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1.  Interferon-γ promotes gastric lymphoid follicle formation but not gastritis in Helicobacter-infected BALB/c mice 
Gut Pathogens  2016;8:61.
Background
Mouse infection studies have shown that interferon-γ (IFN-γ), a T helper 1 (Th1) cytokine, is required for the development of severe pathology induced by chronic Helicobacter infection. This finding is largely based on studies performed using mice that have polarised Th1 responses i.e. C57BL/6 animals. The current work aims to investigate the role of IFN-γ in Helicobacter-induced inflammation in BALB/c mice which have Th2-polarised immune responses.
Results
At 7 months post-infection with Helicobacter felis, IFN-γ deficiency in BALB/c mice had no significant effect on H. felis colonisation levels in the gastric mucosa, nor on humoral responses, or gastritis severity. Ifng −/− animals with chronic H. felis infection did, however, develop significantly fewer lymphoid follicle lesions, as well as increased IL-4 splenocyte responses, when compared with infected Ifng +/+ mice (P = 0.015 and P = 0.0004, respectively).
Conclusions
The work shows that in mice on a BALB/c background, IFN-γ is not required for bacterial clearance, antibody responses, nor gastric inflammation. Conversely, IFN-γ appears to play a role in the development of gastric lymphoid follicles, which are precursor lesions to mucosa-associated lymphoid tissue (MALT) lymphoma. This study highlights the importance of mouse host background on the susceptibility to Helicobacter-induced pathologies.
doi:10.1186/s13099-016-0142-0
PMCID: PMC5117576  PMID: 27895717
Helicobacter; Interferon-gamma; Lymphoid follicle; Mucosa-associated lymphoid tissue; MALT lymphoma; BALB/c; T helper response; Gastric inflammation
2.  Cytokine and Chemokine Expression in Kidneys during Chronic Leptospirosis in Reservoir and Susceptible Animal Models 
PLoS ONE  2016;11(5):e0156084.
Leptospirosis is caused by pathogenic spirochetes of the genus Leptospira. Humans can be infected after exposure to contaminated urine of reservoir animals, usually rodents, regarded as typical asymptomatic carriers of leptospires. In contrast, accidental hosts may present an acute form of leptospirosis with a range of clinical symptoms including the development of Acute Kidney Injury (AKI). Chronic Kidney Disease (CKD) is considered as a possible AKI-residual sequela but little is known about the renal pathophysiology consequent to leptospirosis infection. Herein, we studied the renal morphological alterations in relation with the regulation of inflammatory cytokines and chemokines, comparing two experimental models of chronic leptospirosis, the golden Syrian hamster that survived the infection, becoming carrier of virulent leptospires, and the OF1 mouse, a usual reservoir of the bacteria. Animals were monitored until 28 days after injection with a virulent L. borgpetersenii serogroup Ballum to assess chronic infection. Hamsters developed morphological alterations in the kidneys with tubulointerstitial nephritis and fibrosis. Grading of lesions revealed higher scores in hamsters compared to the slight alterations observed in the mouse kidneys, irrespective of the bacterial load. Interestingly, pro-fibrotic TGF-β was downregulated in mouse kidneys. Moreover, cytokines IL-1β and IL-10, and chemokines MIP-1α/CCL3 and IP-10/CXCL-10 were significantly upregulated in hamster kidneys compared to mice. These results suggest a possible maintenance of inflammatory processes in the hamster kidneys with the infiltration of inflammatory cells in response to bacterial carriage, resulting in alterations of renal tissues. In contrast, lower expression levels in mouse kidneys indicated a better regulation of the inflammatory response and possible resolution processes likely related to resistance mechanisms.
doi:10.1371/journal.pone.0156084
PMCID: PMC4878748  PMID: 27219334
3.  Rainbow trout (Oncorhynchus mykiss) muscle satellite cells are targets of salmonid alphavirus infection 
Veterinary Research  2016;47:9.
Sleeping disease in rainbow trout is characterized by an abnormal swimming behaviour of the fish which stay on their side at the bottom of the tanks. This sign is due to extensive necrosis and atrophy of red skeletal muscle induced by the sleeping disease virus (SDV), also called salmonid alphavirus 2. Infections of humans with arthritogenic alphaviruses, such as Chikungunya virus (CHIKV), are global causes of debilitating musculoskeletal diseases. The mechanisms by which the virus causes these pathologies are poorly understood due to the restrictive availability of animal models capable of reproducing the full spectrum of the disease. Nevertheless, it has been shown that CHIKV exhibits a particular tropism for muscle stem cells also known as satellite cells. Thus, SDV and its host constitute a relevant model to study in details the virus-induced muscle atrophy, the pathophysiological consequences of the infection of a particular cell-type in the skeletal muscle, and the regeneration of the muscle tissue in survivors together with the possible virus persistence. To study a putative SDV tropism for that particular cell type, we established an in vivo and ex vivo rainbow trout model of SDV-induced atrophy of the skeletal muscle. This experimental model allows reproducing the full panel of clinical signs observed during a natural infection since the transmission of the virus is arthropod-borne independent. The virus tropism in the muscle tissue was studied by immunohistochemistry together with the kinetics of the muscle atrophy, and the muscle regeneration post-infection was observed. In parallel, an ex vivo model of SDV infection of rainbow trout satellite cells was developed and virus replication and persistence in that particular cell type was followed up to 73 days post-infection. These results constitute the first observation of a specific SDV tropism for the muscle satellite cells.
doi:10.1186/s13567-015-0301-1
PMCID: PMC4705810  PMID: 26743565
4.  Disruption of Parasite hmgb2 Gene Attenuates Plasmodium berghei ANKA Pathogenicity 
Infection and Immunity  2015;83(7):2771-2784.
