Probiotics and respiratory and gastrointestinal tract infections in Finnish military conscripts - a randomised placebo-controlled double-blinded study.

Military conscripts are susceptible to respiratory and gastrointestinal tract infections. In previous studies probiotics have shown potency to reduce upper respiratory and gastrointestinal infections. The aim was to study whether probiotic intervention has an impact on seasonal occurrence of upper respiratory and gastrointestinal infections in two different conscript groups. In a randomised, double-blinded, placebo controlled study (https://clinicaltrials.gov NCT01651195), a total of 983 healthy adults were enrolled from two intakes of conscripts. Conscripts were randomised to receive either a probiotic combination of Lactobacillus rhamnosus GG (LGG) and Bifidobacterium animalis ssp. lactis BB12 (BB12) or a control chewing tablet twice daily for 150 days (recruits) or for 90 days (reserve officer candidates). Clinical examinations were carried out and daily symptom diaries were collected. Outcome measures were the number of days with respiratory and gastrointestinal symptoms and symptom incidence, number and duration of infection episodes, number of antibiotic treatments received and number of days out of service because of the infection. Statistically no significant differences were found between the intervention groups either in the risk of symptom incidence or duration. However, probiotic intervention was associated with reduction of specific respiratory infection symptoms in military recruits, but not in reserve officer candidates. Probiotics did not significantly reduce overall respiratory and gastrointestinal infection morbidity.

Prospective clinical and serological follow-up in early childhood reveals a high rate of subclinical RSV infection and a relatively high reinfection rate within the first 3 years of life.

Children encounter repeated respiratory tract infections during their early life. We conducted a prospective clinical and serological follow-up study to estimate the respiratory syncytial virus (RSV) primary infection and reinfection rates in early childhood. Sera were collected from 291 healthy children at the ages of 13, 24 and 36 months and antibody levels against RSV antigens were determined by enzyme immunoassay. The RT-PCR method was also used for identifying the possible presence of RSV in symptomatic patients. At ages 1, 2 and 3 years, 37%, 68% and 86%, respectively, of studied children were seropositive for RSV. In children seropositive at age 1 year, RSV reinfection rate was at least 37%. Only one of reinfected children showed evidence for a third reinfection by age 3 years. Of children who turned RSV seropositive between ages 1 and 2 years, the reinfection rate was 32% during the third year of life. The mean antibody levels at primary infection were very similar in all age groups. The average decrease of antibody levels was 25-30% within a year. In 66 cases RSV infection was identified by RT-PCR. RSV infection rate in early childhood is 86% and reinfection rate is around 35%. This prospective serological follow-up study also provided evidence for the presence of RSV infections in children that did not show clinical signs warranting RSV RNA detection.

Dynamin inhibition interferes with inflammasome activation and cytokine gene expression in Streptococcus pyogenes-infected human macrophages.

In the present study, we have analysed the ability of Streptococcus pyogenes [Group A streptococcus (GAS)] to activate the NACHT-domain-, leucine-rich repeat- and PYD-containing protein 3 (NALP3) inflammasome complex in human monocyte-derived macrophages and the molecules and signalling pathways involved in GAS-induced inflammatory responses. We focused upon analysing the impact of dynamin-dependent endocytosis and the role of major streptococcal virulence factors streptolysin O (SLO) and streptolysin S (SLS) in the immune responses induced by GAS. These virulence factors are involved in immune evasion by forming pores in host cell membranes, and aid the bacteria to escape from the endosome-lysosome pathway. We analysed cytokine gene expression in human primary macrophages after stimulation with live or inactivated wild-type GAS as well as with live SLO and SLS defective bacteria. Interleukin (IL)-1ß, IL-10, tumour necrosis factor (TNF)-α and chemokine (C-X-C motif) ligand (CXCL)-10 cytokines were produced after bacterial stimulation in a dose-dependent manner and no differences in cytokine levels were seen between live, inactivated or mutant bacteria. These data suggest that streptolysins or other secreted bacterial products are not required for the inflammatory responses induced by GAS. Our data indicate that inhibition of dynamin-dependent endocytosis in macrophages attenuates the induction of IL-1ß, TNF-α, interferon (IFN)-ß and CXCL-10 mRNAs. We also observed that pro-IL-1ß protein was expressed and efficiently cleaved into mature-IL-1ß via inflammasome activation after bacterial stimulation. Furthermore, we demonstrate that multiple signalling pathways are involved in GAS-stimulated inflammatory responses in human macrophages.

