STING/MPYS mediates host defense against Listeria monocytogenes infection by regulating Ly6C(hi) monocyte migration.

MPYS (also known as STING, MITA, and TMEM173) is a type I IFN stimulator that is essential for host defense against DNA virus infection and appears important in defense against certain bacteria. The in vivo significance and mechanisms by which MPYS mediates host defense against nonviral pathogens are unknown. Using an MPYS-deficient mouse (Tmem173()), we determined that, distinct from the IFNAR(-/-) mice, MPYS deficiency leads to increased bacterial burden in the liver upon Listeria monocytogenes infection. The increase was correlated with the diminished MCP-1 and MCP-3 chemokine production and decreased blood and liver Ly6C(hi) monocyte frequency. We further demonstrate that MPYS-deficient Ly6C(hi) monocytes are intrinsically defective in migration to the liver. Lastly, adoptive transfer of wild-type Ly6C(hi) monocyte into MPYS-deficient mice decreases their liver bacterial burden. Our findings reveal a novel in vivo function of MPYS that is distinct from its role in activating type I IFN production.

The TRPM2 ion channel, an oxidative stress and metabolic sensor regulating innate immunity and inflammation.

TRPM2 (transient receptor potential melastatin 2) is the unique fusion of a Ca(2+)-permeable pore with an enzymatic domain that binds the NAD(+)-metabolite ADP-ribose (ADPR), resulting in channel opening. ADPR formation is a metabolic corollary of cellular stress, but can also be elicited enzymatically through NAD glycohydrolases like CD38. TRPM2 thus functions as a metabolic and oxidative stress sensor and translates this information into ion fluxes that can affect Ca(2+) signaling and the membrane potential. TRPM2 is strongly represented in immune cells of the phagocytic lineage, themselves professional generators of oxidants. The recent characterization of TRPM2-deficient mouse models has revealed the involvement of this channel in various aspects of immunity. Monocytes lacking TRPM2 show reduced production of the CXCL2 chemokine, resulting in diminished neutrophilic influx to the colon in chemically induced colitis, and thus protection against tissue ulceration in TRPM2(-/-) mice. However, the insufficient production of proinflammatory cytokines leads to high morbidity and lethality of the TRPM2(-/-) mice following infection with the bacterial pathogen Listeria monocytogenes. In the context of endotoxin-induced pulmonary inflammation, TRPM2's absence was found to promote inflammation and ROS production. TRPM2 acts thereby as a negative feedback loop by interfering through membrane depolarization with ROS generation by NADPH oxidases. In dendritic cells, TRPM2 is a lysosomal Ca(2+)-release channel that promotes chemokine responsiveness and cell migration, which is reminiscent of CD38-mediated functions. The discovery of TRPM2 has unveiled an unsuspected signaling pathway and established ADPR as a novel second messenger. Understanding TRPM2's complex involvement in inflammation is crucial to evaluating the potential of manipulating TRPM2 activity and ADPR metabolism for therapeutic intervention.

MPYS is required for IFN response factor 3 activation and type I IFN production in the response of cultured phagocytes to bacterial second messengers cyclic-di-AMP and cyclic-di-GMP.

Cyclic-di-GMP and cyclic-di-AMP are second messengers produced by bacteria and influence bacterial cell survival, differentiation, colonization, biofilm formation, virulence, and bacteria-host interactions. In this study, we show that in both RAW264.7 macrophage cells and primary bone marrow-derived macrophages, the production of IFN-ß and IL-6, but not TNF, in response to cyclic-di-AMP and cyclic-di-GMP requires MPYS (also known as STING, MITA, and TMEM173). Furthermore, expression of MPYS was required for IFN response factor 3 but not NF-κB activation in response to these bacterial metabolites. We also confirm that MPYS is required for type I IFN production by cultured macrophages infected with the intracellular pathogens Listeria monocytogenes and Francisella tularensis. However, during systemic infection with either pathogen, MPYS deficiency did not impact bacterial burdens in infected spleens. Serum IFN-ß and IL-6 concentrations in the infected control and MPYS(-/-) mice were also similar at 24 h postinfection, suggesting that these pathogens stimulate MPYS-independent cytokine production during in vivo infection. Our findings indicate that bifurcating MPYS-dependent and -independent pathways mediate sensing of cytosolic bacterial infections.

Transient Receptor Potential Melastatin 2 (TRPM2) ion channel is required for innate immunity against Listeria monocytogenes.

