Transgenic Expression of CXCR3 on T Cells Enhances Susceptibility to Cutaneous Leishmania major Infection by Inhibiting Monocyte Maturation and Promoting a Th2 Response

Cutaneous leishmaniasis, caused mainly by Leishmania major, an obligate intracellular parasite, is a disfiguring disease characterized by large skin lesions and is transmitted by a sand fly vector. We previously showed that the chemokine receptor CXCR3 plays a critical role in mediating resistance to cutaneous leishmaniasis caused by Leishmania major. Furthermore, T cells from L. major-susceptible BALB/c but not L. major-resistant C57BL/6 mice fail to efficiently upregulate CXCR3 upon activation. We therefore examined whether transgenic expression of CXCR3 on T cells would enhance resistance to L. major infection in susceptible BALB/c mice. We generated BALB/c and C57BL/6 transgenic mice, which constitutively overexpressed CXCR3 under a CD2 promoter, and then examined the outcomes with L. major infection. Contrary to our hypothesis, transgenic expression of CXCR3 (CXCR3^Tg) on T cells of BALB/c mice resulted in increased lesion sizes and parasite burdens compared to wild-type (WT) littermates after L. major infection. Restimulated lymph node cells from L. major-infected BALB/c-CXCR3^Tg mice produced more interleukin-4 (IL-4) and IL-10 and less gamma interferon (IFN-γ). Cells in draining lymph nodes from BALB/c-CXCR3^Tg mice showed enhanced Th2 and reduced Th1 cell accumulation associated with increased neutrophils and inflammatory monocytes. However, monocytes displayed an immature phenotype which correlated with increased parasite burdens. Interestingly, transgenic expression of CXCR3 on T cells did not impact the outcome of L. major infection in C57BL/6 mice, which mounted a predominantly Th1 response and spontaneously resolved their infection similar to WT littermates. Our findings demonstrate that transgenic expression of CXCR3 on T cells increases susceptibility of BALB/c mice to L. major.     

Immunomodulatory Properties of Borage (<Emphasis Type="BoldItalic">Echium amoenum</Emphasis>) on BALB/c Mice Infected with <Emphasis Type="BoldItalic">Leishmania major</Emphasis>

Leishmaniasis is caused by parasitic protozoa transmitted by the bite of a female sand fly and is currently endemic in 88 countries. BALB/c mice are highly susceptible to the infection with the parasite Leishmania major, and this susceptibility has been attributed, in part, to the expansion of Th2 cells, production of their cytokines, and downregulation of Th1 cytokine, interferon gamma (IFN-γ). In this report, we used both aqueous and alcoholic extracts of Iranian borage (Echium amoenum Fisch & C.A. Mey) for treatment of L. major infection in BALB/c mice. We found that both extracts had immunomodulatory properties and increased the level of IFN-γ and lowered the parasite burden in the proximal lymph nodes and prevented the necrosis of the footpad as compared with the untreated infected mice. These results may provide a basis for further studies directed toward the use of the Iranian borage against L. major infection.     

Early parasite containment is decisive for resistance to Leishmania major infection

We investigated the early spread of Leishmania major in various mouse strains. In BALB/c mice, which are extremely vulnerable to L. major infection, the parasites disseminated within 10-24 h from the site of subcutaneous footpad infection in to the popliteal lymph node, spleen, lung, liver and bone marrow. Application of recombinant (r)IL-12 prior to infection prevented the early dissemination of parasites into visceral organs and the animals healed the infection. In three mouse strains tested, C57BL/6, CBA/J and C3H/HeJ, which are all resistant to L. major infection, the parasites remained localized in the footpad and in the draining LN for 3 days without evidence of dissemination. In C57BL/6 mice, depletion of NK1.1⁺ cells or neutralization of interferon (IFN)-γ prior to infection led to rapid parasite spreading with kinetics similar to those seen in susceptible animals. Depletion of either CD4⁺ or CD8⁺ T cells in vivo prior to infection did not alter the kinetics of dissemination in any mouse strain tested. Experiments with severe-combined immunodeficient mice provided further evidence that parasite containment depends on natural killer cells and IFN-γ, but is independent of T cells. The finding that all resistant mouse strains restrict the spread of the parasites within the first 24 h after infection strongly suggests that early parasite containment is closely associated with a resistant phenotype. The data show that local restriction of parasites in the pre-T cell phase of the infection is mediated by the innate immune system and suggest that this function plays an important role in the development of a protective T cell response.     

