Mast cell/IL-4 control of Francisella tularensis replication and host cell death is associated with increased ATP production and phagosomal acidification.

Mast cells are now recognized as effective modulators of innate immunity. We recently reported that mast cells and secreted interleukin-4 (IL-4) effectively control intramacrophage replication of Francisella tularensis Live Vaccine Strain (LVS), and that mice deficient in mast cells or IL-4 receptor (IL-4R(-/-)) exhibit greater susceptibility to pulmonary challenge. In this study, we further evaluated the mechanism(s) by which mast cells/IL-4 control intramacrophage bacterial replication and host cell death, and found that IL-4R(-/-) mice exhibited significantly greater induction of active caspase-3 within lung macrophages than wild-type animals following intranasal challenge with either LVS or the human virulent type A strain SCHU S4. Treatment of LVS-infected bone-marrow-derived macrophages with a pancaspase inhibitor (zVAD) did not alter bacterial replication, but minimized active caspase-3 and other markers (Annexin V and propidium iodide) of cell death, whereas treatment with both rIL-4 and zVAD resulted in concomitant reduction of both parameters, suggesting that inhibition of bacterial replication by IL-4 was independent of caspase activation. Interestingly, IL-4-treated infected macrophages exhibited significantly increased ATP production and phagolysosomal acidification, as well as enhanced mannose receptor upregulation and increased internalization with acidification, which correlated with observations in mast cell-macrophage co-cultures, with resultant decreases in F. tularensis replication.

Proteolytic bacteria in the lower digestive tract of poultry may affect avian influenza virus pathogenicity.

Proteolytic cleavage of hemagglutinin is required for cell entry by receptor-mediated endocytosis and plays a key role in pathogenicity of the influenza virus. Despite several studies describing relationships between bacterial proteases and influenza A viral activation in mammals, very little is known about the role of the normal bacterial flora of birds on hemagglutinin activation. We examined the indigenous intestinal microflora of 100 mixed-sex, 27-d-old Ross chickens from a commercial poultry facility for protease-secreting bacteria. Protease-secreting bacteria were isolated from 82 of 100 chickens with 50 birds exhibiting 2 or more protease-secreting bacterial species. A total of 20 protease-secreting bacterial species were identified: 17 gram-positive cocci, 2 gram-positive rods, and 1 gram-negative rod. Enterococcus faecalis, Enterococcus gallinarum, and Proteus mirabilis were the most frequently observed protease-secreting bacterial species. The presence of proteolytic bacteria in the intestinal tract of poultry in this study suggests the possibility of yet-to-be-described role(s) in cleavage of hemagglutinin that may alter the pathogenicity of avian influenza viruses.

Molecular analysis of isolates from influenza B outbreaks in the U.S. and Nepal, 2005.

Currently circulating influenza B viruses can be divided into two antigenically and genetically distinct lineages referred to by their respective prototype strains, B/Yamagata/16/88 and B/Victoria/2/87, based on amino acid differences in the hemagglutinin surface glycoprotein. During May and July 2005, clinical specimens from two early season influenza B outbreaks in Arizona and southeastern Nepal were subjected to antigenic (hemagglutinin inhibition) and nucleotide sequence analysis of hemagglutinin (HA1), neuraminidase (NA), and NB genes. All isolates exhibited little reactivity with the B/Shanghai/361/2002 (B/Yamagata-like) vaccine strain and significantly reduced reactivity with the previous 2003/04 B/Hong Kong/330/2001 (B/Victoria-like) vaccine strain. The majority of isolates were antigenically similar to B/Hawaii/33/2004, a B/Victoria-like reference strain. Sequence analysis indicated that 33 of 34 isolates contained B/Victoria-like HA and B/Yamagata-like NA and NB proteins. Thus, these outbreak isolates are both antigenically and genetically distinct from the current Northern Hemisphere vaccine virus strain as well as the previous 2003-04 B/Hong Kong/330/2001 (B/Victoria lineage) vaccine virus strain but are genetically similar to B/Malaysia/2506/2004, the vaccine strain proposed for the coming seasons in the Northern and Southern Hemispheres. Since these influenza B outbreaks occurred in two very distant geographical locations, these viruses may continue to circulate during the 2006 season, underscoring the importance of rapid molecular monitoring of HA, NA and NB for drift and reassortment.

