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Biostimulants improve yield, quality, and stress acclimation in crops. In this work, we tested the possibility of using phenolics-rich extracts from spelt (Triticum dicoccum L.) husks to attenuate the effects of salt stress (100–200 mM NaCl) in maize. Two methanolic extracts were prepared from the soluble-conjugated (SC), and the insoluble-bound (IB) phenolic acid fractions of the spelt husk, and their effects were investigated on several stress-associated biochemical parameters, such as proline, lipid peroxidation, H2O2, GSH levels, and ion content. Results show that SC and IB fractions of husk extracts behaved very differently, no doubt due to their greatly divergent chemical composition, as revealed by both GC-MS and HPLC analyses. The efficacy of treatments in mitigating salt stress was also dose- and timing-dependent. IB, even at the lower concentration tested, was able to recover the performance of stressed plants in terms of growth, photosynthetic pigments content, and levels of salt stress markers. Recovery of shoot growth to control levels and reduction of stress-induced proline accumulation occurred regardless of whether plants were pre-treated or post-treated with IB, whereas only pre-treatment with the higher dose of IB was effective in mitigating oxidative stress. Although in some cases SC and even methanol alone exerted some positive effects, they could also be deleterious whereas IB never was. Overall, results indicate that a polyphenol-containing extract obtained from spelt by-products can behave as biostimulant in maize plants and can mitigate their response to salt stress, by acting on different biochemical targets.

Host-directed therapy using drugs that target cellular pathways required for virus lifecycle or its clearance might represent an effective approach for treating infectious diseases. Changes in redox homeostasis, including intracellular glutathione (GSH) depletion, are one of the key events that favor virus replication and contribute to the pathogenesis of virus-induced disease. Redox homeostasis has an important role in maintaining an appropriate Th1/Th2 balance, which is necessary to mount an effective immune response against viral infection and to avoid excessive inflammatory responses. It is known that excessive production of reactive oxygen species (ROS) induced by viral infection activates nuclear factor (NF)-kB, which orchestrates the expression of viral and host genes involved in the viral replication and inflammatory response. Moreover, redox-regulated protein disulfide isomerase (PDI) chaperones have an essential role in catalyzing formation of disulfide bonds in viral proteins. This review aims at describing the role of GSH in modulating redox sensitive pathways, in particular that mediated by NF-kB, and PDI activity. The second part of the review discusses the effectiveness of GSH-boosting molecules as broad-spectrum antivirals acting in a multifaceted way that includes the modulation of immune and inflammatory responses.

Glutathione (GSH) has poor pharmacokinetic properties; thus, several derivatives and biosynthetic precursors have been proposed as GSH-boosting drugs. I-152 is a conjugate of N-acetyl-cysteine (NAC) and S-acetyl-β-mercaptoethylamine (SMEA) designed to release the parent drugs (i.e., NAC and β-mercaptoethylamine or cysteamine, MEA). NAC is a precursor of L-cysteine, while MEA is an aminothiol able to increase GSH content; thus, I-152 represents the very first attempt to combine two pro-GSH molecules. In this review, the in-vitro and in-vivo metabolism, pro-GSH activity and antiviral and immunomodulatory properties of I-152 are discussed. Under physiological GSH conditions, low I-152 doses increase cellular GSH content; by contrast, high doses cause GSH depletion but yield a high content of NAC, MEA and I-152, which can be used to resynthesize GSH. Preliminary in-vivo studies suggest that the molecule reaches mouse organs, including the brain, where its metabolites, NAC and MEA, are detected. In cell cultures, I-152 replenishes experimentally depleted GSH levels. Moreover, administration of I-152 to C57BL/6 mice infected with the retroviral complex LP-BM5 is effective in contrasting virus-induced GSH depletion, exerting at the same time antiviral and immunomodulatory functions. I-152 acts as a pro-GSH agent; however, GSH derivatives and NAC cannot completely replicate its effects. The co-delivery of different thiol species may lead to unpredictable outcomes, which warrant further investigation.

Ataxia telangiectasia (A-T) is a rare incurable neurodegenerative disease caused by biallelic mutations in the gene for ataxia-telangiectasia mutated (ATM). The lack of a functional ATM kinase leads to a pleiotropic phenotype, and oxidative stress is considered to have a crucial role in the complex physiopathology. Recently, steroids have been shown to reduce the neurological symptoms of the disease, although the molecular mechanism of this effect is largely unknown. In the present study, we have demonstrated that dexamethasone treatment of A-T lymphoblastoid cells increases the content of two of the most abundant antioxidants [glutathione (GSH) and NADPH] by up to 30%. Dexamethasone promoted the nuclear accumulation of the transcription factor nuclear factor (erythroid-derived 2)-like 2 to drive expression of antioxidant pathways involved in GSH synthesis and NADPH production. The latter effect was via glucose 6-phosphate dehydrogenase activation, as confirmed by increased enzyme activity and enhancement of the pentose phosphate pathway rate. This evidence indicates that glucocorticoids are able to potentiate antioxidant defenses to counteract oxidative stress in ataxia telangiectasia, and also reveals an unexpected role for dexamethasone in redox homeostasis and cellular antioxidant activity.

