Acute Conditioning of Antigen-Expanded CD8+ T Cells via the GSK3ß-mTORC Axis Differentially Dictates Their Immediate and Distal Responses after Antigen Rechallenge.

CD8+ T cells protect against tumors and intracellular pathogens. The inflammatory cytokines IL-2, IL-15, and IL-7 are necessary for their expansion. However, elevated serum levels of these cytokines are often associated with cancer, poorer prognosis of cancer patients, and exhaustion of antigen-expanded CD8+ T cells. The impact of acute conditioning of antigen-expanded CD8+ T cells with these cytokines is unknown. Here, we generated antigen-expanded CD8+ T cells using dendritic cells and PC-3 cells. The cells were acutely (18-24 h) conditioned with IL-2 and either the GSK3ß inhibitor TWS119, the mTORC1 inhibitor rapamycin, or the mTORC1/2 inhibitor Torin1, then their immediate and post-re-expansion (distal) cytokine responses after antigen rechallenge were evaluated. We found that acute IL-2 conditioning upregulated the immediate antigen-induced cytokine response of the tested cells. Following their re-expansion, however, the cells showed a decreased cytokine response. These IL-2 conditioning-mediated impacts were counteracted with TWS119 or rapamycin but not with Torin1. Our data revealed that the acute conditioning of antigen-expanded CD8+ T cells with IL-2 modulates the GSK3ß-mTORC signaling axis. This modulation differentially affected the immediate and distal cytokine responses of the cells. The acute targeting of this signaling axis could, therefore, represent a novel strategy for the modulation of antigen-expanded CD8+ T cells.

The Ability of Prostate Health Index (PHI) to Predict Gleason Score in Patients With Prostate Cancer and Discriminate Patients Between Gleason Score 6 and Gleason Score Higher Than 6-A Study on 320 Patients After Radical Prostatectomy.

AIM: The purpose of this study was to investigate the Prostate Health Index as a marker for tumor aggressiveness in prostate biopsy and the optimization of indication for treatment options. METHODS: Our cohort consisted of 320 patients indicated for radical prostatectomy with preoperative measurements of total prostate-specific antigen, free prostate-specific antigen, [-2]proPSA, calculated %freePSA, and Prostate Health Index. The Gleason score was determined during biopsy and after radical prostatectomy. Using the Gleason score, we divided the group of patients into the 2 subgroups: Gleason score ≤6 and Gleason score >6. This division was performed according to the biopsy Gleason score and according to the postoperative Gleason score. We compared total prostate-specific antigen, [-2]proPSA, %freePSA, and Prostate Health Index in the subgroups Gleason score ≤6 and Gleason score >6 after biopsy and the definitive score. RESULTS: On evaluation of the subgroups created by Gleason score ≤6 and Gleason score >6, we observed agreement between biopsy Gleason score and definitive Gleason score in only 45.3% of cases. Of the calculated biopsy, Gleason score ≤6 and Gleason score >6 subgroups, [-2]proPSA, and Prostate Health Index ( P = .0003 and P = .0005) were statistically significant. Of the definitive Gleason score ≤6 and Gleason score >6 subgroups, Prostate Health Index, [-2]proPSA, %freePSA, and PSA ( P < .0001, P < .0001, P = .0003, and P = .0043) were statistically significant. The best area under the curve value (0.7496) was achieved by Prostate Health Index when the subgroups were established according to the postoperative Gleason score. CONCLUSION: Prostate Health Index is the best of the tested markers for the categorization of Gleason score 6 tumors and for facilitating the management of patients with prostate cancer. Prostate Health Index can be a helpful marker for indication of active surveillance or radical prostatectomy. Prostate health index can also simplify the decision of whether to perform nerve-sparing radical prostatectomy.

Inter strand crosslinks in DNA induced in vivo by percutaneous application of sulphur mustard to rats and mice.

