A cluster of interferon-γ-inducible p65 GTPases plays a critical role in host defense against Toxoplasma gondii.

Interferon-γ (IFN-γ) is essential for host defense against intracellular pathogens. Stimulation of innate immune cells by IFN-γ upregulates ∼2,000 effector genes such as immunity-related GTPases including p65 guanylate-binding protein (Gbp) family genes. We show that a cluster of Gbp genes was required for host cellular immunity against the intracellular parasite Toxoplasma gondii. We generated mice deficient for all six Gbp genes located on chromosome 3 (Gbp(chr3)) by targeted chromosome engineering. Mice lacking Gbp(chr3) were highly susceptible to T. gondii infection, resulting in increased parasite burden in immune organs. Furthermore, Gbp(chr3)-deleted macrophages were defective in IFN-γ-mediated suppression of T. gondii intracellular growth and recruitment of IFN-γ-inducible p47 GTPase Irgb6 to the parasitophorous vacuole. In addition, some members of Gbp(chr3) restored the protective response against T. gondii in Gbp(chr3)-deleted cells. Our results suggest that Gbp(chr3) play a pivotal role in anti-T. gondii host defense by controlling IFN-γ-mediated Irgb6-dependent cellular innate immunity.

Mask Ventilation during Induction of General Anesthesia: Influences of Obstructive Sleep Apnea.

BACKGROUND: Depending on upper airway patency during anesthesia induction, tidal volume achieved by mask ventilation may vary. In 80 adult patients undergoing general anesthesia, the authors tested a hypothesis that tidal volume during mask ventilation is smaller in patients with sleep-disordered breathing priorly defined as apnea hypopnea index greater than 5 per hour. METHODS: One-hand mask ventilation with a constant ventilator setting (pressure-controlled ventilation) was started 20 s after injection of rocuronium and maintained for 1 min during anesthesia induction. Mask ventilation efficiency was assessed by the breath number needed to initially exceed 5 ml/kg ideal body weight of expiratory tidal volume (primary outcome) and tidal volumes (secondary outcomes) during initial 15 breaths (UMIN000012494). RESULTS: Tidal volume progressively increased by more than 70% in 1 min and did not differ between sleep-disordered breathing (n = 42) and non-sleep-disordered breathing (n = 38) patients. In post hoc subgroup analyses, the primary outcome breath number (mean [95% CI], 5.7 [4.1 to 7.3] vs. 1.7 [0.2 to 3.2] breath; P = 0.001) and mean tidal volume (6.5 [4.6 to 8.3] vs. 9.6 [7.7 to 11.4] ml/kg ideal body weight; P = 0.032) were significantly smaller in 20 sleep-disordered breathing patients with higher apnea hypopnea index (median [25th to 75th percentile]: 21.7 [17.6 to 31] per hour) than in 20 non-sleep disordered breathing subjects with lower apnea hypopnea index (1.0 [0.3 to 1.5] per hour). Obesity and occurrence of expiratory flow limitation during one-hand mask ventilation independently explained the reduction of efficiency of mask ventilation, while the use of two hands effectively normalized inefficient mask ventilation during one-hand mask ventilation. CONCLUSIONS: One-hand mask ventilation is difficult in patients with obesity and severe sleep-disordered breathing particularly when expiratory flow limitation occurs during mask ventilation.

Ifit1 inhibits Japanese encephalitis virus replication through binding to 5' capped 2'-O unmethylated RNA.

The interferon-inducible protein with tetratricopeptide (IFIT) family proteins inhibit replication of some viruses by recognizing several types of RNAs, including 5'-triphosphate RNA and 5' capped 2'-O unmethylated mRNA. However, it remains unclear how IFITs inhibit replication of some viruses through recognition of RNA. Here, we analyzed the mechanisms by which Ifit1 exerts antiviral responses. Replication of a Japanese encephalitis virus (JEV) 2'-O methyltransferase (MTase) mutant was markedly enhanced in mouse embryonic fibroblasts and macrophages lacking Ifit1. Ifit1 bound 5'-triphosphate RNA but more preferentially associated with 5' capped 2'-O unmethylated mRNA. Ifit1 inhibited the translation of mRNA and thereby restricted the replication of JEV mutated in 2'-O MTase. Thus, Ifit1 inhibits replication of MTase-defective JEV by inhibiting mRNA translation through direct binding to mRNA 5' structures.