Injection of the LP-BM5 murine leukemia virus into mice causes murine AIDS, a disease characterized by many dysfunctions of immunocompetent cells. To establish whether the disease is characterized by glutathione imbalance, reduced glutathione (GSH) and cysteine were quantified in different organs. A marked redox imbalance, consisting of GSH and/or cysteine depletion, was found in the lymphoid organs, such as the spleen and lymph nodes. Moreover, a significant decrease in cysteine and GSH levels in the pancreas and brain, respectively, was measured at 5 weeks postinfection. The Th2 immune response was predominant at all times investigated, as revealed by the expression of Th1/Th2 cytokines. Furthermore, investigation of the activation status of peritoneal macrophages showed that the expression of genetic markers of alternative activation, namely, Fizz1, Ym1, and Arginase1, was induced. Conversely, expression of inducible nitric oxide synthase, a marker of classical activation of macrophages, was detected only when Th1 cytokines were expressed at high levels. In vitro studies revealed that during the very early phases of infection, GSH depletion and the downregulation of interleukin-12 (IL-12) p40 mRNA were correlated with the dose of LP-BM5 used to infect the macrophages. Treatment of LP-BM5-infected mice with N -( N -acetyl- l -cysteinyl)- S -acetylcysteamine (I-152), an N -acetyl-cysteine supplier, restored GSH/cysteine levels in the organs, reduced the expression of alternatively activated macrophage markers, and increased the level of gamma interferon production, while it decreased the levels of Th2 cytokines, such as IL-4 and IL-5. Our findings thus establish a link between GSH deficiency and Th1/Th2 disequilibrium in LP-BM5 infection and indicate that I-152 can be used to restore the GSH level and a balanced Th1/Th2 response in infected mice. IMPORTANCE The first report of an association between Th2 polarization and alteration of the redox state in LP-BM5 infection is presented. Moreover, it provides evidence that LP-BM5 infection causes a decrease in the thiol content of peritoneal macrophages, which can influence IL-12 production. The restoration of GSH levels by GSH-replenishing molecules can represent a new therapeutic avenue to fight this retroviral infection, as it reestablishes the Th1/Th2 balance. Immunotherapy based on the use of pro-GSH molecules would permit LP-BM5 infection and probably all those viral infections characterized by GSH deficiency and a Th1/Th2 imbalance to be more effectively combated.

Excessive production of immunoglobulins (Ig) causes endoplasmic reticulum (ER) stress and triggers the unfolded protein response (UPR). Hypergammaglobulinemia and lymphadenopathy are hallmarks of murine AIDS that develops in mice infected with the LP-BM5 murine leukemia retrovirus complex. In these mice, Th2 polarization and aberrant humoral response have been previously correlated to altered intracellular redox homeostasis. Our goal was to understand the role of the cell's redox state in Ig secretion and plasma cell (PC) maturation. To this aim, LP-BM5-infected mice were treated with I-152, an N-acetyl-cysteine and cysteamine supplier. Intraperitoneal I-152 administration (30 μmol/mouse three times a week for 9 weeks) decreased plasma IgG and increased IgG/Syndecan 1 ratio in the lymph nodes where IgG were in part accumulated within the ER. PC containing cytoplasmic inclusions filled with IgG were present in all animals, with fewer mature PC in those treated with I-152. Infection induced up-regulation of signaling molecules involved in the UPR, i.e. CHAC1, BiP, sXBP-1 and PDI, that were generally unaffected by I-152 treatment except for PDI and sXBP-1, which have a key role in protein folding and PC maturation, respectively. Our data suggest that one of the mechanisms through which I-152 can limit hypergammaglobulinemia in LP-BM5-infected mice is by influencing IgG folding/assembly as well as secretion and affecting PC maturation.

Despite early treatment with antimycobacterial combination therapy, drug resistance continues to emerge. Maintenance of redox homeostasis is essential for Mycobacterium avium (M. avium) survival and growth. The aim of the present study was to investigate the antimycobacterial activity of two pro-glutathione (pro-GSH) drugs that are able to induce redox stress in M. avium and to modulate cytokine production by macrophages. Hence, we investigated two molecules shown to possess antiviral and immunomodulatory properties: C4-GSH, an N-butanoyl GSH derivative; and I-152, a prodrug of N-acetyl-cysteine (NAC) and β-mercaptoethylamine (MEA). Both molecules showed activity against replicating M. avium, both in the cell-free model and inside macrophages. Moreover, they were even more effective in reducing the viability of bacteria that had been kept in water for 7 days, proving to be active both against replicating and non-replicating bacteria. By regulating the macrophage redox state, I-152 modulated cytokine production. In particular, higher levels of interferon-gamma (IFN-γ), interleukin 1 beta (IL-1β), IL-18 and IL-12, which are known to be crucial for the control of intracellular pathogens, were found after I-152 treatment. Our results show that C4-GSH and I-152, by inducing perturbation of redox equilibrium, exert bacteriostatic and bactericidal activity against M. avium. Moreover, I-152 can boost the host response by inducing the production of cytokines that serve as key regulators of the Th1 response.