Anticancer compound ABT-263 accelerates apoptosis in virus-infected cells and imbalances cytokine production and lowers survival rates of infected mice.

ABT-263 and its structural analogues ABT-199 and ABT-737 inhibit B-cell lymphoma 2 (Bcl-2), BCL2L1 long isoform (Bcl-xL) and BCL2L2 (Bcl-w) proteins and promote cancer cell death. Here, we show that at non-cytotoxic concentrations, these small molecules accelerate the deaths of non-cancerous cells infected with influenza A virus (IAV) or other viruses. In particular, we demonstrate that ABT-263 altered Bcl-xL interactions with Bcl-2 antagonist of cell death (Bad), Bcl-2-associated X protein (Bax), uveal autoantigen with coiled-coil domains and ankyrin repeats protein (UACA). ABT-263 thereby activated the caspase-9-mediated mitochondria-initiated apoptosis pathway, which, together with the IAV-initiated caspase-8-mediated apoptosis pathway, triggered the deaths of IAV-infected cells. Our results also indicate that Bcl-xL, Bcl-2 and Bcl-w interact with pattern recognition receptors (PRRs) that sense virus constituents to regulate cellular apoptosis. Importantly, premature killing of IAV-infected cells by ABT-263 attenuated the production of key pro-inflammatory and antiviral cytokines. The imbalance in cytokine production was also observed in ABT-263-treated IAV-infected mice, which resulted in an inability of the immune system to clear the virus and eventually lowered the survival rates of infected animals. Thus, the results suggest that the chemical inhibition of Bcl-xL, Bcl-2 and Bcl-w could potentially be hazardous for cancer patients with viral infections.

Lactobacillus rhamnosus GG and Streptococcus thermophilus induce suppressor of cytokine signalling 3 (SOCS3) gene expression directly and indirectly via interleukin-10 in human primary macrophages.

In the present study we have characterized T helper type 2 (Th2) [interleukin (IL)-10]/Th1 (IL-12) cytokine expression balance in human primary macrophages stimulated with multiple non-pathogenic Gram-positive bacteria used in the food industry and as probiotic substances. Bacteria representing Lactobacillus, Bifidobacterium, Lactococcus, Leuconostoc, Propionibacterium and Streptococcus species induced anti-inflammatory IL-10 production, although quantitative differences between the bacteria were observed. S. thermophilus was able to induce IL-12 production, while the production of IL-12 induced by other bacteria remained at a low level. The highest anti-inflammatory potential was seen with bifidobacteria, as evidenced by high IL-10/IL-12 induction ratios. All studied non-pathogenic bacteria were able to stimulate the expression of suppressor of cytokine signalling (SOCS) 3 that controls the expression of proinflammatory cytokine genes. Lactobacillus and Streptococcus species induced SOCS3 mRNA expression directly in the absence of protein synthesis and indirectly via bacteria-induced IL-10 production, as demonstrated by experiments with cycloheximide (CHX) and anti-IL-10 antibodies, respectively. The mitogen-activated protein kinase (MAPK) p38 signalling pathway played a key role in bacteria-induced SOCS3 gene expression. Enhanced IL-10 production and SOCS3 gene expression induced by live non-pathogenic Lactobacillus and Streptococcus is also likely to contribute to their immunoregulatory effects in vivo.

High frequency of cross-reacting antibodies against 2009 pandemic influenza A(H1N1) virus among the elderly in Finland.

Since May 2009, the pandemic influenza A(H1N1) virus has been spreading throughout the world. Epidemiological data indicate that the elderly are underrepresented among the ill individuals. Approximately 1,000 serum specimens collected in Finland in 2004 and 2005 from individuals born between 1909 and 2005, were analysed by haemagglutination-inhibition test for the presence of antibodies against the 2009 pandemic influenza A(H1N1) and recently circulating seasonal influenza A viruses. Ninety-six per cent of individuals born between 1909 and 1919 had antibodies against the 2009 pandemic influenza virus, while in age groups born between 1920 and 1944, the prevalence varied from 77% to 14%. Most individuals born after 1944 lacked antibodies to the pandemic virus. In sequence comparisons the haemagglutinin (HA) gene of the 2009 pandemic influenza A(H1N1) virus was closely related to that of the Spanish influenza and 1976 swine influenza viruses. Based on the three-dimensional structure of the HA molecule, the antigenic epitopes of the pandemic virus HA are more closely related to those of the Spanish influenza HA than to those of recent seasonal influenza A(H1N1) viruses. Among the elderly, cross-reactive antibodies against the 2009 pandemic influenza virus, which likely originate from infections caused by the Spanish influenza virus and its immediate descendants, may provide protective immunity against the present pandemic virus.

