Disulfide bond-mediated dimerization of HLA-G on the cell surface

HLA-G is a nonclassical class I MHC molecule with an unknown function and with unusual characteristics that distinguish it from other class I MHC molecules. Here, we demonstrate that HLA-G forms disulfide-linked dimers that are present on the cell surface. Immunoprecipitation of HLA-G from surface biotinylated transfectants using the anti-beta2-microglobulin mAb BBM.1 revealed the presence of an approximately equal 78-kDa form of HLA-G heavy chain that was reduced by using DTT to a 39-kDa form. Mutation of Cys-42 to a serine completely abrogated dimerization of HLA-G, suggesting that the disulfide linkage formed exclusively through this residue. A possible interaction between the HLA-G monomer or dimer and the KIR2DL4 receptor was also investigated, but no interaction between these molecules could be detected through several approaches. The cell-surface expression of dimerized HLA-G molecules may have implications for HLA-Greceptor interactions and for the search for specific receptors that bind HLA-G.     

Subcellular trafficking of HPMA copolymer-Tat conjugates in human ovarian carcinoma cells.

One of the main obstacles to efficient intracellular delivery of therapeutic macromolecules is the barrier posed by the plasma membrane. In this study, the cell penetrating peptide Tat was conjugated to a synthetic macromolecule based on N-(2-hydroxypropyl)methacrylamide (HPMA) and its subcellular distribution in human ovarian carcinoma cell lines was studied. The Tat peptide mediated uptake resulted in cytoplasmic and nuclear localization and was found to be energy independent. Time and concentration studies verified the rapidity and dependence of the transport process on these parameters. Enhanced uptake of a polymer bound anticancer drug doxorubicin was also demonstrated. These results were corroborated independently by subcellular fractionation.     

Eight‐channel transceiver RF coil array tailored for 1H/19F MR of the human knee and fluorinated drugs at 7.0 T

The purpose of this study was to evaluate the feasibility of an eight‐channel dual‐tuned transceiver surface RF coil array for combined ¹H/¹⁹F MR of the human knee at 7.0 T following application of ¹⁹F‐containing drugs. The ¹H/¹⁹F RF coil array includes a posterior module with two ¹H loop elements and two anterior modules, each consisting of one ¹H and two ¹⁹F elements. The decoupling of neighbor elements is achieved by a shared capacitor. Electromagnetic field simulations were performed to afford uniform transmission fields and to be in accordance with RF safety guidelines. Localized ¹⁹F MRS was conducted with 47 and 101 mmol/L of flufenamic acid (FA) – a ¹⁹F‐containing non‐steroidal anti‐inflammatory drug – to determine T₁ and T₂ and to study the ¹⁹F signal‐to‐dose relationship. The suitability of the proposed approach for ¹H/¹⁹F MR was examined in healthy subjects. Reflection coefficients of each channel were less than ?17 dB and coupling between channels was less than ?11 dB. Q_L/Q_U was less than 0.5 for all elements. MRS results demonstrated signal stability with 1% variation. T₁ and T₂ relaxation times changed with concentration of FA: T₁/T₂ = 673/31 ms at 101 mmol/L and T₁/T₂ = 616/26 ms at 47 mmol/L. A uniform signal and contrast across the patella could be observed in proton imaging. The sensitivity of the RF coil enabled localization of FA ointment administrated to the knee with an in‐plane spatial resolution of (1.5 × 1.5) mm² achieved in a total scan time of approximately three minutes, which is well suited for translational human studies. This study shows the feasibility of combined ¹H/¹⁹F MRI of the knee at 7.0 T and proposes T₁ and T₂ mapping methods for quantifying fluorinated drugs in vivo. Further technological developments are necessary to promote real‐time bioavailability studies and quantification of ¹⁹F‐containing medicinal compounds in vivo. Copyright © 2015 John Wiley & Sons, Ltd.     

Rapid detection and differentiation of the exfoliative toxin A-producing Staphylococcus aureus strains based on ϕETA prophage polymorphisms

The exfoliative toxin A (ETA) is encoded by the gene located on Staphylococcus aureus prophages. We have developed a single-reaction multiplex polymerase chain reaction (PCR) assay for rapid and specific detection of various ?ETA prophages of serogroup B responsible for dissemination of eta gene and ETA production in clinical strains. This PCR strategy enabled to classify the ETA-positive strains into 6 groups designated ETA-B1, ETA-B2, ETA-B3, ETA-B4, ETA-B5, and ETA-B6. The method was tested on a diverse set of 101 ETA and/or ETB-positive S. aureus strains isolated in 22 Czech maternity hospitals and 1 Slovak maternity hospital between 1998 and 2009. This novel PCR strategy is reliable in the rapid identification of yet undescribed ETA-converting B prophages and differentiation of the closely related ETA-positive strains, and it is a convenient tool for hospital epidermolytic infection control.     

Use of polysaccharide hemostatic agent (HaemoCer™) in breast cancer surgery to reduce postoperative complications: A randomised controlled trial

Postoperative wound-site bleeding, tissue inflammation and seroma formation are well-known complications in the field of breast surgery. Hemostatic agents consisting of polysaccharides may be used intra-operatively to minimise postoperative complications. We conducted a prospective randomised-controlled, single-centre study including 136 patients undergoing breast-conserving surgery for invasive or intraductal breast cancer. Of these, 68 patients were randomised to receive an absorbable polysaccharide hemostatic agent into the wound site during surgery, while 68 patients were randomised to the control group and did not receive any hemostatic agent. Primary outcome was the total volume of postoperative drained fluid from the surgical site. Secondary outcomes were the number of days until drain removal and rate of immediate postoperative surgical site infection Patients in the intervention group had significantly higher drainage output volumes compared with the control group 85 mL (IQR 46.25–110) versus 50 mL (IQR 30–75), respectively; (P = .003). Univariable linear regression analyses showed a significant association between the surgical specimen and the primary outcome (P < .001). After multivariable analysis, the use of absorbable polysaccharide hemostatic product was no longer significantly associated with a higher drainage output and only the size of the surgical specimen remained a significant predictor. The number of days until drainage removal and the postoperative seroma formation were higher in the intervention group (P = .004) and (P = .003), respectively. In our study, intraoperative application of polysaccharide hemostatic agent during breast-conserving surgery did not decrease postoperative fluid production. Only the size of the surgical specimen was significantly associated with postoperative drainage volume.