During aging, glutathione (GSH) content declines and the immune system undergoes a deficiency in the induction of Th1 response. Reduced secretion of Th1 cytokines, which is associated with GSH depletion, could weaken the host defenses against viral infections. We first evaluated the concentration of GSH and cysteine in organs of old mice; then, the effect of the administration of the N‐butanoyl GSH derivative (GSH‐C4) on the response of aged mice infected with influenza A PR8/H1N1 virus was studied through the determination of GSH concentration in organs, lung viral titer, IgA and IgG1/IgG2a production, and Th1/Th2 cytokine profile. Old mice had lower GSH than young mice in organs. Also the gene expression of endoplasmic reticulum (ER) stress markers involved in GSH metabolism and folding of proteins, that is, Nrf2 and PDI, was reduced. Following infection, GSH content remained low and neither infection nor GSH‐C4 treatment affected Nrf2 expression. In contrast, PDI expression was upregulated during infection and appeared counterbalanced by GSH‐C4. Moreover, the treatment with GSH‐C4 increased GSH content in organs, reduced viral replication and induced a predominant Th1 response. In conclusion, GSH‐C4 treatment could be used in the elderly to contrast influenza virus infection by inducing immune response, in particular the Th1 profile.

A novel two-step synthesis of N-butanoyl-l-glutathione (C4-GSH) was developed involving the chemical modification of the commercially available starting material l-glutathione (GSH). This process n...

An imbalance in GSH/GSSG ratio represents a triggering event in pro-inflammatory cytokine production and inflammatory response. However, the molecular mechanism(s) through which GSH regulates macrophage and cell autonomous inflammation remains not deeply understood. Here, we investigated the effects of a derivative of GSH, the N-butanoyl glutathione (GSH-C4), a cell permeable compound, on lipopolisaccharide (LPS)-stimulated murine RAW 264.7 macrophages, and human macrophages. LPS alone induces a significant production of pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α and a significant decrement of GSH content. Such events were significantly abrogated by treatment with GSH-C4. Moreover, GSH-C4 was highly efficient in buffering cell autonomous inflammatory status of aged C2C12 myotubes and 3T3-L1 adipocytes by suppressing the production of pro-inflammatory cytokines. We found that inflammation was paralleled by a strong induction of the phosphorylated form of NFκB, which translocates into the nucleus; a process that was also efficiently inhibited by the treatment with GSH-C4. Overall, the evidence suggests that GSH decrement is required for efficient activation of an inflammatory condition and, at the same time, GSH-C4 can be envisaged as a good candidate to abrogate such process, expanding the anti-inflammatory role of this molecule in chronic inflammatory states.

Stimulus-responsive cleavage reactions have found broad use to direct drug release at a particular target disease area. Increased levels of reactive oxygen species (ROS) have been associated with the development and progression of cancer and several other disease states, motivating the development of drug conjugates that can undergo a chemoselective ROS-triggered release. Melatonin (MLT) and the reactive electrophile p-benzoquinone methide ( p-QM) have evidenced either cytoprotective or cytotoxic effects in biological systems, depending on the dose, cellular targets, and time of exposure. In this study, we report the synthesis and biological activity of two MLT derivatives linked to ROS-responsive arylboronate triggers (P1 and P2), which can be activated by endogenously generated hydrogen peroxide (H2O2) to release MLT, or 5-methoxytryptamine (5-MeOT), and p-QM-intermediates. Their H2O2-induced activation mechanism was studied by HPLC-DAD-MS. P1, which rapidly releases MLT and p-QM, was able to strongly induce the Nrf2 antioxidant signaling pathway, but was ineffective to provide protection against H2O2-mediated oxidative damage. By contrast, P1 exhibited strong toxic effects in HeLa cancer cells, without causing significant toxicity to normal NCTC-2544 cells. Similar, although more limited, effects were exerted by P2. In both cases, cytotoxicity was accompanied by depletion of cellular glutathione (GSH), probably as a consequence of p-QM release, and increased ROS levels. A role for MLT in toxicity was also observed, suggesting that the P1 released products, MLT and p-QM, contributed additively to promote cell death.

The aim of this study was to determine whether GSH-C4, a hydrophobic glutathione derivative, affects in vitro and in vivo influenza virus infection by interfering with redox-sensitive intracellular pathways involved in the maturation of viral hemagglutinin (HA).GSH-C4 strongly inhibited influenza A virus replication in cultured cells and in lethally infected mice, where it also reduced lung damage and mortality. In cell-culture studies, GSH-C4 arrested viral HA folding; the disulfide-rich glycoprotein remained in the endoplasmic reticulum as a reduced monomer instead of undergoing oligomerization and cell plasma-membrane insertion. HA maturation depends on the host-cell oxidoreductase, protein disulfide isomerase (PDI), whose activity in infected cells is probably facilitated by virus-induced glutathione depletion. By correcting this deficit, GSH-C4 increased levels of reduced PDI and inhibited essential disulfide bond formation in HA. Host-cell glycoprotein expression in uninfected cells was unaffected by glutathione, which thus appears to act exclusively on glutathione-depleted cells.All currently approved anti-influenza drugs target essential viral structures, and their efficacy is limited by toxicity and by the almost inevitable selection of drug-resistant viral mutants. GSH-C4 inhibits influenza virus replication by modulating redox-sensitive pathways in infected cells, without producing toxicity in uninfected cells or animals. Novel anti-influenza drugs that target intracellular pathways essential for viral replication ("cell-based approach") offer two important potential advantages: they are more difficult for the virus to adapt to and their efficacy should not be dependent on virus type, strain, or antigenic properties.Redox-sensitive host-cell pathways exploited for viral replication are promising targets for effective anti-influenza strategies.