Inter-strand crosslinks (ICL) in the DNA are regarded to be the main toxic lesions induced by sulphur mustard (SM). We have followed the induction of ICL in the DNA of different organs of Wistar rats and Balb/c or NMRI mice by the percutaneous application of SM using the modified (reverse) comet assay. Significant amounts of ICL were found in Balb/C lymphocytes, in bone marrow and liver cells after the dose of 80 mg/kg. A dose-dependent amount of ICL was induced in rats, with efficient induction in lymphocytes and spleen cells already after 5 mg SM/kg, indicating a higher susceptibility of rats to the DNA-damaging effect of SM compared with mice. A significant induction of ICL in other tested tissues (liver, bone marrow, colon epithelium) was seen at the dose of 20 mg/kg. The induced ICL were removed from the DNA during 48 h except for rats at the dose of 80 mg/kg. In fact, we observed that ICL are almost completely repaired in tissues of rats receiving high lethal doses. Results suggest that the unhooking of ICL, which we followed with the comet assay, may lead to the formation of another toxic DNA lesion during the repair process.

The impact of sesquiterpenes ß-caryophyllene oxide and trans-nerolidol on xenobiotic-metabolizing enzymes in mice in vivo.

1. Sesquiterpenes, constituents of plant essential oil, are popular bioactive compounds due to the positive effect on human health, but their potential toxicity and possible herb-drug interactions are often omitted. In our in vivo study, we followed up the effect of p.o. administration of two sesquiterpenes ß-caryophyllene oxide (CAO) and trans-nerolidol (NER) on various xenobiotic-metabolizing enzymes in mice liver and small intestine. 2. To spot the early effect of studied compounds, enzymatic activity and mRNA levels were assessed 6 and 24 h after single dose. 3. CAO and NER markedly increased cytochromes P450 (CYP2B, 3A, 2C) activity and mRNA levels in both tissues. Liver also showed elevated activity of aldo-ketoreductase 1C and carbonyl reductase after treatment. Contrary, sesquiterpenes decreased NAD(P)H:quinone oxidoreductase 1 activity in small intestine. Among conjugation enzymes, only liver sulfotransferase activity was increased by sesquiterpenes. 4. Our results document that single dose of sesquiterpenes modulate activities and expression of several xenobiotic-metabolizing enzymes.

The effects of ß-caryophyllene oxide and trans-nerolidol on the efficacy of doxorubicin in breast cancer cells and breast tumor-bearing mice.

BACKGROUND: One approach to improve effect of chemotherapy is combination of classical cytostatic drugs with natural compounds, e. g. sesquiterpenes. In our previous study, sesquiterpenes ß-caryophyllene oxide (CAO) and trans-nerolidol (NER) improved the anti-proliferative effect of doxorubicin (DOX) in intestinal cancer cell lines. PURPOSE: The present study was designed to evaluate the effect of CAO and NER on DOX efficacy, focusing on cell proliferation, migration, apoptosis and DOX accumulation in breast cancer cells MDA-MB-231 and MCF7 in vitro and in mice bearing solid Ehrlich tumors (EST) in vivo. METHODS: The impact of cytotoxic effect was assessed by the neutral red uptake test. The ability to migrate was tested using real-time measurement in x-CELLigence system. Expressions of molecules were examined using western blot analysis. The accumulation of DOX inside the cells using time lapse microscopy was observed. The mice with inoculated EST cells were treated repeatedly with DOX and DOX+CAO or DOX+NER and the growth of tumors were monitored. DOX concentrations in plasma and tumor were assayed using HPLC. RESULTS: In MDA-MB-231, combination of DOX with CAO enhanced anti-proliferative effect and acted strongly synergistic. NER increased accumulation of DOX inside the cells; moreover combination DOX with NER suppressed migration ability in vitro. In vivo, apoptosis was activated especially in group treated with DOX and CAO. However, none of tested sesquiterpenes was able to improve DOX accumulation in tumors and DOX-mediated inhibition of tumor growth. CONCLUSION: In conclusion, sesquiterpenes CAO and NER increased the efficacy of DOX in breast cancer cells in vitro, but did not improve its effect in vivo, in Ehrlich solid tumor bearing mice.

