Fibronectin-binding proteins and clumping factor A in Staphylococcus aureus experimental endocarditis: FnBPA is sufficient to activate human endothelial cells.

Surface molecules of Staphylococcus aureus are involved in the colonization of vascular endothelium which is a crucial primary event in the pathogenesis of infective endocarditis (IE). The ability of these molecules to also launch endothelial procoagulant and proinflammatory responses, which characterize IE, is not known. In the present study we investigated the individual capacities of three prominent S. aureus surface molecules; fibronectin-binding protein A (FnBPA) and B (FnBPB) and clumping factor A (ClfA), to promote bacterial adherence to cultured human endothelial cells (ECs) and to activate phenotypic and functional changes in these ECs. Non-invasive surrogate bacterium Lactococcus lactis, which, by gene transfer, expressed staphylococcal FnBPA, FnBPB or ClfA molecules were used. Infection of ECs increased 50- to 100-fold with FnBPA- or FnBPB-positive recombinant lactococci. This coincided with EC activation, interleukin-8 secretion and surface expression of ICAM-1 and VCAM-1 and concomitant monocyte adhesion. Infection with ClfA-positive lactococci did not activate EC. FnBPA-positive L. lactis also induced a prominent tissue factor-dependent endothelial coagulation response that was intensified by cell-bound monocytes. Thus S. aureus FnBPs, but not ClfA, confer invasiveness and pathogenicity to non-pathogenic L. lactis organisms indicating that bacterium-EC interactions mediated by these adhesins are sufficient to evoke inflammation as well as procoagulant activity at infected endovascular sites.

Fibrinogen and fibronectin binding cooperate for valve infection and invasion in Staphylococcus aureus experimental endocarditis.

The expression of Staphylococcus aureus adhesins in Lactococcus lactis identified clumping factor A (ClfA) and fibronectin-binding protein A (FnBPA) as critical for valve colonization in rats with experimental endocarditis. This study further analyzed their role in disease evolution. Infected animals were followed for 3 d. ClfA-positive lactococci successfully colonized damaged valves, but were spontaneously eradicated over 48 h. In contrast, FnBPA-positive lactococci progressively increased bacterial titers in vegetations and spleens. At imaging, ClfA-positive lactococci were restricted to the vegetations, whereas FnBPA-positive lactococci also invaded the adjacent endothelium. This reflected the capacity of FnBPA to trigger cell internalization in vitro. Because FnBPA carries both fibrinogen- and fibronectin-binding domains, we tested the role of these functionalities by deleting the fibrinogen-binding domain of FnBPA and supplementing it with the fibrinogen-binding domain of ClfA in cis or in trans. Deletion of the fibrinogen-binding domain of FnBPA did not alter fibronectin binding and cell internalization in vitro. However, it totally abrogated valve infectivity in vivo. This ability was restored in cis by inserting the fibrinogen-binding domain of ClfA into truncated FnBPA, and in trans by coexpressing full-length ClfA and truncated FnBPA on two separate plasmids. Thus, fibrinogen and fibronectin binding could cooperate for S. aureus valve colonization and endothelial invasion in vivo.

Comparative transcriptomics of extreme phenotypes of human HIV-1 infection and SIV infection in sooty mangabey and rhesus macaque.

High levels of HIV-1 replication during the chronic phase of infection usually correlate with rapid progression to severe immunodeficiency. However, a minority of highly viremic individuals remains asymptomatic and maintains high CD4+ T cell counts. This tolerant profile is poorly understood and reminiscent of the widely studied nonprogressive disease model of SIV infection in natural hosts. Here, we identify transcriptome differences between rapid progressors (RPs) and viremic nonprogressors (VNPs) and highlight several genes relevant for the understanding of HIV-1-induced immunosuppression. RPs were characterized by a specific transcriptome profile of CD4+ and CD8+ T cells similar to that observed in pathogenic SIV-infected rhesus macaques. In contrast, VNPs exhibited lower expression of interferon-stimulated genes and shared a common gene regulation profile with nonpathogenic SIV-infected sooty mangabeys. A short list of genes associated with VNP, including CASP1, CD38, LAG3, TNFSF13B, SOCS1, and EEF1D, showed significant correlation with time to disease progression when evaluated in an independent set of CD4+ T cell expression data. This work characterizes 2 minimally studied clinical patterns of progression to AIDS, whose analysis may inform our understanding of HIV pathogenesis.

Retroviral infection of neonatal Peyer's patch lymphocytes: the mouse mammary tumor virus model.

