Cleverballoon: An integrated approach for developing a drug-coated balloon with everolimus.

Coronary artery disease (CAD) is a chronic disease associated with high mortality and morbidity. Although treatment with drug-eluting stents is the most frequent interventional approach for coronary artery disease, drug-coated balloons (DCBs) constitute an innovative alternative, especially in the presence of certain anatomical conditions in the local coronary vasculature. DCBs allow the fast and homogenous transfer of drugs into the arterial wall, during the balloon inflation. Their use has been established for treating in-stent restenosis caused by stent implantation, while recent clinical trials have shown a satisfactory efficacy in de novo small-vessel disease. Several factors affect DCBs performance including the catheter design, the drug dose and formulation. Cleverballoon focuses on the design and development of an innovative DCB with everolimus. For the realization of the development of this new DCB, an integrated approach, including in- vivo, in-vitro studies and in-silico modelling towards the DCB optimization, is presented.Clinical Relevance-The proposed study introduces the integration of in- vivo, in-vitro and in silico approaches in the design and development process of a new DCB, following the principles of 3R's for the replacement, reduction, and refinement of animal and clinical studies.

Safety and Efficacy of an Innovative Everolimus-Coated Balloon in a Swine Coronary Artery Model.

BACKGROUND: Drug-coated balloons have been used as a non-stenting treatment in coronary and peripheral artery disease. Until recently, only sirolimus- and paclitaxel-coated balloons have been investigated in clinical trials. We evaluated the safety and efficacy of an innovative everolimus-coated balloon (ECB) in a swine coronary artery model. METHODS: thirty-two swine coronary arteries were prepared through dilatation with a non-coated angioplasty balloon in a closed-chest model. During a period of 90 days, the following four groups (four animals per group, two coronary arteries per animal) were compared for safety and efficacy: A, Rontis ECB with 2.5 µg/mm2 of drug per balloon surface; B, Rontis ECB with 7.5 µg/mm2; C, Rontis Europa Ultra bare balloon; and D, Magic Touch, Concept Medical, sirolimus-coated balloon with a drug load of 1.3 µg/mm2. RESULTS: Differences in local biological effects (arterial reaction scores) and surface of intimal area (mm2) were not statistically significant between the treatment groups. Numerically, group A showed the lowest intimal area and intimal mean thickness, while group B showed the lowest stenosis among all groups. CONCLUSIONS: ECB was safe and effective in a porcine coronary artery model. The dose of everolimus may play a role in the biocompatibility of the balloon.

Histopathological evaluation of a retinoic acid eluting stent in a rabbit iliac artery model.

This study aimed to evaluate the safety and efficacy of innovative retinoic acid (RA) eluting stents with bioabsorbable polymer. Sixty stents divided in ten groups were implanted in the iliac arteries of 30 rabbits. Two polymers ("A", poly (lactic-co-glycolic acid) and "B", polylactic acid), and three doses ("Low", "Medium" and "High") of RA (groups: AL, AM, AH, BL, BM, BH) were used on cobalt chromium stents (Rontis Corporation), one group of bare stent (C), one group (D) of Everolimus eluting stent (Xience-Pro, Abbot Vascular), and two groups of Rontis Everolimus eluting stents coated with polymer A (EA) and B (EB). Treated arteries were explanted after 4 weeks, processed by methyl methacrylate resin and evaluated by histopathology. None of the implanted stents was related with thrombus formation or extensive inflammation. Image analysis showed limited differences between groups regarding area stenosis (BH, D and EB groups had the lower values). Group BH had lower intimal mean thickness than AH (105.1 vs 75.3 µm, p = 0.024). Stents eluting RA, a non-cytotoxic drug, were not related with thrombus formation and had an acceptable degree of stenosis 4 weeks post implantation. RA dose and type of polymer may play role in the biocompatibility of the stents.

Screening for TSC1 and TSC2 mutations using NGS in Greek children with tuberous sclerosis syndrome.

