Permissive and protective roles for neutrophils in leishmaniasis.

Leishmania parasites are the causative agents of leishmaniasis, a neglected tropical disease that causes substantial morbidity and considerable mortality in many developing areas of the world. Recent estimates suggest that roughly 10 million people suffer from cutaneous leishmaniasis (CL), and approximately 76,000 are afflicted with visceral leishmaniasis (VL), which is universally fatal without treatment. Efforts to develop therapeutics and vaccines have been greatly hampered by an incomplete understanding of the parasite's biology and a lack of clear protective correlates that must be met in order to achieve immunity. Although parasites grow and divide preferentially in macrophages, a number of other cell types interact with and internalize Leishmania parasites, including monocytes, dendritic cells and neutrophils. Neutrophils appear to be especially important shortly after parasites are introduced into the skin, and may serve a dual protective and permissive role during the establishment of infection. Curiously, neutrophil recruitment to the site of infection appears to continue into the chronic phase of disease, which may persist for many years. The immunological impact of these cells during chronic leishmaniasis is unclear at this time. In this review we discuss the ways in which neutrophils have been observed to prevent and promote the establishment of infection, examine the role of anti-neutrophil antibodies in mouse models of leishmaniasis and consider recent findings that neutrophils may play a previously unrecognized role in influencing chronic parasite persistence.

CC chemokine receptor (CCR)2 is required for langerhans cell migration and localization of T helper cell type 1 (Th1)-inducing dendritic cells. Absence of CCR2 shifts the Leishmania major-resistant phenotype to a susceptible state dominated by Th2 cytokines, b cell outgrowth, and sustained neutrophilic inflammation.

There is growing evidence that chemokines and their receptors regulate the movement and interaction of antigen-presenting cells such as dendritic cells (DCs) and T cells. We tested the hypothesis that the CC chemokine receptor (CCR)2 and CCR5 and the chemokine macrophage inflammatory protein (MIP)-1alpha, a ligand for CCR5, influence DC migration and localization. We found that deficiency of CCR2 but not CCR5 or MIP-1alpha led to distinct defects in DC biology. Langerhans cell (skin DC) density in CCR2-null mice was normal, and their ability to migrate into the dermis was intact; however, their migration to the draining lymph nodes was markedly impaired. CCR2-null mice had lower numbers of DCs in the spleen, and this was primarily due to a reduction in the CD8alpha(1) T helper cell type 1 (Th1)-inducing subset of DCs. Additionally, there was a block in the Leishmania major infection-induced relocalization of splenic DCs from the marginal zone to the T cell areas. We propose that these DC defects, in conjunction with increased expression of B lymphocyte chemoattractant, a B cell-specific chemokine, may collectively contribute to the striking B cell outgrowth and Th2 cytokine-biased nonhealing phenotype that we observed in CCR2-deficient mice infected with L. major. This disease phenotype in mice with an L. major-resistant genetic background but lacking CCR2 is strikingly reminiscent of that observed typically in mice with an L. major-susceptible genetic background. Thus, CCR2 is an important determinant of not only DC migration and localization but also the development of protective cell-mediated immune responses to L. major.

Preformed membrane-associated stores of interleukin (IL)-12 are a previously unrecognized source of bioactive IL-12 that is mobilized within minutes of contact with an intracellular parasite.

The prevailing paradigm is that production of the interleukin (IL)-12 p70 heterodimer, a critical T helper cell type 1 (Th1)-inducing cytokine, depends on the induced transcription of the p40 subunit. Concordant with this paradigm, we found that dendritic cells (DCs) produced IL-12 p70 only after at least 2-4 h of stimulation with lipopolysaccharide plus interferon gamma. However, using several complementary experimental approaches, including electron and confocal microscopy, we now show that resting murine and human myeloid cells, including macrophages/DCs and DC-rich tissues, contain a novel source of bioactive IL-12 that is preformed and membrane associated. These preformed, membrane-associated IL-12 p70 stores are released within minutes after in vitro or in vivo contact with Leishmania donovani, an intracellular pathogen. Our findings highlight a novel source of bioactive IL-12 that is readily available for the rapid initiation of Th1 host responses to pathogens such as Leishmania species.

Molecular diagnosis of protozoan parasites by Recombinase Polymerase Amplification.

