Comparative vascular responses three months after paclitaxel and everolimus-eluting stent implantation in streptozotocin-induced diabetic porcine coronary arteries.

BACKGROUND: Diabetes remains a significant risk factor for restenosis/thrombosis following stenting. Although vascular healing responses following drug-eluting stent (DES) treatment have been characterized previously in healthy animals, comparative assessments of different DES in a large animal model with isolated features of diabetes remains limited. We aimed to comparatively assess the vascular response to paclitaxel-eluting (PES) and everolimus-eluting (EES) stents in a porcine coronary model of streptozotocin (STZ)-induced type I diabetes. METHOD: Twelve Yucatan swine were induced hyperglycemic with a single STZ dose intravenously to ablate pancreatic ß-cells. After two months, each animal received one XIENCE V® (EES) and one Taxus Liberte (PES) stent, respectively, in each coronary artery. After three months, vascular healing was assessed by angiography and histomorphometry. Comparative in vitro effects of everolimus and paclitaxel (10-5 M-10-12 M) after 24 hours on carotid endothelial (EC) and smooth muscle (SMC) cell viability under hyperglycemic (42 mM) conditions were assayed by ELISA. Caspase-3 fluorescent assay was used to quantify caspase-3 activity of EC treated with everolimus or paclitaxel (10-5 M, 10-7 M) for 24 hours. RESULTS: After 3 months, EES reduced neointimal area (1.60 ± 0.41 mm, p < 0.001) with trends toward reduced % diameter stenosis (11.2 ± 9.8%, p = 0.12) and angiographic late-loss (0.28 ± 0.30 mm, p = 0.058) compared to PES (neointimal area: 2.74 ± 0.58 mm, % diameter stenosis: 19.3 ± 14.7%, late loss: 0.55 ± 0.53 mm). Histopathology revealed increased inflammation scores (0.54 ± 0.21 vs. 0.08 ± 0.05), greater medial necrosis grade (0.52 ± 0.26 vs. 0.0 ± 0.0), and persistently elevated fibrin scores (1.60 ± 0.60 vs. 0.63 ± 0.41) with PES compared to EES (p < 0.05). In vitro, paclitaxel significantly increased (p < 0.05) EC/SMC apoptosis/necrosis at high concentrations (≥ 10-7 M), while everolimus did not affect EC/SMC apoptosis/necrosis within the dose range tested. In ECs, paclitaxel (10-5 M) significantly increased caspase-3 activity (p < 0.05) while everolimus had no effect. CONCLUSION: After 3 months, both DES exhibited signs of delayed healing in a STZ-induced diabetic swine model. PES exhibited greater neointimal area, increased inflammation, greater medial necrosis, and persistent fibrin compared to EES. Differential effects of everolimus and paclitaxel on vascular cell viability may potentially be a factor in regulating delayed healing observed with PES. Further investigation of molecular mechanisms may aid future development of stent-based therapies in treating coronary artery disease in diabetic patients.

Sociodemographic patterning of dietary profiles among Inuit youth and adults in Nunavik, Canada: a cross-sectional study.

OBJECTIVES: Country (traditional) foods are integral to Inuit culture, but market food consumption is increasing. The Qanuilirpitaa? 2017 Nunavik Health Survey (Q2017) reported similar country food consumption frequency compared to that in 2004; however, examining food items individually does not account for diet patterns, food accessibility, and correlations between food items. Our objective was to identify underlying dietary profiles and compare them across sex, age, ecological region, and food insecurity markers, given the links among diet, health, and sociocultural determinants. METHODS: Food frequency and sociodemographic data were derived from the Q2017 survey (N = 1176). Latent profile analysis identified dietary profiles using variables for the relative frequencies of country and market food consumption first, followed by an analysis with those for country food variables only. Multinomial logistic regression examined the associations among dietary profiles, sociodemographic factors, and food insecurity markers (to disassociate between food preferences and food access). RESULTS: Four overall dietary profiles and four country food dietary profiles were identified characterized by the relative frequency of country and market food in the diet. The patterns were stable across several sensitivity analyses and in line with our Inuit partners' local knowledge. For the overall profiles, women and adults aged 30-49 years were more likely to have a market food-dominant profile, whereas men and individuals aged 16-29 and 50+ years more often consumed a country food-dominant profile. In the country food profiles, Inuit aged 16-29 years were more likely to have a moderate country food profile whereas Inuit aged 50+ were more likely to have a high country food-consumption profile. A low country and market food-consumption profile was linked to higher prevalence of food insecurity markers. CONCLUSION: We were able to identify distinct dietary profiles with strong social patterning. The profiles elucidated in this study are aligned with the impact of colonial influence on diet and subsequent country food promotion programs for Inuit youth. These profiles will be used for further study of nutritional status, contaminant exposure, and health to provide context for future public health programs.

