Comparative Analysis of Apoptosis and Inflammation Genes of Mice and Humans

Apoptosis (programmed cell death) plays important roles in many facets of normal mammalian physiology. Host-pathogen interactions have provided evolutionary pressure for apoptosis as a defense mechanism against viruses and microbes, sometimes linking apoptosis mechanisms with inflammatory responses through NFκB induction. Proteins involved in apoptosis and NFκB induction commonly contain evolutionarily conserved domains that can serve as signatures for identification by bioinformatics methods. Using a combination of public (NCBI) and private (RIKEN) databases, we compared the repertoire of apoptosis and NFκB-inducing genes in humans and mice from cDNA/EST/genomic data, focusing on the following domain families: (1) Caspase proteases; (2) Caspase recruitment domains (CARD); (3) Death Domains (DD); (4) Death Effector Domains (DED); (5) BIR domains of Inhibitor of Apoptosis Proteins (IAPs); (6) Bcl-2 homology (BH) domains of Bcl-2 family proteins; (7) Tumor Necrosis Factor (TNF)-family ligands; (8) TNF receptors (TNFR); (9) TIR domains; (10) PAAD (PYRIN; PYD, DAPIN); (11) nucleotide-binding NACHT domains; (12) TRAFs; (13) Hsp70-binding BAG domains; (14) endonuclease-associated CIDE domains; and (15) miscellaneous additional proteins. After excluding redundancy due to alternative splice forms, sequencing errors, and other considerations, we identified cDNAs derived from a total of 227 human genes among these domain families. Orthologous murine genes were found for 219 (96%); in addition, several unique murine genes were found, which appear not to have human orthologs. This mismatch may be due to the still fragmentary information about the mouse genome or genuine differences between mouse and human repertoires of apoptotic genes. With this caveat, we discuss similarities and differences in human and murine genes from these domain families.     

Intracellular Trafficking of Bordetella pertussis in Human Macrophages

Although Bordetella pertussis has been observed to survive inside macrophages, its ability to resist or evade degradation in phagolysosomes has not been defined. We here investigated the trafficking of B. pertussis upon entry into human macrophages. During the first hours following phagocytosis, a high percentage of bacteria were destroyed within acidic compartments positive for the lysosome-associated membrane proteins (LAMP). However, roughly one-fourth of the bacteria taken up evade this initial killing event, remaining in nonacidic compartments. Forty-eight hours after infection, the number of intracellular bacteria per cell increased, suggesting that B. pertussis is capable of replicating in this type of compartment. Viable bacteria accumulated within phagosomal compartments positive for the early endosomal marker Rab5 but not the late endosomal marker LAMP. Moreover, B. pertussis-containing phagosomes acquired exogenously added transferrin, indicating that intracellular bacteria have access to extracellular components and essential nutrients via the host cell recycling pathway. Overall, these results suggest that B. pertussis survives and eventually replicates in compartments with characteristics of early endosomes, potentially contributing to its extraordinary ability to persist within hosts and populations.Bordetella pertussis colonizes the human respiratory tract, causing a disease known as whooping cough or pertussis, which affects around 4 million people worldwide and causes more than 300,000 deaths each year. Despite high vaccination rates, whooping cough remains a serious threat to human health and its incidence has been increasing in