Eukaryotic high-mobility-group-box (HMGB) proteins are nuclear factors involved in chromatin remodeling and transcription regulation. When released into the extracellular milieu, HMGB1 acts as a proinflammatory cytokine that plays a central role in the pathogenesis of several immune-mediated inflammatory diseases. We found that the Plasmodium genome encodes two genuine HMGB factors, Plasmodium HMGB1 and HMGB2, that encompass, like their human counterparts, a proinflammatory domain. Given that these proteins are released from parasitized red blood cells, we then hypothesized that Plasmodium HMGB might contribute to the pathogenesis of experimental cerebral malaria (ECM), a lethal neuroinflammatory syndrome that develops in C57BL/6 (susceptible) mice infected with Plasmodium berghei ANKA and that in many aspects resembles human cerebral malaria elicited by P. falciparum infection. The pathogenesis of experimental cerebral malaria was suppressed in C57BL/6 mice infected with P. berghei ANKA lacking the hmgb2 gene (Δhmgb2 ANKA), an effect associated with a reduction of histological brain lesions and with lower expression levels of several proinflammatory genes. The incidence of ECM in pbhmgb2-deficient mice was restored by the administration of recombinant PbHMGB2. Protection from experimental cerebral malaria in Δhmgb2 ANKA-infected mice was associated with reduced sequestration in the brain of CD4+ and CD8+ T cells, including CD8+ granzyme B+ and CD8+ IFN-γ+ cells, and, to some extent, neutrophils. This was consistent with a reduced parasite sequestration in the brain, lungs, and spleen, though to a lesser extent than in wild-type P. berghei ANKA-infected mice. In summary, Plasmodium HMGB2 acts as an alarmin that contributes to the pathogenesis of cerebral malaria.
doi:10.1128/IAI.03129-14
PMCID: PMC4468538  PMID: 25916985
5.  Dissociation of Tissue Destruction and Bacterial Expansion during Bubonic Plague 
PLoS Pathogens  2015;11(10):e1005222.
Activation and/or recruitment of the host plasmin, a fibrinolytic enzyme also active on extracellular matrix components, is a common invasive strategy of bacterial pathogens. Yersinia pestis, the bubonic plague agent, expresses the multifunctional surface protease Pla, which activates plasmin and inactivates fibrinolysis inhibitors. Pla is encoded by the pPla plasmid. Following intradermal inoculation, Y. pestis has the capacity to multiply in and cause destruction of the lymph node (LN) draining the entry site. The closely related, pPla-negative, Y. pseudotuberculosis species lacks this capacity. We hypothesized that tissue damage and bacterial multiplication occurring in the LN during bubonic plague were linked and both driven by pPla. Using a set of pPla-positive and pPla-negative Y. pestis and Y. pseudotuberculosis strains in a mouse model of intradermal injection, we found that pPla is not required for bacterial translocation to the LN. We also observed that a pPla-cured Y. pestis caused the same extensive histological lesions as the wild type strain. Furthermore, the Y. pseudotuberculosis histological pattern, characterized by infectious foci limited by inflammatory cell infiltrates with normal tissue density and follicular organization, was unchanged after introduction of pPla. However, the presence of pPla enabled Y. pseudotuberculosis to increase its bacterial load up to that of Y. pestis. Similarly, lack of pPla strongly reduced Y. pestis titers in LNs of infected mice. This pPla-mediated enhancing effect on bacterial load was directly dependent on the proteolytic activity of Pla. Immunohistochemistry of Pla-negative Y. pestis-infected LNs revealed extensive bacterial lysis, unlike the numerous, apparently intact, microorganisms seen in wild type Y. pestis-infected preparations. Therefore, our study demonstrates that tissue destruction and bacterial survival/multiplication are dissociated in the bubo and that the primary action of Pla is to protect bacteria from destruction rather than to alter the tissue environment to favor Y. pestis propagation in the host.
Author Summary
The hallmark of bubonic plague, a disease that ravaged Medieval Europe and is still prevalent in several countries, is the bubo, a highly inflammatory and painful lymph node, which is characterized by high concentrations of bacteria within a severely damaged organ. Yersinia pestis, the causative agent, expresses a surface protease, Pla, critical to the development of bubonic plague. This multitarget protease has the potential to activate the fibrinolytic pathway and to promote destruction of extracellular protein networks within tissues. Hence, it was expected that Pla was responsible for the tissue destructions of the bubo, and consequently, for bacterial propagation and virulence. However, we found, using various engineered Yersinia strains in a mouse model of bubonic plague, that Pla proteolytic activity was dispensable for lymph node alteration, but was required to achieve high bacterial loads in the organ. Further analysis showed that Pla is essential for preventing the bacteria from being destroyed in the host. Therefore, the role of Pla as a virulence factor is to protect Y. pestis survival and integrity in the host, rather than to assist its spread through tissue destruction.
doi:10.1371/journal.ppat.1005222
PMCID: PMC4615631  PMID: 26484539
6.  Hyperactivation of Alk induces neonatal lethality in knock-in AlkF1178L mice 
Oncotarget  2014;5(9):2703-2713.