Mouse mammary tumor virus is known to infect newborn mice via mother's milk. A proposed key step for viral spread to the mammary gland is by the infection of lymphocytes. We show here that although in suckling mice retroviral proteins are found in all epithelial cells of the gut, viral DNA is exclusively detectable in the Peyer's patches. As early as 5 d after birth the infection leads to a superantigen response in the Peyer's patches but not in other lymphoid organs draining the intestine. Viral DNA can be detected before the superantigen response and becomes first evident in the Peyer's patches followed by mesenteric lymph nodes and finally all lymphoid organs.

Adaptation of a Gaussia princeps Luciferase reporter system in Candida albicans for in vivo detection in the Galleria mellonella infection model.

For the past 10 years, mini-host models and in particular the greater wax moth Galleria mellonella have tended to become a surrogate for murine models of fungal infection mainly due to cost, ethical constraints and ease of use. Thus, methods to better assess the fungal pathogenesis in G. mellonella need to be developed. In this study, we implemented the detection of Candida albicans cells expressing the Gaussia princeps luciferase in its cell wall in infected larvae of G. mellonella. We demonstrated that detection and quantification of luminescence in the pulp of infected larvae is a reliable method to perform drug efficacy and C. albicans virulence assays as compared to fungal burden assay. Since the linearity of the bioluminescent signal, as compared to the CFU counts, has a correlation of R(2) = 0.62 and that this method is twice faster and less labor intensive than classical fungal burden assays, it could be applied to large scale studies. We next visualized and followed C. albicans infection in living G. mellonella larvae using a non-toxic and water-soluble coelenterazine formulation and a CCD camera that is commonly used for chemoluminescence signal detection. This work allowed us to follow for the first time C. albicans course of infection in G. mellonella during 4 days.

Mouse Model of Respiratory Tract Infection Induced by Waddlia chondrophila.

Waddlia chondrophila, an obligate intracellular bacterium belonging to the Chlamydiales order, is considered as an emerging pathogen. Some clinical studies highlighted a possible role of W. chondrophila in bronchiolitis, pneumonia and miscarriage. This pathogenic potential is further supported by the ability of W. chondrophila to infect and replicate within human pneumocytes, macrophages and endometrial cells. Considering that W. chondrophila might be a causative agent of respiratory tract infection, we developed a mouse model of respiratory tract infection to get insight into the pathogenesis of W. chondrophila. Following intranasal inoculation of 2 x 108 W. chondrophila, mice lost up to 40% of their body weight, and succumbed rapidly from infection with a death rate reaching 50% at day 4 post-inoculation. Bacterial loads, estimated by qPCR, increased from day 0 to day 3 post-infection and decreased thereafter in surviving mice. Bacterial growth was confirmed by detecting dividing bacteria using electron microscopy, and living bacteria were isolated from lungs 14 days post-infection. Immunohistochemistry and histopathology of infected lungs revealed the presence of bacteria associated with pneumonia characterized by an important multifocal inflammation. The high inflammatory score in the lungs was associated with the presence of pro-inflammatory cytokines in both serum and lungs at day 3 post-infection. This animal model supports the role of W. chondrophila as an agent of respiratory tract infection, and will help understanding the pathogenesis of this strict intracellular bacterium.

Transgenic mice overexpressing neuropeptide Y: An experimental model of metabolic and cardiovascular diseases

Neuropeptide Y (NPY) is an abundant neurotransmitter in the brain and sympathetic nervous system (SNS). Hypothalamic NPY is known to be a key player in food intake and energy expenditure. NPY’s role in cardiovascular regulation has also been shown. In humans, a Leucine 7 to Proline 7 single nucleotide polymorphism (p.L7P) in the signal peptide of the NPY gene has been associated with traits of metabolic syndrome. The p.L7P subjects also show increased stress-related release of NPY, which suggests that more NPY is produced and released from SNS. The main objective of this study was to create a novel mouse model with noradrenergic cell-targeted overexpression of NPY, and to characterize the metabolic and vascular phenotype of this model. The mouse model was named OE-NPY^DBH mouse. Overexpression of NPY in SNS and brain noradrenergic neurons led to increased adiposity without significant weight gain or increased food intake. The mice showed lipid accumulation in the liver at young age, which together with adiposity led to impaired glucose tolerance and hyperinsulinemia with age. The mice displayed stress-related increased mean arterial blood pressure, increased plasma levels of catecholamines and enhanced SNS activity measured by GDP binding activity to brown adipose tissue mitochondria. Sexual dimorphism in NPY secretion pattern in response to stress was also seen. In an experimental model of vascular injury, the OE-NPY^DBH mice developed more pronounced neointima formation compared with wildtype controls. These results together with the clinical data indicate that NPY in noradrenergic cells plays an important role in the pathogenesis of metabolic syndrome and related diseases. Furthermore, new insights on the role of the extrahypothalamic NPY in the process have been obtained. The OE-NPY^DBH model provides an important tool for further stress and metabolic syndrome-related studies.