Infections caused by protozoan parasites affect millions of people around the world. Traditionally, diagnosis was made by microscopy, which is insensitive and in some cases not specific. Molecular methods are highly sensitive and specific, but equipment costs and personnel training limit its availability only to specialized centers, usually far from populations with the highest risk of infection. Inexpensive methods that can be applied at the point of care (POC), especially in places with limited health infrastructure, would be a major advantage. Isothermal amplification of nucleic acids does not require thermocyclers and is relatively inexpensive and easy to implement. Among isothermal methods, recombinase polymerase amplification (RPA) is sensitive and potentially applicable at POC. We and others have developed RPA diagnostic tests to detect protozoan parasites of medical importance. Overall, our results have shown high specificity with limits of detection similar to PCR. Currently, the optimization of RPA for use at the POC is under development, and in the near future the tests should become available to detect protozoan infections in the field. In this review we discuss the current status, challenges, and future of RPA in the field of molecular diagnosis of protozoan parasites.

Profile of human T cell response to leishmanial antigens. Analysis by immunoblotting.

Control and resolution of leishmanial infection depends primarily on T cell-mediated immune mechanisms. The nature of the leishmanial antigens involved in eliciting T cell immunity is unknown. We have examined the pattern of peripheral blood lymphocyte responses in patients with active, healed, or subclinical leishmanial infection to fractionated leishmanial antigens using a T cell immunoblotting method in which nitrocellulose-bound leishmanial antigen, resolved by one or two dimensional electrophoresis, are incorporated into lymphocyte cultures. The proliferative and IFN-gamma responses of cells from patients with healed mucosal or cutaneous leishmaniasis were remarkably heterogeneous and occurred to as many as 50-70 distinct antigens. In contrast, responses from subjects with active, nonhealing, diffuse cutaneous leishmaniasis were either absent or present to only a small number of antigens. Control and resolution of leishmaniasis, and resistance to reinfection, is therefore associated with a T cell response to a large and diverse pool of parasite antigens. The method of T cell immunoblotting appears to offer a powerful, rapid, and relatively simple approach to the identification of antigens involved in eliciting a T cell response in human leishmaniasis.

Quantitative measurement of human cytokine gene expression by polymerase chain reaction.

We have developed a method in which human cytokine gene expression can be quantitated using gene amplification technology. Total RNA was extracted from a human T cell line, reverse transcribed to cDNA, and amplified using the polymerase chain reaction (PCR). Inclusion of a radiolabeled nucleotide in the PCR reaction mixture followed by electrophoresis and quantitative imaging of the amplification product with the BetaScope imager and software enabled quantitation of the input cDNA. Linear standard curves within the exponential phase of DNA amplification using purified cytokine cDNA templates were generated over a several log concentration range of input DNA. A 10-67-fold increase in interleukin-2 (IL-2), IL-3, IL-4, IL-10, and interferon-gamma gene expression following cell activation could be identified by interpolation from the standard curve. The lower limit of linearity on the standard curve was as little as 0.01 fg of input DNA which corresponded to approximately 20 cells. This very sensitive methodology is a valuable tool in the detection and quantitation of cytokine gene expression when only small amounts of tissue or cells are available.

Overexpression of cardiotrophin-1 and gp130 during experimental acute Chagasic cardiomyopathy.

Cardiotrophin-1 (CT-1), a cytokine with structural similarities to interleukin-6, has been shown to signal through gp130-dependent pathways. In vitro, CT-1 promotes the survival and induces hypertrophy of neonatal cardiomyocytes. Since acute Chagas' disease involves an inflammatory response followed by chamber dilation, with subsequent compensatory hypertrophy, we hypothesized CT-1 and gp130 may participate in this disease process. Thus, we investigated expression and localization of these moieties during acute Chagasic cardiomyopathy. Lewis rats (n = 6/group) were either inoculated with cell culture-derived T. cruzi trypomastigotes or saline, and sacrificed 15 days later. Hearts were collected for histology, immunohistochemistry (IHC), mRNA, and protein analyses. Histology showed dense myocardial infection with amastigotes and diffuse mononuclear cell infiltrate. Northern blot analysis showed low level expression of CT-1 mRNA in controls, which was markedly elevated in infected animals (2.5-fold; P < 0.001). Similarly, Western blotting showed a twofold elevation of CT-1 protein in infected animals (P < 0.025). Likewise, levels of both gp130 mRNA and protein were low in controls, but were approximately threefold higher in infected animals. IHC showed weak and diffuse staining for CT-1 in control myocardium, while intense staining especially localized to the cytoplasmic region of cardiomyocytes, was found in infected animals. Although gp130 immunoreactivity was observed in both normal and infected myocardium, more intense staining was found in infected animals. Unlike CT-1, gp130 staining was granular, and was present in both the cytoplasm as well as in the perinuclear region. These data suggest that there is substantial overexpression of both CT-1 and gp130 in the heart during acute Chagasic carditis. Their overexpression may provide a mechanism for myocyte protection, and for development of compensatory cardiac hypertrophy following myocardial damage in this form of cardiomyopathy.

