Diane McMahon-Pratt

Disruption of CD40–CD40 Ligand Interactions Results in an Enhanced Susceptibility to Leishmania amazonensis Infection

To study the role of CD40 ligand (CD40L) in the host immune responses against intracellular pathogens, we infected CD40L knockout (CD40L-/-) mice with Leishmania amazonensis. Although wild-type mice were susceptible to infection and developed progressive ulcerative lesions, tissue parasite burdens in CD40L-/- mice were significantly higher. This heightened susceptibility to infection was associated with an impaired T cell and macrophage activation and altered inflammatory response, as reflected by low levels of IFN gamma, lymphotoxin-tumor necrosis factor (LT-TNF), and nitric oxide (NO) production. Furthermore, CD40L-/- mice failed to generate a protective immune response after immunization. These results indicate an essential role of cognate CD40-CD40L interactions in the generation of cellular immune responses against an intracellular parasite.

Does the Leishmania major paradigm of pathogenesis and protection hold for New World cutaneous leishmaniases or the visceral disease

Summary:  Parasitic protozoa of the genus Leishmania have provided a useful perspective for immunologists in terms of host defense mechanisms critical for the resolution of infection caused by intracellular pathogens. These organisms, which normally reside in a late endosomal, major histocompatibility complex (MHC) class II+ compartment within host macrophages cells, require CD4+ T‐cell responses for the control of disease. The paradigm for the CD4+ T‐helper 1 (Th1)/Th2 dichotomy is largely based on the curing/non‐curing responses, respectively, to Leishmania major infection. However, this genus of parasitic protozoa is evolutionarily diverse, with the cutaneous disease‐causing organisms of the Old World (L. major) and New World (Leishmania mexicana/ Leishmania amazonensis) having diverged 40–80 million years ago. Further adaptations to survive within the visceral organs (for Leishmania donovani, Leishmania chagasi, and Leishmania infantum) must have been required. Consequently, significant differences in host–parasite interactions have evolved. Different virulence factors have been identified for distinct Leishmania species, and there are profound differences in the immune mechanisms that mediate susceptibility/resistance to infection and in the pathology associated with disease. These variations not only point to interesting features of the host–pathogen interaction and immunobiology of this genus of parasitic protozoa, but also have important implications for immunotherapy and vaccine development.

Intradermal infection model for pathogenesis and vaccine studies of murine visceral leishmaniasis

ABSTRACT The levels of protection found in vaccine studies of murine visceral leishmaniasis are significantly lower than for cutaneous leishmaniasis; whether this is due to the high-challenge murine model employed and/or is a consequence of differences required in tissue-specific local immune responses is not understood. Consequently, an intradermal murine model of visceral leishmaniasis has been explored. Intradermal inoculation established a chronic infection in susceptible mice which was associated with a pattern of parasite clearance with time postinfection in the liver and skin; in contrast, parasite persistence and expansion was observed in lymphoid tissue (spleen and draining lymph node). The course of disease found appears to be similar to those reported for subclinical canine and human visceral leishmaniasis. Clearance of parasites from the skin was correlated with an inflammatory response and the infiltration and activation of CD4+ and CD8+ T cells. In contrast, in lymphoid tissue (lymph node or spleen), the production of Th1/Th2 cytokines (interleukin-4 [IL-4], IL-10, and gamma interferon) appeared to correlate with parasite burden and pathogenesis. In vaccination experiments employing the Leishmania infantum D-13 (p80) antigen, significantly higher levels of protection were found with the intradermal murine model (29 to 7,500-fold more than naive controls) than were found with a low-dose intravenous infection model (9 to 173-fold). Thus, this model should prove useful for further investigation of disease pathogenesis as well as vaccine studies of visceral leishmaniasis.