RéSUMé: OBJECTIFS: Les aliments traditionnels font partie intégrante de la culture inuite, mais la consommation d'aliments du marché est en augmentation. L'enquête de santé des Inuit Qanuilirpitaa? réalisée en 2017 (Q2017) a mis en évidence que la fréquence de consommation d'aliments traditionnels était similaire à celle rapportée en 2004. Or, les fréquences de consommation des aliments pris individuellement ne tiennent pas compte des habitudes alimentaires, de l'accessibilité des aliments et des corrélations entre les aliments consommés. Notre objectif était d'identifier les profils alimentaires sous-jacents et de les comparer selon le sexe, l'âge, la région écologique et les marqueurs d'insécurité alimentaire, étant donné le lien entre l'alimentation, la santé et les déterminants socioculturels. MéTHODES: Les données sur les fréquences alimentaires et sociodémographiques sont issues de l'enquête Q2017 (N=1176). L'analyse des profils latents a permis d'identifier des profils alimentaires en utilisant les variables pour les fréquences relatives de la consommation d'aliments traditionnels et du marché et uniquement celles pour les aliments traditionnels. Des régressions logistiques multinomiales ont été utilisées pour examiner les associations entre les profils alimentaires, les facteurs sociodémographiques et les marqueurs d'insécurité alimentaire (pour dissocier les préférences alimentaires de l'accès aux aliments). RéSULTATS: Quatre profils alimentaires globaux et quatre profils alimentaires spécifiques à la consommation d'aliments traditionnels ont été identifiés en fonction de la fréquence relative des aliments traditionnels et des aliments du marché dans l'alimentation. Les profils étaient en accord avec les connaissances locales de nos partenaires Inuit. Pour les profils alimentaires globaux, les femmes et les adultes âgés de 30 à 49 ans étaient plus susceptibles d'avoir un profil dominé par les aliments du marché, tandis que les hommes et les personnes âgées de 16 à 29 ans et celles de 50 ans et plus avaient plus fréquemment un profil dominé par les aliments traditionnels. En ce qui concerne les profils de consommation d'aliments traditionnels, les Inuit âgés de 16 à 29 ans étaient plus susceptibles d'avoir un profil modéré de consommation d'aliments traditionnels, tandis que les Inuit âgés de 50 ans et plus étaient plus susceptibles d'avoir un profil élevé de consommation d'aliments traditionnels. Un profil bas de consommation d'aliments traditionnels et de marché était associé à une prévalence plus élevée de marqueurs d'insécurité alimentaire. CONCLUSION: Nous avons identifié différents profils alimentaires et ces derniers étaient associés à des caractéristiques socio-démographiques distinctes. Les profils alimentaires mis en lumière dans cette étude concordent avec l'impact du colonialisme sur l'alimentation au Nunavik et aux programmes subséquents de promotion des aliments traditionnels auprès des jeunes Inuit. Ces profils seront utilisés pour une étude plus approfondie du statut nutritionnel, de l'exposition aux contaminants et des issues de santé afin d'identifier des pistes de solutions pour les futurs programmes de santé publique.

Discordant phenotypes in a mother and daughter with mosaic supernumerary ring chromosome 19 explained by a de novo 7q36.2 deletion and 7p22.1 duplication.