Protective potential of different compounds and their combinations with MESNA against sulfur mustard-induced cytotoxicity and genotoxicity.

The purpose of this study was to evaluate the efficacy of potential candidate molecules or their combinations against strong alkylation agent sulfur mustard (SM) on the human lung alveolar epithelial cell line A-549. Candidate molecules were chosen on the basis of their previously observed protective effects in vitro. The tested compounds, including antioxidants, sulfhydryl or other sulfur-containing molecules, nitrogen-containing molecules, PARP inhibitors and a NO synthase inhibitor, were applicated 30min before SM treatment. The efficiency of candidate molecules to protect cells against DNA damage and cell death induced by SM was determined using single-cell gel electrophoresis (comet assay) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction by viable cells. The damage of DNA was assessed 1 and 24h after dose 50µM SM. Cell survival was assessed 24 and 72h after the exposure. To achieve maximal cytoprotection, combinations of selected compounds with sodium 2-mercaptoethane sulphonate (MESNA) were tested. We found significant protective effects by several drugs used individually and also in combination with MESNA. High protection was achieved by sodium thiosulphate, which was further potentiated when combined with MESNA. Most of the selected compounds or mixture provided only moderate genoptotection without having any effect towards cell viability.

Induction and repair of DNA cross-links induced by sulfur mustard in the A-549 cell line followed by a comet assay.

Sulfur mustard is a highly toxic chemical warfare agent with devastating impact on intoxicated tissues. DNA cross-links are probably the most toxic DNA lesions induced in the cell by sulfur mustard. The comet assay is a very sensitive method for measuring DNA damage. In the present study using the A-549 lung cell line, the comet assay protocol was optimized for indirect detection of DNA cross-links induced by sulfur mustard. The method is based on the additional treatment of the assayed cells containing cross-links with the chemical mutagen, styrene oxide. Alkali-labile adducts of styrene oxide cause DNA breaks leading to the formation of comets. A significant dose-dependent reduction of DNA migration of the comet's tail was found after exposing cells to sulfur mustard, indicative of the amount of sulfur mustard induced cross-links. The remarkable decrease of % tail DNA could be observed as early as 5min following exposure to sulfur mustard and the maximal effect was found after 30min, when DNA migration was reduced to the minimum. Sulfur mustard preincubated in culture medium without cells lost its ability to induce cross-links and had a half-life of about 15min. Pre-incubation longer than 30min does not lead to a significant increase in cross-links when applied to cells. However, the amount of cross-links is decreased during further incubation due to repair. The current modification of the comet assay provides a useful tool for detecting DNA cross-links induced by sulfur mustard and could be used for detection of other DNA cross-linking agents such as chemotherapeutic drugs.

Sulfur mustard induced oxidative stress and its alteration using asoxime (HI-6).

Sulfur mustard (SM) is a blister agent with cytotoxic mechanism of action. There is no suitable treatment based on administration of an antidote. In this study, Wistar rats were exposed to SM in doses of 0-40 mg/kg body weight and treated with the compound HI-6. The treatment provided no significant effect on ferric reducing antioxidant power of blood and plasma. However, HI-6 caused an increase in the level of thiobarbituric acid reactive substances. This stressogenic response was presumably the cause of the significant elevation of the blood level of both glutathione reductase and reduced glutathione. HI-6 appears to be suitable for enhancing prophylactically oxidative stress protection from small oxidative insult.

Sulfur mustard causes oxidative stress and depletion of antioxidants in muscles, livers, and kidneys of Wistar rats.