The protective Th1 response in mice is induced in the T-cell zone only three weeks after infection with <Emphasis Type="Italic">Leishmania major</Emphasis> and not during early T-cell activation

The protozoan parasite Leishmania spp. causes clinical pictures ranging in severity from spontaneously healing skin ulcers to systemic disease. The immune response associated with healing involves the differentiation of IFNγ-producing Th1 cells, whereas the non-healing phenotype is associated with IL4-producing Th2 cells. The widespread assumption has been that the T-cell differentiation that leads to a healing or non-healing phenotype is established at the time of T-cell activation early after infection. By selectively analyzing the expression of cytokine genes in the T-cell zones of lymph nodes of resistant (Th1) C57BL/6 mice and susceptible (Th2) BALB/c mice during an infection with Leishmania major in vivo, we show that the early T-cell response does not differ between C57BL/6 mice and BALB/c mice. Instead, Th1/Th2 polarization appears suddenly 3 weeks after infection. At the same time point, the number of parasites increases in lymph nodes of both mouse strains, but about 100-fold more in susceptible BALB/c mice. We conclude that the protective Th1 response in C57BL/6 mice is facilitated by the capacity of their innate effector cells to keep parasite numbers at low levels.     

Different patterns of the L-histidine decarboxylase (HDC) gene expression in mice resistant and susceptible to experimental cutaneous leishmaniasis

Objective and design: In the present study the experimental murine Leishmania major (L. major) infection model was used to investigate the role of histamine biosynthesis in cutaneous leishmaniasis.Subjects, treatment and methods: A novel RNase Protection Assay (RPA) was developed and applied for the assessment of L-histidine decarboxylase (HDC) gene expression in organs of resistant C57BL/6 and susceptible BALB/c mice after infection with L. major.Results: In the acute phase of infection a rapid but transient induction of HDC expression was observed in the infected lymph nodes of both strains correlating both temporally and spatially with parasite spread. The signal was present in the draining popliteal lymph nodes of both hosts, however, only susceptible mice known to be unable to control parasite dissemination showed induction of HDC in their distant periaortic lymph nodes as well. During the chronic phase of infection only the heavily parasitized organs of BALB/c mice showed high HDC gene expression.Conclusions: These data suggest that expression of the histamine-producing enzyme HDC in the decisive acute phase of leishmaniasis is not coupled with development of either appropriate Th1 or inadequate Th2 responses to L. major. We hypothesize however, that during the chronic phase of infection elevated HDC levels, possibly of mast cell origin, are associated with Th2-dominated responses and serious disease development.Received 12 May 2003; returned for revision 3 July 2003; returned for final revision 11 September 2003; accepted by M. Parnham 24 September 2003     

Cytokine responses in mice infected with<Emphasis Type="Italic"> Clonorchis sinensis</Emphasis>

FVB and BALB/c mice show different morbidity, development of Clonorchis sinensis, and pathological changes following C. sinensis infection. FVB mice are susceptible and BALB/c mice are relatively more resistant to C. sinensis infection. To investigate the relationship between cytokine reaction and susceptibility to C. sinensis infection in FVB and BALB/c mice, we described both the patterns and kinetics of Th1 cytokines and Th2 cytokines in spleen cell culture. Interleukin (IL)-4 and IL-10 cytokine production in the culture supernatants of the concanavalin-A-stimulated spleen cells increased at 2–3 weeks post-infection in both strains. IL-5 production increased between 2 and 5 weeks post-infection in both strains, and reached a peak level at 2 weeks post-infection in BALB/c mice and 4 weeks post-infection in FVB mice. In contrast, gamma interferon (IFN-) production decreased between 2 and 4 weeks in both strains. IL-2 production increased slightly in BALB/c mice following infection, but was unchanged in FVB mice. IL-4 production over preinfection levels was significantly higher in FVB mice, whereas IFN-, IL-2, and IL-10 production were significantly higher in BALB/c mice. The levels of serum immunoglobulin E (IgE) and blood eosinophils in both mouse strains significantly increased between 3 and 6 weeks postinfection. Serum IgE levels were significantly higher in FVB mice than in BALB/c mice. The results of this study suggest that susceptibility to C. sinensis infection is associated with Th2 cytokine production, especially IL-4 which is predominant in relatively susceptible FVB mice.     

