Diclofenac Interacts with Photosynthetic Apparatus: Isolated Spinach Chloroplasts and Thylakoids as a Model System.

Diclofenac, often detected in environmental samples, poses a potential hazard to the aquatic environment. The present study aimed to understand the effect of this drug on photosynthetic apparatus, which is a little-known aspect of its phytotoxicity. Chloroplasts and thylakoids isolated from spinach (Spinacia oleracea) were used for this study and treated with various concentrations of diclofenac (from 125 to 4000 µM). The parameters of chlorophyll a fluorescence (the OJIP test) as measurements for both the intact chloroplasts and the thylakoid membranes revealed that isolated thylakoids showed greater sensitivity to the drug than chloroplasts. The relatively high concentration of diclofenac that is required to inhibit chloroplast and thylakoid functions suggests a narcotic effect of that drug on photosynthetic membranes, rather than a specific interaction with a particular element of the electron transport chain. Using confocal microscopy, we confirmed the degradation of the chloroplast structure after DCF treatment, which has not been previously reported in the literature. In conclusion, it can be assumed that diclofenac's action originated from a non-specific interaction with photosynthetic membranes, leading to the disruption in the function of the electron transport chain. This, in turn, decreases the efficiency of photosynthesis, transforming part of the PSII reaction centers into heat sinks and enhancing non-photochemical energy dissipation.

Does diclofenac act like a photosynthetic herbicide on green algae? Chlamydomonas reinhardtii synchronous culture-based study with atrazine as reference.

The non-steroidal anti-inflammatory drug diclofenac (DCF) is one of the commonly used and frequently detected drugs in water bodies, and several studies indicate its toxic effect on plants and algae. Studies performed with asynchronous Chlamydomonas reinhardtii cultures indicated that DCF inhibit the growth of population of the algae. Here, a synchronous population of C. reinhardtii, in which all cells are in the same developmental phase, is used. Following changes in cells size, photosynthetic activity and gene expression, we could compare, at the level of single cell, DCF-mediated effects with the effects caused by atrazine, a triazine herbicide that inhibits photosynthesis and triggers oxidative stress. Application of DCF and atrazine at the beginning of the cell cycle allowed us to follow the changes occurring in the cells in the subsequent stages of their development. Synchronized Chlamydomonas reinhardtii cultures (strain CC-1690, wild type) were exposed to diclofenac sodium salt (135 mg/L) or atrazine (77.6 µg/L). The cell suspension was sampled hourly (0-10 h) in the light period of the cell cycle to determine cell number and volume, photosynthetic pigment content, chlorophyll a fluorescence (OJIP test) in vivo, and selected gene expression (real-time qPCR), namely psbA, psaA, FSD1, MSD3 and APX1. The two toxicants differently influenced C. reinhardtii cells. Both substances decreased photosynthetic "vitality" (PI - performance index) of the cells, albeit for different reasons. While atrazine significantly disrupted the photosynthetic electron transport, resulting in excessive production of reactive oxygen species (ROS) and limited cell growth, DCF caused silencing of photosystem II (PSII) reaction centers, transforming them into "heat sinks", thus preventing significant ROS overproduction. Oxidative stress caused by atrazine was the probable reason for the rapid appearance of phytotoxic action soon after entering the cells, while the effects of DCF could only be seen several hours after treatment. A comparison of DCF-caused effects with the effects caused by atrazine led us to conclude that, although DCF cannot be regarded as typical photosynthetic herbicide, it exhibits an algicidal activity and can be potentially dangerous for aquatic plants and algae.

Diclofenac and atrazine restrict the growth of a synchronous Chlamydomonas reinhardtii population via various mechanisms.