Immunohistochemical detection of Tritrichomonas foetus in experimentally infected mice

The need to intensify knowledge of the pathogenesis of bovine genital trichomoniasis (BGT) led to the use of alternative animal models such as the mouse. Nevertheless, it is necessary to elucidate the dynamics of the infection in this animal species, evaluating different stages of the colonization and evolution of the pathological alterations. The immunohistochemistry (IHC) offers advantages over the routine histopathological staining techniques for the detection of the protozoan in tissues, cellular detritus and inside the macrophages. The goal of the present study was to demonstrate the presence of Tritrichomonas foetus in the reproductive tract of infected mice using an IHC technique. Female BALB/c mice were infected with a suspension of T. foetus by intravaginal route, in the estrum phase, detected by exfoliative vaginal cytology. After 10 weeks, the animals were sacrificed; uterus and vagina were fixed and histologically processed. Some slides were stained with HE. The rest of the slides were processed for IHC. An immunoadsorbed polyclonal serum against T. foetus was used. The avidine-biotine technique (HistoMouse, Zymed™) was employed. The histopathological studies showed a dilation of the uterine glands, presence of macrophages in the lumen of the organ and inner part of the endometrial glands. No T. foetus was identified using this method. The IHQ allowed additionally the identification of the protozoan in the endometrium, endometrial glands, uterine lumen and inside neutrophils and macrophages. The cytological studies stained with IHC showed either isolated T. foetus adhered to epithelial cells or inside macrophages. This technique proves to be a useful tool for the study of the pathogenesis of bovine genital trichomoniasis (BGT) in an experimental model.

Effects of infection with Toxoplasma gondii oocysts on the intestinal wall and the myenteric plexus of chicken (Gallus gallus)

This paper aims to analyze the effects of the Toxoplasma gondii infection in the intestinal wall and myenteric plexus of chicken (Gallus gallus). Ten 36-day-old chickens were separated into two groups: control and experimental, orally inoculated with oocysts of the T. gondii strain M7741 genotype III. After 60 days the birds were submitted to euthanasia and had their duodenum removed. Part of the intestinal segments was submitted to histological routine, HE staining, PAS histochemical technique, and Alcian Blue. Qualitative analysis of the intestinal wall and comparative measurements among the groups with respect to total wall thickness, muscle tunic, mucosa, and tunica mucosa were carried out. Caliciform cells were quantified. The other part of the intestinal segments was fixed in formol acetic acid and dissected having the tunica mucosa and the tela submucosa removed. Neurons were stained with Giemsa, counted, and measured. Chickens from the experimental group presented diarrhea and inflammatory infiltrates in the tunica mucosa, thickness reduction of all the parameters assessed in the intestinal wall, and an increase of the number of caliciform cells. There was a ~70% reduction regarding the intensity of myenteric neurons; and the remaining cells presented a reduction of ~2.4% of the perikarion and ~40.5% of the nucleus (p<0.05). Chronic infection induced by T. gondii oocysts resulted in intestinal wall atrophy, mucin secretion increase, death and atrophy of chicken myenteric plexus neurons. Death and atrophy of myenteric plexus neurons may be related with the causes of diarrhea observed in chickens with toxoplasmosis.

Infection of sparrows (Passer domesticus) and different mice strains with Lawsonia intracellularis