Novel current-conducting composite substrates for exposing osteoblasts to alternating current stimulation.

The present study demonstrates that novel nanocomposites consisting of blends of polylactic acid and carbon nanotubes effectively can be used to expose cells to electrical stimulation. When osteoblasts cultured on the surfaces of these nanocomposites were exposed to electric stimulation (10 microA at 10 Hz) for 6 h/day for various periods of time, there was a 46% increase in cell proliferation after 2 days, a 307% increase in the concentration of extracellular calcium after 21 consecutive days, and upregulation of mRNA expression for collagen type-I after both 1 and 21 consecutive days. These results provide evidence that electrical stimulation delivered through novel, current-conducting polymer/nanophase composites promotes osteoblast functions that are responsible for the chemical composition of the organic and inorganic phases of bone. Furthermore, this evidence elucidates aspects of the cellular/molecular-level mechanisms involved in new bone formation under electrical stimulation.

Frequency- and duration-dependent effects of cyclic pressure on select bone cell functions.

The present study demonstrated unique correlations between characteristic parameters of mechanical loading and osteoblast functions. Specifically, osteoblast proliferation was dependent on the frequency and on the duration of the applied cyclic pressure stimulus: decreased cell proliferation was only observed when these cells were exposed to cyclic pressure at 1.0-Hz (but not at 0.25-Hz) frequency for 1 h (but not for 20 min) daily for 5 days. In contrast, endothelial cells were not responsive to cyclic pressure, whereas fibroblast proliferation increased under similar test conditions. Most important, cyclic pressure affected various osteoblast genes differently: exposure of osteoblasts to cyclic pressure (at 1.0-Hz frequency for 1 h daily) resulted in enhanced transcription and translation of alkaline phosphatase after 5 days; the same mechanical stimulus, however, did not affect osteopontin mRNA expression during the same time periods. These findings provide cellular and molecular level information, which is not only important in elucidating the correlation between mechanical loading and bone homeostasis, but can be useful in development of new technology in skeletal tissue engineering.

Intranasal vaccination with pneumococcal surface protein A and interleukin-12 augments antibody-mediated opsonization and protective immunity against Streptococcus pneumoniae infection.

Streptococcus pneumoniae is a major pathogen in humans that enters the host primarily through the respiratory tract. Targeting mucosal surfaces directly may therefore be an optimal approach for vaccination to prevent bacterial colonization and invasive disease. We have previously demonstrated the effectiveness of interleukin-12 (IL-12) delivered intransally (i.n.) as an antiviral respiratory adjuvant. In this study, we examined the effects of i.n. IL-12 treatment on induction of protective humoral immunity against S. pneumoniae. Immunization i.n. with pneumococcal surface protein A (PspA) and IL-12 resulted in enhanced lung IL-10 mRNA expression and marked augmentation of respiratory and systemic immunoglobulin G1 (IgG1), IgG2a, and IgA antibody levels compared to those in animals receiving PspA alone. In addition, i.n. vaccination with PspA and IL-12 provided increased protection against nasopharyngeal carriage. Flow cytometric analysis revealed a threefold increase in antibody-mediated, complement-independent opsonic activity in the sera of PspA- and IL-12-treated animals, which was mainly contributed by IgG2a and, to a lesser extent, IgA. Passive transfer of these immune sera conferred complete protection from death upon systemic pneumococcal challenge. These findings demonstrate the effectiveness of combining PspA and IL-12 at mucosal sites to achieve optimal antibody-mediated opsonization and killing of S. pneumoniae.

IL-12-mediated increases in protection elicited by pneumococcal and meningococcal conjugate vaccines.

