Accelerated control of visceral Leishmania donovani infection in interleukin-6-deficient mice

In patients with visceral leishmaniasis, increased levels of circulating interleukin-6 (IL-6) regularly accompany fully expressed, progressive infections (kala-azar). To experimentally test the role of IL-6, responses to an intracellular Leishmania donovani infection in the livers of IL-6(-/-) and wild-type mice were compared. IL-6(-/-) mice showed an enhanced control of the infection and earlier, rapid parasite killing along with additional evidence of a stimulated antileishmanial Th1-cell-type response: increased levels of circulating gamma interferon, accelerated granuloma assembly, and heightened responsiveness to chemotherapy. In this model of visceral leishmaniasis, IL-6 appears to act in a suppressive, macrophage-deactivating fashion, which identifies it as a potential target for therapeutic blockade.     

Identification and characterization of a ribose transport system in Leishmania donovani promastigotes

A transport system for ribose in Leishmania donovani promastigotes was identified and characterized by measuring the uptake of radioisotope-labeled ribose. The pentoses arabinose, 2-deoxyribose and xylose inhibited ribose uptake, whereas hexoses (glucose, alpha-methylglucoside, thioglucose, galactose, lactose, maltose, mannose), adenosine, and proline did not inhibit uptake, indicating that the transporter exhibited substrate specificity. Intracellular ribose exchanged with 2-deoxyribose. Uptake of ribose showed saturation kinetics with an apparent Km = 2 mM and Vmax = 11 nmol (mg protein)-1 min-1. Both N-ethylmaleimide and p-hydroxymercuribenzoate inhibited ribose uptake which was prevented by dithiothreitol. The uncoupling agents 2,4-dinitrophenol and carbonylcyanide p-(trifluoromethoxy)phenylhydrazone and a variety of inhibitors of energy-driven transport had no significant effect on ribose uptake. Following transport, the intracellular ribose pool contained two-thirds of the sugar in the phosphorylated form and one-third in the neutral form. These cumulative results indicate that a specific carrier mediates ribose uptake via a facilitated diffusion system in L. donovani promastigotes.     

Identification of an immunodominant 32-kilodalton membrane protein of Leishmania donovani infantum promastigotes suitable for specific diagnosis of Mediterranean visceral leishmaniasis.

Sera from 35 patients suffering from Mediterranean visceral leishmaniasis (caused by Leishmania donovani infantum) and 59 patients with various forms of cutaneous leishmaniasis prevalent in the sub-Mediterranean countries (caused by Leishmania major, L. donovani infantum, or Leishmania tropica) were tested by immunoblotting and enzyme-linked immunosorbent assay (ELISA) with both membrane and soluble antigens prepared from L. donovani infantum parasites. Control sera were from healthy children (n = 41), adults with nonleishmanial diseases (n = 40), and patients with Chagas' disease (n = 12). A P32 antigen present in the membrane preparation from L. donovani infantum parasites was recognized by 95% of serum specimens from patients with Mediterranean visceral leishmaniasis but not by serum specimens from patients with cutaneous leishmaniasis or sera from control individuals. An ELISA with electroeluted P32 antigen was found to have a specificity and sensitivity of 94% in the serodiagnosis of Mediterranean visceral leishmaniasis. Healthy children with asymptomatic Leishmania infection were seronegative for the P32 antigen by ELISA. These results suggest that antibodies to P32 antigen develop only in patients with visceral leishmaniasis and that the P32 ELISA may be useful in areas where the disease is endemic for discriminating between patients with this disease and those with other clinical conditions.     