Non-steroidal anti-inflammatory drug diclofenac (DCF) is commonly found in freshwater bodies and can have adverse effects on non-target organisms. Among the studies on DCF toxicity, several ones have reported its harmful effects on plants and algae. To gain a better understanding of the mechanisms of DCF toxicity towards green algae, we used a synchronous Chlamydomonas reinhardtii cc-1690 culture and compared DCF (135 mg/L) effects with effects caused by atrazine (ATR; 77.6 µg/L), an herbicide with a well-known mechanism of toxic action. To achieve our goal, cell number and size, photosynthetic oxygen consumption/evolution, chlorophyll a fluorescence in vivo, H2O2 production by the cells, antioxidative enzymes encoding genes expression were analyzed during light phase of the cell cycle. We have found, that DCF and ATR affect C. reinhardtii through different mechanisms. ATR inhibited the photosynthetic electron transport chain and induced oxidative stress in chloroplast. Such chloroplastic energetics disruption indirectly influenced respiration, the intensification of which could partially mitigate low efficiency of photosynthetic energy production. As a result, ATR inhibited the growth of single cell leading to limitation in C. reinhardtii population development. In contrast to ATR-treated algae, in DCF-treated cells the fraction of active PSII reaction centers was diminished without drastic changes in electron transport or oxidative stress symptoms in chloroplast. However, significant increase in transcript level of gene encoding for mitochondria-located catalase indicates respiratory processes as a source of H2O2 overproduced in the DCF-treated cells. Because the single cell growth was not strongly affected by DCF, its adverse effect on progeny cell number seemed to be related rather to arresting of cell divisions. Concluding, although the DCF phytotoxic action appeared to be different from the action of the typical herbicide ATR, it can act as algal growth-inhibiting factor in the environment.

Implementation of Dietary Reference Intake Standards in Prison Menus in Poland.

Adequate nutrition in prisons should constantly be monitored due to the limited possibilities of external control as well as the low catering budget for prison meals and poorly defined requirements in this regard. The aim of the study was to assess the nutritional value of meals served in Polish prisons. Using a computer program, 14-day regular and bland diets from 30 prisons were analyzed. The energy value of the meals and the percentage of energy provided by protein, fat, and carbohydrate contained therein were found to meet the recommendations of the Polish National Food and Nutrition Institute. The amount of minerals supplied with the diet did not cover the recommended dietary allowance (RDA) in the case of calcium and magnesium. Particularly disturbing was the excessive supply of sodium in the regular and bland diets, which covered 537% and 311% of the dietary reference intake (DRI), respectively, as well as phosphorus (194 and 192% of RDA). The largest vitamin deficiencies were recorded for vitamins D and C and folate. An especially excessive supply was observed for vitamins A and B12. The type of diet significantly differentiated the average content of over half of the analyzed components, whereas the season of the year turned out to be statistically insignificant. The results of the present investigations indicate a need for development of more accurate legal provisions to regulate the nutrition in Polish prisons in terms of not only the energy value and macronutrient supply but also the intake of minerals and vitamins.

Influence of onion skin powder on nutritional and quality attributes of wheat pasta.

Onion skin is a waste produced during onion bulb processing. Recent studies have reported that it contains large amounts of bioaccessible and bioavailable compounds thus it can be used to design of novel food products. The objective of the study was an attempt to substitute semolina with onion skin (OS) powder in pasta at 2.5, 5 and 7.5 g/100 g levels. The effects on the chemical composition, antioxidant potential, technological and sensory properties of the fortified pasta samples were evaluated compared with a control sample. Fortification with OS resulted in a significant (P < 0.05) improvement in nutritional properties, which was demonstrated by an increase in the content of dietary fibre, ash, total phenolic compounds, flavonoids content and antioxidant activity (FRAP and DPPH). Cooking loss increased with increasing levels of OS, however, all pasta samples were in the acceptable range (8 g/100 g). Onion skin incorporation decreased optimal cooking time, water solubility index and increase redness (a*), compared to the control sample. Results of sensory evaluation suggest that pasta, in which 2.5% of the flour was replaced by this plant component, showed the highest value of the "overall quality". Our study indicates that onion skin powder can be a potential alternative for the food industry to provide nutritional enriched pasta.

