Recombinant cysteine proteinase from Leishmania (Leishmania) chagasi implicated in human and dog T-cell responses

High in vitro lymphoproliferative responses were induced in humans and dogs by a recombinant Leishmania (Leishmania) chagasi cysteine proteinase, with secretion of IFN-gamma in asymptomatic subjects or of IFN-gamma, interleukin 4 (IL-4), and IL-10 in oligosymptomatic subjects. In contrast, responses of symptomatic patients and dogs were lower, with production of IL-4 and IL-10.     

Insights into the tracking of the cysteine proteinase B COOH-terminal polypeptide of Leishmania (Leishmania) amazonensis by surface plasmon resonance

Parasitology Research - Leishmania (Leishmania) amazonensis has adaptive mechanisms to the host environment that are guided by its proteinases, including cysteine proteinase B (CPB), and primarily...     

Characterisation of three groups of cysteine proteinases in the amastigotes of Leishmania mexicana mexicana

The multiple cysteine proteinases characteristic of the amastigote forms of Leishmania mexicana mexicana have been shown to be of three types. The groups of enzymes are distinguished by their substrate specificities and physical properties and have been purified free from other proteinases and most other proteins. One group (A) comprises at least four enzymes that bind to Con A. The cysteine proteinases comprising the other two groups (B and C) were separated by ion exchange chromatography. These last two groups of enzymes show different specificities towards a range of peptidyl aminomethylcoumarins. Notably, the two group C proteinases are more active towards compounds with a basic amino acid in the P1 position, whereas the three enzymes of group B are as active towards substrates with tyrosine in this position. All the cysteine proteinases show preference for compounds with bulky amino acids at positions P2 and P3 and all are equally susceptible to a range of inhibitors characteristically active against cysteine proteinases. Other proteinases present in amastigotes of L. m. mexicana were shown to differ significantly from the cysteine proteinases with respect to their substrate preferences and susceptibility to inhibitors.     

Evaluation of amastigote reactive cells in human cutaneous leishmaniasis caused by Leishmania aethiopica

Lymphoproliferative responses to three affinity chromatography purified amastigote antigens of Leishmania pifanoi, P-2, P-4 and P-8, were evaluated in peripheral blood mononuclear cells (PBMC) from patients with Ethiopian cutaneous leishmaniasis. Antigen-stimulated cells were analysed for the percentage of CD4+, CD8+ and CD16/56+ cells and the expressed levels of gamma interferon (IFNgamma) and interleukin (IL)-10 were determined in culture supernatants. The amastigote antigens induced cellular responses in leishmaniasis patients with heterologous Leishmania parasite infection. These responses were compared to those of freeze-thawed L. aethiopica promastigote antigen stimulation. The membrane protein (P-8), and to a lesser extent the megasomal/cytoplasmic cysteine proteinase(P-2), induced proliferation with high levels of IFNgamma and IL-10 production in cells from patients with active L. aethiopica lesions. CD16/56+ NK cells were the main cell types induced to proliferate in response to P-8 and P-2 stimulation, followed by CD8+ cell populations. P-4 had no such effect. This contrasts from previous studies of New World human leishmaniasis where P-4 and P-8 were stimulatory. The success of a particular molecule in the induction of a response with a protective phenotype may be dependent on the infecting Leishmania spp. To our knowledge, there are no studies that directly compare the New versus Old World cutaneous leishmaniasis in respect of NK cell and IL-10 responses. Our studies indicate that some leishmanial molecules are recognized across the species, while others are apparently more species specific.     