Interleukin-12 (IL-12) may be a beneficial adjuvant for augmenting vaccine efficacy against encapsulated bacteria such as Streptococcus pneumoniae and Neisseria meningitidis since it can stimulate production of interferon-gamma (IFN-gamma) and secretion of antibody isotypes that are efficient at mediating complement fixation and opsonophagocytosis. In this study, we demonstrate the ability of IL-12 to enhance murine antibody responses, particularly IgG2a levels, to both pneumococcal and meningococcal conjugate vaccines. Transfer of immune serum from mice immunized with the meningococcal conjugate vaccine and IL-12 resulted in increased survival times, whereas transfer of serum from mice immunized with the pneumococcal conjugate and IL-12 resulted in protection from death upon bacterial challenge. Although treatment with vaccine and IL-12 increased levels of IFN-gamma mRNA, IL-12-mediated enhancement of antibody responses still occurred in IFN-gamma(-/-) mice. The results demonstrate the effectiveness of IL-12 as an adjuvant for polysaccharide conjugate vaccines, especially the pneumococcal conjugate vaccine.

Fcgamma-receptor signaling augments the LPS-stimulated increase in serum tumor necrosis factor-alpha levels.

The phagocytosis of IgG-coated erythrocytes (EIgG) has been shown to augment the bacterial lipopolysaccharide (LPS)-stimulated increase in serum tumor necrosis factor-alpha (TNF-alpha) levels. The present study evaluated the role of Fcgamma-receptor (FcgammaR) signaling and complement activation in the effect of EIgG on the TNF-alpha response to LPS. The role of FcgammaR was determined using FcR gamma-chain knockout mice that lack functional FcgammaRI and FcgammaRIII. In wild-type animals, EIgG caused a 16-fold augmentation of the serum TNF-alpha response to LPS, whereas there was no augmentation in the FcgammaR-deficient animals. Heat-damaged erythrocytes also augmented the TNF-alpha response to LPS. This effect was absent in FcgammaR-deficient animals. An IgG antibody against heated erythrocytes was detected in mouse serum. The complement activation caused by EIgG had little effect on the LPS-stimulated increase in serum TNF-alpha levels as indicated by activation of complement with cobra venom factor or IgM-coated erythrocytes as well as studies with C5-deficient mice. These results indicate that FcgammaR signaling primarily mediates the augmented serum TNF-alpha response to LPS caused by EIgG.

IgA immunodeficiency leads to inadequate Th cell priming and increased susceptibility to influenza virus infection.

IgA is considered to be the principal Ab involved in defense against pathogens in the mucosal compartment. Using mice with a targeted disruption in IgA gene expression (IgA(-/-) mice), we have examined the precise role of IgA in protective anti-influenza responses after intranasal vaccination. IgA(-/-) mice immunized intranasally with soluble hemagglutinin (hemagglutinin subtype 1) and neuraminidase (neuraminidase subtype 1) vaccine in the absence of adjuvant were found to be more susceptible to influenza virus infection than IgA(+/+) mice (13 vs 75% survival after virus challenge). Inclusion of IL-12 during immunization restored the protective efficacy of the vaccine to that seen in IgA(+/+) animals. IgA(-/-) mice had no detectable IgA expression, but displayed enhanced serum and pulmonary IgM and IgG Ab levels after IL-12 treatment. Assessment of T cell function revealed markedly depressed splenic lymphoproliferative responses to PHA in IgA(-/-) animals compared with IgA(+/+) mice. Furthermore, IgA(-/-) animals displayed impaired T cell priming to the H1N1 subunit vaccine, with concomitant reduction in recall memory responses due to a defect in APC function. Collectively, these results provide evidence that a major role of IgA is to facilitate presentation of Ag to mucosal T cells. IL-12 treatment can overcome IgA deficiency by providing adequate T cell priming during vaccination.

Neonatal administration of IL-12 enhances the protective efficacy of antiviral vaccines.