Induction of IRF-3 and IRF-7 phosphorylation following activation of the RIG-I pathway.

The induction of type I interferon (IFN) and the development of the innate antiviral response are mediated by the activation of interferon regulatory factor (IRF)-3 and IRF-7 under the control of the non-canonical kinases TBK-1 and IKKepsilon. The initial sensing of infection by RNA viruses is mediated by the cytoplasmic, retinoic acid inducible gene I (RIG-I), via a Toll-like receptor (TLR) independent signaling pathway. In the present study, we identify key residues involved in IRF-3 and IRF-7 phosphorylation using TAP-tag purification of TBK-1 and IKKepsilon proteins. Based on the identification of an extended sequence motif--SxSxxxS--common to both IRF-3 and IRF-7, an IRF-7 pSer477/479 phosphospecific antibody was generated. Virus infection, TBK-1/IKKepsilon expression or co-expression of different signaling adaptors such as RIG-I, MAVS and TRIF, all stimulated pSer477/479 phosphorylation. Furthermore, the newly identified adaptor of the RIG-I pathway (MAVS/IPS-1/VISA/Cardif) was able to induce IRF and NF-kappaB dependent promoter activity as efficiently as the constitutively active form of RIG-I (DeltaRIG-I). Co-expression of the NS3/4A protease activity of hepatitis C virus however blocked MAVS-mediated gene activation in a dose dependent manner. These studies link RIG-I sensing of viral RNA to downstream kinase signaling and phosphorylation of IRF-3 and IRF-7 via the MAVS/IPS/VISA/Cardif adaptor.

Measles virus enhances the expression of cellular immediate-early genes and DNA-binding of transcription factor AP-1 in lung epithelial A549 cells.

In this work we investigated the effect of measles virus (MV) infection on the expression of immediate-early genes junB, c-jun and c-fos mRNA as well as AP-1 DNA-binding activity in the lung epithelial-like adenocarcinoma cell line A549. The transcription factor AP-1, which is a group of dimeric complexes of the Fos and Jun family proteins, is an important regulator in many cellular responses to different extracellular stimuli. Membrane cofactor protein CD46, which acts as a receptor for laboratory-adapted and vaccine strains of MV, has been reported to associate with beta1 integrin molecules, which are known to trigger signaling events and activate immediate-early genes. The expression of junB and c-jun mRNA was rapidly induced by MV. It was observed already at 1 h postinfection and detected again at the later phase of infection. Moreover, the expression of c-fos mRNA seemed to be weak and transient. The early induction was apparently associated with MV binding and CD46 clustering, whereas the later induction coincided with virus replication. MV infection also enhanced the activation of AP-1 DNA-binding. Our results suggest that changes in the expression of immediate-early genes and in the activation of AP-1 DNA-binding may have an important role in many cellular events detected in MV-infected cells.

Inhibition of human natural killer cell activity by cereulide, an emetic toxin from Bacillus cereus.

The lipophilic toxin, cereulide, emitted by emetic food poisoning causing strains of Bacillus cereus, is a powerful mitochondria toxin. It is highly lipophilic and rapidly absorbed from the gut into the bloodstream. We tested how this toxin influences natural killer (NK) cells, which are important effectors in defence against infections and malignancy. Cereulide inhibited cytotoxicity and cytokine production of natural killer cells, caused swelling of natural killer cell mitochondria, and eventually induced natural killer cell apoptosis. The suppressive effect on cytotoxicity was fast and toxic concentration low, 20-30 microg/l. As the emesis causing concentration of cereulide is around 10 microg/kg of total body mass, our results suggest that emesis causing or even lower doses of cereulide may also have a systemic natural killer cell suppressive effect.

