Primary and malignant cholangiocytes undergo CD40 mediated Fas dependent Apoptosis, but are insensitive to direct activation with exogenous fas ligand

Introduction Cholangiocarcinoma is a rare malignancy of the biliary tract, the incidence of which is rising, but the pathogenesis of which remains uncertain. No common genetic defects have been described but it is accepted that chronic inflammation is an important contributing factor. We have shown that primary human cholangiocyte and hepatocyte survival is tightly regulated via co-operative interactions between two tumour necrosis family (TNF) receptor family members; CD40 and Fas (CD95). Functional deficiency of CD154, the ligand for CD40, leads to a failure of clearance of biliary tract infections and a predisposition to cholangiocarcinoma implying a direct link between TNF receptor-mediated apoptosis and the development of cholangiocarcinoma. Aims To determine whether malignant cholangiocytes display defects in CD40 mediated apoptosis. By comparing CD40 and Fas-mediated apoptosis and intracellular signalling in primary human cholangiocytes and three cholangiocyte cell lines. Results Primary cholangiocytes and cholangiocyte cell lines were relatively insensitive to direct Fas-mediated killing with exogenous FasL when compared with Jurkat cells, which readily underwent Fas-mediated apoptosis, but were extremely sensitive to CD154 stimulation. The sensitivity of cells to CD40 activation was similar in magnitude in both primary and malignant cells and was STAT-3 and AP-1 dependent in both. Conclusions 1) Both primary and malignant cholangiocytes are relatively resistant to Fas–mediated killing but show exquisite sensitivity to CD154, suggesting that the CD40 pathway is intact and fully functional in both primary and malignant cholangiocytes 2) The relative insensitivity of cholangiocytes to Fas activation demonstrates the importance of CD40 augmentation of Fas dependent death in these cells. Agonistic therapies which target CD40 and associated intracellular signalling pathways may be effective in promoting apoptosis of malignant cholangiocytes

Proapoptotic genes BAX and CD40L are predictors of survival in transitional cell carcinoma of the bladder

The purpose of the study was to investigate the effects of expression of a range of genes involved in apoptosis on outcome in bladder cancer. Immunohistochemistry was used to examine expression of BCL2, BAX, P53, CD40 and CD40L in archival tissues of patients included in various treatment trials for transitional cell carcinoma (TCC) of the bladder. Data were collected on 94 patients who first presented with either invasive or superficial bladder cancer. Median follow-up for alive patients was 83 months (m) (range 12-195 m). Median survival was 80 m (95% CI=56-128 m). Median survivals for the various markers were as follows: BAX-positive patients 110 m vs BAX-negative patients 18 m (P=0.0002); CD40L-positive patients 95 m vs CD40L-negative patients 45 m (P=0.04); BCL2-positive patients 44 m and BCL2-negative patients 74 m, (P=0.64); CD40-positive patients 110 m and CD40 negative patients 45 m (P=0.12); and P53 positive patients 80 m and P53 negative patients 45 m (P=0.58). In conclusion, it was seen that overexpressions of BAX and CD40L are prognostic of better survival in TCC of the bladder. Our results also raise the possibility of the future development of CD40- and CD40 ligand-based immunotherapy for bladder cancer. This study links proapoptotic and antiapoptotic markers to overall survival

A redox state-dictated signalling pathway deciphers the malignant cell specificity of CD40-mediated apoptosis