Neonates are highly susceptible to infectious agents and are known to display polarized expression of Th2-like cytokines and Abs. This neonatal immune bias has important implications for the development of vaccine strategies, particularly against viral infections. We now report that coadministration of IL-12 and an influenza subunit vaccine at birth enhances the protective efficacy of antiviral vaccination. Immunization and treatment with IL-12 within 24 h of birth resulted in elevated expression of IFN-gamma, IL-10, and IL-15 mRNA in the spleens of newborn mice compared with animals exposed to vaccine only. In addition, these animals showed dramatic increases in IFN-gamma-, IL-2-, and IL-4-secreting cells, and in IgG2a Ab levels upon adult challenge compared with mice primed with vaccine alone. Most importantly, animals vaccinated and simultaneously treated with IL-12 at birth displayed enhanced survival after lethal challenge with infectious influenza virus as adults compared with infected animals that had been primed with vaccine alone. This augmented protection required B cells and could be transferred to naive mice by immune serum. Collectively, these results provide evidence that administration of IL-12 to neonates induces a Th1-like response in newborns and elicits protective antiviral immune memory.

Intranasal interleukin-12 is a powerful adjuvant for protective mucosal immunity.

The use of interleukin (IL)-12 as a new vaccine adjuvant for stimulating protective antiviral mucosal immunity has been examined. Mice were immunized intranasally (in) with an influenza vaccine consisting of soluble hemagglutinin (H1) and neuraminidase (N1) plus IL-12. This treatment resulted in elevated levels of lung and splenic interferon-gamma and IL-10 mRNA. Total and IgG2a anti-H1N1 antibody levels in serum were significantly elevated, as were total, IgG1, IgG2a, and secretory IgA antibody levels in bronchoalveolar lavage (BAL) fluids compared with animals receiving vaccine alone. Mice immunized in with vaccine and IL-12 also exhibited decreased weight loss and dramatically enhanced survival after lethal challenge with infectious influenza virus. Protection was dependent upon the presence of B cells and could be transferred to naive mice by inoculation of either serum or BAL fluid from IL-12-treated mice. These findings show for the first time that soluble IL-12 delivered in serves as a powerful respiratory adjuvant for protective antiviral immunity.

IL-12 is a potent neonatal vaccine adjuvant.

Neonatal animals show generally poor responsiveness to foreign antigens and are known to display polarized expression of Th2-like cytokines and antibody responses. We now report that newborn mice display a reduction in peripheral expression of the Th1-inducing cytokine, IL-12. Attempts to overcome this decrease by immunization and treatment with IL-12 within 24 h of birth resulted in elevated levels of IFN-gamma and IL-10 mRNA in the spleens of mice compared to animals exposed to antigen only. Moreover, such animals showed dramatic enhancement of IgG2a and IgG2b antibody levels upon adult challenge compared to mice primed with antigen alone. These effects appeared to be due to induction of neonatal B cell memory. IgG1 antibody levels, a measure of Th2 activity, were unaffected or even somewhat enhanced by neonatal IL-12 treatment. Taken together, these results provide evidence that IL-12 administration induces a Th1-like cytokine response in newborns and causes priming for heightened memory antibody responses in vivo. Our findings suggest the use of IL-12 as a vaccine adjuvant in neonates for inducing protection against common childhood pathogens.

Modulation of mucosal and systemic immunity by intranasal interleukin 12 delivery.

Interleukin-12 (IL-12) is an important mediator of both cell-mediated and humoral immunity. We have now utilized a noninvasive intranasal (i.n.) delivery system to evaluate the ability of IL-12 to modulate both mucosal and systemic components of the immune system. Mice immunized i.n. with dinitrophenyl conjugated to ovalbumin (DNP-OVA) in combination with cholera toxin B subunit and IL-12 were found to have elevated levels of IFN-gamma and IL-10 mRNA transcripts in both lungs and spleens compared with mice not receiving IL-12. In addition, expression of lung IL-5 mRNA was inhibited. Analysis of bronchoalveolar lavage fluid after IL-12 treatment revealed a significant increase in IgG2a and unaltered IgG1 and IgA anti-OVA antibody levels. Serum IgG2a, IgG2b and IgG3 anti-DNP antibody levels were significantly increased by IL-12 given i.n., while serum IgG1 antibody levels were suppressed, results that are similar to those seen after systemic antigen plus IL-12 administration. Delivery of IL-12 i.n. also enhanced faecal IgG2a and suppressed IgA levels, in contrast to parenteral treatment which increased both faecal IgG2a and IgA antibody expression. These results provide evidence that i.n. IL-12 treatment can effectively modulate antigen-specific immune responses and enhance immunization strategies for mucosal vaccines.

