Gingival cell growth with antiresorptive treatment combined with corticosteroids or antiestrogen

Objectives: Antiresorptive treatment has been shown to impair mucosal cell proliferation, migration, and viability. However, in the clinic, antiresorptives are often used in combination with other drugs. We studied the effect of antiresorptives combined with a corticosteroid or antiestrogen on oral mucosal keratinocytes and fibroblasts. Material and methods: Human gingival keratinocyte and fibroblast cell lines were exposed to bisphosphonates (BPs) and denosumab in different concentrations and durations together with an antiestrogen or corticosteroid. Changes in cell viability, proliferation and migration after exposures were measured. Data were evaluated with hierarchical linear mixed model for repeated measurements. Results: Bisphosphonate exposure suppressed keratinocyte and fibroblast cell viability, proliferation, and migration in a time-dependent manner. Combining a corticosteroid or antiestrogen with BPs further increased this negative effect. Denosumab alone had a mild positive effect on keratinocyte and fibroblast growth. When denosumab was combined with a corticosteroid or antiestrogen, cell growth was suppressed. Conclusions: Our results show that coexisting medications may increase the negative impact of BPs or denosumab on oral mucosal cells.Peer reviewe

Spacer length, label moiety interchange and probe pair orientation in a homogeneous solid-phase hybridization assay utilizing lanthanide chelate complementation

We have studied parameters affecting DNA hybridization and lanthanide chelate complementation based signal formation in a separation-free solid-phase assay. This binary probe assay system consists of two probes labeled either with a europium carrier chelate or a light harvesting antenna ligand. One probe was immobilized on the microtiter well bottom in spot format while the other probe was free in solution. The probe concentration used in spotting, spacer length, and the choice and orientation of the either 3&acute; or 5&acute;-end immobilized probe had significant impact on signal-to-background (S/B) ratios. The highest ratio was achieved by saturating the spot with the 5&acute;-end immobilized antenna ligand probe separated from the solid support with a 25 nucleotide poly dT spacer. The obtained detection limit of 18 pM for synthetic Pseudomonas aeruginosa heat shock protein groES gene sequence was close to a 20-fold improvement compared to the previous assay. The dynamic range of the assay was three orders of magnitude.</p

Simultaneous detection of Human Immunodeficiency Virus 1 and Hepatitis B virus infections using a dual-label time-resolved fluorometric assay

A highly specific and novel dual-label time-resolved immunofluorometric assay was developed exploiting the unique emission wavelengths of the intrinsically fluorescent terbium (Tb3+) and europium (Eu3+) tracers for the simultaneous detection of human immunodeficiency virus 1 (HIV-1) and hepatitis B virus (HBV) infections, respectively. HIV-1 infection was detected using a double antigen sandwich format wherein anti-HIV-1 antibodies were captured using an in vivo biotinylated version of a chimeric HIV-1 antigen and revealed using the same antigen labeled with Tb3+ chelate. Hepatitis B surface antigen (HBsAg), which served as the marker of HBV infection, was detected in a double antibody sandwich using two monoclonal antibodies (mAbs), one chemically biotinylated to capture, and the other labeled with Eu3+ nanoparticles, to reveal. The performance of the assay was evaluated using a collection (n = 60) of in-house and commercially available human sera panels. This evaluation showed the dual-label assay to possess high degrees of specificity and sensitivity, comparable to those of commercially available, single analyte-specific kits for the detection of HBsAg antigen and anti-HIV antibodies. This work demonstrates the feasibility of developing a potentially time- and resource-saving multiplex assay for screening serum samples for multiple infections in a blood bank setting

Deep neck space infections: an upward trend and changing characteristics

Purpose This study reviews our experience with deep neck space infections (DNIs) requiring surgical intervention, includingcervical necrotizing fasciitis. The aim of the study was to identify predisposing and aggravating factors of the disease andrecognize the possible factors that can lead to life-threatening complications and slow down the healing process.Methods We compare the results to previous data from 1985 to 2005 to fnd possible alterations and changing trends. Thecharacteristics of four lethal cases are described. This retrospective analysis includes patient data from 2004 to 2015 intertiary referral hospital and in total, 277 patients were found.Results Surgical drainage through a neck opening±intraoral incision was made in 215 (77.6%) patients, an intraoral incision was only made in 62 patients (22.4%). ICU care was needed in 66 (23.8%) cases. Odontogenic etiology (44.8%) wasthe most common origin. The most common comorbidity was a psychiatric disorder and/or dementia and occurred in 55(19.9%) patients. Patients with underlying illnesses were more likely to be admitted to the ICU (p=0.020), required a longerICU stay (p=0.004) and repeated surgery (p=0.009). Gas formation seemed to be predictive of a more severe course ofinfection. Early extraction of the odontogenic foci was related to a lower length of stay (LOS) (p=0.039).Conclusion The annual numbers have risen from 14 to 24 cases per year when compared to previous data. DNIs remain cause of lethal complications; the mortality was 1.4% and overall complications occurred in 61 (22.0%) patients.</p

Escherichia coli–expressed near full length HIV-1 envelope glycoprotein is a highly sensitive and specific diagnostic antigen

