After initially polyclonal engraftment we observed oligoclonal regenerative nodules derived from transplanted RGB-marked FH-hTERT. Some mice developed monochromatic invasive liver tumors; their clonal origin was confirmed both on the molecular level, based on specific lentiviral-vector insertion sites, and by serial transplantation of one tumor. Vector insertions in proximity to the proto-oncogene MCF2 and the transcription factor MITF resulted in strong upregulation of mRNA expression in the respective tumors. Notably, upregulated MCF2 and MITF expression was also observed in 21% and 33% of 24 human hepatocellular carcinomas analyzed. In conclusion, liver repopulation with RGB-marked FH-hTERT is a useful tool to study clonal progression of liver tumors caused by insertional mutagenesis in vivo and will help identifying genes involved in liver cancer.We recently introduced red-green-blue (RGB) marking for clonal cell tracking based on individual color-coding. Here, we applied RGB marking to study clonal development of liver tumors. Immortalized, non-tumorigenic human fetal hepatocytes expressing the human telomerase reverse transcriptase (FH-hTERT) were RGB-marked by simultaneous transduction with lentiviral vectors encoding mCherry, Venus, and Cerulean. Multi-color fluorescence microscopy was used to analyze growth characteristics of RGB-marked FH-hTERT  To this end, more comprehensive understanding of the molecular pathogenesis of HCC is required. In addition, preclinical research on targeted HCC therapies has been hampered by the absence of slow-growing humanized Although many risk factors, such as hepatitis B virus (HBV) infection, are well known, processes of molecular HCC evolution and its malignant progression remain to be elucidated. This includes a potential role of virus-insertion mediated oncogene dysregulation. HBV, which is a pararetrovirus, replicates via reverse transcription with stable integration of subgenomic fragments in up to 90% of HCC . These iin vitro and identified RIPK4 as a tumor suppressor gene .Based on the positive results of MCF2/DBL and MITF upregulation in FH-hTERT-derived tumors in transplanted mice, we finally addressed a potential role of overexpression of both genes in human hepatocarcinogenesis. First we assessed potential dysregulation of the two genes in human HCC cell lines exploiting CellMinerHCC, a microarray-based expression database for HCC cell lines established by Teufel and colleagues . For MCFNext, expression levels of MCF2/DBL and MITF were measured by qPCR in 24 human HCC samples as well as in the respective surrounding non-tumorous tissue of the same surgically resected patient livers . UpregulRetroviral including lentiviral vectors stably integrate as proviruses into the host cell genome. As a consequence, vector integration can influence expression of cellular genes, which in the worst case may result in malignant transformation as seen in mouse models , 8, 35, There has been strong evidence that IM also plays a role in HCC. For example, stable integration of subgenomic HBV sequences in hepatocytes affecting regulatory mechanisms has been suggested to be involved in HCC development . Recent in vitro there was no definite proof for proliferative activity in vivo. It is reasonable to assume that only a limited number of additional “hits”, potentially just one, are necessary for malignant transformation of these cells. Therefore, FH-hTERT represent an interesting target for IM-mediated hepatocarcinogenesis. We here combined IM with a chronic liver failure/regeneration model, i.e. the uPA/SCID mouse [In the present study we aimed at using human-derived cells (FH-hTERT) in an orthotopic xenograft model, since species differences between murine and human cells have to be taken into account when such studies are used as preclinical models for human hepatocarcinogenesis. FH-hTERT cells immortalized by expression of the proto-oncogene hTERT have been previously shown to engraft and survive in the liver of immunodeficient mice . At the ID mouse , 20. TheFor the first time we demonstrate stable engraftment and liver repopulation by FH-hTERT cells. Although FH-hTERT did not adopt typical hepatocyte-like morphology eight weeks after transplantation, remaining in their original rather spindle-cell shaped form, cells produced albumin as a characteristic hepatocyte-specific protein. Quantitative-PCR data on genomic DNA showed that repopulation rates were at least in the range of 0.5% of total liver mass. Although mice homozygous for the uPA transgene would potentially allow higher repopulation rates , 47, we For labeling of FH-hTERT we applied multi-color RGB marking that enables clonality assessment of liver tumors in murine xenograft models . The thrIn our first experimental setting of long-term follow-up of RGB-marked FH-hTERT-transplanted mice, we detected one liver tumor in one out of seven mice observed long-term. In the other six mice, repopulated areas consisted of mono- or oligoclonal FH-hTERT without signs of malignant transformation. Although tumor formation was a singular event, this was the first demonstration of malignancy development after FH-hTERT transplantation . MoleculTo confirm IM-mediated malignant transformation of FH-hTERT, we repeated the transplantation experiments. However, in order to increase the likelihood of insertional mutagenesis, we introduced two modifications: (i) We transduced more cells per group to increase the probability of a malignant “hit” and (ii) we used novel RGB vectors containing several enhancers and promotors aiming to force IM. In addition we studied two further experimental groups of transplanted mice; receiving either FH-hTERT transduced with lentiviral RGB vectors containing an EFS promoter or FH-hTERT without lentiviral transduction (“control”). Engraftment of transplanted FH-hTERT was similar to the first experiment. None of the control animals developed a tumor. In contrast, three out of seven mice that received lentiviral-transduced FH-hTERT developed tumors, where it unexpectedly appeared to be irrelevant which of the above-mentioned vectors was used. While this finding confirms the initial proof-of-concept result, numbers of transplanted mice were too low to imply statistically sound conclusions regarding differences between the two vector types.ex vivo and prove its malignant phenotype and monoclonality by serial transplantation.Two of the three observed tumors were monoclonal, whereas the third one was polyclonal consisting of at least six different clones. In addition, we were able to sustain one of the tumors For both monoclonal tumors, we identified insertion sites of the integrated LeGO vectors. Notably, in both cases proto-oncogenes were located in close proximity to vector insertions. The first, MCF2/DBL is a guanine exchange factor (GEF) that activates GTP-binding proteins such as Cdc42, Rac, and Rho. This gene has been involved in carcinogenesis and progression of a variety of malignant diseases –30. The Since none of the two genes had been previously associated with liver cancer, we assessed their transcription levels in human HCC cell lines and HCC samples. For cell-line analysis we made use of the CellMinerHCC , a comprAltogether, our data indicates that MCF2/DBL and MITF could play a role during malignant transformation of hepatocytes, although their actual impact needs to be evaluated in larger studies.In conclusion, our findings demonstrate that human-derived FH-hTERT can be successfully used for repopulation of mouse livers, and that RGB marking of these cells is applicable to study their malignant transformation and progression on a clonal level. We have provided proof-of-principle that the joint use of RGB marking and insertional mutagenesis opens new possibilities to induce liver tumors and to study underlying molecular mechanisms. We therefore propose that this approach will be useful to explore molecular cancerogenesis as well as clonality of tumor growth in various malignancies.4 or 1×106 cells were transduced at a multiplicity of infection (MOI) of 1 for each vector. In order to eliminate untransduced FH-hTERT, fluorescence-positive cells were sorted by fluorescence-activated cell sorting (FACS) on a FACS AriaIIIu , using three different BP Filters . Flow-cytometry analysis was performed using FACS Canto II and BD FACS Diva Software 6.1.3 (both BD Biosciences).Cell characteristics and culture conditions of FH-hTERT were described previously . Generat6 FH-hTERT in 40 μl DMEM+GlutaMAX with 2% FBS were injected into splenic pulp  mice were housed and maintained under pathogen-free conditions. To permeabilize the sinusoidal endothelium, mice received 200 mg/kg monocrotaline 16-24 hours before cell transplantation , 23. MicREAL EnVision Kit (DAKO) according to the manufacturer’s instruction. PFA-fixed cryosections were used for molecular analyses of lentiviral integration sites. Quantitative PCR for human beta globin gene was performed as described [Fixation of excised livers with paraformaldehyde (PFA), tissue dehydration in sucrose, snap freezing, and cryotome sectioning were essentially performed as described . To visuescribed .nested-PCR. The following primers were used: PCR: AGCTTGCCTTGAGTGCTTCA; TGCAGTTTTCTGGCTCTCCT, nested-PCR: AGTAGTGTGTGCCCGTCTGT; CAGTCTCTATGCAGTGGCTTT.Isolation of DNA and RNA from PFA-fixed liver-cryosections was done following the manufacturer’s instructions . For detection of lentiviral insertion sites in the genome of FH-hTERT, we made use of ligation-mediated PCR (LM-PCR) as previously described , 54 usinSYBR Green based real-time RT-PCR Kit and gene-specific QuantiTect primer assays (Qiagen) according to the manufacturer’s instructions: For expression of MCF2/DBL proto-oncogene in transplanted mouse livers and patient samples we applied human Hs_MCF2_1_SG QuantiTect Primer Assay (QT00051429) amplifying exon 20/21 expressed in all existing splice variants [Hs_MITF_1_SG QuantiTect Primer Assay , amplifying exons 3/4/5 expressed in all existing splice variants, which were characterized before [Hs_GAPDH_1_SG QuantiTect Primer Assay (QT00079247), Qiagen).Expression levels of targeted genes were measured using the variants . For expd before . Human GFor detection of liver tumor development, MRI measurements of xenografted mice were carried out 4 months after cell transplantation and 3 to 5 weeks after retransplantation, respectively. Mice were imaged with a clinical 3.0 Tesla magnetic resonance scanner and a commercially available small-animal receiver coil as previously described , except mRNA was isolated from 24 human HCC samples and the respective surrounding non-tumorous liver tissue. Patients gave their written informed consent and the local ethical committee of “Ärztekammer Hamburg” approved the use of resected tissue for research purpose . Quantitative RT-PCR for MCF2/DBL and MITF expression was performed as described above. Patient data is summarized in