IMP dehydrogenase deficiency in Leishmania donovani causes a restrictive growth phenotype in promastigotes but is not essential for infection in mice

Leishmania cannot synthesize purines de novo and therefore must scavenge purines from its host for survival and growth. Biochemical and genomic analyses have indicated that Leishmania species express three potential routes for the synthesis of guanylate nucleotides: (1) a two-step pathway that converts IMP to GMP; (2) a three-step pathway that starts with the deamination of guanine to xanthine, followed by phosphoribosylation to XMP and then conversion to GMP; or (3) direct guanine phosphoribosylation by HGPRT. To determine the role of the first of these pathways to guanylate nucleotide synthesis, an L. donovani line deficient in IMP dehydrogenase (IMPDH), the first step in the IMP to GMP pathway, was constructed by targeted gene replacement. The Δimpdh lesion triggered a highly restrictive growth phenotype in promastigotes in culture but did not impact parasitemias in mice. The dispensability of IMPDH in vivo is the first definitive demonstration that intracellular L. donovani amastigotes have access to a sufficient pool of guanine, xanthine, or guanylate precursors from the host.     

Susceptibilities of macrophage populations to infection in vitro by Leishmania donovani.

Many studies have demonstrated differences in the resistance of strains of mice to infection by Leishmania donovani, Salmonella typhimurium, and Mycobacterium bovis BCG; this resistance/susceptibility phenotype seems to be controlled by a single gene. The present study investigated the susceptibility of liver, lung, peritoneal, and spleen macrophages to infection by L. donovani promastigotes in vitro; the objective was to determine if the susceptibility of animals was expressed by their macrophages when infected in vitro. This study indicated that the Lsh phenotype was only expressed by liver macrophages. The liver macrophages of the susceptible C57BL/6J strain were significantly more phagocytic than those of the resistant C57L/J strain; infection affected the phagocytic activity of the macrophage population. These results indicated that only liver macrophages can express the Lsh gene. Recognition of expression is in part due to its effect on the phagocytic activity of the macrophages.     

Failure of hamster macrophages to discriminate between infective and noninfective promastigotes of Leishmania donovani during attachment in vitro.

The attachment of infective and noninfective promastigotes of Leishmania donovani to hamster macrophages derived from spleen, lymph nodes, and peritoneal exudate was studied in vitro. Regardless of macrophage origin, no significant differences were observed between attachment of promastigotes that were infective and those that were noninfective for hamsters.     

Immunoblot analysis of the humoral immune response to Leishmania donovani infantum polypeptides in human visceral leishmaniasis.

Using the immunoblot technique, we have compared the reactions of Leishmania donovani infantum polypeptides with the immunoglobulin G of human sera from patients with parasitologically proven L. d. infantum infection, with suspected visceral leishmaniasis, and with other leishmaniases, protozoiases, helminthiases, and fungal or bacterial diseases. A 94-kDa component reacted with all L. d. infantum-infected sera and with 75% of sera from patients with clinical and serological but no parasitological diagnoses. No reaction was observed with sera from patients in the other disease groups or with control sera. Studies of eight different isolates, subspecies, and species of the genus Leishmania demonstrated that the 94-kDa component was expressed in all strains examined.     

Neutrophils contribute to development of a protective immune response during onset of infection with Leishmania donovani

Neutrophils are key components of the inflammatory response and as such contribute to the killing of microorganisms. In addition, recent evidence suggests their involvement in the development of the immune response. The role of neutrophils during the first weeks post-infection with Leishmania donovani was investigated in this study. When L. donovani-infected mice were selectively depleted of neutrophils with the NIMP-R14 monoclonal antibody, a significant increase in parasite numbers was observed in the spleen and bone marrow and to a lesser extent in the liver. Increased susceptibility was associated with enhanced splenomegally, a delay in the maturation of hepatic granulomas, and a decrease in inducible nitric oxide synthase expression within granulomas. In the spleen, neutrophil depletion was associated with a significant increase in interleukin 4 (IL-4) and IL-10 levels and reduced gamma interferon secretion by CD4⁺ and CD8⁺ T cells. Increased production of serum IL-4 and IL-10 and higher levels of Leishmania-specific immunoglobulin G1 (IgG1) versus IgG2a revealed the preferential induction of Th2 responses in neutrophil-depleted mice. Altogether, these data suggest a critical role for neutrophils in the early protective response against L. donovani, both as effector cells involved in the killing of the parasites and as significant players influencing the development of a protective Th1 immune response.     

