Quantitative Proteomics of the Endothelial Secretome Identifies RC0497 as Diagnostic of Acute Rickettsial Spotted Fever Infections.

Mediterranean spotted fever is a reemerging acute tick-borne infection produced by the α-proteobacterium, Rickettsia conorii. Rickettsia conorii infects vascular endothelial cells producing disseminated plasma leakage, manifesting as nonspecific fever, headache, and maculopapular rash. Because there are no available tests of early infection, Mediterranean spotted fever is often undiagnosed and untreated, resulting in significant mortality. To address this critical need, we have applied a quantitative proteomics pipeline for analyzing the secretome of primary human umbilical vein endothelial cells. Of the 104 proteins whose abundance changed significantly in the R. conorii-infected human umbilical vein endothelial cells' secretome, 46 proteins were up-regulated: 45 were host secreted proteins (including cytokines), and 1 was a rickettsial protein, the putative N-acetylmuramoyl-l-alanine amidase RC0497. Proteins with sequence highly homologous to RC0497 were found to be shared by many species of the spotted fever group rickettsiae, but not typhus group rickettsiae. Quantitative targeted proteomics studies of plasma from a mouse model of sublethal and lethal R. conorii identified RC0497 in the blood, and its circulating levels were proportionally associated with infection outcome. Finally, the presence of RC0497 in the serum samples from a cohort of humans presenting with acute rickettsioses was confirmed. The detection of RC0497 has the potential to be a sensitive and specific marker for acute rickettsial spotted rickettsioses.

Endothelial Cell Proteomic Response to Rickettsia conorii Infection Reveals Activation of the Janus Kinase (JAK)-Signal Transducer and Activator of Transcription (STAT)-Inferferon Stimulated Gene (ISG)15 Pathway and Reprogramming Plasma Membrane Integrin/Cadherin Signaling.

Rickettsia conorii is the etiologic agent of Mediterranean spotted fever, a re-emerging infectious disease with significant mortality. This Gram-negative, obligately intracellular pathogen is transmitted via tick bites, resulting in disseminated vascular endothelial cell infection with vascular leakage. In the infected human, Rickettsia conorii infects endothelial cells, stimulating expression of cytokines and pro-coagulant factors. However, the integrated proteomic response of human endothelial cells to R. conorii infection is not known. In this study, we performed quantitative proteomic profiling of primary human umbilical vein endothelial cells (HUVECs) with established R conorii infection versus those stimulated with endotoxin (LPS) alone. We observed differential expression of 55 proteins in HUVEC whole cell lysates. Of these, we observed induction of signal transducer and activator of transcription (STAT)1, MX dynamin-like GTPase (MX1), and ISG15 ubiquitin-like modifier, indicating activation of the JAK-STAT signaling pathway occurs in R. conorii-infected HUVECs. The down-regulated proteins included those involved in the pyrimidine and arginine biosynthetic pathways. A highly specific biotinylated cross-linking enrichment protocol was performed to identify dysregulation of 11 integral plasma membrane proteins that included up-regulated expression of a sodium/potassium transporter and down-regulation of α-actin 1. Analysis of Golgi and soluble Golgi fractions identified up-regulated proteins involved in platelet-endothelial adhesion, phospholipase activity, and IFN activity. Thirty four rickettsial proteins were identified with high confidence in the Golgi, plasma membrane, or secreted protein fractions. The host proteins associated with rickettsial infections indicate activation of interferon-STAT signaling pathways; the disruption of cellular adhesion and alteration of antigen presentation pathways in response to rickettsial infections are distinct from those produced by nonspecific LPS stimulation. These patterns of differentially expressed proteins suggest mechanisms of pathogenesis as well as methods for diagnosis and monitoring Rickettsia infections.

A human lung xenograft mouse model of Nipah virus infection.

