Implementation study of a 5-component pediatric early warning system (PEWS) in an emergency department in British Columbia, Canada, to inform provincial scale up.

BACKGROUND: The rapid identification of deterioration in the pediatric population is complex, particularly in the emergency department (ED). A comprehensive multi-faceted Pediatric Early Warning System (PEWS) might maximize early recognition of clinical deterioration and provide a structured process for the reassessment and escalation of care. The objective of the study was to evaluate the implementation fidelity, effectiveness, and utility of a 5-component PEWS implemented in the ED of an urban public general hospital in British Columbia, Canada, and to guide provincial scale up. METHODS: We used a before-and-after design to evaluate the implementation fidelity, effectiveness, and utility of a 5-component PEWS (pediatric assessment flowsheet, PEWS score, situational awareness, escalation aid, and communication framework). Sources of data included patient medical records, surveys of direct care staff, and key-informant interviews. Data were analyzed using mixed-methods approaches. RESULTS: The majority of medical records had documented PEWS scores at triage (80%) and first bedside assessment (81%), indicating that the intervention was implemented with high fidelity. The intervention was effective in increasing vital signs documentation, both at first beside assessment (84% increase) and throughout the ED stay (> 100% increase), in improving staff's self-perceived knowledge and confidence in providing pediatric care, and self-reported communication between staff. Satisfaction levels were high with the PEWS scoring system, flowsheet, escalation aid, and to a lesser extent with the situational awareness tool and communication framework. Reasons for dissatisfaction included increased paperwork and incidence of false-positives. Overall, the majority of providers indicated that implementation of PEWS and completing a PEWS score at triage alongside the Canadian Triage and Acuity Scale (CTAS) added value to pediatric care in the ED. Results also suggest that the intervention is aligned with current practice in the ED. CONCLUSION: Our study shows that high-fidelity implementation of PEWS in the ED is feasible. We also show that a multi-component PEWS can be effective in improving pediatric care and be well-accepted by staff. Results and lessons learned from this pilot study are being used to scale up implementation of PEWS in ED settings across the province of British Columbia.

Continuing Trauma Care After Discharge, the Experience of Outpatient Trauma Clinics in British Columbia.

The recovery process from traumatic injuries, and the potential for complications, extends beyond the time of hospital discharge. In 2014, the Fraser Health Trauma Network established outpatient clinics to provide follow-up care for trauma patients after discharge from hospital. The following research questions were asked: Which services were commonly performed by our trauma clinics and how satisfied were patients with the care they received at our clinics? A survey was distributed to patients after their clinic visit to assess overall satisfaction and areas for improvement. A retrospective medical record review was performed to illustrate and quantify the interventions provided during clinic visits. During the first 22 months of clinic operation, a total of 412 appointments were scheduled and the attendance rate was 88%. The provided services included obtaining additional imaging (41% of visits), providing wound and brace care (16%), and initiating referrals to specialists (12%). Seventy-seven patient satisfaction surveys were returned during the study period, 34 in 2014 and 43 in 2015. Seventy-four percent of respondents strongly agreed, and 21% agreed that they were satisfied with the care they received in the clinic. Ninety percent found their visit helpful, and only 10% reported having additional medical issues that were not addressed during the appointment. At trauma clinic follow-up, discharged patients have ongoing care requirements, including a need for further investigation, specialist referral, and wound or brace issues that are likely to benefit from specialist trauma care. Patients were satisfied with the care provided by a postdischarge trauma clinic.

Leishmania exosomes and other virulence factors: Impact on innate immune response and macrophage functions.

Leishmania parasites are the causative agents of the leishmaniases, a collection of vector-borne diseases that range from simple cutaneous to fatal visceral forms. Employing potent immune modulation mechanisms, Leishmania is able to render the host macrophage inactive and persist inside its phagolysosome. In the last few years, the role of exosomes in Leishmania-host interactions has been increasingly investigated. For instance, it was reported that Leishmania exosome release is augmented following temperature shift, a condition mimicking parasite's entry into its mammalian host. Leishmania exosomes were found to strongly affect macrophage cell signaling and functions, similarly to whole parasites. Importantly, these vesicles were shown to be pro-inflammatory, capable to recruit neutrophils at their inoculation site exacerbating the pathology. In this review, we provide the most recent insights on the role of exosomes and other virulence factors, especially the surface protease GP63, in Leishmania-host interactions, deepening our knowledge on leishmaniasis and paving the way for the development of new therapeutics.