The susceptibility of sparrows (Passer domesticus) and strains of mice (Swiss, BALB/c, C-57 and DB-A) to Lawsonia intracellularis infection was studied. Thirty-two sparrows were inoculated with pure culture of L. intracellularis and eleven received sham inoculum. Feces were collected on -1, 7, 14 and 21 days post infection (dpi) for detection of L. intracellularis by PCR. After 21 days, all sparrows were euthanized and the tissues processed for histology and immunohistochemistry (IHC). One hundred sixty mice of four different strains (n=40, per strain) were used. For each mouse strain, 16 animals received mucosa homogenate from a pig infected with L. intracellularis, 16 received pure culture of L. intracellularis and eight animals received sham inoculum. Two control and four inoculated mice from each group were euthanized on 7, 14, 21 and 28 dpi. Sections of intestine were collected for histologic analysis and IHC and pooled feces were collected for L. intracellularis PCR. None of the sparrows had any histologic lesions characteristic of proliferative enteropathy or antigen labeling by IHC. All sparrow fecal samples were negative by PCR. All mice strains studied had histopathological lesions typical of PE and IHC labeling consistent with L. intracellularis infection, especially those animals inoculated with pure culture. The most severe lesions were observed in DB-A and Swiss mice. Fecal shedding was detected in all mice strains, with peak at 14 dpi. We conclude that sparrows do not seem to be relevant in the epidemiology of L. intracellularis. The results showed variations in the lesions among the four mice strains used.

Regulation of lymphocyte response in vitro and in vivo

CD4+ T helper (Th) cells have an important role in the defence against diverse pathogens. Th cells can differentiate into several functionally distinct subtypes including Th1 and Th2 cells. Th1 cells are important for eradicating intracellular pathogens, whereas Th2 cells pro¬tect our body against extracellular parasites. However if uncontrolled, Th cells can mediate immunopathology such as asthma or allergies, but inappropriate Th response can also lead to autoimmune diseases such as multiple sclerosis or type 1 diabetes. Deeper knowledge of the regulation of the lymphocyte response both in vitro and in vivo is important for un¬derstanding the pathogenesis of immune-mediated diseases and microbe-host interactions. In the work presented in this thesis, the first goal was to elucidate the role of novel factors, PIM kinases and c-FLIP in the regulation of human Th cell differentiation. The oncogenic serine-threonine kinases of the PIM family were shown to be preferentially expressed in Th1 cells and in addition, by using RNA interference, they were also shown to be positive regulators of Th1 differentiation. The PIM depletion experiments suggest that PIM kinases promote the expression of the hallmark cytokine of Th1 cells, IFNγ, and influence the IL12/STAT4 pathway during the early Th1 cell differentiation. In addition to cytokine and T cell receptor (TCR) induced pathways, caspase activity has been shown to regulate Th cell proliferation. In the work presented in this thesis, the two isoforms of the caspase regulator protein, c-FLIP, were shown to be differentially ex¬pressed in Th1 and Th2 cells. Both of the isoforms were up-regulated in response to TCR activation, but the expression of the short isoform was selectively induced by IL4, the Th2 inducing cytokine. Furthermore, the c-FLIP isoforms had distinct and opposite roles during the early differentiation of Th1 and Th2 cells. The knockdown of the long isoform of c-FLIP led to the induction of Th1 marker genes, such as IFNγ and TBET, whereas the depletion of c-FLIP short down-regulated Th2 marker genes IL-4 and GATA3. The third goal was to elucidate the gene expression profiles characterizing the T- and B-lymphocyte responses in vivo during experimental infection by intracellular bacte¬rium Chlamydia pneumoniae. Previously, it has been shown that CD8+ and CD4+ T cells are important for the protection against Chlamydia pneumoniae. In this study, the analysis revealed up-regulation of interferon induced genes during recurrent infection underlining the importance of IFNγ secreted by Th1 and CD8+ T cells in the protection against this pathogen. Taken together, in this study novel regulators of Th cell differ¬entiation were discovered and in addition the gene expression profiles of lymphocytes induced by Chlamydia pneumoniae infection were characterized.

Bronchoalveolar lavage as a tool for evaluation of cellular alteration during Aelurostrongylus abstrusus infection in cats

Bronchoalveolar lavage (BAL) is a procedure that retrieves cells and other elements from the lungs for evaluation, which helps in the diagnosis of pulmonary diseases. The aim of this study was to perform this procedure for cellular analysis of BAL fluid alterations during experimental infection with Aelurostrongylus abstrusus in cats. Fourteen cats were individually inoculated with 800 third stage larvae of A. abstrusus and five non-infected cats lined as a control group. The BAL procedure was performed through the use of an endotracheal tube on the nineteen cats with a mean age of 18 months, on 0, 30, 60, 90, 120, 180 and 270 days after infection. Absolute cell counts in the infected cats revealed that alveolar macrophages and eosinophils were the predominant cells following infection. This study shows that the technique allows us to retrieve cells and first stage larvae what provides information about the inflammatory process caused by aelurostrongylosis.

