Inhibition of murine AIDS by pro-glutathione (GSH) molecules.

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.

Characterization and complementation of a Fus3/Kss1 type MAPK from Tuber borchii, TBMK.

Mitogen-activated protein kinases (MAPK) are used by organisms to transduce extra cellular signals from the environment in cellular events such as proliferation and differentiation. In the present study, we have characterized the first MAPK from the ectomycorrhizal fungus Tuber borchii (TBMK) which belongs to the YERK1 (yeast extra cellular regulated kinase) subfamily. TBMK is present as a single copy in the genome and the codified protein was phosphorylated during the interaction with the host plant, Tilia americana. Complementation studies showed that TBMK restores pheromone signaling in Saccharomyces cerevisiae and partially restores invasive growth of Fusarium oxysporum that lack the fmk1 gene. This suggests a protein kinase activity and its involvement in the infection processes. Hence, TBMK could play an important role during the pre-symbiotic phase of T. borchii with its host plant in the modulation of genes necessary for the establishment of symbiosis leading to the synthesis of functional ectomycorrhizae.

Red blood cell-mediated delivery of recombinant HIV-1 Tat protein in mice induces anti-Tat neutralizing antibodies and CTL.

The immunotherapeutic potential of biologically active HIV-1 Tat protein coupled to autologous red blood cells (RBCs) was evaluated in a mouse model. HIV-1 Tat expressed in Escherichia coli and purified to homogeneity was found to be active in viral trans activation and efficiently internalised by monocyte-derived dendritic cells (MDDCs). The product of HIV-Tat biotinylation and coupling to RBCs by means of a biotin-avidin-biotin bridge, (RBC-Tat), showed no trans activation activity and was still efficiently internalized by MDDCs as compared to uncoupled Tat.Balb/c mice were then immunized with 10 microg of soluble Tat in complete Freund's adjuvant or with 40 ng of Tat coupled on RBCs surface and boosted at week 3, 6 and 25 with 5 microg soluble Tat in incomplete Freund's adjuvant or with 20 ng of RBC-coupled Tat, respectively. Anti-Tat antibody response was similar in both groups; however, 2/6 animals immunized with soluble Tat and 6/6 animals immunized with RBC-Tat developed anti-Tat neutralizing antibodies. In addition, at week 28 cytolytic anti-Tat CTLs were detected in all animals although they were slightly higher in mice immunized with RBC-Tat. These results indicate that RBC-mediated delivery of HIV-1 Tat, in amounts 250 times lower than soluble Tat, is safe and induces specific CTL responses and neutralizing antibodies.

Macrophage protection by addition of glutathione (GSH)-loaded erythrocytes to AZT and DDI in a murine AIDS model.

Monocyte-macrophages play a central role in HIV-1 infection because they are among the first cells to be infected and because later they are important reservoirs for the virus. Thus, newly designed therapies should take into account the protection of this cell compartment. Herein, we report the results obtained in a murine AIDS model, by the addition to AZT+DDI of a system (GSH-loaded erythrocytes) able to protect macrophages against HIV-1 infection. Five groups of LP-BM5-infected mice were treated as follows: one group was treated by AZT, one group was treated by DDI, one group was treated by the combination of both, another by GSH-loaded erythrocytes, and finally, one by the combination of all three. After 10 weeks of infection the parameters of the disease were studied and the proviral DNA content in different organs and in macrophages of bone marrow and of the peritoneal cavity was quantified. The results obtained show that mice treated with AZT+DDI+GSH-loaded erythrocytes showed proviral DNA content in the brain and in macrophages of bone marrow that was significantly lower than in mice treated with AZT+DDI. This study may help developing strategies aimed at blocking HIV-1 replication in its reservoirs in the body.

Erythrocyte-mediated delivery of drugs, peptides and modified oligonucleotides.