Neutralizing antibodies reduce MxA protein induction in interferon-beta-1a-treated MS patients.

BACKGROUND: Neutralizing antibodies (NAb) during interferon-beta (IFNbeta) treatment of MS are associated with reduced clinical and MR efficacy. NAb inhibit the IFN- inducible MxA gene expression and neutralize the capability of IFNbeta to inhibit virus growth in vitro. Presently, there is no clear concept of the biologic importance of IFNbeta antibodies; most of the tests applied for the detection of NAb in previous publications are not widely available, and the results are not fully comparable. METHODS: A 1-year prospective study of the development of binding antibodies (BAb) and NAb and their relationship to IFN-inducible MxA protein levels in peripheral blood leukocytes in 20 IFNbeta-1a-treated patients with relapsing-remitting MS was conducted. RESULTS: In seven of nine NAb-positive patients, IFNbeta-1a was unable to induce MxA protein. BAb were detected in 11 patients, and they preceded or paralleled the development of NAb in all the patients. The titer of NAb correlated positively with BAb titer and negatively with MxA expression level. There was also a weaker but clear correlation between BAb titers and MxA levels. CONCLUSIONS: NAb, in most but not all cases, inhibited the in vivo function of IFNbeta. Analysis of MxA protein in lymphocytes together with analysis of NAb is a promising marker for evaluating the biologic effects of IFNbeta treatment in MS patients.

IFNs activate toll-like receptor gene expression in viral infections.

Toll-like receptors (TLRs) mediate innate immune responses to microbes. TLR2, TLR5, TLR6, and TLR9 have been implicated in responses to bacterial components, and TLR4 is the receptor for Gram-negative bacteria. Recently, TLR4 was described to function in respiratory syncytial virus-induced NF-kappaB activation. Here we have analyzed TLR1-9 mRNA expression in human primary macrophages infected with influenza A and Sendai viruses. TLR1, TLR2, TLR4, TLR6, and TLR8 mRNAs were expressed at basal levels in macrophages. Viral infection enhanced TLR1, TLR2, TLR3, and TLR7 mRNA expression, and neutralizing anti-IFN-alpha/beta antibodies downregulated gene expression of these TLRs. Exogenously added IFN-alpha upregulated TLR1, TLR2, TLR3, and TLR7 mRNA expression in macrophages, as well as TLR3 mRNA expression in epithelial and endothelial cell lines. IFN-gamma enhanced the expression of TLR1 and TLR2 mRNA in macrophages, and TLR3 in epithelial and endothelial cells. The data suggests a novel role for IFNs in the activation of innate immunity.

IFN-alpha and IL-18 synergistically enhance IFN-gamma production in human NK cells: differential regulation of Stat4 activation and IFN-gamma gene expression by IFN-alpha and IL-12.

IFN-gamma, a product of NK and T cells, is a key cytokine contributing innate and adaptive immunity. IFN-gamma production is induced via direct cell-cell contacts with APC and IFN-gamma -producing cells or by cytokines. During microbial infections macrophage-derived IFN-alpha, IL-12, and IL-18 enhance IFN-gamma production and Th1 response. Here we show that IFN-alpha in combination with IL-18 very efficiently induces IFN-gamma expression also in primary, nonactivated NK cells and in NK-92 cell line. Comparison of the kinetics of IFN-gamma mRNA expression in nonactivated NK cells, NK-92 cells and activated T cells stimulated with IFN-alpha or IL-12 revealed that, although both of these cytokines directly up-regulate IFN-gamma mRNA expression, its levels remain elevated much longer with IL-12 stimulation. In both NK cells and T cells, Stat4 is known to be critical in IL-12 and IFN-alpha signaling. We show that Stat4 activation is transient in cells stimulated with IFN-alpha, whereas IL-12 induces more long-lasting activation of the transcription factor. This prolonged activation of IFN-gamma gene by IL-12 may result in more efficient IFN-gamma production compared to that of IFN-alpha. Our results demonstrate that IFN-alpha and IL-18 are important innate cytokines in inducing NK cell IFN-gamma production.

Infection of human macrophages and dendritic cells with Mycobacterium tuberculosis induces a differential cytokine gene expression that modulates T cell response.