JAK2 mutations are observed in more than 95% of PV patients, and about half of ET and PMF patients. About two-thirds of JAK2 mutation-negative ET and PMF patients harbor Calreticulin (CALR) mutations. CALR mutations with a 52-bp deletion or a 5-bp insertion in exon 9 occur in more than 80% of patients with CALR mutations, and cause frameshifts that result in proteins with novel C-terminal domains composed of many positively charged amino acids [CALR mutations are exclusively observed in conjunction with JAK2 mutations in MPN patients, mutant CALR is speculated to have a driver role in MPNs.Polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) are categorized as myeloproliferative neoplasms (MPNs). All are characterized by the autonomous growth of one or more lineages of hematopoietic cells, splenomegaly, constitutional symptoms such as fatigue, night sweating, itching, and weight loss, and is frequently complicated by thrombosis and hemorrhage. no acids . As CALRJAK2 mutation, JAK2 and STAT5 are constitutively activated without cytokine stimulation, and cause autonomous cell growth , thrombopoietin (TPO), and granulocyte colony-stimulating factor bind to their specific cell surface receptors, commonly activate the JAK-STAT signaling cascade, and induce cytokine-dependent transient hematopoiesis . In caseh Figure . As CALRCALR mutations are knocked-in human hematopoietic cell lines expressing MPL using the CRISPR-Cas9 system, these cell lines demonstrate increased growth or acquire cytokine-independent cell growth accompanied by STAT5 phosphorylation. These observations indicate that mutant CALR induces cytokine-independent activation of the JAK-STAT signaling cascade in cells expressing MPL, which results in increased cell growth or cytokine-independent cell growth . TPO stimulation is reported to increase HSC numbers  of MPNs .CALR mutation knock-in mice via the deletion of base pairs in the murine CALR C-terminal . This murine mutant CALR possesses many positively charged amino acids, similar to the human mutant CALR, but the precise amino acid sequences are different from their human counterpart. Murine CALR mutation knock-in mice do not develop ET, which suggests that newly generated amino acids in human CALR may be essential in the development of ET.Mutant CALR plays a driver role in MPNs, however the precise mechanism whereby mutant CALR activates STAT5 has not been fully clarified. Araki et al. reported that mutant CALR, but not WT CALR, binds to MPL, and that the novel C-terminal domains of the mutant protein are required for this interaction . Elf et


Oncolytic virotherapy has recently emerged as a promising strategy for inducing tumor-specific cell death. Adenoviruses are widely and frequently used in oncolytic virotherapy. The mechanism of oncolytic adenovirus-mediated tumor suppression involves virus-induced activation of the autophagic machinery in tumor cells. Autophagy is a cytoprotective process that produces energy via lysosomal degradation of intracellular components as a physiologic response to various stresses, including hypoxia, nutrient deprivation, and disruption of growth signaling. However, infection with oncolytic adenoviruses induces autophagy and subsequent death of tumor cells rather than enhancing their survival. In this review, we summarize the beneficial role of autophagy in oncolytic adenoviral therapy, including the roles of infection, replication, and cell lysis. Numerous factors are involved in the promotion and inhibition of oncolytic adenovirus-mediated autophagy. Furthermore, recent evidence has shown that oncolytic adenoviruses induce autophagy-related immunogenic cell death (ICD), which enhances the antitumor immune response by inducing the activation of danger signal molecules and thus represents a novel cancer immunotherapy. Understanding the precise role of oncolytic adenovirus-induced autophagy and ICD could enhance the therapeutic potential of oncolytic adenoviral therapy for treating various cancers. E1 gene is the most critical factor, as E1-deletion adenoviruses cannot replicate. Therefore, the wild-type E1 promoter has been biogenetically modified using several different tumor-specific promoters, including the human telomerase reverse transcriptase (hTERT) [hTERT promoter is a useful tool for tumor-specific regulation of viral gene expression [Oncolytic virotherapy has emerged as a novel antitumor strategy for inducing the lytic death of tumor cells as a result of rapid viral replication . Althoug (hTERT) ,3,4,5,6, (hTERT) ,8, cyclo (hTERT) , and sur (hTERT)  promoter (hTERT) , the hTEpression . Tumor-sE1A and E1B genes under control of the hTERT promoter for tumor-specific viral replication, and we found that OBP-301 induces lytic death of tumor cells with telomerase activity [The mechanism of oncolytic adenovirus-mediated tumor suppression involves virus-induced activation of the autophagic machinery in tumor cells . Autophaactivity . OBP-301activity ,23. ThusRecent evidence suggests that antitumor therapy-induced autophagy is associated with immunogenic cell death (ICD), which involves the induction of antitumor immune responses via the release of damage-associated molecular pattern (DAMP) molecules and tumor-associated antigens (TAAs) . DAMP moIn this review, we summarize the beneficial role of autophagy in oncolytic adenovirus-mediated tumor suppression processes, including virus infection, replication, and cell lysis. Factors that modulate autophagy are also described in the context of oncolytic adenoviral therapy. Moreover, the preclinical and clinical relevance of autophagy-mediated ICD in oncolytic adenoviral therapy are discussed in terms of antitumor immunotherapy.E1 promoter and intact fiber knobs induce only a mild form of autophagy that promotes the survival of tumor cells. Thus, the intracellular level of autophagy is a crucial factor in determining whether oncolytic adenoviral therapy leads to tumor cell death or survival.A number of different oncolytic adenoviruses have been shown to induce autophagy in tumor cells. The relationship between the type of oncolytic adenovirus and role of autophagy in the cell is summarized in our previous review . A varieE1 promoter to a tumor-specific promoter enhances the replication rate of oncolytic adenoviruses in target tumor cells. Replication of the oncolytic adenovirus within the tumor cell in turn contributes to the induction of autophagy.The intracellular level of autophagy increases in tumor cells during the life cycle of oncolytic adenoviruses . To infeSeveral adenovirus proteins, including E1A, E1B, and E4, are expressed and accumulate in the cytoplasm of target cells during viral replication . OncolytThe expression of various autophagy-related biomarker proteins is associated with induction of the autophagic machinery by oncolytic adenoviruses, such as autophagy related 5 (Atg5) , microtuWhen extensive autophagy is induced following rapid viral replication, oncolytic adenoviruses primarily induce the lysis of tumor cells, resulting in further virus spread . As exteE1A region, induces autophagic death of human cancer cells [A variety of factors can promote autophagy during the process of oncolytic adenoviral therapy, including infection, replication, and cell lysis . The effer cells . EnhanceE1 promoter is a promising strategy for improving the level of viral replication because the expression level of E1 is crucial for the replication of oncolytic adenoviruses. The tumor-specific promoter enhances the viral replication level only in tumor cells, without affecting normal cells. The hTERT promoter is one of the most useful tools for inducing tumor-specific cell death. For example, a conditionally replicating oncolytic adenovirus, hTERT-Ad, in which a 255-bp hTERT promoter fragment is inserted into the wild-type E1A promoter region, induces the autophagic death of malignant brain tumor cells [hTERT promoter, induces the autophagic death of a variety of tumor cells with telomerase activities [hTERT promoter-driven oncolytic adenovirus OBP-301 can replicate more efficiently than wild-type Ad5, even in hypoxic tumor microenvironments [The replication of oncolytic adenoviruses also affects the intracellular level of autophagy. Modification of the wild-type or cells . OBP-301tivities ,23. Moreronments .The adenoviral proteins E1A and E1B play roles in the induction of pro-autophagic signaling pathways. Adenoviral E1A binds to the tumor suppressor Rb, resulting in release of the transcription factor E2F1 from the Rb-E2F1 complex . E2F1 acp53 is a multifunctional transcription factor that regulates diverse cellular processes, including autophagy, for tumor suppression [hTERT promoter-driven OBP-301 variant (OBP-702) that expresses p53 [miR-7 is involved in OBP-301-mediated autophagic death of human lung cancer cells through suppression of the anti-autophagic factor, epidermal growth factor receptor [miR-93 and miR-106 suppresses the expression of anti-autophagic p21 in OBP-702-mediated autophagy in human osteosarcoma cells [The activation of therapeutic transgenes is another useful strategy for enhancing the antitumor effect of oncolytic adenoviruses through the induction of autophagy. Although there are many types of therapeutic transgenes, those that specifically induce autophagy are the best candidates for enhancing antitumor effects in oncolytic adenoviral therapy. For example, activation of pro-autophagic Beclin-1 expression by Beclin-1-armed oncolytic adenoviruses enhances the autophagic death of malignant tumor cells . In addipression . We genesses p53 ,62. OBP-sses p53 . OBP-702sses p53 . We demosses p53 ,63. E2F1receptor . E2F1-mema cells ,63. ThesCAR gene promoter attenuates the infectivity of oncolytic adenoviruses for tumor cells [A number of factors can also inhibit autophagy by suppressing the infection, replication, or autophagy-inducing activity of oncolytic adenoviruses . Low infor cells . The preor cells . In addior cells ,67,68. Tor cells , inhibitor cells . Adenovior cells  and subsor cells  during vor cells . Bafilomor cells . Thus, tRecent evidence has shown that chemotherapeutic agents induce ICD in association with programmed cell death pathways such as apoptosis, necrosis, and autophagy . ICD is The non-histone nuclear factor HMGB1, the most abundant non-histone protein in the nucleus, is another DAMP molecule. HMGB1 is passively released from tumor cells after infection. Ad5/3-D24-GM-CSF induces the release of HMGB1 as well as ATP in virus-infected tumor cells . The relCRT is a DAMP molecule that functions as a chaperone in the endoplasmic reticulum (ER). During apoptotic ICD induction, ecto-CRT is translocated from the cytoplasmic ER to the cell surface . AlthougUric acid, which is the primary end metabolite of purine catabolism, is another DAMP molecule. Uric acid is passively released from tumor cells after infection. We demonstrated that OBP-301 causes lytic cell death with the release of uric acid, which results in the activation of DCs to produce high amounts of IFN-γ and IL-12 . MoreoveOncolytic adenoviral therapy is a promising strategy for inducing tumor-specific cell death via the activation of autophagy. Recent reports have demonstrated the beneficial role of autophagy in oncolytic adenoviral therapy. Persistent and extensive autophagy induced by oncolytic adenoviruses plays a crucial role in the death of tumor cells. Stimulation of CAR expression, fiber modification, insertion of tumor-specific promoters, induction of therapeutic transgenes, and the use of autophagy-inducing reagents are useful strategies for inducing extensive autophagy through enhancement of viral infection, replication, and cell lysis. However, the precise roles of oncolytic adenovirus-induced autophagy and ICD in antitumor immunity remain to be elucidated. Therefore, exploring the functional role of oncolytic adenovirus-induced autophagy and ICD could improve the therapeutic potential of oncolytic adenoviral anticancer therapy.


