Incorporation of human recombinant tropoelastin into silk fibroin membranes with the view to repairing Bruch’s membrane

Bombyx mori silk fibroin membranes provide a potential delivery vehicle for both cells and extracellular matrix (ECM) components into diseased or injured tissues. We have previously demonstrated the feasibility of growing retinal pigment epithelial cells (RPE) on fibroin membranes with the view to repairing the retina of patients afflicted with age-related macular degeneration (AMD). The goal of the present study was to investigate the feasibility of incorporating the ECM component elastin, in the form of human recombinant tropoelastin, into these same membranes. Two basic strategies were explored: (1) membranes prepared from blended solutions of fibroin and tropoelastin; and (2) layered constructs prepared from sequentially cast solutions of fibroin, tropoelastin, and fibroin. Optimal conditions for RPE attachment were achieved using a tropoelastin-fibroin blend ratio of 10 to 90 parts by weight. Retention of tropoelastin within the blend and layered constructs was confirmed by immunolabelling and Fourier-transform infrared spectroscopy (FTIR). In the layered constructs, the bulk of tropoelastin was apparently absorbed into the initially cast fibroin layer. Blend membranes displayed higher elastic modulus, percentage elongation, and tensile strength (p < 0.01) when compared to the layered constructs. RPE cell response to fibroin membranes was not affected by the presence of tropoelastin. These findings support the potential use of fibroin membranes for the co-delivery of RPE cells and tropoelastin.

Clinical factors associated with the humoral immune response to influenza vaccination in chronic obstructive pulmonary disease

Background and objective Individuals with chronic obstructive pulmonary disease (COPD) are at a high risk of developing significant complications from infection with the influenza virus. It is therefore vital to ensure that prophylaxis with the influenza vaccine is effective in COPD. The aim of this study was to assess the immunogenicity of the 2010 trivalent influenza vaccine in persons with COPD compared to healthy subjects without lung disease, and to examine clinical factors associated with the serological response to the vaccine. Methods In this observational study, 34 subjects (20 COPD, 14 healthy) received the 2010 influenza vaccine. Antibody titers at baseline and 28 days post-vaccination were measured using the hemagglutination inhibition assay (HAI) assay. Primary endpoints included seroconversion (≥4-fold increase in antibody titers from baseline) and the fold increase in antibody titer after vaccination. Results Persons with COPD mounted a significantly lower humoral immune response to the influenza vaccine compared to healthy participants. Seroconversion occurred in 90% of healthy participants, but only in 43% of COPD patients (P=0.036). Increasing age and previous influenza vaccination were associated with lower antibody responses. Antibody titers did not vary significantly with cigarette smoking, presence of other comorbid diseases, or COPD severity. Conclusion The humoral immune response to the 2010 influenza vaccine was lower in persons with COPD compared to non-COPD controls. The antibody response also declined with increasing age and in those with a history of prior vaccination.

The enigma of IgE+ B-cell memory in human subjects

Our understanding of the origin and fate of the IgE-switched B cell has been markedly improved by studies in mouse models. The immediate precursor of the IgE-switched B cell is either a relatively naive nonswitched B cell or a mature IgG-switched B cell. These 2 routes are referred to as the direct and indirect pathways, respectively. IgE responses derived from each pathway differ significantly, largely reflecting the difference in time spent in a germinal center and thus time for clonal expansion, somatic hypermutation, affinity maturation, and acquisition of a memory phenotype. The clinical and therapeutic implications for IgE responses in human subjects are still a matter of debate, largely because the immunization procedures used in the animal models are significantly different from classical atopic sensitization to allergens from pollen and mites. On the basis of the limited information available, it seems likely that these atopic IgE responses are characterized by a relatively low IgG/IgE ratio, low B-cell memory, and modest affinity maturation, which fits well with the direct switching pathway. It is still unresolved how the IgE response evolves to cover a wide epitope repertoire involving many epitopes per allergen, as well as many different allergens from a single allergen source. © 2013 American Academy of Allergy, Asthma & Immunology.