The Impact of Drying of Wild-Growing Mushrooms on the Content and Antioxidant Capacity of Water- Soluble Polysaccharides.

Water-soluble polysaccharides (WSPs) were isolated from freeze-dried and hot-air-dried fruiting bodies of five wild-growing edible species: Armillaria mellea, Lactarius deliciosus, Leccinum aurantiacum, Suillus luteus, and Boletus badius. The concentrations of WSPs ranged from 36.3 ± 0.7 mg/g dw to 105.9 ± 3.9 mg/g dw. The method of drying substantially affected the quantity of WSP. The loss of WSP depended on species and varied between ~ 19% and ~ 48%. The extracted WSP contained varied amounts of carbohydrate, protein, and phenolics. The samples exerted antioxidant properties measured with the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) assay (11.5 ± 2.0 to 38.4 ± 3.6 µmol Trolox/g dw) and the Ferric reducing antioxidant power (FRAP) assay (9.1 ± 1.3 to 40.6 ± 1.4 µmol Trolox/g dw). In most cases, hot-air drying slightly increased the antioxidant potential of WSP.

Phytotoxic activity of diclofenac: Evaluation using a model green alga Chlamydomonas reinhardtii with atrazine as a reference substance.

Human activities have caused increasing inputs of pharmaceuticals to the environment and diclofenac (DF) is one of the most commonly detected in freshwater systems. The aim of this study was to determine the impact of DF on a freshwater green alga as a non-target organism. For DF toxicity evaluation, its effects on a model organism Chlamydomonas reinhardtii were compared with effects caused by the herbicide atrazine (AT). EC50 values were about 135 mg/L for DF and 78 mg/L for AT, respectively. Both toxicants enhanced H2O2 production by the cells (144% and 178% of control for AT and DF, respectively) and stimulated catalase activity (≈200% of control). Activity of ascorbate peroxidase was elevated in AT-cells but not in DF-treated cells. DF did not influence dark respiration of the cells, whereas AT inhibited this process by about 50% compared to the control. Both toxicants caused photosynthesis inhibition. Analysis of parameters of chlorophyll a fluorescence in vivo showed diminishment of a performance index (PI) in both DF- and AT-treated cells (≈50% of control), but the reasons for the changes detected were different. AT diminished the efficiency of electron transport between PS II and PS I without significant inhibition of PS II or PS I reaction centers (RCs). In contrast to AT, DF seemed to influence directly PS II RCs. The fraction of active PS II RCs was lowered in DF-treated cells, but energy flux per active RC increased. Our study indicates that DF phytotoxicity results mainly from photosynthesis inhibition due to "silencing" of a fraction of PS II RCs.

Antioxidative properties of milk protein preparations fermented by Polish strains of Lactobacillus helveticus.