We report on a patient with severe intellectual disability, microcephaly, short stature, and dysmorphic features who, based on standard karyotyping, has two cytogenetic abnormalities: an apparently balanced paracentric inversion of chromosome 7, inv(7)(q31.2q36), and a small supernumerary ring chromosome derived entirely of material from chromosome 19. While the inversion was detected in all cells, mosaicism was observed for the ring chromosome. Interestingly, apparently identical cytogenetic abnormalities were detected in the patient's mother, who presented with normal stature, few dysmorphic features, and normal cognition without microcephaly. While the level of mosaicism could not adequately explain the phenotypic discordance, comparative genome hybridization revealed a de novo terminal deletion of chromosome 7, del(7)(q36.2), and a terminal duplication of chromosome 7, dup(7)(p22.1) in the patient. Additional cytogenetic investigation revealed that the patient inherited a recombinant chromosome derived from a cryptic maternal pericentric inversion: inv(7)(p22q36). The patient's distinctive features are consistent with the wide phenotypic spectrum reported in 7p duplication and 7q terminal deletion syndromes. These chromosomal regions contain several candidate genes of clinical significance, including SHH, EN2, and FAM20C. Our findings strongly suggest that our patient's phenotype is largely attributable to partial 7pter trisomy and partial 7qter monosomy rather than mosaic supernumerary ring chromosome 19.

Equity and inclusivity in research: co-creation of a digital platform with representatives of marginalized populations to enhance the involvement in research of people with limited literacy skills.

To improve health equity, as well as equity in research, community-engaged research and participatory research needs to be inclusive. Equity in health research refers to the principle that anyone affected by research or who can benefit from its outcomes should have equal opportunities to contribute to it. Many researchers advocate the importance of promoting equity in research and engage in processes that foster the research involvement of lay persons, patients, and community members who are otherwise "absent" or "silent". Still, people with limited literacy skills who experience unwarranted structural barriers to healthcare access have little involvement in research. Low literacy is a major barrier to equity in health research. Yet there exist approaches and methods that promote the engagement in research of people with literacy challenges. Building on our previous research projects conducted with community members using participatory visual and sound methods (participatory mapping, photovoice, digital storytelling, etc.), we embarked on the co-creation of a digital platform in 2017. Our aim in this commentary is to report on this co-creation experience that was based on a social justice-oriented partnership. The development of the online platform was overseen by a steering committee made up of workers from community organizations involved with people with limited literacy skills, students, and researchers. In the development process, the co-creation steps included a literature review, informal interviews with key informants, and discussion and writing sessions about format and content. After numerous challenges raised and addressed during co-creation, the Engage digital platform for engagement in research went live in the winter of 2020. This platform presents, on an equal footing, approaches and methods from academic research as well as from the literacy education community engaged with people with limited literacy skills.

People with limited literacy skills are often excluded from health research. Engaging patients and community members with limited literacy in research requires tailored approaches and methods that have been tried and tested with them. In 2017, building on an existing partnership between researchers well-versed in using participatory visual and sound methods and community partners, we undertook the co-creation of a digital platform. Our aim was to empower both academic researchers and community researchers and partners (lay persons, clinicians, stakeholders, community organizations) to engage in research projects with people with limited literacy skills. The result was a digital platform ( https://www.engageplus.org ) comprising several modules and resources available in French and English and accessible on the Web. In this commentary, we share our experience in co-creating this digital platform and discuss the facilitators and challenges encountered.

Both t-Darpp and DARPP-32 can cause resistance to trastuzumab in breast cancer cells and are frequently expressed in primary breast cancers.

