Intralesional regulatory T-cell suppressive function during human acute and chronic cutaneous leishmaniasis due to Leishmania guyanensis.

The levels of regulatory T cells (Treg cells), analyzed by Foxp3 mRNA expression, were determined in lesions from patients with acute cutaneous leishmaniasis (ACL) and chronic cutaneous leishmaniasis (CCL). We demonstrated that Treg cells preferentially accumulate in lesions from ACL patients during the early phase of infection (lesion duration of less than 1 month). In addition, levels of Foxp3 mRNA transcripts were significantly higher in specimens from patients with CCL than in those from patients with ACL, suggesting a critical role of intralesional Treg cells in CCL. Intralesional Treg cells from both ACL and CCL patients were shown to have suppressive functions in vitro, since they inhibited the gamma interferon (IFN-gamma) produced by CD4(+) CD25(-) T cells purified from peripheral blood mononuclear cells from the same patient in response to Leishmania guyanensis stimulation. Intralesional 2,3-indoleamine dioxygenase (IDO) mRNA expression was associated with that of Foxp3, suggesting a role for IDO in the suppressive activity of intralesional Treg cells. In addition, a role, albeit minor, of interleukin-10 (IL-10) was also demonstrated, since neutralization of IL-10 produced by intralesional T cells increased IFN-gamma production by effector cells in an in vitro suppressive assay. These results confirm the role of intralesional Treg cells in the immunopathogenesis of human Leishmania infection, particularly in CCL patients.

A novel cellular RNA helicase, RH116, differentially regulates cell growth, programmed cell death and human immunodeficiency virus type 1 replication.

In an effort to define novel cellular factors regulating human immunodeficiency virus type 1 (HIV-1) replication, a differential display analysis has been performed on endogenously infected cells stimulated with the HIV-suppressive immunomodulator Murabutide. In this study, the cloning and identification of a Murabutide-downregulated gene, named RH116, bearing classical motifs that are characteristic of the DExH family of RNA helicases, are reported. The 116 kDa encoded protein shares 99.9 % similarity with MDA-5, an inducible RNA helicase described recently. Ectopic expression of RH116 in HeLa-CD4 cells inhibited cell growth and cell proliferation but had no measurable effect on programmed cell death. RH116 presented steady state cytoplasmic localization and could translocate to the nucleus following HIV-1 infection. Moreover, the endogenous expression of RH116, at both the transcript and protein levels, was found to be considerably upregulated after infection. Overexpression of RH116 in HIV-1-infected HeLa-CD4 cells also resulted in a dramatic increase in the level of secreted viral p24 protein. This enhancement in virus replication did not stem from upregulated proviral DNA levels but correlated with increased unspliced and singly spliced viral mRNA transcripts. These findings implicate RH116 in the regulation of HIV-1 replication and point to an apoptosis-independent role for this novel helicase in inducing cell growth arrest.

Discordant effects of interleukin-2 on viral and immune parameters in human immunodeficiency virus-1-infected monocyte-derived mature dendritic cells.

Use of interleukin-2 (IL-2) in the immunotherapy of human immunodeficiency virus (HIV) has frequently resulted in the restoration of CD4 lymphocyte counts but not of virus-specific responses. We reasoned that the absence of reconstituted functional immune parameters could be related to the inability of IL-2 to correct HIV-induced dysfunctions in antigen-presenting cells. In this study, we used in vitro-differentiated monocyte-derived macrophages (MDMs) and mature dendritic cells (MDDCs), acutely infected with primary HIV-1 isolates, to analyse the effects of IL-2 on virus replication, co-receptor expression, and cytokine or chemokine release. Stimulation of MDMs with IL-2 had no measurable effect on HIV-1 replication, on cytokine secretion, or on CD4 and CXCR4 gene expression. Moreover, although a significant down-regulation of CCR5 mRNA expression could be repeatedly detected in MDMs, this IL-2-mediated effect was not of substantial magnitude to affect virus replication. On the other hand, IL-2 stimulation of MDDCs dramatically increased HIV-1 replication and this effect was highly evident on low-replicating, CXCR4-dependent isolates. Nevertheless, the HIV-enhancing activity of IL-2 in MDDCs was not accompanied by any measurable change in cytokine or chemokine release, in virus receptor and co-receptor mRNA accumulation, or in the surface expression of a battery of receptors implicated in virus entry, cell activation or costimulatory function. Taken together, these findings point to a role for IL-2 in inducing virus purging from dendritic cell reservoirs but indicate no relevant potential of the cytokine in restoring defective elements of innate immunity in HIV infection.