NRASQ61K and BRAFV600E, causes expression of the major histocompatibility class II antigen presentation apparatus, via secreted IL-1ß signaling and expression of CIITA, a master regulator of major histocompatibility class II gene transcription. In vitro, OIS melanocytes activate T-cell proliferation. In vivo, nonproliferating oncogene-expressing melanocytes localize to skin-draining lymph nodes, where they induce T-cell proliferation and an antigen presentation gene expression signature. In patients, expression of major histocompatibility class II in melanoma is linked to favorable disease outcome. We propose that OIS in melanocytes is accompanied by an antigen presentation phenotype, likely to promote tumor suppression via activation of the adaptive immune system.On acquisition of an oncogenic mutation, primary human and mouse cells can enter oncogene-induced senescence (OIS). OIS is characterized by a stable proliferation arrest and secretion of proinflammatory cytokines and chemokines, the senescence-associated secretory phenotype. Proliferation arrest and the senescence-associated secretory phenotype collaborate to enact tumor suppression, the former by blocking cell proliferation and the latter by recruiting immune cells to clear damaged cells. However, the interactions of OIS cells with the immune system are still poorly defined. Here, we show that engagement of OIS in primary human melanocytes, specifically by melanoma driver mutations  BRAF and NRAS (oncogene-induced senescence (OIS)  . Nevus mce (OIS) . Aggregace (OIS) .NrasQ61K oncogene was also shown to depend on activation of adaptive immunity, specifically on CD4+ T cells (+ T cells by major histocompatibility (MHC) class II-mediated antigen presentation to naïve T cells.OIS is also characterized by a secretory program, the senescence-associated secretory phenotype . The sen T cells . Typical T cells . DendritBrafV600E or NrasQ61K mutations re-localize specifically to skin-draining lymph nodes in mouse models. We also present functional evidence that in the nodes, oncogene-expressing nonproliferating melanocytes enact an antigen presentation function to activate the adaptive immune system.How senescent cells can activate the adaptive immune systems has been a mystery. Here we show that OIS in melanocytes caused by activation of the RAS/mitogen-activated protein kinase pathway is accompanied by dramatic up-regulation of the MHC class II antigen presentation complex. Furthermore, melanocytes carrying either As we and others previously showed , ectopicWe previously investigated the transcriptional changes of melanocytes undergoing OIS by whole-genome microarray analysis and RNA sequencing (RNA-seq) analysis . CompariUp-regulation of the major components of MHC class II mRNAs HLA-DRA and HLA-DRB was confirmed by quantitative real-time reverse transcriptase–PCR d. ExpresTo establish whether BRAFV600E-associated MHC class II expression is cell-type restricted, we transduced melanocytes, primary human fibroblasts (IMR90), and primary human epidermal keratinocytes with BRAFV600E or control vector Aa. In coreplicative senescence, which results from excess rounds of cell division . This showed that, across all these datasets, expression of both HLA-DRA and HLA-DRB correlated most strongly with other HLA molecules ; other molecules involved in antigen presentation ; and CIITA, a transcription factor already known to drive expression of HLA-DRA and HLA-DRB in dendritic cells   i. The re < 0.05) j. Togeth < 0.05) . This suTyr-NrasQ61K) was shown to be hyperpigmented because of an excess of melanocytes in the skin but also to contain melanocytes in the lymph nodes (Tyr-NrasQ61K (but not wild-type) mice showed cells expressing the melanocyte marker DCT and exhibiting dendritic features characteristic of melanocytes predominantly localized adjacent to the subcapsular sinus of the lymph node    that 4 wafV600E) b.Figure Tyr-NrasQ61K mice were enlarged (Tyr-NrasQ61K mice exhibited more melanocytes in the nodes than the Tyr-Cre-Er:LSL-BrafV600E mice (compare NrasQ61K and BrafV600E), paralleling the relative numbers of melanocytes in the skin of each model  (T-cell activation and proliferation can be stimulated by MHC class II-mediated antigen presentation . To inve 0.0001) f, 5g. AlTyr-NRasQ61K mice exhibited features of increased antigen presentation, we harvested RNA from lymph nodes and performed RNA-seq. This showed up- and down-regulation of approximately 577 and 423 genes, respectively . Third, oncogene-expressing primary melanocytes appear to promote activation of the immune system. Oncogene-expressing senescent melanocytes stimulated T-cell proliferation in vitro in the mixed leukocyte reaction assay, and localization of melanocytes to the nodes was accompanied by a large increase in node-resident CD3+ T cells. RNA-seq analysis of lymph nodes also showed gene expression signatures characteristic of increased antigen presentation in the melanocyte-containing lymph nodes of Tyr-NrasQ61K mice. The increase in T cells was not accounted for by an overt increase in FoxP3-expressing tolerance-inducing regulatory T cells. In sum, although we cannot, of course, formally rule out the possibility that the node melanocytes are very early and/or failed micrometastases, the collective data from mice and humans at least suggest the possibility that oncogene-expressing premalignant melanocytes might be programmed to activate the adaptive immune system.Several observations suggest that MHC class II expression in oncogene-expressing melanocytes has a dedicated function. First, melanocytes are restricted to the skin-draining nodes, suggesting that they reach the nodes specifically via the lymphatics, the normal route for migration of antigen-presenting dendritic cells, not nonspecifically via the blood. Second, these cells do not appear to be malignant, as judged by the absence of proliferation and their routine occurrence in mice lacking any detectable melanoma. Both the n a year . In bothn a year   IMR90 fibroblasts  were cultured according to supplier instructions. Infections with lentiviral vectors were performed as described . In all NrasQ61K gene (Tyr-NrasQ61K) have been described (BrafV600E gene under control of tyrosinase driven CRE-ER (Tyr-CRE-ER:LSL-BrafV600E) have also been described  under project license 60/4079.Mice carrying a tyrosinase promoter driven escribed . Mice con CRE-ER  .Microarray and RNA-seq analysis of melanocytes transduced with BRAF600E expression or control vectors has been described ; sequencThe authors state no conflict of interest.


Among the TKIs, dasatinib is distinctive in that it inhibits a broad spectrum of tyrosine kinases, including key regulators of immune responses [in vitro. Meanwhile, a unique immunological phenomenon, expansion of large granular lymphocytes (LGLs), has been frequently observed in Ph+ leukemia patients treated with dasatinib. Such expansion does not occur with other TKIs, imatinib or nilotinib. Importantly, LGL expansion during dasatinib treatment is associated with superior therapeutic responses [+ leukemia.Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL1 kinase have revolutionized the treatment strategy for Philadelphia chromosome-positive  in the LGL expansion, since it is predominantly observed in CMV-seropositive (CMV+) patients [Here is an enigma; dasatinib is immunosuppressive patients . However+ T cells, γδT cells, and NK cells, collectively cytotoxic lymphocytes. We first identified NK cells as the dominant LGLs expanding in dasatinib-treated patients. All the patients with NK cell expansion were CMV+. NK cells express a complex mosaic of inhibitory and activating receptors with large variation among individuals. In order to assess the accurate status of such variegated cells, we performed multiparametric phenotyping of NK cells from healthy donors and Ph+ leukemia patients treated with imatinib, nilotinib or dasatinib. We then analyzed the data using principal component analysis (PCA), a mathematical algorithm that reduces dimensionality of multiparametric data by defining a novel parameter  as a combination of the parameters to preserve the most essential characteristics of the dataset. PCA revealed that NK cells from CMV+ dasatinib-treated patients underwent phenotypic progression that reflects CMV-associated differentiation (such as NKG2Chigh CD57high LIR-1high NKp30low NKp46low). NK cells from CMV+ imatinib- or nilotinib-treated patients and CMV+ healthy donors had an intermediate phenotype, and those from CMV-seronegative individuals did not have the CMV-associated phenotype. Seven of 30 CMV+ dasatinib-treated patients developed CMV reactivation as defined by an elevation of CMV-IgM or a positive result of PCR. Notably, the CMV-associated highly differentiated status of NK cells was already observed at leukemia diagnosis in the majority of CMV+ patients, and was further enhanced after starting dasatinib in virtually all CMV+ patients. CMV reactivation was detected in 8 of 20 CMV+ patients at leukemia diagnosis. Importantly, the patients with a higher degree of NK cell differentiation at diagnosis underwent significantly greater NK cell expansion and faster decreases in BCR-ABL1 mRNA after starting dasatinib. Thus, pretreatment differentiated status of NK cells predicts robust expansion of NK cells and an earlier clinical response after starting dasatinib in CMV+ patients.LGLs are composed of CD8+ patients exhibited enhancement of CMV-associated NK cell differentiation during dasatinib treatment, regardless of the presence or absence of documented CMV reactivation. We assume that a low level of continuous CMV reactivation, often below detection levels of assay, is responsible for the NK cell differentiation. Furthermore, similar enhancement of NK cell differentiation was observed with mere presence of leukemia, exclusively again in CMV+ patients. Based on these comprehensive analyses, we propose CMV as an initiation factor followed by leukemia and dasatinib as enhancing factors in the NK cell activation  [Notably, the CMV-associated NKG2C EAT-2-) . It is p EAT-2-) , the CMVAn important issue in CML treatment is whether TKIs can be discontinued after achieving a deep molecular response. A substantial proportion of such patients maintains the response for more than 12 months after discontinuation of TKIs. Notably, increases in NK cells , 7 but n+ leukemia. Furthermore, the findings may illustrate exploitation of immunity against resident viruses, virobiota, for the control of malignancies [Our study using PCA suggests that the distinctive NK cell subset, that is, CMV-associated adaptive NK cells, is responsible for the dasatinib-induced NK cell expansion, which likely occurs through subclinical CMV reactivation. Intriguingly, the CMV-associated highly differentiated status is already observed at leukemia diagnosis in a large proportion of patients, and predicts prompt expansion of NK cells after starting dasatinib. These findings provide a rationale for the exploitation of CMV-associated NK cell activation to improve the treatment outcome for Phgnancies .