The clinical use of trastuzumab (Herceptin), a humanized antibody against the HER2 growth factor receptor, has improved survival in patients with breast tumors with ERBB2 amplification and/or over-expression. However, most patients with advanced ERBB2 amplified breast cancers whose tumors initially respond to trastuzumab develop resistance to the drug, leading to tumor progression. To identify factors responsible for acquired resistance to trastuzumab, gene expression profiling was performed on subclones of an ERBB2 amplified breast cancer cell line, BT474, which had acquired resistance to trastuzumab. The most overexpressed gene in these subclones was PPP1R1B, encoding the DARPP-32 phosphatase inhibitor. Western analysis revealed that only the truncated isoform of the DARPP-32 protein, t-Darpp, was overexpressed in the trastuzumab resistant cells. Using gene silencing experiments, we confirmed that t-Darpp over-expression was required for trastuzumab resistance in these cells. Furthermore, transfecting t-Darpp in parental BT-474 cells conferred resistance to trastuzumab, suggesting that t-Darpp expression was sufficient for trastuzumab resistance. We also found that t-Darpp over-expression was associated with Akt activation and that the T75 residue in t-Darpp was required for both Akt activation and trastuzumab resistance. Finally, we found that full-length DARPP-32 and t-Darpp are expressed in a majority of primary breast tumors. Over-expression of full-length DARPP-32 can also confer resistance to trastuzumab and, moreover, is associated with a poor prognostic value in breast cancers. Thus, t-Darpp and DARPP-32 expression are novel prognostic and predictive biomarkers in breast cancer.

A patient with de-novo partial deletion of Xp (p11.4-pter) and partial duplication of 22q (q11.2-qter).

We report on a girl with partial deletion of Xp and partial duplication of 22q. Family studies demonstrate that both the patient's mother and her nonidentical twin sister carry the corresponding balanced translocation; 46,X,t(X;22)(p11.4;q11.2). This girl has developmental delay, microcephaly, mild dysmorphisms and hearing loss but otherwise shows few of the features described in individuals with duplications of the long arm of chromosome 22. She does manifest characteristics, such as short stature and biochemical evidence of ovarian failure, which are seen in partial or complete Xp deletions and Turner's syndrome.

Both TRIM5alpha and TRIMCyp have only weak antiviral activity in canine D17 cells.

BACKGROUND: TRIM5alpha, which is expressed in most primates and the related TRIMCyp, which has been found in one of the New World monkey species, are antiviral proteins of the TRIM5 family that are able to intercept incoming retroviruses early after their entry into cells. The mechanism of action has been partially elucidated for TRIM5alpha, which seems to promote premature decapsidation of the restricted retroviruses. In addition, through its N-terminal RING domain, TRIM5alpha may sensitize retroviruses to proteasome-mediated degradation. TRIM5alpha-mediated restriction requires a physical interaction with the capsid protein of targeted retroviruses. It is unclear whether other cellular proteins are involved in the inhibition mediated by TRIM5alpha and TRIMCyp. A previous report suggested that the inhibition of HIV-1 by the rhesus macaque orthologue of TRIM5alpha was inefficient in the D17a canine cell line, suggesting that the cellular environment was important for the restriction mechanism. Here we investigated further the behavior of TRIM5alpha and TRIMCyp in the D17 cells. RESULTS: We found that the various TRIM5alpha orthologues studied (human, rhesus macaque, African green monkey) as well as TRIMCyp had poor antiviral activity in the D17 cells, despite seemingly normal expression levels and subcellular distribution. Restriction of both HIV-1 and the distantly related N-tropic murine leukemia virus (N-MLV) was low in D17 cells. Both TRIM5alpharh and TRIMCyp promoted early HIV-1 decapsidation in murine cells, but weak levels of restriction in D17 cells correlated with the absence of accelerated decapsidation in these cells and also correlated with normal levels of cDNA synthesis. Fv1, a murine restriction factor structurally unrelated to TRIM5alpha, was fully functional in D17 cells, showing that the loss of activity was specific to TRIM5alpha/TRIMCyp. CONCLUSION: We show that D17 cells provide a poor environment for the inhibition of retroviral replication by proteins of the TRIM5 family. Because both TRIM5alpha and TRIMCyp are poorly active in these cells, despite having quite different viral target recognition domains, we conclude that a step either upstream or downstream of target recognition is impaired. We speculate that an unknown factor required for TRIM5alpha and TRIMCyp activity is missing or inadequately expressed in D17 cells.