Sulfur mustard (SM) is a chemical warfare agent with cytotoxic effect and a tight link to oxidative stress (OS). Depletion of antioxidants is considered as a cause of detrimental consequence and belongs to the important steps leading to cell death. The oxidative injury appearing after SM exposure is not well understood. Nevertheless, identification of the pathological processes would be a good opportunity to establish an efficient therapy. Here, we focused our effort on an estimation of reactive oxygen species homeostasis and apoptotic processes in Wistar rats exposed to 0-160 mg/kg of SM. We assayed antioxidant activity, thiobarbituric acid reactive substances, reduced glutathione/oxidized glutathione, metallothionein, glutathione reductase, glutathione peroxidase, glutathione S-transferase, caspase 3, and glucose in the livers, kidneys, and muscles of the animals. Significant OS, depletion of low-molecular-mass antioxidants, increase in caspase activity, and some other processes related to SM action were determined. Moreover, we infer a principal role of OS in the tested organs.

Polyamine conjugates of stigmasterol.

Three new polyamine conjugates with stigmasterol [(3ß,22E)-stigmasta-5,22-dien-3-ol] were synthesized and subjected to basic antimicrobial and cytotoxic tests. The conjugate derived from spermine, (3ß,22E)-stigmasta-5,22-dien-3-yl 4(12-amino-4,9-diaza-dodecylamino)-4-oxobutanoate (5c), displayed considerable antimicrobial activity on Staphylococcus aureus at low concentration (50 µg mL(-1)). The cytotoxic activity was tested on cells of human T-lymfoblastic leukemia (IC(50)=35.8 ± 10.3 µM (5c) and IC(50)=35.9 ± 5.7 µM (5b)) and normal human fibroblasts (IC(50)=38.0 ± 2.8 µM (5c) and IC(50)=45.5 ± 1.9 µM (5b)). Conjugate 5a displayed no activity in both tests.

Acetylcholine and an acetylcholinesterase inhibitor neostigmine can aggravate tularemia progress in BALB/c mice.

The present experiment was aimed at assessing the application of neostigmine, an acetylcholinesterase (AChE) pseudo-irreversible inhibitor with poor penetration through the hematoencephalitic barrier, and the neurotransmitter acetylcholine (ACh). The experiment was done to evaluate their ability to modulate an infectious disease: tularemia. Mice infected with Franciselle tularensis and exposed to either ACh or neostigmine had a higher mortality and spleen bacterial burden when compared to infected mice exposed to saline solution only. The activated cholinergic anti-inflammatory pathway suppressed pathways necessary for tularemia resolution. Administration of AChE inhibitors to the individuals suffering from tularemia is contra-indicatory. Drugs based on AChE inhibition should be restricted when tularemia or disease with a similar pathogenesis is suspected.

Cytotoxicity and genotoxicity evaluation of antidote oxime HI-6 tested on eight cell lines of human and rodent origin.

Oxime HI-6 is an efficient reactivator of the acetylcholinesterase inhibited by organophosphorous nerve agents. In this study we have estimated cytotoxicity of HI-6 by the colony forming assay and genotoxicity by the comet assay on human and rodent cell lines. IC50 of HI-6 assessed by the colony forming capacity was 3.59 mM for HeLa cells and 5.18 mM for a mouse cell line L929. Small difference in cytotoxicity was found among other cell lines tested: IC50 was 1.61 mM for human A549 cells, 1.14 mM for UROtse line, 1.96 mM and 1.71 mM for Chinese hamster cells AA8 and UV-20, respectively. The A549 cell viability measured with the MTT test was 5 times decreased comparing 2 and 24 hours of HI-6 oxime treatment. The 5 mM HI-6 concentration reduced the viability within 2 hours to 95% only, however, it induced a significant number of DNA breaks in mouse cells L929, and also in human UROtse and HepG2 cells. 1-ß-D-arabinofuranosylcytosine (10(-4) M) and hydroxyurea (10(-2) M), supplemented to the cultivation medium, did not cause any significant accumulation of DNA breaks during treatment, which indicated that the nucleotide excision repair was not acting on the induced DNA damage.

Design, synthesis and stimuli responsive gelation of novel stigmasterol-amino acid conjugates.