Macrophages and dendritic cells (DC) play an essential role in the initiation and maintenance of immune response to pathogens. To analyze early interactions between Mycobacterium tuberculosis (Mtb) and immune cells, human peripheral blood monocyte-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC) were infected with Mtb. Both cells were found to internalize the mycobacteria, resulting in the activation of MDM and maturation of MDDC as reflected by enhanced expression of several surface Ags. After Mtb infection, the proinflammatory cytokines TNF-alpha, IL-1, and IL-6 were secreted mainly by MDM. As regards the production of IFN-gamma-inducing cytokines, IL-12 and IFN-alpha, was seen almost exclusively from infected MDDC, while IL-18 was secreted preferentially by macrophages. Moreover, Mtb-infected MDM also produce the immunosuppressive cytokine IL-10. Because IL-10 is a potent inhibitor of IL-12 synthesis from activated human mononuclear cells, we assessed the inhibitory potential of this cytokine using soluble IL-10R. Neutralization of IL-10 restored IL-12 secretion from Mtb-infected MDM. In line with these findings, supernatants from Mtb-infected MDDC induced IFN-gamma production by T cells and enhanced IL-18R expression, whereas supernatants from MDM failed to do that. Neutralization of IFN-alpha, IL-12, and IL-18 activity in Mtb-infected MDDC supernatants by specific Abs suggested that IL-12 and, to a lesser extent, IFN-alpha and IL-18 play a significant role in enhancing IFN-gamma synthesis by T cells. During Mtb infection, macrophages and DC may have different roles: macrophages secrete proinflammatory cytokines and induce granulomatous inflammatory response, whereas DC are primarily involved in inducing antimycobacterial T cell immune response.

Molecular pathogenesis of influenza A virus infection and virus-induced regulation of cytokine gene expression.

Despite vaccines and antiviral substances influenza still causes significant morbidity and mortality world wide. Better understanding of the molecular mechanisms of influenza virus replication, pathogenesis and host immune responses is required for the development of more efficient means of prevention and treatment of influenza. Influenza A virus, which replicates in epithelial cells and leukocytes, regulates host cell transcriptional and translational systems and activates, as well as downregulates apoptotic pathways. Influenza A virus infection results in the production of chemotactic (RANTES, MIP-1 alpha, MCP-1, MCP-3, and IP-10), pro-inflammatory (IL-1 beta, IL-6, IL-18, and TNF-alpha), and antiviral (IFN-alpha/beta) cytokines. Cytokine gene expression is associated with the activation of NF-kappa B, AP-1, STAT and IRF signal transducing molecules in influenza A virus-infected cells. In addition of upregulating cytokine gene expression, influenza A virus infection activates caspase-1 enzyme, which is involved in the proteolytic processing of proIL-1 beta and proIL-18 into their biologically active forms. Influenza A virus-induced IFN-alpha/beta is essential in host's antiviral defence by activating the expression of antiviral Mx, PKR and oligoadenylate synthetase genes. IFN-alpha/beta also prolongs T cell survival, upregulates IL-12 and IL-18 receptor gene expression and together with IL-18 stimulates NK and T cell IFN-gamma production and the development of Th1-type immune response.

Virus infection induces proteolytic processing of IL-18 in human macrophages via caspase-1 and caspase-3 activation.

There is increasing evidence that IL-18 is a key pro-inflammatory cytokine and an important mediator of Th1 immune response. The main source of IL-18 is macrophage-like cells. In the present study we have investigated IL-18 protein expression in primary human macrophages in response to influenza A and Sendai virus infections. Macrophages constitutively expressed proIL-18 but produced biologically active IL-18 only after virus infection. The IL-18 release was due to virus infection-induced proteolytic processing of 24-kDa proIL-18 into its mature 18-kDa form. ProIL-18 processing required active caspase-1 enzyme and the release of mature IL-18 was blocked with a caspase-1-specific inhibitor. Caspase-3 inhibitor also reduced IL-18 production in response to virus infection. Inactive proforms of caspase-1 and caspase-3 were basally expressed in macrophages, and virus infection induced the cleavage of procaspases into their mature forms. Besides increasing the expression of caspase proteins, virus infection enhanced caspase mRNA expression in macrophages. The enhancement of caspase gene expression was abrogated by anti-IFN-alpha antibody. Furthermore, IFN-alpha and IFN-gamma could induce caspase gene expression. These results imply that interferons are involved in virus-induced caspase activation that leads to proIL-18 processing and subsequent release of mature IL-18.