Mimicry between HTLV-I and myelin basic protein: No response in HTLV-I-associated myelopathy patients

The reactivity to a peptide from the HTLV-I polyprotein (FKLPGLNSR) and a similar sequence from myelin basic protein (MBP) (FKLGGRDSR) was examined in relation to the proposal that mimicry of MBP by HTLV-I could be involved in autoimmune responses in HTLV-I-associated myelopathy (HAM). It was found that rabbit antibodies raised against the HTLV-I peptide recognised both peptides, with a titre of 1/10240 to the HTLV-I peptide and 1/5220 to the MBP peptide. Human sera from HAM patients and a HTLV-I carrier without HAM showed slightly higher responses to the HTLV-I peptide compared to the responses from uninfected human sera. HAM patients had greater responses to the HTLV-I peptide than to the similar MBP peptide and an unrelated bovine MBP peptide. There was no recognition of the peptides by peripheral blood lymphocytes from HAM patients or a HTLV-I carrier without HAM. It was concluded that although cross-reactivity was demonstrated in rabbits and the HTLV-I peptide was recognised by sera from HAM patients, the epitope does not appear to evoke a mimicking response to the similar region in MBP. Hence it is not likely to be involved in the pathogenesis of HAM through molecular mimicry.

Infection and cellular defense dynamics in a novel 17beta-estradiol murine model of chronic human group B streptococcus genital tract colonization reveal a role for hemolysin in persistence and neutrophil accumulation

Genital tract carriage of group B streptococcus (GBS) is prevalent among adult women; however, the dynamics of chronic GBS genital tract carriage, including how GBS persists in this immunologically active host niche long term, are not well defined. To our knowledge, in this study, we report the first animal model of chronic GBS genital tract colonization using female mice synchronized into estrus by delivery of 17β-estradiol prior to intravaginal challenge with wild-type GBS 874391. Cervicovaginal swabs, which were used to measure bacterial persistence, showed that GBS colonized the vaginal mucosa of mice at high numbers (106–107 CFU/swab) for at least 90 d. Cellular and histological analyses showed that chronic GBS colonization of the murine genital tract caused significant lymphocyte and PMN cell infiltrates, which were localized to the vaginal mucosal surface. Long-term colonization was independent of regular hormone cycling. Immunological analyses of 23 soluble proteins related to chemotaxis and inflammation showed that the host response to GBS in the genital tract comprised markers of innate immune activation including cytokines such as GM-CSF and TNF-α. A nonhemolytic isogenic mutant of GBS 874391, Δcyle9, was impaired for colonization and was associated with amplified local PMN responses. Induction of DNA neutrophil extracellular traps, which was observed in GBS-infected human PMNs in vitro in a hemolysin-dependent manner, appeared to be part of this response. Overall, this study defines key infection dynamics in a novel murine model of chronic GBS genital tract colonization and establishes previously unknown cellular and soluble defense responses to GBS in the female genital tract.

Click functionalization of methacrylate-based hydrogels and their cellular response

Methacrylate-based hydrogels, such as homo- and copolymers of 2-hydroxyethyl methacrylate (HEMA), have demonstrated significant potential for use in biomedical applications. However, many of these hydrogels tend to resist cell attachment and growth at their surfaces, which can be detrimental for certain applications. In this article, glycidyl methacrylate (GMA) was copolymerized with HEMA to generate gels functionalized with epoxide groups. The epoxides were then functionalized by two sequential click reactions, namely, nucleophilic ring opening of epoxides with sodium azide and then coupling of small molecules and peptides via Huisgen's copper catalyzed 1,3-dipolar cycloaddition of azides with alkynes. Using this strategy it was possible to control the degree of functionalization by controlling the feed ratio of monomers during polymerization. In vitro cell culture of human retinal pigment epithelial cell line (ARPE-19) with the hydrogels showed improved cell adhesion, growth and proliferation for hydrogels that were functionalized with a peptide containing the RGD sequence. In addition, the cell attachment progressively decreased with increasing densities of the RGD containing peptide. In summary, a facile methodology has been presented that gives rise to hydrogels with controlled degrees of functionality, such that the cell response is directly related to the levels and nature of that functionality.

The ovarian response to controlled stimulation in IVF cycles may be predictive of the age at menopause

STUDY QUESTION Can the number of oocytes retrieved in IVF cycles be predictive of the age at menopause? SUMMARY ANSWER The number of retrieved oocytes can be used as an indirect assessment of the extent of ovarian reserve to provide information on the duration of the reproductive life span in women of different ages. WHAT IS KNOWN ALREADY Menopause is determined by the exhaustion of the ovarian follicular pool. Ovarian reserve is the main factor influencing ovarian response in IVF cycles. As a consequence the response to ovarian stimulation with the administration of gonadotrophins in IVF treatment may be informative about the age at menopause. STUDY DESIGN, SIZE, DURATION In the present cross-sectional study, participants were 1585 infertile women from an IVF clinic and 2635 menopausal women from a more general population. PARTICIPANTS/MATERIALS, SETTING, METHODS For all infertile women, the response to ovarian stimulation with gonadotrophins was recorded. For menopausal women, relevant demographic characteristics were available for the analysis. MAIN RESULTS AND THE ROLE OF CHANCE A cubic function described the relationship between mean numbers of oocytes and age, with all terms being statistically significant. From the estimated residual distribution of the actual number of oocytes about this mean, a distribution of the age when there would be no oocytes retrieved following ovarian stimulation was derived. This was compared with the distribution of the age at menopause from the menopausal women, showing that menopause occurred about a year later. LIMITATIONS, REASONS FOR CAUTION The retrieved oocyte data were from infertile women, while the menopausal ages were from a more general population. WIDER IMPLICATIONS OF THE FINDINGS In the present study, we have shown some similarity between the distributions of the age when no retrieved oocytes can be expected after ovarian stimulation and the age at menopause. For a given age, the lower the ovarian reserve, the lower the number of retrieved oocytes would be and the earlier the age that menopause would occur.

