Psychometric validation of the Croatian version of the Quality of Life in Epilepsy Inventory (QOLIE-31)

The primary goals of this study were to adapt the Quality of Life in Epilepsy Inventory-31 items (QOLIE-31) questionnaire to the Croatian language and to assess the translated questionnaire's psychometric properties. Translation/retranslation of the English version of the QOLIE-31 was done, and all steps for cross-cultural adaptation process were performed properly by an expert committee. Later, QOLIE-31 questionnaires and previously validated Short Form-36 (SF-36) outcome instruments were given to 200 patients with epilepsy. 172 patients (86%) responded to the first set of questionnaires, and 114 of the first time respondents (66%) returned their second survey. The two measures of reliability as internal consistency and reproducibility were determined by Cronbach alpha statistics and intraclass correlation coefficient, respectively. Concurrent validity was measured by comparing with a SF-36 questionnaire, and measurement was made using the Pearson correlation coefficient (r). The study demonstrated satisfactory internal consistency with high Cronbach a values for all of the corresponding domains (seizure worry 0.84, medication effects 0.80, emotional well-being 0.73, energy/fatigue 0.76, cognitive functioning 0.71, social functioning 0.77, overall quality of life 0.65). The intraclass correlation coefficient for six domains of QOLIE-31 questionnaire demonstrated excellent test/retest reproducibility (ICC > or = 0.75), and good test/retest reproducibility (ICC 0.71) in one domain (cognitive functioning). Considering concurrent validity, three domains had excellent correlation (r = 0.75-1), while 11 had good correlation (r = 0.50 to 0.75), and 3 had moderate correlation (r = 0.25-0.50). This study demonstrated that, if measures are to be used across cultures, the items must not only be translated well linguistically but also must be culturally adapted to maintain the content validity of the instrument at a conceptual level across different cultures. Croatian version of QOLIE-31 will be a valuable contribution to outcome measurement in epilepsy patients, particularly in the context of treatment trials, but als in a wider research context.     

Cys-Ph-TAHA: a lanthanide binding tag for RDC and PCS enhanced protein NMR

Here we present Cys-Ph-TAHA, a new nonadentate lanthanide tag for the paramagnetic labelling of proteins. The tag can be easily synthesized and is stereochemically homogenous over a wide range of temperatures, yielding NMR spectra with a single set of peaks. Bound to ubiquitin, it induced large residual dipolar couplings and pseudocontact shifts that could be measured easily and agreed very well with the protein structure. We show that Cys-Ph-TAHA can be used to label large proteins that are biochemically challenging such as the Lac repressor in a 90 kDa ternary complex with DNA and inducer.     

Protective effect of olive leaf extract on hippocampal injury induced by transient global cerebral ischemia and reperfusion in Mongolian gerbils

The beneficial effects of antioxidant nutrients, as well as complex plant extracts, in cerebral ischemia/reperfusion brain injury are well known. Mediterranean diet, rich in olive products, is associated with lower incidence of cardiovascular disease, cancer, inflammation and stroke. In this study, the possible neuroprotective effect of standardized dry olive leaf extract (OLE) is investigated for the first time. Transient global cerebral ischemia in Mongolian gerbils was used to investigate the OLE effects on different parameters of oxidative stress and neuronal damage in hippocampus. The biochemical measurements took place at different time points (80 min, 2, 4 and 24 h) after reperfusion. The effects of applied OLE were compared with effects of quercetin, a known neuroprotective plant flavonoid. Pretreatment with OLE (100 mg/kg, per os) significantly inhibited production of superoxide and nitric oxide, decreased lipid peroxidation, and increased superoxide dismutase activity in all time points examined. Furthermore, OLE offered histological improvement as seen by decreasing neuronal damage in CA1 region of hippocampus. The effects of applied OLE were significantly higher than effects of quercetin (100 mg/kg, per os). Our results indicate that OLE exerts a potent neuroprotective activity against neuronal damage in hippocampus after transient global cerebral ischemia, which could be attributed to its antioxidative properties.     

Sterically congested quinone methides in photodehydration reactions of 4-hydroxybiphenyl derivatives and investigation of their antiproliferative activity

In aqueous media, photochemical excitation (S(1)) of hydroxyadamantyl, diphenylhydroxymethyl, and hydroxypropyl derivatives of 4-phenylphenol 5-9 leads to solvent-assisted deprotonation of the phenol OH, and protonation of the benzyl alcohol coupled with dehydration, that delivers quinone methides (QMs) 14-18. The QMs react with CH(3)OH converting them in high quantum yields to the photosolvolysis products (overall Φ～ 0.1-0.5). QMs were characterized by laser flash photolysis in CH(3)CN-H(2)O and TFE. In TFE, the zwitterionic QM 15 has a lifetime of 250 ns, whereas para QMs 16 and 17 have lifetimes of 500 μs and 1.1 s, respectively. Introduction of the steric hindrance to the parent QM structure (with the adamantyl moiety), or additional stabilization by two phenyl rings, results in an increase of QM lifetimes and selectivity in the reactions with nucleophiles. In vitro studies of the antiproliferative activity of photochemically generated QMs 15-17 were carried out on one human cancer cell line. Irradiation of cells incubated with 7 showed enhanced antiproliferative activity compared to cells that were not irradiated, in accordance with the activity being due to the formation of QM 16.     