The induction of a protective response in Leishmania major-infected BALB/c mice with anti-CD40 mAb

A protective immune response to the intracellular parasite Leishmania major requires the development of a Th1 CD4⁺ T cell phenotype. We demonstrate herein that BALB/c mice, which normally develop a susceptible Th2 response to L. major infection, are protected when co-injected with an agonistic anti-murine CD40 mAb. Anti-CD40 mAb-mediated protection in this system was found to be T cell dependent, since it was not observed in C57BL/ 6 × 129 mice that were rendered T cell deficient (TCR β^–/– × TCR δ^–/–) and L. major susceptible. Anti-CD40 mAb stimulation of L. major-infected BALB/c mice was accompanied by increased IL-12 and IFN-γ production in draining lymphnodes, analyzed either by direct expression, or in an antigen-specific in vitro recall assay. The protective role of these cytokines was indicated by the finding that anti-CD40 mAb-mediated protection of L. major-infected BALB/c mice could be reversed by co-treating the animals with neutralizing anti-IL-12 and/or anti-IFN-γ mAb. Collectively, these data suggest that BALB/c mice develop a protective Th1 CD4⁺ T cell response to L. major infection when co-injected with anti-CD40 mAb. While the CD40-CD40L interaction has been previously shown to be vital in the control of murine Leishmaniasis, the current study establishes in vivo that anti-CD40 mAb treatment alone is sufficient to protect BALB/c mice from L. majorinfection and raises the possibility of utilizing this approach for vaccination strategies.     

Natural killer cells participate in the early defense against Leishmania major infection in mice

In this study the role of natural killer (NK) cells in the course of experimental Leishmania major infection was investigated. NK cells in genetically resistant C57BL/6 mice were depleted by in vivo administration of anti-asialo-GM1 or anti-NK1.1 antibodies. A marked exacerbation of the infection was found in the NK-depleted mice within the first two weeks of infection. Both the local tissue swelling and the number of parasites in the lesions were significantly higher than in normal animals. Lymph node cells taken from infected NK-depleted mice released less interferon-γ (IFN-γ) when cultured in vitro. As an alternate approach we have used poly I: C treatment in order to activate NK cell activity in vivo in BALB/c mice, which are genetically susceptible to L. major infection. Poly I: C treatment led to milder symptoms and to a significantly lower parasite burden in the early course of infection. Lymph node cells from infected and poly I: C-treated BALB/c mice released higher amount of IFN-γ in vitro than cells from control mice. These data show that NK cells are active participants in the non-specific phase of anti-leishmanial activity in the control of parasite multiplication early in the course of L. major infection in mice.     

Comparative Evaluation of Two Vaccine Candidates against Experimental Leishmaniasis Due to Leishmania major Infection in Four Inbred Mouse Strains

