Seronegative visceral leishmaniasis with relapsing and fatal course following rituximab treatment.

Both the presentation and clinical course of visceral leishmaniasis (VL) may be atypical in immunosuppressed subjects, often resulting in delayed diagnosis and treatment. We describe a case of VL characterized by negative serologic testing, a relapsing course, and a fatal outcome 2 years after the patient had been successfully treated for non-Hodgkin's lymphoma with rituximab. Diagnosis of VL may be further delayed or even missed in patients treated with drugs that interfere with specific antibody production unless specific diagnostic methods, such as bone marrow examination and parasite DNA amplification/detection, are routinely employed.

HIV-infected long-term nonprogressors display a unique correlative pattern between the interleukin-7/interleukin-7 receptor circuit and T-cell homeostasis.

BACKGROUND: We hypothesized that there may be a correlation between the interleukin-7 (IL-7)/IL-7 receptor (IL-7R) regulatory system and parameters of T-cell homeostasis in HIV-infected long-term nonprogressors (LTNPs) as compared with patients with disease progression. METHODS: The possibility of a correlation between T-cell homeostatic parameters and IL-7/IL-7R was investigated in 22 LTNPs (CD4 count > or =500 cells/microL for >10 years) vs. HIV-positive patients at different disease stages [12 early: CD4 count > or =400 cells/microL ; 15 late (AIDS-presenters): CD4 count < or =150 cells/microL ]. RESULTS: Compared with early-stage HIV-positive patients, LTNPs displayed a higher circulating IL-7 concentration (P=0.05), which was positively associated with higher IL-7Ralpha expression and a higher T-cell receptor excision circle (TREC) content specifically within CD4 cells (P<0.05). Compared with late-stage disease patients, early-stage disease patients displayed a lower IL-7 concentration (P<0.01) and higher percentages of IL-7Ralpha+ CD4 and CD8 cells (P=0.05). IL-7 was positively correlated with the percentage of TREC+ CD4 cells (P<0.01), which translated into a higher percentage of naïve CD4 cells in early-stage disease patients than in late-stage disease patients; however, the CD4 cells in early-stage disease patients were less enriched in recent thymic emigrants (RTEs) compared with LTNPs (P<0.05). In late-stage AIDS-developing patients, substantially increased IL-7 was correlated with a decreased percentage of IL-7Ralpha+ CD4 cells (P=0.01), which resulted in these patients having a significantly lower percentage of naïve T cells (P<0.01) and a significantly lower content of TREC (P<0.01) than the other patients. CONCLUSIONS: The maintenance of high CD4 cell counts in LTNPs was associated with a specific IL-7/IL-7R pattern characterized by increased IL-7 and highest IL-7Ralpha-expressing CD4 cells relative to other patients. Compared with patients with late-stage disease, LTNPs displayed a phenotypically naïve, less activated CD4 cell pool highly enriched in RTEs, suggesting the existence of a compensatory IL-7-mediated pathway specifically sustaining peripheral CD4 counts.

Effect of macrophage depletion on viral DNA rebound following antiretroviral therapy in a murine model of AIDS (MAIDS).

In the attempt to eradicate HIV-1 infection, a strategy to eliminate macrophages, one of the most important cellular reservoirs in sustaining virus replication during HAART, could be of great benefit in the suppression of viral rebound. Aware of the ability of clodronate to cause macrophage depletion, the effect of the administration of clodronate encapsulated in erythrocytes on disease progression and on viral rebound was evaluated in a murine model of AIDS (MAIDS). One group of LP-BM5 retroviral complex-infected C57BL/6 mice received oral administrations of azidothymidine and dideoxyinosine daily for 12 weeks; two other groups received in addition, either clodronate-loaded erythrocytes or free clodronate at 7-10 day intervals. At the end of the treatment, the three groups maintained parameters characterizing disease progression similar to those of uninfected mice and showed a significantly lower level of BM5d DNA than infected mice in all organs and cells tested. To assess the viral rebound, some animals were left for an additional 4 month period without any treatment. After this time, the BM5d DNA content in blood leukocytes increased in all groups, but the group having received clodronate-loaded erythrocytes, in addition to transcriptase inhibitors, showed a significant delay in viral rebound.