Tuberous Sclerosis Complex (TSC) is a rare neurocutaneous syndrome inherited by an autosomal dominant manner. The disorder is commonly manifested by the presence of multiple benign tumors located in numerous tissues, including the brain, heart, skin and kidneys. Seizures, autism, developmental and behavioral delay, as well as non-neurological phenotypic findings, are suggestive of TSC. The identification of one pathogenic mutation in either the TSC1 or TSC2 genes is considered to be an independent diagnostic criterion. In our study, seventeen Greek patients, 2yo on average, were analyzed for the presence of pathogenic germline mutations in the aforementioned loci by Next-Generation Sequencing. A TSC1/2 gene panel was designed for the molecular diagnosis of the disease. Patients underwent initial diagnosis based on their clinical symptoms, most frequently involving the presence of skin lesions and/or epilepsy. Only one case was familial. Sixteen different genetic alterations were identified in TSC1 and TSC2 genes in fifteen patients, giving a 88% detection rate by employing NGS technology. Overall, most pathogenic mutations (11/15) identified were located in the TSC2 gene with exon 41 being the most frequent. With respect to genotype-phenotype association, no patient TSC1 (+) developed SEGA or renal cysts. No significant differences were observed between different types of TSC2 mutations and any clinical feature. Sequencing also revealed 18 different SNPs across the TSC1 and 20 across the TSC2 genes. This is the first registry of the genetic profile of TSC patients in Greece using a custom-made gene panel as molecular diagnostic tool.

Inherited mutations in 17 breast cancer susceptibility genes among a large triple-negative breast cancer cohort unselected for family history of breast cancer.

PURPOSE: Recent advances in DNA sequencing have led to the development of breast cancer susceptibility gene panels for germline genetic testing of patients. We assessed the frequency of mutations in 17 predisposition genes, including BRCA1 and BRCA2, in a large cohort of patients with triple-negative breast cancer (TNBC) unselected for family history of breast or ovarian cancer to determine the utility of germline genetic testing for those with TNBC. PATIENTS AND METHODS: Patients with TNBC (N = 1,824) unselected for family history of breast or ovarian cancer were recruited through 12 studies, and germline DNA was sequenced to identify mutations. RESULTS: Deleterious mutations were identified in 14.6% of all patients. Of these, 11.2% had mutations in the BRCA1 (8.5%) and BRCA2 (2.7%) genes. Deleterious mutations in 15 other predisposition genes were detected in 3.7% of patients, with the majority observed in genes involved in homologous recombination, including PALB2 (1.2%) and BARD1, RAD51D, RAD51C, and BRIP1 (0.3% to 0.5%). Patients with TNBC with mutations were diagnosed at an earlier age (P < .001) and had higher-grade tumors (P = .01) than those without mutations. CONCLUSION: Deleterious mutations in predisposition genes are present at high frequency in patients with TNBC unselected for family history of cancer. Mutation prevalence estimates suggest that patients with TNBC, regardless of age at diagnosis or family history of cancer, should be considered for germline genetic testing of BRCA1 and BRCA2. Although mutations in other predisposition genes are observed among patients with TNBC, better cancer risk estimates are needed before these mutations are used for clinical risk assessment in relatives.

Genome-wide association study identifies 25 known breast cancer susceptibility loci as risk factors for triple-negative breast cancer.