Triggering receptor expressed on myeloid cells 1 (TREM-1) amplifies inflammatory responses and is upregulated during sepsis and pulmonary infection. The association between serum soluble TREM-1 (sTREM-1) level and pulmonary tuberculosis (PTB) disease deserves investigation. In the present study, patients with PTB, latent TB infection (LTBI), and non-TB, non-LTBI subjects were prospectively enrolled and serum levels of sTREM-1, sTREM-2, and C-reactive protein (CRP) were measured. We correlated serum biomarkers and clinical presentations and treatment outcomes of PTB cases. We also utilized immunohistochemistry (IHC) to visualize TREM-1-expressing cells in lung tissues from PTB patients. A total of 86 PTB, 41 LTBI, and 20 non-TB, non-LTBI subjects were enrolled. Serum levels of sTREM-1 and CRP significantly increased in PTB patients; these higher serum levels were correlated with more advanced involvement in chest films and higher bacteria burden in sputum. In multivariate analysis, serum levels of sTREM-1 >260 pg/mL and CRP >2.6 mg/L were independent predictors for on-treatment mortality. Abundant TREM-1-expressing macrophages were identified in lung tissues from PTB samples. In conclusion, serum levels of sTREM-1 correlated with disease severity and treatment outcomes in PTB patients. Mycobacterium tuberculosis (MTB) in human lungs. The major innate cells in humans include macrophages, neutrophils, dendritic cells, and natural killer cells. Macrophages play a pivotal role among the innate cells, and alveolar macrophages are the first line of defense against MTB. Through a number of receptors on the cell surface, MTB is recognized and ingested by alveolar macrophages, which in turn activate downstream signaling and induce the production of inflammatory cytokines. Although innate immunity is activated early to limit disease dissemination, recent studies demonstrated that myeloid cells also facilitate the replication of MTB2.Despite effective anti-tuberculosis (TB) therapies, TB remains an infectious disease with high morbidity and mortality. Both innate and adaptive immunity are involved in host defense mechanisms against 4. After associating with the adaptor DNAX activation protein (DAP)-12, TREM family proteins trigger a signaling pathway that modulates infection-related inflammatory responses5. Among the TREM family, TREM-1 has the synergistic ability to amplify the signaling of the Toll-like receptors (TLRs) TLR4 or TLR2, which can recognize components of a variety of microorganisms including bacteria, fungi, and viruses7. In mouse models, engagement of TREM-1 on the cell surface enhances inflammatory reactions, such as cytokine/chemokine production, neutrophil degranulation, and macrophage phagocytosis9. Unlike TREM-1, the role of TREM-2 in inflammation is still unclear and is likely to be anti-inflammatory. Activation of the TREM-2 pathway can inhibit the production of pro-inflammatory cytokines and promote phagocytosis in macrophages11. Recent genome-wide studies showed that heterozygous missense mutations in TREM-2 increase the risk of Alzheimer’s disease13.The triggering receptor expressed on myeloid cells (TREM) family is a group of cell surface receptors expressed on myeloid cells, including monocytes/macrophages and neutrophils14. Increased plasma sTREM-1 is also reported to be a useful marker for evaluating disease severity and outcomes of patients with sepsis15. In contrast to bacterial pneumonia and sepsis, information regarding the role of TREM-1 in pulmonary TB is relatively scarce. Considering the potential synergistic effects of TREM-1 in inflammatory reactions mediated by TLR4 and TLR2, which can recognize MTB components, we hypothesize that TREM-1 is also involved in immune responses against MTB. In this study, we measured the serum levels of the soluble forms of TREM-1 and TREM-2 (sTREM-2) in patients with pulmonary TB before initiating anti-TB treatment. The aim of the study was to evaluate the role of sTREMs in clinical presentations and treatment outcomes of pulmonary TB patients. We also used immunohistochemistry (IHC) staining to investigate the presence of TREM-1-expressing cells in lung tissues from patients with pulmonary TB.Several clinical studies have suggested a specific role of TREM-1 in pneumonia and sepsis. Levels of the soluble form of TREM-1 (sTREM-1) in serum and bronchoalveolar lavage fluid can be used to diagnose bacterial lung infections in intensive care unit (ICU) patientsDuring the study period, a total of 147 subjects, including 86 active pulmonary TB patients, 41 LTBI patients, and 20 non-TB, non-LTBI subjects, were prospectively enrolled  significantly increased in patients with ventilator-associated pneumonia, and dynamic changes in sTREM-1 concentration in BALF are prognostically valuable for predicting mortality24. Serum levels of sTREM-1 have also been reported to predict bacteremia in patients with community-acquired pneumonia (CAP)25. A recent meta-analysis evaluated the prognostic values of sTREM-1 levels in the initial stages of infection and concluded that sTREM-1 alone is insufficient to predict mortality as a biomarker in adult patients with infection26. However, elevated sTREM-1 levels were associated with increased risk of death, indicating that TREM-1 is involved in immune responses against infections in humans.The role of sTREM-1 as a potential biomarker to evaluate disease severity and treatment outcomes has been evaluated in patients with sepsis and pneumonia. Oku et al. reported that sTREM-1 was detectable in sputum from pulmonary TB patients, but levels did not differ from those observed in patients with CAP27. Hung et al. analyzed the 6-month mortality in TB patients and demonstrated that serum sTREM-1 level was an independent mortality predictor in multivariate analysis. In the present study, we observed elevated sTREM-1 levels in pulmonary TB patients. We analyzed the clinical characteristics and presentations among pulmonary TB patients with higher and lower sTREM-1 levels, and found that TB patients with higher sTREM-1 levels were older and had trend of more diabetes (data not shown). Furthermore, serum sTREM-1 level was positively correlated with higher bacterial burden in sputum and more advanced lung lesions in chest films. In line with results of previous studies, we found that increased serum sTREM-1 level was an independent predictor for on-treatment mortality. Considering the significant association between increased sTREM1 levels and higher bacterial burden and on-treatment mortality, we speculate that increased sTREM-1 levels in pulmonary TB patients with older age and diabetes may be due to more profound TB diseases in these population. However, the potential causative role of older age and diabetes in escalation of sTREM-1 levels also deserves further evaluation.As compared to sepsis and bacterial pneumonia, studies evaluating the role of sTREM-1 in pulmonary TB are relatively limited. Tintinger Most importantly, we clearly identified TREM-1-expressing macrophages in lung tissues from patients with pulmonary TB. To our knowledge, this is the first study that used IHC staining to demonstrate the presence of TREM-1-expressing macrophages in human lungs infected with MTB. Interestingly, we found that the expression of TREM-1 was stronger in alveolar macrophages than in the macrophages from granulomas. Although the exact mechanism remains unknown, our findings indicate that TREM-1 is highly involved in the initial stages of innate immunity against MTB in the alveolar space, and plays a relatively minor role in the later stages of immune responses, such as granuloma formation. However, our findings should be interpreted with caution. we did not obtain lung tissues from LTBI or non-TB, non-LTBI individuals for comparison in our IHC analysis. The expression of TREM-1 is neither evaluated quantitatively. Therefore, further IHC analysis in lung tissues from non-TB subjects is required to consolidate the potential role of TREM-1 in immune responses against MTB.29. Increased expression of TREM-1 in TAM were reported to predict cancer aggressiveness and associated with worse treatment outcomes. In vitro studies demonstrated that the expression of TREM-1 in macrophages in tumor microenvironment is dependent on COX-2 signaling. It suggested that TREM-1 expression in lung tissue is not specific to infectious disease such as pulmonary TB, and is probably also involved in immunomodulation of lung cancer.It is worth noting that expression of TREM-1 in lung tissue is not specific to pulmonary TB. TREM-1 is also reported to be expressed in tumor-associated macrophages (TAM) in non-small cell lung cancer in previous studieset al. reported elevated cell-associated TREM-2 expression on monocytes and CD8+ lymphocytes from TB patients, but serum sTREM-2 levels were similar between TB patients and healthy controls30. In our study, serum sTREM-2 levels were similar between patients with pulmonary TB, LTBI cases, and non-TB, non-LTBI subjects. Although patients with higher sTREM-2 levels had higher on-treatment mortality, sTREM-2 level was not an independent factor for on-treatment mortality. Unlike TREM-1, the role of TREM-2 in modulating inflammation is much vaguer and further studies are needed to elucidate this issue.Compared to TREM-1, studies evaluating the role of TREM-2 in TB are even scarcer. Hoogendijk This study had several limitations that are worth noting. As a prospective study, the number of patients with pulmonary TB was relatively small, which may overestimate or underestimate the mortality predictive values of the serum biomarkers. Blood samples for biomarker measurement were collected before initiating anti-TB treatment; therefore, the role of the changing patterns of these biomarkers in treatment outcomes cannot be evaluated. The impact of anti-TB treatment in dynamic changes of these biomarkers is also unknown. Patients with HIV and other comorbidities that may affect immune response were excluded in our study design. Therefore, further studies are needed to validate the mortality-predictive value of sTREM-1 in pulmonary TB patients with compromised immune function. Finally, only patients with pulmonary TB were included in our study for analysis. Therefore, our findings cannot be readily applied to patients with extrapulmonary TB.in vivo and in vitro studies are warranted to explore the exact role of TREM-1 in innate immune responses triggered by MTB infection.In conclusion, this prospective observational study demonstrated that serum levels of