Rapid Implementation and Evaluation of Virtual Health Training in a Subspecialty Hospital in British Columbia, in Response to the COVID-19 Pandemic.

Introduction: Adoption of virtual health (VH) solutions in healthcare has been challenging; this changed rapidly after implementation of physical distancing measures due to the COVID-19 pandemic. In response to the pandemic, British Columbia's Children's and Women's sub-specialty hospitals rapidly trained and scaled up support to equip staff and clinicians to use VH. Methods: Ninety-minute live online training workshops and frequently updated online support materials were offered for 6 weeks. Training was monitored via feedback collected at training sessions and a brief post-training survey. After training completion, a second survey was circulated to measure utilization outcomes and experiences with VH. Results: Eight hundred and ninety-five participants representing 82% of staff requiring support were trained through 101 sessions; 348 (38.9%) and 272 (30.4%) responses were collected for the monitoring and outcome surveys, respectively. Overall, 89% agreed that training was relevant to their needs; participants indicated average 58.1% (SD = 26.6) and 60.6% (SD = 25.2) increase in knowledge and confidence in VH after training; 90.1% had booked or conducted VH sessions. Increase in confidence was more pronounced in participants with lesser previous exposure to VH, but number of sessions conducted post-training and percentage of successful sessions were independent of previous exposure. For future training and support, participants suggested subject-tailored trainings, asynchronous trainings, and availability of experienced users. Discussion: Training is key to success of VH implementation. Moving forward, core competencies in VH should be developed to support standardization and allow for evaluation and quality improvement. Incorporation of VH training in continuous professional development and onboarding is also highly recommended.

Comparative study of the ability of Leishmania mexicana promastigotes and amastigotes to alter macrophage signaling and functions.

Leishmania alternates between two morphologically different stages, promastigotes and amastigotes. While the majority of reports focused on how the promastigote form can alter macrophage (Mphi) signaling and function, fewer reports investigated signaling alterations mediated by amastigotes, and there is a lack of comparative studies. In this study, we performed a comparison between the ability of both forms of the parasite to alter Mphi signaling and functions. Here, we show that both promastigotes and amastigotes were able to rapidly activate host protein tyrosine phosphatases (PTPs), importantly the Src homology 2 domain-containing PTP (SHP-1). However, we found that PTP-1B is specifically activated by promastigote but not amastigote infection and that lmcpb(-/-) promastigotes were no longer able to activate PTP-1B. We also show a similarity in the way promastigotes and amastigotes inactivate the transcription factors (TFs) STAT-1alpha and AP-1, but we show differences in the modulation of NF-kappaB, with promastigotes cleaving the p65 subunit, generating a smaller p35 subunit, and amastigotes fully degrading the p65 subunit with no p35 production. Importantly, we show that the cysteine proteinase LmCPb plays a key role in the alteration of NF-kappaB, STAT-1alpha, and AP-1 by promastigote and amastigote infections, ultimately leading to the inability of these TFs to translocate to the nucleus in response to gamma interferon (IFN-gamma) stimulation and thus contributing to the ability of both parasite forms to effectively block IFN-gamma-mediated nitric oxide (NO) production in Mphis.

Implementing Appetite to Play at scale in British Columbia: Evaluation of a Capacity-Building Intervention to Promote Physical Activity in the Early Years.

Childcare is a critical target for promoting children's physical activity (PA) and physical literacy (PL). With emerging evidence about the efficacy of policy and capacity-building strategies, more information about how to bring these strategies to scale is needed. This paper describes implementation at scale of Appetite to Play (ATP), a capacity-building intervention for childcare providers, and examines the implementation and impact on early years providers' capacity to address PA. The ATP implementation evaluation was a natural experiment that utilized a mixed methods concurrent parallel design framed within the Reach, Effectiveness, Adoption, Implementation, Maintenance framework (RE-AIM). Workshop and website tracking assessed reach and adoption. Surveys and interviews with workshop participants and stakeholders assessed satisfaction, implementation, and maintenance. Training reached 60% of British Columbia municipalities and 2700 early years providers. Significant changes in participants' knowledge and confidence to promote PA and PL were achieved (p >0.01-0.001). Childcare level implementation facilitators as reported by early years providers included appropriate resources, planning, indoor space, and equipment, whereas weather and space were reported barriers. The stakeholder advisory group viewed the stakeholder network and Active Play policy as facilitators and adjustments to recent shifts in childcare funding and previous initiatives as barriers to implementation. ATP was scalable and impacted provider knowledge, confidence, and intentions. The impact on actual policies and practices, and children's PA needs to be assessed along with sustainability.

