In Vitro modelling of RSV infection and cytopathogenesis in well-differentiated human primary airway epithelial cells (WD-PAECs).

The choice of model used to study human respiratory syncytial virus (RSV) infection is extremely important. RSV is a human pathogen that is exquisitely adapted to infection of human hosts. Rodent models, such as mice and cotton rats, are semi-permissive to RSV infection and do not faithfully reproduce hallmarks of RSV disease in humans. Furthermore, immortalized airway-derived cell lines, such as HEp-2, BEAS-2B, and A549 cells, are poorly representative of the complexity of the respiratory epithelium. The development of a well-differentiated primary pediatric airway epithelial cell models (WD-PAECs) allows us to simulate several hallmarks of RSV infection of infant airways. They therefore represent important additions to RSV pathogenesis modeling in human-relevant tissues. The following protocols describe how to culture and differentiate both bronchial and nasal primary pediatric airway epithelial cells and how to use these cultures to study RSV cytopathogenesis.

Enteropathogenic Escherichia coli O125:H6 triggers attaching and effacing lesions on human intestinal biopsy specimens independently of Nck and TccP/TccP2

Typical enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) employ either Nck, TccP/TccP2, or Nck and TccP/TccP2 pathways to activate the neuronal Wiskott-Aldrich syndrome protein (N-WASP) and to trigger actin polymerization in cultured cells. This phenotype is used as a marker for the pathogenic potential of EPEC and EHEC strains. In this paper we report that EPEC O125:H6, which represents a large category of strains, lacks the ability to utilize either Nck or TccP/TccP2 and hence triggers actin polymerization in vitro only inefficiently. However, we show that infection of human intestinal biopsies with EPEC O125:H6 results in formation of typical attaching and effacing lesions. Expression of TccP in EPEC O125:H6, which harbors an EHEC O157-like Tir, resulted in efficient actin polymerization in vitro and enhanced colonization of human intestinal in vitro organ cultures with detectable N-WASP and electron-dense material at the site of bacterial adhesion. These results show the existence of a natural category of EPEC that colonizes the gut mucosa using Nck- and TccP-independent mechanisms. Importantly, the results highlight yet again the fact that conclusions made on the basis of in vitro cell culture models cannot be extrapolated wholesale to infection of mucosal surfaces and that the ability to induce actin polymerization on cultured cells should not be used as a definitive marker for EPEC and EHEC virulence.

Genome Wide Association Studies of Phagocytosis and the Cellular Immune Response in Drosophila melanogaster.

Phagocytosis of bacteria by specialized blood cells, known as hemocytes, is a vital component of Drosophila cellular immunity. To identify novel genes that mediate the cellular response to bacteria, we conducted three separate genetic screens using the Drosophila Genetic Reference Panel (DGRP). Adult DGRP lines were tested for the ability of their hemocytes to phagocytose the Gram-positive bacteria Staphylococcus aureus or the Gram-negative bacteria Escherichia coli. The DGRP lines were also screened for the ability of their hemocytes to clear S. aureus infection through the process of phagosome maturation. Genome-wide association analyses were performed to identify potentially relevant single nucleotide polymorphisms (SNPs) associated with the cellular immune phenotypes. The S. aureus phagosome maturation screen identified SNPs near or in 528 candidate genes, many of which have no known role in immunity. Three genes, dpr10, fred, and CG42673, were identified whose loss-of-function in blood cells significantly impaired the innate immune response to S. aureus. The DGRP S. aureus screens identified variants in the gene, Ataxin 2 Binding Protein-1 (A2bp1) as important for the cellular immune response to S. aureus. A2bp1 belongs to the highly conserved Fox-1 family of RNA-binding proteins. Genetic studies revealed that A2bp1 transcript levels must be tightly controlled for hemocytes to successfully phagocytose S. aureus. The transcriptome of infected and uninfected hemocytes from wild type and A2bp1 mutant flies was analyzed and it was found that A2bp1 negatively regulates the expression of the Immunoglobulin-superfamily member Down syndrome adhesion molecule 4 (Dscam4). Silencing of A2bp1 and Dscam4 in hemocytes rescues the fly’s immune response to S. aureus indicating that Dscam4 negatively regulates S. aureus phagocytosis. Overall, we present an examination of the cellular immune response to bacteria with the aim of identifying and characterizing roles for novel mediators of innate immunity in Drosophila. By screening panel of lines in which all genetic variants are known, we successfully identified a large set of candidate genes that could provide a basis for future studies of Drosophila cellular immunity. Finally, we describe a novel, immune-specific role for the highly conserved Fox-1 family member, A2bp1.

