Karolin Hijazi

Llama Antibody Fragments with Cross-Subtype Human Immunodeficiency Virus Type 1 (HIV-1)-Neutralizing Properties and High Affinity for HIV-1 gp120

ABSTRACT Members of the Camelidae family produce immunoglobulins devoid of light chains. We have characterized variable domains of these heavy chain antibodies, the VHH, from llamas immunized with human immunodeficiency virus type 1 (HIV-1) envelope protein gp120 in order to identify VHH that can inhibit HIV-1 infection. To increase the chances of isolating neutralizing VHH, we employed a functional selection approach, involving panning of phage libraries expressing the VHH repertoire on recombinant gp120, followed by a competitive elution with soluble CD4. By immunizing with gp120 derived from an HIV-1 subtype B′/C primary isolate, followed by panning on gp120 from HIV-1 isolates of subtypes A, B, and C, we could select for VHH with cross-subtype neutralizing activity. Three VHH able to neutralize HIV-1 primary isolates of subtypes B and C were characterized. These bound to recombinant gp120 with affinities close to the suggested affinity ceiling for in vivo-maturated antibodies and competed with soluble CD4 for this binding, indicating that their mechanism of neutralization involves interacting with the functional envelope spike prior to binding to CD4. The most potent VHH in terms of low 50% inhibitory concentration (IC50) and IC90 values and cross-subtype reactivity was A12. These results indicate that camelid VHH can be potent HIV-1 entry inhibitors. Since VHH are stable and can be produced at a relatively low cost, they may be considered for applications such as HIV-1 microbicide development. Antienvelope VHH might also prove useful in defining neutralizing and nonneutralizing epitopes on HIV-1 envelope proteins, with implications for HIV-1 vaccine design.

Mucosal Microbiome in Patients with Recurrent Aphthous Stomatitis

Recurrent aphthous stomatitis (RAS) is the most common disease affecting oral mucosae. Etiology is unknown, but several factors have been implicated, all of which influence the composition of microbiota residing on oral mucosae, which in turn modulates immunity and thereby affects disease progression. Although no individual pathogens have been conclusively shown to be causative agents of RAS, imbalanced composition of the oral microbiota may play a key role. In this study, we sought to determine composition profiles of bacterial microbiota in the oral mucosa associated with RAS. Using high-throughput 16S rRNA gene sequencing, we characterized the most abundant bacterial populations residing on healthy and ulcerated mucosae in patients with RAS (recruited using highly stringent criteria) and no associated medical conditions; we also compared these to the bacterial microbiota of healthy controls (HCs). Phylum-level diversity comparisons revealed decreased Firmicutes and increased Proteobacteria in ulcerated sites, as compared with healthy sites in RAS patients, and no differences between RAS patients with healthy sites and HCs. Genus-level analysis demonstrated higher abundance of total Bacteroidales in RAS patients with healthy sites over HCs. Porphyromonadaceae comprising species associated with periodontal disease and Veillonellaceae predominated in ulcerated sites over HCs, while no quantitative differences of these families were observed between healthy sites in RAS patients and HCs. Streptococcaceae comprising species associated with oral health predominated in HCs over ulcerated sites but not in HCs over healthy sites in RAS patients. This study demonstrates that mucosal microbiome changes in patients with idiopathic RAS—namely, increased Bacteroidales species in mucosae of RAS patients not affected by active ulceration. While these changes suggest a microbial role in initiation of RAS, this study does not provide data on causality. Within this limitation, the study contributes to the understanding of the potential role of mucosal microbiome changes in oral mucosal disease.

Expression of Genes for Drug Transporters in the Human Female Genital Tract and Modulatory Effect of Antiretroviral Drugs

Anti-retroviral (ARV) –based microbicides are one of the strategies pursued to prevent HIV-1 transmission. Delivery of ARV drugs to subepithelial CD4+ T cells at concentrations for protection is likely determined by drug transporters expressed in the cervicovaginal epithelium. To define the role of drug transporters in mucosal disposition of topically applied ARV-based microbicides, these must be tested in epithelial cell line-based biopharmaceutical assays factoring the effect of relevant drug transporters. We have characterised gene expression of influx and efflux drug transporters in a panel of cervicovaginal cell lines and compared this to expression in cervicovaginal tissue. We also investigated the effect of dapivirine, darunavir and tenofovir, currently at advanced stages of microbicides development, on expression of drug transporters in cell lines. Expression of efflux ABC transporters in cervical tissue was best represented in HeLa, Ect1/E6E7 and End1/E6E7 cell lines. Expression of influx OCT and ENT transporters in ectocervix matched expression in Hela while expression of influx SLCO transporters in vagina was best reflected in VK2/E6E7 cell line. Stimulation with darunavir and dapivirine upregulated MRP transporters, including MRP5 involved in transport of tenofovir. Dapivirine also significantly downregulated tenofovir substrate MRP4 in cervical cell lines. Treatment with darunavir and dapivirine showed no significant effect on expression of BCRP, MRP2 and P-glycoprotein implicated in efflux of different ARV drugs. Darunavir strongly induced expression in most cell lines of CNT3 involved in cell uptake of nucleotide/nucleoside analogue reverse transcriptase inhibitors and SLCO drug transporters involved in cell uptake of protease inhibitors. This study provides insight into the suitability of cervicovaginal cell lines for assessment of ARV drugs in transport kinetics studies. The modulatory effect of darunavir and dapivirine on expression of drug transporters involved in transport of tenofovir points to the possibility of combining these drugs to improve retention of individual drugs at target tissues.

