BackgroundEvery curriculum needs to be reviewed, implemented and evaluated; it must also comply with the regulatory standards. This report demonstrates the value of curriculum mapping (CM), which shows the spatial relationships of a curriculum, in developing and managing an integrated medical curriculum.MethodsA new medical school developed a clinical presentation driven integrated curriculum that incorporates the active-learning pedagogical practices of many educational institutions worldwide while adhering to the mandated requirements of the accreditation bodies. A centralized CM process was run in parallel as the curriculum was being developed. A searchable database, created after the CM data was uploaded into an electronic curriculum management system, was used to ensure placing, integrating, evaluating and revising the curricular content appropriately.ResultsCM facilitated in a) appraising the content integration, b) identifying gaps and redundancies, c) linking learning outcomes across all educational levels (i.e. session to course to program), c) organizing the teaching schedules, instruction methods, and assessment tools and d) documenting compliance with accreditation standards.ConclusionsCM is an essential tool to develop, review, improve and refine any integrated curriculum however complex. Our experience, with appropriate modifications, should help other medical schools efficiently manage their curricula and fulfill the accreditation requirements at the same time.

Mannheimia haemolytica is a key pathogen in the bovine respiratory disease complex. It produces a leukotoxin (LKT) that is an important virulence factor, causing cell death in bovine leukocytes. The LKT binds to the ␤ 2 integrin CD11a/CD18, which usually activates signaling pathways that facilitate cell survival. In this study, we investigated mechanisms by which LKT induces death in bovine lymphoblastoid cells (BL-3). Incubation of BL-3 cells with a low concentration of LKT results in the activation of caspase-3 and caspase-9 but not caspase-8. Similarly, the proapoptotic proteins Bax and BAD were significantly elevated, while the antiapoptotic proteins Bcl-2, Bcl XL and Akt-1 were downregulated. Following exposure to LKT, we also observed a reduction in mitochondrial cytochrome c and corresponding elevation of cytosolic cytochrome c, suggesting translocation from the mitochondrial compartment to the cytosol. Consistent with this observation, tetramethylrhodamine ethyl ester perchlorate staining revealed that mitochondrial membrane potential was significantly reduced. These data suggest that LKT induces apoptosis of BL-3 cells via a caspase-9-dependent mitochondrial pathway. Furthermore, scanning electron micrographs of mitochondria from LKT-treated BL-3 cells revealed lesions in the outer mitochondrial membrane, which are larger than previous reports of the permeability transition pore through which cytochrome c is usually released.Mannheimia (Pasteurella) haemolytica is the principal bacterial pathogen of the bovine respiratory disease complex (23,49). Among the most important virulence factors produced by this pathogen is a 104-kDa leukotoxin (LKT) that both activates and kills bovine leukocytes (2, 41). The M. haemolytica LKT is a member of the "repeats in toxin" (RTX) family of gram-negative bacterial exotoxins (34). It has been demonstrated that M. haemolytica LKT binds to the ␤ 2 integrin CD11a/CD18 (also known as leukocyte functional antigen 1 [LFA-1]) (2, 15, 21). Paradoxically, LFA-1 is usually a survival receptor whose signaling pathways protect cells against death (31,37,41). It is not clear how binding of LKT to LFA-1 results in apoptosis of bovine leukocytes.The molecular mechanisms leading to apoptosis are complex. There are two major pathways that initiate apoptosis in cells (3,7,14). The first involves ligand binding to a death receptor (e.g., Fas or tumor necrosis factor receptor) that initiates a signaling pathway, which activates caspase-8 (5, 19). The second pathway involves mitochondrial activation of caspase-9. Both activated caspase-8 and caspase-9 can then activate the effector caspase-3 that cleaves macromolecules, leading to apoptotic cell death (35,42). The caspase-9-dependent mitochondrial pathway is induced by cellular stress, which results in deployment of an array of proapoptotic members of the Bcl-2 family of proteins (e.g., BAD and Bax) to the outer mitochondrial membrane (OMM) (4,52,54). This destabilizes the membrane, resulting in pore formation and release of cytochrome c (c...

Mannheimia (Pasteurella) haemolytica A1 is the primary bacterial agent of bovine pneumonic pasteurellosis (shipping fever), which is characterized by acute lobar fibronecrotizing pneumonia with extensive peripheral blood neutrophil (PMN) infiltration in small airways and alveoli (4,39,47). Several virulence factors of M. haemolytica play an important role in the pathogenesis of pasteurellosis (7, 13). Foremost among these is a leukotoxin (LKT), whose effects are specific for ruminant leukocytes and platelets (2, 6, 9, 44). The M. haemolytica LKT is member of the repeats-in-toxin (RTX) family of gram-negative bacterial pore-forming exotoxins (46). Members of the RTX family have similar mechanisms of toxin production, secretion, and target cell intoxication (8, 45). Previously, it has been reported that other members of the RTX family bind to ␤ 2 -integrins on target cells (23). More recently, it has been demonstrated that M. haemolytica LKT binds to lymphocyte function-associated antigen 1 (LFA-1), a ␤ 2 -integrin (CD 11a/CD18) on bovine leukocytes (1,17,25,27). LKT binding to bovine leukocytes induces formation of pore-like structures in the plasma membrane, resulting in both activation of leukocytes and death by necrosis and apoptosis (14,18,24,29,34,40,43,45,53).For reasons that are not well understood, active viral infections can greatly enhance the susceptibility of cattle to M. haemolytica pneumonia (11,28,42,48,49). One mechanism that might be involved is the release of inflammatory cytokines during viral infection (33, 34). Inflammatory cytokines secreted by respiratory tract cells, such as interleukin 1 (IL-1␤), tumor necrosis factor alpha (TNF-␣), and gamma interferon (IFN-␥), can stimulate leukocyte migration and functional activation of ␤ 2 -integrins on lung leukocytes (10,35,38). Once M. haemolytica infection is established in the lung, the continued release of these inflammatory cytokines could be sustained by M. haemolytica virulence factors (i.e., LKT and lipopolysaccharide [LPS]) (15,21,22,30,50,51,52).PMNs are thought to contribute to the lung pathology observed in pneumonic pasteurellosis (4). PMN depletion reduces the severity of lung damage in experimentally infected cattle (4, 39). We hypothesized that inflammatory cytokines released during viral infection might increase surface expression or conformational activation of LFA-1 on bovine PMNs, thus amplifying their interaction with M. haemolytica LKT. In this study, we demonstrated increased expression of LFA-1 on bovine PMNs, as detected by flow cytometry, following incubation of PMNs with IL-1␤, TNF-␣, or IFN-␥. This in turn was reflected in increased LKT binding to, and cytotoxicity for, bovine PMNs. These observations suggest that the ability of inflammatory cytokines to increase surface expression or conformational activation of LFA-1 on bovine PMNs increases their interaction with M. haemolytica LKT. The outcome of the response might increase the severity of bovine pasteurellosis. MATERIALS AND METHODSPMN preparation. Peripheral blood ...

