[Show abstract][Hide abstract] ABSTRACT: Group B Streptococcus (GBS), a commensal organism, can turn into a life-threatening pathogen in neonates and elderly, or in adults with severe underlying diseases such as diabetes. We developed a vaccine targeting the GBS glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme detected at the bacterial surface, which was proven to be effective in a neonatal mouse model of infection. Since this bacterium has emerged as an important pathogen in non-pregnant adults, here we investigated whether this vaccine also confers protection in an adult susceptible and in a diabetic mouse model of infection. For immunoprotection studies, sham or immunized adult mice were infected with GBS serotype Ia and V strains, the two most prevalent serotypes isolated in adults. Sham and vaccinated mice were also rendered diabetic and infected with a serotype V GBS strain. For toxicological (pre-clinical) studies, adult mice were vaccinated three times, with three concentrations of recombinant GAPDH adjuvanted with Allydrogel, and the toxicity parameters were evaluated twenty-four hours after the last immunization. For the stability tests, the vaccine formulations were maintained at 4°C for 6 and 12 months prior immunization. The results showed that all tested doses of the vaccine, including the stability study formulations, were immunogenic and that the vaccine was innocuous. The organs (brain, blood, heart, and liver) of vaccinated susceptible or diabetic adult mice were significantly less colonized compared to those of control mice. Altogether, these results demonstrate that the GAPDH-based vaccine is safe and stable and protects susceptible and diabetic adult mice against GBS infections. It is therefore a promising candidate as a global vaccine to prevent GBS-induced neonatal and adult diseases.
[Show abstract][Hide abstract] ABSTRACT: We have used vascular castings, light microscopy coupled with tracers, and scanning electron microscopy to define the detailed anatomy of the bronchial arteries in the Wistar rat, a rodent often used in experimental research on lung disorders; namely in those that involve vascular alterations. We found that there are two bronchial arteries in the Wistar rat and that they have a cranial origin, either from the subclavian arteries or from their primary branches. The left bronchial artery was always originated from the internal thoracic artery, ran between the thoracic aorta and the left cranial vena cava and offered branches to the thymus, trachea and esophagus. The right bronchial artery was of variable origin, and was located between the right cranial vena cava and the trachea; it gave off branches to the right cranial vena cava, phrenic nerve, trachea, esophagus and, seldomly, to the mediastinic-pericardial pleura, myocardium and caudal vena cava. In more than half of the rats, there were anastomoses between the bronchial and pulmonary arteries. The histological organization of rat bronchial arteries was different from those of humans, suggesting that there are differences in the resistance to blood flow between the two species. Scanning electron microscopy revealed that the bronchial arteries formed two plexuses surrounding the intrapulmonary airways and also supplied the vasa vasorum of pulmonary arteries and veins. We conclude that there are important differences in the arrangement and structure between bronchial arteries in humans and rats and that this should be taken into account whenever data from experimental studies are to be extended to humans.