CD40, a member of the tumour necrosis factor receptor (TNFR) superfamily, has the capacity to cause extensive apoptosis in carcinoma cells, while sparing normal epithelial cells. Yet, apoptosis is only achieved by membrane-presented CD40 ligand (mCD40L), as soluble receptor agonists are but weakly pro-apoptotic. Here, for the first time we have identified the precise signalling cascade underpinning mCD40L-mediated death as involving sequential TRAF3 stabilisation, ASK1 phosphorylation, MKK4 (but not MKK7) activation and JNK/AP-1 induction, leading to a Bak- and Bax-dependent mitochondrial apoptosis pathway. TRAF3 is central in the activation of the NADPH oxidase (Nox)-2 component p40phox and the elevation of reactive oxygen species (ROS) is essential in apoptosis. Strikingly, CD40 activation resulted in down-regulation of Thioredoxin (Trx)-1 to permit ASK1 activation and apoptosis. Although soluble receptor agonist alone could not induce death, combinatorial treatment incorporating soluble CD40 agonist and pharmacological inhibition of Trx-1 was functionally equivalent to the signal triggered by mCD40L. Finally, we demonstrate using normal, ‘para-malignant’ and tumour-derived cells that progression to malignant transformation is associated with increase in oxidative stress in epithelial cells, which coincides with increased susceptibility to CD40 killing, while in normal cells CD40 signalling is cytoprotective. Our studies have revealed the molecular nature of the tumour specificity of CD40 signalling and explained the differences in pro-apoptotic potential between soluble and membrane-bound CD40 agonists. Equally importantly, by exploiting a unique epithelial culture system that allowed us to monitor alterations in the redox-state of epithelial cells at different stages of malignant transformation, our study reveals how pro-apoptotic signals can elevate ROS past a previously hypothesised ‘lethal pro-apoptotic threshold’ to induce death; an observation that is both of fundamental importance and carries implications for cancer therap

The Use of Coconut Shells for the Removal of Dyes from Aqueous Solutions

The main purpose of the work was to check the possibility of using coconut shells for the removal of the dyes popular in the textile industry from aqueous solutions. The sorption abilities of an unconventional sorbent were tested against four anionic dyes: Reactive Black 5, Reactive Yellow 84, Acid Yellow 23, Acid Red 18 as well as two cationic dyes: Basic Violet 10 and Basic Red 46. The scope of research included investigation pertaining to the effect of pH on the effectiveness of sorption of dyes, conducted in order to determine the time of equilibrium of sorption and determine the maximum sorption capacity of coconut shells with respect to pigments. The most favorable pH of sorption for the anionic dyes and Basic Violet 10 was pH 3 and for Basic Red 46 – pH 6. The equilibrium time of sorption was the shortest in the case of acidic dyes (Acid Yellow 23/ Acid Red 18 – 45 min), while the longest in the case of alkaline dyes (Basic Red 46 – 90 min, Basic Violet 10 – 180 min). The sorption capacity of coconut shells in relation to anionic dyes was for Reactive Black 5 – 0.82 mg/g, Reactive Yellow 84 – 0.96 mg/g, Acid Yellow 23 – 0.53 mg/g and for Acid Red 18 – 0.66 mg/g. The tested sorbent showed much higher sorption capacity with respect to the cationic dyes, i.e. Basic Violet 10 (28.54 mg/g) and Basic Red 46 (68.52 mg/g)

