A sensitive photochemical reaction-capacitively coupled contactless conductivity detection system for HPLC and its application in determination of Cyclosporin A.

High performance liquid chromatography (HPLC) post-column photochemical reaction (PR) coupled capacitively coupled contactless conductivity detector (C4D) was used for the first time in analysis of weak ultraviolet (UV)-absorbing, non-fluorescence and nonpolar compound. A series of conditions including the radiation power of light source, the length of the reaction tube and the thickness of detection tube were investigated. HPLC-PR-C4D system was successfully applied to the determination of Cyclosporin A (CsA). Consequently, under optimal conditions, the detection system exhibited a detection limit of 0.04 µg/mL and wide linear range from 0.5 µg/mL to 100 µg/mL for CsA detection. Application of the HPLC-PR- C4D system to pharmaceutical formulation and biological samples revealed the system developed maybe reliably applied to clinical studies.

Degradation of cyclophosphamide and 5-fluorouracil by UV and simulated sunlight treatments: Assessment of the enhancement of the biodegradability and toxicity.

The presence of pharmaceuticals in the environment has triggered concern among the general population and received considerable attention from the scientific community in recent years. However, only a few publications have focused on anticancer drugs, a class of pharmaceuticals that can exhibit cytotoxic, genotoxic, mutagenic, carcinogenic and teratogenic effects. The present study investigated the photodegradation, biodegradation, bacterial toxicity, mutagenicity and genotoxicity of cyclophosphamide (CP) and 5-fluorouracil (5-FU). The photodegradation experiments were performed at a neutral to slight pH range (7-7.8) using two different lamps (medium-pressure mercury lamp and a xenon lamp). The primary elimination of the parent compounds was monitored by means of liquid chromatography tandem mass spectrometry (LC-IT-MS/MS). NPOC (non-purgeable organic carbon) analyses were carried out in order to assess mineralization rates. The Closed Bottle Test (CBT) was used to assess ready biodegradability. A new method using Vibrio fischeri was adopted to evaluate toxicity. CP was not degraded by any lamp, whereas 5-FU was completely eliminated by irradiation with the mercury lamp but only partially by the Xe lamp. No mineralization was observed for the experiments performed with the Xe lamp, and a NPOC removal of only 18% was registered for 5-FU after 256 min using the UV lamp. Not one of the parent compounds was readily biodegradable in the CBT. Photo transformation products (PTPs) resulting from photolysis were neither better biodegradable nor less toxic than the parent compound 5-FU. In contrast, the results of the tests carried out with the UV lamp indicated that more biodegradable and non-toxic PTPs of 5-FU were generated. Three PTPs were formed during the photodegradation experiments and were identified. The results of the in silico QSAR predictions showed positive mutagenic and genotoxic alerts for 5-FU, whereas only one of the formed PTPs presented positive alerts for the genotoxicity endpoint.

Immunosuppressive capabilities of mesenchymal stromal cells are maintained under hypoxic growth conditions and after gamma irradiation.

BACKGROUND AIMS: The discovery of regenerative and immunosuppressive capacities of mesenchymal stromal cells (MSCs) raises hope for patients with tissue-damaging or severe, treatment-refractory autoimmune disorders. We previously presented a method to expand human MSCs in a bioreactor under standardized Good Manufacturing Practice conditions. Now we characterized the impact of critical treatment conditions on MSCs with respect to immunosuppressive capabilities and proliferation. METHODS: MSC proliferation and survival after γ irradiation were determined by 5-carboxyfluorescein diacetate N-succinimidyl ester and annexinV/4',6-diamidino-2-phenylindole (DAPI) staining, respectively. T-cell proliferation assays were used to assess the effect of γ irradiation, passaging, cryopreservation, post-thaw equilibration time and hypoxia on T-cell suppressive capacities of MSCs. Quantitative polymerase chain reaction and ß-galactosidase staining served as tools to investigate differences between immunosuppressive and non-immunosuppressive MSCs. RESULTS: γ irradiation of MSCs abrogated their proliferation while vitality and T-cell inhibitory capacity were preserved. Passaging and long cryopreservation time decreased the T-cell suppressive function of MSCs, and postthaw equilibration time of 5 days restored this capability. Hypoxic culture markedly increased MSC proliferation without affecting their T-cell-suppressive capacity and phenotype. Furthermore, T-cell suppressive MSCs showed higher CXCL12 expression and less ß-galactosidase staining than non-suppressive MSCs. DISCUSSION: We demonstrate that γ irradiation is an effective strategy to abrogate MSC proliferation without impairing the cells' immunosuppressive function. Hypoxia significantly enhanced MSC expansion, allowing for transplantation of MSCs with low passage number. In summary, our optimized MSC expansion protocol successfully addressed the issues of safety and preservation of immunosuppressive MSC function after ex vivo expansion for therapeutic purposes.