Étude du niveau de B Lymphocyte Stimulator (BLyS) et de son impact sur les lymphocytes B en relation avec l’infection et la résistance au virus d’immunodéficience humaine (VIH) chez des travailleuses du sexe au Bénin

L’infection au VIH s’accompagne souvent de dérégulations du compartiment des lymphocytes B qui nuisent à la génération de réponses efficaces. En effet, détectées tôt après l’infection, ces dérégulations perdurent, ne sont pas totalement restaurées par la thérapie, et mènent souvent à des manifestations auto-immunes et lymphomes. Une étude longitudinale de notre groupe, effectuée avec des cellules mononucléées du sang circulant provenant de patients VIH+ avec différents types de progression clinique, a démontré qu’un niveau élevé de BLyS chez des individus VIH+ progresseurs était associé à une dérégulation des fréquences de populations de cellules B avec augmentation de cellules innées de la zone marginale (MZ) présentant des caractéristiques d’immaturité et d’activation. Au contraire, chez des individus VIH+ non-progresseurs avirémiques ou contrôleurs d’élite, les niveaux de BLyS étaient dans la normale et ce sont les fréquences de cellules B MZ plus matures qui étaient diminuées. La résistance au VIH pourrait aussi impliquer le contrôle de BLyS et son impact sur les cellules B. De ce fait, nous avons préalablement recruté une cohorte de travailleuses du sexe (TS) à Cotonou (Bénin) dans laquelle nous avons identifié des femmes qui demeurent séronégatives malgré une exposition soutenue au virus. Nous avons mesuré les niveaux de BLyS dans le sang et dans les lavages cervico-vaginaux (CVL) de TS VIH- et les avons comparés à ceux mesurés chez des TS VIH+ et un groupe contrôle de non-TS VIH- . Nous avons trouvé que les niveaux de BLyS dans le sang et le CVL des TS VIH- étaient inférieurs à ceux des TS VIH+ et des non-TS VIH-. Le niveau d’expression de BLyS à la surface des lymphocytes T, monocytes et cellules dendritiques de TS VIH- était augmenté, mais à un niveau moindre que les TS VIH+. Chez les TS VIH+, les hauts niveaux de BLyS étaient concomitants avec une dérégulation du compartiment B caractérisée par une hyperglobulinémie, une augmentation de la fréquence de populations avec un profil immature/inné et une plus grande proportion de plasmablastes IgG vs IgA. Au contraire, les niveaux inférieurs de BLyS dans le sang des TS VIH- coïncident avec un compartiment B préservé, révélant que les lymphocytes B MZ peuvent être impliqués dans l’immunité naturelle au VIH. Ces résultats démontrent l’importance du contrôle des niveaux de BLyS et du maintien de l’intégrité du compartiment B dans la résistance au VIH.