Comparative analysis of megasomes in members of the Leishmania mexicana complex

Megasomes are large lysosome-like structures, previously described in amastigote forms of Leishmania belonging to the mexicana complex, whose major constituents are the cysteine proteinases. Routine observation of thin sections of amastigotes obtained from species of the mexicana complex revealed variations in size and number of megasomes according to the species, and also between amastigotes obtained from axenic cultures and from infected animals. Three-dimensional reconstruction of amastigotes, stereology and immunocytochemical localization of cysteine proteinase revealed significant differences between the three Leishmania species examined, L. amazonensis, L. mexicana and L. pifanoi. The relative volume of megasomes in lesion-derived amastigotes was higher than in axenic amastigotes of L. amazonensis and L. mexicana. The relative volume of megasomes from lesion-derived amastigotes of L. mexicana was 2-3 times higher than in L. amazonensis. Axenic amastigotes of L. pifanoi showed a small relative volume of megasomes and low cysteine proteinase activity, and were not able to produce lesions in the animals, whereas axenic amastigotes of L. mexicana and L. amazonensis did. There were significant differences in the structural organization, distribution within the cell, size and number of megasomes, and in the characteristics of cysteine proteinases found in the amastigotes of the three Leishmania species. These results suggest that these organelles and their constituents may be involved in the infectivity and virulence of Leishmania species.     

Subunit vaccination of mice against new world cutaneous leishmaniasis: comparison of three proteins expressed in amastigotes and six adjuvants

A mixture of well-defined recombinant antigens together with an adjuvant that preferentially stimulates specific gamma interferon (IFN-gamma)-secreting helper type 1 CD4(+) T cells (Th1 cells) presents a rational option for a vaccine against leishmaniasis. The potential of this approach was investigated in murine infections with Leishmania mexicana, which are characterized by the absence of a parasite-specific Th1 response and uncontrolled parasite proliferation. A mixture of three antigens (glycoprotein 63, cysteine proteinases, and a membrane-bound acid phosphatase), which are all expressed in amastigotes, the mammalian stage of the parasite, were used for the immunization of C57BL/6 mice in combination with six adjuvants (interleukin 12 [IL-12], Detox, 4'-monophosphoryl lipid A, QS-21, Mycobacterium bovis BCG, and Corynebacterium parvum). All six vaccine formulations containing the mixture of recombinant antigens were protective against challenge infections with promastigotes, the insect stage of the parasite, in that mice controlled and healed infections but developed transient and, in certain cases, accentuated disease. The most effective adjuvants were IL-12 followed by Detox. Further studies using these two adjuvants showed that a similar protective effect was observed with a mixture of the corresponding native proteins, and mice which had controlled the infection showed a preponderance of IFN-gamma-secreting CD4(+) T cells in the lymph nodes draining the lesion. Using the recombinant proteins individually, it is shown that the relatively abundant cysteine proteinases and glycoprotein 63, but not the acid phosphatase, are able to elicit a protective response. The results are discussed in comparison to previous studies with subunit vaccines and with respect to cell biological aspects of antigen presentation in Leishmania-infected macrophages.     

Induction of interleukin-6 release from monocytes by serine proteinases and its potential mechanisms

Serine proteinases have been recognized playing an important role in inflammation via proteinase-activated receptors (PAR). However, little is known of the influence of serine proteinases and PAR on interleukin-6 (IL-6) secretion from highly purified monocytes. We challenged monocytes from human peripheral blood with serine proteinases and agonist peptides of PAR and measured the levels of IL-6, IL-1beta and IL-12 in culture supernatants by enzyme-linked immunosorbent assay. The results showed that thrombin, trypsin, tryptase and elastase stimulated approximately up to 2.9-, 2.0-, 1.8- and 2.1-fold increase in IL-6 release from monocytes following 16 h of incubation, respectively. Proteinase inhibitors inhibited the actions of proteinases on monocytes. Agonist peptides of PAR-1 (SFLLR-NH(3)) and PAR-4 (GYPGQV-NH(2)), but not PAR-3 (TFRGAP-NH(2)), also induced IL-6 release from monocytes. The proteinases and agonists of PAR failed to stimulate IL-1beta and IL-12 secretion. In conclusion, the induction of IL-6 secretion by serine proteinases may be through the activation of PAR.     

