George M. Bahr

The effect of two distinct forms of cell-mediated response to mycobacteria on the protective efficacy of BCG.

The previous paper presents evidence that prior exposure to some environmental mycobacteria enhances the protective efficacy of BCG, whereas exposure to other species opposes it, and suggests that these different species act by evoking one of two types of cell-mediated response of different protective efficacy. This paper reviews past evidence for the existence of these two types of response, and suggests that both can be demonstrated in mice. The type of response evoked in mice by environmental species correlates with their effect on the efficacy of BCG in man, and with the type of proliferative response evoked in human peripheral blood lymphocytes by their soluble antigens in vitro. Preimmunisation of mice to give one type of response can block subsequent induction of the other. We therefore present a model, based on this principle, for the interaction of contact with environmental mycobacteria with subsequent BCG vaccination.

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.

Macrophage stimulation with Murabutide, an HIV-suppressive muramyl peptide derivative, selectively activates extracellular signal-regulated kinases 1 and 2, C / EBPβ 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 weakor no activation of STAT3 or STAT5 respectively, could be detected in MB‐stimulated MDM. Using MonoMac6 cells, we observed high C / EBPβ and AP‐1 but weaker and transient NF‐κB activation by MB.Moreover, the truncated form of C / EBPβ, 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.

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.

The Synthetic Immunomodulator Murabutide Controls Human Immunodeficiency Virus Type 1 Replication at Multiple Levels in Macrophages and Dendritic Cells

ABSTRACT 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 β-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.

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.

SS-56, a novel cellular target of autoantibody responses in Sjögren syndrome and systemic lupus erythematosus

Certain autoimmune disorders, including Sjögren syndrome (SS) and systemic lupus erythematosus (SLE), are characterized by autoantibodies against the Ro/SSA and La/SSB cellular antigens. Although the implication of these autoantibodies in disease pathogenesis is still unclear, it is believed that the aberrant responses against autoantigens may extend to other proteins that are not yet well defined. In an attempt to analyze the regulated gene expression in lymphocytes by an HIV-suppressive immunomodulator, we have identified and cloned a novel gene encoding a 56-kDa protein, named SS-56, which is structurally related to the 52-kDa Ro/SSA antigen. The new protein showed primarily perinuclear cytoplasmic localization, and recombinant SS-56 was found to react in ELISA with sera from most patients with SS or SLE. Western blot analysis confirmed the autoantigenic nature of native SS-56 in extracts from HeLa cells. Interestingly, the incidence of antibodies to SS-56 was associated with visceral complications in SLE, and roughly half of the 17 SS or SLE patients with no detectable antibodies to SSA and SSB antigens presented measurable antibodies against recombinant SS-56. Thus, SS-56 represents a new member of the SS family of autoantigens and could become an additional and important diagnostic marker for SS and SLE.

Modulation of cellular and humoral immune responses to a multiepitopic HIV-1 DNA vaccine by interleukin-18 DNA immunization/viral protein boost

In this study, the impact of Th1-inducing cytokine gene co-delivery and antigen boosting on humoral and cellular responses induced by multiepitopic DNA immunization in mice have been investigated. Intramuscular injection of mixed DNA constructs encoding for HIV-1 Gag, Tat and Nef proteins, co-administered with the DNA encoding for interleukin-18 (IL-18) have been used. The effect of boosting with the recombinant proteins was also evaluated on the outcome of the responses in DNA-primed mice. It was demonstrated that at least two DNA immunizations were necessary to generate virus specific Th-1 responses detected by the presence of cytotoxic T lymphocyte (CTL) and by the secretion of IL-2 and IFN-gamma, but not IL-4 and IL-10, in antigen-stimulated splenocyte cultures. Interestingly, co-delivery of Th-1-inducing IL-18 gene was able to shorten by 2 weeks, the CTL induction time, and to increase the antigen-induced secretion of IL-2 and IFN-gamma. Furthermore, IL-18 co-delivery enhanced antigen-specific lymphoproliferative responses, and this was most evident in mice that were primed and boosted with plasmid DNA. However, the induction of detectable antibodies in mice required two DNA vaccinations and a protein boost. In contrast to the effects on cell-mediated immunity, co-administration of IL-18-plasmid resulted in decreased antibody titers against viral proteins.

Interleukin-18 modulates immune responses induced by HIV-1 Nef DNA prime/protein boost vaccine

Many different HIV-1 vaccine strategies have been developed, but as yet none has been completely successful. Promising results from combined DNA prime/protein boost vaccines have been reported. Specific immune responses generated by DNA vaccines can be modulated by the co-delivery of genes coding for cytokines. In this study, we have used the intradermal route by needle injection of a plasmid coding for the HIV-1 Nef accessory protein. We show that DNA prime/protein boost vaccine combinations increase the humoral and cellular immune responses against HIV-1 Nef and that the co-injection of DNA encoding Interleukin-18 (IL-18) modulates the specific immune response towards a Th1 type.

Recombinant human IL-16 inhibits HIV-1 replication and protects against activation-induced cell death (AICD).

The chemoattractant cytokine IL‐16 has been reported to suppress lymphocyte activation and to inhibit HIV‐1 replication in acutely infected T cells. We have cloned and expressed human IL‐16 in Escherichia coli and investigated whether the recombinant protein could regulate the level of lymphocyte apoptosis from HIV‐1‐infected subjects. After purification and refolding, only 2–10% of the recombinant cytokine was present in a biologically active homotetrameric form. This could explain the need for high concentrations of the bacterially derived IL‐16 to induce significant inhibition of HIV‐1 replication. Addition of IL‐16 to unstimulated peripheral blood mononuclear cell (PBMC) cultures from HIV‐1‐infected subjects did not modify the observed level of spontaneous lymphocyte apoptosis. In contrast, IL‐16 added to PBMC cultures stimulated with anti‐CD3, anti‐CD95 or dexamethasone reduced significantly the percentage of lymphocytes undergoing AICD. This effect was found to correlate with the ability of the cytokine to decrease CD95 expression on activated CD4+ T cells. Comparative studies on PBMC from healthy individuals indicated that the regulation of apoptosis levels by IL‐16 is a complex phenomenon and could depend on the nature of the activator used and/or the immune status of lymphocytes tested. The outcome of CD4 cross‐linking on T cells by various ligands is discussed in the context of the observed beneficial activities of IL‐16 and its potential role in the treatment of HIV disease.