Bovine respiratory disease (BRD) often occurs when active respiratory virus infections (BHV-1, etc.) impair resistance to Mannheimia haemolytica infection in the lower respiratory tract. The interactions that occur when the respiratory epithelium encounters these viral and bacterial pathogens are poorly understood. We used Agilent bovine gene microarray chips containing 44,000 transcripts to elucidate bovine bronchial epithelial cell (BBEC) responses following in vitro exposure to BHV-1 alone, M. haemolytica alone, or both BHV-1 and M. haemolytica. Microarray analysis revealed differential regulation (>2-fold) of 978 transcripts by BHV-1 alone, 2040 transcripts by M. haemolytica alone, and 2189 genes by BHV-1 and M. haemolytica in combination. M. haemolytica treatment produced significantly greater inductions (>10-fold) of several inflammation associated genes, such as CXCL2, IL-6, IL-1α, e-selectin, and IL-8, than to BHV-1 alone. Functional analysis of the microarray data revealed a significant upregulation of genes involved in important biological processes such as inflammation (TNF-α, IL-8, Tlr-2, IL-1, CXCL2, CSF2), vascular functions (VEGF, EDN2) and leukocyte migration (ICAM1, IL-16) during a co-infection with BHV-1 and M. haemolytica compared to either pathogen alone. This study provides evidence to support that lung epithelial cells are a source of mediators that may promote inflammatory changes observed during bovine respiratory disease.

Exotoxins which belong to the family containing the RTX toxins (repeats in toxin) contribute to a variety of important human and animal diseases. One example of such a toxin is the potent leukotoxin (LKT) produced by the bovine respiratory pathogen Mannheimia haemolytica. LKT binds to CD18, resulting in the death of bovine leukocytes. In this study, we showed that internalized LKT binds to the outer mitochondrial membrane, which results in the release of cytochrome c and collapse of the mitochondrial membrane potential ( M. haemolytica is the principal bacterial pathogen in the bovine respiratory disease complex (19, 58). The most important virulence factor of M. haemolytica is the LKT, which has potent cytotoxic effects on ruminant leukocytes but not on leukocytes from other species (38). M. haemolytica LKT is a member of the RTX toxin (repeats in toxin) family, which includes LKTs and hemolysins produced by a number of gramnegative bacteria. It was originally thought that M. haemolytica LKT damages cells by inserting into the cell membrane, resulting in pore formation and necrosis (53). At lower LKT concentrations, however, susceptible cells die via caspase-dependent apoptotic pathways (8,10,45,46). LKT was first reported to bind to the ␤ 2 integrin CD18/CD11a, also known as leukocyte functional antigen 1 (LFA-1) (1,24,28). Recent studies, however, have shown that Mac-1 (CD18/CD11b) also binds LKT and that CD18 is the functional receptor for LKT (9, 30). After binding to LFA-1, LKT induces apoptosis of bovine leukocytes as a result of activation of caspase 1, caspase 3, and caspase 9 (4, 10, 31, 46). The LFA-1-dependent cytotoxicity of LKT is a conundrum, because signaling through LFA-1 generally results in cell adhesion and promotes cell survival (34,44,51).In this study we hypothesized that mitochondrial damage and caspase 9 activation could be due to direct binding of LKT to the mitochondrial outer membrane (MOM). In this paper we show that LKT directly targets mitochondria in BL-3 cells, creating lesions in the MOM that can lead to release of proapoptotic proteins, culminating in cell death. We also show that LKT targeting to mitochondria is dynamin-2 dependent and that mitochondria appear to be the principal source of dynamin-2 in BL-3 cells. Treating BL-3 cells with cyclosporine (CSA) depleted mitochondrial dynamin-2, thereby inhibiting LKT transport to mitochondria and cell death. MATERIALS AND METHODSLKT production and purification. Crude LKT was prepared and purified as described previously and was stored at Ϫ80°C until it was used in experiments (4). Inactive toxin from an lktC mutant of M. haemolytica strain A1 (SH 1562), which produces an LKT protein with no biological activity (LKT inact ) (generously provided by S. K. Highlander, Baylor College of Medicine, Houston, TX), was prepared in a similar manner.Cell lines, cell cultures, and antibodies. Nonadherent bovine lymphoblastoid cells (BL-3 cells) and adherent murine macrophages (RAW 264.7 cells) (kindly provided by Ronald Schultz, University of...