An important determinant for the success of every new therapy is the ability to deliver the molecules of interest to the target cells or organ. This selective delivery is even more complex when the therapeutic agents are peptides, modified oligonucleotides or genes. In this paper we summarize the possibility of using autologous erythrocytes for the delivery and targeting of new and conventional therapeutics. In fact, a number of macromolecules can be encapsulated by different procedures into human erythrocytes. These modified cells can then be re-infused into the same or a compatible recipient where they can circulate for several weeks. However, drug-loaded erythrocytes can also be modified to be selectively recognized by tissue macrophages. These phagocyte cells recognize the modified drug-loaded erythrocytes which are able to release their content into the macrophage. The feasibility and safety of the use of erythrocytes as drug delivery systems was evaluated in 10 cystic fibrosis patients, where a sustained release of corticosteroids from dexamethasone 21-phosphate-loaded erythrocytes was obtained. In vitro human erythrocytes were found to be able to deliver ubiquitin analogues and modified oligonucleotides to macrophages. Thus, drug-loaded erythrocytes are safe and useful carriers of new and conventional therapeutics and can be advantageous delivery systems for new clinical applications where proteins and oligonucleotides are therapeutic agents.

Heat stress-activated, calcium-dependent nitric oxide synthase in sponges.

The presence of Ca(2+)-dependent, heat-stress-activated nitric oxide synthase (NOS) activity in peculiarly shaped, fusiform, and dendritic sponge cells is described for the first time. The NOS activity was evidenced evaluating the conversion of radioactive citrulline from [(14)C]arginine in intact cells from two different species that are phylogenetically unrelated in the class of Demospongiae: Axinella polypoides and Petrosia ficiformis. The production of nitrogen monoxide (NO) was confirmed by electron paramagnetic resonance analysis, and the histochemistry technique of NADPH diaphorase showed a specific localization of NOS activity in a particular network of dendritic cells in the sponge parenchyma. Sponges are the most primitive metazoan group; their evolution dates back 600 million years. The presence of environmental stress-activated NOS activity in these organisms may prove to be the most ancient NO-dependent signaling network in the animal kingdom.

New drug combinations for the treatment of murine AIDS and macrophage protection.

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.

Inhibition of murine AIDS by alternate administration of azidothymidine and fludarabine monophosphate.

Anti-HIV-1 combination therapies, including protease and reverse transcriptase inhibitors, can reduce plasma viremia to undetectable levels within the first 2 weeks of treatment. This reduction is followed by a slower decline that primarily results from the presence of viral reservoirs such as CD4+ memory cells, dendritic cells, and macrophages. For this reason, we evaluated a new drug combination therapy that includes a lympholytic drug: (2-fluoro-ara-AMP, fludarabine) to eliminate cells already infected and an antiviral drug (azidothymidine [AZT]) to protect cells not yet infected. We used C57BL/6 mice infected with the retroviral complex LP-BM5, which developed severe immunodeficiency (i.e., murine AIDS), to select the most effective fludarabine regimen to inhibit disease progression, and then to evaluate the efficacy and toxicity of the fludarabine and AZT combinations. The results obtained show that intraperitoneal administration of fludarabine at 3 mg/mouse twice a day for 4 weeks is the most effective regimen in reducing splenomegaly, lymphadenopathy, hypergammaglobulinemia, and proviral DNA content in spleen and lymph nodes and in restoring the architecture of lymph nodes. Subsequently, we evaluated the combined or sequential administration of fludarabine and AZT. The data reported in this paper show that the sequential administration of the two drugs provides additive antiviral effects that reduce spleen and lymph node weights to normal values and proviral DNA content by approximately 95% in all infected organs; the phenotypes of blood T and B cells moved toward control values, although the number of B cells was significantly reduced by fludarabine treatment. Finally, we evaluated the outcome of the disease after suspension or continuation of different treatment regimens. In all treatment groups, the disease progressed and increased proviral DNA content was found in infected organs, but animals receiving the sequential administration of fludarabine and AZT were less affected than those receiving only fludarabine or the simultaneous administration of both. The results obtained suggest that fludarabine could be part of a new therapeutic approach aiming at eradicating HIV from those cells that have been already infected and that are not protected by highly active antiretroviral therapy (HAART).

New treatment protocol including lympholytic and antiretroviral drugs to inhibit murine AIDS.

