Safety, Efficacy and Pharcacokinetics of Targeted Therapy with The Liposomal RNA Interference Therapeutic Atu027 Combined with Gemcitabine in Patients with Pancreatic Adenocarcinoma. A Randomized Phase Ib/IIa Study.

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 suppress STING expression via miR-24 to enhance infection.

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: New Horizons for Diagnosis and Effective Management.

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.

Brucella Peptide Cross-Reactive Major Histocompatibility Complex Class I Presentation Activates SIINFEKL-Specific T Cell Receptor-Expressing T Cells.

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 was 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 gamma interferon (IFN-γ) 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 those of the host and may also lead to the activation of autoreactive T cells.

Genetic and Functional Associations with Decreased Anti-inflammatory Tumor Necrosis Factor Alpha Induced Protein 3 in Macrophages from Subjects with Axial Spondyloarthritis.

OBJECTIVE: 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. METHODS: 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 TNFAIP3 gene and other auto-inflammation-implicated genes were sequenced by next-generation sequencing and variants identified. RESULTS: Mean TNFAIP3 was significantly lower in spondyloarthritis macrophages than controls (p = 0.0085). Spondyloarthritis subject macrophages correspondingly produced more TNF-α in response to lipopolysaccharide (LPS, p = 0.015). Subjects with the highest TNFAIP3 produced significantly less TNF-α in response to LPS (p = 0.0023). Within AxSpA subjects, those on TNF blockers or with shorter duration of disease expressed lower levels of TNFAIP3 (p = 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 TNFAIP3 displayed differences in TNFAIP3 (p = 0.03-0.004). Nominal pQTL associations with genetic variants outside TNFAIP3 were identified. CONCLUSION: 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.

The Bacterial Second Messenger Cyclic di-GMP Regulates Brucella Pathogenesis and Leads to Altered Host Immune Response.

Brucella species are facultative intracellular bacteria that cause brucellosis, a chronic debilitating disease significantly impacting global health and prosperity. Much remains to be learned about how Brucella spp. succeed in sabotaging immune host cells and how Brucella spp. respond to environmental challenges. Multiple types of bacteria employ the prokaryotic second messenger cyclic di-GMP (c-di-GMP) to coordinate responses to shifting environments. To determine the role of c-di-GMP in Brucella physiology and in shaping host-Brucella interactions, we utilized c-di-GMP regulatory enzyme deletion mutants. Our results show that a ΔbpdA phosphodiesterase mutant producing excess c-di-GMP displays marked attenuation in vitro and in vivo during later infections. Although c-di-GMP is known to stimulate the innate sensor STING, surprisingly, the ΔbpdA mutant induced a weaker host immune response than did wild-type Brucella or the low-c-di-GMP guanylate cyclase ΔcgsB mutant. Proteomics analysis revealed that c-di-GMP regulates several processes critical for virulence, including cell wall and biofilm formation, nutrient acquisition, and the type IV secretion system. Finally, ΔbpdA mutants exhibited altered morphology and were hypersensitive to nutrient-limiting conditions. In summary, our results indicate a vital role for c-di-GMP in allowing Brucella to successfully navigate stressful and shifting environments to establish intracellular infection.

Molecular testing for familial hypercholesterolaemia-associated mutations in a UK-based cohort: development of an NGS-based method and comparison with multiplex polymerase chain reaction and oligonucleotide arrays.

Background Detection of disease-associated mutations in patients with familial hypercholesterolaemia is crucial for early interventions to reduce risk of cardiovascular disease. Screening for these mutations represents a methodological challenge since more than 1200 different causal mutations in the low-density lipoprotein receptor has been identified. A number of methodological approaches have been developed for screening by clinical diagnostic laboratories. Methods Using primers targeting, the low-density lipoprotein receptor, apolipoprotein B, and proprotein convertase subtilisin/kexin type 9, we developed a novel Ion Torrent-based targeted re-sequencing method. We validated this in a West Midlands-UK small cohort of 58 patients screened in parallel with other mutation-targeting methods, such as multiplex polymerase chain reaction (Elucigene FH20), oligonucleotide arrays (Randox familial hypercholesterolaemia array) or the Illumina next-generation sequencing platform. Results In this small cohort, the next-generation sequencing method achieved excellent analytical performance characteristics and showed 100% and 89% concordance with the Randox array and the Elucigene FH20 assay. Investigation of the discrepant results identified two cases of mutation misclassification of the Elucigene FH20 multiplex polymerase chain reaction assay. A number of novel mutations not previously reported were also identified by the next-generation sequencing method. Conclusions Ion Torrent-based next-generation sequencing can deliver a suitable alternative for the molecular investigation of familial hypercholesterolaemia patients, especially when comprehensive mutation screening for rare or unknown mutations is required.

Cascade Screening for Familial Hypercholesterolemia: PCR Methods with Melting-Curve Genotyping for the Targeted Molecular Detection of Apolipoprotein B and LDL Receptor Gene Mutations to Identify Affected Relatives.

