Ovarian cancer is the leading cause of death among gynecologic diseases in the USA and Europe. High-grade serous carcinoma (HGSC) of the ovary, the most aggressive type of ovarian cancer, is ...typically diagnosed at advanced stages when the 5-year survival is dismal. Since the cure rate for stage I HGSC is high, early detection of localized initial disease may improve patient outcomes. Serous tubal intraepithelial carcinoma (STIC) is considered to be a precursor lesion of HGSC. Discovery of biomarkers associated with STIC could aid in the development of an HGSC screening algorithm. Using immunohistochemical staining, we have demonstrated overexpression of UCHL1, ADAMTS13, and GAPDH in patients' STIC lesions, but not in cancer-free fallopian tubes. We additionally demonstrated a marked increase of T cells in perineoplastic stroma surrounding STIC lesions (largely CD4 + cells), but not in normal fallopian tubes and HGSC. FOXP3 + T regulatory cells are absent in STIC lesions but are present in HGSC. These observations indicate the microenvironment surrounding a STIC lesion may be immune promoting in contrast to the immune suppressive microenvironment of invasive carcinoma. In summary, we have identified UCHL1, ADAMTS13, and GAPDH as novel potentially useful markers associated with early stages of HGSC tumorigenesis and possibly contribute to STIC immunogenicity. The lack of immune suppression in the STIC microenvironment indicates that the immune system can still recognize and keep STIC controlled at this stage of the tumor development.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Severe hepatic inflammation is a common cause of acute liver injury following systemic infection with Ehrlichia, obligate Gram-negative intracellular bacteria that lack lipopolysaccharide (LPS). We ...have previously shown that type I IFN (IFN-I) and inflammasome activation are key host-pathogenic mediators that promote excessive inflammation and liver damage following fatal Ehrlichia infection. However, the underlying signals and mechanisms that regulate protective immunity and immunopathology during Ehrlichia infection are not well understood. To address this issue, we compared susceptibility to lethal Ixodes ovatus Ehrlichia (IOE) infection between wild type (WT) and MyD88-deficient (MyD88-/-) mice. We show here that MyD88-/- mice exhibited decreased inflammasome activation, attenuated liver injury, and were more resistant to lethal infection than WT mice, despite suppressed protective immunity and increased bacterial burden in the liver. MyD88-dependent inflammasome activation was also dependent on activation of the metabolic checkpoint kinase mammalian target of rapamycin complex 1 (mTORC1), inhibition of autophagic flux, and defective mitophagy in macrophages. Blocking mTORC1 signaling in infected WT mice and primary macrophages enhanced bacterial replication and attenuated inflammasome activation, suggesting autophagy promotes bacterial replication while inhibiting inflammasome activation. Finally, our data suggest TLR9 and IFN-I are upstream signaling mechanisms triggering MyD88-mediated mTORC1 and inflammasome activation in macrophages following Ehrlichia infection. This study reveals that Ehrlichia-induced liver injury and toxic shock are mediated by MyD88-dependent inflammasome activation and autophagy inhibition.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Insight into the establishment and maintenance of HIV-1 infection in resting CD4
T cell subsets is critical for the development of therapeutics targeting the HIV-1 reservoir. Although the frequency ...of HIV-1 infection, as quantified by the frequency of HIV-1 DNA, is lower in CD4
naive T cells (T
) than in the memory T cell subsets, recent studies have shown that T
harbor a large pool of replication-competent virus. Interestingly, however, T
are highly resistant to direct (
) HIV-1 infection
, in particular to R5-tropic HIV-1, as T
do not express CCR5. In this study, we investigated whether T
could be efficiently HIV-1
infected by professional antigen-presenting B lymphocytes and myeloid dendritic cells (DC) in the absence of global T cell activation. We found that B cells, but not DC, have a unique ability to efficiently
infect T
In contrast, both B cells and DC mediated HIV-1
infection of memory and activated CD4
T cells. Moreover, we found that T
isolated from HIV-1-infected nonprogressors (NP) harbor significantly disproportionately lower levels of HIV-1 DNA than T
isolated from progressors. This is consistent with our previous finding that antigen-presenting cells (APC) derived from NP do not efficiently
infect CD4
T cells due to alterations in APC cholesterol metabolism and cell membrane lipid raft organization. These findings support that B cell-mediated
infection of T
with HIV-1 has a more profound role than previously considered in establishing the viral reservoir and control of HIV-1 disease progression.