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.     

Multiple host defense defects in failure of C57BL/6 ep/ep (pale ear) mice to resolve visceral Leishmania donovani infection.

Euthymic C57BL/L ep/ep (pale ear [PE]) mice halt the visceral replication of intracellular Leishmania donovani but fail to properly resolve infection. A previous study identified an isolated defect in tissue granuloma formation in these mice; CD4+ and CD8+ cell number, gamma interferon (IFN-gamma) production, and macrophage antimicrobial activity in vitro were all intact. New in vivo results reported here suggest a considerably more complex immune defect, with evidence indicating (i) enhanced control over L. donovani after transfer of normal C57BL/6 spleen cells, (ii) a partially suppressive Th2 cell-associated response mediated by interleukin-4 (IL-4) but not reversed by CD4+ cell depletion, (iii) absent responses to endogenous Th1 cell lymphokines (IFN-gamma and IL-2) but preserved responsiveness to endogenous tumor necrosis factor alpha, (iv) absent responses to exogenous treatment with recognized antileishmanial cytokines (IFN-gamma, IL-2, IL-12, and granulocyte-macrophage colony-stimulating factor [GM-CSF]) not corrected by transfer of C57BL/6 spleen cells, and (v) a deficient response to antimony chemotherapy. Defective hepatic granuloma formation was not corrected by transfer of C57BL/6 spleen cells or by anti-IL-4 administration. While treatment with IL-2 and GM-CSF modified the tissue reaction and induced selected effector cells to encase tissue macrophages, no antileishmanial activity resulted. Together, these observations suggest that the failure of PE mice to resolve visceral L. donovani infection likely represents expression of multiple suboptimal immune responses and/or partial defects, probably involving a combination of T-cell dysfunction, a Th2 cell response, and target cell (macrophage) hyporesponsiveness.     

Transfer of innate resistance and susceptibility to Leishmania donovani infection in mouse radiation bone marrow chimaeras.

Reciprocal radiation bone marrow chimaeras were made between H-2-compatible strains of mice innately resistant or susceptible to visceral leishmaniasis. In initial experiments, susceptibility but not resistance to Leishmania donovani could be transferred with donor bone marrow into irradiated recipients. In subsequent experiments it was possible to transfer both resistance and susceptibility. This was achieved either by selecting more radiosensitive mouse strains as susceptible recipients, or alternatively by increasing the irradiation dose for the susceptible recipients used in the initial experiments. Using the higher irradiation dose, successful transfer of resistance and susceptibility between congenic mice carrying the Lshr and Lshs alleles on the more radioresistant B10 genetic background provided firm evidence that the results obtained in this study were specifically related to expression of the Lsh gene. We conclude that Lsh gene-controlled resistance and susceptibility to L. donovani is determined by bone marrow-derived cells. The cell type(s) involved is likely to be of the macrophage lineage.     

Immunoblot analysis of the humoral immune response to Leishmania donovani polypeptides in cases of human visceral leishmaniasis: its usefulness in prognosis

Sera from Indian patients with parasitologically confirmed visceral leishmaniasis were studied by immunoblot analysis in order to identify a specific pattern for Leishmania infection. A soluble extract of Leishmania donovani was used as antigen. At diagnosis the sera from patients with visceral leishmaniasis specifically recognized fractions represented by bands of 201 kDa (50% of serum samples), 193 kDa (60%), 147 kDa (50%), 120 kDa (60%), 100 kDa (50%), 80 kDa (80%), 70 kDa (70%), 65 kDa (100%), 50 kDa (50%), 36 kDa (50%), 20 kDa (70%), and 18 kDa (50%). The 65-kDa band, common to all patients infected with Leishmania parasites, was found at the time of diagnosis. However, the immunoblot pattern changed after patients were treated and cured with sodium antimony gluconate (SAG; n =10) or miltefosine (n =10), as was evident from blots of sera obtained pretreatment and at 1, 3, and 6 months posttreatment. At 6 months posttreatment, immunoblots of sera from patients on the SAG regimen showed the disappearance of all bands except the 70-kDa band. Similarly, sera from those on the miltefosine regimen showed the disappearance of all bands except the 65- and 70-kDa bands. This study shows that Western blot analysis is a sensitive test for detection of anti-Leishmania antibodies. Moreover, the persistence of reactivity with the 65- and 70-kDa bands in the sera of all groups shows its promise as a diagnostic and prognostic tool.     