BACKGROUND: The increasing significance of food products containing substances with antioxidative activi- ties is currently being observed. This is mainly due to the fact that pathogenic changes underlying some diseases are related to the carcinogenic effects of free radicals. Antioxidative compounds play an important role in supporting and enhancing the body’s defense mechanisms, which is useful in preventing some civili- zation diseases. Unfortunately, it has been already proved that some synthetic antioxidants pose a potential risk in vivo. Therefore, antioxidant compounds derived from a natural source are extremely valuable. Milk is a source of biologically active precursors, which when enclosed in structural protein sequences are inactive. The hydrolysis process, involving bacterial proteolytic enzymes, might release biopeptides that act in various ways, including having antioxidant properties. The objective of this study was to determine the antioxidant properties of milk protein preparations fermented by Polish strains of L. helveticus. The research also focused on evaluating the dynamics of milk acidification by these strains and analyzing the textural properties of the skim milk fermented products obtained. METHODS: The research studied Polish strains of L. helveticus: B734, 141, T80 and T105, which have not yet been used industrially. The antioxidant properties of 1% (w/v) solutions of milk protein preparations (skim milk powder, caseinoglycomacropeptide and α-lactoalbumin) fermented by these strains were determined by neutralizing the free radicals with 2,2-diphenyl-1-picrylhydrazyl (DPPH˙). Moreover, solutions of skim milk powder (SMP) fermented by the microorganisms being tested were analyzed on gel electrophoresis (SDS-PAGE). The dynamics of milk acidification by these microorganisms was also analyzed L. helveticus strains were used to prepare fermented regenerated skim milk products that were subjected to texture profile analysis (TPA) performed using a TA-XT2i (Stable Micro Systems, Godalming, UK). RESULTS: The results suggest that the antioxidant activity of fermented milk protein preparations depended on the type of milk protein preparation and was also related to the strain that conducted the fermentation process. The process of caseinoglycomacropeptide (CGMP) fermentation by DSMZ 20075, T105 and 141 signifi- cantly (p < 0.05) influenced the increase in the antioxidant activities of the protein preparation, the highest values of parameter were obtained in samples fermented by L. helveticus T105 (64.82 ±0.013%), while in the case of α-lactoalbumin (α-la), the strongest free radical scavenging activity (66.67 ±0.020%) was noted for unfermented samples (control). CONCLUSIONS: The greatest increase in DPPH scavenging activity (% of inhibition) was noted for fermented SMP solutions. The highest values of the parameter measured were recorded for SMP fermented by the reference strain (85.98 ±0.009%) and T80 (81.66 ±0.013%). Strain T105 demonstrated the most desirable properties with respect to milk acidifying dynamic and texture properties of fermented skim milk products, while the reference strain (L. helveticus DSMZ 20075) and L. helveticus T80 seem to be more desirable in terms of the possibility of obtaining fermented protein preparations with the best antioxidant properties. The Polish strains analyzed here might find application in dairy products and also in developing functional food products. Furthermore, the preparations of milk protein that were fermented by the strains being tested may be a natural source dietary antioxidants.

Effect of jasmonic acid elicitation on the yield, chemical composition, and antioxidant and anti-inflammatory properties of essential oil of lettuce leaf basil (Ocimum basilicum L.).

The effect of elicitation with jasmonic acid (JA) on the plant yield, the production and composition of essential oils of lettuce leaf basil was evaluated. JA-elicitation slightly affected the yield of plants and significantly increased the amount of essential oils produced by basil - the highest oil yield (0.78±0.005mL/100gdw) was achieved in plants elicited with 100µM JA. The application of the tested elicitor also influenced the chemical composition of basil essential oils - 100µM JA increased the linalool, eugenol, and limonene levels, while 1µM JA caused the highest increase in the methyl eugenol content. Essential oils from JA-elicited basil (especially 1µM and 100µM) exhibited more effective antioxidant and anti-inflammatory potential; therefore, this inducer may be a very useful biochemical tool for improving production and composition of herbal essential oils.

Study on vitamin D2 stability in dried mushrooms during drying and storage.

The main objective of this work was to determine the stability of vitamin D2 in dried mushrooms Agaricus bisporus, Pleurotus ostreatus and Lentinula edodes during storage, as well as to examine the possibility of inducing vitamin D2 production in dried mushrooms by UVB irradiation. After 1.5 year storage of dried mushrooms, the level of vitamin D2 in button mushrooms was found to be 6.90 µg/g dw, which is a 48.32% of initial level of vitamin D2. In the case of dried oyster and shiitake mushrooms there was a decrease to the level of 66.90% and 68.40%, respectively. It was determined that dried mushrooms can produce ergocalciferol under UVB irradiation. The highest content of vitamin D2 was observed in A. bisporus. Freeze-dried A. bisporus contained from 42.08 to 119.21 µg/g dw and hot-air dried mushrooms contained from 21.51 to 81.17 µg/g dw vitamin D2.

The effect of addition of selected milk protein preparations on the growth of Lactobacillus acidophilus and physicochemical properties of fermented milk.