The ALK (Anaplastic Lymphoma Kinase) gene encodes a tyrosine kinase receptor preferentially expressed in the central and peripheral nervous systems. A syndromic presentation associating congenital neuroblastoma with severe encephalopathy and an abnormal shape of the brainstem has been described in patients harbouring de novo germline F1174V and F1245V ALK mutations. Here, we investigated the phenotype of knock-in (KI) mice bearing the AlkF1178L mutation (F1174L in human). Although heterozygous KI mice did not reproduce the severe breathing and feeding difficulties observed in human patients, behavioral tests documented a reduced activity during dark phases and an increased anxiety of mutated mice. Matings of heterozygotes yielded the expected proportions of wild-type, heterozygotes and homozygotes at birth but a high neonatal lethality was noticed for homozygotes. We documented Alk expression in several motor nuclei of the brainstem involved in the control of sucking and swallowing. Evaluation of basic physiological functions 12 hours after birth revealed slightly more apneas but a dramatic reduced milk intake for homozygotes compared to control littermates. Overall, our data demonstrate that Alk activation above a critical threshold is not compatible with survival in mice, in agreement with the extremely severe phenotype of patients carrying aggressive de novo ALK germline mutations.
PMCID: PMC4058038  PMID: 24811761
ALK; brainstem; neonatal lethality; plethysmography; feeding difficulties
7.  Interleukin-22 Reduces Lung Inflammation during Influenza A Virus Infection and Protects against Secondary Bacterial Infection 
Journal of Virology  2013;87(12):6911-6924.
Interleukin-22 (IL-22) has redundant, protective, or pathogenic functions during autoimmune, inflammatory, and infectious diseases. Here, we addressed the potential role of IL-22 in host defense and pathogenesis during lethal and sublethal respiratory H3N2 influenza A virus (IAV) infection. We show that IL-22, as well as factors associated with its production, are expressed in the lung tissue during the early phases of IAV infection. Our data indicate that retinoic acid receptor-related orphan receptor-γt (RORγt)-positive αβ and γδ T cells, as well as innate lymphoid cells, expressed enhanced Il22 transcripts as early as 2 days postinfection. During lethal or sublethal IAV infections, endogenous IL-22 played no role in the control of IAV replication and in the development of the IAV-specific CD8+ T cell response. During lethal infection, where wild-type (WT) mice succumbed to severe pneumonia, the lack of IL-22 did not accelerate or delay IAV-associated pathogenesis and animal death. In stark contrast, during sublethal IAV infection, IL-22-deficient animals had enhanced lung injuries and showed a lower airway epithelial integrity relative to WT littermates. Of importance, the protective effect of endogenous IL-22 in pulmonary damages was associated with a more controlled secondary bacterial infection. Indeed, after challenge with Streptococcus pneumoniae, IAV-experienced Il22−/− animals were more susceptible than WT controls in terms of survival rate and bacterial burden in the lungs. Together, IL-22 plays no major role during lethal influenza but is beneficial during sublethal H3N2 IAV infection, where it limits lung inflammation and subsequent bacterial superinfections.
doi:10.1128/JVI.02943-12
PMCID: PMC3676141  PMID: 23596287
8.  Expression of follicle-stimulating hormone receptor by the vascular endothelium in tumor metastases 
BMC Cancer  2013;13:246.
Background
The Follicle Stimulating Hormone receptor (FSHR) is expressed by the vascular endothelium in a wide range of human tumors. It was not determined however if FSHR is present in metastases which are responsible for the terminal illness.
Methods
We used immunohistochemistry based on a highly FSHR-specific monoclonal antibody to detect FSHR in cancer metastases from 6 major tumor types (lung, breast, prostate, colon, kidney, and leiomyosarcoma ) to 6 frequent locations (bone, liver, lymph node, brain, lung, and pleura) of 209 patients.
Results
In 166 patients examined (79%), FSHR was expressed by blood vessels associated with metastatic tissue. FSHR-positive vessels were present in the interior of the tumors and some few millimeters outside, in the normally appearing tissue. In the interior of the metastases, the density of the FSHR-positive vessels was constant up to 7 mm, the maximum depth available in the analyzed sections. No significant differences were noticed between the density of FSHR-positive vessels inside vs. outside tumors for metastases from lung, breast, colon, and kidney cancers. In contrast, for prostate cancer metastases, the density of FSHR-positive vessels was about 3-fold higher at the exterior of the tumor compared to the interior. Among brain metastases, the density of FSHR-positive vessels was highest in lung and kidney cancer, and lowest in prostate and colon cancer. In metastases of breast cancer to the lung pleura, the percentage of blood vessels expressing FSHR was positively correlated with the progesterone receptor level, but not with either HER-2 or estrogen receptors. In normal tissues corresponding to the host organs for the analyzed metastases, obtained from patients not known to have cancer, FSHR staining was absent, with the exception of approx. 1% of the vessels in non tumoral temporal lobe epilepsy samples.
Conclusion
FSHR is expressed by the endothelium of blood vessels in the majority of metastatic tumors.
doi:10.1186/1471-2407-13-246
PMCID: PMC3663659  PMID: 23688201
Breast cancer; Colon cancer; Kidney cancer; Lung cancer; Prostate cancer; Endothelial cells; Leiomyosarcoma; Follicle-stimulating hormone receptor; Metastasis; Tumor blood vessels
9.  A novel murine model of rhinoscleroma identifies Mikulicz cells, the disease signature, as IL-10 dependent derivatives of inflammatory monocytes 
EMBO Molecular Medicine  2013;5(4):516-530.