The transcriptional response of human macrophages to murabutide reflects a spectrum of biological effects for the synthetic immunomodulator.

The synthetic immunomodulator murabutide (MB) presents multiple biological activities with minimal toxicity in animals and in man. Although MB is known to target cells of the reticuloendothelial system and to regulate cytokine synthesis, the molecular mechanisms underlying several of its biological effects are still largely unknown. In an effort to define cellular factors implicated in the immunomodulatory and HIV-suppressive activities of MB, we have undertaken profiling the regulated expression of genes in human monocyte-derived macrophages (MDM) following a 6-h stimulation with this synthetic glycopeptide. Oligonucleotide microarray analysis was performed on RNA samples of differentiated MDM from four separate donors, using probe sets corresponding to 1081 genes. We have identified, in a reproducible fashion, the enhanced expression of 40 genes and the inhibition of 16 others in MB-treated MDM. These regulated genes belonged to different families of immune mediators or their receptors, transcription factors and kinases, matrix proteins and their inhibitors, ion channels and transporters, and proteins involved in cell metabolic pathways. Additional verification of the regulated expression of selected genes was carried out using Northern blots or the quantification of released proteins in MDM cultures. The profile of MB-regulated genes in MDM provides a molecular basis for some of its previously reported biological activities, and reveals new set of genes targeted by the immunomodulator suggesting potential application in novel therapeutic indications.

Variations in serum IL-7 and 90K/Mac-2 binding protein (Mac-2 BP) levels analysed in cohorts of HIV-1 patients and correlated with clinical changes following antiretroviral therapy.

Serum levels of interleukin-7 (IL-7), a non-redundant cytokine that plays a crucial role in lymphopoiesis, are known to be elevated in HIV-1-infected subjects. To examine further the association between levels of IL-7, CD4+ cell counts and viraemia, we analysed these parameters in a large cohort of HIV-1 patients along with serum levels of 90K, a marker of disease severity but with no established involvement in lymphopoiesis. While IL-7 levels were only found to correlate with CD4+ cell counts, 90K levels presented strong correlations with both CD4+ cell numbers and with plasma viral loads (VLs). These correlations were maintained in patients naive to treatment with antiretrovirals (n = 38) but were abolished when the analysis was restricted to the group receiving highly active antiretroviral therapy (HAART, n = 82). Moreover, although 90K levels were significantly reduced in patients on HAART, IL-7 levels continued to be elevated despite successful treatment. The influence of HAART on the variations in these serum parameters was further assessed in a longitudinal study on 32 subjects. The HAART-induced decrease in VLs and increase in CD4+ counts were found to correlate with a reduced serum level of 90K and IL-7, respectively. Nevertheless, following a median period of 33 months of immunological and virological successful HAART, serum levels of IL-7 continued to be significantly elevated compared with those detected in healthy controls. These findings suggest that immunotherapy with IL-7, aimed to replenish T-cell stock in HAART-treated subjects, may have a limited impact on the process of immune reconstitution.

Clinical tolerance and profile of cytokine induction in healthy volunteers following the simultaneous administration of ifn-alpha and the synthetic immunomodulator murabutide.