Critical role of AIM2 in Mycobacterium tuberculosis infection.

Absent in melanoma 2 (AIM2) is a sensor of cytosolic DNA that is responsible for activation of the inflammasome and host immune responses to DNA viruses and intracellular bacteria. However, the role of AIM2 in host defenses against Mycobacterium tuberculosis is unknown. Here, we show that AIM2-deficient mice were highly susceptible to intratracheal infection with M. tuberculosis and that this was associated with defective IL-1ß and IL-18 production together with impaired T (h) 1 responses. Macrophages from AIM2-deficient mice infected with M. tuberculosis showed severely impaired secretion of IL-1ß and IL-18 as well as activation of the inflammasome, determined by caspase-1 cleavage. Genomic DNA extracted from M. tuberculosis (Mtb DNA) induced caspase-1 activation and IL-1ß/IL-18 secretion in an AIM2-dependent manner. Mtb DNA, which was present in the cytosol, co-localized with AIM2. Taken together, these findings demonstrate that AIM2 plays an important role in M. tuberculosis infection through the recognition of Mtb DNA.

Effects and Limitations of Naldemedine for Opioid-Induced Urinary Retention: A Case Report.

A 78-year-old man with postoperative recurrence of esophageal cancer was admitted to the hospital due to chest pain and dyspnea. Oral short-acting opioids provided some relief, but chest pain persisted and worsened, leading to the initiation of a transdermal fentanyl patch. However, the patient developed opioid-induced urinary retention, which was treated with a naldemedine, a medication used for opioid-induced constipation and urinary retention. Opioid switching led to recurrent urinary retention, requiring placement of a urinary catheter. The patient ultimately required continuous deep sedation for refractory symptoms and died several days later.

[Factors and prosesses associated with weight loss in male workers in a specific health guidance program. A qualitative analysis of in-depth interviews].

OBJECTIVES: A qualitative analysis was conducted to identify factors important for weight loss through a specific health guidance program and to understand the processes that were crucial in achieving success. METHODS: Twenty-six male workers aged 41-59 years from five corporate health insurance societies in four prefectures who had lost > or = 4% weight by attending the six-month specific health guidance program were invited to participate in the in-depth interviews. Data were collected between October and December 2009. We audio taped the 30-minute interviews and performed qualitative analysis on the transcripts using a grounded theory. The discussion by the expert panel strengthened the validity of the analysis. RESULTS: The mean age was 49.9 +/- 5.6 years, and the average weight loss was 6.8 +/- 2.5%. All subjects were somewhat concerned about their health status and body shape before the first appointment, but two major prosesses, "critical feeling" and "sense of obligation," were identified after the first appointment. We also identified innovative efforts in all subjects during the process. Those who reported a "sense of obligation" at the beginning and those who had a negative perception during the program were found to have higher risks of weight rebound after the program was over. We considered personality, values, attitudes toward the program, and support from both family and workplace as the intervening conditions for behavior modification. CONCLUSION: Since everyone aged 40-74 years with a certain risk of metabolic syndromes is obligated by law to participate in the specific health guidance program, weight loss is challenging for those who are not motivated enough to change their behaviors. Therefore, the initial assessment of one's motivations, followed by interventions taken in consideration of one's lifestyle and social background, are crucial for the success of a weight loss program, as is the use of a client-centered approach.

NFATc1 mediates Toll-like receptor-independent innate immune responses during Trypanosoma cruzi infection.