Rhinoscleroma is a human specific chronic disease characterized by the formation of granuloma in the airways, caused by the bacterium Klebsiella pneumoniae subspecies rhinoscleromatis, a species very closely related to K. pneumoniae subspecies pneumoniae. It is characterized by the appearance of specific foamy macrophages called Mikulicz cells. However, very little is known about the pathophysiological processes underlying rhinoscleroma. Herein, we characterized a murine model recapitulating the formation of Mikulicz cells in lungs and identified them as atypical inflammatory monocytes specifically recruited from the bone marrow upon K. rhinoscleromatis infection in a CCR2-independent manner. While K. pneumoniae and K. rhinoscleromatis infections induced a classical inflammatory reaction, K. rhinoscleromatis infection was characterized by a strong production of IL-10 concomitant to the appearance of Mikulicz cells. Strikingly, in the absence of IL-10, very few Mikulicz cells were observed, confirming a crucial role of IL-10 in the establishment of a proper environment leading to the maturation of these atypical monocytes. This is the first characterization of the environment leading to Mikulicz cells maturation and their identification as inflammatory monocytes.
doi:10.1002/emmm.201202023
PMCID: PMC3628109  PMID: 23554169
IL-10; inflammatory monocytes; Klebsiella; Mikulicz cell; rhinoscleroma
10.  Visualizing Non Infectious and Infectious Anopheles gambiae Blood Feedings in Naive and Saliva-Immunized Mice 
PLoS ONE  2012;7(12):e50464.
Background
Anopheles gambiae is a major vector of malaria and lymphatic filariasis. The arthropod-host interactions occurring at the skin interface are complex and dynamic. We used a global approach to describe the interaction between the mosquito (infected or uninfected) and the skin of mammals during blood feeding.
Methods
Intravital video microscopy was used to characterize several features during blood feeding. The deposition and movement of Plasmodium berghei sporozoites in the dermis were also observed. We also used histological techniques to analyze the impact of infected and uninfected feedings on the skin cell response in naive mice.
Results
The mouthparts were highly mobile within the skin during the probing phase. Probing time increased with mosquito age, with possible effects on pathogen transmission. Repletion was achieved by capillary feeding. The presence of sporozoites in the salivary glands modified the behavior of the mosquitoes, with infected females tending to probe more than uninfected females (86% versus 44%). A white area around the tip of the proboscis was observed when the mosquitoes fed on blood from the vessels of mice immunized with saliva. Mosquito feedings elicited an acute inflammatory response in naive mice that peaked three hours after the bite. Polynuclear and mast cells were associated with saliva deposits. We describe the first visualization of saliva in the skin by immunohistochemistry (IHC) with antibodies directed against saliva. Both saliva deposits and sporozoites were detected in the skin for up to 18 h after the bite.
Conclusion
This study, in which we visualized the probing and engorgement phases of Anopheles gambiae blood meals, provides precise information about the behavior of the insect as a function of its infection status and the presence or absence of anti-saliva antibodies. It also provides insight into the possible consequences of the inflammatory reaction for blood feeding and pathogen transmission.
doi:10.1371/journal.pone.0050464
PMCID: PMC3521732  PMID: 23272060
11.  Type II Secretion System of Pseudomonas aeruginosa: In Vivo Evidence of a Significant Role in Death Due to Lung Infection 
The Journal of Infectious Diseases  2011;203(10):1369-1377.
Background. The role of toxins secreted by the type II secretion system (T2SS) of Pseudomonas aeruginosa during lung infection has been uncertain despite decades of research.
Methods. Using a model of pneumonia in Toll-like receptor (TLR) 2,4−/− mice, we reexamined the role of the T2SS system. Flagellin-deficient mutants of P. aeruginosa, with mutations in the T2SS and/or T3SS, were used to infect mice. Mice were followed up for survival, with some killed at different intervals to study bacterial clearance, inflammatory responses, and lung pathology.
Results. Strains carrying either secretion system were lethal for mice. Double mutants were avirulent. The T3SS+ strains killed mice within a day, and the T2SS+ strains killed them later. Mice infected with a strain that had only the T2SS were unable to eradicate the organism from the lungs, whereas those infected with a T2SS-T3SS double deletion were able to clear this mutant. Death caused by the T2SS+ strain was accompanied by a >50-fold increase in bacterial counts and higher numbers of viable intracellular bacteria.
Conclusions. The T2SS of P. aeruginosa may play a role in death from pneumonia, but its action is delayed. These data suggest that antitoxin strategies against this organism will require measures against the toxins secreted by both T2SS and T3SS.
doi:10.1093/infdis/jir045
PMCID: PMC3080898  PMID: 21502078
12.  Docosahexaenoic Acid Inhibits Helicobacter pylori Growth In Vitro and Mice Gastric Mucosa Colonization 
PLoS ONE  2012;7(4):e35072.
H. pylori drug-resistant strains and non-compliance to therapy are the major causes of H. pylori eradication failure. For some bacterial species it has been demonstrated that fatty acids have a growth inhibitory effect. Our main aim was to assess the ability of docosahexaenoic acid (DHA) to inhibit H. pylori growth both in vitro and in a mouse model. The effectiveness of standard therapy (ST) in combination with DHA on H. pylori eradication and recurrence prevention success was also investigated. The effects of DHA on H. pylori growth were analyzed in an in vitro dose-response study and n in vivo model. We analized the ability of H. pylori to colonize mice gastric mucosa following DHA, ST or a combination of both treatments. Our data demonstrate that DHA decreases H. pylori growth in vitro in a dose-dependent manner. Furthermore, DHA inhibits H. pylori gastric colonization in vivo as well as decreases mouse gastric mucosa inflammation. Addition of DHA to ST was also associated with lower H. pylori infection recurrence in the mouse model. In conclusion, DHA is an inhibitor of H. pylori growth and its ability to colonize mouse stomach. DHA treatment is also associated with a lower recurrence of H. pylori infection in combination with ST. These observations pave the way to consider DHA as an adjunct agent in H. pylori eradication treatment.
doi:10.1371/journal.pone.0035072
PMCID: PMC3328494  PMID: 22529974
13.  An Encapsulated Yersinia pseudotuberculosis Is a Highly Efficient Vaccine against Pneumonic Plague 
Background
Plague is still a public health problem in the world and is re-emerging, but no efficient vaccine is available. We previously reported that oral inoculation of a live attenuated Yersinia pseudotuberculosis, the recent ancestor of Yersinia pestis, provided protection against bubonic plague. However, the strain poorly protected against pneumonic plague, the most deadly and contagious form of the disease, and was not genetically defined.