Developmental Life Cycle of Leishmania—Cultivation and Characterization of Cultured Extracellular Amastigotes

ABSTRACT. The biochemistry and immunology of Leishmania promastigotes has been extensively studied; this is due primarily to the facility with which this stage, in contrast to the amastigotes stage, can be maintained in axenic culture. Several attempts to axenically culture lines of Leishmania amastigotes have been reported in the literature. This paper summarizes methods of adaptation (low pH, elevated temperature and culture medium) and characterization of several axenic lines of Leishmania amastigotes. Based on morphological, biological, immunological and biochemical evidence, these organisms appear to resemble amastigotes from infected macrophages or tissue. The axenically cultured amastigotes appear to be distinct from shocked (heat, serum deprivation, stressed) Leishmania promastigotes in the plethora of proteins synthesized, growth (multiplication) in culture, and developmental regulation observed. These data suggest that Leishmania organisms have a significant developmental response to certain signals (pH, temperature) mimicking their in vivo macrophage milieu. The response to other environmental parameters characteristic of the host‐macrophage remain to be determined. These axenically cultured amastigotes should be of interest for further immunological, biochemical and developmental investigations of the disease‐maintaining stage of this parasite.

A Leishmania Ortholog of Macrophage Migration Inhibitory Factor Modulates Host Macrophage Responses

Parasitic organisms have evolved specialized strategies to evade immune defense mechanisms. We describe herein an ortholog of the cytokine, macrophage migration inhibitory factor (MIF), which is produced by the obligate intracellular parasite, Leishmania major. The Leishmania MIF protein, Lm1740MIF, shows significant structural homology with human MIF as revealed by a high-resolution x-ray crystal structure (1.03 Å). Differences between the two proteins in the N-terminal tautomerization site are evident, and we provide evidence for the selective, species-specific inhibition of MIF by small-molecule antagonists that target this site. Lm1740MIF shows significant binding interaction with the MIF receptor, CD74 (Kd = 2.9 × 10−8 M). Like its mammalian counterpart, Lm1740MIF induces ERK1/2 MAP kinase activation in a CD74-dependent manner and inhibits the activation-induced apoptosis of macrophages. The ability of Lm1740MIF to inhibit apoptosis may facilitate the persistence of Leishmania within the macrophage and contribute to its evasion from immune destruction.

Identification of two distinct cysteine proteinase genes of Leishmania pifanoi axenic amastigotes using the polymerase chain reaction

A developmentally regulated cysteine proteinase associated with an unique lysosomal organelle, the megasome, has been described for the intracellular amastigotes of the Leishmania mexicana complex; this proteinase appears to be important in the survival of the parasite. Degenerate primers encoding the active sites residues have been used to amplify cysteine proteinase cDNA sequences from axenically cultured amastigotes of Leishmania pifanoi, a member of the L. mexicana complex. Based on sequence data, two distinct genes (Lpcys1 and Lpcys2) were identified. Although both genes are preferentially transcribed in the amastigote stage, each is distinct in genomic arrangement and chromosome location, with Lpcys2 showing evidence for the presence of 8-20 tandemly arrayed copies and mRNA levels 10-fold higher than Lpcys1. Related forms of the Lpcys1 and Lpcys2 genes exist in other species of the genus Leishmania, including Leishmania braziliensis, Leishmania major and Leishmania donovani. The protein sequence of an abundant immunoaffinity purified amastigote cysteine proteinase (A-2) is identical to that predicted for the product of Lpcys2; immunofluorescence studies show an intracellular pattern/distribution for the A-2 proteinase consistent with a putative megasomal association. The DNA sequence of a genomic copy of Lpcys2 predicts a C-terminal extension for the proteinase; comparative sequence analyses of the C-terminal extensions found for Trypanosoma cruzi and Trypanosoma brucei reveal the selective conservation of cysteine, as well as proline and glycine residues, suggesting that conservation of folding and secondary structure may be required for biological function.

Heterologous Prime-Boost Vaccination with the LACK Antigen Protects against Murine Visceral Leishmaniasis

ABSTRACT This study reports the efficacy of a heterologous prime-boost vaccination using DNA and vaccinia viruses (Western Reserve [WR] virus and modified [attenuated] vaccinia virus Ankara [MVA]) expressing the LACK antigen (Leishmania homologue of receptors for activated C kinase) and an intradermal murine infection model employing Leishmania infantum. At 1 month postinfection, vaccinated mice showed high levels of protection in the draining lymph node (240-fold reduction in parasite burden) coupled with significant levels of gamma interferon (20 to 200 ng/ml) and tumor necrosis factor alpha/lymphotoxin (8 to 134 pg/ml). Significant but lower levels of protection (6- to 30-fold) were observed in the spleen and liver. Comparable levels of protection were found for mice boosted with either LACK-WR or LACK-MVA, supporting the use of an attenuated vaccinia virus-based vaccine against human visceral leishmaniasis.