As the therapeutic use of interferon-alpha (IFN-alpha) is limited by a dose-dependent toxicity and variable efficacy, ways of improving the therapeutic index of the cytokine are being sought. Murabutide (N-acetyl muramyl-L-alanyl-D-glutamine-O-n-butyl-ester) (ISTAC Biotechnology, Lille, France) is a safe synthetic and clinically acceptable immunomodulator that enhances the biologic activities of IFN-alpha in different experimental models. We evaluated in healthy human volunteers tolerance of the coadministration of Murabutide with increasing doses of IFN-alpha. The simultaneous administration of the two drugs was well tolerated without any increased or prohibiting toxicity, and all recipients experienced side effects that were similar to those observed after the administration of IFN-alpha alone. We also profiled the serum levels of cytokines induced following coinjection of the two drugs. We mostly detected an induction of anti-inflammatory cytokines and of human immunodeficiency virus type 1 (HIV-1)-suppressive beta-chemokines, in the absence of release of key proinflammatory cytokines. Therefore, the simultaneous administration of Murabutide and IFN-alpha is well tolerated and does not lead to increased toxicity. In addition, the selectivity in the profile of cytokines and chemokines induced following the coadministration of Murabutide and IFN-alpha points to the potential use of this combination in the treatment of inflammatory diseases and chronic viral infections.

Macrophage stimulation with Murabutide, an HIV-suppressive muramyl peptide derivative, selectively activates extracellular signal-regulated kinases 1 and 2, C/EBPbeta and STAT1: role of CD14 and Toll-like receptors 2 and 4.

The smallest unit of bacterial peptidoglycans known to be endowed with biological activities is muramyl dipeptide (MDP). A clinically acceptable synthetic derivative of MDP, namely murabutide (MB), has been found to present interesting pharmacological properties and to suppress HIV-1 replication in monocyte-derived macrophages (MDM). We have addressed the signaling events activated in MDM following stimulation with either MB or the potent immunostimulant LPS. We also examined whether signaling by muramyl peptides involves the use of cell surface receptors, including CD14 and Toll-like receptor 2 (TLR2) or TLR4 that are known to be signal-transducing receptors for other bacterial cell wall components. We demonstrate that, unlike LPS, the safe immunomodulator MB selectively activates extracellular signal-regulated kinases (Erk) 1/2, in the absence of detectable Jun N-terminal kinase (JNK) or p38 mitogen-activated kinase activation. Furthermore, STAT1 activation but weak or no activation of STAT3 or STAT5 respectively, could be detected in MB-stimulated MDM. Using MonoMac6 cells, we observed high C/EBPbeta and AP-1 but weaker and transient NF-kappaB activation by MB.Moreover, the truncated form of C/EBPbeta, known to repress HIV-1 transcription, was detected in extracts from MB-treated THP-1 cells. Surprisingly, neither MB nor MDP were able to transduce signals via CD14 and TLR2 or 4. These findings present major differences in the early cell activation process between LPS and muramyl peptides, and strongly argue for the implication of co-receptors other than TLR2 and TLR4 in mediating the signaling events induced by defined subunits of bacterial peptidoglycans.

Selective regulation of human immunodeficiency virus-infected CD4(+) lymphocytes by a synthetic immunomodulator leads to potent virus suppression in vitro and in hu-PBL-SCID mice.