Previous exposure to a low infectious dose of Leishmania major exacerbates infection with Leishmania infantum in the susceptible BALB/c mouse

The geographic distribution of Leishmania major overlaps with several other species of Leishmania. This study seeks to examine what effect previous exposure to L. major has on the outcome of infection with Leishmania infantum, the agent of virulent visceral leishmaniasis. The L. major immune response is well characterized by a strong Th1 response leading to resolution and protection against subsequent re-infection. A contrasting Th2 immune response leads to disseminated disease, while the role Th17 cytokines may play in Leishmania infection is still being explored. The cytokine profile, antibody titer, and parasite burden were evaluated in the susceptible BALB/c mouse after L. infantum infection in either naïve mice or those previously infected with a low/self-healing dose of L. major. Only IL-4 expression in mice previously exposed to L. major was found to be significantly increased over controls, a cytokine with an ambiguous role in L. infantum infection. However, disease exacerbation, with a notably higher parasite burden, was observed in the L. major exposed mice compared to the L. infantum only. Cross-reactive antibodies were seen in both groups of infected mice regardless of their immune history. Studies have shown a role for opsonizing antibodies leading to increased disease in visceral leishmaniasis. We speculate that cross-reactive antibodies may be playing a role in augmenting visceral disease in mice with immunological memory to L. major.     

Leishmania donovani infection of a susceptible host results in CD4+ T-cell apoptosis and decreased Th1 cytokine production

The disease visceral leishmaniasis is caused by a protozoan parasite, Leishmania donovani and is characterized by depressed cell-mediated immunity (CMI) and unhindered parasite growth in a susceptible host. The opposite trend is observed in a resistant host. However, the mechanism of this loss of CMI during the progressive disease is unknown as yet. In this report, we demonstrate that more than 40% of CD4+ T cells from a susceptible host undergo apoptosis resulting in a significant decrease in interleukin (IL)-2 and interferon (IFN)-gamma secretion, leaving IL-4 secretion unaffected. These changes are not apparent in the case of CD4+ T cells derived from a resistant host. The data reported here suggest that experimental Leishmania donovani infection leads to selective deletion of the IL-2 and IFN-gamma-secreting cells but not Th2-like cells in a susceptible but not a resistant host.     

Immunoregulation of genetically controlled acquired responses to Leishmania donovani infection in mice: demonstration and characterization of suppressor T cells in noncure mice

On a B10 genetic background, genes in the I region of H-2 influence the development of acquired T-cell mediated immunity to Leishmania donovani infection in mice. In previous studies, noncure in H-2d mice could be abrogated by pretreatments with cyclophosphamide or sublethal irradiation. The prophylactic effect of these pretreatments was consistent with deletion of the precursors of suppressor T cells suppressing T-cell-mediated immune responses. In this study, cell transfer experiments provide direct evidence for the role of suppressor T cells in the noncure response. T-cell-enriched populations isolated from the spleens of B10.D2/n mice infected 30, 61, or 85 days previously reversed the prophylactic effect of sublethal irradiation when injected before infection into B10.D2/n mice that had received 550 rads. B-cell-enriched populations failed to transfer suppression in this manner, and T-cell-enriched populations from the spleens of normal B10.D2/n mice had only a transient effect on liver parasite loads. Transfer of suppression with the T-cell-enriched populations from infected donors was abrogated by pretreatment with anti-Thy-1.2 and anti-Lyt-1.2 antisera plus complement but not by pretreatment with anti-Lyt-2.2 plus complement, indicating that the suppressor T cell involved has an Lyt-1+2- surface phenotype. Results are discussed in relation to the possible mechanism of H-2-linked control.     