5,6,11,12,17,18,23,24-Octahydrocyclododeca[1,2-b:4,5-b':7,8-b'':10,11- b''']tetrai ndole (CTet), an indole-3-carbinol (I3C) metabolite endowed with anticancer properties, is poorly soluble in the solvents most frequently used in biological tests. This study indicates that the use of gamma-cyclodextrin (gamma-CD) avoids this problem. Formulated with gamma-CD CTet is a potent inhibitor of DNA synthesis in both estrogen receptor positive (MCF-7) and estrogen receptor negative (MDA-MB-231) human breast cell lines (IC50 = 1.20 +/- 0.04 microM and 1.0 +/- 0.1 microM, respectively).

The failure of highly active antiretroviral therapies (HAART) is mainly due to the existence of latent infected reservoirs, such as macrophages and resting CD4+ T cells. In this paper, we report the results that we obtained in a murine model of AIDS by alternating the administration of the lympholitic drug 2-Fluoro-ara-AMP (Fludarabine) to eliminate the infected cells, with that of Azidothymidine (AZT) plus reduced glutathione (GSH) encapsulated in erythrocytes, to protect lymphocytes and macrophages not yet infected, respectively. Two groups of infected mice were treated as follows: one group was treated by alternating the administration of Fludarabine and AZT (treatment A), while the other group received the same treatment plus GSH-loaded erythrocytes given with AZT (treatment A + L-RBC). Fludarabine was administered intraperitoneally, AZT in the drinking water and GSH was encapsulated in erythrocytes by a procedure of hypotonic dialysis and isotonic resealing. The results obtained show that GSH-loaded erythrocytes provide additive effects in all the parameters examined. Alternation of a lympholitic drug and antiretroviral drug is effective in reducing the progression of murine AIDS. Addition of a system to protect macrophages provides additive effects in almost all the parameters considered, confirming that combination therapies aimed at protecting different infectable cell compartments are better than treatments protecting mainly lymphocytes.

Macrophages are an important defense against in vivo herpes simplex virus (HSV) infection by early cytokine secretion; however, they can be infected by HSV-1 and they may be compromised in their ability to produce cytokines. In this paper, we studied the expression of two Th1 cytokines, interleukin (IL)-12 and IL-27, upon HSV-1 infection of human macrophages, and how it is regulated by treatment with two antiviral drugs exerting their anti-HSV-1 activity through different mechanisms of action. We found that infection does not alter intra-macrophage thiol content, while it induces mRNA expression of IL-12 p35 and IL-12 p40 as well as of IL-27 p28 and IL-27 EBI3, as revealed by RT-PCR. The increased expression of mRNA is accompanied by increased production of IL-12 p40 and IL-27 p28 protein, as detected in the culture supernatants by ELISA. The two antiviral drugs tested were acyclovir (ACV), commonly used to treat herpes virus infections, and an N-butanoyl glutathione (GSH) derivative, GSH-C4. While ACV inhibits viral DNA polymerase, GSH-C4 inhibits virus replication by interfering with protein folding and maturation of viral particles. Indeed, GSH-C4, altering the intracellular redox state, may modulate the Th1/Th2 balance favoring Th1-type response. Our data show that both drugs inhibit HSV-1 replication in macrophages, without significantly affecting cytokine mRNA levels. Nonetheless, lower levels of IL-12 p40 and IL-27 p28 proteins were found in the supernatants of macrophages treated with either GSH-C4 or ACV, likely as an indirect consequence of inhibited HSV-1 replication.

The imbalance of the redox state in cells and body fluids in HIV-1-infected patients may result in progression of the disease as well as in immunologic disfuctions. In this report, we have evaluated whether the direct administration of high doses of reduced glutathione (GSH) exerts any antiviral activity and/or improves immune functions in a murine immunodeficiency animal model. Intramuscular administration of 50 or 100 mg GSH/mouse for five consecutive days weekly to LP-BM5-infected mice did not show local or systemic signs of acute toxicity. During the first 3 weeks from infection, a period in which clinical signs of disease were not yet detectable, GSH significantly reduced the viral load in lymph nodes and spleen as evaluated by a PCR semiquantitative assay of the proviral DNA content. At 10 weeks a GSH concentration-dependent reduction of splenomegaly, lymphadenopathy and hypergammaglobulinemia was evident in all treated mice. Evaluation of proviral DNA content showed that GSH was effective in inhibiting LP-BM5 infectivity in lymph nodes, spleen, and bone marrow at 100 mg/day, while it was less effective when administered at 50 mg/day. At 10 weeks some animals receiving the highest GSH dose died, thus only the mice receiving 50 mg GSH were followed up to 15 weeks without signs of toxicity. In this case, almost not significant differences among infected untreated or treated animals were observed. Thus, GSH is effective in reducing the proviral DNA load in the first period of infection. These data and the failure of sulfhydril supplementation to further counteract the progression of disease after 10 weeks of infection suggest that combinations of GSH and other antiviral agents may be useful for improving current antiviral therapies.