Étude du niveau de B Lymphocyte Stimulator (BLyS) et de son impact sur les lymphocytes B en relation avec l’infection et la résistance au virus d’immunodéficience humaine (VIH) chez des travailleuses du sexe au Bénin

L’infection au VIH s’accompagne souvent de dérégulations du compartiment des lymphocytes B qui nuisent à la génération de réponses efficaces. En effet, détectées tôt après l’infection, ces dérégulations perdurent, ne sont pas totalement restaurées par la thérapie, et mènent souvent à des manifestations auto-immunes et lymphomes. Une étude longitudinale de notre groupe, effectuée avec des cellules mononucléées du sang circulant provenant de patients VIH+ avec différents types de progression clinique, a démontré qu’un niveau élevé de BLyS chez des individus VIH+ progresseurs était associé à une dérégulation des fréquences de populations de cellules B avec augmentation de cellules innées de la zone marginale (MZ) présentant des caractéristiques d’immaturité et d’activation. Au contraire, chez des individus VIH+ non-progresseurs avirémiques ou contrôleurs d’élite, les niveaux de BLyS étaient dans la normale et ce sont les fréquences de cellules B MZ plus matures qui étaient diminuées. La résistance au VIH pourrait aussi impliquer le contrôle de BLyS et son impact sur les cellules B. De ce fait, nous avons préalablement recruté une cohorte de travailleuses du sexe (TS) à Cotonou (Bénin) dans laquelle nous avons identifié des femmes qui demeurent séronégatives malgré une exposition soutenue au virus. Nous avons mesuré les niveaux de BLyS dans le sang et dans les lavages cervico-vaginaux (CVL) de TS VIH- et les avons comparés à ceux mesurés chez des TS VIH+ et un groupe contrôle de non-TS VIH- . Nous avons trouvé que les niveaux de BLyS dans le sang et le CVL des TS VIH- étaient inférieurs à ceux des TS VIH+ et des non-TS VIH-. Le niveau d’expression de BLyS à la surface des lymphocytes T, monocytes et cellules dendritiques de TS VIH- était augmenté, mais à un niveau moindre que les TS VIH+. Chez les TS VIH+, les hauts niveaux de BLyS étaient concomitants avec une dérégulation du compartiment B caractérisée par une hyperglobulinémie, une augmentation de la fréquence de populations avec un profil immature/inné et une plus grande proportion de plasmablastes IgG vs IgA. Au contraire, les niveaux inférieurs de BLyS dans le sang des TS VIH- coïncident avec un compartiment B préservé, révélant que les lymphocytes B MZ peuvent être impliqués dans l’immunité naturelle au VIH. Ces résultats démontrent l’importance du contrôle des niveaux de BLyS et du maintien de l’intégrité du compartiment B dans la résistance au VIH.

Sovereign wealth funds and state immunity

The study sheds light on the application of the rule of state immunity to sovereign wealth funds (SWFs). SWFs are Janus-faced investment vehicles established by their parent states to invest public resources in financial markets, with the aim of increasing long-term returns and pursuing macroeconomic goals. The ultimate purpose of the study is to assess if the hybrid nature of SWFs results in changes to the rule of state immunity when applied to them, and whether a generally accepted standard in this regard can be deduced from state practice. The research is conducted through a comparative analysis. It is based on the provisions of the UN Convention on Jurisdictional Immunities of States and Their Property (UNCSI), as well as on six domestic jurisdictions (US, UK, France, Germany, Italy and China) among those that have contributed most significantly to the international debate on state immunity and which host the largest amount of SWF investments.

Candida albicans in patients with oronasal communication and obturator prostheses

Patients using obturator prostheses often present denture-induced stomatitis. In order to detect the presence of oral Candida albicans in patients with oronasal communications and to evaluate the effectiveness of a topical antifungal treatment, cytological smears obtained from the buccal and palatal mucosa of 10 adult patients, and from the nasal acrylic surface of their obturator prostheses were examined. A therapeutic protocol comprising the use of oral nystatin (Mycostatin®) and prosthesis disinfection with sodium hypochlorite was prescribed for all patients. Seven patients were positive for C. albicans in the mucosa, with 1 negative result for the prosthetic surface in this group of patients. Post-treatment evaluation revealed the absence of C. albicans on prosthesis surface and on the oral mucosa of all patients. The severity of the candidal infection was significantly higher in the palatal mucosa than in the buccal mucosa, but similar in the palatal mucosa and prosthesis surface, indicating that the mucosa underlying the prosthesis is more susceptible to infection. The therapeutic protocol was effective in all cases, which emphasizes the need for denture disinfection in order to avoid reinfection of the mucosa.

