Biomedical subjects

R Elez

Publications and source records attributed to R Elez.

7 recordsLinked to original sources

PubMed2004

Electroporation of subcutaneous mouse tumors by rectangular and trapezium high voltage pulses.

The artificial electrotransfer of bioactive agents such as drugs, peptides or therapeutical nucleic acids and oligonucleotides by membrane electroporation (MEP) into single cells and tissue cells requires knowledge of the optimum ranges of the voltage, pulse duration and frequency of the applied pulses. For clinical use, the classical electroporators appear to necessitate some tissue specific presetting of the pulse parameters at the high voltage generator, before the actual therapeutic pulsing is applied. The optimum pulse parameters may be derived from the kinetic normal mode analysis of the current relaxations due to a voltage step (rectangular pulse). Here, the novel method of trapezium test pulses is proposed to rapidly assess the current (I)/voltage (U) characteristics (IUC). The analysis yields practical values for the voltage U(app) between a given electrode distance and pulse duration t(E) of rectangular high voltage (HV) pulses, to be preset for an effective in vivo electroporation of mouse subcutaneous tumors, clamped between two planar plate electrodes of stainless steel. The IUC of the trapezium pulse compares well with the IUC of rectangular pulses of increasing amplitudes. The trapezium pulse phase (s) of constant voltage and 3 ms duration, following the rising ramp phase (r), yields a current relaxation which is similar to the current relaxation during a rectangular pulse of similar duration. The fit of the current relaxation of the trapezium phase (s) to an exponential function and the IUC can be used to estimate the maximum current at a given voltage. The IUC of the falling edge (phase f) of the trapezium pulse serves to estimate the minimum voltage for the exploration of the long-lived electroporation membrane states with consecutive low-voltage (LV) pulses of longer duration, to eventually enhance electrophoretic uptake of ionic substances, initiated by the preceding HV pulses.

Animals↗

PubMed2004

Mutations in the putative HCV-E2 CD81 binding regions and correlation with cell surface CD81 expression.

The hepatitis C virus (HCV) envelope (E)2 protein interacts with the cellular receptor CD81 leading to modulation of B and T cell function. Recently, a higher binding affinity of subtype 1a in comparison with 1b derived E2 proteins for CD81 in vitro was described. The importance of mutations within the putative CD81 binding regions of different HCV geno-/subtypes in correlation with CD81 expression is unknown. In the present study, CD81 expression on blood lymphocytes of patients with chronic hepatitis C infected with different HCV geno-/subtypes were analysed by fluorescence activated cell sorter analyses. In addition, the putative CD81 binding regions on the E2 gene comprising the hypervariable region (HVR)2 were analysed by direct sequencing. CD81 expression on CD8(+) T-lymphocytes from patients infected with subtype 1a (n = 6) was significantly higher in comparison with subtype 1b (n = 12) and 3 (n = 5) infected patients before and during antiviral therapy (P = 0.006; P = 0.021, respectively). Sequencing of the putative CD81 binding regions in the E2 protein comprising the HVR2 (codon 474-495 and 522-552 according to the HCV-1a prototype HCV-H) showed a highly conserved motif within HVR2 for subtype 1a isolates and an overall low number of mutations within the putative CD81 binding regions, whereas numerous mutations were detected for subtype 1b isolates (12.0 vs 23.6%). HCV-3 isolates showed an intermediate number of mutations within the putative binding sites (19.2%; P = 0.022). In conclusion, the highly conserved sequence within HVR2 and putative CD81 binding sites of subtype 1a isolates previously associated with a high CD81 binding affinity in vitro is correlated with high CD81 expression on CD8(+) T-lymphocytes in vivo.

Adult↗

PubMed2001

Interferon alfa down-regulates CD81 in patients with chronic hepatitis C.

CD81 protein has been shown to bind hepatitis C virus (HCV) envelope 2 (E2) glycoprotein in vitro and may act as a (co)receptor for HCV. Regulation of CD81 expression by interferon alfa (IFN-alpha) and ribavirin could thereby affect the response to antiviral therapy. In the present study, the effects of IFN-alpha and ribavirin on CD81 protein and CD81 mRNA were assessed in peripheral blood lymphocytes (PBL) and isolated human hepatocytes by fluorescence-activated cell sorter (FACS) analysis and real-time polymerase chain reaction (PCR), respectively. In addition, regulation of CD81 in PBL was investigated in 10 patients treated with combination therapy. Incubation with IFN-alpha (50 U/mL) down-regulated total CD81 in PBL (81.7 +/- 11.6% of control; P =.003) and in isolated human hepatocytes (91.6 +/- 8.1% of control; P =.034). Incubation with IFN-alpha with and without ribavirin (2.2 microg/mL) significantly reduced cell surface-associated CD81 protein (83.9 +/- 10.3% of control; P =.003). PBL of untreated patients chronically infected with HCV had significantly higher levels of total CD81 protein compared with PBL obtained from healthy donors (631.1 +/- 93.3 vs. 538.9 +/- 95.2 relative fluorescence units [RFU]; P =.030). Pretreatment cell surface-associated CD81 protein was lower in patients infected with genotype HCV-3 than those infected with HCV-1 (111.8 +/- 15.0 vs. 162.0 +/- 41.3 RFU; P =.019). Furthermore, cell surface-associated CD81 protein was lower 4 weeks after initiation of therapy in patients with an initial virologic response compared with initial virologic nonresponders (110.5 +/- 8.5 vs. 139.8 +/- 27.5 RFU; P =.057). In conclusion, IFN-alpha and ribavirin regulate CD81 expression in vitro and in vivo. CD81 expression correlates with HCV genotype and initial virologic response in patients with chronic hepatitis C.