Capacity building and mentorship among pan-Canadian early career researchers in community-based primary health care.

AIM: To describe activities and outcomes of a cross-team capacity building strategy that took place over a five-year funding period within the broader context of 12 community-based primary health care (CBPHC) teams. BACKGROUND: In 2013, the Canadian Institutes of Health Research funded 12 CBPHC Teams (12-Teams) to conduct innovative cross-jurisdictional research to improve the delivery of high-quality CBPHC to Canadians. This signature initiative also aimed to enhance CBPHC research capacity among an interdisciplinary group of trainees, facilitated by a collaboration between a capacity building committee led by senior researchers and a trainee-led working group. METHODS: After the committee and working group were established, capacity building activities were organized based on needs and interests identified by trainees of the CBPHC Teams. This paper presents a summary of the activities accomplished, as well as the outcomes reported through an online semistructured survey completed by the trainees toward the end of the five-year funding period. This survey was designed to capture the capacity building and mentorship activities that trainees either had experienced or would like to experience in the future. Descriptive and thematic analyses were conducted based on survey responses, and these findings were compared with the existing core competencies in the literature. FINDINGS: Since 2013, nine webinars and three online workshops were hosted by trainees and senior researchers, respectively. Many of the CBPHC Teams provided exposure for trainees to innovative methods, CBPHC content, and showcased trainee research. A total of 27 trainees from 10 of the 12-Teams responded to the survey (41.5%). Trainees identified key areas of benefit from their involvement in this initiative: skills training, networking opportunities, and academic productivity. Trainees identified gaps in research and professional skill development, indicating areas for further improvement in capacity building programs, particularly for trainees to play a more active role in their education and preparation.

Disturbed gut microbiota and bile homeostasis in Giardia-infected mice contributes to metabolic dysregulation and growth impairment.

Although infection with the human enteropathogen Giardia lamblia causes self-limited diarrhea in adults, infant populations in endemic areas experience persistent pathogen carriage in the absence of diarrhea. The persistence of this protozoan parasite in infants has been associated with reduced weight gain and linear growth (height-for-age). The mechanisms that support persistent infection and determine the different disease outcomes in the infant host are incompletely understood. Using a neonatal mouse model of persistent G. lamblia infection, we demonstrate that G. lamblia induced bile secretion and used the bile constituent phosphatidylcholine as a substrate for parasite growth. In addition, we show that G. lamblia infection altered the enteric microbiota composition, leading to enhanced bile acid deconjugation and increased expression of fibroblast growth factor 15. This resulted in elevated energy expenditure and dysregulated lipid metabolism with reduced adipose tissue, body weight gain, and growth in the infected mice. Our results indicate that this enteropathogen's modulation of bile acid metabolism and lipid metabolism in the neonatal mouse host led to an altered body composition, suggesting how G. lamblia infection could contribute to growth restriction in infants in endemic areas.

Regulation of macrophage nitric oxide production by the protein tyrosine phosphatase Src homology 2 domain phosphotyrosine phosphatase 1 (SHP-1).