sTREM-1 significantly increased in patients with pulmonary TB. Elevated serum levels of sTREM-1 and CRP correlated with more advanced pulmonary involvement, higher bacteria burden in sputum, and were independent factors associated with on-treatment mortality in patients with pulmonary TB. Moreover, for the first time, we demonstrated the high expression of TREM-1 in macrophages from lung tissues of patients with pulmonary TB, although LTBI or non-TB, non-LTBI individuals were not included for IHC analysis. Our findings provided serological and pathological evidence suggesting that TREM-1 is potentially involved in the host immune responses against MTB infection. Further This was a prospective observational study conducted in a tertiary medical center in northern Taiwan. Newly diagnosed, culture-proven pulmonary tuberculosis patients from August 2014 to July 2016 were eligible for enrollment. We also included individuals of active TB contact for comparison. Active TB patients without pulmonary involvement; individuals younger than 20 years; and patients with active malignancies, human immunodeficiency virus (HIV) infection, autoimmune diseases, and organ transplants were excluded. The demographic characteristics and clinical symptoms/signs were determined based on participant statements obtained in the enrollment interview. The chest plain films of each pulmonary TB patient were reviewed by a pulmonologists and a radiologist, and the radiographic presentations, including extent of lung involvement and the presence of cavitary lesions, were recorded accordingly. This study was approved by the Institutional Review Board of Taipei Veterans General Hospital (IRB number 2014-09-007 A), and written informed consent was obtained from each participant or their authorized representative(s) before enrollment. The methods were carried out in accordance with the approved guidelines.31. Individuals of active TB contacts were categorized into subgroups of latent TB infection (LTBI) cases and non-TB, non-LTBI subjects based on IGRA results and analyzed accordingly.The Interferon-γ (IFN-γ) release assay (IGRA) was performed in all participants with whole-blood-based QuantiFERON-TB Gold In-Tube  as previously prescribedPeripheral blood samples were collected from the participants on the day of enrollment, which was before the initiation of anti-TB or LTBI prophylactic treatment. We used commercialized enzyme-linked immunosorbent assay kits to measure serum levels of sTREM-1  and sTREM-2  in duplicate. Serum C-reactive protein (CRP) levels were measured by the core facility of Taipei Veterans General Hospital using an Abbott Architect C16000 analyzer . The measurement of serum biomarkers levels by commercialized ELISA kits were performed in bio-containment level 1 laboratory. Based on the median serum levels of sTREM-1, sTREM2, and CRP, patients with pulmonary TB were categorized into subgroups of high and low serum biomarker levels for further analysis.32. On-treatment mortality was defined as patients who died for any reason during the course of anti-TB treatment, which means the observation ended when anti-TB treatment is completed33. The treatment duration of the enrolled TB patients ranged from 174 days to 368 days and the median treatment duration was 245 days.To investigate the role of serum biomarker levels in pulmonary TB disease severity, we analyzed differences in clinical presentations and treatment outcomes of patients with pulmonary TB possessing high and low serum biomarker levels. The clinical presentations we analyzed included constitutional symptoms/signs, respiratory symptoms/signs, radiological presentations, and bacteria burden evaluated by the results of sputum smears. The treatment outcomes evaluated in the present study included two-month smear/culture conversion and on-treatment mortality. Two-month smear/culture conversion was defined as all collected sputum at two months after initiation of anti-TB treatment being smear/culture negative for MTB, and there being no positive cultures thereafter until the completion of anti-TB treatmentTo identify TREM-1-expressing inflammatory cells in lung tissues, we obtained lung specimens from two patients with pulmonary TB that were diagnosed based on pathologic characteristics and positive TB cultures of lung specimens. Both their lung specimens showed necrotizing granulomatous inflammation with the presence of mycobacterium, and the in-house TB polymerase chain reaction results were positive. IHC staining for TREM-1 expression analysis was carried out in the core facility of Taipei Veterans General Hospital. In short, 4-μm sections were prepared from formalin-fixed, paraffin-embedded tissues and the IHC staining was performed on a Leica Bond-MAX system (automatic IHC staining system). After deparaffinization, sections were pre-treated using heat-mediated antigen retrieval with sodium citrate buffer  for 30 minutes. The section was then incubated with a human anti-TREM-1 antibody  at 10 µg/mL (diluted 1/100) for 60 minutes at room temperature, and detected using an HRP-conjugated compact polymer system (Anti-Rabbit IgG–Poly-HRP). The section was blocked with peroxide block  for 5 minutes. DAB was used as the chromogen. IHC staining with anti-CD68 antibody  was also performed to identify the presence of human macrophages in lung specimens.t tests, and categorical variables were compared using Pearson’s chi-square or Fisher’s exact test. Comparisons of serum biomarker levels were carried out using a Mann-Whitney U test. For treatment outcome analysis, patients with pulmonary TB were stratified into subgroups of high and low biomarker levels based on the median serum biomarker level. The optimal cut-off values of serum biomarkers levels for predicting mortality were determined by Receiver Operating Characteristic curves. The Kaplan-Meier method was used to estimate on-treatment survival times of patients with pulmonary TB. A log-rank test was used to compare on-treatment mortality between TB patients with higher and lower serum biomarkers levels. A multivariate Cox proportional hazards regression model with forward stepwise selection procedures was used to identify the risk factors for on-treatment mortality. The parameters included in the model were clinical characteristics, comorbidities, and serum biomarkers levels. A p value less than 0.1 in the univariate analysis was required for a variable to be entered into the multivariate model. All tests were two-tailed, and p values less than 0.05 were considered to be statistically significant.Statistical analysis was carried out using statistical software package . Continuous variables between subgroups were compared with independent The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary Table 1


TERT gene promoter (hTERT) and the antioxidant responseelement (ARE) from the human GCLM gene promoter. The hybridpromoter retains the tumor specificity of the basal hTERTpromoter but is characterized by an enhanced transcriptional activityin cancer cells with abnormal activation of the Nrf2 transcription factor andupon induction of oxidative stress. In the in vitroenzyme-prodrug cancer gene therapy scheme, ARE-hTERT promoter-drivenexpression of CD : UPRT (yeast cytosine deaminase : uracilphosphoribosyltransferase) chimeric protein induced a more pronounced death ofcancer cells either upon treatment with 5-fluorouracil (5FC) alone or when 5FCwas combined with chemotherapeutic drugs as compared to the hTERT promoter. Thedeveloped hybrid promoter can be considered a better alternative to the hTERTpromoter in cancer gene therapy schemes.describe a novel hybrid tumor-specific promoter, ARE-hTERT, composed of thehuman  Gene therapy is a strategy that is witnessing dynamic development; severaldrugs have already been approved for clinical use, many are undergoing variousphases of clinical trials, and a vast number of drugs are under development atlaboratories. Various approaches to provide specific activity of gene therapyagents in cancer cells have been proposed and validated, includingpost-transcriptional regulation of the therapeutic transgene expression levelin cancer cells via the selective stabilization of the transcript in tumorcells  or destaTumor-specific promoters have been successfully validated in many cases, buttheir usage is associated with several drawbacks, including a far from absolutetumor specificity and low promoter activity, which affects the transgeneexpression level and, consequently, the therapeutic effect. In particular, thehuman telomerase reverse transcriptase (hTERT) gene promoter is one of the bestcharacterized tumor-specific promoters and is active in a wide variety oftumors, providing the advantage of targeting cancer cells of different origins-7.Nevercis-acting regulatory elements that provide a transcriptionalresponse to oxidative stress or hypoxia, which are the hallmarks of manytumors. In particular, antioxidant response elements (ARE), the binding sitesfor the Nrf2 transcription factor, which is a master activator of the oxidativestress response, suffice to support tumor-specific transgene expression whenlinked to a basal promoter [Many promoters like hTERT show tumor-specific activity due to reactivation intumor cells, and the tumor specificity of transgene expression can be furtherincreased by exploiting genetic regulatory elements that respond to a perturbedtumor microenvironment or are abnormally active due to somatic mutations intumor cells. This strategy can be exemplified by exploitingpromoter . In suchIn this paper, we show that combining the tumor-specific hTERT promoter withARE results in increased activity of the hybrid promoter in tumor cellscompared to the hTERT promoter. At the same time, this modification did notaffect promoter activity in non-cancerous cells, in which Nrf2 is not activatedunder normal conditions. This approach can be used to increase the transgeneexpression level and activity of therapeutic proteins in tumor cells without anappreciable loss of tumor specificity.Cell cultureHuman lung epidermoid carcinoma Calu-1 (ECACC #93120818), nonsmall cell lungcarcinomas NCI-H1299 (ATCC #CRL-5803) and A549 (ATCC #CRL-185), and nonsmallcell lung bronchioalveolar carcinoma NCI-H358 (ATCC #CRL-5807) cell lines werecultured in a DMEM/F12 (1 : 1) medium  supplemented with 10%fetal bovine serum (HyClone), penicillin (100 U/ml), and streptomycin (100µg/ml) . Human bronchial epithelial cells HBEpC (ECACC #502-05)were cultured in the Bronchial Epithelial Growth Medium .For cell viability or luciferase reporter gene assays, cells were seeded into24-well plates at an indicated density  and transfected with a Unifectin-56 transfectionreagent  the next day.PlasmidshTERT promoter was described earlier[GCLM) gene promoter to the5’-end of the hTERT promoter in phTERT-Luc plasmid[The plasmid phTERT-Luc encoding firefly luciferase under the control of a-206…+37 nt  earlier. Plasmid plasmid. phTERT- plasmid. The SV4ChemicalsTert-butylhydroquinone (tBHQ), doxorubicin, cisplatin,etoposide, and 5-fluorocytosine (5FC) were purchased from Sigma-Aldrich (USA).Luciferase reporter gene assayRenilla luciferase reporter gene under the control of the CMVimmediate early enhancer/promoter). If indicated, cells were treated with 100µM tBHQ for 24 hrs prior to harvesting for luciferase activity analysis.Luciferase activities were quantified 2 days after transfection using theDual-Luciferase® Reporter Assay System (Promega). Firefly luciferaseactivity was normalized to the Renilla luciferase activity,and the average values of relative light units (RLU) and standard deviation(SD) were calculated.Cells were transfected in triplicate for each plasmid combination by a mixtureof the firefly reporter plasmid ) with pRL-CMV  plasmid . 