The latent human immunodeficiency virus type 1 (HIV-1) reservoir in persons on antiretroviral therapy (ART) represents a major barrier to a cure. Although most studies have focused on the HIV-1 reservoir in the memory T cell subset, replication-competent HIV-1 has been isolated from T
, and CCR5-tropic HIV-1 has been recovered from CCR5
T
from ART-suppressed HIV-1-infected individuals. In this study, we showed that CCR5
T
are efficiently
infected with R5-tropic HIV-1 by B lymphocytes, but not by myeloid dendritic cells. Furthermore, we found that T
isolated from NP harbor no or significantly fewer copies of HIV-1 DNA than those from ART-suppressed progressors. These findings support that B cell-mediated
infection of T
with HIV-1 has a more profound role than previously considered in establishing the viral reservoir and control of HIV-1 disease progression. Understanding the establishment and maintenance of the HIV-1 latent reservoir is fundamental for the design of effective treatments for viral eradication.
The ribonuclease H (RNase H) active site of HIV-1 reverse transcriptase (RT) is the only viral enzyme not targeted by approved antiretroviral drugs. Using a fluorescence-based in vitro assay, we ...screened 65,239 compounds at a final concentration of 10 µM to identify inhibitors of RT RNase H activity. We identified 41 compounds that exhibited 50% inhibitory concentration (i.e., IC50) values < 1.0 µM. Two of these compounds, 2-(4-methyl-3-(piperidin-1-ylsulfonyl)phenyl)benzodisothiazol-3(2H)-one (1) and ethyl 2-(2-(3-oxobenzodisothiazol-2(3H)-yl)thiazol-4-yl)acetate (2), which both share the same benzisothiazolone pharmacophore, demonstrate robust antiviral activity (50% effective concentrations of 1.68 ± 0.94 µM and 2.68 ± 0.54, respectively) in the absence of cellular toxicity. A limited structure–activity relationship analysis identified two additional benzisothiazolone analogs, 2-methylbenzodisothiazol-3(2H)-one (3) and N,N-diethyl-3-(3-oxobenzodisothiazol-2(3H)-yl)benzenesulfonamide (4), which also resulted in the inhibition of RT RNase H activity and virus replication. Compounds 1, 2 and 4, but not 3, inhibited the DNA polymerase activity of RT (IC50 values~1 to 6 µM). In conclusion, benzisothiazolone derivatives represent a new class of multifunctional RT inhibitors that warrants further assessment for the treatment of HIV-1 infection.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Summary
Pseudomonas aeruginosa infection is a serious complication in immunocompromised individuals and in patients with cystic fibrosis. We have previously shown that the type III secreted effector ...ExoS triggers apoptosis in various cultured cell lines via its ADP‐ribosyltransferase (ADPRT) activity. The apoptosis process was further shown to involve intrinsic signalling pathway requiring c‐Jun N‐terminal kinase (JNK)‐initiated mitochondrial pathway. In the present study, we investigated the role of Fas pathway activation in P. aeruginosa‐induced apoptosis. P. aeruginosa infection resulted in caspase 8 cleavage in HeLa cells, which was inhibited by overexpression of a dominant negative version of Fas‐associated death domain (FADD), suggesting that Fas pathway was activated. In fact, confocal laser scanning microscopy showed that P. aeruginosa induced clustering of FasR. In addition, the ADPRT activity of the ExoS was required for the induction of FasR clustering and caspase 8 cleavage. However, blocking the FasR–FasL interaction by antagonistic antibodies to FasR or to FasL had no effect on P. aeruginosa‐induced caspase 8 and caspase 3 activation, neither did the silencing of FasR by small interfering RNA (siRNA), suggesting that caspase 8 activation through the FADD bypasses FasR/FasL‐mediated signalling. Thus, FADD‐mediated caspase 8 activation involves intracellular ExoS in an ADPRT‐dependent manner. Furthermore, silencing of caspase 8 by siRNA did not interfere with P. aeruginosa‐induced apoptosis, whereas it rendered HeLa cells markedly increased resistance towards FasL‐induced apoptosis. In conclusion, our findings indicate that ExoS of P. aeruginosa induces apoptosis through a mechanism that is independent of Fas receptor/caspase 8 pathway.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