Triple-negative (TN) breast cancer is an aggressive subtype of breast cancer associated with a unique set of epidemiologic and genetic risk factors. We conducted a two-stage genome-wide association study of TN breast cancer (stage 1: 1529 TN cases, 3399 controls; stage 2: 2148 cases, 1309 controls) to identify loci that influence TN breast cancer risk. Variants in the 19p13.1 and PTHLH loci showed genome-wide significant associations (P < 5 × 10(-) (8)) in stage 1 and 2 combined. Results also suggested a substantial enrichment of significantly associated variants among the single nucleotide polymorphisms (SNPs) analyzed in stage 2. Variants from 25 of 74 known breast cancer susceptibility loci were also associated with risk of TN breast cancer (P < 0.05). Associations with TN breast cancer were confirmed for 10 loci (LGR6, MDM4, CASP8, 2q35, 2p24.1, TERT-rs10069690, ESR1, TOX3, 19p13.1, RALY), and we identified associations with TN breast cancer for 15 additional breast cancer loci (P < 0.05: PEX14, 2q24.1, 2q31.1, ADAM29, EBF1, TCF7L2, 11q13.1, 11q24.3, 12p13.1, PTHLH, NTN4, 12q24, BRCA2, RAD51L1-rs2588809, MKL1). Further, two SNPs independent of previously reported signals in ESR1 [rs12525163 odds ratio (OR) = 1.15, P = 4.9 × 10(-) (4)] and 19p13.1 (rs1864112 OR = 0.84, P = 1.8 × 10(-) (9)) were associated with TN breast cancer. A polygenic risk score (PRS) for TN breast cancer based on known breast cancer risk variants showed a 4-fold difference in risk between the highest and lowest PRS quintiles (OR = 4.03, 95% confidence interval 3.46-4.70, P = 4.8 × 10(-) (69)). This translates to an absolute risk for TN breast cancer ranging from 0.8% to 3.4%, suggesting that genetic variation may be used for TN breast cancer risk prediction.

Review: Diagnostic and therapeutic applications of rat basophilic leukemia cells.

The research into understanding of the immunological processes is often difficult due to several factors complicating the isolation and culturing of primary degranulating cells like mast cells and basophils. The establishment of rat basophilic leukemia (RBL) cell line as an efficient and reliable experimental research tool was considered a major advance toward the understanding of the wild-type mast cell population's biology. The development of sub-clone RBL-IV (HR+) led to the isolation of histamine-secreting RBL-2H3 cell line. Since then, RBL-2H3 cells have been extensively used for studying the IgE high affinity receptor (FcɛRI) interactions with their ligand, the IgE antibody. This cell line has been employed for generating human and more recently canine and equine FcɛRIα-transfected RBL cell lines facilitating an assessment of the residues involved in the complementary interaction between the IgE molecules from these species and their cognate high affinity receptor. A proteomics-based approach to the definition of IgE-receptor-mediated signaling pathways was also carried out using this cell line. Furthermore, RBL-2H3 cells have the potential of being used to assess the potential allergenicity of antigens to humans and other animals like dogs and horses which are known to suffer from similar allergic manifestations.

Identification of two novel mutations in the RET proto-oncogene in the same family.

BACKGROUND: Activating germline mutations of the RET gene cause multiple endocrine neoplasia type 2 and familial medullary thyroid carcinoma (FMTC), conditions that are inherited in an autosomal dominant manner. In addition, somatic RET mutations have been identified in a variable proportion (about 30-70%) of sporadic (nonfamilial) MTC cases. METHODS: We describe a Greek family with two novel likely pathogenic sequence variants of the RET gene. The first is a C to T transition at position 2458 (c.2458C>T) that causes an arginine to cysteine substitution (p.R820C) in exon 14 in the intracellular region of the kinase. This sequence variant was identified in an apparently healthy woman who had a recently deceased sister with confirmed aggressive MTC (age of onset 37 years). To assess the pathogenicity of this novel missense sequence variant, screening was performed on all available relatives: her two sons, the mother, and a second sister, including an MTC tumor sample from the deceased sister of the proband. At the time of the investigation, no clinical symptoms suggestive of multiple endocrine neoplasia type 2 or MTC were present in any of the individuals screened. RESULTS: The c.2458C>T transition was found in one son, the living sister, and the mother. Interestingly, it was not present in the tumor sample from the deceased sister. Instead, an in-frame deletion of 54 nt in exon 10 resulting in a protein missing 18 amino acids from I590 to G608 (c.1766_1819del 54) was found. Both genetic alterations were present in heterozygous state. CONCLUSIONS: These data suggest that the novel in-frame deletion was the disease-causing mutation in the deceased sister. The effect of the 2458C>T mutation on the activity of the kinase is under investigation.