Highly active antiretroviral therapy (HAART), although very efficient in reducing viral load to undetectable levels within 2 weeks, does not eradicate HIV-1 infection and after the suspension of therapy, HIV RNA rebounds to pretherapy levels. This limited efficacy is mainly due to the existence of viral reservoirs such as CD4+ T cells, macrophages, and dendritic cells in which the virus can remain latent. Elimination of these latent reservoirs would be a possible solution to this problem and various efforts are now being directed to this end. With this goal in mind, we investigated a lympholytic drug with known activity against lymphoproliferative malignancies, 2-fluoro-ara-AMP (fludarabine). The murine model of AIDS was used to evaluate the efficacy of alternating administration of fludarabine and azidothymidine (AZT). The aim of this experiment was to eliminate infected cells with fludarabine and protect noninfected cells with AZT. LP-BM5-infected mice were treated with two different therapeutic protocols: one group was treated with two alternating 3-week cycles of fludarabine and AZT (treatment A), whereas the other was treated with three alternating 2-week cycles of fludarabine and AZT (treatment B); both treatments lasted 12 weeks and the animals in the two groups received the same amount of drug. At different times of infection, disease-related findings (i.e., splenomegaly, lymphadenopathy, hypergammaglobulinemia, T-cell and B-cell spleen cell proliferative index, and phenotypes of peripheral blood lymphocytes) were analyzed and the content of proviral DNA in the lymph nodes was quantified. The results obtained show that treatment B was more effective in inhibiting disease progression than treatment A. In fact, all parameters investigated were almost within control values. These results were also confirmed by the quantification of proviral DNA content in the lymph nodes, which after 12 weeks of treatment A declined by approximately 50%, whereas treatment B decreased proviral DNA content by approximately 85% with respect to infected/untreated mice. The data obtained suggest that a therapeutic protocol including three cycles rather than two of a lympholytic drug and antiretroviral drugs is more advantageous. The efficacy of the treatment could likely increase if other drugs were used in addition to AZT and more cycles of fludarabine were added. This approach appears to be of potential interest in an HIV-1 eradication protocol.

Metabolism, mitochondrial uptake and toxicity of 2', 3'-dideoxycytidine.

2',3'-Dideoxycytidine (ddCyd) is a prescription anti-retroviral drug that causes mitochondrial toxicity and peripheral neuropathy. ddCyd is actively phosphorylated by cytosolic deoxycytidine kinase and nucleoside (di)phosphate kinase to the 5'-triphosphate derivative. However, 2',3'-dideoxycytidine 5'-diphosphocholine (ddCDP-choline) was also found in human cells incubated with ddCyd. In this paper we show that ddCDP-choline is produced from dideoxyCTP (ddCTP) and phosphocholine by phosphocholine cytidylyltransferase. dCTP and CTP appear to activate this synthesis in a concentration-dependent manner. Although ddCTP and ddCDP-choline can both enter the mitochondria, ddCDP-choline uptake is more efficient than ddCTP uptake. These data suggest that ddCDP- choline is the ddCyd metabolite that is probably responsible for mitochondrial toxicity. The uptake of ddCTP and ddCDP-choline by mitochondria is inhibited by 3.0 mM l-carnitine in the cell-free system investigated; when added to U937 cells grown in the presence of 0.25 microM ddCyd, 3.0 mM l-carnitine partially abrogated the mitochondrial toxicity of ddCyd.

Liposomes as carriers of the antiretroviral agent dideoxycytidine-5'-triphosphate.

The presence and replication of the human immunodeficiency virus (HIV) in cells of the mononuclear phagocyte system (MPS) together with the preferential uptake of liposomes in macrophages suggest that liposomes can become a valuable carrier of anti-HIV agents. Moreover, liposomes reduce toxicity of encapsulated drugs and protect encapsulated drugs against rapid degradation in the blood circulation. To overcome problems associated with the administration of free nucleosides and to improve targeting to the MPS, dideoxycytidine-5'-triphosphate (ddCTP) was encapsulated in liposomes. Liposomes were stable with regard to retention of the entrapped drug, particle size and chemical stability of ddCTP. Results obtained with liposome encapsulated ddCTP in the murine acquired immunodeficiency syndrome (MAIDS) model indicate that ddCTP encapsulated in liposomes can reduce proviral DNA in cells of the mononuclear phagocyte system (MPS) in both spleen and bone marrow.