Nitric oxide (NO) is a potent molecule involved in the cytotoxic effects mediated by macrophages (MØ) against microorganisms. We previously reported that Src homology 2 domain phosphotyrosine phosphatase 1 (SHP-1)-deficient cells generate a greater amount of NO than wild-type cells in response to interferon-gamma (IFN-gamma). We also reported that the Leishmania-induced MØ SHP-1 activity is needed for the survival of the parasite within phagocytes through the attenuation of NO-dependent and NO-independent mechanisms. In the present study, we investigated the role of SHP-1 in regulating key signalling molecules important in MØ NO generation. Janus tyrosine kinase 2 (JAK2), mitogen-activated extracellular signal-regulated protein kinase kinase (MEK), extracellular signal-regulated kinases 1 and 2 (Erk1/Erk2) mitogen-activated protein kinases, p38 and stress-activated mitogen-activated protein kinases/c-Jun NH(2)-terminal kinase (SAPK/JNK) were examined in immortalized bone marrow-derived MØ (BMDM) from both SHP-1-deficient motheaten mice (me-3) and their respective littermates (LM-1). The results indicated that Erk1/Erk2 and SAPK/JNK are the main kinases regulated by SHP-1 because the absence of SHP-1 caused an increase in their phosphorylation. Moreover, only Apigenin, the specific inhibitor of Erk1/Erk2, was able to block IFN-gamma-induced inducible nitric oxide synthase (iNOS) transcription and translation in me-3 cells. Transcription factor analyses revealed that in the absence of SHP-1, activator protein-1 (AP-1) was activated. The activation of AP-1, and not nuclear factor-kappaB (NF-kappaB) or signal transducer and activator of transcription-1 alpha (STAT-1 alpha), may explain the enhanced NO generation in SHP-1-deficient cells. These observations emphasize the involvement of the MAPKs Erk1/Erk2 and SAPK/JNK in NO generation via AP-1 activation. Collectively, our findings suggest that SHP-1 plays a pivotal role in the negative regulation of signalling events leading to iNOS expression and NO generation. Furthermore, our observations underline the importance of SHP-1-mediated negative regulation in maintaining NO homeostasis and thus preventing the abnormal generation of NO that can be detrimental to the host.

Contextual factors influencing the implementation of innovations in community-based primary health care: the experience of 12 Canadian research teams.

The objectives of this paper are to: (1) identify contextual factors such as policy that impacted the implementation of community-based primary health care (CBPHC) innovations among 12 Canadian research teams and (2) describe strategies used by the teams to address contextual factors influencing implementation of CBPHC innovations. In primary care settings, consideration of contextual factors when implementing change has been recognized as critically important to success. However, contextual factors are rarely recorded, analyzed or considered when implementing change. The lack of consideration of contextual factors has negative implications not only for successfully implementing primary health care (PHC) innovations, but also for their sustainability and scalability. For this evaluation, data collection was conducted using self-administered questionnaires and follow-up telephone interviews with team representatives. We used a combination of directed and conventional content analysis approaches to analyze the questionnaire and interview data. Representatives from all 12 teams completed the questionnaire and 11 teams participated in the interviews; 40 individuals participated in this evaluation. Four themes representing contextual factors that impacted the implementation of CBPHC innovations were identified: (I) diversity of jurisdictions (II) complexity of interactions and collaborations (III) policy, and (IV) the multifaceted nature of PHC. The teams used six strategies to address these contextual factors including: (1) conduct an environmental scan at the beginning (2) maintaining engagement among partners and stakeholders by encouraging open and inclusive communication; (3) contextualizing the innovation for different settings; (4) anticipating and addressing changes, delays, and the need for additional resources; (5) fostering a culture of research and innovation among partners and stakeholders; and (6) ensuring information about the innovation is widely available. Implementing CBPHC innovations across jurisdictions is complex and involves navigating through multiple contextual factors. Awareness of the dynamic nature of context should be considered when implementing innovations.

Promoting cross-jurisdictional primary health care research: developing a set of common indicators across 12 community-based primary health care teams in Canada.

AimTo describe the process by which the 12 community-based primary health care (CBPHC) research teams worked together and fostered cross-jurisdictional collaboration, including collection of common indicators with the goal of using the same measures and data sources. BACKGROUND: A pan-Canadian mechanism for common measurement of the impact of primary care innovations across Canada is lacking. The Canadian Institutes for Health Research and its partners funded 12 teams to conduct research and collaborate on development of a set of commonly collected indicators. METHODS: A working group representing the 12 teams was established. They undertook an iterative process to consider existing primary care indicators identified from the literature and by stakeholders. Indicators were agreed upon with the intention of addressing three objectives across the 12 teams: (1) describing the impact of improving access to CBPHC; (2) examining the impact of alternative models of chronic disease prevention and management in CBPHC; and (3) describing the structures and context that influence the implementation, delivery, cost, and potential for scale-up of CBPHC innovations.FindingsNineteen common indicators within the core dimensions of primary care were identified: access, comprehensiveness, coordination, effectiveness, and equity. We also agreed to collect data on health care costs and utilization within each team. Data sources include surveys, health administrative data, interviews, focus groups, and case studies. Collaboration across these teams sets the foundation for a unique opportunity for new knowledge generation, over and above any knowledge developed by any one team. Keys to success are each team's willingness to engage and commitment to working across teams, funding to support this collaboration, and distributed leadership across the working group. Reaching consensus on collection of common indicators is challenging but achievable.