Indole-3-carbinol (I3C) and its oligomeric derivatives have been widely studied for their chemopreventive and chemotherapeutic properties. We have previously shown that the I3C cyclic tetrameric derivative CTet inhibits breast cancer cell proliferation in vitro and in xenotrasplanted tumor. Here we report the antitumoral activity of a mixture of tri- and tetrameric cyclic I3C derivatives (CTr/CTet) both in vitro (MCF-7 and MDA-MB-231 breast cancer cell lines) and in vivo in a tumor xenograft model. CTr/CTet mixture avoids the low solubility drawbacks of CTet, thus favouring its solubilization, and reducing purification process, time and costs. CTr/CTet mixture has been shown to inhibit breast cancer cell proliferation (IC50 = 1.3 and 1.6 μg/ml in MCF-7 and MDAMB- 231, respectively) inducing the G0/1 cell cycle phase accumulation. The main molecular events related to CTr/CTet activity are the overexpression of p21, p27 and GADD45A, nuclear translocation of FOXO3a, inhibition of Akt activity and downregulation of estrogen receptor. In vivo, the growth of xenotransplanted tumor has been inhibited and the pro-tumoral low molecular weight cyclin E downregulation has been detected. Our data indicate that CTr/CTet is a potential anticancer combination agent for both hormone-responsive and triple-negative breast tumors.

Hexokinase inactivating antibodies were loaded into human erythrocytes using an encapsulation procedure based on hypotonic haemolysis, isotonic resealing and reannealing. Red blood cells loaded with anti-hexokinase IgG showed 20 percent residual hexokinase activity and reduced glycolytic activity. Incubation of these cells in the presence of an oxidizing agent such as terbutyl hydroperoxide (TBH) and then in autologous plasma, promoted opsonization by autologous IgG and complement deposition, but not haemolysis. Furthermore, the antihexokinase IgG loaded cells treated with TBH were actively recognized and phagocytosed by macrophages. Thus, metabolic impairment of human erythrocytes promotes autologous IgG binding, C3 deposition and phagocytosis, a mechanism already reported for the removal of senescent erythrocytes.

Introduction: Indole-3-carbinol (I3C), an autolysis product of glucosinolates present in cruciferous vegetables, and its dimeric derivative (3,3’-DIM) have been indicated as promising agents in preventing the development and progression of breast cancer. We have recently shown that I3C cyclic tetrameric derivative CTet formulated in gcyclodextrin (g-CD) efficiently inhibited cellular proliferation in breast cancer cell lines. This study aims to analyze the mechanisms involved in the in vitro inhibition of cell proliferation and to evaluate the in vivo antitumor activity of CTet in a xenograft study. Methods: Estrogen receptor-positive MCF-7 and triple-negative MDA-MB-231 breast cancer cell lines were exposed to CTet to evaluate cell cycle perturbation (propidium iodide staining and cytofluorimetric acquisition), induction of autophagic morphological features (co-localization of LC3b autophagosome marker and LAMP2a lysosome marker by immunofluorescence) and changes in protein expression (immunoblot and microarray-based gene expression analyses). To test the in vivo efficacy of CTet, female athymic nude mice inoculated with MCF-7 cells were i.p. treated with 5 mg/kg/day of CTet for five days/week for two weeks and the tumor mass was externally monitored. Results: CTet induced accumulation in G2/M phase without evidence of apoptotic response induction in both cell lines tested. In triple-negative MDA-MB-231 the autophagic lysosomal activity was significantly up-regulated after exposure to 4 μM of CTet for 8 hours, while the highest CTet concentration was necessary to observe autophagic features in MCF-7 cells. The inhibition of Akt activity and p53-independent p21/CDKN1A and GADD45A overexpression were identified as the main molecular events responsible for CTet activity in MCF-7 and p53-mutant MDA-MB-231 cells. In vivo, CTet administration was able to significantly inhibit the growth of MCF-7 xenotransplanted into nude mice, without adverse effect on body weight or on haematological parameters. Conclusions: Our data support CTet formulated with g-CD as a promising and injectable anticancer agent for both hormone-responsive and triple-negative breast tumors.

In the present work, the capacity of new pro-GSH molecules to increase the intra-macrophage thiol content in vitro and in vivo as well as to shift the immune response to Th1 in ovalbumin (Ova)-sensitized mice were examined. The molecules were the N-butanoyl GSH derivative, GSH-C4, and a pro-drug of N-acetylcysteine (NAC) and beta-mercaptoethylamine (MEA), I-152. In vitro, 2 h-incubation with both molecules was found to increase intra-macrophage thiol content; in vivo, Ova-sensitized mice pre-treated by intraperitoneal administration of the pro-GSH molecules showed an increase in plasma anti-Ova IgG2a and IgG2b, characterizing Th1 immune response, and a decrease in IgG1, typical of the Th2 response. Such findings were connected to a shift to a Th1 response also involving splenocyte IFN-γ production as revealed by ELISPOT assay and higher levels of IL-12 in circulation. Although immune responses are in vivo mediated both by dendritic cells and macrophages, the data reported in this paper corroborate the suggestion that the pro-GSH molecules, increasing the intra-cellular thiol pool, modulate the Th1/Th2 balance favouring Th1-type responses and may be employed as Th1-directing adjuvants in new vaccination protocols and as immunomodulators in those diseases where Th1 response patterns are compromised in favour of Th2.