The cytolethal distending toxin (CDT) of the oral pathogen Aggregatibacter actinomycetemcomitans induces cell cycle arrest and apoptosis in various cell types. Western analysis, pharmacological ...inhibition and siRNA silencing were performed in human immortalized gingival keratinocytes (HIGK) to dissect the functional role of the ataxia telangiectasia mutated (ATM) pathway in the signal transduction steps triggered by the CDT. Infection of HIGK was associated with a time-dependent induction of cytoplasmic histone-associated DNA fragmentation. However, in the absence of CDT, infected HIGK underwent reversible DNA strand breaks but not apoptosis, while caspase 3 activity, p21 levels, and HIGK viability were unaffected. Caspase 9 activity was attenuated in the CDT mutant-infected HIGK compared to wild-type infected cells. Pharmacological inhibition and siRNA-silencing of the ATM downstream effector, the protein kinase checkpoint kinase 2 (Chk2), significantly impacted CDT-mediated apoptosis. Together, these findings provide insight on the specificity of the ATM-Chk2 pathway in response to the CDT of A. actinomycetemcomitans in oral epithelial cells, which ultimately leads to apoptosis. We further propose the existence of an unidentified factor that is distinct from the CDT, and involved with a reversible DNA fragmentation that does not trigger terminal apoptosis in oral epithelial cells. This model potentially explains conflicting reports on the biological activity of the A. actinomycetemcomitans CDT.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Lipopolysaccharide (LPS) positive pathogens trigger autophagy and inflammasome activation. Autophagy is an innate anti-microbial host defense that maintains tissue homeostasis and is ...regulated by the nutrient-sensing mTORC1 complex. However it is unknown how the LPS-negative, Gram negative Ehrlichia elicits the same response. Monocytic Ehrlichia triggers Type I IFN-dependent NLRP3 inflammasome activation, which detrimentally effects host response to Ehrlichia. In this study, we examined the regulatory mechanisms of autophagy and inflammasome activation. With toll like receptor (TLR)-adaptor molecule MYD88 deficient mice, we reveal a crucial role for MYD88 in mediation of Ehrlichia-dependent inflammasome activation and hepatic inflammation. This role is demonstrated by decreased Caspase 1 and IL-1β levels in infected liver tissues, and decreased inflammatory cytokines (TNF-α and IL-6) when compared to wild type. Ehrlichia-induced inflammasome activation in wild type mice correlated with inhibition of autophagy. Ehrlichia-induced inhibition of autophagy in infected macrophages was dependent on TLR9, TLR2, and their adaptor MYD88. Inhibition was enhanced by IFN-β stimulation. We also demonstrate that mTORC1 autophagy inhibition acts downstream of MYD88. These data suggest that LPS-negative Ehrlichia inhibit autophagy and activate the inflammasome through mTORC1 activation and MYD88 signaling, contributing to Ehrlichia-induced inflammatory disregulation and liver injury.
Abstract
Ovarian cancer is the fifth leading cause of cancer death in women and the most lethal gynecologic malignancy. High-grade serous ovarian carcinoma (HGSC) originates mainly from the fallopian ...tube epithelium, and is characterized by TP53 mutation identified in all HGSCs as well as the fallopian tubal precursor of serous tubal intraepithelial carcinomas (STICs). The loss of a functional TP53 is an early event in the malignant transformation of fallopian tube epithelium. We have established 4 primary cell cultures of normal fallopian tube epithelial (FTE) cells, and further generated immortalized FTE cells using SV40 large plus small T antigens (FTE-TAg) to disrupt TP53 function. This model mimics the primary lesions of ovarian cancer and is suitable to identify biomarkers and molecular targets of ovarian cancer. Therefore, we compared the secretomes of primary FTE cells with that of their parental isogenic pairs FTE-TAg cells using mass spectrometry analysis. We identified 813 proteins, 228 of which were differentially expressed. Ingenuity Pathway Analysis (IPA) core analysis revealed glycolysis as a major canonical pathway. Overexpression of key enzymes of glycolytic pathway was confirmed by IHC in human STIC lesions. Molecular mechanisms of glycolysis regulation in premalignant lesion of ovarian cancer were determined. Role of glycolysis in the establishment of premalignant ovarian cancer microenvironment (PME) was characterized. Prevention approaches targeting glycolysis were tested in transgenic animal model of ovarian cancer.