Identification and characterization of major allergens in excretory/secretory products of the worm Paragonimus ohirai

Allergens present in the excretory/secretory (ES) products of adult Paragonimus ohirai were biochemically identified. Immunoblot analysis using sera from P. ohirai-infected rats revealed only two allergens to be major proteins in the ES products, with apparent molecular masses (M(r)) of 27 and 29 kD. As the ES products contained a high proportion of acidic and neutral cysteine proteinases, we examined whether or not the allergens and the cysteine proteinases were identical. The acidic and neutral cysteine proteinases were biochemically purified from the ES products and showed M(r) of 27 and 29 kD, respectively. The two cysteine proteinases had almost identical N-terminal amino acid sequences and were reactive with specific IgE in sera from the infected rats. The allergenicity of the cysteine proteinases was confirmed by 48-hour homologous passive cutaneous anaphylaxis. Immunoblot and immunocapture assays using anti-human IgE monoclonal antibody showed that the proteinase allergens were reactive with specific IgE of patients with paragonimiasis westermani. Also, the cysteine proteinases were reactive with specific IgG of both the infected rats and the patients. Therefore the acidic and neutral cysteine proteinases prepared from the ES products of P. ohirai will be useful allergens and antigens for the immunodiagnosis of paragonimiasis.     

Antigen presentation by Leishmania mexicana-infected macrophages: Activation of helper T cells specific for amastigote cysteine proteinases requires intracellular killing of the parasites

Leishmania mexicana amastigotes proliferate in the phagolysosomes of mammalian macrophages. The parasites abundantly synthesize lysosomal cysteine proteinases, which are encoded by the lmcpb gene family. One of these genes was overexpressed in Escherichia coli, and the purified recombinant protein was used as an antigen to induce and establish a T helper 1 (T_h1) cell line. The T cells recognize epitopes shared by the native cysteine proteinases and the recombinant protein. Infected bone marrow-derived macrophages induced to express major histocompatibility complex class II molecules by interferon (IFN)-γ do not affect parasite viability. These macrophages fail to stimulate the proliferation of the T cell line. In contrast, strong T cell stimulation is observed after the parasites are killed by treatment with L-leucine methylester, or after activation of macrophages by IFN-γ and tumor necrosis factor-α. It is concluded that infected macrophages efficiently present this lysosomal Leishmania antigen once the parasites are inactivated and degraded. This observation may be of considerable relevance for the outcome of Leishmania infections provided that it can be extended to other parasite antigens.     

A radioiodinated peptidyl diazomethane detects similar cysteine proteinases in amastigotes and promastigotes ofLeishmania (L.) mexicana andL. (L.) amazonensis

Cysteine proteinase activities were examined in lesion amastigotes as well as in stationary-phase promastigotes ofLeishmania (L.) mexicana andLeishmania (L.) amazonensis isolates. Enzyme detection in gelatin gels revealed that amastigotes of threeL. (L.) mexicana isolates (M379, IOC-0561, and IP) shared similar proteinases, including the multiple low-molecular-weight (25–35 kDa) cysteine proteinases. High cysteine proteinase activity was also observed inL. (L.) amazonensis amastigotes, but the banding profile was different in two of the isolates examined. Promastigotes displayed fewer low-molecular-weight proteinase bands, and these were much less intense as compared with those of lesion amastigotes. Independently of theLeishmania isolates and developmental stages examined, incubation of the parasites for 2 h with 0.2 M radioiodinatedN-benzyloxycarbonyl-tyrosyl-alanyl diazomethane (Z-Tyr[¹²⁵I]-AlaCHN₂) markedly and selectively labeled bands comigrating with the 28- and 31-kDa cysteine proteinases. Under reducing conditions, labeling was associated with four similar polypeptides (29–34 kDa), which were also detected when incubation with Z-Tyr[¹²⁵I]-AlaCHN₂ was carried out after cell lysis. Labeling was completely abolished if lysates were first incubated with 20 M E-64 and then exposed to the¹²⁵I-tagged inhibitor, thus confirming the specificity of the compound toward cysteine proteinases.     