Reduced glutathione (GSH) is the most prevalent non-protein thiol in animal cells. Its de novo and salvage synthesis serves to maintain a reduced cellular environment. GSH is the most powerful intracellular antioxidant and plays a role in the detoxification of a variety of electrophilic compounds and peroxides via catalysis by glutathione-S-transferases (GST) and glutathione peroxidases (GPx). As a consequence, the ratio of reduced and oxidized glutathione (GSH:GSSG) serves as a representative marker of the antioxidative capacity of the cell. A deficiency in GSH puts the cell at risk for oxidative damage. An imbalance in GSH is observed in a wide range of pathologies, such as cancer, neurodegenerative diseases, cystic fibrosis (CF), several viral infections including HIV-1, as well as in aging. Several reports have provided evidence for the use of GSH and molecules able to replenish intracellular GSH levels in antiviral therapy. This non-conventional role of GSH and its analogs as antiviral drugs is discussed in this chapter.

PNA(PR2) is a peptide nucleic acid (PNA) complementary to a sequence of the viral protease-encoding gene, effective in blocking HIV release, when used at high doses. Erythrocytes (RBC) were used to target PNA(PR2) to the macrophage compartment. The antiviral activity was assessed in human HIV-infected macrophages both as inhibition of p24 production and reduction of HIV DNA content. PNA(PR2), either added to the medium at a concentration of 100 microM or loaded into RBC at about 40 microM, inhibited p24 production approximately 80% compared with infected samples and reduced HIV DNA content by 83% and 90%, respectively. The results show that (1) a stronger anti-HIV effect is achievable with higher doses of PNA(PR2), both when given free and encapsulated into RBC; (2) the antiviral effect obtained by free PNA(PR2) at a concentration of 100 microM is achievable by encapsulating it into RBC at a concentration of 40 microM, suggesting that RBC can be used as a delivery system to increase the antisense effect of PNA(PR2).

The accumulation of chromium by germinating kiwifruit pollen appears to be significantly affected by Cr species, Cr concentration and calcium vailability. Cr(III) accumulation always occurred in a linear manner while Cr(VI) uptake followed a logarithmic model. In the absence of exogenous calcium, Cr(III) accumulation was much higher than that of Cr(VI). It was observed that, as the Cr(III) concentration increased, there was a significant decrease in the endogenous calcium content of pollen, ultimately leading to complete calcium depletion after 90 min of incubation at 150 lM Cr(III). This loss of calcium could be responsible for the strong inhibition of tube emergence and growth following exposure of pollen to Cr(III). Indeed, when exogenous calcium was added to the kiwifruit pollen culture medium, significant growth recovery and reduced Cr(III) uptake occurred; the opposite was true in Cr (VI)-treatments. A significant rise in lipid peroxide production occurs in the presence of both Cr species; the effect was more pronounced following Cr(VI) exposure. Finally, glutathione pool dynamics appears to be differentially affected by chromium species and concentrations. In conclusion, results of the present study have provided important information regarding the different activity profi les of Cr(III) and Cr(VI) in relation to kiwifruit pollen performance, and have also demonstrated differences in some biochemical esponses of pollen to metal stress. © 2008 Elsevier Ltd. All rights reserved doi:10.1016/j.chemosphere.2008.07.083

Antioxidant molecules can be used both to replenish the depletion of reduced glutathione (GSH) occurring during HIV infection, and to inhibit HIV replication. The purpose of this work was to assess the efficacy of two pro-GSH molecules able to cross the cell membrane more easily than GSH. We used an experimental animal model consisting of C57BL/6 mice infected with the LP-BM5 viral complex; the treatments were based on the intramuscular administration of I-152, a pro-drug of N-acetylcysteine and S-acetyl-beta-mercaptoethylamine, and S-acetylglutathione, an acetylated GSH derivative. The results show that I-152, at a concentration of 10.7 times lower than GSH, caused a reduction in lymph node and spleen weights of about 55% when compared to infected animals and an inhibition of about 66% in spleen and lymph node virus content. S-acetylglutathione, at half the concentration of GSH, caused a reduction in lymph node weight of about 17% and in spleen and lymph node virus content of about 70% and 30%, respectively. These results show that the administration of pro-GSH molecules may favorably substitute for the use of GSH as such.