Serine and metalloprotease signaling through PAR1 in arterial thrombosis and vascular injury

Thrombin-dependent platelet activation has been shown to be important in the setting of angioplasty and stenting, which may cause ischemic complications including acute myocardial infarction and death. Inhibitors of the high-affinity thrombin receptor, protease-activated receptor 1 (PAR1), are now being evaluated in clinical trials for safety and efficacy in patients with atherothrombotic disease. However, it is unknown whether chronic inhibition of PAR1 in these large patient populations will have beneficial or possibly adverse effects on other biologic processes involved in blood vessel homeostasis and the response to vascular injury. Most recently, PAR1 was found to be cleaved at a distinct site by matrix metalloprotease-1 (MMP-1) to create a longer tethered ligand, which activates a distinct spectrum of G protein pathways in platelets. The differential activation by serine proteases such as thrombin and the metalloprotease MMP-1, places the protease receptor PAR1 at the junction of two major protease classes critically involved in thrombosis, matrix remodeling, and the response to vascular injury.     

EPS8 Inhibition Increases Cisplatin Sensitivity in Lung Cancer Cells

Cisplatin, a commonly used chemotherapeutic, is associated with ototoxicity, renal toxicity and neurotoxicity, thus identifying means to increase the therapeutic index of cisplatin may allow for improved outcomes. A SNP (rs4343077) within EPS8, discovered through a genome wide association study of cisplatin-induced cytotoxicity and apoptosis in lymphoblastoid cell lines (LCLs), provided impetus to further study this gene. The purpose of this work was to evaluate the role of EPS8 in cellular susceptibility to cisplatin in cancerous and non-cancerous cells. We used EPS8 RNA interference to determine the effect of decreased EPS8 expression on LCL and A549 lung cancer cell sensitivity to cisplatin. EPS8 knockdown in LCLs resulted in a 7.9% increase in cisplatin-induced survival (P = 1.98×10⁻⁷) and an 8.7% decrease in apoptosis (P = 0.004) compared to control. In contrast, reduced EPS8 expression in lung cancer cells resulted in a 20.6% decrease in cisplatin-induced survival (P = 5.08×10⁻⁵). We then investigated an EPS8 inhibitor, mithramycin A, as a potential agent to increase the therapeutic index of cisplatin. Mithramycin A decreased EPS8 expression in LCLs resulting in decreased cellular sensitivity to cisplatin as evidenced by lower caspase 3/7 activation following cisplatin treatment (42.7%±6.8% relative to control P = 0.0002). In 5 non-small-cell lung carcinoma (NSCLC) cell lines, mithramycin A also resulted in decreased EPS8 expression. Adding mithramycin to 4 NSCLC cell lines and a bladder cancer cell line, resulted in increased sensitivity to cisplatin that was significantly more pronounced in tumor cell lines than in LCL lines (p<0.0001). An EGFR mutant NSCLC cell line (H1975) showed no significant change in sensitivity to cisplatin with the addition of mithramycin treatment. Therefore, an inhibitor of EPS8, such as mithramycin A, could improve cisplatin treatment by increasing sensitivity of tumor relative to normal cells.     

Regulation of cytoskeletal mechanics and cell growth by myosin light chain phosphorylation

The role ofmyosin light chain phosphorylation in regulating the mechanicalproperties of the cytoskeleton was studied in NIH/3T3 fibroblastsexpressing a truncated, constitutively active form of smooth musclemyosin light chain kinase (tMK). Cytoskeletal stiffness determined byquantifying the force required to indent the apical surface of adherentcells showed that stiffness was increased twofold in tMK cells comparedwith control cells expressing the empty plasmid (Neo cells).Cytoskeletal stiffness quantified using magnetic twisting cytometryshowed an ~1.5-fold increase in stiffness in tMK cells compared withNeo cells. Electronic volume measurements on cells in suspensionrevealed that tMK cells had a smaller volume and are more resistant toosmotic swelling than Neo cells. tMK cells also have smaller nuclei,and activation of mitogen-activated protein kinase (MAP kinase) andtranslocation of MAP kinase to the nucleus are slower in tMK cells thanin control cells. In tMK cells, there is also lessbromodeoxyuridine incorporation, and the doubling time isincreased. These data demonstrate that increased myosin light chainphosphorylation correlates with increased cytoskeletal stiffness andsuggest that changing the mechanical characteristics of thecytoskeleton alters the intracellular signaling pathways that regulatecell growth and division.

