Triage of Women with Minor Cervical Lesions: Data Suggesting a “Test and Treat” Approach for HPV E6/E7 mRNA Testing

Background

Human papillomavirus (HPV) testing is included in the cervical cancer screening program in the triage of women with equivocal (ASC-US) or low-grade (LSIL) cytological lesions. These women have an increased risk for developing high grade dysplasia and cancer (CIN2+) compared to women with normal cytology. However, in order to avoid unnecessary follow-up, as well as overtreatment, a high positive predictive value (PPV) of the triage test is important.

Methodology/Principal Findings

The HPV test PreTect HPV-Proofer, detecting E6/E7 mRNA from the HPV types 16, 18, 31, 33 and 45, is used as triage test together with repeat cytology. PPV data for HPV E6/E7 mRNA testing during the period from January 2006 up to June 2009 are reported. In total, 406 of 2099 women (19.3%) had a positive HPV test result. Of the women with a positive test result and with a histological diagnosis (n = 347), 243 women had histological high-grade dysplasia or cancer (CIN2+), giving a PPV of 70.0% (95% confidence interval [CI], 65.2%–74.8%). For HPV 16 or HPV 33 positive women above 40 years of age, the PPV was 83.7% (95% CI, 73.3%–94.0%) and 84.6% (95% CI, 65.0%–100.0%) respectively. The PPV of test positive women with HSIL cytology was 94.2% (95% CI, 88.7%–99.7%).

Conclusions

When the result in triage is HPV mRNA positive, our data suggest direct treatment for women above 40 years of age or for women with a concurrent cytological HSIL diagnosis, contributing to better clinical safety for these women. In addition, by decreasing the time to treatment, thereby reducing the number of recalls, the patient management algorithm will be considerably improved, in turn reducing follow-up costs as well as unnecessary psychological stress among patients.     

Binding of cell-penetrating penetratin peptides to plasma membrane vesicles correlates directly with cellular uptake

Cell-penetrating peptides (CPPs) gain access to intracellular compartments mainly via endocytosis and have capacity to deliver macromolecular cargo into cells. Although the involvement of various endocytic routes has been described it is still unclear which interactions are involved in eliciting an uptake response and to what extent affinity for particular cell surface components may determine the efficiency of a particular CPP. Previous biophysical studies of the interaction between CPPs and either lipid vesicles or soluble sugar-mimics of cell surface proteoglycans, the two most commonly suggested CPP binding targets, have not allowed quantitative correlations to be established. We here explore the use of plasma membrane vesicles (PMVs) derived from cultured mammalian cells as cell surface models in biophysical experiments. Further, we examine the relationship between affinity for PMVs and uptake into live cells using the CPP penetratin and two analogs enriched in arginines and lysines respectively. We show, using centrifugation to sediment PMVs, that the amount of peptide in the pellet fraction correlates linearly with the degree of cell internalization and that the relative efficiency of all-arginine and all-lysine variants of penetratin can be ascribed to their respective cell surface affinities. Our data show differences between arginine- and lysine-rich variants of penetratin that has not been previously accounted for in studies using lipid vesicles. Our data also indicate greater differences in binding affinity to PMVs than to heparin, a commonly used cell surface proteoglycan mimic. Taken together, this suggests that the cell surface interactions of CPPs are dependent on several cell surface moieties and their molecular organization on the plasma membrane.     

Binding of the mannose-specific lectin, griffithsin, to HIV-1 gp120 exposes the CD4-binding site

The glycans on HIV-1 gp120 play an important role in shielding neutralization-sensitive epitopes from antibody recognition. They also serve as targets for lectins that bind mannose-rich glycans. In this study, we investigated the interaction of the lectin griffithsin (GRFT) with HIV-1 gp120 and its effects on exposure of the CD4-binding site (CD4bs). We found that GRFT enhanced the binding of HIV-1 to plates coated with anti-CD4bs antibodies b12 and b6 or the CD4 receptor mimetic CD4-IgG2. The average enhancement of b12 or b6 binding was higher for subtype B viruses than for subtype C, while for CD4-IgG2, it was similar for both subtypes, although lower than observed with antibodies. This GRFT-mediated enhancement of HIV-1 binding to b12 was reflected in synergistic neutralization for 2 of the 4 viruses tested. The glycan at position 386, which shields the CD4bs, was involved in both GRFT-mediated enhancement of binding and neutralization synergism between GRFT and b12. Although GRFT enhanced CD4bs exposure, it simultaneously inhibited ligand binding to the coreceptor binding site, suggesting that GRFT-dependent enhancement and neutralization utilize independent mechanisms. This study shows for the first time that GRFT interaction with gp120 exposes the CD4bs through binding the glycan at position 386, which may have implications for how to access this conserved site.     

