we tested zeaxanthin effects in preventing progression of liver injury in a model of NASH. Mongolian gerbils, fed a methionine-choline-deficient diet, were treated with different doses of zeaxanthin. We assessed histopathological changes by hematoxylin-eosin and Masson trichrome staining and determined oxidative stress by measuring lipid peroxidation. The obtained results show that zeaxanthin significantly prevented NASH progression by decreasing oxidative stress and liver fibrosis, thus suggesting a potential therapeutic application for this carotenoid in the management of NASH.

Presence of lymph node vasculature: a new EUS criterion for benign nodes?

OBJECTIVES: Lymph nodes normally have prominent centrally located blood vessels, which may become obliterated with tumor infiltration. The presence of intranodal vasculature has been noted to coincide with benign cytology. We sought to determine the test characteristics of the presence of intranodal mediastinal vasculature during endoscopic ultrasound (EUS). METHODS: 67 mediastinal lymph nodes evaluated by EUS in 66 patients over a 1-year period were evaluated for the presence of intranodal vasculature, which was considered benign when it traversed through the node without disruption. RESULTS: Of the 67 lymph nodes evaluated, 29 (43%) were found to be malignant on cytopathologic review. Benign vascular markings were present in 15/67 (22.4%) lymph nodes evaluated. All 15 (100%) of these nodes were found to have benign fine-needle aspiration (FNA) results. The presence of benign vasculature had a negative predictive value of 100%. CONCLUSIONS: The presence of intranodal vasculature was universally associated with a benign diagnosis. The addition of this EUS finding improves the ability to characterize lymph nodes and predict the likelihood of malignant involvement.

Infected-host-cell repertoire and cellular response in the lung following inhalation of Francisella tularensis Schu S4, LVS, or U112.

Francisella tularensis causes systemic disease in humans and other mammals, with high morbidity and mortality associated with inhalation-acquired infection. F. tularensis is a facultative intracellular pathogen, but the scope and significance of cell types infected during disease is unknown. Using flow cytometry, we identified and quantified infected-cell types and assessed the impact of infection on cell populations following inhalation of F. tularensis strains U112, LVS, and Schu S4. Initially, alveolar macrophages comprised over 70% of Schu S4- and LVS-infected cells, whereas approximately 51% and 27% of U112-infected cells were alveolar macrophages and neutrophils, respectively. After 3 days, roughly half of Schu S4- and LVS- and nearly 80% of U112-infected cells were neutrophils. All strains infected CD11b(high) macrophages, dendritic cells, monocytes, and alveolar type II cells throughout infection. Macrophage, monocyte, and dendritic-cell populations were reduced during U112 infection but not Schu S4 or LVS infection. These results demonstrate directly that F. tularensis is a promiscuous intracellular pathogen in the lung that invades and replicates within cell types ranging from migratory immune cells to structural tissue cells. However, the proportions of cell types infected and the cellular immune response evoked by the human pathogenic strain Schu S4 differ from those of the human avirulent U112.

RipA, a cytoplasmic membrane protein conserved among Francisella species, is required for intracellular survival.

Francisella tularensis is a highly virulent bacterial pathogen that invades and replicates within numerous host cell types, including macrophages and epithelial cells. In an effort to better understand this process, we screened a transposon insertion library of the F. tularensis live vaccine strain (LVS) for mutant strains that invaded but failed to replicate within alveolar epithelial cell lines. One such strain isolated from this screen contained an insertion in the gene FTL_1914, which is conserved among all sequenced Francisella species yet lacks significant homology to any gene with known function. A deletion strain lacking FTL_1914 was constructed. This strain did not replicate in either epithelial or macrophage-like cells, and intracellular replication was restored by the wild-type allele in trans. Based on the deletion mutant phenotype, FTL_1914 was termed ripA (required for intracellular proliferation, factor A). Following uptake by J774.A1 cells, F. tularensis LVS Delta ripA colocalized with LAMP-1 then escaped the phagosome at the same rate and frequency as wild-type LVS-infected cells. Electron micrographs of the F. tularensis LVS Delta ripA mutant demonstrated the reentry of the mutant bacteria into double membrane vacuoles characteristic of autophagosomes in a process that was not dependent on replication. The F. tularensis LVS Delta ripA mutant was significantly impaired in its ability to persist in the lung and in its capacity to disseminate and colonize the liver and spleen in a mouse model of pulmonary tularemia. The RipA protein was expressed during growth in laboratory media and localized to the cytoplasmic membrane. Thus, RipA is a cytoplasmic membrane protein conserved among Francisella species that is required for intracellular replication within the host cell cytoplasm as well as disease progression, dissemination, and virulence.

Expression of the Helicobacter pylori adhesin SabA is controlled via phase variation and the ArsRS signal transduction system.

Adaptation to the acidic microenvironment, and adherence to mucosal epithelium, are essential for persistent colonization of the human stomach by Helicobacter pylori. The expression of SabA, an adhesin implicated in the ability of H. pylori to adhere to the host gastric epithelium, can be modulated by phase variation via slipped-strand mispairing in repetitive nucleotide tracts located in both the promoter region and the coding region. This study demonstrates the occurrence of phase variation at the sabA locus within individual strains of H. pylori, and among multiple isolates from a single patient. In addition, transcription of sabA is repressed by the acid-responsive ArsRS two-component signal transduction system in vitro. Our results demonstrate that isogenic inactivation of the arsS (jhp0151/HP0165) histidine kinase locus results in a 10-fold SabA-dependent increase in adherence to gastric epithelial cells in strain J99 (contains an in-frame sabA allele), but not in strain 26695 (out-of-frame sabA allele). The combination of transcriptional regulation of the sabA locus by the ArsRS two-component signal-transduction system and the generation of subpopulations harbouring alternate sabA alleles by slipped-strand mispairing during chromosomal replication could permit H. pylori to rapidly adapt to varying microenvironments or host immune responses. As a pathogen with a paucity of regulatory proteins, this dual regulation indicates that SabA expression is a tightly regulated process in H. pylori infection.

Francisella tularensis invasion of lung epithelial cells.

Francisella tularensis, a gram-negative facultative intracellular bacterial pathogen, causes disseminating infections in humans and other mammalian hosts. Macrophages and other monocytes have long been considered the primary site of F. tularensis replication in infected animals. However, recently it was reported that F. tularensis also invades and replicates within alveolar epithelial cells following inhalation in a mouse model of tularemia. TC-1 cells, a mouse lung epithelial cell line, were used to study the process of F. tularensis invasion and intracellular trafficking within nonphagocytic cells. Live and paraformaldehyde-fixed F. tularensis live vaccine strain organisms associated with, and were internalized by, TC-1 cells at similar frequencies and with indistinguishable differences in kinetics. Inhibitors of microfilament and microtubule activity resulted in significantly decreased F. tularensis invasion, as did inhibitors of phosphatidylinositol 3-kinase and tyrosine kinase activity. Collectively, these results suggest that F. tularensis epithelial cell invasion is mediated by a preformed ligand on the bacterial surface and driven entirely by host cell processes. Once internalized, F. tularensis-containing endosomes associated with early endosome antigen 1 (EEA1) followed by lysosome-associated membrane protein 1 (LAMP-1), with peak coassociation frequencies occurring at 30 and 120 min postinoculation, respectively. By 2 h postinoculation, 70.0% (+/- 5.5%) of intracellular bacteria were accessible to antibody delivered to the cytoplasm, indicating vacuolar breakdown and escape into the cytoplasm.