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Key Points Summary The proto-oncogene c- MYC encodes a transcription factor that is implicated in various cellular processes ? cell growth, proliferation, loss of differentiation and apoptosis. c-MYC activates a variety of known target genes as part of a heterodimeric complex with the protein MAX. For example, cyclin D2 and CDK2 are essential for cell-cycle progression, and translation initiation factors eIF4 and eIF2 are important in cell growth. MYC?MAX heterodimers regulate gene activation through chromatin remodelling: association with co-activator TRRAP, which contains HAT activity, leads to acetylation of nucleosomal histones. c-MYC inhibits the differentiation of many cell types. Conversely, MAD/MXI1 transcription factors promote differentiation by antagonizing c-MYC function by forming dimers with MAX. MAD?MAX dimers recruit corepressors (such as SIN3) and HDACs to target DNA, leading to histone deacetylation and subsequent repression of MYC target genes. c-MYC sensitizes cells to a wide range of pro-apoptotic stimuli in vitro via cytochrome c release from mitochondria and subsequent formation of the apoptosome with APAF1 and procaspase-9. Oncogenic c-MYC implies constitutive or deregulated expression of c- MYC that is no longer dependent on external signals and is associated with many human cancers. Conditional transgenic mice, which allow regulated activation of c-MYC in distinct tissues (epidermis and pancreatic islets), have highlighted which cellular response attributed to c-MYC alone (proliferation or apoptosis) predominates in intact tissues in vivo . Regulatable c- MYC transgenic mouse models of cancer have highlighted oncogenic properties of c-MYC in vivo when its apoptotic pathway is blocked, which include induction of angiogenesis, loss of cell?cell contacts and local tissue invasion. Conditional mouse transgenic systems have ascertained when cancer-initiating oncogenic mutations (such as c- MYC and RAS ) remain essential for maintenance of the established tumour in vivo . These findings are important for the development of candidate drug molecules that are directed against the oncoprotein. Deregulated expression of c- MYC occurs in a broad range of human cancers and is often associated with poor prognosis, indicating a key role for this oncogene in tumour progression. However, as established human tumours often bear multiple genetic lesions, it is difficult to determine whether c-MYC is instrumental in the initiation/progression of the tumour, or indeed whether inactivating c-MYC would lead to tumour regression. Regulatable transgenic mouse models of oncogenesis have shed light on these issues and provide hope for effective cancer therapies.

Brucella melitensis is a facultative intracellular bacterium that causes brucellosis, the most prevalent zoonosis worldwide. The Brucella intracellular replicative niche in macrophages and dendritic cells thwarts immune surveillance and complicates both therapy and vaccine development. Currently, host-pathogen interactions supporting Brucella replication are poorly understood. Brucella fuses with the endoplasmic reticulum (ER) to replicate, resulting in dramatic restructuring of the ER. This ER disruption raises the possibility that Brucella provokes an ER stress response called the Unfolded Protein Response (UPR). In this study, B. melitensis infection up regulated expression of the UPR target genes BiP, CHOP, and ERdj4, and induced XBP1 mRNA splicing in murine macrophages. These data implicate activation of all 3 major signaling pathways of the UPR. Consistent with previous reports, XBP1 mRNA splicing was largely MyD88-dependent. However, up regulation of CHOP, and ERdj4 was completely MyD88 independent. Heat killed Brucella stimulated significantly less BiP, CHOP, and ERdj4 expression, but induced XBP1 splicing. Although a Brucella VirB mutant showed relatively intact UPR induction, a TcpB mutant had significantly compromised BiP, CHOP and ERdj4 expression. Purified TcpB, a protein recently identified to modulate microtubules in a manner similar to paclitaxel, also induced UPR target gene expression and resulted in dramatic restructuring of the ER. In contrast, infection with the TcpB mutant resulted in much less ER structural disruption. Finally, tauroursodeoxycholic acid, a pharmacologic chaperone that ameliorates the UPR, significantly impaired Brucella replication in macrophages. Together, these results suggest Brucella induces a UPR, via TcpB and potentially other factors, that enables its intracellular replication. Thus, the UPR may provide a novel therapeutic target for the treatment of brucellosis. These results also have implications for other intracellular bacteria that rely on host physiologic stress responses for replication.

