Carboplatin-induced upregulation of pan ß-tubulin and class III ß-tubulin is implicated in acquired resistance and cross-resistance of ovarian cancer.

Resistance to platinum- and taxane-based chemotherapy represents a major obstacle to long-term survival in ovarian cancer (OC) patients. Here, we studied the interplay between acquired carboplatin (CBP) resistance using two OC cell models, MES-OV CBP and SK-OV-3 CBP, and non-P-glycoprotein-mediated cross-resistance to paclitaxel (TAX) observed only in MES-OV CBP cells. Decreased platination, mesenchymal-like phenotype, and increased expression of α- and γ-tubulin were observed in both drug-resistant variants compared with parental cells. Both variants revealed increased protein expression of class III ß-tubulin (TUBB3) but differences in TUBB3 branching and nuclear morphology. Transient silencing of TUBB3 sensitized MES-OV CBP cells to TAX, and surprisingly also to CBP. This phenomenon was not observed in the SK-OV-3 CBP variant, probably due to the compensation by other ß-tubulin isotypes. Reduced TUBB3 levels in MES-OV CBP cells affected DNA repair protein trafficking and increased whole-cell platination level. Furthermore, TUBB3 depletion augmented therapeutic efficiency in additional OC cells, showing vice versa drug-resistant pattern, lacking ß-tubulin isotype compensation visible at the level of total ß-tubulin (TUBB) in vitro and ex vivo. In summary, the level of TUBB in OC should be considered together with TUBB3 in therapy response prediction.

Grapevine Leafroll-Associated Virus 3 Replication in Grapevine Hosts Changes through the Dormancy Stage.

Grapevine leafroll-associated virus 3 (GLRaV-3) is a graft-transmissible virus present in every viticultural region of the world and poses a large threat to grapevine production. Frequent coinfections with other viruses, the large number of grapevine varieties, the complexity of processes involved in plant response to virus infection, and the lack of studies on GLRaV-3 replication limit our knowledge of GLRaV-3 damaging effects and their background. In this study, five different inocula, one containing GLRaV-3 and others containing GLRaV-3 in combination with different grapevine viruses were green grafted to 52 different grapevine plants of four varieties to analyze the influence of the phenological stage and virus composition on GLRaV-3 replication. Relative concentration analysis by quantitative PCR conducted over a 16-month period revealed that other viruses as well as plant stage had a significant effect on GLRaV-3 replication and symptoms expression. The replication was most pronounced in the deep dormancy stage at the beginning of the infection, and the least at the exit of the dormancy stage. This study brings new insight into GLRaV-3 replication and discusses about viral interactions in one of the most economically important perennial plants, the grapevine.

Isolation of MDCK cells with low expression of mdr1 gene and their use in membrane permeability screening.

The Madin-Darby canine kidney (MDCK) cell line is frequently used for permeability screening in drug discovery. It contains endogenous transporters, most prominently canine multidrug resistance P-glycoprotein (Mdr1), which can interfere with studies of P-glycoprotein substrate assessment and permeability measurements. Because MDCK wild type (WT) is genetically heterogeneous, an isolation procedure was investigated in this study to obtain the subclonal line with low P-glycoprotein expression. The best clone obtained had up to 3-fold lower amprenavir efflux and P-glycoprotein expression in comparison to WT. Of 12 standard compounds tested that exhibited active efflux in WT cells, 11 showed a decrease in efflux in the isolated clone. However, the decrease was not below the cut-off value of 2, indicating residual P--glycoprotein activity. Clone isolation via the limiting dilution method, combined with bidirectional amprenavir permeability for clone selection, successfully identified MDCK clones with substantially lower P-glycoprotein efflux and has been demonstrated as a useful tool for assessing passive permeability in early drug discovery.

Claudins: Beyond Tight Junctions in Human IBD and Murine Models.