Methodology and Principal Findings
The sequenced Y. pseudotuberculosis IP32953 has been irreversibly attenuated by deletion of genes encoding three essential virulence factors. An encapsulated Y. pseudotuberculosis was generated by cloning the Y. pestis F1-encoding caf operon and expressing it in the attenuated strain. The new V674pF1 strain produced the F1 capsule in vitro and in vivo. Oral inoculation of V674pF1 allowed the colonization of the gut without lesions to Peyer's patches and the spleen. Vaccination induced both humoral and cellular components of immunity, at the systemic (IgG and Th1 cells) and the mucosal levels (IgA and Th17 cells). A single oral dose conferred 100% protection against a lethal pneumonic plague challenge (33×LD50 of the fully virulent Y. pestis CO92 strain) and 94% against a high challenge dose (3,300×LD50). Both F1 and other Yersinia antigens were recognized and V674pF1 efficiently protected against a F1-negative Y. pestis.
Conclusions and Significance
The encapsulated Y. pseudotuberculosis V674pF1 is an efficient live oral vaccine against pneumonic plague, and could be developed for mass vaccination in tropical endemic areas to control pneumonic plague transmission and mortality.
Author Summary
Plague, among the most deadly infections of mankind's history, is present in Africa, Asia and America, and is currently re-emerging, recently causing cases in areas from where it had disappeared for decades. Pneumonic plague, its most deadly and contagious form, is responsible for human-to-human spreading of the infection. Vaccination would be an effective means to control the disease, but no efficient vaccine is currently available. Because live vaccines are potent inducers of protective immunity, our strategy was to use a Yersinia pseudotuberculosis, closely related to Y. pestis but genetically more stable, to make it suitable for use as live oral vaccine. We have developed a genetically defined Y. pseudotuberculosis strain strongly attenuated by deletion of virulence factors genes, which was also induced to produce the Y. pestis F1 pseudocapsule. A single oral dose was harmless and provided high- level protection against pneumonic plague. Such a candidate vaccine offers promising perspectives to control pneumonic plague mortality and transmission.
doi:10.1371/journal.pntd.0001528
PMCID: PMC3279354  PMID: 22348169
14.  Evolutionary History of Helicobacter pylori Sequences Reflect Past Human Migrations in Southeast Asia 
PLoS ONE  2011;6(7):e22058.
The human population history in Southeast Asia was shaped by numerous migrations and population expansions. Their reconstruction based on archaeological, linguistic or human genetic data is often hampered by the limited number of informative polymorphisms in classical human genetic markers, such as the hypervariable regions of the mitochondrial DNA. Here, we analyse housekeeping gene sequences of the human stomach bacterium Helicobacter pylori from various countries in Southeast Asia and we provide evidence that H. pylori accompanied at least three ancient human migrations into this area: i) a migration from India introducing hpEurope bacteria into Thailand, Cambodia and Malaysia; ii) a migration of the ancestors of Austro-Asiatic speaking people into Vietnam and Cambodia carrying hspEAsia bacteria; and iii) a migration of the ancestors of the Thai people from Southern China into Thailand carrying H. pylori of population hpAsia2. Moreover, the H. pylori sequences reflect iv) the migrations of Chinese to Thailand and Malaysia within the last 200 years spreading hspEasia strains, and v) migrations of Indians to Malaysia within the last 200 years distributing both hpAsia2 and hpEurope bacteria. The distribution of the bacterial populations seems to strongly influence the incidence of gastric cancer as countries with predominantly hspEAsia isolates exhibit a high incidence of gastric cancer while the incidence is low in countries with a high proportion of hpAsia2 or hpEurope strains. In the future, the host range expansion of hpEurope strains among Asian populations, combined with human motility, may have a significant impact on gastric cancer incidence in Asia.
doi:10.1371/journal.pone.0022058
PMCID: PMC3139604  PMID: 21818291
16.  Pulmonary Bacteriophage Therapy on Pseudomonas aeruginosa Cystic Fibrosis Strains: First Steps Towards Treatment and Prevention 
PLoS ONE  2011;6(2):e16963.
