HIV-1 Inhibits Leishmania-lnduced Cell Proliferation but not Production of Interleukin-6 and Tumour Necrosis Factor Alpha

The immune response of normal human peripheral blood mononuclear cells (PBMC) after stimulation with human immunodeficiency virus-1 (HIV-1) antigens plus Letshmania donovani promastigotes in vitro was investigated. HIV-I-antigen stimulation of PBMC did not induce the intracellular accumulation of interleukin-6 (IL-6),. tumour necrosis factor-alpha (TNF-α), or Interferon-gamma (IFN-γ). However, cells stimulated with L. donovani antigens exhibited the production of IL-6 and TNF-α, but not IFN-γ. Furthermore, co-stimulation of PBMC with HIV-1 antigen plus L. donovani resulted in the intracellular accumulation of IL-6 and TNF-α comparable to that of cells that were activated with L. donovani antigen alone. Heat-inactivated HIV-1 antigen did not appear to induce or suppress cytokine production by PBMC. However, the same HIV antigens did suppress L. donovani-induced proliferation as well as PPD-induced proliferation in a dose-dependent fashion. Elevated levels of serum cytokines have been demonstrated in patients with HIV infection indicating their role in the pathogenesis of HIV-associated immunosuppression. The results may partially support the idea that the abnormally increased cytokine levels in the sera of HIV-infected subjects is due to the various opportunistic pathogens that these patients contract, rather than a response to HIV antigens. As cytokines have been shown to up-regulatc HIV replication, the data suggest a role for opportunistic infections in cytokine-induced transactivation of HIV-1 and disease progression.     

γδ and αβ T Cells are Equally Susceptible to Apoptosis

Gamma/delta TCR bearing T lymphocytes represent a T-cell subset whose functional relevance remains unclear. Nevertheless these T cells may play a role in the early immune reponse against bacteria. Until now the regulatory mechanisms on this response have not been investigated. The study described here evaluated the immunoregulatory effects of Interleukin-10 on γ/δ and α/β TCR-positive T-cell clones and freshly isolated peripheral-blood mononuclear cells (PBMC). IL-10 has been shown previously to inhibit lectin and antigen-induced proliferation and cytokine production by α/β T cells. The results outlined below show that rhIL-10 strongly inhibits lectin-induced production of IFN-γ, TNF-α. IL-2, and to a lesser degree proliferation and IL-4 production of both T-cell subsets. As IL-10 did not inhibit proliferation but at the same time strongly suppressed cytokine production in various experiments, the hypothesis that it could function as a growth factor for human T cells as has been described for murine thymoeytes was tested. The data demonstrate that, although the γ/δ T-cell clones tested do not produce IL-10 they can use it as a growth factor in combination with IL-2, IL-4 or alone. Furthermore, IL-10 has the same properties on human α/β T-cell clones and PBMC. In summary, it is shown that IL-10 has pleiotropic effects on γ/δ and α/β TCR⁺ T cells by inhibiting lectin-induced cytokine production and by acting as a growth factor for these cells alone or in combination with IL-2 or IL-4.     

Augmented Lung Adenocarcinoma Cytotoxicity by the Combination of a Genetically Modified Anti-Lewis Y Antibody and Antibodies to Complement Regulatory Proteins

Nine vervet monkeys ( Cercopithecus aethiops ) were infected intradermally with 8 × 10⁷ virulent L. donovani promastigotes. Four animals developed clinical visceral leishmaniasis and died over a period of 18 months. The remaining five animals have remained asymptomatic for a period of 3 years now. Attempts to isolate parasites from spleen and liver through biopsies were fruitless. Immunological respotises of these subclinically infected animals were examined. Enzyme-linked itnmunosorbent assay ( ELISA ) and western blot analyses demonstrated Leishmania specific antibodies in these animals, but the antibody titres were low. When proliferation of peripheral blood monocytes ( PBMC ) to Con-cunavalin A (Con A) of these animals was compared with control 'disease free animals' there were no significant differences iti response. However L. donovani antigen (fixed promastigotes) specific proliferation was demonstrated in the five subclinically infected animals. High and varying levels of interferon gamma (IFN-γ) were secreted in PBMC cultures from the five vervet monkeys when stimulated with either Con A or L. donovani antigens. In control animals, IFN-γ was only detected wheti PBMC were stimulated with Con A. Marked delayed-type hypersensitivity ( DTH ) responses were demonstrated in the five subclinically infected animals 48 h after injection with formalin fixed promastgotes.
It was concluded that the visceral Leishmania disease spectrum due to L. donovani observed in humans could be induced in vervet monkeys and that L. donovani asymptomatic/cryptic infected animals have competent humoral and cellular responses to homologous parasites.     