Models of care for traumatically injured patients at trauma centres in British Columbia: variability and sustainability.

BACKGROUND: Successful trauma systems employ a network of variably-resourced hospitals, staffed by experienced providers, to deliver optimal care for injured patients. The "model of care"-the manner by which inpatients are admitted and overseen, is an important determinant of patient outcomes. OBJECTIVES: To describe the models of inpatient trauma care at British Columbia's (BC's) ten adult trauma centres, their sustainability, and their compatibility with accreditation guidelines. METHODS: Questionnaires were distributed to the trauma medical directors at BC's ten Level I-III adult trauma centres. Follow-up semi-structured interviews clarified responses. RESULTS: Three different models of inpatient trauma care exist within BC. The "admitting trauma service" was a multidisciplinary team providing exclusive care for injured patients. The "on-call consultant" assisted with Emergency Department (ED) resuscitation before transferring patients to a non-trauma admitting service. The single "short-stay trauma unit" employed on-call consultants who also oversaw a 48-hour short-stay ward. Both level I trauma centres utilized the admitting trauma service model (2/2). All Level II sites employed an on-call consultant model (3/3), deviating from Level II trauma centre accreditation standards. Level III sites employed all three models in similar proportions. None of the on-call consultant sites believed their current care model was sustainable. Inadequate compensation, insufficient resources, and difficulty recruiting physicians were cited barriers to sustainability and accreditation compliance. CONCLUSIONS: Three distinct models of care are distributed inconsistently across BC's Level I-III trauma hospitals. Greater use of admitting trauma service and short-stay trauma unit models may improve the sustainability and accreditation compliance of our trauma system.

Assessment of scalability of evidence-based innovations in community-based primary health care: a cross-sectional study.

BACKGROUND: In 2013, the Canadian Institutes of Health Research funded 12 community-based primary health care research teams to develop evidence-based innovations. We aimed to explore the scalability of these innovations. METHODS: In this cross-sectional study, we invited the 12 teams to rate their evidence-based innovations for scalability. Based on a systematic review, we developed a self-administered questionnaire with 16 scalability assessment criteria grouped into 5 dimensions (theory, impact, coverage, setting and cost). Teams completed a questionnaire for each of their innovations. We analyzed the data using simple frequency counts and hierarchical cluster analysis. We calculated the mean number and standard deviation (SD) of innovations that met criteria within each dimension that included more than 1 criterion. The analysis unit was the innovation. RESULTS: The 11 responding teams evaluated 33 evidence-based innovations (median 3, range 1-8 per team). The innovations focused on access to care and chronic disease prevention and management, and varied from health interventions to methodological innovations. Most of the innovations were health interventions (n = 21), followed by analytical methods (n = 4), conceptual frameworks (n = 4), measures (n = 3) and strategies to build research capacity (n = 1). Most (29) met criteria in the theory dimension, followed by impact (mean 22.3 [SD 5.6] innovations per dimension), setting (mean 21.7 [SD 8.5]), cost (mean 17.5 [SD 2.1]) and coverage (mean 14.0 [SD 4.1]). On average, the innovations met 10 of the 16 criteria. Adoption was the least assessed criterion (n = 9). Most (20) of the innovations were highly ranked for scalability. INTERPRETATION: Scalability varied among innovations, which suggests that readiness for scale up was suboptimal for some innovations. Coverage remained largely unaddressed; further investigation of this critical dimension is necessary.

Exosome Secretion by the Parasitic Protozoan Leishmania within the Sand Fly Midgut.