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.

Antiviral and immunomodulatory properties of new pro-glutathione (GSH) molecules.

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.

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.

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.

Protein degradation and apoptotic death in lymphocytes during Fiv infection: activation of the ubiquitin-proteasome proteolytic system.

The movement of a cell through the sequential phases of apoptosis is accompanied by a progressive decrease in cell size with loss in protein mass. In lymphocytes from Hiv-infected persons, protein loss during apoptosis is due to increased protein degradation rather than decreased synthesis. To identify and characterize the proteolytic enzymes or enzyme systems involved in this process, we studied several features of protein turnover in lymphocytes from peripheral blood and lymph nodes during the natural and experimental infection by feline immunodeficiency virus (Fiv). This animal model allowed us to integrate in vivo results with in vitro observations of protein damage. Here we report that protein breakdown in apoptotic cells is concomitant with the activation of the ATP and ubiquitin-dependent multicatalytic system (proteasome). We suggest that proteasome activation is part of the proteolytic cascade in the execution phases of apoptosis in AIDS.

Competitive PCR for quantification of BM5d proviral DNA in mice with AIDS.

Murine AIDS in C57BL/6 mice is caused by a unique mixture of murine leukemia viruses. We report the use of a competitive PCR to detect and quantitate BM5d proviral DNA. This assay allowed discrimination among endogenous wild-type murine retroviruses and BM5d sequences. Furthermore, the method was subsequently used to evaluate the amount of BM5d in infected mice and in infected AZT (zidovudine)-treated mice, providing an effective way to quantitatively evaluate drug efficacy in the murine AIDS model.

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.

2',3'-Dideoxycytidine cytotoxicity in human macrophages.

Human macrophages when cultured for several weeks in the presence of therapeutically relevant 2',3'-dideoxycytidine (ddC) concentrations show a time-dependent decay in mitochondrial DNA content. This decay is associated with a reduction of Rhodamine 123 fluorescence, a marker for mitochondrial membrane potential suggesting that impairment of mitochondrial functions occurs. Mitochondrial metabolic impairment was confirmed by direct evaluation of lactate production, which is markedly increased in cells treated with ddC. The activity of protein kinase C and intracellular free Ca2+ upon addition of phorbol 12-myristate 13-acetate (PMA) were lower in the drug-treated cells compared to controls. A 50% reduction in O2-release was also found upon PMA stimulation. Fluorescent latex beads, yeast and bacteria phagocytosis were normal, but intracellular bacteria killing was markedly impaired in ddC-exposed macrophages. Thus, ddC exerts a delayed mitochondrial toxicity also on differentiated macrophages with impairment of several metabolic properties and O2 production causing a reduced ability of these phagocytic cells to kill phagocytosed bacteria.

Up-regulation of the ubiquitin-conjugating and proteolytic systems in murine acquired immunodeficiency syndrome.

Lymphoproliferation, chronic B-cell activation resulting in hypergammaglobulinemia, and profound immunodeficiency are prominent features of retrovirus-induced murine acquired immunodeficiency syndrome (murine AIDS). Here we demonstrate that in murine AIDS the ATP-dependent and ubiquitin-dependent proteolytic system is strongly affected, at least in the lymph nodes of infected mice. Solid-phase immunochemical assays show that the ubiquitin-conjugate pools increase by about threefold 10 weeks after infection, then decline slightly 15 weeks after infection to a twofold increase. Accumulation of ubiquitin conjugates is accompanied by induction of the ubiquitin-conjugating pathway, involving several carrier-protein isozymes (E2), mainly 14-kDa E2 and 17-kDa E2. Furthermore, accumulation of ubiquitin conjugates and induction of the conjugating system are coincident with an increase in the proteolytic activity supported by the 26S proteolytic complex. However, 15 weeks after infection, when the conjugation rate and levels of ubiquitin conjugates decrease, proteasome activity returns to values similar to those of the control, suggesting that a higher proteosomal activity is no longer needed. The concerted induction of the ubiquitin-conjugating and proteolytic systems in murine AIDS apparently does not involve the breakdown of viral products nor is it supported by virus-coded events, but probably arises as a cellular response to viral infection.