Preview · Article · Mar 2015 · European Journal of Anatomy
[Show abstract][Hide abstract] ABSTRACT: The adipose tissue can have important contributions for immune functions. Nevertheless, only a limited number of reports investigated in lean hosts the immune response elicited in this tissue upon infection. Previous studies suggested that the intracellular protozoan Neospora caninum might affect adipose tissue physiology. Therefore, we investigated in mice challenged with this protozoan if immune cell populations within adipose tissue of different anatomical locations could be differently affected. Early upon infection, parasites were detected in the adipose tissue and by seven days of infection increased numbers of macrophages, regulatory T cells and T-bet+ cells were observed in gonadal, mesenteric, omental and subcutaneous adipose tissue. Increased expression of Interferon-γ was also detected in gonadal adipose tissue of infected mice. Two months after infection parasitic DNA was no longer detected in these tissues, but Th1 cell numbers remained above control levels in the infected mice. Moreover, the Th1/Treg cell ratio was higher than that of controls in the mesenteric and subcutaneous adipose tissue. Interestingly, chronically infected mice presented a marked increase of serum leptin, a molecule that plays a role in energy balance regulation as well as in promoting Th1-type immune responses. Altogether, we show that an apicomplexa parasitic infection influences immune cellular composition of adipose tissue throughout the body as well as adipokine production, still noticed at a chronic phase of infection when parasites were already cleared from that particular tissue. This strengthens the emerging view that infections can have long-term consequences for the physiology of adipose tissue.This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Rabbit Haemorrhagic Disease (RHD) is caused by a calicivirus (RHDV) that kills 90% of infected adult European rabbits within 3 days. Remarkably, young rabbits are resistant to RHD. We induced immunosuppression in young rabbits by treatment with methylprednisolone acetate (MPA) and challenged the animals with RHDV by intramuscular injection. All of these young rabbits died within 3 days of infection due to fulminant hepatitis, presenting a large number of RHDV-positive dead or apoptotic hepatocytes, and a significant seric increase in cytokines, features that are similar to those of naive adult rabbits infected by RHDV. We conclude that MPA-induced immunosuppression abrogates the resistance of young rabbits to RHD, indicating that there are differences in the innate immune system between young and adult rabbits that contribute to their distinct resistance/susceptibility to RHDV infection.
Preview · Article · Feb 2014 · Veterinary Research
[Show abstract][Hide abstract] ABSTRACT: Sepsis is the third most common cause of neonatal death, with Group B Streptococcus (GBS) being the leading bacterial agent. The pathogenesis of neonatal septicemia is still unsolved. We described previously that host susceptibility to GBS infection is due to early IL-10 production. In this study, we investigated whether triggering TLR2 to produce IL-10 is a risk factor for neonatal bacterial sepsis. We observed that, in contrast to wild-type (WT) pups, neonatal TLR2-deficient mice were resistant to GBS-induced sepsis. Moreover, if IL-10 signaling were blocked in WT mice, they also were resistant to sepsis. This increased survival rate was due to an efficient recruitment of neutrophils to infected tissues that leads to bacterial clearance, thus preventing the development of sepsis. To confirm that IL-10 produced through TLR2 activation prevents neutrophil recruitment, WT pups were treated with the TLR2 agonist Pam3CSK4 prior to nebulization with the neutrophil chemotactic agent LTB4. Neutrophil recruitment into the neonatal lungs was inhibited in pups treated with Pam3CSK4. However, the migration was restored in Pam3CSK4-treated pups when IL-10 signaling was blocked (either by anti-IL-10R mAb treatment or by using IL-10-deficient mice). Our findings highlight that TLR2-induced IL-10 production is a key event in neonatal susceptibility to bacterial sepsis.
Preview · Article · Sep 2013 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: Group B Streptococcus (GBS) is the leading cause of meningitis in neonates. We have previously shown that plasminogen, once recruited to the GBS cell surface and converted into plasmin by host-derived activators, leads to an enhancement of bacterial virulence. Here, we investigated whether plasmin(ogen) bound at the GBS surface contributes to blood-brain barrier penetration and invasion of the central nervous system. For that purpose, GBS strain NEM316 preincubated with or without plasminogen plus tissue type plasminogen activator was analyzed for the capacity to adhere to, invade and transmigrate the human brain microvascular endothelial cell (hBMEC) monolayer, and to penetrate the central nervous system using a neonatal mouse model. At earlier times of infection, plasmin(ogen)-treated GBS exhibited a significant increase in adherence to and invasion of hBMECs. Later, injury of hBMECs were observed with plasmin(ogen)-treated GBS that displayed a plasmin-like activity. The same results were obtained when hBMECs were incubated with whole human plasma and infected with untreated GBS. To confirm that the observed effects were due to the recruitment and activation of plasminogen on GBS surface, the bacteria were first incubated with epsilon-aminocaproic acid (εACA), an inhibitor of plasminogen binding, and thereafter with plasmin(ogen). A significant decrease in the hBMECs injury that was correlated with a decrease of the GBS surface proteolytic activity was observed. Furthermore, plasmin(ogen)-treated GBS infected more efficiently the brain of neonatal mice than the untreated bacteria, indicating that plasmin(ogen) bound to GBS surface may facilitate the traversal of the blood-brain barrier. A higher survival rate was observed in offspring born from εACA-treated mothers, compared to untreated mice, and no brain infection was detected in these neonates. Our findings suggest that capture of the host plasmin(ogen) by the GBS surface promotes the crossing of the blood-brain barrier and contributes to the establishment of meningitis.