We have previously observed that the synthetic immunomodulator Murabutide inhibits human immunodeficiency virus type 1 (HIV-1) replication at multiple levels in macrophages and dendritic cells. The present study was designed to profile the activity of Murabutide on CD8-depleted phytohemagglutinin-activated lymphocytes from HIV-1-infected subjects and on the outcome of HIV-1 infection in severe combined immunodeficiency mice reconstituted with human peripheral blood leukocytes (hu-PBL-SCID mice). Maintaining cultures of CD8-depleted blasts from 36 patients in the presence of Murabutide produced dramatically reduced levels of viral p24 protein in the supernatants. This activity correlated with reduced viral transcripts and proviral DNA, was evident in cultures harboring R5, X4-R5, or X4 HIV-1 isolates, was not linked to inhibition of cellular DNA synthesis, and did not correlate with beta-chemokine release. Moreover, c-myc mRNA expression was down-regulated in Murabutide-treated cells, suggesting potential interference of the immunomodulator with the nuclear transport of viral preintegration complexes. On the other hand, daily treatment of HIV-1-infected hu-PBL-SCID mice with Murabutide significantly reduced the viral loads in plasma and the proviral DNA content in human peritoneal cells. These results are the first to demonstrate that a clinically acceptable synthetic immunomodulator with an ability to enhance the host's nonspecific immune defense mechanisms against infections can directly regulate cellular factors in infected lymphocytes, leading to controlled HIV-1 replication.

The synthetic immunomodulator murabutide controls human immunodeficiency virus type 1 replication at multiple levels in macrophages and dendritic cells.

Macrophages and dendritic cells are known to play an important role in the establishment and persistence of human immunodeficiency virus (HIV) infection. Besides antiretroviral therapy, several immune-based interventions are being evaluated with the aim of achieving better control of virus replication in reservoir cells. Murabutide is a safe synthetic immunomodulator presenting a capacity to enhance nonspecific resistance against viral infections and to target cells of the reticuloendothelial system. In this study, we have examined the ability of Murabutide to control HIV type 1 (HIV-1) replication in acutely infected monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs). Highly significant suppression of viral replication was consistently observed in Murabutide-treated cultures of both cell types. Murabutide did not affect virus entry, reverse transcriptase activity, or early proviral DNA formation in the cytoplasm of infected cells. However, treated MDMs and MDDCs showed a dramatic reduction in nuclear viral two-long terminal repeat circular form and viral mRNA transcripts. This HIV-1-suppressive activity was not mediated by inhibiting cellular DNA synthesis or by activating p38 mitogen-activated protein kinase. Furthermore, Murabutide-stimulated cells expressed reduced CD4 and CCR5 receptors and secreted high levels of beta-chemokines, although neutralization of the released chemokines did not alter the HIV-1-suppressive activity of Murabutide. These results provide evidence that a clinically acceptable immunomodulator can activate multiple effector pathways in macrophages and in dendritic cells, rendering them nonpermissive for HIV-1 replication.

Human chemokine receptors CCR5, CCR3 and CCR2B share common polarity motif in the first extracellular loop with other human G-protein coupled receptors implications for HIV-1 coreceptor function.

Chemokine receptors (CRs) are 7-helix membrane proteins from the family of G-protein coupled receptors (GPCRs). A few human CRs act as cofactors for macrophage-tropic (M-tropic) human immunodeficiency virus type-1 (HIV-1) entry into cells, while others do not. In this study, we describe an application of molecular modeling techniques to delineate common molecular determinants that might be related to coreceptor activity, and the use of the data to identify other GPCRs as putative cofactors for M-tropic HIV-1 entry. Subsequently, the results were confirmed by an experimental approach. The sequences of extracellular domains (ECDs) of CRs were employed in a compatibility search against a database of environmental profiles derived for proteins with known spatial structure. The best-scoring sequence-profile alignments obtained for each ECD were compared in pairs to check for common patterns in residue environments, and consensus sequence-profile fits for ECDs were also derived. Similar hydrophobicity motifs were found in the first extracellular loops of the CRs CCR5, CCR3, and CCR2B, and are all used by M-tropic HIV-1 for cell entry. In contrast, other CRs did not reveal common motifs. However, the same environmental pattern was also delineated in the first extracellular loop of some human GPCRs showing either high (group 1) or low (group 2) degree of similarity of their polarity patterns with those in HIV-1 coreceptors. To address the question of whether the delineated molecular determinant plays a critical role in the receptor-virus binding, three of the identified GPCRs, bradykinin receptor (BRB2) and G-protein receptor (GPR)-CY6 from group 1, and GPR8 from group 2, were cloned and transfected into HeLa-CD4 cells, which are nonpermissive to M-tropic HIV-1 infection. We demonstrate that, similar to CCR5, the two selected GPCRs from group 1 were capable of mediating M-tropic HIV-1 entry, whereas GPR8 from group 2 did not serve as HIV-1 coreceptor. The potential biological significance of the identified structural motif shared by the human CCR5, CCR3, CCR2B and other GPCRs is discussed.