Reduced glutathione (GSH) is present in millimolar concentrations in mammalian cells. It is involved in many cellular functions such as detoxification, amino acid transport, production of coenzymes, and the recycling of vitamins E and C. GSH acts as a redox buffer to preserve the reduced intracellular environment. Decreased glutathione levels have been found in numerous diseases such as cancer, viral infections, and immune dysfunctions. Many antioxidant molecules, such as GSH and N-acetylcysteine (NAC), have been demonstrated to inhibit in vitro and in vivo viral replication through different mechanisms of action. Accumulating evidence suggests that intracellular GSH levels in antigen-presenting cells such as macrophages, influence the Th1/Th2 cytokine response pattern, and more precisely, GSH depletion inhibits Th1-associated cytokine production and/or favours Th2 associated responses. It is known that GSH is not transported to most cells and tissues in a free form. Therefore, a number of different approaches have been developed in the last years to circumvent this problem. This review discusses the capacity of some new molecules with potent pro-GSH effects either to exert significant antiviral activity or to augment GSH intracellular content in macrophages to generate and maintain the appropriate Th1/Th2 balance. The observations reported herein show that pro-GSH molecules represent new therapeutic agents to treat antiviral infections and Th2-mediated diseases such as allergic disorders and AIDS.

Peptide nucleic acids (PNAs) provide a powerful tool to study the mechanism of transcription and translation, an innovative strategy to regulate target gene expression. They have been successfully used in antisense technology, for their ability to specifically bind to messenger RNA (mRNA) targets and to inhibit translation of the target genes. However, unlike most of the DNA and RNA oligonucleotides, PNAs are poorly penetrated through the cell membrane, partially due to their uncharged property. To enhance the efficiency in PNA delivery, many strategies have been explored. We here compare the efficacy of three different delivery strategies for antisense PNA: 1) conjugation to hydrophobic peptides, 2) adsorption onto polymeric microspheres and 3) encapsulation in autologous erythrocytes. To this purpose, we designed and prepared PNA sequences able to inhibit the expression of macrophage enzymes involved in inflammatory process, i.e. nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) and tested their antisense activity in a murine macrophage cellular model. Both delivery through polymeric microspheres and encapsulation into erythrocytes allowed the antisense activity of unmodified PNAs at nanomolar concentration.

A real-time PCR assay using SYBR Green I for quantification of provirus load in a murine model of AIDS (i.e., LP-BM5 infection) was developed and validated. In this method, data are normalized against the 18S rRNA gene. The method has a dynamic range of 8 logs and a sensitivity of one copy.

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A combination of antiretroviral drugs acting at different points in the virus replication cycle was evaluated in a murine retrovirus-induced immunodeficiency model of AIDS (MAIDS). Intramuscular administration of high doses of reduced glutathione (GSH, 100 mg/mouse/day) and AZT (0.25 mg/ml in drinking water) was found to reduce lymphoadenopathy (92%), splenomegaly (80%), and hypergammaglobulinemia (90%) significantly more than AZT alone. Combined treatment resulted in a reduction in proviral DNA content of 69, 66, and 60%, respectively, in lymph nodes, spleen, and bone marrow. Furthermore, the stimulation index of B cells was also significantly higher in animals receiving GSH and AZT whereas additional responses were not observed in the T cell stimulation index and blood lymphocyte phenotype analyses. In conclusion, the administration of high doses of GSH and AZT, a new combination of antiviral drugs, seems to provide additional advantages compared to single-agent therapy.

International audience; BACKGROUND: The aim of this study was to investigate the molecular mechanisms involved in the production of Th1 cytokines, namely IL-12 and IL-27, when the intra-macrophage redox state was altered by different chemical entities such as GSH-C4, which is reduced glutathione carrying an aliphatic chain, or I-152, a pro-drug of N-acetyl-cysteine (NAC) and beta-mercaptoethylamine. We had already demonstrated that GSH-C4 and I-152 could shift the immune response towards Th1 in Ovalbumin-immunized mice as well as enhance Th1 response in HIV-1 Tat-immunized mice. METHODOLOGY/PRINCIPAL FINDINGS: By a new high performance liquid chromatography method, we found that 20 mM GSH-C4 provided a number of thiol species in the form of GSH, while 20 mM I-152 decreased GSH and increased the thiols in the form of NAC and I-152. Under these experimental conditions, GSH-C4 and I-152 enhanced and suppressed respectively the mRNA expression levels of IL-12 p40 induced by LPS/IFN-γ as assessed by Real-Time PCR. The protein production of IL-12 p40 was increased by GSH-C4 and decreased by I-152 as determined by Enzyme-linked immunosorbent assay. Western immunoblot and electrophoretic mobility shift assays revealed that Nuclear Factor -kB (NF-kB) activation was inhibited by I-152 and prolonged by GSH-C4. Twenty mM I-152 stimulated IL-27 p28 gene expression and sustained Signal Transducer and Activator of Transcription (STAT)-mediated interferon regulator factor 1 (IRF-1) de novo synthesis. By contrast, 20 mM GSH-C4 did not exert any effect on IL-27 p28 gene expression. CONCLUSIONS AND SIGNIFICANCE: an increase in the intra-macrophage redox state by GSH-C4 and I-152 enhances Th1 cytokine production although the chemical structure and the intra-cellular metabolism influence differently signalling pathways involved in IL-27 or IL-12 production. GSH-C4 and I-152 may be used as Th1 immunomodulators in some pathologies and in ageing where GSH depletion may contribute to the Th1/Th2 imbalance, and in new immunization strategies.