Identification of Leishmania major cysteine proteinases as targets of the immune response in humans

In this study, we report the identification of two parasite polypeptides recognized by human sera of patients infected with Leishmania major. Isolation and sequencing of the two genes encoding these polypeptides revealed that one of the genes is similar to the L. major cathepsin L-like gene family CPB, whereas the other gene codes for the L. major homologue of the cysteine proteinase a (CPA) of L. mexicana. By restriction enzyme digestion of genomic DNA, we show that the CPB gene is present in multiple copies in contrast to the cysteine proteinase CPA gene which could be unique. Specific antibodies directed against the mature regions of both types expressed in Escherichia coli were used to analyze the expression of these polypeptides in different stages of the parasite's life cycle. Polypeptides of 27 and 40 kDa in size, corresponding to CPA and CPB respectively, were detected at higher level in amastigotes than in stationary phase promastigotes. Purified recombinant CPs were also used to examine the presence of specific antibodies in sera from either recovered or active cases of cutaneous leishmaniasis patients. Unlike sera from healthy uninfected controls, all the sera reacted with recombinant CPA and CPB. This finding indicates that individuals having recovered from cutaneous leishmaniasis or with clinically apparent disease have humoral responses to cysteine proteinases demonstrating the importance of these proteinases as targets of the immune response and also their potential use for serodiagnosis.     

LACK-specific CD4(+) T cells that induce gamma interferon production in patients with localized cutaneous leishmaniasis during an early stage of infection

The profile of cytokines induced by soluble leishmania antigen (SLA) and the Leishmania homologue of the mammalian receptor for activated C kinase (LACK), a candidate vaccine against leishmaniasis, and the cellular source of the cytokines produced in response to these antigens were analyzed in patients infected with Leishmania guyanensis. Gamma interferon (IFN-gamma) and interleukin-10 (IL-10) were produced in response to LACK. Although LACK-specific CD4(+) cells producing IFN-gamma were isolated only during the early phase of infection (less than 30 days following the onset of infection), cells producing IL-10 in response to LACK were detected in all patients. CD4(+) T cells producing IFN-gamma and IL-13 were produced in response to SLA in all patients. SLA- and LACK-specific T cells are effector memory cells, as they are CD45RA(-) CCR7(-) CD4(+) T cells. CD4(+) T cells producing IFN-gamma are CD62L(-), and CD4(+) T cells producing IL-10 are CD62L(+), indicating that these cells have different tissue-homing capacities. These findings show that SLA and LACK induce both type 1 (IFN-gamma) and type 2 (IL-10 or IL-13) cell responses.     

Flow cytometric determination of cellular sources and frequencies of key cytokine-producing lymphocytes directed against recombinant LACK and soluble Leishmania antigen in human cutaneous leishmaniasis

Leishmaniasis, caused by infection with the protozoan parasite Leishmania, affects millions of individuals worldwide, causing serious morbidity and mortality. This study directly determined the frequency of cells producing key immunoregulatory cytokines in response to the recombinant antigen Leishmania homolog of receptors for activated kinase C (LACK) and soluble leishmania antigen (SLA), and it determined relative contributions of these antigens to the overall cytokine profile in individuals infected for the first time with Leishmania braziliensis. All individuals presented with the cutaneous clinical form of leishmaniasis and were analyzed for proliferative responses to LACK antigen and SLA, frequency of lymphocyte subpopulations (analyzed ex vivo), and antigen-induced (LACK and SLA) cytokine production at the single-cell level (determined by flow cytometry). The following were determined. (i) The Th1-type response previously seen in patients with cutaneous leishmaniasis is due to gamma interferon (IFN-gamma) production by several different sources, listed in order of contribution: CD4(+) T lymphocytes, CD4(-), CD8(-) lymphocytes, and CD8(+) T lymphocytes. (ii) SLA induced a higher frequency of lymphocytes producing IFN-gamma and tumor necrosis factor alpha (TNF-alpha) than did LACK. (iii) LACK induced an activation of monocyte populations as reflected by an increased percentage of CD14-positive cells. (iv) Neither SLA nor LACK induced detectable frequencies of cells producing interleukin-4 (IL-4) or IL-5. These data demonstrated a multifaceted immune response to SLA in human leishmaniasis involving Th1 CD4(+) T lymphocytes (IFN-gamma(+) and IL-10(-)/IL-4(-)), Tc1 CD8(+) T cells (IFN-gamma(+), and IL-10(-)/IL-4(-)), and a high frequency of TNF-alpha-producing lymphocytes. Moreover, it was determined that the recombinant antigen LACK acts as a weak inducer of Th1-type lymphocyte responses compared to SLA.     