Nipah virus (NiV) is a member of the genus Henipavirus (family Paramyxoviridae) that causes severe and often lethal respiratory illness and encephalitis in humans with high mortality rates (up to 92%). NiV can cause Acute Lung Injury (ALI) in humans, and human-to-human transmission has been observed in recent outbreaks of NiV. While the exact route of transmission to humans is not known, we have previously shown that NiV can efficiently infect human respiratory epithelial cells. The molecular mechanisms of NiV-associated ALI in the human respiratory tract are unknown. Thus, there is an urgent need for models of henipavirus infection of the human respiratory tract to study the pathogenesis and understand the host responses. Here, we describe a novel human lung xenograft model in mice to study the pathogenesis of NiV. Following transplantation, human fetal lung xenografts rapidly graft and develop mature structures of adult lungs including cartilage, vascular vessels, ciliated pseudostratified columnar epithelium, and primitive "air" spaces filled with mucus and lined by cuboidal to flat epithelium. Following infection, NiV grows to high titers (10(7) TCID50/gram lung tissue) as early as 3 days post infection (pi). NiV targets both the endothelium as well as respiratory epithelium in the human lung tissues, and results in syncytia formation. NiV infection in the human lung results in the production of several cytokines and chemokines including IL-6, IP-10, eotaxin, G-CSF and GM-CSF on days 5 and 7 pi. In conclusion, this study demonstrates that NiV can replicate to high titers in a novel in vivo model of the human respiratory tract, resulting in a robust inflammatory response, which is known to be associated with ALI. This model will facilitate progress in the fundamental understanding of henipavirus pathogenesis and virus-host interactions; it will also provide biologically relevant models for other respiratory viruses.

Behavioural and neurological symptoms accompanied by cellular neuroinflammation in IL-10-deficient mice infected with Plasmodium chabaudi.

BACKGROUND: Cerebral malaria is one of the most severe complications of Plasmodium falciparum infection and occurs mostly in young African children. This syndrome results from a combination of high levels of parasitaemia and inflammation. Although parasite sequestration in the brain is a feature of the human syndrome, sequestering strains do not uniformly cause severe malaria, suggesting interplay with other factors. Host genetic factors such as mutations in the promoters of the cytokines IL-10 and TNF are also clearly linked to severe disease. Plasmodium chabaudi, a rodent malaria parasite, leads to mild illness in wildtype animals. However, IL-10(-/-) mice respond to parasite with increased levels of pro-inflammatory cytokines IFN-γ and TNF, leading to lethal disease in the absence of sequestration in the brain. These mice also exhibit cerebral symptoms including gross cerebral oedema and haemorrhage, allowing study of these critical features of disease without the influence of sequestration. METHODS: The neurological consequences of P. chabaudi infection were investigated by performing a general behavioural screen (SHIRPA). The immune cell populations found in the brain during infection were also analysed using flow cytometry and confocal microscopy. RESULTS: IL-10(-/-) mice suffer significant declines in behavioural and physical capacities during infection compared to wildtype. In addition, grip strength and pain sensitivity were affected, suggestive of neurological involvement. Several immune cell populations were identified in the perfused brain on day 7 post-infection, suggesting that they are tightly adherent to the vascular endothelium, or potentially located within the brain parenchyma. There was an increase in both inflammatory monocyte and resident macrophage (CD11b(hi), CD45(+), MHCII(+), Ly6C(+/-)) numbers in IL-10(-/-) compared to wildtype animals. In addition, the activation state of all monocytes and microglia (CD11b(int), CD45(-), MHC-II(+)) were increased. T cells making IFN-γ were also identified in the brain, but were localized within the vasculature, and not the parenchyma. CONCLUSIONS: These studies demonstrate exacerbated neuroinflammation concurrent with development of behavioural symptoms in P. chabaudi infection of IL-10(-/-) animals.

Generation and Characterization of a Humanized Lung Xenograft Mouse Model for Studying Henipavirus Pathogenesis.

The development of humanized mouse models has recently opened new avenues in the field of infectious diseases. These models allow research on many human viruses that were once difficult to study, because finding suitable animal models of infection can be challenging, cost prohibitive, and often do not entirely recapitulate all parameters of the disease. Here, we describe the procedure of human immune system reconstitution (humanization) of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice by the bone marrow, liver, and thymus (BLT) reconstitution method as well as the process of human lung engraftment. We then describe how to infect these human lung grafts with the paramyxovirus Nipah virus (NiV) that can cause lethal respiratory disease in humans, and for which there is only limited understanding of pathogenesis to acute lung injury.

Outbreak of Rocky Mountain spotted fever in Córdoba, Colombia.