Leishmania spp. parasite isolation through inoculation of patient biopsy macerates in interferon gamma knockout mice

Isolation of Leishmania parasite and species identification are important for confirmation and to help define the epidemiology of the leishmaniasis. Mice are often used to isolate pathogens, but the most common mouse strains are resistant to infection with parasites from the Leishmania (Viannia) subgenus. In this study we tested the inoculation of interferon gamma knockout (IFNγ KO) mice with biopsy macerates from Leishmania-infected patients to increase the possibility of isolating parasites. Biopsies from twenty five patients with clinical signs of leishmaniasis were taken and tested for the presence of parasites. Immunohistochemical assay (IHC) and conventional histopathology detected the parasite in 88% and 83% of the patients, respectively. Leishmania sp. were isolated in biopsy macerates from 52% of the patients by culture in Grace's insect medium, but 13% of isolates were lost due to contamination. Inoculation of macerates in IFNγ KO mice provides isolation of parasites in 31.8% of the biopsies. Most isolates belong to L. (Viannia) subgenus, as confirmed by PCR, except one that belongs to L. (Leishmania) subgenus. Our preliminary results support the use of IFNγ KO mice to improve the possibility to isolate New World Leishmania species.

High prevalence of drug-resistant tuberculosis and other mycobacteria among HIV-infected patients in Brazil: a systematic review

There is a little-noticed trend involving human immunodeficiency virus (HIV)-infected patients suspected of having tuberculosis: the triple-treatment regimen recommended in Brazil for years has been potentially ineffective in over 30% of the cases. This proportion may be attributable to drug resistance (to at least 1 drug) and/or to infection with non-tuberculous mycobacteria. This evidence was not disclosed in official statistics, but arose from a systematic review of a few regional studies in which the diagnosis was reliably confirmed by mycobacterial culture. This paper clarifies that there has long been ample evidence for the potential benefits of a four-drug regimen for co-infected patients in Brazil and it reinforces the need for determining the species and drug susceptibility in all positive cultures from HIV-positive patients.

Identification of a Highly Antigenic Linear B Cell Epitope within Plasmodium vivax Apical Membrane Antigen 1 (AMA-1)

Apical membrane antigen 1 (AMA-1) is considered to be a major candidate antigen for a malaria vaccine. Previous immunoepidemiological studies of naturally acquired immunity to Plasmodium vivax AMA-1 (PvAMA-1) have shown a higher prevalence of specific antibodies to domain II (DII) of AMA-1. In the present study, we confirmed that specific antibody responses from naturally infected individuals were highly reactive to both full-length AMA-1 and DII. Also, we demonstrated a strong association between AMA-1 and DII IgG and IgG subclass responses. We analyzed the primary sequence of PvAMA-1 for B cell linear epitopes co-occurring with intrinsically unstructured/ disordered regions (IURs). The B cell epitope comprising the amino acid sequence 290-307 of PvAMA-1 (SASDQPTQYEEEMTDYQK), with the highest prediction scores, was identified in domain II and further selected for chemical synthesis and immunological testing. The antigenicity of the synthetic peptide was identified by serological analysis using sera from P. vivax-infected individuals who were knowingly reactive to the PvAMA-1 ectodomain only, domain II only, or reactive to both antigens. Although the synthetic peptide was recognized by all serum samples specific to domain II, serum with reactivity only to the full-length protein presented 58.3% positivity. Moreover, IgG reactivity against PvAMA-1 and domain II after depletion of specific synthetic peptide antibodies was reduced by 18% and 33% (P = 0.0001 for both), respectively. These results suggest that the linear epitope SASDQPTQYEEEMTDYQK is highly antigenic during natural human infections and is an important antigenic region of the domain II of PvAMA-1, suggesting its possible future use in pre-clinical studies.