recent years in vaccinated populations. Although some potential contributors to initial colonization have been described, the mechanisms that allow this pathogen to evade immune clearance and to cause the extraordinarily prolonged disease known in China as Bai Ri Ke (100-day cough) are not known.B. pertussis expresses a number of potent virulence factors, adhesins, and toxins (23) with known or predicted roles during infection. Although B. pertussis is described as an extracellular pathogen, several studies indicate that the immunomodulatory properties of several of these virulence factors enable the bacterium to persist within epithelial cells and leukocytes (1, 3, 22, 24), leading to speculation that the infection might also comprise an intracellular stage. The dual extra- and intracellular locations of B. pertussis are also consistent with the reported need for both cellular and humoral immune responses for bacterial elimination from the respiratory tract (12, 17, 33, 38).It is presumed that macrophages play an important role in the clearance of B. pertussis (21). However, in vitro studies indicated that B. pertussis is capable of surviving intracellularly in human macrophages for several days in the absence of opsonins (11). Moreover, B. pertussis was found viable in alveolar macrophage cells of mice for more than 21 days after infection (16). These observations have led to speculation that alveolar macrophages might represent an intracellular niche for B. pertussis (16, 41). The recovery of viable B. pertussis from human hosts several weeks after infection (19, 30) and the observation of B. pertussis within pulmonary alveolar macrophages of HIV-infected children (7) and in infants with confirmed B. pertussis pneumonia (28) provide support for this theory.Efforts to characterize the interaction between B. pertussis and human macrophages have been mainly focused on B. pertussis adherence. Several B. pertussis virulence factors facilitate interaction with phagocytes. B. pertussis fimbriae mediate the binding to the very late antigen 5 receptor on monocytes and macrophages, inducing the upregulation of complement receptor 3 (CR3: CD11b/CD18) (14). CR3 expression is further upregulated by pertussis toxin and filamentous hemagglutinin (FHA) (18, 42). It has been demonstrated that CR3 serves as a docking molecule for B. pertussis binding by FHA (18, 31), which eventually leads to B. pertussis uptake in a nonbactericidal way (15). However, little is known about the fate of B. pertussis inside macrophages. Recent studies by our group showed that neutrophil uptake of B. pertussis in the absence of specific antibodies leads to the failure of lysosomal maturation and bacterial clearance (20). These observations are intriguing evidence that B. pertussis has mechanisms that can allow for evasion of phagolysosome biogenesis. However, short-lived neutrophils are unlikely to provide a prolonged reservoir of bacteria, and evasion of phagolysosome biogenesis in macrophages appears to be an important aspect of the persistence of many other pathogens (9, 10, 29, 40).The aim of this study was to determine the fate of B. pertussis following phagocytosis by macrophages. Although many ingested bacteria were rapidly killed by macrophages, a significant fraction of internalized B. pertussis was capable of evading phagosome-lysosome fusion, surviving for days and eventually replicating in nonacidic compartments with characteristics of early endosomes. These results reveal a pathway that may contribute to both the extraordinarily long persistence of the coughing illness caused by B. pertussis and its ability to persist within largely immune populations.     