Development and delivery of patient treatment in the Trondheim Hip Fracture Trial. A new geriatric in-hospital pathway for elderly patients with hip fracture

ABSTRACT: BACKGROUND: Hip fractures are common among frail elderly persons and often have serious consequences on function, mobility and mortality. Traditional treatment of these patients is performed in orthopedic departments without additional geriatric assessment. However, studies have shown that interdisciplinary geriatric treatment may be beneficial compared to traditional treatment. The aim of the present study is to investigate whether treatment of these patients in a Department of Geriatrics (DG) during the entire hospital stay gives additional benefits as compared to conventional treatment in a Department of Orthopaedic Surgery (DOS). FINDINGS: A new clinical pathway for in-hospital treatment of hip fracture patients was developed. In this pathway patients were treated pre-and postoperatively in DG. Comprehensive geriatric assessment was performed as an interdisciplinary, multidimensional, systematic assessment of all patients focusing on each patient's capabilities and limitations as recommended in guidelines and systematic reviews. Identification and treatment of co-morbidities, pain relief, hydration, oxygenation, nutrition, elimination, prevention and management of delirium, assessment of falls and osteoporosis were emphasized. Discharge planning started as early as possible. Initiation of rehabilitation with focus on early mobilisation and development of individual plans was initiated in hospital and continued after discharge from hospital. Fracture specific treatment was based upon standard treatment for the hospital, expert opinions and a review of the literature. CONCLUSION: A new treatment program for old hip fracture patients was developed, introduced and run in the DG, the potential benefits of which being compared with traditional care of hip fracture patients in the DOS in a randomised clinical trial.     

Stabilizing mutations increase secretion of functional soluble TCR-Ig fusion proteins

Background  

Whereas T cell receptors (TCRs) detect peptide/major histocompatibility complexes (pMHCs) with exquisite specificity, there are challenges regarding their expression and use as soluble detection molecules due to molecular instability. We have investigated strategies for the production of TCR-immunoglobulin (Ig) fusion proteins. Two different TCRs that are characteristic of a mouse model for idiotype (Id) dependent immune regulation were engineered. They are structurally unrelated with different variable (V), diversity (D) and joining (J) segments, but each share one V gene segment, either V_α or V_β, with the well characterized murine TCR, 2C.     

Are Pharmaceuticals with Evolutionary Conserved Molecular Drug Targets More Potent to Cause Toxic Effects in Non-Target Organisms?

The ubiquitous use of pharmaceuticals has resulted in a continuous discharge into wastewater and pharmaceuticals and their metabolites are found in the environment. Due to their design towards specific drug targets, pharmaceuticals may be therapeutically active already at low environmental concentrations. Several human drug targets are evolutionary conserved in aquatic organisms, raising concerns about effects of these pharmaceuticals in non-target organisms. In this study, we hypothesized that the toxicity of a pharmaceutical towards a non-target invertebrate depends on the presence of the human drug target orthologs in this species. This was tested by assessing toxicity of pharmaceuticals with (miconazole and promethazine) and without (levonorgestrel) identified drug target orthologs in the cladoceran Daphnia magna. The toxicity was evaluated using general toxicity endpoints at individual (immobility, reproduction and development), biochemical (RNA and DNA content) and molecular (gene expression) levels. The results provide evidence for higher toxicity of miconazole and promethazine, i.e. the drugs with identified drug target orthologs. At the individual level, miconazole had the lowest effect concentrations for immobility and reproduction (0.3 and 0.022 mg L⁻¹, respectively) followed by promethazine (1.6 and 0.18 mg L⁻¹, respectively). At the biochemical level, individual RNA content was affected by miconazole and promethazine already at 0.0023 and 0.059 mg L⁻¹, respectively. At the molecular level, gene expression for cuticle protein was significantly suppressed by exposure to both miconazole and promethazine; moreover, daphnids exposed to miconazole had significantly lower vitellogenin expression. Levonorgestrel did not have any effects on any endpoints in the concentrations tested. These results highlight the importance of considering drug target conservation in environmental risk assessments of pharmaceuticals.     

Staphylococcal surface display of immunoglobulin A (IgA)- and IgE-specific in vitro-selected binding proteins (affibodies) based on Staphylococcus aureus protein A.

An expression system designed for cell surface display of hybrid proteins on Staphylococcus carnosus has been evaluated for the display of Staphylococcus aureus protein A (SpA) domains, normally binding to immunoglobulin G (IgG) Fc but here engineered by combinatorial protein chemistry to yield SpA domains, denoted affibodies, with new binding specificities. Such affibodies, with human IgA or IgE binding activity, have previously been selected from a phage library, based on an SpA domain. In this study, these affibodies have been genetically introduced in monomeric or dimeric forms into chimeric proteins expressed on the surface of S. carnosus by using translocation signals from a Staphylococcus hyicus lipase construct together with surface-anchoring regions of SpA. The recombinant surface proteins, containing the IgA- or IgE-specific affibodies, were demonstrated to be expressed as full-length proteins, localized and properly exposed at the cell surface of S. carnosus. Furthermore, these chimeric receptors were found to be functional, since recombinant S. carnosus cells were shown to have gained IgA and IgE binding capacity, respectively. In addition, a positive effect in terms of IgA and IgE reactivity was observed when dimeric versions of the affibodies were present. Potential applications for recombinant bacteria with redirected binding specificity in their surface proteins are discussed.