Role of macrophage protection in the development of murine AIDS.

Macrophages play a key role in AIDS pathogenesis and thus controlling infectivity and viral replication in these cells is a key issue in any antiretroviral therapy. In the present study, using a murine model of AIDS, we evaluated new therapeutic approaches specifically designed for the protection of macrophages. Based on previous observations, we took advantage of the unique ability of autologous erythrocytes to deliver drugs selectively to macrophages. The antiviral drugs selected were a new homodimer of AZT (AZTp2AZT) and reduced glutathione (GSH). The addition of an oral drug for the protection of lymphocytes (i.e., AZT) was also investigated. C57BL/6 mice infected with the retroviral complex LP-BM5 were treated with GSH-loaded erythrocytes, GSH-loaded erythrocytes plus oral AZT, or GSH/AZTp2AZT-loaded erythrocytes plus oral AZT. The treatments including AZT and erythrocytes loaded with GSH alone or with GSH plus AZTp2AZT provided similar results and were most effective in inhibiting the progression of MAIDS; they reduced splenomegaly, lymphadenopathy, and hypergammaglobulinemia by about 70%, 90% and 83%, respectively, when compared with infected animals at 10 weeks postinfection. Evaluation of BM5d proviral DNA content in infected organs revealed that both treatments were able to almost completely protect most infected animals. They were also able to normalize the blood lymphocyte phenotype and to restore the responses of T and B cells to mitogens significantly. Treatment with GSH-loaded erythrocytes alone did not provide significant results for most parameters investigated, but a marked reduction in proviral DNA content was obtained in infected organs, including the brain. The results reported in this paper confirm the important role of macrophages in retroviral infection and moreover prove that erythrocytes, by selectively protecting these cells, strongly affect MAIDS progression. Furthermore, the combination of GSH- or GSH/AZTp2AZT-loaded erythrocytes with an oral nucleoside analogue (AZT) for the protection of lymphocytes provides additive responses in all the parameters investigated.

Bioreactivity and biocompatibility of a vitamin E-modified multi-layer hemodialysis filter.

BACKGROUND: The present study was designed to test the biocompatibility of a new vitamin E-modified multi-layer membrane (CL-E filter), as well as its ability to protect against oxygen free radicals during hemodialysis (HD). METHODS: We investigated, both in vitro and in vivo, the bioreactivity of the filter with respect to the blood antioxidants and its ability to prevent lipoperoxidation. The effects on the leukocyte respiratory burst were also studied. Cuprammonium rayon was used as a comparison material (CL-S filter). RESULTS: The in vitro results demonstrated that, under controlled conditions, CL-E is able to preserve blood antioxidants, and particularly vitamin E, from the spontaneous consumption observed in the incubation with CL-S filters and in control incubations. In accordance with this observation, the rate of the oxidative demolition of lipids either in plasma and red blood cells (RBC) or from rat brain homogenate decreased after the exposure to CL-E filters in comparison with the CL-S filter. Moreover, in the absence of any significant cytotoxic effects due to both the types of material studied, the production of oxygen free radicals and nitric oxide (NO) by leukocytes was higher after their in vitro exposure to CL-S, but was quite similar to that of the control leukocytes after exposure to CL-E. In vivo, a one-month treatment with the CL-E filter increased plasma vitamin E by 84.3% with respect to treatment with CL-S; this gain slightly decreased to 68.9% when CL-E treatment was prolonged to three months. In the RBC, vitamin E was found to have increased by 76.7% and 113.4% at one and three months, respectively. Plasma glutathione (GSH) levels determined at three months were significantly increased from 0.10 +/- 0.02 to 0.33 +/- 0.12 mumol/ml, while the erythrocyte GSH was only slightly increased. The leukocyte function estimated as responsiveness to soluble chemical stimuli in CL-S-treated patients was significantly improved both qualitatively and quantitatively after CL-E treatment. The presence of an increased number of mononuclear cells undergoing programmed cell death (apoptosis) in CL-S-treated patients (18.8 +/- 1.7% vs. a control value of 6.5 +/- 2.3%) as well as the apoptogenic effect of their plasma in vitro on U937 cells was significantly corrected after CL-E treatment (mean decrease in apoptotic mononuclear cells at 24 hours of culture, 25.5% and 27.1% at 1 and 3 months, respectively). The anti-apoptogenic effect of CL-E treatment showed a close dependence on the increase in vitamin E in the blood cell compartment. CONCLUSIONS: This study suggests that this vitamin E-modified membrane can be considered a highly biocompatible material, the antioxidant properties of which can exert a site-specific and timely scavenging function against oxygen free radicals in synergy with a hypostimulatory action on the PMN respiratory burst.