Brucellosis, caused by a number of Brucella species, remains the most prevalent zoonotic disease worldwide. Brucella establish chronic infections within host macrophages despite triggering cytosolic innate immune sensors, including Stimulator of Interferon Genes (STING), which potentially limit infection. In this study, STING was required for control of chronic Brucella infection in vivo . However, early during infection, Brucella down-regulated STING mRNA and protein. Down-regulation occurred post-transcriptionally, required live bacteria, the Brucella type IV secretion system, and was independent of host IRE1-RNase activity. STING suppression occurred in MyD88 -/- macrophages and was not induced by Toll-like receptor agonists or purified Brucella lipopolysaccharide (LPS). Rather, Brucella induced a STING-targeting microRNA, miR-24-2, in a type IV secretion system-dependent manner. Furthermore, STING downregulation was inhibited by miR-24 anti-miRs and in Mirn23a locus-deficient macrophages. Failure to suppress STING expression in Mirn23a -/- macrophages correlated with diminished Brucella replication, and was rescued by exogenous miR-24. Mirn23a -/- mice were also more resistant to splenic colonization one week post infection. Anti-miR-24 potently suppressed replication in wild type, but much less in STING -/- macrophages, suggesting most of the impact of miR-24 induction on replication occurred via STING suppression. In summary, Brucella sabotages cytosolic surveillance by miR-24-dependent suppression of STING expression; post-STING activation “damage control” via targeted STING destruction may enable establishment of chronic infection.

Familial hypercholesterolemia (FH) is a common genetic cause of premature cardiovascular disease (CVD). The reported prevalence rates for both heterozygous FH (HeFH) and homozygous FH (HoFH) vary significantly, and this can be attributed, at least in part, to the variable diagnostic criteria used across different populations. Due to lack of consistent data, new global registries and unified guidelines are being formed, which are expected to advance current knowledge and improve the care of FH patients. This review presents a comprehensive overview of the pathophysiology, epidemiology, manifestations, and pharmacological treatment of FH, whilst summarizing the up-to-date relevant recommendations and guidelines. Ongoing research in FH seems promising and novel therapies are expected to be introduced in clinical practice in order to compliment or even substitute current treatment options, aiming for better lipid-lowering effects, fewer side effects, and improved clinical outcomes.

Tumor necrosis factor alpha-induced protein 3 (TNFAIP3) is an anti-inflammatory protein implicated in multiple autoimmune and rheumatologic conditions. We hypothesized that lower levels of TNFAIP3 contributes to excessive cytokine production in response to inflammatory stimuli in axial spondyloarthritis (AxSpA). A further aim was to determine the immune signaling and genetic variation regulating TNFAIP3 expression in individual subjects. Blood-derived macrophages from 50 AxSpA subjects and 30 healthy controls were assessed for TNFAIP3 expression. Cell lysates were also analyzed for NF-κB, mitogen-activated protein (MAP) kinase and STAT3 phosphorylation, and supernatants for cytokine production. Coding and regulatory regions in the gene and other auto-inflammation-implicated genes were sequenced by next-generation sequencing and variants identified. Mean TNFAIP3 was significantly lower in spondyloarthritis macrophages than controls (  = 0.0085). Spondyloarthritis subject macrophages correspondingly produced more TNF-α in response to lipopolysaccharide (LPS,  = 0.015). Subjects with the highest TNFAIP3 produced significantly less TNF-α in response to LPS (  = 0.0023). Within AxSpA subjects, those on TNF blockers or with shorter duration of disease expressed lower levels of TNFAIP3 (  = 0.0011 and 0.0030, respectively). TNFAIP3 expression correlated positively with phosphorylated IκBα, phosphorylated MAP kinases, and unstimulated phosphorylated STAT3, but negatively with LPS or TNF-α-stimulated fold induction of phosphorylated STAT3. Further, subjects with specific groups of variants within displayed differences in TNFAIP3 (  = 0.03-0.004). Nominal pQTL associations with genetic variants outside were identified. Our results suggest that both immune functional and genetic variations contribute to the regulation of TNFAIP3 levels in individual subjects. Decreased expression of TNFAIP3 in AxSpA macrophages correlated with increased LPS-induced TNF-α, and thus, TNFAIP3 dysregulation may be a contributor to excessive inflammatory responses in spondyloarthritis subjects.