Wykorzystanie niekonwencjonalnych systemów do usuwania barwnika RB5

Around 7·10 tons of dyes are produced annually to supply for the needs of the global textile, tanning and paper industry. Due to the low susceptibility of some materials to dyeing process and high solubility of dyes, up to 50% get into sewage, about 15% of which goes to the natural environment. Colored substances are mostly difficult to biodegrade, so decolorization of wastewater using biological methods used on conventional wastewater treatment plants is not effective. One of the most effective, cheap and environmentally friendly methods of removing dyes from wastewater is the adsorption process. However, the practical application of this process encounters a number of limitations, mainly due to the high cost of commercial sorbents and difficulties in their regeneration. Therefore, many studies focus on the search for cheap and effective sorbents. Recently, chitin, which is one of the most widespread biopolymers in nature, is becoming more and more popular among sorbents. According to literature data, the annual global production of chitin by living organisms reaches up to 1011 tons and 120,000-200,000 tons of chitin are obtained in the world from the waste of marine invertebrates processing annually. However, not all kinds of waste from various industry sectors are characterized by a high sorption capacity for dyes. That encourages seeking various methods of modifying potential sorbents to improve this ability. The study investigated the possibility of increasing the adsorption capacity of chitin in relation to the RB5 dye by initial modification of the sorbent. Modifying factors such as ultrasound, microwaves, magnetic field and ozone were used. Due to appropriate modification, the maximum sorption capacity of chitin in relation to the anionic dye Reactive Black 5 has changed. It was found that the effectiveness of the RB5 dye on chitin was influenced by the initial concentration of the solution and the type of modifying agent. Among the tested sorbents, the highest sorption capacity of 38.02 mg/dm3 was obtained for chitin modified with magnetic field at an induction of 15 mT. The efficiency of removing the anionic dye on this sorbent was higher than the maximum sorption capacity of unmodified chitin by 25%. In order to determine the exact effect of the magnetic field on chitin, additional and more detailed studies would have to be carried out. The lowest sorption capacity was obtained for chitin treated with ozone in the amount of 2 g of O3, where the result of 13.17 mg/dm3 was obtained. Ozonation affected negatively the sorption capacity of chitin by its partial depolymerisation and disruption of bonds, such as hydrogen bonds, responsible for the sorption of impurities in the form of anions.Na potrzeby światowego przemysłu włókienniczego, garbarskiego i papierniczego wytwarzanych jest rocznie około 7·105 ton barwników. Z powodu niskiej podatności niektórych materiałów na barwienie i wysokiej rozpuszczalności barwników nawet do 50% z nich po procesie barwienia przedostaje się do ścieków, z czego koło 15% trafia do środowiska naturalnego. Substancje barwne w większości są trudno-biodegradowalne, więc dekoloryzacja ścieków za pomocą metod biologicznych stosowanych na konwencjonalnych oczyszczalniach ścieków nie jest skuteczna. Jedną z efektywniejszych, tańszych i przyjaznych dla środowiska metod usuwania barwników ze ścieków jest proces adsorpcji. Pomimo tego jej praktyczne zastosowanie napotyka na szereg ograniczeń, głównie z uwagi na wysoki koszt komercyjnych sorbentów i trudności w ich regeneracji. W związku z tym wiele badań koncentruje się na poszukiwaniu sorbentów tanich a zarazem efektywnych. W ostatnim czasie coraz większym zainteresowaniem wśród sorbentów cieszy się chityna, która jest jednym z najbardziej rozpowszechnionych biopolimerów w przyrodzie. Według danych literaturowych roczna światowa produkcja chityny przez organizmy żywe osiąga nawet 1011 ton, a z odpadów przetwórstwa bezkręgowców morskich rocznie na świecie pozyskuje się 120 000-200 000 ton chityny. Jednak wszelkiego rodzaju odpady z różnych sektorów przemysłu nie zawsze charakteryzują się wysoką pojemnością sorpcyjną względem barwników. W związku z tym poszukuje się różnych metod modyfikacji potencjalnych sorbentów w celu poprawienia tej zdolności. W pracy zbadano możliwość zwiększenia zdolności adsorpcyjnej chityny względem barwnika RB5 poprzez wstępną modyfikację sorbentu. Jako czynnik modyfikujący chitynę przed sorpcją wykorzystano ultradźwięki, mikrofale, pole magnetyczne i ozonowanie W wyniku odpowiedniej modyfikacji maksymalna pojemność sorpcyjna chityny względem barwnika anionowego Reactive Black 5 uległa zmianie. Stwierdzono, iż na efektywność sorpcji barwnika RB 5 na chitynie miały wpływ początkowe stężenie roztworu oraz rodzaj czynnika modyfikującego. Spośród testowanych sorbentów największą pojemnością sorpcyjną wynoszącą 38,02 mg/ dm3 charakteryzowała się chityna modyfikowana polem magnetycznym o indukcji 15 mT. Efektywność usuwania barwnika anionowego na tym sorbencie była wyższa od maksymalnej pojemności sorpcyjnej chityny niemodyfikowanej o 25%. W celu określenia dokładnego wpływu pola magnetycznego na chitynę, należałoby przeprowadzić dodatkowe i bardziej szczegółowe badania. Natomiast najniższą pojemność sorpcyjną odnotowano dla chityny poddanej działaniu ozonu w ilości 2 g O3, gdzie uzyskano wynik 13,17 mg/ dm3. Ozonowanie negatywnie wpłynęło na zdolność sorpcyjną chityny poprzez jej częściową depolimeryzację oraz rozrywanie wiązań, takich jak wiązania wodorowe, odpowiedzialnych za sorpcję zanieczyszczeń w formie anionów