Leishmaniasis in Texas: epidemiology and clinical aspects of human cases.

Twenty-seven autochthonous cases of cutaneous leishmaniasis in Texas were identified by contact with dermatologists and State Health Department officials, and by a review of medical records, pathology reports, and previously published case reports. Fifteen cases were previously unreported. Although the date of onset of the first recognized case was 1903, in 20 of the cases the date of onset of the lesion(s) was in 1980-1989. Twelve cases were female; 15 were male. Age at diagnosis ranged from two to 86 (median 37) years. The disease was identified significantly more frequently in younger males and older females. The distribution of cases closely followed the distribution of Neotoma micropus, a rodent host for Leishmania mexicana. The most common risk factor appeared to be residence or activity in close proximity to woodrat habitat. Two cases lived in central Texas; the remainder had a residence in, or history of travel to, southern Texas. A majority of cases were first recognized during the cooler months of the year. Most lesions began as papules or nodules that subsequently ulcerated. In 20 cases, a single lesion was present. Five cases had resolution of their lesions without receiving specific anti-leishmanial therapy; lesions of 17 resolved after treatment with a variety of therapies. One life-long case of disseminated disease failed to respond to treatment, and four cases were lost to follow-up. A Leishmania-specific lymphocyte proliferation assay gave a positive response for four of five cases tested. Screening of 13 family members found no evidence of subclinical infection. These 27 cases, and two recently recognized cases reported in a note added in proof, indicate that cutaneous leishmaniasis may be more common in Texas than previously thought.

Analysis of the peripheral immune response in patients with neurocysticercosis: evidence for T cell reactivity to parasite glycoprotein and vesicular fluid antigens.

In neurocysticercosis (NCC), it is thought that the long-term survival of the parasite within the human brain is due in part to the ability of the cestode to suppress the local immune response. When the parasite dies, the immunosuppression is apparently lost and a strong local inflammatory response then develops. In contrast, little is known about the immunologic response that may occur in the peripheral immune system of these patients. In this study, the status of the peripheral (extracerebral) cellular and humoral response was evaluated in patients with a history of NCC. The in vitro proliferation of peripheral blood mononuclear cells to mitogens and foreign antigens was similar in patients and controls. Importantly, a substantive response was elicited by two Taenia solium metacestode antigens. In addition, 8 of 10 patients had a detectable humoral response to the antigenic glycoproteins of the cestode. Considering both the cellular and humoral response, all of the patients with NCC presented an active peripheral immunity.

In situ expression of interleukin-10 and interleukin-12 in active human cutaneous leishmaniasis.

Th1-type cellular immune responses (interferon-gamma) play a critical role in protection against Leishmania spp. infection, whereas Th2-type cytokines (interleukin (IL)-4, IL-10) have a counter-protective effect. IL-12, a potent inducer of Th1-type cellular immune responses, may play a pivotal role in the development of a protective response. We found that IL-10 and IL-12 mRNAs were expressed in most lesions of individuals with active cutaneous leishmaniasis. The quantity of IL-12 mRNA was highly variable but correlated strongly with the level of interferon-gamma expression. IL-12 expression also paralleled the expression of IL-10, a potent in vitro suppressor of IL-12 and interferon-gamma production. The more chronic, non-healing lesions generally had higher levels of IL-12 mRNA indicating that the expression of this cytokine alone was not sufficient to induce healing. Although the in situ production of IL-10 did not appear to block IL-12 expression, IL-10 may still promote disease by direct suppression of macrophage activation.

Induction of macrophage killing of Leishmania donovani by human CD4+ T cell clones.