Recombinant interleukin-16 selectively modulates surface receptor expression and cytokine release in macrophages and dendritic cells.

Interleukin-16 (IL-16), a natural ligand for the CD4 receptor, has been found to modulate T-lymphocyte function and to inhibit human immunodeficiency virus type 1 (HIV-1) replication. Antigen-presenting cells (APC), including macrophages and dendritic cells, are known to express functional surface CD4 molecules, to be susceptible to HIV-1 infection and to play a critical role in different immune processes. Therefore, we evaluated the ability of recombinant IL-16 (rIL-16) to regulate receptor expression and cytokine release in monocyte-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC). Recombinant IL-16 was found to up-regulate CD25 and CD80 but to down-regulate CD4 and CD86 surface expression in MDM cultures. However, no change could be observed on the level of CD4, CD80 and CD86 expression in IL-16-stimulated MDDC, although a significant up-regulation of CD25 and CD83 was consistently detected. Furthermore, the level of gene expression of the chemokine receptors CCR5 and CXCR4 was significantly reduced in rIL-16-treated MDM and costimulation with IL-2 did not modify the activity of the recombinant cytokine. The effects on chemokine receptor gene expression were less evident in MDDC and only a transient down-regulation of weak intensity could be detected following stimulation with rIL-16. Analysis of supernatants from rIL-16-stimulatedcultures revealed a different profile of released cytokines/chemokines among the two cell populations studied. These findings establish an important role for IL-16 in modulating the activity of APC and may have relevance regarding the protection of reservoir cells against HIV-1 infection.

Interleukin-16 inhibits human immunodeficiency virus type 1 entry and replication in macrophages and in dendritic cells.

Recombinant interleukin-16 (rIL-16) has been found to inhibit human immunodeficiency virus type 1 (HIV-1) replication in acutely or endogenously infected CD4(+) T cells. However, the effect of rIL-16 on HIV-1 replication in antigen-presenting cells (APCs) is still unknown. We show here a potent HIV-suppressive activity of rIL-16 in acutely infected monocyte-derived macrophages and dendritic cells determined by the levels of viral RNA transcripts or of viral reverse transcriptase in culture supernatants. The observed effect was dependent on the presence of rIL-16 early after infection and could not be induced by a 24-h treatment of cells with the cytokine prior to infection. Using macrophage-tropic and dually tropic primary isolates, we also showed that the addition of rIL-16 to cell cultures only during the infection period was effective in blocking virus entry and reducing proviral DNA levels in APCs. However, the anti-HIV activity of rIL-16 could not be linked to the induction of virus-suppressive concentrations of beta-chemokines or to the inhibition of HIV-enhancing cytokines. These findings establish a critical role for rIL-16 in protecting APCs against HIV-1 infection and lend further support to its potential use in the treatment of HIV disease.

Interleukin-16 (IL-16) inhibits human immunodeficiency virus replication in cells from infected subjects, and serum IL-16 levels drop with disease progression.