Antigens, CD↗

PubMed2000

Polo-like kinase1, a new target for antisense tumor therapy.

The Polo-like kinase 1 (Plk1) is a highly conserved mitotic serine/threonine kinase which is commonly overexpressed in cancer cell lines. Plk1 positively regulates mitotic progression by activating the CDC25C-CDK1 amplification loop and by regulating late mitotic events, primarily the ubiquitin-dependent proteolysis. In the present study, an antisense strategy against Plk1 mRNA was developed to specifically inhibit cell proliferation of cancer cells in cell culture and in the nude-mouse tumor model. Among 41 phosphorothioate antisense oligodeoxynucleotides tested, the 20-mer JWG2000 strongly inhibited expression of Plk1 in cultured A549 cells, leading to loss of cell viability, and exhibited anti-tumor activity in nude mice A549 xenograft. JWG2000 did not inhibit growth and viability of primary human mesangial cells and human amnion fibroblasts.

Animals↗

PubMed2000

Pertussis toxin inhibits cholecystokinin- and epidermal growth factor-induced mitogen-activated protein kinase activation by disinhibition of the cAMP signaling pathway and inhibition of c-Raf-1.

Pertussis toxin (PTx), which inactivates G(i/o) type G proteins, is widely used to investigate the involvement of G(i/o) proteins in signal transduction. Activation of extracellular-regulated kinases 1 and 2 (ERK1/2) by G protein-coupled receptors has been described to occur either through a PTx-insensitive pathway involving activation of phospholipase C and protein kinase C (PKC), or through a PTx-sensitive pathway involving G(i)betagamma-mediated activation of Src. Cholecystokinin (CCK) activates ERK1/2 by a PKC-dependent, and thus presumably PTx-insensitive, pathway. However, CCK has recently been shown to induce activation of G(i) proteins in addition to G(q/11). In the present study, PTx partially inhibited CCK-induced ERK1/2 activation in pancreatic AR42J cells, although activation of phospholipase C was not reduced. PTx also inhibited ERK1/2 activation in response to the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) as well as activation of c-Raf-1 by EGF and CCK. In contrast, PTx, CCK, and EGF had only minor effects on A-Raf and B-Raf activity. Forskolin, a direct activator of adenylyl cyclase, inhibited CCK- and EGF-induced activation of c-Raf-1 and ERK1/2 in a manner similar to that of PTx. In PTx-treated cells, the cAMP content was increased and forskolin did not further inhibit CCK- and EGF-induced activation of c-Raf-1 or ERK1/2. In conclusion, the present study shows that PTx-sensitivity of receptor-induced ERK1/2 activation could be a consequence of disinhibition of the adenylyl cyclase signaling pathway, which in turn causes inhibition of c-Raf-1 activation rather than indicating involvement of a PTx-sensitive G protein in this signaling pathway.

Adenylate Cyclase Toxin↗

PubMed1999

Prognostic significance of polo-like kinase (PLK) expression in squamous cell carcinomas of the head and neck.

Previously, we demonstrated that the mammalian polo-like kinase (PLK), which participates in the regulation of the cell cycle, is a novel marker of cellular proliferation. Because current prognostic tools for the evaluation of patients with head and neck squamous cell cancer (HNSCC) need to be improved, we analyzed 89 patients and found elevated PLK expression in most tumors. Nodal stage as a crucial prognostic factor in HNSCC also correlated to PLK transcript levels (P = 0.0043). A Kaplan-Meier analysis showed that HNSCC patients with moderate versus high PLK expression survived significantly longer (5-year survival rates, 43% versus 12%; P = 0.0047). Interestingly, a combination of nodal stage and PLK expression contributed to discriminate patients with a better prognosis in the pN(0/1) and pN(2/3) groups, which could improve the definition of a suitable therapy.

Adult↗

PubMed1997

Prognostic significance of polo-like kinase (PLK) expression in non-small cell lung cancer.

Our previous data indicate that the expression of the PLK gene which codes for a serine/threonine kinase is restricted to proliferating cells. In Northern blot experiments PLK mRNA expression was at the limit of detection in normal lung tissue but elevated in most samples of non-small cell lung cancer (NSCLC). A very low frequency of PLK transcripts was only found in bronchiolo-alveolar carcinomas. NSCLC patients whose tumors showed moderate PLK expression survived significantly longer (5 year survival rate=51.8%) than those with high levels of PLK transcripts (24.2%, P=0.001). No statistically significant correlation was found between PLK mRNA expression and age, sex, TNM status, histological type or degree of differentiation. Interestingly, the prognosis of patients in post-surgical stages I and II was correlated with PLK expression (5 year survival rates in stage I: 69.1% (moderate PLK) - 43.5% (high PLK), P=0.03 or in stage II: 51.9% (moderate PLK) - 9.9% (high PLK), P=0.006). These results suggest that PLK mRNA expression provides a new independent prognostic indicator for patients with NSCLC.

Adenocarcinoma, Bronchiolo-Alveolar↗