Multidrug-resistant bacteria are the cause of an increasing number of deadly pulmonary infections. Because there is currently a paucity of novel antibiotics, phage therapy—the use of specific viruses that infect bacteria—is now more frequently being considered as a potential treatment for bacterial infections. Using a mouse lung-infection model caused by a multidrug resistant Pseudomonas aeruginosa mucoid strain isolated from a cystic fibrosis patient, we evaluated bacteriophage treatments. New bacteriophages were isolated from environmental samples and characterized. Bacteria and bacteriophages were applied intranasally to the immunocompetent mice. Survival was monitored and bronchoalveolar fluids were analysed. Quantification of bacteria, bacteriophages, pro-inflammatory and cytotoxicity markers, as well as histology and immunohistochemistry analyses were performed. A curative treatment (one single dose) administrated 2 h after the onset of the infection allowed over 95% survival. A four-day preventive treatment (one single dose) resulted in a 100% survival. All of the parameters measured correlated with the efficacy of both curative and preventive bacteriophage treatments. We also showed that in vitro optimization of a bacteriophage towards a clinical strain improved both its efficacy on in vivo treatments and its host range on a panel of 20 P. aeruginosa cystic fibrosis strains. This work provides an incentive to develop clinical studies on pulmonary bacteriophage therapy to combat multidrug-resistant lung infections.
doi:10.1371/journal.pone.0016963
PMCID: PMC3039662  PMID: 21347240
17.  Molecular Detection and Identification of Zygomycetes Species from Paraffin-Embedded Tissues in a Murine Model of Disseminated Zygomycosis: a Collaborative European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Fungal Infection Study Group (EFISG) Evaluation ▿  
Journal of Clinical Microbiology  2010;48(6):2043-2046.
The present study was performed to assess the interlaboratory reproducibility of the molecular detection and identification of species of Zygomycetes from formalin-fixed paraffin-embedded kidney and brain tissues obtained from experimentally infected mice. Animals were infected with one of five species (Rhizopus oryzae, Rhizopus microsporus, Lichtheimia corymbifera, Rhizomucor pusillus, and Mucor circinelloides). Samples with 1, 10, or 30 slide cuts of the tissues were prepared from each paraffin block, the sample identities were blinded for analysis, and the samples were mailed to each of seven laboratories for the assessment of sensitivity. A protocol describing the extraction method and the PCR amplification procedure was provided. The internal transcribed spacer 1 (ITS1) region was amplified by PCR with the fungal universal primers ITS1 and ITS2 and sequenced. As negative results were obtained for 93% of the tissue specimens infected by M. circinelloides, the data for this species were excluded from the analysis. Positive PCR results were obtained for 93% (52/56), 89% (50/56), and 27% (15/56) of the samples with 30, 10, and 1 slide cuts, respectively. There were minor differences, depending on the organ tissue, fungal species, and laboratory. Correct species identification was possible for 100% (30 cuts), 98% (10 cuts), and 93% (1 cut) of the cases. With the protocol used in the present study, the interlaboratory reproducibility of ITS sequencing for the identification of major Zygomycetes species from formalin-fixed paraffin-embedded tissues can reach 100%, when enough material is available.
doi:10.1128/JCM.02319-09
PMCID: PMC2884487  PMID: 20375233
18.  The crucial role of the Aspergillus fumigatus siderophore system in interaction with alveolar macrophages 
Microbes and Infection / Institut Pasteur  2010;12(12-13):1035-1041.
Iron plays a central role in manifestation of infections for a variety of pathogens. To ensure an adequate supply with iron, Aspergillus fumigatus employs extra- and intracellular siderophores (low-molecular mass iron chelators), which are of importance for fungal growth in particular during iron starvation. Here we show that the lack of extracellular siderophores, and especially, the lack of the entire siderophore system cause in immunosuppressed mice in vivo (i) a reduced extracellular growth rate, (ii) a reduced intracellular growth rate in alveolar macrophages, and (iii) an increased susceptibility to conidial growth inhibition by alveolar macrophages. These data underline the crucial role of the fungal siderophore system not only for extracellular growth but also in the interaction with the host immune cells. Moreover, the hyphal growth rate within alveolar macrophages compared to extracellular lavage fluid was significantly decreased indicating that, besides elimination of fungal conidia, inhibition of pathogenic growth is a function of macrophages.
doi:10.1016/j.micinf.2010.07.005
PMCID: PMC2977081  PMID: 20659583
Aspergillus fumigatus; Alveolar macrophage; Aspergillosis; Siderophore; Conidial killing; Inflammation
19.  Comparative investigation of the pathogenicity of three Mycobacterium tuberculosis mutants defective in the synthesis of p-hydroxybenzoic acid derivatives 
p-Hydroxybenzoic acid derivatives (p-HBADs) are glycoconjugates secreted by all Mycobacterium tuberculosis isolates whose contribution to pathogenicity remains to be determined. The pathogenicity of three transposon mutants of M. tuberculosis deficient in the biosynthesis of some or all forms of p-HBADs was studied. Whilst the mutants grew similarly to the wild-type strain in macrophages and C57BL/6 mice, two of the mutants induced a more severe and diffuse inflammation in the lungs. The lack of production of some or all forms of p-HBADs in these two mutants also correlated with an increased secretion of the pro-inflammatory cytokines tumour-necrosis factor α, interleukin 6 and interleukin 12 in vivo. We propose that the loss of production of p-HBADs by tubercle bacilli results in their diminished ability to suppress the pro-inflammatory response to infection and that this ultimately provokes extensive pulmonary lesions in the C57BL/6 model of tuberculosis infection.
doi:10.1016/j.micinf.2006.04.008
PMCID: PMC2964916  PMID: 16782391
Mycobacterium; Tuberculosis; Phenolic glycolipids; p-Hydroxybenzoic acid derivatives
20.  Differential Protein Modulation in Midguts of Aedes aegypti Infected with Chikungunya and Dengue 2 Viruses 
PLoS ONE  2010;5(10):e13149.