Folic Acid Modified Poly(lactide‐co‐glycolide) Nanoparticles,Layer‐by‐Layer Surface Engineered for Targeted Delivery

The layer‐by‐layer (LbL) assembly technique was applied for the surface modification of biodegradable poly(lactide‐co‐glycolide) nanoparticles (NPs), employing poly(acrylic acid) (PAA), and polyethylenimine (PEI) as building blocks. Amino terminated poly(ethylene glycol) (PEG) and folate decorated PEG (PEG‐FA) were grafted onto the multilayers via condensation between carboxylic groups and amine groups from PEG or PEG‐FA. The LbL assembly and the covalent functionalization were monitored by means of ζ‐potential measurements and the quartz crystal microbalance with dissipation technique (QCM‐D). Protein adsorption after incubation of the NPs in culture medium containing optionally the serum proteins was investigated and related to cellular uptake. Experiments on cellular uptake showed that after PEGylation the uptake ratio of the NPs decreased significantly, but became three times larger when PEG‐FA was grafted on the NPs instead of the PEG.

     

A role for CD4+CD25+ T cells in regulation of the immune response during human tuberculosis

Active tuberculosis (TB) is associated with prolonged suppression of Mycobacterium tuberculosis (MTB)-specific immune responses, but mechanisms involved are understood incompletely. We investigated a potential role for CD4+CD25+ regulatory T cells in depressed anti-MTB immunity by evaluating serially CD4 cell phenotype and interferon (IFN)-gamma production by mononuclear cells from patients with TB. At diagnosis, frequencies of CD4+CD25+ T cells were increased in blood from TB patients compared to healthy purified protein derivative (PPD)-positive controls (with a history of prior TB exposure), and remained elevated at completion of therapy (6 months). By contrast, expression of another activation marker, CD69, by CD4 T cells was increased at diagnosis, but declined rapidly to control levels with treatment. Among CD4+CD25+ T cells from TB patients at diagnosis those expressing high levels of CD25, probably representing regulatory T cells, were increased 2.9-fold when compared to control subjects, while MTB-stimulated IFN-gamma levels in whole blood supernatants were depressed. A role for CD4+CD25+ T cells in depressed IFN-gamma production during TB was substantiated in depletion experiments, where CD25+-depleted CD4 T cells produced increased amounts of IFN-gamma upon MTB stimulation compared to unseparated T cells. At follow-up, IFN-gamma production improved most significantly in blood from TB patients with high baseline frequencies of CD4+CD25+ T cells (more than threefold higher than controls for both total and CD25hi+ CD4 T cells), who also had a significant drop in frequencies of both total and 'regulatory' CD4+CD25+ T cells in response to treatment. Expansion of CD4+CD25+ regulatory T cells during active TB may play a role in depressed T cell IFN-gamma production.     

How nonclinical are community samples?

Mental health services are underutilized in our society by both adults and children. This finding presents a potential problem for researchers conducting community‐based research. Previous studies have demonstrated that community‐based researchers frequently do not screen participants for the presence of psychopathology nor do they ascertain whether therapeutic services are currently utilized. The present study explored the prevalence of psychopathology and treatment involvement in a sample of families recruited from the community. Results indicated that a fifth of the participants in this community‐based sample met diagnostic criteria for a psychiatric disorder or were in treatment for psychological difficulties at the time of recruitment for this study. Furthermore, mothers, fathers, and adolescents who met the criteria according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM‐IV; American Psychiatric Association, 1994 ) for a psychological disorder had higher symptomatology than those who did not meet criteria. Methodological suggestions are provided. © 2008 Wiley Periodicals, Inc.     

Characterization of rotavirus specific B cells and their relation with serological memory

We quantified circulating total, rotavirus (RV) and Tetanus toxin (TT) memory B cells (mBc) in healthy adults using a limiting dilution assay (LDA) and a flow cytometry assay (FCA) that permit evaluation of both CD27+ and CD27− mBc.RV mBc were enriched in the CD27−, IgG+ and in the CD27+, IgM+ subsets. The numbers of RV mBc were higher by FCA than by LDA and results of the two assays did not correlate. TT IgGmBc and RV IgA mBc determined by FCA and by LDA correlated with TT plasma IgG and RV plasma IgA, respectively. The mean ratio of specific mBc/μg/ml of the corresponding plasma immunoglobulin was lower for TT IgG than for RV IgA mBc.Our studies contribute to understand the relationship between circulating mBc and serological memory, and enhance our capacity to develop better correlates of protection against RV disease.     

TLR2 engagement on CD4+ T cells enhances effector functions and protective responses to Mycobacterium tuberculosis

We have previously demonstrated that mycobacterial lipoproteins engage TLR2 on human CD4⁺ T cells and upregulate TCR‐triggered IFN‐γ secretion and cell proliferation in vitro. Here we examined the role of CD4⁺ T‐cell‐expressed TLR2 in Mycobacterium tuberculosis (MTB) Ag‐specific T‐cell priming and in protection against MTB infection in vivo. Like their human counterparts, mouse CD4⁺ T cells express TLR2 and respond to TLR2 costimulation in vitro. This Th1‐like response was observed in the context of both polyclonal and Ag‐specific TCR stimulation. To evaluate the role of T‐cell TLR2 in priming of CD4⁺ T cells in vivo, naive MTB Ag85B‐specific TCR transgenic CD4⁺ T cells (P25 TCR‐Tg) were adoptively transferred into Tlr2^?/? recipient C57BL/6 mice that were then immunized with Ag85B and with or without TLR2 ligand Pam₃Cys‐SKKKK. TLR2 engagement during priming resulted in increased numbers of IFN‐γ‐secreting P25 TCR‐Tg T cells 1 week after immunization. P25 TCR‐Tg T cells stimulated in vitro via TCR and TLR2 conferred more protection than T cells stimulated via TCR alone when adoptively transferred before MTB infection. Our findings indicate that TLR2 engagement on CD4⁺ T cells increases MTB Ag‐specific responses and may contribute to protection against MTB infection.     