The generation of reactive oxygen species (ROS) is inherent to immune responses. ROS are crucially involved in host defense against pathogens by promoting bacterial killing, but also as signaling agents coordinating the production of cytokines. Transient Receptor Potential Melastatin 2 (TRPM2) is a Ca(2+)-permeable channel gated via binding of ADP-ribose, a metabolite formed under conditions of cellular exposure to ROS. Here, we show that TRPM2-deficient mice are extremely susceptible to infection with Listeria monocytogenes (Lm), exhibiting an inefficient innate immune response. In a comparison with IFNγR-deficient mice, TRPM2(-/-) mice shared similar features of uncontrolled bacterial replication and reduced levels of inducible (i)NOS-expressing monocytes, but had intact IFNγ responsiveness. In contrast, we found that levels of cytokines IL-12 and IFNγ were diminished in TRPM2(-/-) mice following Lm infection, which correlated with their reduced innate activation. Moreover, TRPM2(-/-) mice displayed a higher degree of susceptibility than IL-12-unresponsive mice, and supplementation with recombinant IFNγ was sufficient to reverse the unrestrained bacterial growth and ultimately the lethal phenotype of Lm-infected TRPM2(-/-) mice. The severity of listeriosis we observed in TRPM2(-/-) mice has not been reported for any other ion channel. These findings establish an unsuspected role for ADP-ribose and ROS-mediated cation flux for innate immunity, opening up unique possibilities for immunomodulatory intervention through TRPM2.

A phase I trial to determine the safety, tolerability, and maximum tolerated dose of deforolimus in patients with advanced malignancies.

PURPOSE: This was a phase I trial to determine the maximum tolerated dose and toxicity of deforolimus (AP23573, MK-8669), an inhibitor of mammalian target of rapamycin (mTOR). The pharmacokinetics, pharmacodynamics, and antineoplastic effects were also studied. EXPERIMENTAL DESIGN: Deforolimus was administered intravenously over 30 min every 7 days according to a flat dosing schedule. Dose was escalated according to an accelerated titration design. Patients remained on study until disease progression as long as they tolerated the drug without significant toxicities. RESULTS: Forty-six patients were enrolled on the study. Common side effects included fatigue, anorexia, and mucositis. The maximum tolerated dose was 75 mg and mucositis was the dose-limiting toxicity. Similar to other mTOR inhibitors, deforolimus exhibited nonlinear pharmacokinetics and a prolonged half-life. Among 34 patients evaluable for response, 1 patient had a partial response, 21 patients had stable disease, and 12 had progressed. Percent change in tumor size was significantly associated with AUC (P=0.015). A significant association was also detected for maximum change in cholesterol within the first two cycles of therapy and change in tumor size (r=-0.38; P=0.029). CONCLUSIONS: Deforolimus was well tolerated on the schedule tested in this trial with toxicity and pharmacokinetic profiles that were similar to that of other mTOR inhibitors. Additional phase II studies are needed to determine if deforolimus is superior to other mTOR inhibitors in terms of efficacy. The change in serum cholesterol as a potential biomarker of activity should be studied further.

Phase I trial of the novel mammalian target of rapamycin inhibitor deforolimus (AP23573; MK-8669) administered intravenously daily for 5 days every 2 weeks to patients with advanced malignancies.

PURPOSE: This phase I trial was conducted to determine the safety, tolerability, pharmacokinetics, and pharmacodynamics of deforolimus (previously known as AP23573; MK-8669), a nonprodrug rapamycin analog, in patients with advanced solid malignancies. PATIENTS AND METHODS: Patients were treated using an accelerated titration design with sequential escalating flat doses of deforolimus administered as a 30-minute intravenous infusion once daily for 5 consecutive days every 2 weeks (QDx5) in a 28-day cycle. Safety, pharmacokinetic, pharmacodynamic, and tumor response assessments were performed. RESULTS: Thirty-two patients received at least one dose of deforolimus (3 to 28 mg/d). Three dose-limiting toxicity events of grade 3 mouth sores were reported. The maximum-tolerated dose (MTD) was 18.75 mg/d. Common treatment-related adverse events included reversible mouth sores and rash. Whole-blood clearance increased with dose. Pharmacodynamic analyses demonstrated mammalian target of rapamycin inhibition at all dose levels. Four patients (one each with non-small-cell lung cancer, mixed müllerian tumor [carcinosarcoma], renal cell carcinoma, and Ewing sarcoma) experienced confirmed partial responses, and three additional patients had minor tumor regressions. CONCLUSION: The MTD of this phase I trial using an accelerated titration design was determined to be 18.75 mg/d. Deforolimus was well tolerated and showed encouraging antitumor activity across a broad range of malignancies when administered intravenously on the QDx5 schedule. On the basis of these overall results, a dose of 12.5 mg/d is being evaluated in phase II trials.