The admissions process in a graduate-entry dental school: can we predict academic performance?

Aim To assess the association between the admissions performance and subsequent academic achievement within a graduate-entry dental school.Methods The study was conducted at the University of Aberdeen Dental School. UCAS forms for course applicants were reviewed and assigned a pre-admission score (PAS) and a tariff given for the UCAS personal statement (UCAS). Individuals ranked highest were invited to attend multiple mini-interviews (MMI), which were scored. Data was correlated with academic performance reported as the University Common Assessment Scale (0-20). Comparisons were also made between the first degree and subsequent educational achievement.Statistics Data were analysed by multiple linear regression, Pearson correlation and unstacked ANOVA (IBM® SPSS® Statistics 19).Results Data were obtained for 75 students (F: 50; M: 25). A correlation between performance at MMI and CAS scores was identified (r = 0.180, p = 0.001, df = 538). A correlation was also noted between each students first degree and the CAS scores (F = 4.08, p = 0.001, df = 9).Conclusions This study suggests that candidate performance at MMI might be a stronger predictor of academic and clinical performance of graduate-entry dental students compared to other pre-interview selection criteria. The first degree for such a programme also appears to be significant.

Drug transporter gene expression in human colorectal tissue and cell lines: modulation with antiretrovirals for microbicide optimization

OBJECTIVES The objectives of this study were to comprehensively assess mRNA expression of 84 drug transporters in human colorectal biopsies and six representative cell lines, and to investigate the alteration of drug transporter gene expression after exposure to three candidate microbicidal antiretroviral (ARV) drugs (tenofovir, darunavir and dapivirine) in the colorectal epithelium. The outcome of the objectives informs development of optimal ARV-based microbicidal formulations for prevention of HIV-1 infection. METHODS Drug transporter mRNA expression was quantified from colorectal biopsies and cell lines by quantitative real-time PCR. Relative mRNA expression was quantified in Caco-2 cells and colorectal explants after induction with ARVs. Data were analysed using Pearsons product moment correlation (r), hierarchical clustering and principal component analysis (PCA). RESULTS Expression of 58 of the 84 transporters was documented in colorectal biopsies, with genes for CNT2, P-glycoprotein (P-gp) and MRP3 showing the highest expression. No difference was noted between individual subjects when analysed by age, gender or anatomical site (rectum or recto-sigmoid) (r = 0.95-0.99). High expression of P-gp and CNT2 proteins was confirmed by immunohistochemical staining. Similarity between colorectal tissue and cell-line drug transporter gene expression was variable (r = 0.64-0.84). PCA showed distinct clustering of human colorectal biopsy samples, with the Caco-2 cells defined as the best surrogate system. Induction of Caco-2 cell lines with ARV drugs suggests that darunavir-based microbicides incorporating tenofovir may result in drug-drug interactions likely to affect distribution of individual drugs to sub-epithelial target cells. CONCLUSIONS These findings will help optimize complex formulations of rectal microbicides to realize their full potential as an effective approach for pre-exposure prophylaxis against HIV-1 infection.

Predictive value of the admissions process and the UK Clinical Aptitude Test in a graduate-entry dental school

Aim To assess the association between admissions performance and the UK Clinical Aptitude Test (UKCAT), and subsequent achievement within a graduate-entry dental school.Method The study was conducted at the University of Aberdeen Dental School between 2010 and 2014. Student demographics, pre-admission scores (PAS), Universities and Colleges Admissions Service (UCAS) tariffs, multiple mini-interview (MMI) grades, UKCAT scores and percentiles were correlated with academic performance reported as the University Common Assessment Scale (0–20).Statistics Data were analysed by Pearson correlation and multiple regression (IBM® SPSS® Statistics 21).Results Data were obtained for 71 students (F: 44; M: 27). Student age, MMI, UKCAT scores and UKCAT percentiles demonstrated a correlation with CAS scores (r2 = 0.119, P = 0.001, r2 = 0.136, P = 0.001, r2 = 0.077, P = 0.019 and r2 = 0.118, P = 0.001, respectively).Conclusions This study suggests that student age, candidate performance at MMI and the UKCAT might be a predictor of academic achievement for graduate-entry dental students.