Mixed infection of Sida jamaicensis in Jamaica reveals the presence of three recombinant begomovirus DNA A components

Begomoviruses impose serious constraints on agriculture throughout the temperate, tropical and subtropical regions. Previously, we characterised a sida golden yellow vein virus isolate, SiGYVV-[JM:Lig2:08] (HQ009519-20) from a symptomatic Sida jamaicensis plant. With the aim of establishing whether it was hosting a mixed infection that could facilitate recombination, PCR-RFLP was done on DNA extracted from this plant, and the results suggested the presence of two additional genetically distinct DNA-A molecules. Sequence analysis of these two DNA-A molecules (relying on BLAST searches and the CLUSTAL V algorithm within the DNASTAR MegAlign module) revealed that they belonged to novel species, and we have tentatively named these viruses sida golden mosaic Braco virus-[Jamaica:Liguanea:2008] and sida golden mosaic Liguanea virus-[Jamaica:1:2008]. Using RDP4 (recombination detection program), we determined that all three viruses were recombinant, with bases ~10 to ~440 of both SiGMLigV-[JM:Lig:08] and SiGYVV-[JM:Lig2:08] having been derived from a relative of SiGMBV-[JM:Lig:08] (P < 2.070 × 10^?7 for all seven of the recombination detection methods). SiGMBV-[JM:Lig:08] was itself a product of recombination, deriving bases ~490–1195 from a virus that was ~92 % similar to malvastrum yellow mosaic Helshire virus. Phylogenetically, these DNA-A components are most closely related to those of malvaceous weed-infecting begomoviruses from Jamaica, Cuba, Florida and Mexico. The SiGMBV DNA-A was able to elicit symptomatic infection in N. benthamiana.     

Inflammatory Effects of Edwardsiella ictaluri Lipopolysaccharide Modifications in Catfish Gut

Bacterial lipopolysaccharides (LPS) are structural components of the outer membranes of Gram-negative bacteria and also are potent inducers of inflammation in mammals. Higher vertebrates are extremely sensitive to LPS, but lower vertebrates, like fish, are resistant to their systemic toxic effects. However, the effects of LPS on the fish intestinal mucosa remain unknown. Edwardsiella ictaluri is a primitive member of the Enterobacteriaceae family that causes enteric septicemia in channel catfish (Ictalurus punctatus). E. ictaluri infects and colonizes deep lymphoid tissues upon oral or immersion infection. Both gut and olfactory organs are the primary sites of invasion. At the systemic level, E. ictaluri pathogenesis is relatively well characterized, but our knowledge about E. ictaluri intestinal interaction is limited. Recently, we observed that E. ictaluri oligo-polysaccharide (O-PS) LPS mutants have differential effects on the intestinal epithelia of orally inoculated catfish. Here we evaluate the effects of E. ictaluri O-PS LPS mutants by using a novel catfish intestinal loop model and compare it to the rabbit ileal loop model inoculated with Salmonella enterica serovar Typhimurium LPS. We found evident differences in rabbit ileal loop and catfish ileal loop responses to E. ictaluri and S. Typhimurium LPS. We determined that catfish respond to E. ictaluri LPS but not to S. Typhimurium LPS. We also determined that E. ictaluri inhibits cytokine production and induces disruption of the intestinal fish epithelia in an O-PS-dependent fashion. The E. ictaluri wild type and ΔwibT LPS mutant caused intestinal tissue damage and inhibited proinflammatory cytokine synthesis, in contrast to E. ictaluri Δgne and Δugd LPS mutants. We concluded that the E. ictaluri O-PS subunits play a major role during pathogenesis, since they influence the recognition of the LPS by the intestinal mucosal immune system of the catfish. The LPS structure of E. ictaluri mutants is needed to understand the mechanism of interaction.     

Microglial inhibition of neuroprotection by antagonists of the EP1 prostaglandin E2 receptor

Background  

The EP1 receptor for the prostanoid PGE2 is a G-protein coupled receptor that has been shown to contribute to excitotoxic neuronal death. In this study we examined the influence of non-neuronal cells on neuroprotective properties of EP1 receptor antagonists (Ono 8711 and SC 51089).