A unidimensional measure of Hong's psychological reactance scale.

Research using Hong's Psychological Reactance Scale has been fraught with methodological concerns. Researchers have been unable to find a stable, a nd replicable factor structure. Here, results suggested t hat Hong's Psychological Reactance Scale is a unidimensional one with an average alpha of .74 (SD=.46). This value was attained by first analyzing correlation matrices reproduced from three reports on Hong's Psychological Reactance Scale and then verifying this new factor structure with original data. Tests for internal consistency supported a 1-factor solution. Tests for external consistency supported prior findings in relation to Psychological Reactance and offer evidence that the 1-factor solution is externally valid. While the authors contend that a 1-factor solution is appropriate, further testing is needed for external consistency and refinement of the measure.

Accumulation of free ADP-ribose from mitochondria mediates oxidative stress-induced gating of TRPM2 cation channels.

TRPM2 is a member of the transient receptor potential melastatin-related (TRPM) family of cation channels, which possesses both ion channel and ADP-ribose hydrolase functions. TRPM2 has been shown to gate in response to oxidative and nitrosative stresses, but the mechanism through which TRPM2 gating is induced by these types of stimuli is not clear. Here we show through structure-guided mutagenesis that TRPM2 gating by ADP-ribose and both oxidative and nitrosative stresses requires an intact ADP-ribose binding cleft in the C-terminal nudix domain. We also show that oxidative/nitrosative stress-induced gating can be inhibited by pharmacological reagents predicted to inhibit NAD hydrolysis to ADP-ribose and by suppression of ADP-ribose accumulation by cytosolic or mitochondrial overexpression of an enzyme that specifically hydrolyzes ADP-ribose. Overall, our data are most consistent with a model of oxidative and nitrosative stress-induced TRPM2 activation in which mitochondria are induced to produce free ADP-ribose and release it to the cytosol, where its subsequent accumulation induces TRPM2 gating via interaction within a binding cleft in the C-terminal NUDT9-H domain of TRPM2.

Foxj1 regulates asymmetric gene expression during left-right axis patterning in mice.

Mice with a targeted mutation of the foxj1 gene demonstrate either D- or L-looping of the embryonic cardiac tube. Foxj1 is expressed in ventral cells of the embryonic node prior to asymmetric, left-right expression of other genes. Despite an absence of 9+2 cilia in foxj1(-/-) mice, 9+0 cilia are present in the node of foxj1(-/-) embryos. In foxj1(-/-) embryos, the patterns of expression of the TGF-beta family member nodal and the homeobox family member pitx2 are randomized. No expression of the TGF-beta family member lefty-2 is observed in any foxj1(-/-) early somite stage embryos. Foxj1 thus acts early in left-right axis patterning and regulates asymmetric gene expression. This regulation does not appear to be the result of a direct interaction between Foxj1 and the genes examined.

Upregulation of group 1 CD1 antigen presenting molecules in guinea pigs with experimental autoimmune encephalomyelitis: an immunohistochemical study.

In humans, group 1 CD1 glycoproteins present foreign and self lipid and glycolipid antigens to T-cells. Homologues of these molecules are not found in mice or rats but are present in guinea pigs (GPs). We examined CD1 and MHC class II expression in the central nervous system (CNS) of GPs sensitized for experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. In normal GPs and the uninflamed CNS, low-level MHC class II (MHC II) immunoreactivity occurred on vascular elements, meningeal macrophages and parenchymal microglial cells, whereas immunoreactivity for CD1 was absent. In the inflamed CNS, the majority of infiltrating cells were MHC II+ and microglia showed increased expression. CD1 immunoreactivity was detected on astrocytes and subsets of inflammatory cells Including B cells and macrophages. Minimal CD1 and MHC II co-expression was noted on inflammatory cells or glia. We conclude that group 1 CD1 molecules are strongly upregulated in the inflamed CNS on subsets of cells distinct from the majority of MHC II bearing cells. The expression of CD1 proteins in such lesions broadens the potential repertoire of antigens recognized at these sites and highlights the value of the GP as a model for studies of the relevance of CD1 molecules in host defense and autoimmune diseases.

Real-time three-dimensional ultrasound for guiding surgical tasks.