     

On the Role of Protein Disulfide Isomerase in the Retrograde Cell Transport of Secreted Phospholipases A2

Following the finding that ammodytoxin (Atx), a neurotoxic secreted phospholipase A₂ (sPLA₂) in snake venom, binds specifically to protein disulfide isomerase (PDI) in vitro we show that these proteins also interact in living rat PC12 cells that are able to internalize this group IIA (GIIA) sPLA₂. Atx and PDI co-localize in both differentiated and non-differentiated PC12 cells, as shown by fluorescence microscopy. Based on a model of the complex between Atx and yeast PDI (yPDI), a three-dimensional model of the complex between Atx and human PDI (hPDI) was constructed. The Atx binding site on hPDI is situated between domains b and b’. Atx interacts hPDI with an extensive area on its interfacial binding surface. The mammalian GIB, GIIA, GV and GX sPLA₂s have the same fold as Atx. The first three sPLA₂s have been detected intracellularly but not the last one. The models of their complexes with hPDI were constructed by replacement of Atx with the respective mammalian sPLA₂ in the Atx—hPDI complex and molecular docking of the structures. According to the generated models, mammalian GIB, GIIA and GV sPLA₂s form complexes with hPDI very similar to that with Atx. The contact area between GX sPLA₂ and hPDI is however different from that of the other sPLA₂s. Heterologous competition of Atx binding to hPDI with GV and GX sPLA₂s confirmed the model-based expectation that GV sPLA₂ was a more effective inhibitor than GX sPLA₂, thus validating our model. The results suggest a role of hPDI in the (patho)physiology of some snake venom and mammalian sPLA₂s by assisting the retrograde transport of these molecules from the cell surface. The sPLA₂–hPDI model constitutes a valuable tool to facilitate further insights into this process and into the (patho)physiology of sPLA₂s in relation to their action intracellularly.     

Relay of Herpes Simplex Virus between Langerhans Cells and Dermal Dendritic Cells in Human Skin

The mechanism by which immunity to Herpes Simplex Virus (HSV) is initiated is not completely defined. HSV initially infects mucosal epidermis prior to entering nerve endings. In mice, epidermal Langerhans cells (LCs) are the first dendritic cells (DCs) to encounter HSV, but it is CD103⁺ dermal DCs that carry viral antigen to lymph nodes for antigen presentation, suggesting DC cross-talk in skin. In this study, we compared topically HSV-1 infected human foreskin explants with biopsies of initial human genital herpes lesions to show LCs are initially infected then emigrate into the dermis. Here, LCs bearing markers of maturation and apoptosis formed large cell clusters with BDCA3⁺ dermal DCs (thought to be equivalent to murine CD103⁺ dermal DCs) and DC-SIGN⁺ DCs/macrophages. HSV-expressing LC fragments were observed inside the dermal DCs/macrophages and the BDCA3⁺ dermal DCs had up-regulated a damaged cell uptake receptor CLEC9A. No other infected epidermal cells interacted with dermal DCs. Correspondingly, LCs isolated from human skin and infected with HSV-1 in vitro also underwent apoptosis and were taken up by similarly isolated BDCA3⁺ dermal DCs and DC-SIGN⁺ cells. Thus, we conclude a viral antigen relay takes place where HSV infected LCs undergo apoptosis and are taken up by dermal DCs for subsequent antigen presentation. This provides a rationale for targeting these cells with mucosal or perhaps intradermal HSV immunization.     

Effect of liposomal formulations and immunostimulating peptidoglycan monomer (PGM) on the immune reaction to ovalbumin in mice

The adjuvant activity of liposomes and immunostimulating peptidoglycan monomer (PGM) in different formulations has been studied in mice model using ovalbumin (OVA) as an antigen. PGM is a natural compound of bacterial origin with well-defined chemical structure: GlcNAc-MurNAc-L-Ala-D-isoGln-mesoDpm(epsilonNH2)-D-Ala-D-Ala. It is a non-toxic, non-pyrogenic, and water-soluble immunostimulator. The aim of this study was to investigate the influence of different liposomal formulations of OVA, with or without PGM, on the production of total IgG, as well as of IgG1 and IgG2a subclasses of OVA-specific antibodies (as indicators of Th2 and Th1 type of immune response, respectively). CBA mice were immunized s.c. with OVA mixed with liposomes, OVA with PGM mixed with liposomes, OVA encapsulated into liposomes and OVA with PGM encapsulated into liposomes. Control groups were OVA in saline, OVA with PGM in saline, and OVA in CFA/IFA adjuvant formulation. The entrapment efficacy of OVA was monitored by HPLC method. The adjuvant activity of the mixture of OVA and empty liposomes, the mixture of OVA, PGM, and liposomes and PGM encapsulated with OVA into liposomes on production of total anti-OVA IgG was demonstrated. The mixture of PGM and liposomes exhibited additive immunostimulating effect on the production of antigen-specific IgGs. The analysis of IgG subclasses revealed that encapsulation of OVA into liposomes favors the stimulation of IgG2a antibodies, indicating the switch toward the Th1 type of immune response. When encapsulated into liposomes or mixed with liposomes, PGM induced a switch from Th1 to Th2 type of immune response. It could be concluded that appropriate formulations of antigen, PGM, and liposomes differently affect the humoral immune response and direct the switch in the type of immune response (Th1/Th2).