Interleukin-15 delays human neutrophil apoptosis by intracellular events and not via extracellular factors: role of Mcl-1 and decreased activity of caspase-3 and caspase-8.

Interleukin-15 (IL-15) induces the de novo protein synthesis of intracellular polypeptides and delays neutrophil apoptosis by a mechanism that is still unclear. Herein, we investigated the potential antiapoptotic role of newly synthesized proteins released into the external milieu in IL-15-induced neutrophils. We found that IL-15 induces the de novo synthesis of an approximately 23-kDa protein, representing the predominant protein detected in the milieu, and identified it as IL-1 receptor antagonist (IL-1Ra) by Western blot and immunoprecipitation. We quantified IL-1Ra, IL-1alpha, and IL-1beta concentrations by enzyme-linked immunosorbent assay in intracellular and extracellular fractions from IL-15-induced neutrophils and found that IL-15 does not increase IL-1alpha or IL-1beta production but induces IL-1Ra release. Also, we demonstrated that IL-1Ra does not modulate apoptosis, even at a concentration 250 times greater than that measured in the external milieu. In contrast to granulocyte macrophage-colony stimulating factor, the supernatant harvested from IL-15-induced neutrophils was devoid of antiapoptotic activity. Addition of cycloheximide demonstrates that IL-15 delays apoptosis via de novo synthesis of intracellular proteins and that it increases myeloid cell differentiation factor-1 stability. We demonstrated also that IL-15 decreases the activity of caspase-3 and caspase-8, resulting in an inhibition of vimentin cleavage. Our results indicate that IL-15 can activate an anti-inflammatory loop, based on its ability to induce the synthesis of IL-1Ra by neutrophils. We conclude that IL-15 delays human neutrophil apoptosis by intracellular events and not via extracellular factors.

Sef downregulation by Ras causes MEK1/2 to become aberrantly nuclear localized leading to polyploidy and neoplastic transformation.

Subcellular trafficking of key oncogenic signal pathway components is likely to be crucial for neoplastic transformation, but little is known about how such trafficking processes are spatially controlled. In this study, we show how Ras activation causes aberrant nuclear localization of phosphorylated mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK; MEK) MEK1/2 to drive neoplastic transformation. Phosphorylated MEK1/2 was aberrantly located within the nucleus of primary colorectal tumors and human colon cancer cells, and oncogenic activation of Ras was sufficient to induce nuclear accumulation of phosphorylated MEK1/2 and ERK1/2 in intestinal epithelial cells. Enforced nuclear localization of MEK1 in epithelial cells or fibroblasts was sufficient for hyperactivation of ERK1/2, thereby driving cell proliferation, chromosomal polyploidy, and tumorigenesis. Notably, Ras-induced nuclear accumulation of activated MEK1/2 was reliant on downregulation of the spatial regulator Sef, the reexpression of which was sufficient to restore normal MEK1/2 localization and a reversal of Ras-induced proliferation and tumorigenesis. Taken together, our findings indicate that Ras-induced downregulation of Sef is an early oncogenic event that contributes to genetic instability and tumor progression by sustaining nuclear ERK1/2 signaling.

Cul3 overexpression depletes Nrf2 in breast cancer and is associated with sensitivity to carcinogens, to oxidative stress, and to chemotherapy.

Nrf2 is the key transcription factor for cytoprotective gene programs. Nrf2 is normally maintained at very low concentrations by proteasomal degradation, through its interaction with the adapter protein Keap1 and the Cul3 E3 ligase. Increased Nrf2 concentration resulting from loss of function Keap1 mutations has been described in chemoresistant non-small cell lung cancer. Previous studies in breast cancer showed low levels of some Nrf2-regulated detoxification genes, but the mechanism has not been systematically examined. We found that half of the breast cancer cell lines examined have decreased concentration of Nrf2 compared with normal mammary epithelial cell lines, associated with variable but detectable levels in Keap1 levels, and consistently increased Cul3 mRNA and protein. Immunochemistry showed that 7 of 10 breast cancer specimens examined also have low Nrf2 levels and increased Cul3. Keap1 protein levels are variable. We found no C23Y mutation in Keap1 of any of the cell lines. Using siRNA, we silenced Cul3 in MCF-7 breast cancer cells, and microarray analysis reveals the induction of GCL, NQO1, AKR1C1, UGDH, and TXN by at least 2-fold. The Nrf2-regulated ABCC1 drug transporter was also found to be increased. These Cul3-silenced MCF7 cells are highly resistant to oxidative stress induced by H(2)O(2,) to the carcinogen benzo(a)pyrene, and to both Doxorubicin and Paclitaxel. This high Cul3/low Nrf2 signature may be key to cellular sensitivity to both chemical carcinogeneic stimuli as well as to cytotoxicity of commonly used chemotherapeutic drugs in established breast cancers.