BACKGROUND: A key objective of the UK National Institute for Health and Care Excellence (NICE) pathway for diagnosis of familial hypercholesterolemia (FH) is the identification of affected relatives of index cases through cascade screening. At present, there is no systematic appraisal of available methodological options to identify the appropriate diagnostic testing protocol that would allow cost-effective cascade genetic screening. The majority of FH-causing mutations identified in the LDL receptor (LDLR) or apolipoprotein B (APOB) genes are single-nucleotide changes. This pattern of mutations suggests that PCR methods using melting curve-based genotyping might offer a convenient methodological approach for screening relatives. METHODS: We developed and validated one-tube PCR methods for the mutations APOB c.10580G>A (p.Arg3527Gln), LDLR c.1474G>A (p.Asp492Asn), and c.2054C>T (p.Pro685Leu) and 3 novel LDLR mutations identified in the Coventry and Warwickshire population: LDLR c.1567G>C (p.Val523Leu), c.487dupC (p.Gln163Profs17), and c.647G>C (p.Cys216Ser). RESULTS: These methods successfully amplified target sequence from genomic DNA extracted from either peripheral blood or saliva. They also demonstrated acceptable analytical performance characteristics (specificity of amplification, repeatability, and reproducibility) over a wide range of DNA concentrations and purity. This approach was used for cascade testing of relatives of index FH cases with confirmed mutations and identified family members with high plasma LDL cholesterol as heterozygous for disruptive alleles. CONCLUSIONS: Our study generates proof-of-concept evidence of methods suitable for detecting single nucleotide substitutions and insertions that can deliver reliable, easy, low-cost, and rapid family screening of FH patients and can be adopted by nonspecialist molecular diagnostic laboratories.

CD8+ T cell exhaustion, suppressed gamma interferon production, and delayed memory response induced by chronic Brucella melitensis infection.

Brucella melitensis is a well-adapted zoonotic pathogen considered a scourge of mankind since recorded history. In some cases, initial infection leads to chronic and reactivating brucellosis, incurring significant morbidity and economic loss. The mechanism by which B. melitensis subverts adaptive immunological memory is poorly understood. Previous work has shown that Brucella-specific CD8(+) T cells express gamma interferon (IFN-γ) and can transition to long-lived memory cells but are not polyfunctional. In this study, chronic infection of mice with B. melitensis led to CD8(+) T cell exhaustion, manifested by programmed cell death 1 (PD-1) and lymphocyte activation gene 3 (LAG-3) expression and a lack of IFN-γ production. The B. melitensis-specific CD8(+) T cells that produced IFN-γ expressed less IFN-γ per cell than did CD8(+) cells from uninfected mice. Both memory precursor (CD8(+) LFA1(HI) CD127(HI) KLRG1(LO)) and long-lived memory (CD8(+) CD27(HI) CD127(HI) KLRG1(LO)) cells were identified during chronic infection. Interestingly, after adoptive transfer, mice receiving cells from chronically infected animals were able to contain infection more rapidly than recipients of cells from acutely infected or uninfected donors, although the proportions of exhausted CD8(+) T cells increased after adoptive transfer in both challenged and unchallenged recipients. CD8(+) T cells of challenged recipients initially retained the stunted IFN-γ production found prior to transfer, and cells from acutely infected mice were never seen to transition to either memory subset at all time points tested, up to 30 days post-primary infection, suggesting a delay in the generation of memory. Here we have identified defects in Brucella-responsive CD8(+) T cells that allow chronic persistence of infection.

Brucella induces an unfolded protein response via TcpB that supports intracellular replication in macrophages.

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.

The c-MYC oncoprotein as a treatment target in cancer and other disorders of cell growth.

The c-MYC proto-oncogene is essential for cellular proliferation but, paradoxically, may also promote cell death. Deregulated expression of c-MYC is present in most, if not all, human cancers, and is associated with a poor prognosis. However, given that human tumours at diagnosis generally carry multiple genetic lesions that have accumulated during (although they are not necessarily essential for) tumour progression, it has proved difficult to attribute a specific role to any given single factor or indeed to explore the therapeutic potential of selectively mitigating their biological functions. Regulatable transgenic mouse models of oncogenesis have shed light on these issues, influenced our thinking about cancer and provided encouragement for the future development of cancer therapies based on targeting individual oncogenes such as c-MYC. Although still in its infancy, encouraging results have been reported using antisense oligodeoxynucleotide-based methods, as well as other approaches to interfere with MYC expression both in vitro and in vivo.

The many faces of c-MYC.

The proto-oncogene c-MYC is implicated in various physiological processes-cell growth, proliferation, loss of differentiation, and cell death (apoptosis). Oncogenic c-MYC implies constitutive or deregulated expression of c-MYC and is associated with many human cancers often with poor prognosis. Recently, c-MYC has been implicated in the loss and dysfunction of insulin-producing beta cells in diabetes. Intriguingly, this raises the possibility that c-Myc may be a key contributor to disease, not only by deregulating cell proliferation, which is well established, but also by virtue of its opposing role in engendering apoptosis. However, given the fact that human diseases at diagnosis are generally advanced and pathologically complex, it is generally difficult to attribute a specific pathogenic role to c-MYC, or indeed any given single factor, or to assess the potential of therapies targeting individual such factors. Regulatable transgenic mouse models have shed light on these issues, have influenced our thinking about cancer, and have provided encouragement for the future development of cancer therapies based on targeting individual oncogenes such as c-MYC. Although still in its infancy, encouraging results have been reported for several approaches using gene targeting to interfere with c-MYC expression or activity both in vitro and in vivo.

c-MYC: more than just a matter of life and death.

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.