Hot topics in necrotising skin and soft tissue infections

Please cite this article as: Kordo Saeed , Silvano Esposito , Ian Gould , Tiziana Ascione , Matteo Bassetti , Eric Bonnet , Emilio Bouza , Monica Chan , Joshua S Davis , Giuseppe De Simone , Matthew Dryden , Thomas Gottlieb , Karolin Hijazi , David C Lye , Pasquale Pagliano , Christina Petridou , Elda Righi , John Segreti , Serhet Unal , Ata Nevzat Yalcin , Hot topics in necrotising skin and soft tissue infections, International Journal of Antimicrobial Agents (2018), doi: 10.1016/j.ijantimicag.2018.02.012

PTU-255 Characterisation of drug transporter gene expression in colorectal tissue and cell lines: induction with anti-retrovirals for microbicide optimisation

Introduction Drug transporter expression in the colorectal epithelium is likely to play a role in the mucosal disposition of anti-retrovirals (ARVs) in rectal microbicidal preparations and impact their efficacy in prevention of HIV-1 infection. However, there is limited information on expression levels available. This study aims to assess expression of 84 drug transporter genes in human colorectal tissue and representative cell lines pre and post ARV exposure. Method Drug transporter mRNA expression was quantified from colorectal biopsies (n = 12) and 6 colorectal cell lines using real time PCR. Relative mRNA expression was quantified in Caco-2 cells after induction with tenofovir (TFV; 5 mM), dapivirine (DPV; 10 µM) and darunavir (DRV; 250 µM) for up to 3 days. Data were analysed using Pearson’s correlation (r), hierarchical clustering and principal component analysis. Results Fifty-eight of the 84 transporters were expressed in colorectal tissue. SLC28A2/CNT2 was the most expressed uptake transporter (>25 fold increase) followed by efflux transporters ABCB1/P-gp and ABCC3/MRP3 (4–5 fold increase). No difference was noted between individual patients, either sexes or biopsy sites (rectum or sigmoid) (r = 0.95–0.99). Similarities between tissue and cell lines were low (r values 0.67–0.81). Principal component analysis showed distinct clustering of colorectal tissue and cell lines with Caco-2 cells showing least variance with tissues. Immunohistochemical staining of 7 colorectal biopsy sections confirmed high expression of Pgp and CNT2 drug transporters. TFV stimulation of Caco-2 cells resulted in > 2 fold increase in expression of MRP5, LAT2, OATPE and GLUT1 mRNA. DPV stimulation resulted in > 2 fold increase in expression of GLUT1, PEPT1 and ABCA1. DRV stimulation resulted in > 2 fold increase in expression of SLC3A2, OATPE and MCT3 mRNA. Conclusion This study has enumerated drug transporter mRNA expression in colorectal tissue and cell lines. There was partial correlation between cell lines and tissue limiting cell line use as in vivosurrogate. TFV and DPV did not induce majority of efflux transporters in Caco-2 cells. This would entail increased retention of drug within the mucosa and as a result enhanced delivery to CD4 T cells. Further studies will elucidate whether combination of ARVs will be similarly effective. Disclosure of interest None Declared.

Draft Whole-Genome Sequences of Periodontal Pathobionts Porphyromonas gingivalis, Prevotella intermedia, and Tannerella forsythia Contain Phase-Variable Restriction-Modification Systems.

ABSTRACT Periodontal disease comprises mild to severe inflammatory host responses to oral bacteria that can cause destruction of the tooth-supporting tissue. We report genome sequences for 18 clinical isolates of Porphyromonas gingivalis, Prevotella intermedia, and Tannerella forsythia, Gram-negative obligate anaerobes that play a role in the periodontal disease process.

Transporters for Antiretroviral Drugs in Colorectal CD4+ T Cells and Circulating α4β7 Integrin CD4+ T Cells: Implications for HIV Microbicides.

CD4+ T lymphocytes in the colorectal mucosa are key in HIV-1 transmission and dissemination. As such they are also the primary target for antiretroviral (ARV)-based rectal microbicides for pre-exposure prophylaxis. Drug transporters expressed in mucosal CD4+ T cells determine ARV distribution across the cell membrane and, most likely, efficacy of microbicides. We describe transporters for antiretroviral drugs in colorectal mucosal CD4+ T lymphocytes and compare gene expression with circulating α4β7+CD4+ T cells, which traffic to the intestine and have been shown to be preferentially infected by HIV-1. Purified total CD4+ T cells were obtained from colorectal tissue and blood samples by magnetic separation. CD4+ T cells expressing α4β7 integrin were isolated by fluorescence-activated cell sorting from peripheral blood mononuclear cells of healthy volunteers. Expressions of 15 efflux and uptake drug transporter genes were quantified using Taqman qPCR assays. Expression of efflux transporters MRP3, MRP5, and BCRP and uptake transporter CNT2 were significantly higher in colorectal CD4+ T cells compared to circulating CD4+ T cells (p = 0.01-0.03). Conversely, circulating α4β7+CD4+ T cells demonstrated significantly higher expression of OATPD compared to colorectal CD4+ T cells (p = 0.001). To the best of our knowledge this is the first report of drug transporter gene expression in colorectal CD4+ and peripheral α4β7+CD4+ T cells. The qualitative and quantitative differences in drug transporter gene expression profiles between α4β7+CD4+ T cells and total mucosal CD4+ T cells may have significant implications for the efficacy of rectally delivered ARV-microbicides. Most notably, we have identified efflux drug transporters that could be targeted by selective inhibitors or beneficial drug-drug interactions to enhance intracellular accumulation of antiretroviral drugs.