Functional localization of the human color center by decreased water displacement using diffusion-weighted fMRI

Introduction Decreased water displacement following increased neural activity has been observed using diffusion-weighted functional MRI (DfMRI) at high b-values. The physiological mechanisms underlying the diffusion signal change may be unique from the standard blood oxygenation level-dependent (BOLD) contrast and closer to the source of neural activity. Whether DfMRI reflects neural activity more directly than BOLD outside the primary cerebral regions remains unclear. Methods Colored and achromatic Mondrian visual stimuli were statistically contrasted to functionally localize the human color center Area V4 in neurologically intact adults. Spatial and temporal properties of DfMRI and BOLD activation were examined across regions of the visual cortex. Results At the individual level, DfMRI activation patterns showed greater spatial specificity to V4 than BOLD. The BOLD activation patterns were more prominent in the primary visual cortex than DfMRI, where activation was localized to the ventral temporal lobe. Temporally, the diffusion signal change in V4 and V1 both preceded the corresponding hemodynamic response, however the early diffusion signal change was more evident in V1. Conclusions DfMRI may be of use in imaging applications implementing cognitive subtraction paradigms, and where highly precise individual functional localization is required.

Removal of organic content from diesel exhaust particles alters cellular responses of primary human bronchial epithelial cells cultured at an air-liquid interface

Background Exposure to air pollutants, including diesel particulate matter, has been linked to adverse respiratory health effects. Inhaled diesel particulate matter contains adsorbed organic compounds. It is not clear whether the adsorbed organics or the residual components are more deleterious to airway cells. Using a physiologically relevant model, we investigated the role of diesel organic content on mediating cellular responses of primary human bronchial epithelial cells (HBECs) cultured at an air-liquid interface (ALI). Methods Primary HBECs were cultured and differentiated at ALI for at least 28 days. To determine which component is most harmful, we compared primary HBEC responses elicited by residual (with organics removed) diesel emissions (DE) to those elicited by neat (unmodified) DE for 30 and 60 minutes at ALI, with cigarette smoke condensate (CSC) as the positive control, and filtered air as negative control. Cell viability (WST-1 cell proliferation assay), inflammation (TNF-α, IL-6 and IL-8 ELISA) and changes in gene expression (qRT-PCR for HO-1, CYP1A1, TNF-α and IL-8 mRNA) were measured. Results Immunofluorescence and cytological staining confirmed the mucociliary phenotype of primary HBECs differentiated at ALI. Neat DE caused a comparable reduction in cell viability at 30 or 60 min exposures, whereas residual DE caused a greater reduction at 60 min. When corrected for cell viability, cytokine protein secretion for TNF-α, IL-6 and IL-8 were maximal with residual DE at 60 min. mRNA expression for HO-1, CYP1A1, TNF-α and IL-8 was not significantly different between exposures. Conclusion This study provides new insights into epithelial cell responses to diesel emissions using a physiologically relevant aerosol exposure model. Both the organic content and residual components of diesel emissions play an important role in determining bronchial epithelial cell response in vitro. Future studies should be directed at testing potentially useful interventions against the adverse health effects of air pollution exposure.

Convergence of regenerative medicine and synthetic biology to develop standardized and validated models of human diseases with clinical relevance

In order to progress beyond currently available medical devices and implants, the concept of tissue engineering has moved into the centre of biomedical research worldwide. The aim of this approach is not to replace damaged tissue with an implant or device but rather to prompt the patient's own tissue to enact a regenerative response by using a tissue-engineered construct to assemble new functional and healthy tissue. More recently, it has been suggested that the combination of Synthetic Biology and translational tissue-engineering techniques could enhance the field of personalized medicine, not only from a regenerative medicine perspective, but also to provide frontier technologies for building and transforming the research landscape in the field of in vitro and in vivo disease models.