[Show abstract][Hide abstract] ABSTRACT: Young rabbits (i.e. up to 4 weeks of age) are naturally resistant to infection by rabbit haemorrhagic disease virus (RHDV), the same calicivirus that kills more than 90% of adult rabbits in 3 days or less. To characterize this fascinating model of age-related natural resistance to viral infection, we have studied the kinetics (from 6h up to 7 days) of cytokines and of leukocyte subpopulations in the liver (the target organ for calicivirus replication) and spleen (host systemic response) of RHDV infected young rabbits. Infection was associated with early (6h) elevation of proinflammatory cytokines (TNF-α, IL-1, IFN-α, IFN-γ, IL-6, IL-8). We found that all three major leukocyte subpopulations (macrophages, B and T lymphocytes) were increased in the liver 48h after the RHDV inoculation. At 7 days of infection, B and T lymphocytes were still elevated in the liver of the rabbits. In the spleen, both macrophages and B lymphocytes (but not T cells) were also enhanced. At 7 days, anti-RHDV specific antibodies were present in sera of all young rabbits infected by the virus. We conclude that natural resistance of young rabbits to RHDV infection is associated with a rapid and effective inflammatory response by the liver, with few hepatocytes being infected, and also with a sustained elevation in local and systemic B and T cells.
No preview · Article · Oct 2012 · Veterinary Immunology and Immunopathology
[Show abstract][Hide abstract] ABSTRACT: Rabbit hemorrhagic disease virus (RHDV) is the etiologic agent of rabbit hemorrhagic disease (RHD), an acute lethal infection that kills 90% of adult rabbits due to severe acute liver inflammation. Interestingly, young rabbits are naturally resistant to RHDV infection. Here, we have compared naturally occurring CD4(+)Foxp3(+) regulatory T cells (Tregs) between young and adult rabbits after infection by RHDV. The number and frequency of Tregs was decreased in the spleen of adult rabbits 24h after the RHDV infection; this was in contrast with the unchanged number and frequency of splenic Tregs found in young rabbits after the same infection. Also, serum levels of IL-10 and TGF-β were enhanced in the infected adult rabbits whereas no alteration was observed in infected young rabbits. However, this increase is accompanied by a burst of pro-inflammatory cytokines, but seems not able to prevent the death of the animals with severe acute liver inflammation in few days after infection. Since Tregs downregulate inflammation, we conclude that their decrease may contribute to the natural susceptibility of adult rabbits to RHDV infection.
No preview · Article · May 2012 · Veterinary Immunology and Immunopathology
[Show abstract][Hide abstract] ABSTRACT: Glyceraldehyde 3-phosphate dehydrogenases (GAPDH) are cytoplasmic glycolytic enzymes that, despite lacking identifiable secretion signals, have been detected at the surface of several prokaryotic and eukaryotic organisms where they exhibit non-glycolytic functions including adhesion to host components. Group B Streptococcus (GBS) is a human commensal bacterium that has the capacity to cause life-threatening meningitis and septicemia in newborns. Electron microscopy and fluorescence-activated cell sorter (FACS) analysis demonstrated the surface localization of GAPDH in GBS. By addressing the question of GAPDH export to the cell surface of GBS strain NEM316 and isogenic mutant derivatives of our collection, we found that impaired GAPDH presence in the surface and supernatant of GBS was associated with a lower level of bacterial lysis. We also found that following GBS lysis, GAPDH can associate to the surface of many living bacteria. Finally, we provide evidence for a novel function of the secreted GAPDH as an inducer of apoptosis of murine macrophages.