Rocky Mountain spotted fever (RMSF) is a tick-borne disease caused by the obligate intracellular bacterium Rickettsia rickettsii. Although RMSF was first reported in Colombia in 1937, it remains a neglected disease. Herein, we describe the investigation of a large cluster of cases of spotted fever rickettsiosis in a new area of Colombia.

Improved detection of Bartonella DNA in mammalian hosts and arthropod vectors by real-time PCR using the NADH dehydrogenase gamma subunit (nuoG).

We used a whole-genome scanning technique to identify the NADH dehydrogenase gamma subunit (nuoG) primer set that is sensitive and specific enough to detect a diverse number of Bartonella species in a wide range of environmental samples yet maintains minimal cross-reactivity to mammalian host and arthropod vector organisms.

Burkholderia mallei cellular interactions in a respiratory cell model.

Burkholderia mallei is a facultative intracellular pathogen that survives and replicates in phagocytic cell lines. The bacterial burden recovered from naïve BALB/c mice infected by intranasal delivery indicated that B. mallei persists in the lower respiratory system. To address whether B. mallei invades respiratory non-professional phagocytes, this study utilized A549 and LA-4 respiratory epithelial cells and demonstrated that B. mallei possesses the capacity to adhere poorly to, but not to invade, these cells. Furthermore, it was found that B. mallei was taken up by the murine alveolar macrophage cell line MH-S following serum coating, an attribute suggestive of complement- or Fc receptor-mediated uptake. Invasion/intracellular survival assays of B. mallei-infected MH-S cells demonstrated decreased intracellular survival, whilst a type III secretion system effector bopA mutant strain survived longer than the wild-type. Evaluation of the potential mechanism(s) responsible for efficient clearing of intracellular organisms demonstrated comparable levels of caspase-3 in both the wild-type and bopA mutant with characteristics consistent with apoptosis of infected MH-S cells. Furthermore, challenge of BALB/c mice with the bopA mutant by the intranasal route resulted in increased survival. Overall, these data suggest that B. mallei induces apoptotic cell death, whilst the BopA effector protein participates in intracellular survival.

Inquiry in the Medical Curriculum: A Pedagogical Conundrum and a Proposed Solution.

Habits of inquiry are considered an essential component of the modern physician's profile. These habits drive physicians to recognize and address the continuous challenges inherent to the practice of medicine; consequently, they meet the aims of better patient-centered care, better health of communities, and improved functioning of the health system. Many medical schools have endeavored to integrate inquiry into their curricula as a means of supporting development of adaptive expertise, a construct that encompasses habits of inquiry. However, the diversity of conceptualizations of inquiry has resulted in correspondingly diverse instructional implementations. Much of the emphasis has been on inquiry methods (e.g., engagement in research projects, courses in research methods and statistics), but the learners' inquiry disposition and its essential attitude component have received little attention in instruction and assessment. The authors propose that both inquiry methods and attitude need to be developed explicitly and simultaneously to prepare physicians to successfully be willing and able to address the challenges of today's health care environment. Because attitudes are established predictors of behavior, a positive inquiry attitude may be the ultimate determinant of physicians' engagement in behaviors of adaptive expertise (i.e., recognizing when learned procedures do not apply, and learning or inventing effective solutions). Addressing the attitude toward inquiry as early as possible in medical school is critical because strong attitudes are difficult to modify. Thus, a curriculum that supports positive inquiry attitude formation and strengthening will carry well beyond medical school and residency training.

Whole-Genome Sequence of Rickettsia parkeri Strain Atlantic Rainforest, Isolated from a Colombian Tick.

Rickettsia parkeri is classified as a member of the alphaproteobacterial microorganisms, genus Rickettsia Here, we report the complete genome sequence of Rickettsia parkeri strain Atlantic Rainforest, which was isolated from an Amblyomma ovale tick collected in the municipality of Necoclí, Colombia.

Murine typhus in Caldas, Colombia.

In the north of Caldas, Colombia, febrile syndromes with positive Weil-Felix reactions have been reported as Murine typhus to the national health authorities. We used indirect immunofluorescence assay (IFA) of serial paired samples to confirm the diagnosis of murine typhus in 14 of 120 patients with a compatible febrile syndrome.