Host defense against the intracellular protozoan parasite Trypanosoma cruzi depends on Toll-like receptor (TLR)-dependent innate immune responses. Recent studies also suggest the presence of TLR-independent responses to several microorganisms, such as viruses, bacteria, and fungi. However, the TLR-independent responses to protozoa remain unclear. Here, we demonstrate a novel TLR-independent innate response pathway to T. cruzi. Myd88(-/-)Trif(-/-) mice lacking TLR signaling showed normal T. cruzi-induced Th1 responses and maturation of dendritic cells (DCs), despite high sensitivity to the infection. IFN-gamma was normally induced in T. cruzi-infected Myd88(-/-)Trif(-/-) innate immune cells, and further was responsible for the TLR-independent Th1 responses and DC maturation after T. cruzi infection. T. cruzi infection induced elevation of the intracellular Ca(2+) level. Furthermore, T. cruzi-induced IFN-gamma expression was blocked by inhibition of Ca(2+) signaling. NFATc1, which plays a pivotal role in Ca(2+) signaling in lymphocytes, was activated in T. cruzi-infected Myd88(-/-)Trif(-/-) innate immune cells. T. cruzi-infected Nfatc1(-/-) fetal liver DCs were impaired in IFN-gamma production and DC maturation. These results demonstrate that NFATc1 mediates TLR-independent innate immune responses in T. cruzi infection.

A novel in vivo inducible dendritic cell ablation model in mice.

Dendritic cells (DCs) are involved in T cell activation via their uptake and presentation of antigens. In vivo function of DCs was analyzed using transgenic mouse models that express diphtheria toxin receptor (DTR) or the diphtheria toxin-A subunit (DTA) under the control of the CD11c/Itgax promoter. However, CD11c+ cells are heterogeneous populations that contain several DC subsets. Thus, the in vivo function of each subset of DCs remains to be elucidated. Here, we describe a new inducible DC ablation model, in which DTR expression is induced under the CD11c/Itgax promoter after Cre-mediated excision of a stop cassette (CD11c-iDTR). Crossing of CD11c-iDTR mice with CAG-Cre transgenic mice, expressing Cre recombinase under control of the cytomegalovirus immediate early enhancer-chicken beta-actin hybrid promoter, led to the generation of mice, in which DTR was selectively expressed in CD11c+ cells (iDTRDelta mice). We successfully deleted CD11c+ cells in bone marrow-derived DCs in vitro and splenic CD11c+ cells in vivo after DT treatment in iDTRDelta mice. This mouse strain will be a useful tool for generating mice lacking a specific subset of DCs using a transgenic mouse strain, in which the Cre gene is expressed by a DC subset-specific promoter.

Lipocalin 2-dependent inhibition of mycobacterial growth in alveolar epithelium.

Mycobacterium tuberculosis invades alveolar epithelial cells as well as macrophages. However, the role of alveolar epithelial cells in the host defense against M. tuberculosis remains unknown. In this study, we report that lipocalin 2 (Lcn2)-dependent inhibition of mycobacterial growth within epithelial cells is required for anti-mycobacterial innate immune responses. Lcn2 is secreted into the alveolar space by alveolar macrophages and epithelial cells during the early phase of respiratory mycobacterial infection. Lcn2 inhibits the in vitro growth of mycobacteria through sequestration of iron uptake. Lcn2-deficient mice are highly susceptible to intratracheal infection with M. tuberculosis. Histological analyses at the early phase of mycobacterial infection in Lcn2-deficient mice reveal increased numbers of mycobacteria in epithelial cell layers, but not in macrophages, in the lungs. Increased intracellular mycobacterial growth is observed in alveolar epithelial cells, but not in alveolar macrophages, from Lcn2-deficient mice. The inhibitory action of Lcn2 is blocked by the addition of endocytosis inhibitors, suggesting that internalization of Lcn2 into the epithelial cells is a prerequisite for the inhibition of intracellular mycobacterial growth. Taken together, these findings highlight a pivotal role for alveolar epithelial cells during mycobacterial infection, in which Lcn2 mediates anti-mycobacterial innate immune responses within the epithelial cells.

Control of cell wall assembly by a histone-like protein in Mycobacteria.

Bacteria coordinate assembly of the cell wall as well as synthesis of cellular components depending on the growth state. The mycobacterial cell wall is dominated by mycolic acids covalently linked to sugars, such as trehalose and arabinose, and is critical for pathogenesis of mycobacteria. Transfer of mycolic acids to sugars is necessary for cell wall biogenesis and is mediated by mycolyltransferases, which have been previously identified as three antigen 85 (Ag85) complex proteins. However, the regulation mechanism which links cell wall biogenesis and the growth state has not been elucidated. Here we found that a histone-like protein has a dual concentration-dependent regulatory effect on mycolyltransferase functions of the Ag85 complex through direct binding to both the Ag85 complex and the substrate, trehalose-6-monomycolate, in the cell wall. A histone-like protein-deficient Mycobacterium smegmatis strain has an unusual crenellated cell wall structure and exhibits impaired cessation of glycolipid biosynthesis in the growth-retarded phase. Furthermore, we found that artificial alteration of the amount of the extracellular histone-like protein and the Ag85 complex changes the growth rate of mycobacteria, perhaps due to impaired down-regulation of glycolipid biosynthesis. Our results demonstrate novel regulation of cell wall assembly which has an impact on bacterial growth.