[Show abstract][Hide abstract] ABSTRACT: Group B Streptococcus (GBS) is the leading cause of neonatal pneumonia, septicemia, and meningitis. We have previously shown that in adult mice GBS glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an extracellular virulence factor that induces production of the immunosuppressive cytokine interleukin-10 (IL-10) by the host early upon bacterial infection. Here, we investigate whether immunity to neonatal GBS infection could be achieved through maternal vaccination against bacterial GAPDH. Female BALB/c mice were immunized with rGAPDH and the progeny was infected with a lethal inoculum of GBS strains. Neonatal mice born from mothers immunized with rGAPDH were protected against infection with GBS strains, including the ST-17 highly virulent clone. A similar protective effect was observed in newborns passively immunized with anti-rGAPDH IgG antibodies, or F(ab')(2) fragments, indicating that protection achieved with rGAPDH vaccination is independent of opsonophagocytic killing of bacteria. Protection against lethal GBS infection through rGAPDH maternal vaccination was due to neutralization of IL-10 production soon after infection. Consequently, IL-10 deficient (IL-10(-/-)) mice pups were as resistant to GBS infection as pups born from vaccinated mothers. We observed that protection was correlated with increased neutrophil trafficking to infected organs. Thus, anti-rGAPDH or anti-IL-10R treatment of mice pups before GBS infection resulted in increased neutrophil numbers and lower bacterial load in infected organs, as compared to newborn mice treated with the respective control antibodies. We showed that mothers immunized with rGAPDH produce neutralizing antibodies that are sufficient to decrease IL-10 production and induce neutrophil recruitment into infected tissues in newborn mice. These results uncover a novel mechanism for GBS virulence in a neonatal host that could be neutralized by vaccination or immunotherapy. As GBS GAPDH is a structurally conserved enzyme that is metabolically essential for bacterial growth in media containing glucose as the sole carbon source (i.e., the blood), this protein constitutes a powerful candidate for the development of a human vaccine against this pathogen.
[Show abstract][Hide abstract] ABSTRACT: Rabbit Haemorrhagic Disease Virus (RHDV), a member of the Caliciviridae family, is the etiologic agent of Rabbit Haemorrhagic Disease (RHD); this viral disease is highly contagious and kills more than 90% of infected adult rabbits. Research on experimental calicivirus infection uses inocula obtained from livers of rabbits dying from calicivirus infection. This implies that caliciviruses have to be purified from liver homogenates. Current methods to isolate caliciviruses from rabbit livers are time consuming. We propose here a new procedure for fast purification of rabbit caliciviruses from liver homogenates that uses centrifugation through an iodixanol gradient. This method offers in approximately 2 h a sample with a high degree of calicivirus purity, as shown by its biochemical and immunocytochemistry analysis, which is also able to kill adult rabbits from RHD within 48 h of inoculation.
Full-text · Article · Aug 2011 · Research in Veterinary Science
[Show abstract][Hide abstract] ABSTRACT: Therapeutic vaccination with Streptococcus sobrinus recombinant enolase (rEnolase) protects rats from dental caries. Here, we investigated the effect that maternal rEnolase vaccination before pregnancy had on the offspring's immune response to S. sobrinus oral infection and dental caries progression. Female Wistar rats were immunized by intranasal and subcutaneous routes with rEnolase adsorbed onto aluminum hydroxide as adjuvant or similarly treated with the adjuvant alone (sham-immunized). Ten days after the last administration, the immunized females were paired with a male rat. The oral immune responses to S. sobrinus infection and dental caries in the offspring were evaluated. The results showed that pups born from rEnolase-immunized mothers had higher levels of rEnolase-specific salivary IgA and IgG antibodies (indicating a placental antibody transfer) and lower sulcal and proximal enamel caries scores than rats born from sham-immunized mothers. In conclusion, rEnolase maternal immunization before pregnancy provides offspring with protection against S. sobrinus-induced dental caries.