Prevalence of antibodies against spotted fever group rickettsiae in a rural area of Colombia.

We recently rediscovered Rocky Mountain spotted fever in Villeta, Colombia, near the same locality (Tobia) where it was first recognized in 1937. To have a better idea of the magnitude of this problem, sera from 392 randomly recruited healthy adults from Villeta were analyzed by indirect immunofluorescent antibody assay to detect IgG against Rickettsia rickettsii as antigen. The seropositivity rate for spotted fever group rickettsiae was 40.3%. We did not find any association between the presence of antibodies to spotted fever group rickettsiae and several demographic and epidemiologic variables, which could be a reflection of unique features of this area.

Human prevalence of the spotted fever group (SFG) rickettsiae in endemic zones of Northwestern Colombia.

In February 2006, an outbreak of human rickettsiosis occurred in the municipality of Necoclí Colombia, with 35% of lethality. This episode was, followed by two more, one in the municipality of Los Cordobas in 2007 with a 54% of lethality and the other one in the municipality of Turbo in 2008 with 27% of lethality. The aim of this study was to perform serological tests in healthy persons to determine the seroprevalence of antibodies against spotted fever group (SFG) rickettsiae and develop a survey to study some infection risk-related factors. A cross-sectional study was performed in 2011 and 2012. A blood sample and survey of associated factors was performed in healthy persons. A prevalence of 32%-41% was found in healthy people. From the multivariate analysis, we found that people living more than 16 years in these sites had a 79% higher risk of being seropositive and a 46% higher risk when they reported having birds in their houses if the variable of having a horse was included in the model. In conclusion, this study shows endemicity of at least one spotted fever group Rickettsia in the study zone.

Wild and domestic animals likely involved in rickettsial endemic zones of Northwestern Colombia.

Between 2006 and 2008, three outbreaks of human rickettsiosis occurred in Northwestern Colombia (municipalities of Necoclí, Los Córdobas and Turbo), with case fatality rates between 27% and 54%. The aim of this study was to determine previous exposure of wild and domestic animals to spotted fever group (SFG) rickettsiae through serological tests, to detect rickettsial evidence in their ectoparasites, and to analyze their possible role in the epidemiology of rickettsial diseases in this zone of the country. A cross-sectional association study was performed from 2010 to 2011. Blood and ectoparasite samples were collected from domestic animals and small mammals. A statistically significant association (p<0.05) between seropositive animals and the study zones was observed. A total of 2937 ticks, 672 fleas and 74 lice were collected and tested in pools by PCR. The minimum infection rate (MIR) of the positive pools was 5% in ticks, 4% in fleas, and 0% in lice. Phylogenetic analyses showed circulation of three 4.Rickettsia species: R. felis in fleas, and R. bellii and Rickettsia sp. strain Atlantic rainforest, both in Amblyomma ovale ticks. In conclusion, this study demonstrated the occurrence of SFG rickettsiae in domestic, synanthropic and wild animals, and suggests the use of equines and canines as good sentinels of infection, in the study zone. We speculate that a transmission cycle exist involving rodents in the areas where these outbreaks have occurred. Tomes' spiny rats (Proechimys semispinosus) and common opossums (Didelphis marsupialis) could be good candidates as amplifier hosts for SFG rickettsiae in enzootic/endemic zones.

Immune Cell Targets of Infection at the Tick-Skin Interface during Powassan Virus Transmission.

Powassan virus (POWV) is a tick-borne flavivirus that can result in a severe neuroinvasive disease with 50% of survivors displaying long-term neurological sequelae. Human POWV cases have been documented in Canada, the United States, and Russia. Although the number of reported POWV human cases has increased in the past fifteen years, POWV remains one of the less studied human pathogenic flaviviruses. Ixodes ticks are the vectors for POWV, and the virus is transmitted to a host's skin very early during the tick feeding process. Central to the successful transmission of a tick-borne pathogen are complex interactions between the host immune response and early tick-mediated immunomodulation, all of which initially occur at the skin interface. In our prior work, we examined the cutaneous immune gene expression during the early stages of POWV-infected Ixodes scapularis feeding. The present study serves to further investigate the skin interface by identifying early cell targets of infection at the POWV-infected tick feeding site. An in vivo infection model consisting of POWV-infected ticks feeding on mice for short durations was used in this study. Skin biopsies from the tick feeding sites were harvested at various early time points, enabling us to examine the skin histopathology and detect POWV viral antigen in immune cells present at the tick feeding site. The histopathology from the present study demonstrates that neutrophil and mononuclear cell infiltrates are recruited earlier to the feeding site of a POWV-infected tick versus an uninfected tick. This is the first report demonstrating that macrophages and fibroblasts contain POWV antigens, which suggests that they are early cellular targets of infection at the tick feeding site. These data provide key insights towards defining the complex interactions between the host immune response and early tick-mediated immunomodulation.