Immunity in leishmaniasis is mediated by T cells, but protective responses in humans have not been fully defined. In this study, the functional activity of CD4+ T cell clones derived from an immune individual was investigated to identify potentially protective responses. The T cells proliferated and produced interferon-gamma (IFN-gamma) in response to a soluble Leishmania donovani antigen extract and live amastigotes. There was considerable variation in the anti-leishmanial activity of the T cell clones when they were co-cultured with L. donovani infected monocytes isolated from an HLA-DR,DQ matched donor. All of the clones which demonstrated antigen specific reactivity by proliferation or cytokine production induced some degree of inhibition of intracellular parasite replication, but only a few of the clones induced pronounced leishmanicidal activity. There was strong correlation between the level of amastigote-induced IFN-gamma secretion and anti-leishmanial activity. This approach enables the identification of potentially protective immune responses in humans at the clonal level, and offers a means for the identification of the relevant antigen(s).

Malnutrition promotes prostaglandin over leukotriene production and dysregulates eicosanoid-cytokine crosstalk in activated resident macrophages.

We previously described a murine model of malnutrition that mimicked features of moderate human malnutrition, and led to increased dissemination of Leishmania donovani. In this study, we investigated the effect of malnutrition on macrophage production of cytokines, prostaglandins (PGs), and leukotrienes (LTs). Using either LPS or calcium ionophore A23187 as a stimulus, macrophages from the malnourished mice produced a 3-fold higher PG/LT ((PGE(2)+6-keto-PGF(1alpha))/(LTB(4)+cysteinyl leukotrienes)) ratio than macrophages from well-nourished mice. LPS-stimulated macrophages from the malnourished mice produced decreased levels of TNF-alpha, GM-CSF, and IL-10, but similar levels of IL-6 and NO compared to well-nourished mice. A complex crosstalk between the eicosanoids and cytokines in the LPS-stimulated macrophages from the malnourished mice was evident by the following: (1) high levels of PG secretion despite low levels of TNF-alpha; (2) supplemental IL-10 modulated the excessive PG production; (3) GM-CSF rectified the PG/LT ratio, but did not correct the abnormal cytokine profile; and (4) inhibitors of cyclooxygenase decreased the PG/LT ratio, but did not affect TNF-alpha. Thus, in this model of malnutrition, there is a relative increase in anti-inflammatory PGs compared to pro-inflammatory LTs, which may contribute to immunodeficiency.

Experimental leishmaniasis in humans: review.

Experimental infection of humans with Leishmania parasites has contributed significantly to the understanding of the etiology, transmission, and pathogenesis of leishmaniasis and the immunity associated with it. Leishmania organisms recovered from human and animal tissue, insect vectors, and in vitro cultures have all produced cutaneous or visceral leishmaniasis in human subjects who were voluntarily inoculated with them. Volunteers bitten by infected Phlebotomine sandflies also developed cutaneous or visceral disease. In these experiments, it appeared that the parasite must undergo certain developmental changes within the sandfly for it to become infective and that the parasites in sandflies were far more efficient in causing full-blown infection than were cultured Leishmania organisms. The clinical manifestations of these experimental infections did not differ from infections that were acquired naturally. Natural or experimental infections appeared to confer resistance to subsequent leishmanial infection. This immunity was best documented to be a species-specific phenomenon; however, a small number of studies have demonstrated cross protection between some Leishmania species. In this review article, data from human experimental infections are summarized and discussed in light of recent advances in the field.

Identification of antigens recognized by T cells in human leishmaniasis: analysis of T-cell clones by immunoblotting.

Immunity in human leishmaniasis is mediated by sensitized T lymphocytes; however, the antigens involved in eliciting this immunity have not been defined. We describe the generation of human T-lymphocyte clones derived from two patients with healed leishmaniasis. By use of one- and two-dimensional cellular immunoblotting techniques, we directly identified the parasite antigens recognized by these clones. To our knowledge, these are the first leishmanial antigens identified to which CD4+, gamma interferon-producing T cells from immune individuals have been shown to respond, the strategy may be of general use for the identification of antigens involved in immunity in this disease.

Animal models for the analysis of immune responses to leishmaniasis.

This unit focuses on the murine model of cutaneous leishmaniasis and models of visceral leishmaniasis in mice and hamsters. Each basic protocol describes the methods used to inoculate parasites and to evaluate infections with regard to lesion progression and visceralization, and quantification of parasite load. Five support protocols are provided; two for the growth and isolation of metacyclic promastigotes from in vitro culture, one for isolation of tissue amastigotes from infected animals, one for cryopreservation of parasites, and one for the preparation of blood agar plates for quantitation of parasite numbers in infected tissue.

Adaptation to the edge of chaos in the self-adjusting logistic map.