The role of recombinant interleukin-16 (rIL-16) in regulating human immunodeficiency virus type 1 (HIV-1) replication in endogenously infected cells has been investigated. Cultures of CD8 cell-depleted mitogen-activated lymphocytes from 22 of 26 HIV-1-infected subjects presented variable levels of secreted p24 antigen. The presence of rIL-16 throughout the 14-day culture period dramatically inhibited p24 release into the culture supernatants. This effect was found to be mediated through inhibition of viral transcription but to be independent of the induced levels of other cytokines or chemokines known to regulate viral replication. Analysis of serum samples from HIV-1-infected subjects over a period of 8 years showed maintained or even increased IL-16 levels during the whole asymptomatic phase and a significant drop on progression to disease. These results strongly support a potential therapeutic value of rIL-16 in HIV-1 infection and the use of serum IL-16 levels to monitor disease progression.

Selective potentiation of cytokine expression in human whole blood by murabutide, a muramyl dipeptide analogue.

Murabutide is a synthetic muramyl peptide which is in clinical stage of development. Its effect on cytokine production was analysed in human whole blood to reproduce the natural environment. Induced gene transcription within 2 h was associated with the release of cytokines such as tumour necrosis factor (TNF), interleukin-1 beta (IL-1 beta), IL-6, IL-8, and also the anti-inflammatory mediator IL-1ra. This synthesis was not associated with the release of IL-4, IL-12, interferon gamma (IFN-gamma), the three colony-stimulating factors (CSFs) or the soluble TNF receptors. The same series of cytokines were assayed to determine the effect of some recombinant cytokines in association with murabutide. Thus, in the presence of IL-2, IL-6, IL-3 or granulocyte-macrophage colony-stimulating factor (GM-CSF), the level of cytokines induced by murabutide was enhanced with no change in the other cytokines profile. IL-3 and GM-CSF were more potent in increasing the murabutide-induced response, eliciting synergistic effects on IL-8 and IL-1Ra production, at both the mRNA accumulation and the protein release. Although neither IL-12 nor IFN-gamma were produced in cells stimulated with murabutide alone, some mRNA expression was found with combined treatments. The results indicate that association of murabutide with a cytokine could exert synergistic effects, thus reducing effective doses of the recombinant protein, increasing the release of anti-inflammatory mediators, and triggering efficient cellular immunity.

Selective induction of CD11a,b,c/CD18 and CD54 expression at the cell surface of human leukocytes by muramyl peptides.

Muramyl dipeptide (MDP), murametide, and murabutide which belong to the family of the immunoadjuvant muramyl dipeptides were applied directly to fresh human whole blood and the expression of some surface markers involved in cell adherence in distinct leukocyte populations was investigated. CD11a,b,c/CD18, CD54, CD49d were selected for their involvement in cell adherence, and transferrin receptor (CD71) and low-affinity IgE receptor (CD23) were selected as markers for activated cells. Whereas CD11a was increased only on monocytes, CD11b, CD11c, and CD18 were strongly enhanced on monocytes and polymorphonuclear cells (PMNs) after treatment with MDPs. This increase in membrane expression of integrins, such as CD11b, was not associated with mRNA synthesis, suggesting a mobilization of the CD11b,c/CD18 intracellular pools present in these cells. In contrast, treatment with MDP, murametide, or murabutide enhanced ICAM-1 (CD54) expression on monocyte and PMN cell surface in association with ICAM-1 mRNA synthesis. No variation of CD49d expression was detected on leukocyte surface after incubation with MDPs. Transferrin receptor (CD71) expression and low-affinity receptor for IgE (CD23) expression were increased on monocyte only after incubation with LPS used as positive control. Moreover, no observable change in the selected markers was detected on lymphocyte after MDPs or LPS treatment. These results indicate that MDPs seem to act preferentially on monocytes and PMNs in increasing the level of molecules involved in cellular adhesion process, either in provoking the expression of preformed molecules or in inducing their synthesis. This contributes to understanding the mechanism of the activities of muramyl peptides on specific and nonspecific immunity.

Modulation of expression of class II MHC and CD40 molecules in murine B cells by various muramyl dipeptides.