An Intradermal Inoculation Mouse Model for Immunological Investigations of Acute Scrub Typhus and Persistent Infection.

Scrub typhus is a neglected tropical disease, caused by Orientia tsutsugamushi, a Gram-negative bacterium that is transmitted to mammalian hosts during feeding by Leptotrombidium mites and replicates predominantly within endothelial cells. Most studies of scrub typhus in animal models have utilized either intraperitoneal or intravenous inoculation; however, there is limited information on infection by the natural route in murine model skin or its related early host responses. Here, we developed an intradermal (i.d.) inoculation model of scrub typhus and focused on the kinetics of the host responses in the blood and major infected organs. Following ear inoculation with 6 x 104 O. tsutsugamushi, mice developed fever at 11-12 days post-infection (dpi), followed by marked hypothermia and body weight loss at 14-19 dpi. Bacteria in blood and tissues and histopathological changes were detected around 9 dpi and peaked around 14 dpi. Serum cytokine analyses revealed a mixed Th1/Th2 response, with marked elevations of MCP-1/CCL2, MIP-1α/CCL3 and IL-10 at 9 dpi, followed by increased concentrations of pro-inflammatory markers (IL-6, IL-12, IFN-γ, G-CSF, RANTES/CCL5, KC/CCL11, IL-1α/ß, IL-2, TNF-α, GM-CSF), as well as modulatory cytokines (IL-9, IL-13). Cytokine levels in lungs had similar elevation patterns, except for a marked reduction of IL-9. The Orientia 47-kDa gene and infectious bacteria were detected in several organs for up to 84 dpi, indicating persistent infection. This is the first comprehensive report of acute scrub typhus and persistent infection in i.d.-inoculated C57BL/6 mice. This is a significant improvement over current murine models for Orientia infection and will permit detailed studies of host immune responses and infection control interventions.

Pulmonary Tuberculosis in Humanized Mice Infected with HIV-1.

Co-infection with HIV increases the morbidity and mortality associated with tuberculosis due to multiple factors including a poorly understood microbial synergy. We developed a novel small animal model of co-infection in the humanized mouse to investigate how HIV infection disrupts pulmonary containment of Mtb. Following dual infection, HIV-infected cells were localized to sites of Mtb-driven inflammation and mycobacterial replication in the lung. Consistent with disease in human subjects, we observed increased mycobacterial burden, loss of granuloma structure, and increased progression of TB disease, due to HIV co-infection. Importantly, we observed an HIV-dependent pro-inflammatory cytokine signature (IL-1ß, IL-6, TNFα, and IL-8), neutrophil accumulation, and greater lung pathology in the Mtb-co-infected lung. These results suggest that in the early stages of acute co-infection in the humanized mouse, infection with HIV exacerbates the pro-inflammatory response to pulmonary Mtb, leading to poorly formed granulomas, more severe lung pathology, and increased mycobacterial burden and dissemination.

Expression of CX3CL1 (fractalkine) in mice with endothelial-target rickettsial infection of the spotted-fever group.

Fractalkine (CX3CL1) is a chemokine expressed mainly by endothelial cells, which are the major cellular targets of rickettsiae. We used immunohistochemistry to investigate the normal expression of CX3CL1 in mice and the kinetics of expression of this chemokine throughout the course of lethal and sublethal rickettsial infections in a mouse model of spotted-fever group rickettsioses. The peak of expression of fractalkine on day 3 of infection coincided with the time of infiltration of macrophages into infected tissues and preceded the peak of rickettsial content in tissues.