BACKGROUND: The intake of fermented milk products, especially yoghurts, has been systematically increasing for a few decades. The purpose of this work was to obtain milk products fermented with a mix of bacterial cultures (yoghurt bacteria and Lactobacillus acidophillus LA-5) and enriched with selected milk protein preparations. Secondly, the aim of the work was to determine physiochemical and rheological properties of the obtained products. METHODS: The following additives were applied in the experiment: whey protein concentrate (WPC 65), whey protein isolate (WPI), demineralised whey powder (SPD), caseinoglycomacropeptide (CGMP), α-lactalbumin (α-la), sodium caseinate (KNa) and calcium caseinate (KCa). Milk was fermented using probiotic strain Lactobacillus acidophillus LA-5 and a typical yoghurt culture. The products were analysed in terms of the survivability of bacterial cells during refrigerated storage, rheological properties and syneresis. Fermented milk products were obtained using blends of bacterial strains: ST-B01:Lb-12 (1:1), ST-B01:Lb-12:LA-5 (1:1:2). RESULTS: Milk beverages fermented with typical yoghurt bacteria and LA-5 strain showed intensive syneresis. The addition of LA-5 strain caused formation of harder acid gels, comparing to typical yoghurts. Milk products which were prepared from skimmed milk possessed higher values of hardness and consistency coefficient. The increase of concentrations of milk preparations (except of WPI) did not cause significant differences in the hardness of acidic gels obtained by fermentation of mixed culture with a probiotic strain. CONCLUSIONS: The applied preparations improved physiochemical properties of the milk beverages which were prepared with a probiotic strain. The increase of protein milk preparations concentration resulted in a gradual decrease of the secreted whey. Among the products that were made of full milk powder and were subjected to three weeks of refrigerated storage the highest survivability of Lb. acidophilus LA-5 was noticed in the samples fortified with 1% WPC.

Hymenolepis diminuta: experimental studies on the antioxidant system with short and long term infection periods in the rats.

Many helminths cause long-lasting infections, living for several years in mammalian hosts reflecting a well balanced coexistence between host and parasite. There are many possible explanations as to how they can survive for lengthy periods. One possibility is their antioxidant systems, which can serve as defence mechanisms against host-generated oxygen radicals. Therefore, the aim of this experimental study was to examine the antioxidant system in Hymenolepisdiminuta during short (1.5 months young tapeworms) and long (1.5 years old tapeworms) term infection in the rat small intestine. The strobilae of H. diminuta tapeworms (14 young and three old) were divided into three pieces: the anterior part, containing the genital primordiae in the immature segments; the medial part, containing the early uterus in the mature, hermaphroditic proglottids and the terminal part with the mature gravid uterus in the gravid segments. Supernatants of these fragments were used for determination of markers of oxidative stress: concentration of thiobarbiturate reactive substances (TBARS) and of reduced glutathione (GSH), and the activity of antioxidant enzymes: superoxide dismutase (SOD1 and SOD2), catalase (CAT), glutathione peroxidases (GSHPxs), glutathione transferase (GST) and glutathione reductase (GSHR). The results indicated changes in levels of oxidative stress markers and antioxidant enzyme activity in both the young and old forms of H. diminuta. Relatively high activity of SOD (particularly in the anterior part of young tapeworms) was observed, as was increased activity of total GSHPx and a relatively high concentration of GSH in all parts of the tapeworms. These are caused by exposure to increased amount of ROS, which are produced during the inflammatory state. Due to the high activity of antioxidant enzymes, the anterior section of young and old tapeworms is equipped with a very effective antioxidant system. Old organisms also effectively resist oxidative stress due to reduced levels of lipid peroxidation and the high activity of GST, all of which suggest good adaptation to the hostile environment in the host's intestine.

Changes in the expression of insulin-like growth factor 1 variants in the postnatal brain development and in neonatal hypoxia-ischaemia.