Background: Atu027 is a liposomally formulated short interfering RNA with anti-metastatic activity, which silences the expression of protein kinase N3 (PKN3) in the vascular endothelium. This trial was designed to assess the safety, pharmacokinetics and efficacy of Atu027 in combination with gemcitabine in advanced pancreatic carcinoma (APC). Methods: In total, 23 patients (pts) with inoperable APC were randomly assigned to gemcitabine combined with two different Atu027 schedules (0.235 mg/kg once weekly vs. 0.235 mg/kg twice weekly). ClinicalTrials.gov Identifier: NCT01808638. Results: The treatment was well-tolerated. There were Grade 3 adverse events (AEs) in 9/11 pts (arm 1) and 11/12 pts (arm 2), while Grade 4 AEs were reported for two pts in each arm. The AEs were mainly laboratory abnormalities without clinical significance. The median progression-free survival reached statistical significance in patients who had metastatic disease (1.6 vs. 2.9 months, p = 0.025). Disease control during treatment was achieved in 4/11 pts (arm 1) and in 7/12 pts (arm 2). Pts in arm 1 experienced stable global health status while pts in arm 2 reported improvement. Conclusions: Combining Atu027 with gemcitabine is safe and well tolerated. In pts with metastatic APC, twice-weekly Atu027 is associated with significantly improved outcomes. Our clinical results support the significant involvement of the vascular endothelium in the spread of cancer, and thus the further investigation of its target role.

Brucella species are Gram-negative, intracellular pathogens which cause brucellosis, the most common zoonosis worldwide. Human brucellosis is a severely debilitating disease with a major socioeconomic impact. Brucellosis has eluded systematic attempts at eradication for more than a century in even the most developed countries. Currently, no human vaccine exists and vaccines used on animals present many difficulties. Brucella spp. invade many cell and tissue types including reproductive tissues, neurons, epithelial, and non-professional and professional phagocytes. The intracellular lifestyle of Brucella limits exposure to the host innate and adaptive immune responses and sequesters the organisms from the effects of antibiotics. The mechanisms by which Brucella subverts the host to enable chronic infection remain unclear. To better design future vaccines, further knowledge of these mechanisms is critical. The goal of this thesis was to elucidate mechanisms from both the host and the pathogen that contribute to the intracellular lifestyle of Brucella. In Chapter II, we describe how Brucella triggers the host Unfolded Protein Response, which supports its intracellular survival. In Chapter III, we describe the effects of the ubiquitous bacterial second messenger, cyclic-di-GMP, on Brucella virulence and host immune pathways. Finally, in Chapter IV, we describe the active evasion of innate immunity by Brucella through the up-regulation of microRNA 24. Taken together, these data identify novel host and bacterial mechanisms that contribute to the long-term survival of Brucella as well as provide novel targets for future vaccines and therapeutics.