Claudins are transmembrane proteins constituting one of three tight junction protein families. In patients with inflammatory bowel disease (IBD), disease activity-dependent changes in expression of certain claudins have been noted, thus making certain claudin family members potential therapy targets. A study was undertaken with the aim of exploring expression of claudins in human disease and two different animal models of IBD: dextrane sulfate sodium-induced colitis and adoptive transfer model of colitis. The expression of sealing claudin-1, claudin-3, claudin-4, and claudin-8, and pore-forming claudin-2 in humans and rodents has been evaluated by immunohistochemistry and quantitative polymerase chain reaction. Claudins were expressed by epithelial and cells of mesodermal origin and were found to be situated at the membrane, within the cytoplasm, or within the nuclei. Claudin expression by human mononuclear cells isolated from lamina propria has been confirmed by Western blot and flow cytometry. The claudin expression pattern in uninflamed and inflamed colon varied between species and murine strains. In IBD and both animal models, diverse alterations in claudin expression by epithelial and inflammatory cells were recorded. Tissue mRNA levels for each studied claudin reflected changes within cell lineage and, at the same time, mirrored the ratio between various cell types. Based on the results of the study, it can be concluded that 1) claudins are not expressed exclusively by epithelial cells, but by certain types of cells of mesodermal origin as well; 2) changes in the claudin mRNA level should be interpreted in the context of overall tissue alterations; and 3) both IBD animal models that were analyzed can be used for investigating claudins as a therapy target, respecting their similarities and differences highlighted in this study.

Sudden bilateral hearing loss in gastric cancer as the only symptom of disease.

This paper reports a case of sudden bilateral deafness as the first symptom of gastric cancer, an extremely rare and atypical clinical situation. Because common signs of stomach cancer were absent, the patient was first evaluated in the Department of Otolaryngology, University Hospital Center, Zagreb. Only after expanded diagnostic evaluation and rapid progression of the disease in such a case is a malignant tumor suspected. Treatment is mostly ineffective. The unusual presentation of the disease and the rapid course may indicate a hereditary predisposition. Inactivation of tumor suppressor gene DFNA5 was found in 50% of gastric cancers, but of a non-metastasized phenotype. Inactivated DFNA5, otherwise described in hereditary bilateral deafness, perhaps favors the development of deafness in patients with gastric cancer. Our patient had a positive multiple viral antibody titer in serum, inactivated DFNA5 in both gastric cancer tissues and cerebellar metastases, and a metastatic form of the disease. If sudden deafness occurs in elderly patients, the possibility of malignant tumor should be taken into consideration. The link between gastric cancer and the DFNA5 gene is unclear and requires further research.

Immunomodulatory effects of azithromycin on the establishment of lipopolysaccharide tolerance in mice.

Recent reports suggest that azithromycin can shift macrophage polarization towards the alternatively activated M2 phenotype. In order to investigate its immunomodulatory activity in vivo, the influence of azithromycin on survival and cytokine production was assessed in the LPS tolerance model which is characterized by an M2 skewed response. For induction of tolerance, mice received an intraplantar injection of 30 µg LPS, 24 h prior to intravenous challenge with 350 µg LPS. Azithromycin (100 mg/kg) was administered orally, 2 h before LPS application. Influence of treatment on survival and cytokine concentration in serum was monitored. Azithromycin alone, instead of LPS, could not induce an LPS tolerant state. However, when administered before LPS priming it significantly increased survival, which was enhanced by concomitant azithromycin before LPS challenge. Azithromycin had no effect on survival when administered only prior to the LPS challenge. Tolerance induction by LPS priming was associated, upon LPS challenge, with decreased serum concentrations of pro-inflammatory cytokines, TNFα, IL-12p40 and CCL5, and increased serum concentrations of the anti-inflammatory cytokines, IL-10 and IL-1ra. Azithromycin treatment, prior to LPS priming, further reduced serum TNFα and CCL5, yielding the greatest inhibition when the macrolide was also given prior to LPS challenge. Serum concentrations of the anti-inflammatory cytokines, IL-10 and IL-1ra, were unchanged following azithromycin treatment. In summary, we have confirmed the immunomodulatory activity of azithromycin, as reflected in its ability to augment tolerance induction to LPS, promoting increased survival and reduced pro-inflammatory cytokine production, without affecting overt inflammation to LPS or anti-inflammatory cytokine production.