AIDS vaccination studies using an ex vivo feline immunodeficiency virus model: homologous erythrocytes as a delivery system for preferential immunization with putative protective antigens.

Feline immunodeficiency virus (FIV) is a useful model for testing of criteria for AIDS vaccine development. In the protocol we adopted, we used a primary isolate of FIV as a source of antigen and, for challenge, plasma from cats infected with the homologous virus never passaged in vitro. Cat erythrocytes (RBC) were coated with the surface components of freshly harvested and purified FIV by means of biotin-avidin-biotin bridges and used to immunize specific-pathogen-free cats (four doses at monthly intervals; total amount of FIV antigen administered per cat, approximately 14 microg). Immunized cats developed moderate levels of antibodies directed mainly to surface components of the virion and clearly evident lymphoproliferative responses. Four months after the last dose of immunogen, FIV-immunized cats and control cats immunized with bovine serum albumin-coated RBC were challenged. Judged from the results of the subsequent 12-month follow-up, FIV-immunized cats exhibited at least some degree of protection. However, following rechallenge, most of the FIV-immunized animals became virus positive in spite of a booster immunogen dose given 2 months before the second challenge.

Inhibition of murine AIDS by a new azidothymidine homodinucleotide.

A new antiretroviral drug (azidothymidine homodinucleotide, AZTp2AZT), designed for the protection of macrophages against retroviral infection, was evaluated in a murine retrovirus-induced immunodeficiency model of AIDS (MAIDS) alone and in combination with oral azidothymidine (AZT). C57BL/6 mice were infected with the retroviral complex LP-BM5 and treated for 3 months by weekly administrations of 15 nmol of AZTp2AZT encapsulated into autologous erythrocytes for macrophage protection. AZTp2AZT treatment was found to reduce lymphoadenopathy (48%), splenomegaly (26%), and BM5d proviral DNA content in lymph nodes, spleen, and brain of 37%, 40%, and 36%, respectively, compared with untreated animals. AZT administration in drinking water (0.25 mg/ml) was more effective than administration of AZTp2AZT encapsulated into erythrocytes in reducing lymphoadenopathy, splenomegaly, gammaglobulinemia, and proviral DNA content in lymph nodes, but it caused a reduction in erythrocyte count and hematocrit levels. Although combined treatments do not provide additive responses in the several parameters investigated, they were found to be much more effective in reducing the proviral DNA content in brain (67%) than were monotherapies. Furthermore, no apparent signs of hematotoxicity were observed. Thus, macrophage delivery of antiviral drugs may contribute to brain protection from retroviral infections by mechanisms other than those exerted by oral AZT administration.

Antiretroviral effect of combined zidovudine and reduced glutathione therapy in murine AIDS.

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.

Red blood cells as delivery system for recombinant HSV-1 glycoprotein B: immunogenicity and protection in mice.

The immunotherapeutic potential of autologous red blood cells (RBC) coupled to the secretory form of herpes simplex virus type 1 (HSV-1) glycoprotein B (gB1s) was examined with a mouse model of HSV-1 infection. C57BL/6 mice were immunized intraperitoneally with gB1s (0.05 microgram per dose) linked to RBC, or mixed with Freund's complete or bound to AlPO4 adjuvants (0.5 microgram per dose). Mice immunized with RBC coupled gB1s were protected against lethal and latent HSV-1 infection, and developed an anti-HSV antibody response, as measured by ELISA and HSV-1 neutralization assays, similar or higher than that elicited by the same antigen in Freund's complete adjuvant, which suggested that autologous RBC coupled to gB1s may provide an effective and safe method of immunization against HSV infection.

Inhibition of murine AIDS by reduced glutathione.

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.