No preview · Article · Mar 2011 · Journal of dental research
[Show abstract][Hide abstract] ABSTRACT: Rabbit Haemorrhagic Disease (RHD) is a lethal infection caused by calicivirus that kills 90% of the infected adult rabbits within 3 days. The calicivirus replicates in the liver and causes a fulminant hepatitis. Most studies on the pathology of RHD have been focused on the fulminant liver disease. This may not be the only mechanism in the pathogenesis of RHD: calicivirus infection may also induce leukopenia in the infected adult rabbits. We show now by flow cytometry analysis that the calicivirus induces an early decrease in B and T cells, in both spleen and liver. The depletion of B and T cells was associated with apoptosis labelled by annexin V. These changes occurred in rabbits before they showed enzymatic evidence of liver damage and persisted after liver transaminase values were very high. We conclude that depletion of lymphocytes caused by the calicivirus infection precedes or attends liver damage. The relative contribution of this lymphocyte depletion for the pathogenesis of the fatal calicivirus infection of rabbits remains to be investigated.
Full-text · Article · Dec 2010 · Veterinary Research Communications
[Show abstract][Hide abstract] ABSTRACT: Streptococcus agalactiae is a contagious, mastitis-causing pathogen that is highly adapted to survive in the bovine mammary gland. This study used a BALB/c mouse model of Streptococcus agalactiae mastitis to evaluate leukocyte populations in regional lymph nodes and cytokine expression in the mammary gland involved in the immune response against Streptococcus agalactiae. It was found that the bacteria replicated efficiently in the mammary gland, peaking after 24 h and increasing by 100-fold. Dissemination of bacteria to systemic organs was observed 6 h after infection. At the same time, a massive infiltration of polymorphonuclear cells and an increase in the inflammatory cytokines interleukin (IL)-1beta, IL-6 and tumour necrosis factor-alpha were detected in mammary glands, indicating an early inflammatory response. A decrease in the levels of inflammatory cytokines in mammary glands was observed 72 h after infection, accompanied by an increase in the levels of IL-12 and IL-10, which were related to a gradual decrease in bacterial load. An increase in the number of macrophages and B220(+) lymphocytes and similar increases in both CD4(+) and CD8(+) T cells in regional lymph nodes were observed, being most pronounced 5 days after infection. Moreover, increased levels of anti-Streptococcus agalactiae antibodies in the mammary gland were observed 10 days after infection. Overall, these data suggest that the host exhibits both innate and acquired immune responses in response to Streptococcus agalactiae mastitis.
Full-text · Article · Aug 2009 · Journal of Medical Microbiology
[Show abstract][Hide abstract] ABSTRACT: Caliciviruses cause rabbit haemorrhagic disease (RHD) that kills more than 90% of the infected adult animals within 1 a 3 days of infection. The virus replicates in the liver and causes a fulminant hepatitis in adult rabbits leading to RHD. A mystery of the calicivirus infection is that young rabbits (less than 8-weeks old) are resistant to the infection, in spite of undergoing viral replication in the liver and of expressing transient hepatitis. Heterophils were the predominant inflammatory cells seen in hepatic tissue of infected adult rabbits, whereas mononuclear cells dominated the inflammatory infiltrates of the infected young rabbits (4-weeks-old). In order to define the role of inflammation in the pathogenesis of the calicivirus infection, we have studied the cellular inflammatory response in young rabbits experimentally infected by calicivirus. For this, we have used transmission electron microscopy (TEM) and flow cytometry to identify the inflammatory cells that infiltrate the hepatic tissue of young rabbits at 48 hours of calicivirus infection. In same infected rabbits, lymphoid organs (spleen and thymus) were used to quantify by flow cytometry the total number of leukocytes seen inside these organs.