Molecular properties of the proteasome activator PA28 family proteins and γ-interferon regulation

Background : Recent cDNA cloning of two homologous proteasome activators, PA28α and PA28β, indicated the presence of a structurally related third protein, Ki antigen, but a functional relationship between Ki antigen and the two PA28 proteins is unknown. Accumulating evidence has implicated an important role for PA28 in the major histocompatibility complex (MHC) class I-restricted antigen processing pathway. Recently, an immunomodulatory cytokine γ-interferon (γ-IFN) was found to increase greatly the messages for PA28α and PA28β, but not Ki antigen, in human cells.
Results : Ki antigen was co-immunoprecipitated with the 20S proteasome by anti-proteasome antibody, and associated reversibly with the 20S proteasome, as observed for PA28α and PA28β. Therefore, Ki antigen was renamed PA28γ. Anti-PA28γ antibody, however, did not immunoprecipitate PA28α and PA28β. γ-IFN caused an almost complete loss of the PA28γ protein in cells without affecting its mRNA level, whereas the levels of both mRNA and protein for PA28α and PA28β were coordinately up-regulated by γ-IFN. Finally we showed that the human chromosomal genes of PA28α and PA28γ were located on 14q11.2 and 17q21.32-21.33, respectively.
Conclusion : PA28γ (equivalent to Ki antigen) is a new member of the PA28 family proteins. It exists as a unique homopolymer under non-denaturing conditions. γ-IFN was found to induce the expression of PA28α and PA28β, whereas it caused almost complete loss of the PA28γ protein in cells. The reciprocal expression of the PA28 family proteins may imply their involvement in distinct biological processes.     

Interleukin-2 (IL-2) Receptor α, β and γ Subunit Expression as a Function of B-Cell Lineage Ontogeny: the Use of IL-2-PE664Glu to Characterize Internalization via IL-2 Receptor Subunits

Interleukin-2 (IL-2) is a pluripotent cytokine which plays a crucial role in the immune system response. Although the IL-2/IL-2 receptor (IL-2R) system has been well characterized in cells of the T lineage it is less known in B lymphocytes. The authors therefore studied the expression of the IL-2Rα, β and γ subunits in human B-cell lines at different stages of maturation, by the polymerase chain reaction technique. The authors found that the α and β subunits are expressed in the final stages of B-cell lineage maturation, whereas the γ subunit is constitutively expressed during B-lymphocyte differentiation. The results indicate that the IL-2/IL-2R system, most probably, does not have a role in the early stages of B-cell differentiation, but may be involved only in the final stages of B-cell lineage ontogeny. Moreover, the ability of the different forms of IL-2R to internalize the IL-2 ligand was investigated, using the chimeric protein IL-2-PE66^4Glu. Cell lines bearing the αγ, βγ and αβγ forms of IL-2R were inhibited by the chimeric protein, while those bearing the γ subunit alone did not respond to the chimera. Thus, internalization of IL-2 is most likely mediated via the αγ form of the IL-2R, as shown here for the first time, as well as through the βγ and αβγ IL-2R forms. However, IL-2 cannot be internalized through the IL-2R γ subunit alone.     

Structure of the T-Cell Receptor in a Tiαβ, Tiγδ Double Positive T-Cell Line

The multichain T-cell receptor is composed of at least six different polypeptide chains. The clonotypic Ti heterodimer (Tiαβ or Tiγδ) is non-covalently associated with the CD3 chains (CD3γδɛζ). The exact number of subunits constituting the T-cell receptor is still not known. It has been suggested that each T-cell receptor contains two Ti dimers. To gain insight into the structure of the T-cell receptor we constructed a Tiαβ, Tiγδ double positive T-cell line which contained four functional Ti chains (Tiαβ, β, γ, and δ). The data demonstrated an absence of Ti dimers containing mixtures of chains other than the typical Tiαβ and Tiγδ combinations. Furthermore, by co-modulation experiments we demonstrated that the Tiαβ and the Tiγδ dimers were not expressed in the same T-cell receptor. Our data indicate that the T-cell receptor does not contain two Ti dimers.     