Despite several studies describing the secretion of exosomes by Leishmania in vitro, observation of their formation and release in vivo has remained a major challenge. Herein, we show that Leishmania constitutively secretes exosomes within the lumen of the sand fly midgut through a mechanism homologous to the mammalian pathway. Through egestion experiments, we demonstrate that Leishmania exosomes are part of the sand fly inoculum and are co-egested with the parasite during the insect's bite, possibly influencing the host infectious process. Indeed, co-inoculation of mice footpads with L. major plus midgut-isolated or in-vitro-isolated L. major exosomes resulted in a significant increase in footpad swelling. Notably, co-injections produced exacerbated lesions through overinduction of inflammatory cytokines, in particular IL-17a. Our data indicate that Leishmania exosomes are an integral part of the parasite's infectious life cycle, and we propose to add these vesicles to the repertoire of virulence factors associated with vector-transmitted infections.

Active suppression of intestinal CD4(+)TCRαß(+) T-lymphocyte maturation during the postnatal period.

Priming of the mucosal immune system during the postnatal period substantially influences host-microbial interaction and susceptibility to immune-mediated diseases in adult life. The underlying mechanisms are ill defined. Here we show that shortly after birth, CD4 T cells populate preformed lymphoid structures in the small intestine and quickly acquire a distinct transcriptional profile. T-cell recruitment is independent of microbial colonization and innate or adaptive immune stimulation but requires ß7 integrin expression. Surprisingly, neonatal CD4 T cells remain immature throughout the postnatal period under homeostatic conditions but undergo maturation and gain effector function on barrier disruption. Maternal SIgA and regulatory T cells act in concert to prevent immune stimulation and maintain the immature phenotype of CD4 T cells in the postnatal intestine during homeostasis. Active suppression of CD4 T-cell maturation during the postnatal period might contribute to prevent auto-reactivity, sustain a broad TCR repertoire and establish life-long immune homeostasis.

Absence of metalloprotease GP63 alters the protein content of Leishmania exosomes.

Protozoan parasites of Leishmania genus are able to successfully infect their host macrophage due to multiple virulence strategies that result in its deactivation. Recent studies suggest Leishmania GP63 to be a critical virulence factor in modulation of many macrophage molecules, including protein tyrosine phosphatases (PTPs) and transcription factors (TFs). Additionally, we and others recently reported that Leishmania-released exosomes can participate in pathogenesis. Exosomes are 40-100 nm vesicles that are freed by many eukaryotic cells. To better understand the GP63-dependent immune modulation of the macrophage by Leishmania parasites and their exosomes, we compared the immunomodulatory properties of Leishmania major (WT) and L. major gp63-/- (KO) as well as their exosomes in vitro and in vivo. Importantly, we observed that Leishmania exosomes can modulate macrophage PTPs and TFs in a GP63-dependent manner. In addition, our qRT-PCR analyses showed that WT parasites were able to downregulate multiple genes involved in the immune response, especially cytokines and pattern recognition receptors. KO parasites showed a strongly reduced modulatory capacity compared to WT parasites. Furthermore, comparison of WT versus KO exosomes also showed divergences in alteration of gene expression, especially of chemokine receptors. In parallel, studying the in vivo inflammatory recruitment using a murine air pouch model, we found that exosomes have stronger proinflammatory properties than parasites and preferentially induce the recruitment of neutrophils. Finally, comparative proteomics of WT and KO exosomes surprisingly revealed major differences in their protein content, suggesting a role for GP63 in Leishmania exosomal protein sorting. Collectively our data clearly establish the crucial role of GP63 in dampening the innate inflammatory response during early Leishmania infection, and also provides new insights in regard to the role and biology of exosomes in Leishmania host-parasite interactions.

Immunomodulatory impact of leishmania-induced macrophage exosomes: a comparative proteomic and functional analysis.