Insulin-like growth factor-1 (IGF-1) is a multifunctional peptide of which numerous isoforms exist. The predominant form, IGF-1Ea is involved in physiological processes while IGF-1Ec (mechano-growth factor, MGF) is expressed in response to a different set of stimuli. We have identified specific changes in the expression patterns of these IGF-1 variants in brain development in normal rats and following neonatal hypoxia-ischaemia (HI). Both IGF-1Ea and IGF-1Ec are expressed during normal postnatal brain development, albeit with highly specific temporal distributions. In contrast, HI produced increased and prolonged expression of the IGF-1Ec isoform only. Importantly, hypoxia alone stimulated the expression of IGF-1Ec as well. Thus, IGF-1Ec may play a role in HI pathology. Neonatal hypoxia-ischaemia occurs in approximately 1:4000-1:10,000 newborns and causes neurological deficits in approximately 75% of those affected. Unfortunately, no specific treatment is available. IGF-1 is known to have neuroprotective activity and its IGF-1Ec variant appears to be an endogenous protective factor in hypoxia-ischaemia. Therefore, IGF-1Ec could potentially be developed into a therapeutic modality for the attenuation or prevention of neuronal damage in this and related disorders.

[Lipid peroxidation level in gastrointestinal tract tumors]. / Poziom peroksydacji lipidów w nowotworach przewodu pokarmowego.

UNLABELLED: Oxygen free radicals and their reactive derivatives participate in formation of chronic inflammation states, which facilitate development of gastrointestinal tract tumors. Oxidative stress is one of the main causes of damage to cell membranes in result of exacerbated lipid peroxidation process. End products of lipid peroxidation (aldehydes, organic peroxides) react with important biological macromolecules such as DNA and proteins, cause changes in cell membrane structure and properties leading to loss of its integrity. Intensification of the lipid peroxidation process is a factor which may also lead to a malfunction in the antioxidant barrier, which further weakens the defense of cells against oxygen free radicals and promotes the onset and development of cancer. The aim of the study was the determination of lipid peroxidation level in gastrointestinal tract tumors (stomach, liver, colon, and colorectal cancer to liver metastases). MATERIAL AND METHODS: Materials for studies were obtained from 150 patients with gastrointestinal tract tumors: 10 with stomach cancer, 30 with malignant and benign liver cancers, 60 with primary colorectal cancer, and 50 with metachronous colorectal cancer liver metastases. We also investigated 25 patients with liver cirrhosis, which was treated as a pre-cancerous condition. In total, 175 patients were examined. Tumor specimens, and normal adjacent tissues (6-7 cm from the edge of the tumor), which served as control tissue in studies, were collected from patients (with their consent) during surgery. Additionally, liver specimens were collected from patients with liver cirrhosis. Lipid peroxidation level was determined spectrophotometrically as a concentration of final lipid peroxidation products, which in reaction with tiobarbituric acid (TBA) form colour complex (thiobarbituric acid-reactive substances - TBARS). RESULTS: The study showed the highest concentration of TBARS in benign, and the lowest in malignant liver tumors. Other types of gastrointestinal tumors studied, were characterized by similar levels of lipid peroxidation. TBARS concentration in these tumors was approximately 2-fold higher than in malignant liver tumors and much lower than in benign tumors. In all cancers of the digestive tract with the exception of malignant liver tumors increased level of TBARS was found, comparing with control tissue. The concentration of TBARS in cirrhotic liver was lower than in control. The level of lipid peroxidation in liver cirrhosis and malignant liver tumors was similar. There were no significant differences in TBARS concentration in the tumors of particular sections of the intestine and normal colon. The highest concentration of TBARS was found in G1 grade of colorectal cancer. In subsequent grades of cells differentiation (G2 and G3) its concentration was lower. The highest level of lipid peroxidation, expressed as the concentration of TBARS was found in the I stage of colorectal cancer clinical advancement. The significantly lowest concentration of TBARS was shown for stage II (UICC). CONCLUSIONS: The level of lipid peroxidation in cancerous cells of gastrointestinal tract indicates increased oxidative stress. The changes of lipid peroxidation level--a marker of oxidative stress in gastrointestinal tumors appear to be closely associated with their development stages (liver cirrhosis/malignant liver cancer; colorectal cancer/colorectal cancer liver metastases) and are likely to create such conditions, in which cancerous cells may proliferate, undergo gradual dedifferentiation and malignancy, and generate metastases.