Augmented photoswitching modulates immune signaling.

Reversible and non-invasive photoswitching of the immunosuppressive effect of a drug would be a very valuable tool for precisely regulating the immune system. Using a combination of protein borrowing and two-photon photoisomerization, we designed and synthesized derivatives of cyclosporin A. Here we demonstrate photoswitching of the local conformation within small molecules, which we used to modulate inhibitory potencies for cyclophilin, influence ternary and quaternary complex formations and regulate T-cell transcriptional activation in situ.

A novel accelerated in vitro release method for biodegradable coating of drug eluting stents: Insight to the drug release mechanisms.

The major objective of the present study was to develop an accelerated in vitro release method for everolimus/poly(lactic-co-glycolic acid) (PLGA) biodegradable DES that reflects and discriminates between many different sources of variations in the manufacturing process by introducing organic solvents in the release medium. To get further insight into the underlying drug release mechanisms, alongside release studies, the surface changes of the coated stents and the molecular weight changes of the polymer upon immersion in the selected release media were examined by scanning electron microscopy and size exclusion chromatography. The incorporation of acetonitrile in the release medium resulted in an increase in the drug release rate due to an increment in total porosity of the matrices. The developed method reflected and discriminated between different sources of variations in the manufacturing process and correlated with the real-time release. Over 80% of everolimus release occurred within 24h. The molecular and gravimetric weights of PLGA remained unchanged throughout the dissolution period, suggesting that the polymer does not undergo degradation through cleavage of its backbone ester linkages. It is likely that the drug release occurred mainly through its diffusion. The method can be employed as a rapid quality control test during development or commercial manufacturing.

Exposure to ultraviolet radiation causes dendritic cells/macrophages to secrete immune-suppressive IL-12p40 homodimers.

UV-induced immune suppression is a risk factor for sunlight-induced skin cancer. Exposure to UV radiation has been shown to suppress the rejection of highly antigenic UV-induced skin cancers, suppresses delayed and contact hypersensitivity, and depress the ability of dendritic cells to present Ag to T cells. One consequence of UV exposure is altered activation of T cell subsets. APCs from UV-irradiated mice fail to present Ag to Th1 T cells; however, Ag presentation to Th2 T cells is normal. While this has been known for some time, the mechanism behind the preferential suppression of Th1 cell activation has yet to be explained. We tested the hypothesis that this selective impairment of APC function results from altered cytokine production. We found that dendritic cells/macrophages (DC/Mphi) from UV-irradiated mice failed to secrete biologically active IL-12 following in vitro stimulation with LPS. Instead, DC/Mphi isolated from the lymphoid organs of UV-irradiated mice secreted IL-12p40 homodimer, a natural antagonist of biologically active IL-12. Furthermore, when culture supernatants from UV-derived DC/Mphi were added to IL-12-activated T cells, IFN-gamma secretion was totally suppressed, indicating that the IL-12p40 homodimer found in the supernatant fluid was biologically active. We suggest that by suppressing DC/Mphi IL-12p70 secretion while promoting IL-12p40 homodimer secretion, UV exposure preferentially suppress the activation of Th1 cells, thereby suppressing Th-1 cell-driven inflammatory immune reactions.

Evidence for functional relevance of CTLA-4 in ultraviolet-radiation-induced tolerance.