Several compounds of the MDP (muramyl dipeptide) series which have the capacity to enhance the immune response to antigens exerted a comitogenic effect on murine splenic B cells. The expression of surface class II major histocompatibility and CD40 antigens was used to more accurately evaluate the comparative influence of the synthetic agents on mature B cells and on the pre-B cell line 70Z/3. MDP and two adjuvant-active analogs enhanced expression of both surface molecules and increased the response to lipopolysaccharide (LPS) or interferon-gamma (IFN-gamma) in splenic B cells. The three synthetic adjuvants alone did not lead to expression of cell-surface I-Ad or CD40 in 70Z/3 cells, indicating that they were unable by themselves to achieve differentiation of pre-B cells to a mature B cell phenotype. However, they increased the CD40 level induced by treatment with LPS. In this cell line, the response (CD40 protein and mRNA) to IFN-gamma was strongly increased by MDP but not by the two other compounds. Actually, MDP was the only adjuvant among the three compounds to functionally activate the transcription factor NF-kappaB, to induce kappa transcription, and to stimulate surface kappa light-chain expression in 70Z/3 cells. The response to muramyl dipeptides in mature splenic B cells could appear independent of the transcription factor.

Synergistic effects between recombinant interleukin-2 and the synthetic immunomodulator murabutide: selective enhancement of cytokine release and potentiation of antitumor activity.

The use of interleukin-2 (IL-2) in the treatment of cancer has shown limited efficacy and dose-limiting toxicity. Combination therapy with other cytokines and/or chemotherapeutic agents has been attempted to enhance the antitumor activity and to reduce the effective therapeutic dose of IL-2. We recently showed, in vitro and in vivo, a synergistic activity between the synthetic immunomodulator murabutide, which is in clinical stage of development, and another therapeutic cytokine, interferon-alpha (IFN-alpha). The present study was performed to assess a possible potentiation of the biologic activities of IL-2 by its association with murabutide. Human PBMC stimulated in vitro with IL-2 and murabutide showed synergistic levels of induced mRNA accumulation and protein secretion for IFN-gamma, IL-12, and colony-stimulating factors (CSFs). No such effects were obtained on the induction of most inflammatory cytokines, including IL-6, IL-8, and tumor necrosis factor alpha (TNF-alpha). Furthermore, the combined administration of murabutide with IL-2 into Meth-A sarcoma-bearing mice resulted in a very significant tumor inhibition as well as in complete tumor regression in nearly 70% of the treated mice. Under the same conditions, treatment with either compound separately had little or no antitumor effect. These preclinical findings will be pursued by the evaluation of the clinical tolerance and biologic activity of the murabutide/IL-2 combination therapy in cancer patients.

Immunopharmacological activities and clinical development of muramyl peptides with particular emphasis on murabutide.

Certain immunopharmacological activities of muramyl peptides have been associated with inflammatory and undesirable side-effects typically observed following the administration of the prototype molecule muramyl dipeptide. This activity is now demonstrated not to be linked to a direct activation of inflammatory processes in endothelial cells. Neither MDP nor other structural derivatives were able to induce inflammatory cytokines release or E-selectin gene expression in cultured human umbilical vein endothelial cells. However, oral administration of muramyl peptides has been reported to induce certain biological effects, including the downregulation of anamnestic, antigen-specific IgE responses, which are not observed following parenteral administration. We elaborate on these findings and extend them to show the efficacy of a new muramyl peptide in suppressing polyclonally induced serum IgE levels in anti-IgD-treated mice. The comparative effects of muramyl peptides, selected for clinical development, on the induction of cytokines in human whole blood are then presented at the level of mRNA accumulation and protein secretion. Moreover, the cytokine profile induced in vitro and in vivo by the combination of the safe immunostimulant, Murabutide, with interferon-alpha is examined. This combination reveals a selective and beneficial synergistic activity and induces anti-inflammatory cytokines in the absence of synergistic toxicity. The potential and the implications for the use of a therapeutic combination of an immunostimulant with a cytokine are discussed.