Released by many eukaryotic cells, the exosomes are 40-100 nm vesicles shown to operate over the complex processes of cell-cell communication. Among the metazoan cell lineages known to generate exosomes is the mononuclear phagocyte lineage, a lineage that parasites such as Leishmania are known to subvert as host cells. We previously reported that mouse macrophage signaling and functions are modified once co-incubated with exoproteome of Leishmania promastigotes. Using mass spectrometry analysis, we were curious to further compare the content of purified exosomes released by the J774 mouse macrophage cell line exposed or not to either LPS or to stationary phase Leishmania mexicana promastigotes. Collectively, our analyses resulted in detection of 248 proteins, ∼50-80% of which were shared among the three sources studied. Using exponentially modified protein abundance index (emPAI) and network analyses, we found that the macrophage exosomes display unique signatures with respect to composition and abundance of many functional groups of proteins, such as plasma membrane-associated proteins, chaperones and metabolic enzymes. Moreover, for the first time, L. mexicana surface protease GP63 is shown to be present in exosomes released from J774 macrophages exposed to stationary phase promastigotes. We observed that macrophage exosomes are able to induce signaling molecules and transcription factors in naive macrophages. Finally, using qRT-PCR, we monitored modulation of expression of multiple immune-related genes within macrophages exposed to exosomes. We found all three groups of exosomes to induce expression of immune-related genes, the ones collected from macrophages exposed to L. mexicana sharing properties with exosomes collected from macrophage left unexposed to any agonist. Overall, our results allowed depicting that protein sorting into macrophage-derived exosomes depends upon the cell status and how such distinct protein sorting can in turn impact the functions of naive J774 cells.

Leishmania virulence factors: focus on the metalloprotease GP63.

Parasites of Leishmania genus have developed elegant strategies permitting them to evade the innate immune response upon their initial interaction with macrophages. Their capacity to dodge the induction of macrophages microbicidal functions was found to correlate with the alteration of several signalling pathways regulating those latter. In this review, the role of the Leishmania GP63 as a critical virulence factor influencing macrophage physiology will be discussed.

Host cell signalling and leishmania mechanisms of evasion.

Leishmania parasites are able to secure their survival and propagation within their host by altering signalling pathways involved in the ability of macrophages to kill pathogens or to engage adaptive immune system. An important step in this immune evasion process is the activation of host protein tyrosine phosphatase SHP-1 by Leishmania. SHP-1 has been shown to directly inactivate JAK2 and Erk1/2 and to play a role in the negative regulation of several transcription factors involved in macrophage activation. These signalling alterations contribute to the inactivation of critical macrophage functions (e.g., Nitric oxide, IL-12, and TNF-α). Additionally, to interfere with IFN-γ receptor signalling, Leishmania also alters several LPS-mediated responses. Recent findings from our laboratory revealed a pivotal role for SHP-1 in the inhibition of TLR-induced macrophage activation through binding to and inactivating IL-1-receptor-associated kinase 1 (IRAK-1). Furthermore, we identified the binding site as an evolutionarily conserved ITIM-like motif, which we named kinase tyrosine-based inhibitory motif (KTIM). Collectively, a better understanding of the evasion mechanisms utilized by Leishmania parasite could help to develop more efficient antileishmanial therapies in the near future.

Temperature-induced protein secretion by Leishmania mexicana modulates macrophage signalling and function.

Protozoan parasites of genus Leishmania are the causative agents of leishmaniasis. These digenetic microorganisms undergo a marked environmental temperature shift (TS) during transmission from the sandfly vector (ambient temperature, 25-26°C) to the mammalian host (37°C). We have observed that this TS induces a rapid and dramatic increase in protein release from Leishmania mexicana (cutaneous leishmaniasis) within 4 h. Proteomic identification of the TS-induced secreted proteins revealed 72 proteins, the majority of which lack a signal peptide and are thus thought to be secreted via nonconventional mechanisms. Interestingly, this protein release is accompanied by alterations in parasite morphology including an augmentation in the budding of exovesicles from its surface. Here we show that the exoproteome of L. mexicana upon TS induces cleavage and activation of the host protein tyrosine phosphatases, specifically SHP-1 and PTP1-B, in a murine bone-marrow-derived macrophage cell line. Furthermore, translocation of prominent inflammatory transcription factors, namely NF-κB and AP-1 is altered. The exoproteome also caused inhibition of nitric oxide production, a crucial leishmanicidal function of the macrophage. Overall, our results provide strong evidence that within early moments of interaction with the mammalian host, L. mexicana rapidly releases proteins and exovesicles that modulate signalling and function of the macrophage. These modulations can result in attenuation of the inflammatory response and deactivation of the macrophage aiding the parasite in the establishment of infection.