Nitric oxide induced expression of stress proteins in virulent and avirulent promastigotes of Leishmania donovani

Intracellular survival and replication of Leishmania donovani inside macrophage is essential for establishment of the disease. Cytokines play an important role in this process through activation or inhibition of macrophage antimicrobial activity. Nitric oxide (NO) has been demonstrated to be the principal effector molecule mediating intracellular killing of Leishmania. We have examined the effect of NO and various other cytokines on stress protein synthesis by promastigotes of L. donovani virulent and avirulent strains. Virulent promastigotes exposed to NO showed appreciable increase in relative synthesis of HSPs 83, 70 and 65. The overexpression of HSPs on exposure of parasite to NO was observed to be more pronounced at 37 degrees C than at 24 degrees C. In contrast, the avirulent promastigotes responded by an increase in relative synthesis of HSP70 alone at 37 degrees C. Furthermore, treatment of promastigotes of L. donovani with gammaIFN, TGF-beta or IL-4 did not significantly alter the stress proteins expression. The overexpression of HSPs in promastigotes of L. donovani in response to sublethal doses of NO suggests that HSPs may be playing a protective role for parasite survival in the mammalian host. This is further supported by the observation that a significantly higher induction of HSPs is seen in the virulent as compared to the avirulent strain of L. donovani.     

The in vivo and in vitro action of 4-amino-5-imidazolecarboxamide in trypanosomatid flagellates

4-Amino-5-imidazolecarboxamide, but not its riboside or ribotide, is inhibitory to the growth of promastigotes of Leishmania donovani, L. braziliensis, L. tarentolae, and L. mexicana, eventually causing cell lysis. Conversely, it is not inhibitory to the growth of epimastigotes of Trypanosoma cruzi. This substituted imidazole proved to be an excellent inhibitor of guanine deaminase from all of the trypanosomatids used in this study, with K_i values in the μM range.     

A Highly Conserved Phenylalanine in the α, β-T Cell Receptor (TCR) Constant Region Determines the Integrity of TCR/CD3 Complexes

In the present study, we have investigated the importance of a phenylalanine (phe¹⁹⁵) in the Tcr-Cα region on Tcr-α, β/CD3 membrane expression. An exchange of phe¹⁹⁵ with a tyrosine residue does not affect Tcr/CD3 membrane expression; however, exchange with aspartic acid, histidine or valine prohibit completely Tcr/CD3 membrane expression. This seems to be due to a lack of interaction between mutated Tcr-α, β/CD3-γɛ, δɛ complexes and ζ₂ homodimers. The Tcr-Cα region around phe¹⁹⁵ seems together with the same region in the Tcr-Cβ region to constitute an interaction site for ζ₂ homodimers. The presence of phe¹⁹⁵ on both Tcr-Cα and Tcr-Cβ causes high avidity interaction with ζ₂ homodimers, whereas his¹⁹⁵ in both Tcr-Cγ and Tcr-Cδ results in an apparently lower avidity interaction with ζ₂ homodimers. It is suggested that the phe¹⁹⁵ region (on β-strand F) and eventually adjacent aromatic amino acid residues on β-strand B region may play an important role in Tcr-α, β/CD3 membrane expression, in Tcr-α, β/CD3 competition with Tcr-γ, δ/CD3 complexes for ζ₂ homodimers and in the control of formation of 'mixed' Tcr heterodimers.     

Purification and enzymatic activity of an NADH-fumarate reductase and other mitochondrial activities of Leishmania parasites

A 65 kD membrane-associated NADH-fumarate reductase subunit, which has a molecular weight similar to that of one of the enzyme subunits from bacteria, was purified from Leishmania donovani promastigotes. NADH-fumarate reductase and other mitochondrial enzymatic activities of L. major and L. donovani promastigotes and amastigotes were investigated. The presence of NADH-fumarate reductase was demonstrated in digitonin-permeabilized L. major promastigotes and mitochondria of L. major and L. donovani promastigotes and amastigotes. The activity of solubilized NADH-fumarate reductase was measured in L. major and L. donovani promastigotes. Succinate exhibited a clear concentration-dependent inhibitory effect on fumarate reductase, whereas fumarate also exhibited a clear concentration-dependent inhibitory effect on succinate dehydrogenase. The data indicate that fumarate reductase is an obligatory component of the respiratory chain of the parasite. Since the enzyme is an important component in the intermediate metabolism in the Leishmania parasite and is absent in mammalian cells, it could be a potential target for antileishmanial drugs.     