No preview · Article · Jul 2009 · Microscopy and Microanalysis
[Show abstract][Hide abstract] ABSTRACT: Streptococcus agalactiae is a common pathogen that causes bovine mastitis. The aims of this study were to evaluate the antibody response against S. agalactiae extracellular proteins in the whey and serum of naturally infected bovines and to identify possible immunodominant extracellular antigens. IgG1 antibodies against S. agalactiae extracellular proteins were elevated in the whey and serum of naturally infected bovines. In the whey, the levels of IgG1 specific for S. agalactiae extracellular proteins were similar in infected and noninfected milk quarters from the same cow, and the production of antibodies specific for S. agalactiae extracellular proteins was induced only by infection with this bacterium. The immunoreactivity of extracellular proteins with bovine whey was clearly different in infected versus control animals. Group B protective surface protein and 5'-nucleotidase family protein were 2 major immunoreactive proteins that were detected only in the whey of infected cows, suggesting that these proteins may be important in the pathogenesis of S. agalactiae-induced mastitis. This information could be used to diagnose S. agalactiae infection. In addition, these antigens may be useful as carrier proteins for serotype-specific polysaccharides in conjugate vaccines.
No preview · Article · Nov 2008 · Canadian Journal of Microbiology
[Show abstract][Hide abstract] ABSTRACT: Dental caries is among the more prevalent chronic human infections for which an effective human vaccine has not yet been achieved.
Enolase from Streptococcus sobrinus has been identified as an immunomodulatory protein. In the present study, we used S. sobrinus recombinant enolase (rEnolase) as a target antigen and assessed its therapeutic effect in a rat model of dental caries. Wistar
rats that were fed a cariogenic solid diet on day 18 after birth were orally infected with S. sobrinus on day 19 after birth and for 5 consecutive days thereafter. Five days after infection and, again, 3 weeks later, rEnolase
plus alum adjuvant was delivered into the oral cavity of the rats. A sham-immunized group of rats was contemporarily treated
with adjuvant alone. In the rEnolase-immunized rats, increased levels of salivary IgA and IgG antibodies specific for this
recombinant protein were detected. A significant decrease in sulcal, proximal enamel, and dentin caries scores was observed
in these animals, compared with sham-immunized control animals. No detectable histopathologic alterations were observed in
all immunized animals. Furthermore, the antibodies produced against bacterial enolase did not react with human enolase. Overall,
these results indicate that rEnolase could be a promising and safe candidate for testing in trials of vaccines against dental
caries in humans.
No preview · Article · Nov 2008 · The Journal of Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: Calicivirus infection of adult rabbits induces the so-called rabbit haemorrhagic disease (RHD) that kills 90% or more of the infected animals; in contrast, young rabbits (up to 8-week-old animals) are resistant to the same infectious agent. We report that calicivirus inoculation of young rabbits induced moderate titres of antiviral antibodies. When these rabbits reached adulthood, a second calicivirus inoculation resulted in resistance to RHD and boosting of antibody titres in half of the rabbits. Adoptive transfer of sera from calicivirus-infected young rabbits to naïve adult rabbits conferred resistance to RHD. We conclude that calicivirus infection of young rabbits induces specific anti-calicivirus antibodies that will protect them from RHD when they reach adulthood.
No preview · Article · Mar 2008 · Veterinary Immunology and Immunopathology
[Show abstract][Hide abstract] ABSTRACT: Interactions of several microbial pathogens with the plasminogen system increase their invasive potential. In this study, we show that Streptococcus agalactiae binds human plasminogen which can be subsequently activated to plasmin, thus generating a proteolytic bacterium. S. agalactiae binds plasminogen via the direct pathway, using plasminogen receptors, and via the indirect pathway through fibrinogen receptors. The glyceraldehyde-3-phosphate dehydrogenase is one of the S. agalactiae proteins that bind plasminogen. Presence of exogenous activators such as uPA and tPA are required to activate bound plasminogen. Results from competitive inhibition assays indicate that binding is partially mediated through the lysine binding sites of plasminogen. Following plasminogen binding and activation, S. agalactiae is able to degrade in vitro fibronectin, one of the host extracellular matrix proteins. Moreover, incubation of S. agalactiae with either plasminogen alone, or plasminogen plus fibrinogen, in the presence of tPA enhanced its virulence in C57BL/6 mice, suggesting that acquisition of plasmin-like activity by the bacteria increase their invasiveness.
Full-text · Article · Oct 2007 · Microbes and Infection