Expression and activity of superoxide dismutase isoenzymes in colorectal cancer.

The aim of the study was an evaluation of changes in protein level and activity of SOD isoenzymes, and the participation of AP-1 and NF-kappaB in subsequent stages of colorectal cancer development. Studies were conducted on 65 colorectal cancers. Controls were unchanged colon regions. Activity of SOD isoenzymes, lipid peroxidation level (TBARS), and protein level of SOD1, SOD2, AP-1 and NF-kappaB were determined. We found that the protein level and activity of SOD isoenzymes and protein level of AP-1 and NF-kappaB change in subsequent stages of clinical advancement of colorectal cancer, according to UICC (I-IV), and in grades of tumor cells differentiation (G(1)-G(3)). These results indicate adaptation of colorectal cancer cells to oxidative stress, and show that the observed changes of SOD activity and protein level depend on gradual progression of colorectal cancer, and suggest an impairment of processes regulated by AP-1 and NF-kappaB which are critical for tumor progression (proliferation, differentiation and apoptosis).

Role of TLR ligands in epicutaneously induced contrasuppression.

Our previous work showed that epicutaneous (EC) immunization in mice with protein antigen (Ag) induced an Ag-independent unresponsiveness mediated by suppressor CD4(+)8(+) T cells (Ts), which inhibited contact hypersensitivity (CS). Simultaneous EC immunization with Ag and various Toll-like receptor (TLR) ligands reversed skin-induced suppression. Our present study shows that this process activates Ag-specific T contrasuppressor (Tcs) cells and leads to the protection of CS effector T cells from suppression. Epicutaneous immunization with Ag and the TLR4 ligand lipopolysaccharide (LPS) led to a significant increase in IFN-gamma production by lymph node and spleen cells. Ag and TLR ligands, like LPS, CpG or lipoteichoic acid did not need to be applied concomitantly to the skin. An identical contrasuppressive effect was observed when the Ag and TLR ligands were deposited on distant skin areas, suggesting that both the generation of Ts and Tcs are independent. To corroborate this finding, we used a model system that uses macrophages (Mf) as Ag-presenting cells. Mf labeled in vitro with Ag (Mf-Ag) induced, upon intravenous (iv) administration, an unresponsiveness reaction that was mediated by Ts cells. When treated simultaneously with LPS-treated Mf (Mf-Ag-LPS), a TLR-ligand could induce CS. Both the Ag and the LPS signal could be uncoupled i.e., Mf-Ag and Mf-LPS given at separate time points (with an 1 h interval between injections) induced immunity.We also found that LPS-treated Mf also produced significant amounts of IL-12, a cytokine that has well-known anti-tolerogenic properties. Our experiments suggest that reversal of EC-induced suppression by TLR-ligands may be a potential tool to increase the immunogenicity of weakly immunogenic antigens.

TCRalphabeta + CD8+ and TCRgammadelta+ lymphocytes inhibit the capability of peritoneal macrophages to induce contact hypersensitivity.