Background
Arthropod borne virus infections cause several emerging and resurgent infectious diseases. Among the diseases caused by arboviruses, dengue and chikungunya are responsible for a high rate of severe human diseases worldwide. The midgut of mosquitoes is the first barrier for pathogen transmission and is a target organ where arboviruses must replicate prior to infecting other organs. A proteomic approach was undertaken to characterize the key virus/vector interactions and host protein modifications that happen in the midgut for viral transmission to eventually take place.
Methodology and Principal Findings
Using a proteomics differential approach with two-Dimensional Differential in-Gel Electrophoresis (2D-DIGE), we defined the protein modulations in the midgut of Aedes aegypti that were triggered seven days after an oral infection (7 DPI) with dengue 2 (DENV-2) and chikungunya (CHIKV) viruses. Gel profile comparisons showed that the level of 18 proteins was modulated by DENV-2 only and 12 proteins were modulated by CHIKV only. Twenty proteins were regulated by both viruses in either similar or different ways. Both viruses caused an increase of proteins involved in the generation of reactive oxygen species, energy production, and carbohydrate and lipid metabolism. Midgut infection by DENV-2 and CHIKV triggered an antioxidant response. CHIKV infection produced an increase of proteins involved in detoxification.
Conclusion/Significance
Our study constitutes the first analysis of the protein response of Aedes aegypti's midgut infected with viruses belonging to different families. It shows that the differentially regulated proteins in response to viral infection include structural, redox, regulatory proteins, and enzymes for several metabolic pathways. Some of these proteins like antioxidant are probably involved in cell protection. On the other hand, we propose that the modulation of other proteins like transferrin, hsp60 and alpha glucosidase, may favour virus survival, replication and transmission, suggesting a subversion of the insect cell metabolism by the arboviruses.
doi:10.1371/journal.pone.0013149
PMCID: PMC2950154  PMID: 20957153
21.  Human Herpesvirus 8 Genotype E in Patients with Kaposi Sarcoma, Peru 
Emerging Infectious Diseases  2010;16(9):1459-1462.
To determine human herpesvirus 8 (HHV-8) K1 genotypes in patients with Kaposi sarcoma (KS) from Peru, we characterized HHV-8 in 25 KS biopsy samples. Our findings of 8 A, 1 B, 14 C, and 2 E subtypes showed high HHV-8 diversity in these patients and association between E genotype and KS development.
doi:10.3201/eid1609.100381
PMCID: PMC3294982  PMID: 20735933
Human herpesvirus 8; HHV-8; Kaposi sarcoma; epidemiology; molecular epidemiology; Peru; viruses; dispatch
22.  Trypanosoma vivax Infections: Pushing Ahead with Mouse Models for the Study of Nagana. I. Parasitological, Hematological and Pathological Parameters 
African trypanosomiasis is a severe parasitic disease that affects both humans and livestock. Several different species may cause animal trypanosomosis and although Trypanosoma vivax (sub-genus Duttonella) is currently responsible for the vast majority of debilitating cases causing great economic hardship in West Africa and South America, little is known about its biology and interaction with its hosts. Relatively speaking, T. vivax has been more than neglected despite an urgent need to develop efficient control strategies. Some pioneering rodent models were developed to circumvent the difficulties of working with livestock, but disappointedly were for the most part discontinued decades ago. To gain more insight into the biology of T. vivax, its interactions with the host and consequently its pathogenesis, we have developed a number of reproducible murine models using a parasite isolate that is infectious for rodents. Firstly, we analyzed the parasitical characteristics of the infection using inbred and outbred mouse strains to compare the impact of host genetic background on the infection and on survival rates. Hematological studies showed that the infection gave rise to severe anemia, and histopathological investigations in various organs showed multifocal inflammatory infiltrates associated with extramedullary hematopoiesis in the liver, and cerebral edema. The models developed are consistent with field observations and pave the way for subsequent in-depth studies into the pathogenesis of T. vivax - trypanosomosis.
Author Summary
While most research efforts have focused on T. b. brucei trypanosomosis, infections caused by T. vivax and T. congolense which predominate in livestock and small ruminants have been subject to little study. In order to circumvent the major constraints inherent to studying T. vivax/host interactions in the field, we developed in vivo murine models of T. vivax trypanosomosis. We show here that the mouse experimental model reproduce most features of the infection in cattle. More than reflecting only the main parasitological parameters of the animal infection, the mouse model can be used to elucidate the immunopathological mechanisms involved in parasite evasion and persistence, and the tissue damage seen during infection and disease. Studies planned for the future will allow us to further investigate T. vivax–induced immunopathology in an experimental context for which all the necessary tools are now available.
doi:10.1371/journal.pntd.0000792
PMCID: PMC2919405  PMID: 20706595
23.  Trypanosoma vivax Infections: Pushing Ahead with Mouse Models for the Study of Nagana. II. Immunobiological Dysfunctions 
Trypanosoma vivax is the main species involved in trypanosomosis, but very little is known about the immunobiology of the infective process caused by this parasite. Recently we undertook to further characterize the main parasitological, haematological and pathological characteristics of mouse models of T. vivax infection and noted severe anemia and thrombocytopenia coincident with rising parasitemia. To gain more insight into the organism's immunobiology, we studied lymphocyte populations in central (bone marrow) and peripherical (spleen and blood) tissues following mouse infection with T. vivax and showed that the immune system apparatus is affected both quantitatively and qualitatively. More precisely, after an initial increase that primarily involves CD4+ T cells and macrophages, the number of splenic B cells decreases in a step-wise manner. Our results show that while infection triggers the activation and proliferation of Hematopoietic Stem Cells, Granulocyte-Monocyte, Common Myeloid and Megacaryocyte Erythrocyte progenitors decrease in number in the course of the infection. An in-depth analysis of B-cell progenitors also indicated that maturation of pro-B into pre-B precursors seems to be compromised. This interferes with the mature B cell dynamics and renewal in the periphery. Altogether, our results show that T. vivax induces profound immunological alterations in myeloid and lymphoid progenitors which may prevent adequate control of T. vivax trypanosomosis.