IL-12 enhances antibody responses to T-independent polysaccharide vaccines in the absence of T and NK cells.

Polysaccharide vaccines to encapsulated bacteria such as Neisseria meningitidis and Streptococcus pneumoniae are weakly immunogenic due to their T-independent (TI) nature. Even when converted to T-dependent forms through conjugation to foreign proteins, polysaccharides induce responses that are deficient in many respects, such as induction of murine IgG2a Ab, the isotype that mediates optimal complement fixation and opsonization. We now show that IL-12 treatment of mice induces significantly increased levels of IgG2a Ab to the model TI-2 Ag, DNP-Ficoll, and to vaccines composed of polysaccharides from pneumococci and meningococci. Use of immunodeficient mice lacking T cells and/or NK cells demonstrated that such cells were not responsible for the observed Ab enhancement. Furthermore, the use of IFN-gamma knockout mice showed that stimulation of TI-2 Ab responses by IL-12 was only partially dependent on IFN-gamma. The ability of IL-12 to dramatically enhance TI Ab responses suggests that IL-12 will be useful as a powerful vaccine adjuvant to induce protective immune responses against encapsulated pathogens.

Norepinephrine-induced expression of the K99 pilus adhesin of enterotoxigenic Escherichia coli.

This study examined whether the provision of norepinephrine, as would be encountered within the highly innervated gastrointestinal system, affected the growth rate of enterotoxigenic Escherichia coli (ETEC) and the expression of the K99 pilus adhesin virulence-related factor. The addition of norepinephrine to serum-containing medium resulted in a 3- to 7-fold increase in the growth rate of the K99+ ETEC strain B44 as compared to growth in vehicle supplemented medium or medium supplemented with normetanephrine, a norepinephrine metabolite that contains one more methyl group than norepinephrine. ELISA analysis revealed that K99 pilus adhesin expression was increased in norepinephrine supplemented culture as compared to normetanephrine and vehicle supplemented controls. This increase occurred from 9 to 15 hours of incubation which represented the exponential growth phase for the norepinephrine supplemented culture. These results indicate that addition of norepinephrine affects both ETEC growth and expression of a specific virulence factor.

Production of Shiga-like toxins by Escherichia coli O157:H7 can be influenced by the neuroendocrine hormone norepinephrine.

To examine whether the neuroendocrine hormone norepinephrine may influence the production of the Shiga-like toxins (SLTs), several Escherichia coli O157:H7 clinical isolates were grown in the presence or absence of norepinephrine. An in vitro culture system consisting of low (<1500 colony-forming units/ml) initial concentrations of inocula into a serum-based medium was used to more closely approximate in vivo conditions. The growth of all isolates was increased several logs in the presence of norepinephrine, as compared with the growth in controls, during a 24-hour growth period. Controls included additional dextrose as well as the use of the norepinephrine metabolite normetanephrine, which contains one more methyl group than norepinephrine and hence would serve as a better energy source for growth if the effect were solely nutritionally mediated. During the 24 hours of growth, the production of cell-associated SLT-I on a protein-equivalent basis was shown to be increased over 100-fold in norepinephrine-cultured bacteria as compared with controls. SLT-II elaboration into culture supernatants was also greatly increased in norepinephrine-cultured bacteria as compared with controls. Maximal detection of cell-associated SLT-II occurred at least 12 hours before maximum levels were achieved in culture supernatants. Because norepinephrine represents one of the largest pools of monoamines present throughout the small intestine, these results suggest that the neuroendocrine environment of the small intestine may play a role in the growth of O157:H7 and the production of SLTs.