Genetic polymorphisms in TLR4, CR1 and Duffy genes are not associated with malaria resistance in patients from Baixo Amazonas region, Brazil

The main purpose of this research was to analyze the relation of the genetic polymorphisms frequently expressed by antigen-presenting cells, erythrocytes and malaria susceptibility/resistance with the human malaria infection cases. The sample used consisted of 23 Plasmodium vivax ( Pv)- and P. falciparum ( Pf)-infected patients, and 21 healthy individuals as a control group, from the Baixo Amazonas population in Para, Brazil. The Asp299Gly polymorphisms in the Toll-like receptor 4 ( TLR4), and Gly42Asp, Arg89Cys, Ala100Thr, and T-33C in the Duffy gene ( FY) were analyzed by restriction fragment length polymorphism-polymerase chain reaction. The Lys1590Glu and Arg1601Gly polymorphisms in the complement receptor type 1 (CR1) were analyzed by DNA sequencing. According to the results obtained and statistical analysis considering a significance level or alpha = 0.01, we conclude that the low heterozygote frequency (2.27%) for the Asp299Gly mutation, detected in the TLR4 gene, is not related to the Pv and Pf infections in the patients analyzed. Also, the promoter region GATA-1 analysis of the FY gene in the Pv-infected patients showed that the heterozygote frequency for the T-33C mutation (11.36% of the infected patients and 20.45% of the control patients) is not related to infection resistance. Regarding the CR1 gene, the observed heterozygote frequency (9.09%) for the Arg1601Gly mutation in Pf-infected patients when compared to heterozygote frequency in the control group (18.18%) suggests that there is no correlation with infection resistance.

Aspirin Treatment of Mice Infected with Trypanosoma cruzi and Implications for the Pathogenesis of Chagas Disease

Chagas disease, caused by infection with Trypanosoma cruzi, is an important cause of cardiovascular disease. It is increasingly clear that parasite-derived prostaglandins potently modulate host response and disease progression. Here, we report that treatment of experimental T. cruzi infection (Brazil strain) beginning 5 days post infection (dpi) with aspirin (ASA) increased mortality (2-fold) and parasitemia (12-fold). However, there were no differences regarding histopathology or cardiac structure or function. Delayed treatment with ASA (20 mg/kg) beginning 60 dpi did not increase parasitemia or mortality but improved ejection fraction. ASA treatment diminished the profile of parasite-and host-derived circulating prostaglandins in infected mice. To distinguish the effects of ASA on the parasite and host bio-synthetic pathways we infected cyclooxygenase-1 (COX-1) null mice with the Brazil-strain of T. cruzi. Infected COX-1 null mice displayed a reduction in circulating levels of thromboxane (TX)A(2) and prostaglandin (PG)F(2 alpha). Parasitemia was increased in COX-1 null mice compared with parasitemia and mortality in ASA-treated infected mice indicating the effects of ASA on mortality potentially had little to do with inhibition of prostaglandin metabolism. Expression of SOCS-2 was enhanced, and TRAF6 and TNF alpha reduced, in the spleens of infected ASA-treated mice. Ablation of the initial innate response to infection may cause the increased mortality in ASA-treated mice as the host likely succumbs more quickly without the initiation of the ""cytokine storm'' during acute infection. We conclude that ASA, through both COX inhibition and other ""off-target'' effects, modulates the progression of acute and chronic Chagas disease. Thus, eicosanoids present during acute infection may act as immunomodulators aiding the transition to and maintenance of the chronic phase of the disease. A deeper understanding of the mechanism of ASA action may provide clues to the differences between host response in the acute and chronic T. cruzi infection.