Author Summary
Trypanosoma vivax is responsible for animal trypanosomosis, or Nagana, in cattle and small ruminants. Under experimental conditions, the outbred mouse model infected with a well studied West African T. vivax isolate reproduces the main characteristics of the infection and pathology observed in livestock. Anemia and non-specific (parasite-directed) polyclonal hypergammaglobulinemia are the most common disorders coincident with the rise in parasitemia. Our results presented here show that the decrease in peripheral B cell populations does not seem to be compensated by newly arriving B cells from the bone marrow. The infection nevertheless prompts intense production of stem cells that mature into myeloid and lymphoid precursors. In spite of this, B cell numbers are specifically reduced in the periphery as the infection progresses. Thus, negative feedback seems to be set in motion by the infection in the bone marrow, more precisely affecting the maturation of B precursors and consequently the output of mature B cells. The origin of these phenomena is unclear but this doubtless creates a homeostatic imbalance that contributes to the inefficient immune response against T. vivax infection.
doi:10.1371/journal.pntd.0000793
PMCID: PMC2919407  PMID: 20711524
24.  Isolation and Characterization of New Leptospira Genotypes from Patients in Mayotte (Indian Ocean) 
Background
Leptospirosis has been implicated as a severe and fatal form of disease in Mayotte, a French-administrated territory located in the Comoros archipelago (southwestern Indian Ocean). To date, Leptospira isolates have never been isolated in this endemic region.
Methods and Findings
Leptospires were isolated from blood samples from 22 patients with febrile illness during a 17-month period after a PCR-based screening test was positive. Strains were typed using hyper-immune antisera raised against the major Leptospira serogroups: 20 of 22 clinical isolates were assigned to serogroup Mini; the other two strains belonged to serogroups Grippotyphosa and Pyrogenes, respectively. These isolates were further characterized using partial sequencing of 16S rRNA and ligB gene, Multi Locus VNTR Analysis (MLVA), and pulsed field gel electrophoresis (PFGE). Of the 22 isolates, 14 were L. borgpetersenii strains, 7 L. kirschneri strains, and 1, belonging to serogoup Pyrogenes, was L. interrogans. Results of the genotyping methods were consistent. MLVA defined five genotypes, whereas PFGE allowed the recognition of additional subgroups within the genotypes. PFGE fingerprint patterns of clinical strains did not match any of the patterns in the reference strains belonging to the same serogroup, suggesting that the strains were novel serovars.
Conclusions
Preliminary PCR screening of blood specimen allowed a high isolation frequency of leptospires among patients with febrile illness. Typing of leptospiral isolates showed that causative agents of leptospirosis in Mayotte have unique molecular features.
Author Summary
Leptospirosis has been recognized as an increasing public health problem affecting poor people from developing countries and tropical regions. However, the epidemiology of leptospirosis remains poorly understood in remote parts of the world. In this study of patients from the island of Mayotte, we isolated 22 strains from the blood of patients during the acute phase of illness. The pathogenic Leptospira strains were characterized by serology and various molecular typing methods. Based on serological data, serogroup Mini appears to be the dominant cause of leptospirosis in Mayotte. Further molecular characterization of these isolates allowed the identification of 10 pathogenic Leptospira genotypes that could correspond to previously unknown serovars. Further progress in our understanding of the epidemiology of Leptospira circulating genotypes in highly endemic regions should contribute to the development of novel strategies for the diagnosis and prevention of this neglected emerging disease.
doi:10.1371/journal.pntd.0000724
PMCID: PMC2889827  PMID: 20582311
25.  Deficiency of the LIM-Only Protein FHL2 Reduces Intestinal Tumorigenesis in Apc Mutant Mice 
PLoS ONE  2010;5(4):e10371.
Background
The four and a half LIM-only protein 2 (FHL2) is capable of shuttling between focal adhesion and nucleus where it signals through direct interaction with a number of proteins including β-catenin. Although FHL2 activation has been found in various human cancers, evidence of its functional contribution to carcinogenesis has been lacking.
Methodology/Principal Findings
Here we have investigated the role of FHL2 in intestinal tumorigenesis in which activation of the Wnt pathway by mutations in the adenomatous polyposis coli gene (Apc) or in β-catenin constitutes the primary transforming event. In this murine model, introduction of a biallelic deletion of FHL2 into mutant ApcΔ14/+ mice substantially reduces the number of intestinal adenomas but not tumor growth, suggesting a role of FHL2 in the initial steps of tumorigenesis. In the lesions, Wnt signalling is not affected by FHL2 deficiency, remaining constitutively active. Nevertheless, loss of FHL2 activity is associated with increased epithelial cell migration in intestinal epithelium, which might allow to eliminate more efficiently deleterious cells and reduce the risk of tumorigenesis. This finding may provide a mechanistic basis for tumor suppression by FHL2 deficiency. In human colorectal carcinoma but not in low-grade dysplasia, we detected up-regulation and enhanced nuclear localization of FHL2, indicating the activation of FHL2 during the development of malignancy.
Conclusions/Significance
Our data demonstrate that FHL2 represents a critical factor in intestinal tumorigenesis.
doi:10.1371/journal.pone.0010371
PMCID: PMC2860980  PMID: 20442768

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