Hapten sensitization through UV-exposed skin induces hapten-specific tolerance that can be adoptively transferred by injecting T lymphocytes into naive recipients. The exact phenotype of T cells responsible for inhibiting the immune response and their mode of action remain unclear. Evidence exists that CTLA-4 negatively regulates T cell activation. We addressed whether CTLA-4 is involved in the transfer of UV-induced tolerance. Injection of lymph node cells from mice that were sensitized with dinitrofluorobenzene (DNFB) through UV-irradiated skin inhibited induction of contact hypersensitivity against DNFB in the recipient animals. When CTLA-4+ cells were depleted, transfer of suppression was lost. Likewise, significantly fewer lymphocytes enriched for CTLA-4+ cells were necessary to transfer suppression than unfractionated cells. Expression of CTLA-4 appears to be functionally relevant, since in vivo injection of a blocking anti-CTLA-4 Ab was able to break UV-induced tolerance and inhibited transfer of suppression. Upon stimulation with dendritic cells in the presence of the water-soluble DNFB analogue, DNBS, CTLA-4+ T cells from DNFB-tolerized mice secreted high levels of IL-10, TGF-beta, and IFN-gamma; low levels of IL-2; and no IL-4, resembling the cytokine pattern of T regulatory 1 cells. Ab blocking of CTLA-4 resulted in inhibition of IL-10 release. Accordingly, transfer of tolerance was not observed when recipients were treated with an anti-IL-10 Ab. Hence we propose that T cells, possibly of the T regulatory 1 type, transfer UV-mediated suppression through the release of IL-10. Activation of CTLA-4 appears to be important in this process.

Cytogenetic effect of chronic low-dose, low-dose-rate gamma-radiation in residents of irradiated buildings.

BACKGROUND: Many people in Taiwan have been living in buildings constructed with cobalt-60-contaminated steel rods. To study the biological effects of chronic low-dose ionising radiation on the residents of one such building, micronucleus formation in these individuals was compared with that in controls. METHODS: The 73 residents had 77 age-and-sex-matched controls: 31 had 31 close relatives as controls (group A controls); eight of the 31 had a second set of close relatives; and the other controls were 38 residents in neighbouring buildings. Two micronucleus assays were used-a cytochalasin B (CBMN) assay and another involving incubation with cytarabine (CBMNA). Assay results are given as "frequency", or the number of binucleate cells containing one micronucleus per 1000 randomly examined binucleate cells. FINDINGS: The CBMN and CBMNA mean (SD) frequencies for 31 exposed individuals (0.016 [0.009] and 0.025 [0.013] respectively) were greater than those for their group A controls (0.009 [0.004] and 0.016 [0.009], respectively) (p = 0.0006 and 0.0002, respectively). The mean CBMN and CBMNA frequencies for all the exposed individuals (0.017 [0.011] and 0.030 [0.014], respectively) were significantly greater than those for all controls (0.011 [0.008] and 0.019 [0.01]; p = 0.0001 for both comparisons). The ranges of the differences in CBMN or CBMNA frequencies between 31 exposed individuals and their group A controls were 0.003 to 0.020 and 0.001 to 0.032, respectively. After adjustment for age, sex, and cigarette smoking, the adjusted relative risks of micronucleus formation from radiation exposure in all 73 residents was 1.58 (95% CI 1.42-1.71; p = 0.0001) by the CBMN assay and 1.64 (1.53-1.77; p = 0.0001) by the CBMNA assay. INTERPRETATION: These findings suggest that chronic low-dose and low-dose-rate gamma-ray environmental exposure may induce cytogenetic damage in human beings.

Epidermal urocanic acid and suppression of contact hypersensitivity by ultraviolet radiation in Monodelphis domestica.

A single specific epidermal photoreceptor for the immunosuppressive action of UV radiation has not been defined, although separate evidence is accruing in favour of each of two candidates, trans-urocanic acid and DNA. In Monodelphis domestica, specific photoreactivation repair of UV radiation-induced pyrimidine dimers has been shown to abrogate the suppression of contact hypersensitivity (CHS), thus suggesting that DNA is the target for this impairment. However, the both haired and hairless mice, immunosuppressive effects of UV radiation have been reproduced by the exogenous administration of the UV photoproduct of urocanic acid, cis-urocanic acid. We show here that the epidermis of M. domestica contains urocanic acid, that UV irradiation of the shaved dorsal skin has resulted in an increase in epidermal cis-urocanic acid and that the topical application of a cis-urocanic acid-containing lotion significantly depressed the capacity of Monodelphis to respond to contact sensitisers, in a manner analogous to these responses in the hairless mouse. Therefore in Monodelphis, suppression of CHS by UV irradiation appears to involve both urocanic acid photo-isomerisation and epidermal DNA damage.