Arginine/lysine-rich structural element is involved in interferon-induced nuclear import of STATs.

Signal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors, which mediate interferon (IFN), interleukin, and some growth factor and peptide hormone signaling in cells. IFN stimulation results in tyrosine phosphorylation, dimerization, and nuclear import of STATs. In response to IFN-gamma stimulation, STAT1 forms homodimers, whereas IFN-alpha induction results in the formation of STAT1.STAT2 heterodimers, which assemble with p48 protein in the nucleus. Phosphorylation as such is not sufficient to target STATs into the nucleus; rather, the dimerization triggered by phosphorylation is essential. Although IFN-induced nuclear import of STATs is mediated by the importin/Ran transport system, no classic nuclear localization signal (NLS) has been found in STATs. In the three-dimensional structure of STAT1, we observed a structural arginine/lysine-rich element within the DNA-binding domain of the molecule. We created a series of point mutations in these elements of STAT1 and STAT2 and showed by transient transfection/IFN stimulation assay that this site is essential for the nuclear import of both STAT1 and STAT2. The results suggest that two arginine/lysine-rich elements, one in each STAT monomer, are required for IFN-induced nuclear import of STAT dimers. Import-defective STAT1 and STAT2 proteins were readily phosphorylated and dimerized, but they functioned as dominant negative molecules inhibiting the nuclear import of heterologous STAT protein.

Measles virus activates NF-kappa B and STAT transcription factors and production of IFN-alpha/beta and IL-6 in the human lung epithelial cell line A549.

Epithelial cells of the respiratory tract are the primary targets of measles virus (MV) infection. In this work we have studied the effect of MV infection on the activation of transcription factors nuclear factor (NF)-kappa B and signal transducer and activator of transcription (STAT) and the production of cytokines in the lung epithelial A549 cell line. NF-kappa B and STAT activation were induced by MV in A549 cells as analyzed by electrophoretic mobility shift assay. NF-kappa B activation was rapid and it was not inhibited by the protein synthesis inhibitor cycloheximide, suggesting that MV directly activates NF-kappa B. In contrast, Stat1, Stat3, and interferon-stimulated gene factor 3 (ISGF3) DNA binding was induced by MV infection with delayed kinetics compared to NF-kappa B activation. MV infection also resulted in an efficient interferon (IFN)-alpha/beta and interleukin-6 production. Cycloheximide and neutralizing anti-IFN-alpha/beta antibodies inhibited MV-induced activation of Stat1, Stat3, and ISGF3 DNA binding in A549 cells. In conclusion, the results suggest that MV infection activates transcription factors involved in the initiation of innate immune responses in epithelial cells by two different mechanisms: directly by leading to NF-kappa B activation and indirectly via IFN-alpha/beta leading to STAT activation.

Identification of distinct signaling pathways leading to the phosphorylation of interferon regulatory factor 3.

Infection of host cells by viruses leads to the activation of multiple signaling pathways, resulting in the expression of host genes involved in the establishment of the antiviral state. Among the transcription factors mediating the immediate response to virus is interferon regulatory factor-3 (IRF-3) which is post-translationally modified as a result of virus infection. Phosphorylation of latent cytoplasmic IRF-3 on serine and threonine residues in the C-terminal region leads to dimerization, cytoplasmic to nuclear translocation, association with the p300/CBP coactivator, and stimulation of DNA binding and transcriptional activities. We now demonstrate that IRF-3 is a phosphoprotein that is uniquely activated via virus-dependent C-terminal phosphorylation. Paramyxoviridae including measles virus and rhabdoviridae, vesicular stomatitis virus, are potent inducers of a unique virus-activated kinase activity. In contrast, stress inducers, growth factors, DNA-damaging agents, and cytokines do not induce C-terminal IRF-3 phosphorylation, translocation or transactivation, but rather activate a MAPKKK-related signaling pathway that results in N-terminal IRF-3 phosphorylation. The failure of numerous well characterized pharmacological inhibitors to abrogate virus-induced IRF-3 phosphorylation suggests the involvement of a novel kinase activity in IRF-3 regulation by viruses.