Comparison of the immune profile of nonhealing cutaneous Leishmaniasis patients with those with active lesions and those who have recovered from infection

Th1-type cellular immune responses play a critical role in protection against infection with Leishmania parasites, whereas activation of Th2-type cells results in progressive disease. Cutaneous leishmaniasis caused by Leishmania major is often a self-healing disease; however, persistent nonhealing forms are also known. In the present study, we have described cell-mediated immune responses in nonhealing patients by measuring T-cell proliferation, cytokine production, and phenotypic characterization of these cells. The responses were compared with those of patients with active lesions, patients who had recovered from infection, and healthy controls. Peripheral blood mononuclear cells from patients with active lesions and recovered donors proliferated vigorously and produced Th1-type cytokine when stimulated with L. major antigens, whereas in nonhealing patients the proliferative responses were significantly lower and showed a Th2-type response to Leishmania antigens. Interleukin-10 (IL-10) production was not a feature of L. major stimulation. Flow cytometric analysis revealed that L. major antigen induced proliferation of the CD4-positive population and that these cells were the major source of gamma interferon and IL-4. These results show a distinct dichotomy in the cytokine response to L. major infection.     

T-cell response in human leishmaniasis

In the present communication we provide evidence for the existence of a Th1/Th2 dichotomy in the T-cell response to Leishmania antigens in human leishmaniasis. Our data suggest that the pattern of IL-4 and IFN-gamma response is polarised in these patients. Lymphocytes from individuals recovered from cutaneous leishmaniasis (CL) responded by IFN-gamma production following stimulation with Leishmania antigens whereas cells from patients recovered from visceral leishmaniasis (VL) showed a mixed pattern of IFN-gamma and IL-4 responses. The cells producing these cytokines were predominantly CD4+. Furthermore, IL-10 plays an important role in the development of post kala azar dermal leishmaniasis (PKDL) from VL. The balance between the parasitic-specific T-cell response plays an important regulatory role in determining the outcome of Leishmania infections in humans.     

Interaction of laminin with Entamoeba histolytica cysteine proteinases and its effect on amebic pathogenesis.

The Entamoeba histolytica 27-kDa cysteine proteinases exhibit striking binding specificities for immobilized laminin over other components of the extracellular matrix, such as collagen and fibronectin. Inactivation of the proteinase with the active-site inhibitor L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane abolishes laminin binding by the enzyme, and conversely, laminin inhibits cleavage of a fluorogenic dipeptide substrate of the amebic cysteine proteinase, suggesting that the substrate binding pocket of the enzyme is involved in the binding of laminin. The addition of laminin but not fibronectin or collagen to E. histolytica trophozoites significantly reduces amebic liver abscess formation in severe combined immunodeficient mice, further supporting the hypothesis that E. histolytica cysteine proteinases play an important role in amebic pathogenesis. The specific interaction of amebic proteinases with laminin may be exploited in designing new inhibitors of these enzymes.     

Induction and abrogation of LACK reactive cells in the evolution of human leishmaniasis.