不同程度脊髓背侧压迫损伤模型大鼠脊髓损伤致伤器的设计

背景：随着对脊髓损伤研究的不断深入,国内外学者不断设计出各种脊髓损伤模型,主要包括脊髓挫伤、重物坠击、脊髓压迫、化学灼伤、放射性损伤及激素横断半横断损伤等,然而各种制备方法都存在一定的局限。 目的：设计脊髓损伤致伤器并建立不同程度脊髓损伤动物模型。 方法：实验自行设计包括致伤部件及传动装置的脊髓损伤致伤器,利用不同致伤质量m1=10 g,m2=20 g,m3=30 g及时间T1=3 s,T2=5 s,造成不同质量×时间暨不同程度SD大鼠脊髓背侧压迫损伤,分为m1T1,m2T1,m3T1,m1T2,m2T2,m3T2共6组,并设假手术组进行对照。 结果与结论：造模后各实验组BBB评分均低于假手术组(P < 0.01),m1T1组与其他各实验组相比差异有显著性意义(P < 0.01),建模后8周m1T2组BBB评分高于与m2T2组及m3T2组(P < 0.05)。建模后8周各组体感诱发电位和运动诱发电位潜伏期明显长于假手术组(P < 0.01)。除了m1T1组与m1T2组外,其他各组间建模后体感诱发电位潜伏期均明显延长(P < 0.05)。所有组建模后运动诱发电位潜伏期均明显延长(P < 0.05)。结果证实,实验所设计的脊髓损伤致伤器可制备出不同程度的脊髓背侧压迫损伤动物模型。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程全文链接：     

The capacity to produce IFN-gamma rather than the presence of interleukin-4 determines the resistance and the degree of susceptibility to Leishmania donovani infection in mice.

The immune response against Leishmania donovani infection has been investigated in one resistant mouse strain (C3H/HeJ) and three susceptible mouse strains (C57BL/6, BALB/c, and B10D2/n). In order to correlate the strain-specific course of infection with the individual T cell response phenotype, the ex vivo cytokine secretion patterns of splenic lymphocytes were assessed by ELISA (interferon-y [IFN-gamma], interleukin-4 [IL-4], IL-10) or by bioassay (IL-2). The strain-dependent differences in the course of infection correlated closely with the potency of T cells to produce IFN-gamma. C3H/HeJ mice produced high amounts of IFN-gamma before and during infection, whereas susceptible mice produced low amounts of IFN-gamma early during L. donovani infection. However, C57BL/6 mice, which recovered from the infection rapidly after the acute stage, developed marked IFN-gamma response within the first 30 days of infection. In contrast, in BALB/c and B10D2/n mice, the IFN-gamma production diminished during the acute stage, and this was associated with a delay in recovery and with subsequent switching into the chronic stage. Interestingly, CD8+ T cells contributed significantly to IFN-gamma production during this phase. In contrast to IFN-y, the levels of IL-4 in response to antigen or mitogen ex vivo were always very low. Moreover, neutralization of endogenous IL-4 in vivo by treatment with soluble murine IL-4 receptor did not result in significant decreases in the parasite burdens in spleen and liver but did cause a decrease in the serum IgE level of L. donovani-infected BALB/c mice. These results confirm that in visceral leishmaniasis a Thl-dominated immune response is protective against the L. donovani parasites and, furthermore, that the capacity to produce IFN-gamma rather than the presence of IL-4 determines the efficacy of the immune response in susceptible mice. The data show that CD8+ T cells represent an important source of IFN-gamma during L. donovani infection in susceptible mice, implying a role for this cell type in healing and development of protective immunity.