The role of CXCR4 receptor expression in breast cancer: a large tissue microarray study.

The chemokine receptor CXCR4 is an important factor in the migration, invasiveness, metastasis and proliferation of breast cancer cells. We have retrospectively analyzed the levels of expression of CXCR4 in a large cohort of breast cancers and pre-invasive breast samples linked to clinical data. A total of 1808 invasive breast carcinomas and 214 pre-invasive breast samples could be analyzed in correlation with basic clinico-pathological data such as hormone receptor status, HER2 status and tumor grade. The majority of breast cancers expressed either nuclear or cytoplasmic staining or both. CXCR4 cytoplasmic expression was associated with parameters of tumor aggressivity (tumor grade and lymph node status) and had prognostic value (age-adjusted hazard ratio=1.73; Confidence Interval: 1.07-2.77) with respect to disease-specific survival. CXCR4 positivity in the cytoplasm but not the nucleus was associated with HER2 expression and amplification as well as with hormone receptor negativity (both ER and PR). The percentage of nuclear staining increased from normal breast tissue (20%) to ductal carcinoma-in-situ DCIS (43%) to invasive cancer (67%) while CXCR4 was expressed in the cytoplasm of 67% of (DCIS) cases (double that in normal breast samples), suggesting an important role in breast tumor progression. The CXCR4 receptor is expressed in many breast cancers, justifying its development as a therapeutic target in breast cancer patients. Its cytoplasmic expression is associated with breast tumor progression, suggesting potential value as a diagnostic marker.

In vivo and in vitro roles of IL-21 in inflammation.

IL-21 is a cytokine known to mediate its biological action via the IL-21R, composed of a specific chain, IL-21Ralpha, and the common gamma-chain (CD132). Recent data suggest that IL-21 possesses proinflammatory properties. However, there is no clear evidence that IL-21 induces inflammation in vivo and, curiously, the interaction between IL-21 and neutrophils has never been investigated, despite the fact that these cells express CD132 and respond to other CD132-dependent cytokines involved in inflammatory disorders. Using the murine air pouch model, we found that IL-21 induced inflammation in vivo, based on recruitment of neutrophil and monocyte populations. In contrast to LPS, administration of IL-21 into the air pouch did not significantly increase the concentration of IL-6, CCL5, CCL3, and CXCL2. We demonstrated that HL-60 cells expressed IL-21Ralpha, which is down-regulated during their differentiation toward neutrophils, and that IL-21Ralpha is not detected in neutrophils. Concomitant with this, IL-21 induced Erk-1/2 phosphorylation in HL-60 cells, but not in neutrophils. To eliminate the possibility that IL-21 could activate neutrophils even in the absence of IL-21Ralpha, we demonstrated that IL-21 did not modulate several neutrophil functions. IL-21-induced Erk-1/2 phosphorylation was not associated with proliferation or differentiation of HL-60 toward neutrophils, monocytes, or macrophages. IL-21Ralpha was detected in human monocytes and monocyte-derived macrophages, but IL-21 increased CXCL8 production only in monocyte-derived macrophages. We conclude that IL-21 is a proinflammatory cytokine, but not a neutrophil agonist. We propose that IL-21 attracts neutrophils indirectly in vivo via a mechanism independent of IL-6, CCL3, CCL5, and CXCL2 production.