OBJECTIVE: As a stand-alone imaging modality, two-dimensional (2D) ultrasound (US) can only guide basic interventional tasks due to the limited spatial orientation information contained in these images. High-resolution real-time three-dimensional (3D) US can potentially overcome this limitation, thereby expanding the applications for US-guided procedures to include intracardiac surgery and fetal surgery, while potentially improving results of solid organ interventions such as image-guided breast, liver or prostate procedures. The following study examines the benefits of real-time 3D US for performing both basic and complex image-guided surgical tasks. MATERIALS AND METHODS: Seven surgical trainees performed three tasks in an acoustic testing tank simulating an image-guided surgical environment using 2D US, biplanar 2D US, and 3D US for guidance. Surgeon-controlled US imaging was also tested. The evaluation tasks were (1) bead-in-hole navigation; (2) bead-to-bead navigation; and (3) clip fixation. Performance measures included completion time, tool tip trajectory, and error rates, with endoscope-guided performance serving as a gold-standard reference measure for each subject. RESULTS: Compared to 2D US guidance, completion times decreased significantly with 3D US for both bead-in-hole navigation (50%, p = 0.046) and bead-to-bead navigation (77%, p = 0.009). Furthermore, tool-tip tracking for bead-to-bead navigation demonstrated improved navigational accuracy using 3D US versus 2D US (46%, p = 0.040). Biplanar 2D imaging and surgeon-controlled 2D US did not significantly improve performance as compared to conventional 2D US. In real-time 3D mode, surgeon-controlled imaging and changes in 3D image presentation made by adjusting the perspective of the 3D image did not diminish performance. For clip fixation, completion times proved excessive with 2D US guidance (> 240 s). However, with real-time 3D US imaging, completion times and error rates were comparable to endoscope-guided performance. CONCLUSIONS: Real-time 3D US can guide basic surgical tasks more efficiently and accurately than 2D US imaging. Real-time 3D US can also guide more complex surgical tasks which may prove useful for procedures where optical imaging is suboptimal, as in fetal surgery or intracardiac interventions.

Involvement of classical and novel protein kinase C isoforms in the response of human V gamma 9V delta 2 T cells to phosphate antigens.

Human gammadelta T cells expressing the Vgamma9Vdelta2 gene segments are activated polyclonally by phosphoantigens found on a wide variety of pathogenic organisms. After ligand exposure, Vgamma9Vdelta2 T cells proliferate and rapidly secrete large amounts of cytokines and chemokines that contribute to the innate immune response to these pathogens. Neither APCs nor costimulatory molecules are required. In this study we examined whether these phosphoantigens activate protein kinase Ctheta (PKCtheta). This novel PKC isoform is essential for Ag signaling through the alphabeta TCR in a costimulation-dependent fashion. The results showed that isopentenyl pyrophosphate (IPP), a soluble phospholigand released by mycobacteria, led to the rapid and persistent activation of PKCtheta in gammadelta T cells, as determined by evidence of translocation and phosphorylation. In contrast, no ligand-dependent response was detected for PKCalpha/beta or PKCdelta. Using the inhibitors Gö6976 and rottlerin, a role for both conventional and novel PKC isoforms in IPP-induced proliferation, CD25 expression, and cytokine and chemokine production was demonstrated. Gel-shift assays indicated that the transcription factors NF-kappaB and AP-1 were downstream targets of PKC activation. IPP also induced the rapid and persistent phosphorylation of extracellular signal-regulated kinases 1 and 2, p38 mitogen-activated kinase, and stress-activated kinase/c-Jun N-terminal kinase, but only an inhibitor of conventional PKCs blocked these responses. We conclude that the gammadelta T cell response to phosphoantigens is regulated by both novel and conventional PKC isoforms, with PKCtheta being more responsive to ligand stimulation and PKCalpha/beta to growth-factor availability.

Full genome screen for Alzheimer disease: stage II analysis.

We performed a two-stage genome screen to search for novel risk factors for late-onset Alzheimer disease (AD). The first stage involved genotyping 292 affected sibling pairs using 237 markers spaced at approximately 20 cM intervals throughout the genome. In the second stage, we genotyped 451 affected sibling pairs (ASPs) with an additional 91 markers, in the 16 regions where the multipoint LOD score was greater than 1 in stage I. Ten regions maintained LOD scores in excess of 1 in stage II, on chromosomes 1 (peak B), 5, 6, 9 (peaks A and B), 10, 12, 19, 21, and X. Our strongest evidence for linkage was on chromosome 10, where we obtained a peak multipoint LOD score (MLS) of 3.9. The linked region on chromosome 10 spans approximately 44 cM from D10S1426 (59 cM) to D10S2327 (103 cM). To narrow this region, we tested for linkage disequilibrium with several of the stage II microsatellite markers. Of the seven markers we tested in family-based and case control samples, the only nominally positive association we found was with the 167 bp allele of marker D10S1217 (chi-square=7.11, P=0.045, df=1).