Targeting Of Her-2 Overexpressing Breast Cancer Cells With Immunoliposomes

Thesis (Ph.D.) University of Alaska Fairbanks, 2010The goal of the research described in this thesis is to develop a liposome based drug delivery system which targets Her-2 overexpressing breast tumors with high specificity. Overexpression of the Her-2 receptor occurs in many cancers but is most prevalent in breast tumors, with 20--30 percent of all cases displaying overexpression of the receptor. In addition, Her-2 overexpressing breast tumors are often aggressive and have a high probability of metastasizing. In the research reported here, a drug delivery system has been created that selectively targets Her-2 overexpressing mammary cells by combining three liposomal technologies. First, a Her-2 targeting antibody was conjugated to the outer surface of the liposomes, resulting in highly specific binding and internalization of liposomes into mammary epithelial cells that overexpress Her-2. Second, the liposomes were designed to be thermosensitive, only releasing their encapsulated cargo in response to mild hyperthermia at 42�C. Finally, the liposomes were attached to a pore-forming protein, listeriolysin O (LLO), which compromises the target cell endosome, allowing for drug delivery directly to the cellular cytoplasm. The liposomes delivered a 22-fold higher concentration of fluorescent marker to cells overexpressing Her-2 than to normal cells, demonstrating the delivery system's potential for targeting Her-2 overexpressing tumors. When a cytotoxin, gelonin, was encapsulated within the liposomes, the delivery system selectively targeted and killed Her-2 overexpressing cells in vitro. To further increase specificity for Her-2 overexpressing cells, the concept of a two-component delivery system was explored. This system would require internalization of two different types of liposomes within a cell endosome for effective drug delivery. Experiments using fluorescent markers show that this method greatly increased targeting specificity for Her-2 overexpressing cells

A single early postnatal estradiol injection affects morphology and gene expression of the ovary and parametrial adipose tissue in adult female rats.

Events during early life can affect reproductive and metabolic functions in adulthood. We evaluated the programming effects of a single early postnatal estradiol injection (within 3h after birth) in female rats. We assessed ovarian and parametrial adipose tissue morphology, evaluated gene expression related to follicular development and adipose tissue metabolism, and developed a non-invasive volumetric estimation of parametrial adipose tissue by magnetic resonance imaging. Estradiol reduced ovarian weight, increased antral follicle size and number of atretic antral follicles, and decreased theca interna thickness in atretic antral follicles. Adult estradiol-injected rats also had malformed vaginal openings and lacked corpora lutea, confirming anovulation. Estradiol markedly reduced parametrial adipose tissue mass. Adipocyte size was unchanged, suggesting reduced adipocyte number. Parametrial adipose tissue lipoprotein lipase activity was increased. In ovaries, estradiol increased mRNA expression of adiponectin, complement component 3, estrogen receptor alpha, and glucose transporter 3 and 4; in parametrial adipose tissue, expression of complement component 3 was increased, expression of estrogen receptor alpha was decreased, and expression of leptin, lipoprotein lipase, and hormone-sensitive lipase was unaffected. These findings suggest that early postnatal estradiol exposure of female rats result in long-lasting effects on the ovary and parametrial adipose tissue at adult age

Gut microbiota of adults with asthma is broadly similar to non-asthmatics in a large population with varied ethnic origins

Bacterial gut communities might predispose children to develop asthma. Yet, little is known about the role of these micro-organisms in adult asthmatics. We aimed to profile the relationship between fecal microbiota and asthma in a large-scale, ethnically diverse, observational cohort of adults. Fecal microbiota composition of 1632 adults (172 asthmatics and 1460 non-asthmatics) was analyzed using 16S ribosomal RNA gene sequencing. Using extremely randomized trees machine learning models, we assessed the discriminatory ability of gut bacterial features to identify asthmatics from non-asthmatics. Asthma contributed 0.019% to interindividual dissimilarities in intestinal microbiota composition, which was not significant (P = .97). Asthmatics could not be distinguished from non-asthmatics based on individual microbiota composition by an extremely randomized trees classifier model (area under the receiver operating characteristic curve = 0.54). In conclusion, there were no prominent differences in fecal microbiota composition in adult asthmatics when compared to non-asthmatics in an urban, large-sized and ethnically diverse cohort

Functional expression and characterization of cytochrome P450 52A21 from Candida albicans