Perforin and gamma interferon are critical CD8+ T-cell-mediated responses in vaccine-induced immunity against Leishmania amazonensis infection.

ABSTRACT Previous studies have demonstrated that protection against New World leishmaniasis caused by Leishmania amazonensis can be elicited by immunization with the developmentally regulated Leishmania amastigote antigen, P-8. In this study, several independent experimental approaches were employed to investigate the protective immunological mechanisms involved. T-cell subset depletion experiments clearly indicate that elicitation of CD8+ (as well as CD4+) effector responses is required for protection. Further, mice lacking β2-microglobulin (and hence deficient in major histocompatibility complex class I antigen presentation) were not able to control a challenge infection after vaccination, indicating an essential protective role for CD8+ T effector responses. Analysis of the events ongoing at the cutaneous site of infection indicated a changing cellular dynamic involved in protection. Early postinfection in protectively vaccinated mice, a predominance of CD8+ T cells, secreting gamma interferon (IFN-γ) and expressing perforin, was observed at the site of infection; subsequently, activated CD4+ T cells producing IFN-γ were primarily found. As protection correlated with the ratio of total IFN-γ-producing cells (CD4+ and CD8+ T cells) to macrophages found at the site of infection, a role for IFN-γ was evident; in addition, vaccination of IFN-γ-deficient mice failed to provide protection. To further assess the effector mechanisms that mediate protection, mice deficient in perforin synthesis were examined. Perforin-deficient mice vaccinated with the P-8 antigen were unable to control infection. Thus, the elicitation of CD8+ T cell effector mechanisms (perforin, IFN-γ) are clearly required in the protective immune response against L. amazonensis infection in vaccinated mice.

A Scaffold Protein, AHNAK1, Is Required for Calcium Signaling during T Cell Activation

Engagement of the T cell antigen receptor (TCR) during antigen presentation initiates a coordinated action of a large number of signaling proteins and ion channels. AHNAK1 is a scaffold protein, highly expressed by CD4+ T cells, and is a critical component for calcium signaling. We showed that AHNAK1-deficient mice were highly susceptible to Leishmania major infection. AHNAK1-deficient CD4+ T cells responded poorly to TCR stimulation in vitro with low proliferation and low Interleukin-2 production. Furthermore, AHNAK1 deficiency resulted in a reduced calcium influx upon TCR crosslinking and subsequent poor activation of the transcription factor NFAT. AHNAK1 was required for plasma membrane expression of L-type calcium channels alpha 1S (Cav1.1), probably through its interaction with the beta regulatory subunit. Thus, AHNAK1 plays an essential role in T cell Ca2+ signaling through Cav1 channels, triggered via TCR activation; therefore, AHNAK1 is a potential target for therapeutic intervention.

The biosynthesis, processing, and immunolocalization of Leishmania pifanoi amastigote cysteine proteinases.

Biosynthesis, enzymatic processing, and immunocytochemical localization of an abundant developmentally regulated cysteine proteinase of Leishmania pifanoi, Lpcys2, were investigated employing axenic cultured amastigotes and monoclonal antibodies specifically recognizing either the mature proteinase or the carboxy-terminal extension domain. Pulse labeling and protein sequence data indicated that a 45-kDa precursor is processed to a 40-kDa intermediate, which is further cleaved to generate the 27-kDa mature enzyme and a 15-kDa COOH-terminal domain. Evidence indicates that proteolytic activity is associated with the intermediate form as well as the mature proteinase. Treatment with selected cysteine but not aspartic acid proteinase inhibitors arrested proteolytic processing of Lpcys2 in vivo and inhibited parasite cell division. Electron microscopic immunolocalization of both catalytic and COOH-terminal domains in L. pifanoi and Leishmania amazonensis amastigotes showed intense labeling of megasomes, indicating that cleavage of the COOH-terminal domain probably occurs in the megasome. A low level of the mature proteinase was also associated with the flagellar pocket and plasma membrane; consistent with this observation, low level secretion of Lpcys2 into the culture medium was detected. Lpcys1, a related, less abundant amastigote-specific cysteine proteinase lacking a comparable COOH-terminal domain, was localized to the flagellar pocket and megasomes. Consequently, enzyme sorting to megasomes does not appear to depend upon the COOH-terminal domain; hence this region of Lpcys2 may not be essential for its intracellular targeting.