2-Fluoro-ara-AMP (fludarabine phosphate) is a purine analogue with anti-neoplastic activity in lymphoproliferative malignancies. Fludarabine phosphate activity and toxicity is schedule-dependent; multiple daily administrations (for five days) are more effective than single dose. We have encapsulated fludarabine phosphate in human erythrocytes and found that it is slowly released as fludarabine for more than four days. Encapsulated fludarabine phosphate does not affect erythrocyte metabolism and is rapidly converted by erythrocyte enzymes both to fludarabine with a Km of 0.4 mM and a Vmax of 20 nmol/min per g hemoglobin and to fludarabine diphosphate and triphosphate. The apparent Km for fludarabine monophosphate in the phosphorylation reaction was 0.4 mM and the Vmax 40 nmol/min per g hemoglobin. In the phosphorylation of 2-fluoro-ara-AMP to the di- and triphosphate derivatives, ATP was the phosphate donor with apparent Km of 0.12 and 1.0 mM, respectively. During incubations of 2-fluoro-ara-AMP-loaded erythrocytes at 37°C fludarabine was found in equilibrium between the erythrocyte and the culture medium suggesting that permeation of the erythrocyte membrane is not rate-limiting. Thus, fludarabine phosphate-loaded erythrocytes might be used as a slow-delivery system for fludarabine administration in the treatment of lymphoid malignancies.

A new Azidothymidine derivative, di-(thymidine-3′-azido-2′,3′-dideoxy-D-riboside)-5′-5′-p 1 -p 2 -pyrophosphate (AZT p2 AZT), was encapsulated in human erythrocytes according to a conservative procedure of hypotonic shock-isotonic resealing and reannealing. Like in erythrocyte lysates supplemented with 1 mM ATP, intact red cells too were found to convert AZT p2 AZT to 3′-Azido-3′-deoxythymidine which was then released linearly in plasma. The major metabolic pathway involved in this conversion was the symmetrical hydrolysis of AZT p2 AZT to yield two 3′-Azido-3′-deoxythymidine-5′-phosphate molecules which were then dephosphorylated to 3′-Azido-3′-deoxythymidine. At late times of incubation, also a limited asymmetrical hydrolysis of AZT p2 AZT became apparent in the intact erythrocytes, yielding 3′-Azido-3′-deoxythymidine-5′-diphosphate that was then converted to the triphosphorylated derivative. Therefore, erythrocytes loaded with AZT p2 AZT act“in vitro” as bioreactors ensuring sustained and potentially useful release of 3′-Azido-3′-deoxythymi-dine.

Overconsumption of abnormally elevated blood metabolites by erythrocyte-encapsulated enzyme proteins has beeen shown to be feasible.1,2 Along this line we examined the possibility of using red blood cells (RBC) loaded with glucose catabolizing enzymes to regulate blood glucose around physiological levels in the hyperglycemic state. In previous studies3 we reported the biochemical properties of human and mice erythrocytes upon encapsulation of glucose oxidase (GOD) alone or coentrapped with hexokinase (HK). The “in vitro” results showed that the cells had an enhanced glucose consumption and acceptable biological properties. In order to establish their use as potential bioreactors for control of hyperglycemias, “in vivo” studies were performed. The results reported in this paper show that erythrocytes glucose oxidase and hexokinase-loaded have a slightly decreased survival and are able to regulate blood glucose around physiological levels in diabetic mice. These results suggest that enzyme-loaded erythrocytes can behave as circulating bioreactors and can provide a new way for the overconsumption of abnormally elevated blood glucose.

2′,3′-Dideoxycytidine (ddC) is a nucleoside analogue that inhibits human immunodeficiency virus type 1 (HIV-1) replication in vitro and is currently used in the therapy of acquired immune deficiency syndrome (AIDS). This compound exerts a delayed cytotoxicity due to inhibition of mitochondrial DNA (mDNA) synthesis. Long-term exposure of U937 human monoblastoid cells to ddC resulted in a time- and concentration-dependent decrease in mDNA content and Rhodamine 123 fluorescence. However, after 2 months on 0.1 microM ddC, a drug-resistant cell line (U937-R) with 66% of the normal amount of mDNA was isolated. ddC transport in U937 and U937-R cell lines was similar. In contrast, U937-R accumulated ddC phosphorylated derivatives at a much lower rate and to a reduced concentration into acid-soluble material. The rate of 2′,3′-dideoxycytidine 5′-triphosphate (ddCTP) formation in U937-R cells was almost one-third of that measured in normal cells, although the rate of ddCTP catabolism was similar in both cell lines. Dideoxyliponucleotide (ddCDP-choline and ddCDP-ethanolamine) formation was also much slower (between one-half and one-third as fast) in U937-R than in control cells, although catabolism occurred at similar rates. ddC was phosphorylated by a cytoplasmic deoxycytidine kinase in both cell lines. This enzyme showed Km values for ddC of 80 +/- 7 and 140 +/- 9 microM in U937 and U937-R cells respectively. Furthermore, Vmax was 12 +/- 1.1 and 7.8 +/- 0.5 pmol/min per mg of protein in U937 and U937-R. Thus resistance to ddC toxicity may be due to cells' decreased ability to accumulate intracellular ddC anabolites, which may depend on cytoplasmic deoxycytidine kinase.