Citation Format: Anna E. Lokshin, Mounia Alaoui El Azher, Liudmila Velikokhatnaya, Denise Prosser. Elevated glycolysis in pre-malignant ovarian cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5142.
Abstract
Background
Immune dysregulation in people with human immunodeficiency virus-1 (PWH) persists despite potent antiretroviral therapy and, consequently, PWH tend to have lower immune responses ...to licensed vaccines. However, limited information is available about the impact of mRNA vaccines in PWH. This study details the immunologic responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines in PWH and their impact on HIV-1.
Methods
We quantified anti-S immunoglobulin G (IgG) binding and neutralization of 3 SARS-CoV-2 variants of concern and complement activation in blood from virally suppressed men with HIV-1 (MWH) and men without HIV-1 (MWOH), and the characteristics that may impact the vaccine immune responses. We also studied antibody levels against HIV-1 proteins and HIV-1 plasma RNA.
Results
MWH had lower anti-S IgG binding and neutralizing antibodies against the 3 variants compared to MWOH. MWH also produced anti-S1 antibodies with a 10-fold greater ability to activate complement and exhibited higher C3a blood levels than MWOH. MWH had decreased residual HIV-1 plasma viremia and anti-Nef IgG approximately 100 days after immunization.
Conclusions
MWH respond to SARS-CoV-2 mRNA vaccines with lower antibody titers and with greater activation of complement, while exhibiting a decrease in HIV-1 viremia and anti-Nef antibodies. These results suggest an important role of complement activation mediating protection in MWH.
In this study we show that the SARS-CoV-2 mRNA vaccines elicit lower S1-specific IgG and lower neutralizing antibodies to SARS-CoV-2, along with greater complement activation and decreased HIV-1 viremia and anti-Nef IgG in men with HIV-1.
Graphical Abstract
Graphical Abstract
Abstract
Ehrlichia is an obligate intracellular gram negative bacterium that target macrophages, and causes the potentially fatal human monocytic ehrlichiosis (HME). In murine models of fatal ...ehrlichiosis (FE), Ixodes ovatus Ehrlichia (IOE) inhibits autophagy, suppresses protective acquired immunity, and causes immunopathology and toxic shock syndrome. Innate Ehrlichia detection by TLR2 mediates host resistance, while NOD2 signaling confers host susceptibility to FE. Recently, we have shown that deficiency of type I interferon receptor (IFNaR) phenocopy NOD2 deficiency in IOE-infected mice, implying a contribution of both NOD2-IFNaR pathways to susceptibility to FE. However, how TLR2 mediates protective function against Ehrlichia is not clearly defined. Here, we investigated the role of MyD88, the main adaptor molecule for the TLRs in host response to virulent IOE using MyD88 knockout mice (MyD88−/−). Our data showed that, MyD88−/− mice have an impaired bacterial clearance, increased liver injury, and a heightened mortality when compared to infected wild type (WT) mice. Increased bacterial load in MYD88−/− mice correlates with decreased levels of TNF-α in both in vivo and in vitro {serum and IOE- infected bone marrow-derived macrophages (BMM)}, and increased frequency of splenic CD4+ T cells producing IL-10 and IL-17. Protective anti-Ehrlichia immunity and minimal pathology in WT mice infected with low virulent Ehrlichia species correlates with a high Th1 response, but a low frequency of IL-10 and IL-17 producing splenic T cells, suggesting that IL-10 and IL-17 may play pathogenic roles in host defense against Ehrlichia. Together these data suggest that MYD88 plays a protective role in ehrlichiosis via suppression of IL-10 and IL-17.
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