Candida albicans contains 10 putative cytochrome P450 (CYP) genes coding for enzymes that appear to play important roles in fungal survival and virulence. Here, we report the characterization of CYP52A21, a putative alkane/fatty acid hydroxylase. The recombinant CYP52A21 protein containing a 6 x (His)-tag was expressed in Eseherichia coli and was purified. The purified protein, reconstituted with rat NADPH-cytochrome P450 reductase, omega-hydroxylated dodecanoic acid to give 12-hydroxydodecanoic acid, but to a lesser extent also catalyzed (omega-1)-hydroxylation to give 11-hydroxydodecanoic acid. When 12,12,12-d(3)-dodecanoic acid was used as the substrate, there was a major shift in the oxidation from the omega- to the (omega-1)-hydroxylated product. The regioselectivity of fatty acid hydroxylation was examined with the 12-iodo-, 12-bromo-, and 12-chlorododecanoic acids. Although all three 12-halododecanoic acids bound to CYP52A21 with similar affinities, the production of 12-oxododecanoic acid decreased as the size of the terminal halide increased. The reeioselectivity of CYP52A21 fatty acid oxidation is thus consistent with presentation of the terminal end of the fatty acid chain for oxidation via a narrow channel that limits access to other atoms of the fatty acid chain. This constricted access, in contrast to that proposed for the CYNA family of enzymes, does not involve covalent binding of the heme to the protein. (c) 2007 Elsevier Inc. All rights reserved

Association between butyrate-producing gut bacteria and the risk of infectious disease hospitalisation : results from two observational, population-based microbiome studies

Background: Microbiota alterations are common in patients hospitalised for severe infections, and preclinical models have shown that anaerobic butyrate-producing gut bacteria protect against systemic infections. However, the relationship between microbiota disruptions and increased susceptibility to severe infections in humans remains unclear. We investigated the relationship between gut microbiota and the risk of future infection-related hospitalisation in two large population-based cohorts. Methods: In this observational microbiome study, gut microbiota were characterised using 16S rRNA gene sequencing in independent population-based cohorts from the Netherlands (HELIUS study; derivation cohort) and Finland (FINRISK 2002 study; validation cohort). HELIUS was conducted in Amsterdam, Netherlands, and included adults (aged 18–70 years at inclusion) who were randomly sampled from the municipality register of Amsterdam. FINRISK 2002 was conducted in six regions in Finland and is a population survey that included a random sample of adults (aged 25–74 years). In both cohorts, participants completed questionnaires, underwent a physical examination, and provided a faecal sample at inclusion (Jan 3, 2013, to Nov 27, 2015, for HELIUS participants and Jan 21 to April 19, 2002, for FINRISK participants. For inclusion in our study, a faecal sample needed to be provided and successfully sequenced, and national registry data needed to be available. Primary predictor variables were microbiota composition, diversity, and relative abundance of butyrate-producing bacteria. Our primary outcome was hospitalisation or mortality due to any infectious disease during 5–7-year follow-up after faecal sample collection, based on national registry data. We examined associations between microbiota and infection risk using microbial ecology and Cox proportional hazards. Findings: We profiled gut microbiota from 10 699 participants (4248 [39·7%] from the derivation cohort and 6451 [60·3%] from the validation cohort). 602 (5·6%) participants (152 [3·6%] from the derivation cohort; 450 [7·0%] from the validation cohort) were hospitalised or died due to infections during follow-up. Gut microbiota composition of these participants differed from those without hospitalisation for infections (derivation p=0·041; validation p=0·0002). Specifically, higher relative abundance of butyrate-producing bacteria was associated with a reduced risk of hospitalisation for infections (derivation cohort cause-specific hazard ratio 0·75 [95% CI 0·60–0·94] per 10% increase in butyrate producers, p=0·013; validation cohort 0·86 [0·77–0·96] per 10% increase, p=0·0077). These associations remained unchanged following adjustment for demographics, lifestyle, antibiotic exposure, and comorbidities. Interpretation: Gut microbiota composition, specifically colonisation with butyrate-producing bacteria, was associated with protection against hospitalisation for infectious diseases in the general population across two independent European cohorts. Further studies should investigate whether modulation of the microbiome can reduce the risk of severe infections. Funding: Amsterdam UMC, Porticus, National Institutes of Health, Netherlands Organisation for Health Research and Development (ZonMw), and Leducq Foundation.Peer reviewe