Purification and characterization of a membrane-bound acid phosphatase of Leishmania mexicana.

As defined by the reaction with monoclonal antibodies, Leishmania mexicana promastigotes contain two acid phosphatases which together comprise about 90% of the cellular activity. A first enzyme recognized by monoclonal antibody AP4 is largely membrane-bound. The protein has an apparent molecular weight of 70,000-72,000, carries about seven N-linked glycan chains and is present in approximately 16,000 copies per cell. The protein is also expressed in the amastigote stage. A second enzyme reactive with monoclonal antibody AP3, that also recognizes lipophosphoglycan and a secreted acid phosphatase, is mainly found in the soluble fraction of promastigote lysates. It is suggested that this enzyme is the precursor of the secreted protein. The N-terminal sequences of the phosphatase recognized by AP4 and the secreted enzyme are similar but not identical. AP4 does not cross-react with phosphatase activity of Leishmania major or Leishmania donovani promastigotes, while AP3 recognizes part of the cellular and all of the secreted phosphatase activity of L. donovani promastigotes but not that of L. major which does not release an acid phosphatase into the culture medium.     

The role of tumor necrosis factor (TNF)-α in the antitumor effect of intrapleural injection of Lactobacillus casei strain Shirota in mice

The involvement of several cytokines in the antitumor effect induced by intrapleural (i.pl.) injection of heat-killed cells of Lactobacillus casei strain Shirota (LC 9018) in mice was investigated. Injection of LC 9018 i.pl. into Meth A fibrosarcoma (Meth A)-bearing mice not only significantly prolonged the survival of the mice, but also effectively inhibited the accumulation of malignant pleural fluid in the thoracic cavity. In the thoracic cavity of tumor-bearing mice treated with LC 9018, we observed large amounts of several cytokines including interleukin (IL)-1β, interferon (IFN)-γ, IL-12 and tumor necrosis factor (TNF)-α. Both anti-IFN-γ and anti-IL-12 monoclonal antibody (mAb) treatments partially diminished the antitumor activity of LC 9018 in vivo, while the treatment of anti-IL-1β mAb did not influence the survival of the mice. However, anti-TNF-α mAb treatment completely abolished the antitumor effect of LC 9018 in vivo, suggesting that in this model LC 9018 has a survival-prolonging effect involving certain cytokines. Moreover, i.pl. injection of mouse recombinant TNF-α into Meth A-bearing mice pretreated with anti-TNF-α mAb partially restored the survival-enhancing effect of LC 9018. These results led us to conclude that TNF-α induced by i.pl. injection of LC 9018 plays an important role in the antitumor effect of LC 9018 in vivo. Received: 22 February 1999     

Cytotoxicity of ester and ether lysophospholipids on Leishmania donovani promastigotes

The cytotoxic activity of four ester lysophospholipids, three ether lysophospholipids, and two radylglycerols on Leishmania donovani promastigotes was determined by measuring the inhibition of cell growth. The 1-acyl lysophospholipids reduced cell growth to 50% of controls at concentrations of 6.4-10.9 microM. In contrast, 1-O-alkenyl-sn-glycero-3-phosphoethanolamine, 1-O-hexadecyl-sn-glycero-3-phosphocholine, and 1-O-hexadecyl-sn-glycerol already showed a 50% inhibition of growth at concentrations between 2.1 and 2.8 microM. Moreover, the unnatural alkyl lysophospholipid analogue 1-O-octadecyl-2-methoxy-sn-glycero-3-phosphocholine was even 10-fold more toxic. Incubations of L. donovani promastigotes with radioactively labelled ether lysophospholipids revealed a rapid uptake of these compounds and their incorporation into cellular lipids at a non-toxic concentration of 1.0 microM. An accumulation of the lysophospholipids in the cell due to insufficient metabolism may be the cause of its cytotoxic effect. The sensitivity of L. donovani cells towards ether lysophospholipids was found to be similar to that reported for tumor cells.     