Background: The Human Immunodeficiency Virus type 1 (HIV-1) envelope glycoprotein gp160, useful in detecting anti-HIV-1 antibodies, is difficult to express in heterologous hosts. The major hurdles are its signal sequence, strong hydrophobic regions and heavy glycosylation. While it has not been possible to express full length recombinant (r)-gp160 in E. coli, it can be expressed in insect and mammalian cells, but at relatively higher cost. In this work, we report E. coli-based over-expression of r-gp160 variant and evaluate its performance in diagnostic immunoassays for the detection of anti-HIV-1 antibodies. Methods: A deletion variant of r-gp160 lacking hydrophobic regions of the parent full length molecule was expressed in E. coli and purified to near homogeneity using single-step Ni (II)-affinity chromatography. Biotinylated and europium (III) chelate-labeled versions of this antigen were used to set up one- and two-step time-resolved fluorometric double antigen sandwich assays. The performance of these assays was evaluated against a collection of well-characterized human sera (n = 131), that included an in-house panel and four commercially procured panels. Results: In-frame deletion of three hydrophobic regions, spanning amino acid residues 1–43, 519–538 and 676–706, of full length HIV-1 gp160 resulted in its expression in E. coli. Both the one- and two-step assays manifested high sensitivity unambiguously identifying 75/77 and 77/77 HIV-1 positive sera, respectively. Both assays also identified all 52 HIV-seronegative sera correctly. Between the two assays, the mean signal-to-cutoff value of the two-step assay was an order of magnitude greater than that of the one-step assay. Both assays were highly specific manifesting no cross-reactivity towards antibodies specific to other viruses like hepatitis B, C and human T cell leukemia viruses. Conclusions: This study has demonstrated the expression of r-gp160 variant in E. coli, by deletion of hydrophobic regions, and its purification in reasonable yields. This underscores the potential for cost saving in antigen production. Evaluation of this antigen in a double antigen sandwich two-step assay showed it to be a highly sensitive and specific HIV-1 diagnostic reagent. The amenability of this assay to the one-step format suggests its potential utility in developing a rapid point-of-care HIV-1 diagnostic test

Upconversion FRET quantitation: the role of donor photoexcitation mode and compositional architecture on the decay and intensity based responses

Lanthanide-doped colloidal nanoparticles capable of photon upconversion (UC) offer long luminescence lifetimes, narrowband absorption and emission spectra, and efficient anti-Stokes emission. These features are highly advantageous for Forster Resonance Energy Transfer (FRET) based detection. Upconverting nanoparticles (UCNPs) as donors may solve the existing problems of molecular FRET systems, such as photobleaching and limitations in quantitative analysis, but these new labels also bring new challenges. Here we have studied the impact of the core-shell compositional architecture of upconverting nanoparticle donors and the mode of photoexcitation on the performance of UC-FRET from UCNPs to Rose Bengal (RB) molecular acceptor. We have quantitatively compared luminescence rise and decay kinetics of Er3+ emission using core-only NaYF4: 20% Yb, 2% Er and core-shell NaYF4: 20% Yb @ NaYF4: 20% Yb, 5% Er donor UCNPs under three photoexcitation schemes: (1) direct short-pulse photoexcitation of Er3+ at 520 nm; indirect photoexcitation of Er3+ through Yb3+ sensitizer with (2) 980 nm short (5-7 ns) or (3) 980 nm long (4 ms) laser pulses. The donor luminescence kinetics and steady-state emission spectra differed between the UCNP architectures and excitation schemes. Aiming for highly sensitive kinetic upconversion FRET-based biomolecular assays, the experimental results underline the complexity of the excitation and energy-migration mechanisms affecting the Er3+ donor responses and suggest ways to optimize the photoexcitation scheme and the architecture of the UCNPs used as luminescent donors

Supersensitive photon upconversion based immunoassay for detection of cardiac troponin I in human plasma

Background and aims: Upconverting nanoparticles (UCNPs) are attractive reporters for immunoassays due to their excellent detectability. Assays sensitive enough to measure baseline level of cardiac troponin I cTnI in healthy population could be used to identify patients at risk for cardiovascular disease. Aiming for a cTnI assay of such sensitivity, the surface chemistry of the nanoparticles as well as the assay reagents and the protocol were optimized for monodispersity of the UCNP antibody conjugates (Mab UCNPs) and to minimize their non-specific interactions with the solid support.Materials and methods: UCNPs were coated with poly(acrylic acid) via two-step ligand exchange and conjugated with monoclonal antibodies. The conjugates were applied in a microplate-based sandwich immunoassay using a combination of two capture antibodies to detect cTnI. Assay was evaluated according to guidelines of Clinical & Laboratory Standards Institute. Results: The limit of detection and limit of blank of the assay were 0.13 ng/L and 0.01 ng/L cTnI, respectively. The recoveries were >90% in spiked plasma in the linear range. The within- and between-run imprecisions were Conclusion: The results demonstrate that UCNPs enable quantification of cTnI concentrations expected in plasma of healthy individuals and could be used to identify patients at risk for cardiovascular disease.</p