Macrophages (Mf) play an important role in induction and regulation of the immune response. It was shown previously that subcutaneous injection of hapten conjugated macrophages (TNP-Mf) induces the contact hypersensitivity (CHS) response, whereas intravenous (i.v.) or intraperitioneal administration of TNP-Mf results in unresponsiveness as a result of induced T suppressor (Ts) cells. The aim of this study was to determine if different T cell populations influence macrophages to become inducers of immunological suppression. Our findings show that indeed i.v. injection of TNP labeled macrophages isolated from control mice into syngenic recipients induces unresponsiveness. However, i.v. administration of TNP substituted macrophages isolated from TCRalpha-/-, TCRdelta-/- and beta2m-/- mice induces strong CHS similar to that observed after skin painting with TNP-C1. Moreover, it was shown that TNP conjugated macrophages isolated from CD1d-/- mice were still able to promote immunosuppression when injected intravenously. This suggests that TCRalphabeta+ CD8+ and TCRgammadelta+ lymphocytes stimulate macrophages to induce immunosuppression instead of a strong CHS reaction, whereas CD1d dependent NKT cells are not involved in negative regulation of macrophage function.

[Contact sensitivity reaction, its mechanism and regulation]. / Reakcja nadwrazliwosci kontaktowej, jej mechanizm i regulacja.

The contact sensitivity (CS) reaction to haptens is a classical example of cell-mediated immune response. In this reaction, two phases can be distinguished: an early component, detectable as early as 2 hr after subsequent contact with the hapten, and a late component, developing approximately 24 hr after challenge and which is mediated by T cells. In the classical CS reaction, CD4+ T helper 1 (Th1) cells act as effector cells, whereas B-1 lymphocytes supported by NKT cells produce antigen-specific IgM antibodies, which play a crucial role in the initiation of CS. The CS reaction is under the precise control of regulatory circuits. The CS response is negatively regulated by T suppressor (Ts) cells induced by treatment with high doses of antigen. On the other hand, the CS response is positively regulated by T contrasuppressor (Tcs) cells that protect Th1 effector lymphocytes from the action of Ts cells. A new view of a negative regulation of Th1-mediated CS response is based on suppression induced by epicutaneous (e.c.) application of protein antigen. This kind of immunization results in the generation of TCR alphabeta+CD4+CD8+ Ts cells that inhibit the CS response via the released TGF-beta. The suppression induced via e.c. immunization with protein antigen can be abrogated by TCR alphabeta+CD4+ Tcs cells induced by simultaneous exposure to protein antigen and Toll-like receptor (TLR) ligands. This method of e.c.-induced tolerance or its reversal by e.c. application of antigen alone or together with TLR ligands may be effective in new therapeutic strategies because of its effectiveness, ease of induction, and noninvasiveness.

Epicutaneous immunization with protein antigen in the presence of TLR4 ligand induces TCR alpha beta+CD4+ T contrasuppressor cells that reverse skin-induced suppression of Th1-mediated contact sensitivity.

Our previous work showed that epicutaneous (EC) immunization of mice with different protein Ags applied on the skin in the form of a patch induces a state of subsequent Ag-nonspecific unresponsiveness due to suppressor CD4+8+ T cells (Ts) that inhibit Th1-mediated contact sensitivity (CS) reactions via released TGF-beta. In the present work we show that EC immunization with Ag together with the TLR4 ligand LPS induced cells that could prevent suppression by the Ag-nonspecific Ts. These up-regulatory cells, called contrasuppressor T cells (Tcs), belong to a population of Ag-specific TCRalphabeta CD4+ lymphocytes and are different from Th1 CD4+ cells that mediate the CS reaction. Experiments using knockout mice showed that EC induced contrasuppression is MyD88, INF-gamma, and IL-12 dependent, whereas IL-6 is not involved in this phenomenon. Additional experiments with anti-IFN-gamma mAb showed that IFN-gamma is required for induction of Tcs cells but does not play a crucial role in the effector phase of contrasuppression. Additionally, treatment of CS effector cells with rIL-12 makes them resistant to EC induced suppression without affecting Ts cells, whereas IL-12 neutralization in vitro abrogates contrasuppression. These data show that IL-12 is indeed involved in the effector phase of EC induced contrasuppression and that this cytokine does not act directly on Ts cells. The mechanism of action of Tcs protects Th1 effector cells mediating CS from the nonspecific Ts, leaving suppression to other Ags intact. Ts and Tcs cells do not influence each other and can be induced simultaneously in the same animal.