Proton modulation of recombinant GABAA receptors: influence of GABA concentration and the β subunit TM2–TM3 domain

Regulation of GABA_A receptors by extracellular pH exhibits a dependence on the receptor subunit composition. To date, the molecular mechanism responsible for the modulation of GABA_A receptors at alkaline pH has remained elusive. We report here that the GABA-activated current can be potentiated at pH 8.4 for both αβ and αβγ subunit-containing receptors, but only at GABA concentrations below the EC₄₀. Site-specific mutagenesis revealed that a single lysine residue, K279 in the β subunit TM2–TM3 linker, was critically important for alkaline pH to modulate the function of both α1β2 and α1β2γ2 receptors. The ability of low concentrations of GABA to reveal different pH titration profiles for GABA_A receptors was also examined at acidic pH. At pH 6.4, GABA activation of αβγ receptors was enhanced at low GABA concentrations. This effect was ablated by the mutation H267A in the β subunit. Decreasing the pH further to 5.4 inhibited GABA responses via αβγ receptors, whereas those responses recorded from αβ receptors were potentiated. Inserting homologous β subunit residues into the γ2 subunit to recreate, in αβγ receptors, the proton modulatory profile of αβ receptors, established that in the presence of β2^H267, the mutation γ2^T294K was necessary to potentiate the GABA response at pH 5.4. This residue, T294, is homologous to K279 in the β subunit and suggests that a lysine at this position is an important residue for mediating the allosteric effects of both acidic and alkaline pH changes, rather than forming a direct site for protonation within the GABA_A receptor.     

Intercellular adhesion molecule-1 on cultured human epithelial cell lines: influence of proinflammatory cytokines

Paolieri F, Battifora M, Riccio AM, Pesce G, Canonica GW, Bagnasco M. Intercellular adhesion molecule-1 on cultured human epithelial cell lines: influence of proinflammatory cytokines.
The expression of intercellular adhesion molecule-1 'CD54 or ICAM-1' on epithelial cells during acute or chronic inflammation may favor the interaction between epithelial cells and leukocytes expressing the natural ligands of ICAM-1, LFA-1 'CD11a/CD18', and Mac-1 'CD11b/CD18'. We have evaluated in vitro the expression of ICAM-1 by a conjunctival 'WK' and an intestinal '1407' human continuous epithelial cell line. Cells were cultured for 24 h in the presence or absence of IFN-γ, TNF-α, IL-1β, IL-4, IL-6, IL-8, IL-10, and TGF-Jβ1. Both epithelial cell lines showed a constitutive expression of ICAM-1. IFN-γ at 500 U/ml and TNF-α at 200 ng/ml upregulated ICAM-1 expression; IL-1β at 100 pg/ml upregulated ICAM-1 on WK cells only. Cells cultured in the presence of both IFN-γ and TNF-α exhibited a mean fluorescence intensity far greater than those cultured with IFN-γ or TNF-α alone. 1407 and WK cells were able to release soluble ICAM-1. IFN-γ and TNF-α enhanced the release of sICAM-1. IL-4, IL-6, IL-8, IL-10, and TGF-β1 did not affect either ICAM-1 expression or sICAM-1 release. In conclusion, continuously cultured human epithelial cells may express ICAM-1 on their surface and release it in culture medium. These phenomena are upregulated by proinflammatory cytokines.     

Methylglyoxal-catabolizing enzymes of Leishmania donovani promastigotes

Methylglyoxal is a toxic metabolite with growth inhibitory properties against Leishmania donovani promastigotes. We have shown in the present study that both log and stationary phase promastigotes of L. donovani can catabolize methylglyoxal to D-lactate as the major end product. The specific activity of methylglyoxal reductase was found to be the highest of all the catabolic enzymes. In contrast, the anabolic pathway for methylglyoxal could not be detected. Moreover, when control promastigotes or promastigotes in which the glycolytic pathway was inhibited were incubated with glucose, glycerol or dihydroxyacetone phosphate as energy source, neither methylglyoxal nor D-lactate could be detected.     