5FC and/or etoposide, cisplatin, ordoxorubicin were added 24 hrs after plating. Cell culture medium containing 5FCand/or chemotherapeutic drugs was changed for a fresh one after 24 and 96 hrsof incubation. If indicated, tBHQ was present in the medium at a concentrationof 100 µM between 24 and 96 hrs of incubation. Cell viability wasdetermined after 120 hrs of incubation using the CellTiter96® AQueous OneSolution Cell Proliferation Assay (Promega) according to themanufacturer’s instruction. Each experimental point was analyzed intriplicate. Cell viabilities were normalized to the viability of cellsincubated in the absence of 5FC and chemotherapeutic drugs, which was taken as 100%.Design of the hybrid ARE-hTERT promoterGCLM gene promoter[GCLM gene promoter and placed it at the 5’-endrelative to the transcription start site of the -206…+37 nt fragment ofthe hTERT promoter, which is sufficient to support tumor-specific transcription[Hurttila et al. previously analyzed the potency of AREs derived from severalNrf2-responsive genes to support transgene expression under oxidative stressconditions and showed that the highest expression level among the tested AREswas provided by ARE from the promoter. Based oActivity of the hybrid ARE-hTERT promoter in cancer and normalcellsFig. 1 “-tBHQ” samples).Importantly, a similar lack of effect onthe transcriptional activity of the hybrid promoter was observed innoncancerous HBEpC cells: relative luciferase activities in pARE-hTERT-Luc- andphTERT-Luc-transfected cells were 1.41 ± 0.45 and 1.00 ± 0.214. Therefore, the ARE-hTERT promoter outperformed theunmodified hTERT promoter in three out of four tested lung cancer cell lines,while modification did not affect the promoter activity in normal cells.We used a luciferase reporter assay to compare the activities of theconventional and the hybrid hTERT promoters in cancer and normal cells. Inthree out of four tested lung cancer cell lines ,the ARE-hTERT promoter showed 2- to 3-fold higher activity compared to theunmodified hTERT promoter, while in NCI-H358 cell line the introduction of AREdid not significantly improve the promoter activity,which is well in line with the responsiveness of GCLM ARE toexternal oxidative stress [ARE-hTERT promoteractivity in Calu-1, A549, and NCI-H1299 cell lines in which ARE-hTERT promoteractivity significantly outperformed that of the unmodified hTERT promoterwithout an external oxidative stressor.Taken together, the hybrid ARE-hTERT promoter possesseshigher activity in cancer cells under basal conditions compared to the hTERTpromoter, likely owing to the activation of the Nrf2 pathway that frequentlyoccurs in cancer cells due to somatic mutations or the increased level ofreactive oxygen species (ROS)[Next, we studied if induction of oxidative stress could increase ARE-hTERTpromoter activity in cancer cells. Treatment of NCI-H358 cells with tBHQresulted in ~2.5-fold induction of luciferase reporter gene activity under thecontrol of the ARE-hTERT promoter, while having no effect on the activity of the hTERT promoter(e stress . At thees (ROS)-19.In cin vitroA hybrid promoter improves the efficiency of enzyme-prodrug suicidecancer gene therapy Fig. 2).Alike, in agreement with the results of a promoter activityanalysis with the reporter gene, ARE modification of the hTERT promoterdirecting CD : UPRT expression did not affect cytotoxicity for NCI-H358 cells.At the same time, simultaneous treatment of NCI-H358 cells with tBHQsignificantly augmented the cytotoxic effect only when the ARE-modifiedpromoter was used,while tBHQ did not increase cytotoxicity when the ARE-hTERT promoter was usedinstead of the unmodified promoter in NCI-H1299, Calu-1, and A549 cells, wherethe hybrid promoter is intrinsically more active than the hTERT promoteraccording to the reporter gene assay (data not shown).The increased activity of the hybrid promoter compared to the conventionalhTERT promoter observed in NCI-H1299, Calu-1, and A549 cells in luciferasereporter gene assay leads one to assume that the modification of the hTERTpromoter with ARE will also improve the performance of cancer gene therapyvectors. In order to directly address this issue, we compared the capacities toinduce cancer cell death in the enzyme-prodrug CD : UPRT–5FC suicidecancer gene therapy scheme when CD : UPRT expression was driven by either anunmodified or ARE-modified hTERT promoter. As expeARE-hTERT promoter-driven enzyme-prodrug suicide cancer gene therapy moreefficiently sensitizes cancer cells to conventional chemotherapeuticdrugs-hTERT promoter-driven CD : UPRT-5FCenzyme-prodrug suicide cancer gene therapy would also result in enhancedcytotoxicity when combined with chemotherapeuticdrugs. Figure 4A demonstratesthat hTERT-driven CD : UPRT expression did not result inNCI-H1299 cell death in the presence of 10 µM 5FC. Also, treatment with0.1 µM doxorubicin resulted only in marginal NCI-H1299 cell death.Notably, under the same settings, application of the ARE-modified hTERTpromoter resulted in substantial cell death (~40%), which was furthersignificantly potentiated by combined treatment with doxorubicin.Similar observations were made for A549 and Calu-1cells treated with doxorubicin, etoposide or cisplatin.Importantly, under our experimental settings, hTERT promoter-drivencancer gene therapy showed no effect alone and failed to potentiate the celldeath elicited by chemotherapeutic drugs, while modification of the promoterwith ARE resulted in pronounced cytotoxicity both in the case of cancer genemonotherapy and when it was combined with chemotherapeutic drugs.ARE-driven enzyme-prodrug suicide cancer gene therapy was reported to increasethe sensitivity of cancer cells to chemotherapeutic drugs: in particular, todoxorubicin . TherefoTERT gene promoter and AREderived from the human GCLM gene promoter showed betterperformance in 3 of 4 tested cancer cell lines both in reporter gene assay andin the CD : UPRT-5FC suicidal cancer gene therapy scheme. In NCI-H358 cells,where hTERT promoter modification did not affect the promoter activity , the activity of the hybridpromoter could be boosted by oxidative stress inducers such as tBHQ. It isimportant to mention that an analysis in primary epithelial HBEpC cells showeda lack of any appreciable increase in promoter activity after the inclusion ofARE in the promoter, thus demonstrating that the introduced modification didnot affect the cancer-cell specificity of transcription.We tested the hypothesis that introduction of ARE into a promoter withintrinsic tumor specificity, which is routinely used to target cancer cells incancer gene therapy, will enhance promoter activity without an appreciable lossof specificity toward tumor cells. Indeed, ARE linked to a nonselective minimalpromoter was previously shown to provide tumor-specific expression owing to anaberrantly activated Nrf2 transcription factor-19or inin vitro and in vivo, which iscaused by several factors, such as altered metabolism and inadequatevascularization [in vivo might furtherpotentiate the overall efficiency of the treatment through the promotion oftherapeutic transgene expression.Our results indicate that the novel hybrid promoter, while retaining a highcancer cell specificity, will outperform the conventional hTERT promoter in asubstantial proportion of tumors where Nrf2 is activated due to a somaticmutation. In addition, cancer cells are generally characterized by an increasedROS level both rization . In addiFig. 4),the hTERT-driven gene therapy failed to result in tumor cell elimination or toenhance the cytotoxic effect of chemotherapeutic drugs. However, under identicalconditions, modification of the hTERT promoter with ARE restored the cytotoxiceffect of the gene therapy and significantly potentiated the chemotherapeuticdrug-induced cytotoxicity. These results explicitly demonstrate that theapplication of a hybrid promoter, instead of the conventional hTERT promoter,would broaden the therapeutic efficiency of gene therapy, thus demonstratingthe advantages of the reported hybrid promoter.The efficiency of a cancer gene therapy is primarily determined by thetherapeutic transgene expression level, which should be high enough to elicit atherapeutic effect. In this work, we used the CD : UPRT-5FC enzyme-prodrugcancer gene therapy approach, in which the overall efficiency of the therapy isdetermined by the efficiency of plasmid delivery into cancer cells, promoteractivity, and cell sensitivity to the cytotoxic agent obtained from the prodrugconversion. These parameters will obviously vary for specific cell types,potentially resulting in a loss of treatment efficiency. Indeed, under theexperimental settings used(We have created a novel tumor-specific promoter that retains the tumorspecificity of the basal hTERT promoter but is characterized by an enhancedtranscriptional activity in cancer cells due to either abnormal Nrf2transcription factor activation or stimulation with ROS inducers. Owing to theabove characteristics, the ARE-hTERT hybrid promoter can be considered a betteralternative to the hTERT promoter in cancer gene therapy schemes. In addition,the combination of ARE with other tumor- or tissue-specific promoters used todevelop vectors for cancer gene therapy can be regarded as a way to improvetheir performance without an appreciable loss of specificity.