Common variants near ATM are associated with glycemic response to metformin in type 2 diabetes

Metformin is the most commonly used pharmacological therapy for type 2 diabetes. We report a genome-wide association study for glycemic response to metformin in 1,024 Scottish individuals with type 2 diabetes with replication in two cohorts including 1,783 Scottish individuals and 1,113 individuals from the UK Prospective Diabetes Study. In a combined meta-analysis, we identified a SNP, rs11212617, associated with treatment success (n = 3,920, P = 2.9 P×-9, odds ratio = 1.35, 95% CI 1.22-1.49) at a locus containing ATM, the ataxia telangiectasia mutated gene. In a rat hepatoma cell line, inhibition of ATM with KU-55933 attenuated the phosphorylation and activation of AMP-activated protein kinase in response to metformin. We conclude that ATM, a gene known to be involved in DNA repair and cell cycle control, plays a role in the effect of metformin upstream of AMP-activated protein kinase, and variation in this gene alters glycemic response to metformin. © 2011 Nature America, Inc. All rights reserved.

The Post-Illumination Pupil Response (PIPR)

Purpose The post-illumination pupil response (PIPR) has been quantified using four metrics, but the spectral sensitivity of only one is known; here we determine the other three. To optimize the human PIPR measurement, we determine the protocol producing the largest PIPR, the duration of the PIPR, and the metric(s) with the lowest coefficient of variation. Methods The consensual pupil light reflex (PLR) was measured with a Maxwellian view pupillometer. - Experiment 1: Spectral sensitivity of four PIPR metrics [plateau, 6 s, area under curve (AUC) early and late recovery] was determined from a criterion PIPR to a 1s pulse and fitted with Vitamin A1 nomogram (λmax = 482nm). - Experiment 2: The PLR was measured as a function of three stimulus durations (1s, 10s, 30s), five irradiances spanning low to high melanopsin excitation levels (retinal irradiance: 9.8 to 14.8 log quanta.cm-2.s-1), and two wavelengths, one with high (465nm) and one with low (637nm) melanopsin excitation. Intra and inter-individual coefficients of variation (CV) were calculated. Results The melanopsin (opn4) photopigment nomogram adequately describes the spectral sensitivity of all four PIPR metrics. The PIPR amplitude was largest with 1s short wavelength pulses (≥ 12.8 log quanta.cm-2.s-1). The plateau and 6s PIPR showed the least intra and inter-individual CV (≤ 0.2). The maximum duration of the sustained PIPR was 83.0±48.0s (mean±SD) for 1s pulses and 180.1±106.2s for 30s pulses (465nm; 14.8 log quanta.cm-2.s-1). Conclusions All current PIPR metrics provide a direct measure of the intrinsic melanopsin photoresponse. To measure progressive changes in melanopsin function in disease, we recommend that the PIPR be measured using short duration pulses (e.g., ≤ 1s) with high melanopsin excitation and analyzed with plateau and/or 6s metrics. Our PIPR duration data provide a baseline for the selection of inter-stimulus intervals between consecutive pupil testing sequences.

Effect of age and refractive error on the melanopsin mediated Post-Illumination Pupil Response (PIPR)

Melanopsin containing intrinsically photosensitive Retinal Ganglion cells (ipRGCs) mediate the pupil light reflex (PLR) during light onset and at light offset (the post-illumination pupil response, PIPR). Recent evidence shows that the PLR and PIPR can provide non-invasive, objective markers of age-related retinal and optic nerve disease, however there is no consensus on the effects of healthy ageing or refractive error on the ipRGC mediated pupil function. Here we isolated melanopsin contributions to the pupil control pathway in 59 human participants with no ocular pathology across a range of ages and refractive errors. We show that there is no effect of age or refractive error on ipRGC inputs to the human pupil control pathway. The stability of the ipRGC mediated pupil response across the human lifespan provides a functional correlate of their robustness observed during ageing in rodent models.

Submission to the External Independent Review of the United Nations Response to Allegations of Sexual Exploitation and Abuse and Other Serious Crimes by Members of Foreign Military Forces Not Under United Nations Command in the Central African Republic

In 2015 the UN Secretary-General established an External Independent Review to review how the United Nations has responded to allegations of child sexual exploitation and child sexual abuse, and to make recommendations concerning how the United Nations should respond to allegations in the future. This submission to the Review Panel draws on literature regarding children's rights, the nature of child sexual abuse, international instruments and policy, the nature of institutional child sexual abuse, and the CAR case itself. It makes recommendations for reform of UN protocols and procedures to better prevent child sexual abuse, and to improve responses to future occurrences.