Increased atherosclerotic lesions and Th17 in interleukin-18 deficient apolipoprotein E-knockout mice fed high-fat diet.

Recent reports show T helper 17 (Th17) cells are involved in the pathogenesis of various chronic inflammatory diseases formerly categorized as Th1-mediated disorders. Interleukin-18 (IL-18) induces Th1 cells to produce interferon-gamma (IFN-gamma) which is proatherogenic, while cholesterol causes atherosclerosis and stimulates intact rat aortae to produce prostaglandin E(2) (PGE(2)), a strong regulator of IL-23 that expands Th17. We wanted to test whether Th17 is proatherogenic and whether cholesterol can induce the alternative Th17 pathway in IL-18 deficient apolipoprotein E-knockout (ApoE(-/-)) mice that have reduced Th1 cells, if they are fed high-cholesterol diet. IL-18(+/+)ApoE(-/-) and IL-18(-/-)ApoE(-/-) mice aged 5 weeks were fed high-cholesterol diet (HCD) and control littermates of IL-18(-/-)ApoE(-/-) low-cholesterol diet (LCD) for 12 weeks. At termination, cryosectioned aortic arches were stained for lesion measurement and immunohistochemistry. We found that serum cholesterol and triglyceride levels were significantly higher in IL-18(-/-)ApoE(-/-) mice on HCD and they also had significantly increased atherosclerosis compared with 18(+/+)ApoE(-/-) mice or IL-18(-/-)ApoE(-/-) mice on LCD. Increased atherosclerosis correlates with enhanced Th17-cells, IL-23-producing vascular smooth muscle cells (VSMC) and macrophages, and thin fibrous cap in lesions, the morphology indicative of unstable plaques prone to rupture. In vitro, cholesterol significantly enhances VSMCs explanted from IL-18(-/-)ApoE(-/-) but not IL-18(+/+)ApoE(-/-) aorta to produce IL-23 and homocysteine mediates secretion. This study suggests that in IL-18 deficiency, cholesterol in HCD synergize mechanistically with homocysteine to accelerate atherosclerosis via the alternative IL-23/Th17 pathway, demonstrating a new role for Th17 in atherosclerosis.

Generation of canine-human Fc IgE chimeric antibodies for the determination of the canine IgE domain of interaction with Fc epsilon RI alpha.

Identification of the domain(s) of canine IgE that interact with Fc epsilon RI alpha may lead to novel therapeutic intervention strategies that inhibit the ability of canine IgE to engage Fc epsilon RI alpha. A panel of canine-human Fc IgE chimeric antibodies was constructed to investigate this interaction by replacing canine IgE-Fc domains with the corresponding human IgE-Fc domains since human IgE-Fc does not recognize canine Fc epsilon RI alpha. beta-Hexosaminidase release assays were performed to assess the ability of the chimeric antibodies to bind to and sensitize a novel RBL cell line transfected with canine Fc epsilon RI alpha for antigen induced mediator release. Replacing canine C epsilon2 with human C epsilon2 resulted in similar levels of release as those elicited by canine Fc IgE from RBL-2H3 cells transfected with either canine Fc epsilon RI alpha or human Fc epsilon RI alpha. Substitution of canine C epsilon4 with human C epsilon4 resulted in approximately 10% lower levels of release compared to cells sensitized with canine Fc IgE. Receptor binding by flow cytometry and cell activation could not be detected when transfected RBL cells were incubated with chimeric constructs where canine C epsilon2 and C epsilon4 were substituted with human C epsilon2 and C epsilon4. However, when this construct was incubated with cognate antigen prior to cell challenge mediator release was observed, albeit at a 20% lower level, indicating that while canine C epsilon3 is the only domain essential for binding to canine or human Fc epsilon RI alpha, species specific residues in canine Cepsilon2 and C epsilon4 inhibit dissociation of the ligand from the receptor.