Expression analyses revealed that miR-466 is under-expressed in PCa compared to normal tissues. Reconstitution of miR-466 in metastatic PCa cell lines impaired their oncogenic functions such as cell proliferation, migration/invasion and induced cell cycle arrest, and apoptosis compared to control miRNA. Conversely, attenuation of miR-466 in normal prostate cells induced tumorigenic characteristics. miR-466 suppressed PCa growth and metastasis through direct targeting of bone-related transcription factor RUNX2. Overexpression of miR-466 caused a marked downregulation of integrated network of RUNX2 target genes such as osteopontin, osteocalcin, ANGPTs, MMP11 including Fyn, pAkt, FAK and vimentin that are known to be involved in migration, invasion, angiogenesis, EMT and metastasis. Xenograft models indicate that miR-466 inhibits primary orthotopic tumor growth and spontaneous metastasis to bone. Receiver operating curve and Kaplan–Meier analyses show that miR-466 expression can discriminate between malignant and normal prostate tissues; and can predict biochemical relapse. In conclusion, our data strongly suggests miR-466-mediated attenuation of RUNX2 as a novel therapeutic approach to regulate PCa growth, particularly metastasis to bone. This study is the first report documenting the anti-bone metastatic role and clinical significance of miR-466 in prostate cancer.MicroRNAs (miRNAs) have emerged as key players in cancer progression and metastatic initiation yet their importance in regulating prostate cancer (PCa) metastasis to bone has begun to be appreciated. We employed multimodal strategy based on in-house PCa clinical samples, publicly available TCGA cohorts, a panel of cell lines,  A number of transcription factors have been identified that play key roles in promoting oncogenesis, tumor growth, metastasis and tissue destruction.Prostate cancer is the second most leading cause of cancer related deaths among American men.αA) is a member of the RUNX family of genes. It is a lineage-specific transcription factor with crucial roles in both bone biology and carcinogenesis.5 High levels of RUNX2 in metastatic prostate cancer emphasize the significance of this master skeletal transcription factor in potentiating tumor cell progression and metastatic bone disease.8 The oncogenic potential of RUNX2 was first documented T-cell lymphoma.10 Since then, a number of independent studies have reported involvement of RUNX2 at early and late stages of tumor progression in various cancers.15 RUNX2 was found to be up-regulated and a predictor of metastasis in prostate cancer.16 Mechanistic studies indicated that RUNX2 accelerated prostate cancer aggressiveness through promotion of cadherin switching, invasion toward collagen I and Akt activation.16 In addition, attenuation of RUNX2 expression in a PC3 subline led to increased adhesion to fibronectin.7 Various reports have convincingly shown that the severity of prostate and mammary cancer cell osteolysis is correlated with levels of RUNX2 expression following injection of neoplastic cells in the medullary cavity of the tibia.14 These findings suggest that RUNX2 can imbue neoplastic cells with the capacity to breakdown the architecture of surrounding tissues, potentially releasing tumor supporting factors indicating that RUNX2 may facilitate a bone-mimetic program that helps cells adapt and thrive in this foreign environment. RUNX2 also directly induced genes associated with angiogenesis, invasiveness, metastasis and stimulated epithelial-to-mesenchymal transition of primary tumors.17 Taken together these studies provide convincing evidence for RUNX2 as a potentially important factor in prostate cancer development and metastasis. Targeting RUNX2 in prostate cancer could interrupt an integrated network of gene expression required to maintain tumor growth and bone metastasis. Here we report use of miR-466 as a potentially novel avenue to regulate RUNX2 and its downstream genes, thereby inhibiting prostate cancer growth and bone metastasis.Runt-domain containing protein RUNX2  although their role in disease pathogenesis is not well documented.MicroRNAs (miRNA) are small (20–25 nucleotides) evolutionarily conserved non-coding RNAs that negatively regulate transcript levels through sequence-dependent recognition mechanisms.in vitro. Importantly, miR-466 has a profound inhibitory effect on prostate cancer growth and bone metastasis in vivo. We further demonstrated that miR-466 partially exerted these effects by directly targeting runt-related RUNX2, a master osteogenic transcription factor and attenuated its downstream target genes that are known to mediate prostate cancer bone metastasis. In addition, our data indicates that miR-466 is potentially a clinically significant prostate cancer biomarker.Here for the first time we show that miR-466 inhibits prostate cancer bone metastasis. miR-466 is significantly under-expressed in a panel of prostate cancer cell lines and clinical tissues compared to normal. Restoration of miR-466 expression in highly metastatic PC3 and Du145 prostate cancer cell lines impaired proliferation, migration, invasion and induced cell cycle arrest and apoptosis 31 we performed preliminary screening to identify differentially expressed miRNAs in prostate cancer cell lines compared to a non-malignant cell line. A set of miRNAs, miR-466, -205, -203, -23b and -34b were found to be significantly downregulated in prostate cancer cells compared to a non-malignant cell line. We validated miR-466 data by miRNA-quantitative real-time PCR (miR qRT-PCR) analysis. The results confirmed that miR-466 was significantly downregulated in a panel of prostate cancer cell lines compared to normal RWPE1 cells  constitutively expressing cont-miR or miR-466 were orthotopically implanted into the posterior prostatic lobe of athymic nude mice  correlates with poor overall survival compared to the high miR-466 expression group  identified three complimentary miR-466 binding sites in the 3′UTR of RUNX2 , Osteocalcin (OC) and MMP11, were downregulated by miR-466 compared with control cells . These rTo assess the biological significance of miR-466 in prostate cancer; we performed parallel experiments using non-cancerous RWPE1 cells. miR-466 was knocked down in RWPE1 cells by transient transfection of anti-miR-466  along with a negative-anti-miRNA-control control; . RWPE1 cP<0.0001). In lower pathological stage (pT2) cases, decreased expression of miR-466 was observed in 75% of cases while 25% of cases showed high miR-466 expression . In the medium group (7), 83% of cases had low-miR-466 expression compared to 17% of cases with high miR-466 expression (P<0.0001). In the high group (>7), 83% of cases had low-miR-466 expression compared to 17% of cases with high miR-466 expression  compared to low-miR-466 expression (Receiver operating curve (ROC) analyses were performed to evaluate the ability of miR-466 expression to discriminate between normal and tumor tissues. An area under the ROC curve (AUC) of 0.915  and so on.35 Thus, therapeutic targeting of RUNX2 may interrupt an integrated network of gene expression that trigger bone metastases and thus deregulate cell survival and migration pathways of invading prostate cancer cells. However, at present transcription factors such as RUNX2 are not considered to be drug targets.33 Here, we show that miR-466-mediated attenuation of RUNX2 may be a novel therapeutic approach to regulate tumor growth and bone metastasis in prostate cancer. Indeed, reconstitution of miR-466 in metastatic prostate cancer cells significantly attenuated RUNX2 protein levels, suggesting a functional role of miR-466 in controlling RUNX2 protein translation.Bone metastases are a major problem in the evolution of prostate cancer. Considerable effort has been devoted to map the requirements of bone lesions in prostate tumors.36 In this study, we have identified miR-466 as a suppressor of prostate cancer growth and metastasis through direct targeting of the bone-related transcription factor RUNX2, which is difficult to target by conventional pharmacologic approaches.37 Our in vivo xenograft studies provide evidence that miR-466 can inhibit primary orthotopic tumor growth and spontaneous metastasis to bone. Expression analyses based on our in-house VAMC patient cohort or the publicly available TCGA data cohort revealed that miR-466 is under-expressed in prostate cancer compared to normal tissues. Conversely RUNX2 has been detected in human prostate cancer tissues but not in normal prostate.34 In this study, we have identified a tumor suppressor role for miR-466 in prostate cancer. Reconstitution of miR-466 in PC3 and Du145 metastatic prostate cancer cell lines inhibited in vitro oncogenic properties such as cell proliferation, migration and invasion compared to controls along with induction of G0/G1 cell cycle arrest and apoptosis in both cancer cell lines. MiRNAs control a wide range of biological functions and may act as tumor suppressors or oncogenes.22 Alteration of their expression plays a critical role in tumorigenesis and cancer progression.38 Our study corroborates other reports that show miRNAs are linked to the biologic activities of RUNX2 in different cancer types.37 Recent studies have demonstrated the potential of miRNAs in the intervention of breast cancer progression, metastatic bone disease and other bone pathologies.39 Interestingly, our study shows that the role of miR-466 in the control of prostate cancer progression and metastasis may be partially attributed to decreased cancer cell proliferation, cell cycle arrest and induced apoptosis, which features the beneficial effects of the miR-466 in regulating prostate cancer progression. However, our study highlights a direct role for miR-466 in reducing the bone metastatic potential of prostate cancer cells. This is evidenced by the fact that reconstitution of miR-466 caused a marked downregulation of RUNX2 target genes such as osteopontin, osteocalcin, ANGPT1, ANGPT4, MMP11 including Fyn, pAkt, FAK and vimentin that are involved in migration, invasion, angiogenesis, EMT and metastasis.35 Thus, miR-466 orchestrates the functional activities of RUNX2 by its convergent action on RUNX2 and its downstream target gene network to control prostate cancer bone metastasis. Furthermore, our study also defines the biological relevance of miR-466 in prostate cancer as indicated by induction of pro-cancerous characteristics in non-malignant RWPE1 cells after inhibition of endogenous expression of miR-466. Thus, our data strongly suggests a miR-466 mediated attenuation of RUNX2 may be a novel approach to prevent bone metastatic disease in prostate cancer  distinguish malignant from normal tissues indicating the powerful diagnostic potential of miR-466 in prostate cancer. In addition, miR-466 expression was predictive of recurrence-free survival (P<0.02) such that patients with higher miR-466 expression levels had better recurrence-free survival. Correlation analyses showed that low-miR-466 expression positively correlated with high pathological T (P=0.0001), Gleason grade (P<0.001) and PSA failure (P<0.0001). Therefore, our data reveals the clinical significance of miR-466 in prostate cancer, although it would benefit from a larger sample cohort.miRNAs have great potential as diagnostic and prognostic biomarkers.In conclusion, for the first time we provide evidence that: (i) miR-466 is an under-expressed metastasis suppressor miRNA in PCa; (ii) miR-466 is biologically relevant and has biomarker potential in PCa; (iii) miR-466 directly regulates RUNX2, a key regulator of bone metastasis; (iv) miR-466 overexpression interrupts RUNX2 integrated network of genes required to maintain PCa growth and bone metastasis. RUNX2, a key transcription factor, is of pivotal significance in the pathogenesis and progression of PCa bone metastases. Therefore, restoration of miR-466 to regulate an integrated network of RUNX2 and its target genes may represent a promising approach to suppress prostate cancer bone metastases.Human prostate cancer cell lines PC3, Du145, LNCaP, MDaPCa2b and a non-malignant cell line (RWPE1) were obtained from the American Type Culture Collection  and grown according to ATCC protocol. These human-derived cell lines were authenticated by DNA short-tandem repeat analysis by ATCC. The experiments with cell lines were performed within 6 months of their procurement/resuscitation. Plasmids pEZX-MT01 miRNA 3′UTR target expression clones for RUNX2 and miRNA Target clone control vector for pEZX-MT01 were purchased from GeneCopoeia . TaqMan probes including mimics, inhibitors and negative controls for hsa-miR-466 (miR-466) were purchased from Applied Biosystems .n=96; 48 pairs) were obtained from the Veterans Affairs Medical Center, San Francisco, CA, USA. A board certified pathologist identified the cancer and normal regions on H&E stained prostate cancer tissue slides. LCM was performed by using AutoPix System (Arcturus) and Arcturus CapSure Macro LCM Caps (Applied Biosystems) following the manufacturer's instructions to isolate pure epithelial cells from normal and cancer areas of prostate tissues. Total RNA was extracted from micro-dissected FFPE tissues or cultured cells using a miRNeasy FFPE Kit  or RNeasy mini kit (Qiagen), respectively, following the manufacturer's instructions. Total RNA was assayed for mature miRNAs using the TaqMan MicroRNA Assays in accordance with the manufacturer's instructions (Applied Biosystems). All RT reactions were run in a 7500 Fast Real Time PCR System (Applied Biosystems). Relative expression was calculated using comparative Ct. Expression of