Peripheral blood mononuclear cells (PBMC) from cutaneous leishmaniasis patients with ongoing Leishmania aethiopica infection and individuals cured/under treatment from L. infantum or L. donovani infection were stimulated in vitro with LACK, the Leishmania homologue of receptors for activated C kinase. The LACK protein is conserved in related leishmanial species and is expressed both in the promastigote and amastigote stages of Leishmania. Our results show that LACK induced marked NK and some CD8+ cell proliferation in PBMC from cutaneous leishmaniasis patients with active disease. These responses were coupled with high levels of IFN-gamma and IL-10 production. At the concentration tested, the proliferative responses to freeze-thawed Leishmania antigen (Ft-Leish) were higher, while the levels of IFN-gamma were consistently lower than that of LACK. Although cells from individuals cured of leishmaniasis could respond to whole Leishmania lysate by proliferation and IFN-gamma production, there was no evident response to LACK. Ethiopian controls tested at the same time also showed LACK induced proliferation with IFN-gamma and IL-10 responses. Thus LACK reactivity in terms of proliferation and cytokine induction were present in cells from some healthy donors and most of the patients with active lesions, while this response was absent in individuals cured of L. infantum or L. donovani leishmaniasis. Since cure from leishmaniasis often results in life-long protection, and active but not cured patients showed in vitro responses to LACK stimulation, questions arose as to how this highly immunodominant molecule functions during human leishmanisasis. Some possible mechanisms are discussed.     

Role of Leishmania donovani and Its Lipophosphoglycan in CD4+ T-cell Activation-Induced Human Immunodeficiency Virus Replication

Chronic immune activation by coinfecting pathogens has been suggested as a cofactor in human immunodeficiency virus (HIV) disease progression, particularly in the setting of developing countries. Here, we used in vivo-infected mononuclear cells to examine the role of the protozoan parasite Leishmania donovani and its major membrane constituent, lipophosphoglycan (LPG), in mediating CD4+ T-lymphocyte activation-induced HIV replication and CD4+ T-cell death. We found that Leishmania antigens upregulated HIV replication in CD8-depleted peripheral blood mononuclear cells from asymptomatic HIV-infected donors compared to unstimulated cells. L. donovani-induced viral replication was associated with cellular proliferation, increased expression of the cellular immune activation markers CD25 and HLA-DR within the CD4+ subpopulation, and enhanced secretion of tumor necrosis factor alpha (TNF-alpha), interleukin 2 (IL-2), and IL-6. LPG induced TNF-alpha secretion in the absence of increased expression of cellular activation markers. Moreover, in a few cases we observed that L. donovani induced HIV replication without significant cellular activation but with cytokine secretion. The rate of apoptosis was accelerated in these latently infected CD4+ T cells primed with Leishmania antigens compared to controls, and TNF-alpha production appeared to be the central event necessary for this effect. Furthermore, we demonstrate that thalidomide inhibited Leishmania-induced virus replication coupled with abrogated Leishmania-induced TNF-alpha secretion but not IL-2 or IL-6 production. Furthermore, thalidomide did not affect Leishmania-induced apoptosis. The results suggest that Leishmania and its product, LPG, up-regulate HIV replication in latently infected cells through distinct antigen-specific and non-antigen-specific cellular immune activation mechanisms and that TNF-alpha secretion is pivotal in this process. The immunomodulatory role of thalidomide raises interest as a potential adjuvant to reduce HIV disease progression in Leishmania-HIV coinfected individuals.     

Hydrolysis of interleukin-12 by Porphyromonas gingivalis major cysteine proteinases may affect local gamma interferon accumulation and the Th1 or Th2 T-cell phenotype in periodontitis

Porphyromonas gingivalis cysteine proteinases (gingipains) have been associated with virulence in destructive periodontitis, a disease process variously considered to represent an unregulated stimulation of either T helper type 1 (Th1)- or Th2-type cells. Critical in maintaining Th1 activity is the response of T lymphocytes to environmental interleukin 12 (IL-12) in the form of up-regulation of gamma interferon (IFN-gamma) production. Here we demonstrate that in the presence or absence of serum, gingipains were able to hydrolyze IL-12 and reduce the IL-12-induced IFN-gamma production from CD4+ T cells. However, the induction of IL-12 receptors on T cells by gingipains did not correlate with the enhancement of IFN-gamma production. The gingipains cleaved IL-12 within the COOH-terminal region of the p40 and p35 subunit chains, which leads to IL-12 inactivity, whereas IL-2 in these assays was not affected. Inactivation of IL-12 by the gingipains could disrupt the cytokine balance or favor Th2 activities in the progression of periodontitis.