ATF6beta is a host cellular target of the Toxoplasma gondii virulence factor ROP18.

The ROP18 kinase has been identified as a key virulence determinant conferring a high mortality phenotype characteristic of type I Toxoplasma gondii strains. This major effector molecule is secreted by the rhoptries into the host cells during invasion; however, the molecular mechanisms by which this kinase exerts its pathogenic action remain poorly understood. In this study, we show that ROP18 targets the host endoplasmic reticulum-bound transcription factor ATF6ß. Disruption of the ROP18 gene severely impairs acute toxoplasmosis by the type I RH strain. Because another virulence factor ROP16 kinase modulates immune responses through its N-terminal portion, we focus on the role of the N terminus of ROP18 in the subversion of host cellular functions. The N-terminal extension of ROP18 contributes to ATF6ß-dependent pathogenicity by interacting with ATF6ß and destabilizing it. The kinase activity of ROP18 is essential for proteasome-dependent degradation of ATF6ß and for parasite virulence. Consistent with a key role for ATF6ß in resistance against this intracellular pathogen, ATF6ß-deficient mice exhibit a high susceptibility to infection by ROP18-deficient parasites. The results reveal that interference with ATF6ß-dependent immune responses is a novel pathogenic mechanism induced by ROP18.

A single polymorphic amino acid on Toxoplasma gondii kinase ROP16 determines the direct and strain-specific activation of Stat3.

Infection by Toxoplasma gondii down-regulates the host innate immune responses, such as proinflammatory cytokine production, in a Stat3-dependent manner. A forward genetic approach recently demonstrated that the type II strain fails to suppress immune responses because of a potential defect in a highly polymorphic parasite-derived kinase, ROP16. We generated ROP16-deficient type I parasites by reverse genetics and found a severe defect in parasite-induced Stat3 activation, culminating in enhanced production of interleukin (IL) 6 and IL-12 p40 in the infected macrophages. Furthermore, overexpression of ROP16 but not ROP18 in mammalian cells resulted in Stat3 phosphorylation and strong activation of Stat3-dependent promoters. In addition, kinase-inactive ROP16 failed to activate Stat3. Comparison of type I and type II ROP16 revealed that a single amino acid substitution in the kinase domain determined the strain difference in terms of Stat3 activation. Moreover, ROP16 bound Stat3 and directly induced phosphorylation of this transcription factor. These results formally establish an essential and direct requirement of ROP16 in parasite-induced Stat3 activation and the significance of a single amino acid replacement in the function of type II ROP16.

Potent antimycobacterial activity of mouse secretory leukocyte protease inhibitor.

Secretory leukocyte protease inhibitor (SLPI) has multiple functions, including inhibition of protease activity, microbial growth, and inflammatory responses. In this study, we demonstrate that mouse SLPI is critically involved in innate host defense against pulmonary mycobacterial infection. During the early phase of respiratory infection with Mycobacterium bovis bacillus Calmette-Guérin, SLPI was produced by bronchial and alveolar epithelial cells, as well as alveolar macrophages, and secreted into the alveolar space. Recombinant mouse SLPI effectively inhibited in vitro growth of bacillus Calmette-Guérin and Mycobacterium tuberculosis through disruption of the mycobacterial cell wall structure. Each of the two whey acidic protein domains in SLPI was sufficient for inhibiting mycobacterial growth. Cationic residues within the whey acidic protein domains of SLPI were essential for disruption of mycobacterial cell walls. Mice lacking SLPI were highly susceptible to pulmonary infection with M. tuberculosis. Thus, mouse SLPI is an essential component of innate host defense against mycobacteria at the respiratory mucosal surface.