Self-adjusting, or adaptive, systems have gathered much recent interest. We present a model for self-adjusting systems which treats the control parameters of the system as slowly varying, rather than constant. The dynamics of these parameters is governed by a low-pass filtered feedback from the dynamical variables of the system. We apply this model to the logistic map and examine the behavior of the control parameter. We find that the parameter leaves the chaotic regime. We observe a high probability of finding the parameter at the boundary between periodicity and chaos. We therefore find that this system exhibits adaptation to the edge of chaos.

Lack of serological specificity of recombinant heat shock protein of Leishmania donovani.

In order to identify a specific recombinant antigen of Leishmania donovani with potential use for diagnosis, a cDNA library was constructed in lambda ZAP II expression vector. On screening the cDNA library using pooled sera from Indian patients with kala azar, 20 antibody reactive clones were identified. These were subcloned into pBluescript phagemid by an in vivo excision procedure. The molecular weights of the expressed recombinant proteins varied from 15 to 70 kDa and the cDNA insert sizes varied from 0.5 kb to the largest size of approximately 2.0 kb which was designated as the E2b clone. The nucleotide sequencing revealed that 50% of the clones had sequence homology to the heat shock protein gene of L. donovani. The serological studies conducted with the kala azar positive sera and sera from healthy laboratory workers using the recombinant protein from the E2b clone and having sequence homology to Ldhsp 70, indicated that although all the kala azar sera was positive, 12 of 20 healthy individuals also showed antibodies against the recombinant hsp70, indicating that this antigen is not suitable for serological diagnosis of kala azar.

Differential regulation of nitric oxide synthase isoforms in experimental acute chagasic cardiomyopathy.

We have previously demonstrated induction and high level expression of IL-1beta, IL-6 and tumour necrosis factor-alpha in the myocardium during the acute stage of experimental Trypanosoma cruzi infection (Chagas' disease). The myocardial depressive effects of these cytokines are mediated in part by the induction of nitric oxide synthase (NOS), production of nitric oxide (NO) and formation of peroxynitrite. In this study we investigated the expression, activity and localization of NOS isoforms, and the levels of NO, malondialdehyde (a measure of oxidative stress), and peroxynitrite in rats at 1.5, 5, 10 and 15 days after infection with T. cruzi trypomastigotes. The myocardial inflammatory infiltrate and number of amastigote nests increased over the course of infection. A significant increase in tissue nitrate + nitrite levels, NOS2 mRNA, and NOS2 enzyme activity was observed at all time points in the infected compared with uninfected animals. The enzyme activity of constitutive NOS, tissue malondialdehyde levels, and NOS3 mRNA levels was only transiently increased after infection. The protein levels of the NOS isoforms paralleled their mRNA expression. While no positive nitrotyrosine immunoreactivity was detected in control myocardium, its levels increased in infected animals over time. Thus, by 1.5 days post-infection, when no parasite or immune cell infiltration could be detected, the myocardium expressed high levels of NOS and NO metabolites. Nevertheless, the early production of NO in the myocardium was not sufficient to clear the parasites.

T-cell responses to infected autologous monocytes in patients with cutaneous and mucocutaneous leishmaniasis.

Although there is strong evidence that the control and resolution of human leishmanial infections depend primarily on activation of parasite-infected macrophages mediated by lymphokines derived from T cells, less is known about the nature of the responding cell type(s) which is protective or the antigen(s) (Ag[s]) that elicits these cells to respond. Studies using preparations of whole soluble Ag ("dead Ag") show that patients respond to a wide range of leishmanial Ags. The objective of the present study was to characterize the response of T cells from patients with healing or healed cutaneous or mucosal infections to Ag expressed by or derived from actively infected autologous monocytes ("live Ag"). Unfractionated T cells proliferated and produced gamma interferon in response to both live and dead Ags. Depletion of CD4+ T cells resulted in the loss of proliferative and gamma interferon responses to both live and dead Ags. The effect of CD8 depletion, although variable and not limited to the cells stimulated by infected monocytes, was clear for some patients. Expansion of T cells specific for live Ags by using amastigote-infected cells followed by restimulation with fast-protein liquid chromatography-fractionated soluble Ags revealed that a diversity of Ags are associated with infected monocytes. There may, however, be quantitative differences in the expression of certain Ags since prestimulation with live Ag induced higher responses to restimulation in mucocutaneous leishmaniasis patients than in localized cutaneous leishmaniasis patients. Prestimulation with dead Ag induced similar secondary responses in both patient groups.