Chitinase and peroxidase activity in different stages of eucalypt leaves after inoculation with Puccinia psidii and acibenzolar-S-metil

Chitinase and peroxidase activity in different stages of eucalypt leaves after inoculation with Puccinia psidii and acibenzolar-S-metil To elucidate some biochemical processes during infection in the pathosystem Puccinia psidii x eucalyptus, the defense metabolism in different-stage leaves was compared between rust-resistant and susceptible clones, respectively. In addition, chitinase and peroxidase activities were assayed. Each treatment consisted of 4 replicates, in a completely randomized design: 2 clones, inoculated and not inoculated with P. psidii; sprayed with acibenzolar-S-methyl (ASM) and distilled water; and represented by the 1(st) leaf pair (size equivalent to 1/5 total leaf development), 2(nd) pair (2/5 total development), and 4(th) pair (4/5 total leaf length). Leaves were harvested in 4 periods: 0, 24, 72 and 96 hours after inoculation. Results indicated that ASM treatment or P. psidii action led to higher chitinase and peroxidase activity level but did not alter the expression of these activities in developed leaves (4(th) pair) during the experiment. Alterations in enzyme levels after inoculation were only observed in developing leaves (1(st) and 2(nd) pairs), which suggests that the response to infection was concomitant to chitinase and peroxidase synthesis. The highest increases in enzymatic activities were observed in resistant clones at 72 hours after inoculation and in susceptible ones previously treated with ASM and later inoculated with the pathogen.

Melatonin enhances pro-inflammatory cytokine levels and protects against Chagas disease

Pro-inflammatory and modulatory cytokines have an essential role in host defense against human and murine Trypanosoma cruzi infection. Control of T. cruzi parasitism during the acute phase of infection is considered to be critically dependent on direct macrophage activation by cytokines. Melatonin has been proposed to regulate the immune system by affecting cytokine production in immunocompetent cells, enhancing the production of several T helper (Th)1 cytokines. The aims of this work were to evaluate in rats, the influences of exogenous melatonin treatment on T. cruzi-infected host`s immune responses. With this in mind, several immunological parameters were analyzed, including tumor necrosis factor-alpha, gamma-interferon, interleukin-12, nitric oxide (NO) and macrophage count. The melatonin therapy was provided in one of two different treatment regimens, that is, either beginning 7 days prior to infection or concomitant with the infection. Both treatments triggered an up-regulation of the immune response, with the concomitant treatment being more effective; in this case all cytokines studied, with exception of NO, displayed enhanced concentrations and there was a higher number of peritoneal macrophages, which displayed reduced concentrations under melatonin therapy. We conclude that melatonin plays a pivotal role in up-regulating the Th1 immune response thus controlling parasite replication.

Changes in neutrophil surface receptor expression, degranulation, and respiratory burst activity following moderate and high intensity exercise

Intense exercise stimulates the systemic release of a variety of factors that alter neutrophil surface receptor expression and functional activity. These alterations may influence resistance to infection after intense exercise. The aim of this study was to examine the influence of exercise intensity on neutrophil receptor expression, degranulation (measured by plasma and intracellular myeloperoxidase concentrations), and respiratory burst activity. Ten well-trained male runners ran on a treadmill for 60 min at 60% [moderate-intensity exercise (MI)] and 85% maximal oxygen consumption [high-intensity exercise (HI)]. Blood was drawn immediately before and after exercise and at 1 h postexercise. Immediately after HI, the expression of the neutrophil receptor CD16 was significantly below preexercise values (P < 0.01), whereas MI significantly reduced CD35 expression below preexercise values (P < 0.05). One hour after exercise at both intensities, there was a significant decline in CD11b expression (P < 0.05) and a further decrease in CD16 expression compared with preexercise values (P < 0.01). CD16 expression was lower 1 h after HI than 1 h after MI (P < 0.01). Immediately after HI, intracellular myeloperoxidase concentration was less than preexercise values (P < 0.01), whereas plasma myeloperoxidase concentration was greater (P < 0.01), indicating that HI stimulated neutrophil degranulation. Plasma myeloperoxidase concentration was higher immediately after HI than after MI (P < 0.01). Neutrophil respiratory burst activity increased after HI (P < 0.01). In summary, both MI and HI reduced neutrophil surface receptor expression. Although CD16 expression was reduced to a greater extent after HI, this reduction did not impair neutrophil degranulation and respiratory burst activity.