RNA interference reveals a role for TLR2 and TLR3 in the recognition of Leishmania donovani promastigotes by interferon-gamma-primed macrophages

Leishmania donovani promastigotes evade the induction of a proinflammatory response during their invasion of naive macrophages. However, their entry into IFN-gamma-primed macrophages is accompanied by the secretion of nitric oxide (NO) and proinflammatory cytokines. In the present study, we addressed the hypothesis that priming with IFN-gamma induces the expression of a receptor that enables mouse macrophages to recognize L. donovani promastigotes. We observed that in IFN-gamma-primed macrophages, L. donovani promastigotes stimulated Interleukin-1 receptor-associated kinase-1 (IRAK-1) activity. We next showed that Toll-like receptor (TLR)3 is barely detectable in naive macrophages but is expressed in IFN-gamma-treated macrophages. Silencing of TLR3, TLR2, IRAK-1 and myeloid differentiation factor 88 (MyD88) expression by RNA interference revealed that both TLR are involved in the secretion of NO and TNF-alpha induced by L. donovani promastigotes. Using L. donovani mutants, we showed that TLR2-mediated responses are dependent on Galbeta1,4Manalpha-PO(4)-containing phosphoglycans, whereas TLR3-mediated responses are independent of these glycoconjugates. Furthermore, our data indicate a participation of TLR2 and TLR3 in the phagocytosis of L. donovani promastigotes and a role for TLR3 in the leishmanicidal activity of the IFN-gamma-primed macrophages. Collectively, our data are consistent with a model where recognition of L. donovani promastigotes depends on the macrophage activation status and requires the expression of TLR3.     

P2X receptor subtype-specific modulation of excitatory and inhibitory synaptic inputs in the rat brainstem

The role of P2 receptors in synaptic transmission to the rat medial nucleus of the trapezoid body (MNTB) was studied in an in vitro brain slice preparation. Whole-cell patch recordings were made and spontaneous synaptic responses studied under voltage clamp during application of P2X receptor agonists. ATPγS (100 μ m ) had no effect on holding current, but facilitated spontaneous excitatory postsynaptic current (sEPSC) frequency in 41% of recordings and facilitated spontaneous inhibitory postsynaptic currents (sIPSCs) in 20% of recordings. These were blocked by the P2 receptor antagonist suramin (100 μ m ). α,β-meATP also facilitated sEPSC and sIPSC frequency, while l -β,γ-meATP facilitated only sIPSCs. The sEPSC facilitation by ATPγS was blocked by TTX (but did not block facilitation of sIPSCs). sEPSC facilitation was blocked by PPADS (30 μ m ) and the selective P2X₃ receptor antagonist A-317491 (3 μ m ), suggesting that modulation of sEPSCs involves P2X₃ receptor subunits. α,β-meATP-facilitated sIPSCs were also recorded in wild-type mouse MNTB neurones, but were absent in the MNTB from P2X₁ receptor-deficient mice demonstrating a functional role for P2X₁ receptors in the CNS.     

Characterization of a mutant Leishmania donovani deficient in adenosine kinase activity

From a mutagenized population of wildtype Leishmania donovani promastigotes, a clonal cell line, TUBA2, was isolated by virtue of its ability to survive and grow in 20 microM tubercidin (7-deazaadenosine). The TUBA2 clone was also 1000-fold less sensitive than the parental line to growth inhibition by formycin A, another cytotoxic adenosine analog. Parental and mutant cells, however, were equally sensitive to growth inhibition by formycin B, allopurinol riboside, and 6-thioguanosine. Mutant cell extracts, unlike those prepared from wildtype cells, did not phosphorylate radiolabelled adenosine, tubercidin, or formycin A. Intact adenosine kinase-deficient cells did not accumulate exogenous tubercidin or formycin A but incorporated [14C]adenosine at rates 25% of those found for parental cells. The uptake data suggest that adenosine kinase plays an important role in the metabolism of adenosine but indicate alternative metabolic pathways for this nucleoside. The metabolism of adenosine to the nucleotide level in TUBA2 cells appears to be initiated via deribosylation to adenine. Significant amounts of both adenosine hydrolytic and adenosine phosphorylytic activities have been detected in L. donovani promastigotes. Furthermore, L. donovani extracts could slowly catalyze the deamination of formycin A. The isolation and characterization of adenosine kinase-deficient cells has provided considerable insight into the function of the purine pathway in L. donovani.