miR-466 was also validated by analyzing an independent prostate adenocarcinoma cohort publicly available at TCGA data portal now available as genomic data commons data portal .Tissue samples from radical prostectomy  according to the manufacturer's protocol. BioCoat control and Matrigel invasion chambers (BD Biosciences) and Cytoselect 24-well cell migration and invasion assay kits  were used for migration and invasion assays according to manufacturer's protocols. Fluorescence activated cell sorting (FACS) analysis was done 72 h post-transfection. The cells were harvested, washed with cold PBS, and resuspended in the nuclear stain DAPI for cell cycle analysis or stained with the 7-AAD and Annexin-V-FITC using ANNEXIN V-FITC/7-AAD KIT (BD Biosciences) for apoptosis analysis according to the manufacturer's protocol. Stained cells were immediately analyzed by FACS .6 cells) constitutively expressing cont-miR or miR-466 were implanted into the posterior prostatic lobe or into the left cardiac ventricle of athymic nude mice  followed by regular monitoring of tumor growth and metastatic dissemination with a live animal bioimaging system . There were ten mice in each group for orthotopic experiment, whereas 8 mice per group for intracardiac implantation. Bioluminescence signal intensity was quantified using the Living Image software (IVIS-PerkinElmer). Orthotopic experiments were performed at PerkinElmer research facility at San Jose, CA, USA.Bioluminescent PC3M-Luc-C6 cells  following the manufacturer's directions. Equal amounts of protein were resolved on 4–20% SDS polyacrylamide gels and transferred to nitrocellulose membrane. The resulting blots were blocked with 5% non-fat dry milk and probed with antibodies. All antibodies were obtained from Cell Signalling Technology Inc. Denver, MA, USA except RUNX2 and GAPDH which were purchased from Santa Cruz Biotech. Blots were visualized using Western blotting luminal reagent .Immunoblotting was performed as described previously.http://www.genecopoeia.com/. For reporter assays, cells were transiently transfected with wild-type or control reporter plasmids and miR-466 or negative miR (control). Firefly luciferase activities were measured using the Dual Luciferase Assay (Promega) 18 h after transfection and the results were normalized with Renilla luciferase. Each reporter plasmid was transfected at least three times (on different days) and each sample was assayed in triplicate.Complimentary miR-466 binding sites from two different miRNA databases  are shown in P-values <0.05 were considered statistically significant. ROC were calculated to determine the potential of miR-466 to discriminate between malignant and non-malignant samples. χ2 tests were performed to determine the correlation between miR-466 expression and the clinicopathological characteristics. For recurrence-free survival analysis Kaplan–Meier curves (log-rank tests) were also performed.Statistical analyses were performed with GraphPad Prism 5  and MedCalc version 10.3.2 . All quantified data represents an average of at least triplicate samples or as indicated. Error bars represent S.D.M. or as indicated. All tests were performed two tailed and


Prevention of mother-to-child HIV transmission (PMTCT) programs usually test pregnant women for HIV without involving their partners. Non-disclosure of maternal HIV status to male partners may deter utilization of PMTCT interventions since partners play a pivotal role in decision-making within the home including access to and utilization of health services.Mothers attending routine 6-week and 9-month infant immunizations were enrolled at 141 maternal and child health (MCH) clinics across Kenya from June–December 2013. The current analysis was restricted to mothers with known HIV status who had a current partner. Multivariate logistic regression models adjusted for marital status, relationship length and partner attendance at antenatal care (ANC) were used to determine correlates of HIV non-disclosure among HIV-uninfected and HIV-infected mothers, separately, and to evaluate the relationship of non-disclosure with uptake of PMTCT interventions. All analyses accounted for facility-level clustering,p < 0.001). HIV-uninfected mothers were more likely to not disclose their HIV status to male partners if they were unmarried , had low (≤KSH 5000) income , experienced intimate partner violence  and if their partner did not attend ANC . Among HIV-infected women, non-disclosure to male partners was less likely if women had salaried employment  and each increasing year of relationship length was associated with decreased likelihood of non-disclosure . HIV-infected women who did not disclose their HIV status to partners were less likely to uptake CD4 testing , to use antiretrovirals (ARVs) during labor , or give their infants ARVs .Overall, 2522 mothers  met inclusion criteria, 420 (17%) were HIV-infected. Non-disclosure of HIV results to partners was higher among HIV-infected than HIV-uninfected women (13% versus 3% respectively, HIV-infected women were less likely to disclose their status to partners than HIV-uninfected women. Non-disclosure was associated with lower use of PMTCT services. Facilitating maternal disclosure to male partners may enhance PMTCT uptake. Worldwide, HIV testing among pregnant women has increased substantially in the last decade . PregnanApproaches to promote male partner involvement such as reorganizing ANC clinic operations to offer male testing during evenings or weekends, fast tracking pregnant women who are accompanied by their male partners and written invitations to male partners to attend ANC have not consistently resulted in marked increase in number of men attending ANC clinics –8. AlterA systematic review in 2014 reported rates of disclosure of HIV status among pregnant and postpartum women in sub-Saharan African vary widely, ranging from 30.2 to 93.3% . HIV-uniThe methodology of the parent study has been described previously . Brieflyn = 30). Nine facilities in the former Nyanza were included in both surveys, thus a total of 141 facilities were sampled between both surveys. Facilities located in the North Eastern province were excluded due to security concerns and logistic feasibility.The PMTCT-MCH survey used probability proportionate to size sampling to randomly sample 120 facilities from among the 540 medium and large facilities across Kenya. The Nyanza Oversample survey included all large facilities in the former Nyanza province  with non-disclosure in univariate analysis. We decided a priori to adjust all multivariate models for marital status, relationship length and male partner attendance at ANC based on previous literature which identified relationship stability and partner engagement in care as predictors of disclosure  5.5) for HIV-infected mothers and 25.8 years (SD 5.5) for HIV uninfected mothers. Ninety-seven percent of both HIV-infected and HIV-uninfected mothers were married or cohabiting with their current parent; the mean relationship duration was 6.8 years (SD 5.2) and 5.3 years (SD 4.7) for HIV-infected and HIV-uninfected mothers, respectively. Among HIV-infected women, frequency of having no formal education was 8 and 9% had salaried employment; 7% of HIV-uninfected women had no formal education and 11% had salaried employment  and male partners of HIV-uninfected women had a mean age 31.2 years (SD 6.8). Few male partners of HIV-infected women (5%) and HIV-uninfected women (3%) had no education and frequency of unemployment among male partners was 13 and 12% for HIV-infected and HIV-uninfected mothers, respectively. Among mothers with male partners who had been tested for HIV, 71% of HIV-infected mothers and 1% of HIV-uninfected mothers had HIV-infected partners.p < 0.001).Overall, 125 of the 2522 (5%) mothers included in the analysis reported non-disclosure of HIV status to their partners. The proportion of HIV-infected women who did not disclose their status was significantly higher 13% (53/420) compared to 3% (72/2102) among HIV-uninfected women . HIV-uninfected mothers who experienced IPV were nearly 4 times as likely to not disclose as mothers who did not experience IPV . Having a partner who did not attend ANC was associated with a 4-fold higher likelihood of non-disclosure compared to having a partner who attended ANC among HIV-uninfected mothers . HIV-uninfected mothers who delivered in healthcare facilities were less likely to not disclose their HIV status .In multivariate analyses, the likelihood of non-disclosure was 4-fold higher among unmarried mothers compared to HIV-uninfected mothers who were married or cohabiting . Having salaried employed was associated with increased likelihood of non-disclosure compared to being unemployed among HIV-infected mothers . Compared to HIV-infected mothers whose partners attended ANC, those with partners who did not attend were nearly 3 times more likely to not disclose . HIV-infected mother whose partners had salaried employment were less likely to not disclose than women whose partners were unemployed .In multivariate models among HIV-infected women, each increasing year of relationship duration was associated with a decreased likelihood of non-disclosure of HIV status to male partners , less likely to use ARVs during birth  and less likely to give their infants ARVs  compared to mothers who disclosed. There were trends towards an association between non-disclosure and decreased likelihood of using HAART , using ARVs during breastfeeding  and receipt of infant PCR testing , though our statistically power was limited to detect associations.Among HIV-infected mothers, those who did not disclose their HIV status to male partners were less likely to not uptake CD4 testing  reported disclosing their HIV status to their male partners in this survey of 141 MCH clinics in Kenya, HIV-infected mothers less frequently reported disclosure than HIV-uninfected mothers. The non-disclosure rate of 13% among HIV-infected mothers that we observed, although markedly higher than among HIV-uninfected mothers in the same clinics, is lower than in previous studies in Kenya and sub-Saharan Africa , 24. TwoIn sub-Saharan Africa, most HIV infections are acquired through heterosexual partnerships . Women wConsistent with previous studies, we found that shorter duration of relationship and employment status were associated with non-disclosure of HIV-positive status, though our estimates for the association between employment and non-disclosure should be interpreted with caution due to imprecise estimations , 36. ConConsistent with other studies we found that women who were unmarried were more likely not to disclose , 36, 37.We found that partner ANC non-attendance was associated with non-disclosure among both HIV-infected and HIV-uninfected mothers. Partner ANC attendance provides an opportunity to counsel and educate men about HIV and PMTCT, offer couple HIV testing and facilitate disclosure of HIV test results. However, male ANC attendance remains low in sub-Saharan African . Low ANCHIV-uninfected mothers who reported frequent IPV were more likely not to disclose. Surprisingly, this association was not seen among HIV- infected women. A recent meta-analysis evaluating risk factors of IPV among pregnant women in Africa reported a positive association between HIV infection and IPV . A studyConsistent with previous studies, we found that HIV-infected women who had not disclosed their results were less likely to use PMTCT services . These fWe found that of the HIV-infected women who had not disclosed their status, approximately 20% reported HIV-uninfected partners who were at risk of HIV acquisition. A modeling study among adults in Zambia and Rwanda estimated that between 55·1% and 92·7% of new heterosexually acquired HIV infections occurred within serodiscordant marital or cohabiting relationships . Non-disThe study had several strengths and limitations. The study enrolled women from across Kenya, except for the former North Eastern Province, where access was limited due to security concerns. However, the region is sparsely populated limiting the impact of excluding this region. We enrolled both HIV-infected and uninfected women allowing for assessment of association of HIV status with non-disclosure. The relatively large sample size ensured that we had adequate power to assess correlates of non-disclosure. Small clinics were excluded and thus our estimates are not representative of small facilities. Oversampling women in Nyanza province allowed for an adequate number of HIV-infected women to assess correlates of non-disclosure among HIV-infected women. Limitations of the study included, recall bias in ascertainment of non-disclosure status, and reliance on self-report for HIV disclosure status, introducing the risk of social desirability bias. The study was not originally designed to primarily assess non-disclosure limiting the depth of our exploration of correlates. Finally, women were selected based on attendance at infant immunizations at participating health facilities, and thus this sample does not include the subset of potentially most at-risk women who do not attend MCH clinic.In conclusion, we found low rates of non-disclosure of maternal HIV status among all mothers, but higher among those who were HIV-infected. Non-disclosure among HIV-infected women was associated with reduced use PMTCT services. Promoting male partner antenatal clinic attendance may be useful to enhance disclosure and optimize PMTCT intervention adherence. There is need for novel strategies to facilitate use of PMTCT intervention by women reluctant to disclose their status.