Cytokine gene expression and molecular detection of Mycobacterium avium subspecies paratuberculosisin organs of experimentally infected mice

AbstractMycobacterium avium subspecies paratuberculosis (MAP) can infect ruminants and remain subclinical for long periods within herds. The identification of organs that are more susceptible to infection and the evaluation of cytokine expression at the site of infection are important to understand the pathogenesis of MAP. In this study, the probability of detection of MAP-DNA and the expression of cytokines in organs of C57BL/6 mice infected intraperitoneally for 120 days were evaluated. Among the evaluated organs, the spleen (85%), colon (75%) and liver (60%) had the highest frequency of positivity. When compared these frequencies between organs, it has been found that the spleen had 1.54 times as likely to be positive in relation to the ileum, and 2.0 times more likely in relation to the Peyer's patches. In addition, at 60 days post-infection, the spleen and the liver were responsible for upregulation of IFN-γ , and the ileum by TNF-α and IL-4. The results indicate that the spleen is the best organ for evaluating an experimental infection by MAP, especially in the initial stages of the infection. Moreover, it showed that the spleen, liver and ileum have a direct role in the inflammatory response in experimental models.

Outcome of human papillomavirus infection among men in the Finnish Family HPV Study

Ihmisen papilloomaviruksen taudinkuva suomalaisperheiden seurantatutkimuksen mieskohortissa Tieto ihmisen papilloomaviruksen (HPV) yhteydestä eri anatomisten alueiden sairauksien syntyyn on lisännyt mielenkiintoa miehen papilloomavirustulehduksen taudinkulun selvittämiseksi. Tämä väitöskirjatyö on osa suomalaista seurantatutkimusta, jossa tutkitaan HPVinfektioiden tartuntareittejä 329 perheessä. Väitöstyössä keskitytään tutkimukseen osallistuneiden 131 miehen aineistoon. Suun limakalvonäytteet otettiin seitsemässä aikapisteessä. Lisäksi otettiin sukuelinalueen näytteet kahdella seurantakäynnillä. Riskitekijöitä kartoittava kyselytutkimus teetettiin tutkimuksen alkutilanteessa sekä viimeisellä seurantakäynnillä. Oireettomat HPV infektiot olivat alkutilanteessa yleisiä molemmilla sukupuolilla (miesten suu 18,3 % ja sukuelinalue 35,9 %, naisten suu 17,2 % ja kohdunsuu 18,8 %), mutta HPV:n genotyyppien vastaavuus partnerien välillä oli vähäinen. Naisen, mutta ei miehen, seksuaalinen riskikäyttäytyminen oli yhteydessä pariskunnan HPV tyyppien vastaavuuteen. Partnerin ja siviilisäädyn vaihtaminen lisäsivät miehen riskiä saada uusia HPV infektioita. Miehen suun limakalvonäytteistä löytyi kaikkiaan 17 eri HPV tyyppiä. Suun HPV-tulehduksen esiintyvyys vaihteli eri aikapisteissä 15 %:sta 31 %:iin. Uusien HPV tulehdusten ilmaantumisaika vaihteli 3,9 ja 25,7 kuukauden välillä. Suun HPV infektio parani valtaosalla miehistä. Suun krooninen HPV-infektio todettiin 14 %:lla miehistä. Näiden infektioiden keskimääräinen kesto vaihteli 6.0:sta 30.7:ään kuukauteen. Tupakointi lisäsi korkean riskin HPV tyyppien aiheuttamien suun kroonisten infektioiden riskiä, kun taas aikaisemmin sairastetut sukuelinten kondyloomat suojasivat siltä. Tuloksemme osoittavat, että miehen oireeton HPV tulehdus on yleinen suussa ja sukuelinten alueella. Vakaa parisuhde suojaa uusilta HPV tulehduksilta. Tupakoinnilla on keskeinen merkitys suun HPV-infektion kroonistumisessa.

The use of non-human primates as animal models for the study of hepatitis viruses

Hepatitis viruses belong to different families and have in common a striking hepatotropism and restrictions for propagation in cell culture. The transmissibility of hepatitis is in great part limited to non-human primates. Enterically transmitted hepatitis viruses (hepatitis A virus and hepatitis E virus) can induce hepatitis in a number of Old World and New World monkey species, while the host range of non-human primates susceptible to hepatitis viruses transmitted by the parenteral route (hepatitis B virus, hepatitis C virus and hepatitis delta virus) is restricted to few species of Old World monkeys, especially the chimpanzee. Experimental studies on non-human primates have provided an invaluable source of information regarding the biology and pathogenesis of these viruses, and represent a still indispensable tool for vaccine and drug testing.