An efficient synthesis of three novel stigmasterol-amino acid (glycine, L-leucine and L-phenylalanine) conjugates as stimuli responsive gelators is reported. The gelation properties of the prepared compounds were investigated in a variety of organic as well as aqueous solvents. The most striking finding of our investigation was that the hydrochloride salts of the prepared conjugates acted as gelators, whereas the neutral conjugates were either non-gelators or formed only a weak gel in anisole. The hydrochloride salts of stigmasteryl glycinate and L-leucinate form gels in n-alcohols (n=4-10) and in ethane-1,2-diol, and that of stigmasteryl L-phenylalaninate forms gels in aromatic solvents and in tetrachloromethane. These unique properties of the gelators were explored to prepare stimuli responsive, "acid-base" triggered reversible sol-gel transitions. The gelators and their gels were characterized by liquid and solid-state NMR as well as FT-IR. The morphology of their corresponding xerogels was investigated by SEM.

Investigation of oxidative stress in blood, brain, kidney, and liver after oxime antidote HI-6 application in a mouse experimental model.

Oxime reactivator HI-6 (asoxime, in some sources) is a potent antidote suitable for treatment of intoxication by nerve agents. Despite the fact that HI-6 is considered for practical application in emergency situations, the impact of HI-6 on patients' bodies has not been established yet. The present experiment was carried out in order to estimate whether HI-6 would be able to trigger or protect from oxidative stress in a BALB/c mice model. HI-6 was applied in doses ranging from 0.2 to 20% of LD50. Ferric-reducing antioxidant power (FRAP), thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH), and glutathione reductase (GR) were assayed in the blood, liver, kidney, and brain of treated animals. It was found that HI-6 does not increase GR or TBARS. On the contrary, TBARS levels in the brain and liver were found to be significantly decreased in HI-6-treated animals. Pertinent antioxidant properties of HI-6 were excluded by the FRAP method. Endogenous antioxidants were unchanged, with the exception of the kidney. Low-molecular-weight antioxidants assayed by the FRAP method were significantly decreased in kidneys of animals treated with HI-6. However, GSH partially recovered the loss of the other low-molecular-weight antioxidants and was significantly increased in the kidney of HI-6-exposed mice. HI-6 potential to produce nephropathy is hypothesized. The achieved conclusions were quite surprising and showed a complex impact of HI-6 on the body.

Steroid conjugates: Synthesis and preliminary biological testing of pro-juvenoids.

A series of 10 new pro-juvenoids (juvenogens, insect hormonogenic compounds, pro-drug-like agents) was synthesized using isomeric synthetic juvenoids (insect juvenile hormone analogs) and steroid molecules as patterns modifying parts of the complex hormonogenic molecules. In addition, several new synthons were prepared, which were required by the designed synthetic protocol to achieve the target molecules. These pro-juvenoids were subjected to the topical screening tests and to the drinking assays on the red firebug (Pyrrhocoris apterus), a convenient model laboratory phytophagous insect. Simple and efficient synthetic procedures for the preparation of the target pro-juvenoids and their synthons are presented. Furthermore, the biological activity of the pro-juvenoids in comparison with the activity of their parent juvenoids and that of several commercially available agents is demonstrated. Juvenoids and pro-juvenoids may replace toxic insecticides persistent in the insect pest control because they have no adverse effects on non-target organisms and/or human.

Preparation and preliminary biological screening of cholic acid-juvenoid conjugates.

Steroidal compounds have been utilized as carriers and for modification of physico-chemical properties of model biologically active secondary alcohols - juvenoids. Juvenoids are juvenile hormone analogues - environmentally safe insecticides, possessing significant biological activity towards different arthropods groups in focus on insect pest species. Structure modification of juvenoids plays important role to control the rate of liberation and decomposition of juvenoid in digestive system and can also play important role in the mode of action towards selected insect. This study presents an approach to the synthesis of steroidal monomers and dimers carrying one and two molecules of a juvenoid in their structures. The prepared compounds were tested for their inhibition activity on reproduction of the blowfly Neobellieria (Sarcophaga) bullata. These steroid-juvenoid conjugates showed promising possibilities in synthesis of new unique biochemical insecticides. Preliminary biological test results of prepared compounds are presented.