Brucella melitensis is a facultative intracellular bacterium that causes brucellosis, the most prevalent zoonosis worldwide. The Brucella intracellular replicative niche in macrophages and dendritic cells thwarts immune surveillance and complicates both therapy and vaccine development. Currently, host-pathogen interactions supporting Brucella replication are poorly understood. Brucella fuses with the endoplasmic reticulum (ER) to replicate, resulting in dramatic restructuring of the ER. This ER disruption raises the possibility that Brucella provokes an ER stress response called the Unfolded Protein Response (UPR). In this study, B. melitensis infection up regulated expression of the UPR target genes BiP, CHOP, and ERdj4, and induced XBP1 mRNA splicing in murine macrophages. These data implicate activation of all 3 major signaling pathways of the UPR. Consistent with previous reports, XBP1 mRNA splicing was largely MyD88-dependent. However, up regulation of CHOP, and ERdj4 was completely MyD88 independent. Heat killed Brucella stimulated significantly less BiP, CHOP, and ERdj4 expression, but induced XBP1 splicing. Although a Brucella VirB mutant showed relatively intact UPR induction, a TcpB mutant had significantly compromised BiP, CHOP and ERdj4 expression. Purified TcpB, a protein recently identified to modulate microtubules in a manner similar to paclitaxel, also induced UPR target gene expression and resulted in dramatic restructuring of the ER. In contrast, infection with the TcpB mutant resulted in much less ER structural disruption. Finally, tauroursodeoxycholic acid, a pharmacologic chaperone that ameliorates the UPR, significantly impaired Brucella replication in macrophages. Together, these results suggest Brucella induces a UPR, via TcpB and potentially other factors, that enables its intracellular replication. Thus, the UPR may provide a novel therapeutic target for the treatment of brucellosis. These results also have implications for other intracellular bacteria that rely on host physiologic stress responses for replication.

Brucellosis, caused by Brucella bacteria species, remains the most prevalent zoonotic disease worldwide. Brucella establish chronic infections within host macrophages despite triggering cytosolic innate immune sensors, including Stimulator of Interferon Genes (STING), which potentially limit infection. In this study, STING was required for control of chronic Brucella infection in vivo. However, early during infection, Brucella down-regulated STING mRNA and protein. Down-regulation occurred post-transcriptionally, required live bacteria, the Brucella type IV secretion system, and was independent of host IRE1-RNase activity. Rather, Brucella induced a STING-targeting microRNA, miR-24-2. Furthermore, STING downregulation was inhibited by miR-24 anti-miRs and in mirn23a locus-deficient macrophages. Failure to suppress STING expression in mirn23a-/- macrophages correlated with diminished Brucella replication, and was rescued by exogenous miR-24. Anti-miR-24 potently suppressed replication in wild type, but much less in STING-/- macrophages, suggesting most of the impact of miR-24 induction on replication occurred via STING suppression. In summary, Brucella sabotages innate immunity by miR-24-dependent suppression of STING expression; post-STING activation damage control via targeted STING destruction may enable establishment of chronic infection.

Brucella spp are intracellular pathogenic bacteria remarkable in their ability to escape immune surveillance and therefore inflict a state of chronic disease within the host. To enable further immune response studies, Brucella were engineered to express the well characterized chicken ovalbumin (OVA). Surprisingly, we found that CD8 T cells bearing T cell receptors (TCR) nominally specific for the OVA peptide SIINFEKL (OT-1) reacted to parental Brucella-infected targets as well as OVA-expressing Brucella variants in cytotoxicity assays. Furthermore, splenocytes from Brucella immunized mice produced IFN- and exhibited cytotoxicity in response to SIINFEKL-pulsed target cells. To determine if the SIINFEKL-reactive OT-1 TCR could be cross-reacting to Brucella peptides, we searched the Brucella proteome using an algorithm to generate a list of near-neighbor nonamer peptides that would bind to H2Kb. Selecting five Brucella peptide candidates, along with controls, we verified that several of these peptides mimicked SIINFEKL resulting in T cell activation through the \"SIINFEKL-specific\" TCR. Activation was dependent on peptide concentration as well as sequence. Our results underscore the complexity and ubiquity of cross-reactivity in T cell recognition. This cross-reactivity may enable microbes such as Brucella to escape immune surveillance by presenting peptides similar to the host, and may also lead to the activation of autoreactive T cells.