Experimental leishmaniasis in BALB/c and C57BL/6 mice are the most investigated murine models that were used for the preclinical evaluation of Leishmania vaccine candidates. We have previously described two new inbred mouse strains named PWK and MAI issued from feral founders that also support the development of experimental leishmaniasis due to L. major. In this study, we sought to determine whether different mouse inbred strains generate concordant or discordant results when used to evaluate the potential of Leishmania proteins to protect against experimental leishmaniasis. To this end, two Leishmania proteins, namely, LACK (for Leishmania homolog of receptor for activated C kinase) and LmPDI (for L. major protein disulfide isomerase) were compared for their capacity to protect against experimental leishmaniasis in PWK, MAI, BALB/c, and C57BL/6 inbred mouse strains. Our data show that the capacity of Leishmania proteins to confer protection depends on the mouse strain used, stressing the important role played by the genetic background in shaping the immune response against the pathogen. These results may have important implications for the preclinical evaluation of candidate Leishmania vaccines: rather than using a single mouse strain, a panel of different inbred strains of various genetic backgrounds should be tested in parallel. The antigen that confers protection in the larger range of inbred strains may have better chances to be also protective in outbred human populations and should be selected for clinical trials.The leishmaniasis are parasitic diseases due to a protozoan of the genus Leishmania that are endemic in 88 countries. Three hundred and fifty million people are exposed to the infection risk and 14 million people are known to be infected. Two million new cases, including 1.5 million of the cutaneous leishmaniasis, are estimated to appear annually (39). The leishmaniasis represent a worldwide major public health problem because of several therapeutic challenges such as drug toxicity, parasite resistance to current drugs, and the high cost of the new treatments. The problem is particularly serious since the disease affects the poorest classes of the developing countries. The cutaneous leishmaniasis are among the rare parasitic diseases that might be potentially vaccine preventable. However, even if theoretically feasible, there is still no human Leishmania vaccine available today (17). One serious obstacle facing such a goal is the lack of experimental animal models that tightly mimic the disease as it occurs in humans.The experimental infection of inbred BALB/c and C57BL/6 mice by Leishmania major parasites has established the functionality of the Th1/Th2 dichotomy of CD4⁺ T helper cells and the contrasted pathogenic roles played in protection or disease promotion by the two Th subsets (33). Thus, C57BL/6 mice infected with L. major develop a Th1 response and efficiently control the disease within few weeks. In contrast, susceptible BALB/c mice mount a Th2 response and develop a severe, unremitting, and ultimately lethal disease (37). The susceptibility of BALB/c mice to L. major infection has been ascribed to the occurrence within the lymph nodes draining the inoculation site, of an early burst (at 16 h postinoculation) of interleukin-4 (IL-4) that polarizes the immune response toward the Th2 pathway (15, 24). The contrasted immunopathogenic mechanisms at work in BALB/c and C57BL/6 strains likely reflect differences in their genetic background. Since the majority of studies evaluating vaccine candidates have been conducted in the BALB/c model, it would be hazardous to extrapolate the conclusions drawn from these experiments to other inbred strains of different genetic backgrounds or to out bred animal models (i.e., primates): one given vaccine could be promising in one strain and still fail to protect in another strain (17). Thus, the criteria that would help to select at the preclinical stage a Leishmania antigen as a promising vaccine candidate worth entering the clinical trial stage are still not clearly defined.We have recently identified two new inbred mouse strains derived from feral founders, named PWK and MAI, that are susceptible to L. major infection (1). MAI mice develop an infiltrated lesion at the site of parasite inoculation that enlarges over time in an unremitted way. In this strain, the primary infection does not induce protection against reinfection. Although the immune response to Leishmania antigens in MAI mice was characterized by a Th2 cytokine profile, IL-4 did not seem to play a dominant role in disease phenotype as in BALB/c mice. In PWK mice, the experimental disease induced by L. major infection is featured by a nodule that develops at the site of parasite inoculation. This nodule is larger and of a much longer duration (30 weeks to complete healing) than the one that develops in C57BL/6. PWK mice acquire a solid immunity after a primary infection and are completely refractory to a secondary challenge. They develop during infection a mixed Th1/Th2 cytokine pattern, with IL-10 playing a disease-promoting role.The diverse disease patterns induced by L. major in PWK, MAI, C57BL/6, and BALB/c mice and the heterogeneity in the immunopathogenic mechanisms at work in each strain are likely shaped by the genetic background of the mice. This assumption led us to explore the effect of the genetic diversity of inbred mouse strains on the protection potentially conferred by Leishmania proteins against L major infection. Two Leishmania promising vaccine candidates were used, namely, the Leishmania homolog of receptor for activated C kinase (LACK) (31) and the L. major protein disulfide isomerase (LmPDI) (5).     

The Xid defect determines an improved clinical course of murine leishmaniasis in susceptible mice

The course ofLeishmania majorinfection in B cell-defective BALB.Xidmice was investigated. Infected BALB.Xid mice showed a significantiyslower lesion development compared with BALB/c controls accompaniedby a 10– to 30– fold lower parasite burden in lymphaticorgans. The B cell immune response, as quantified by anti-leishmanialantibody production and B cell numbers in lymphatic organs,remained significantly lower in BALB.Xid mice as compared withBALB/c control mice. In accordance with disease development,CD4⁺ T cells from lymph nodes of infected BALB.Xid mice produced6– to 10– fold more IFN-; than the respective Tcells of BALB/c mice, when stimulated with leishmanial antigeninvitro. B cells from lymph nodes and the peritoneal cavitiesof BALB/c mice could be induced to produce 3– to 8–fold more IL-10 than the respective cells from B cell-defectiveBALB.Xid mice. The data thus indicate that the Xid mutationallows for the development of T_h1 cells which confer resistanceto infection withL. major. Moreover, the data suggest that Bcells contribute to susceptibility to L. major infection inBALB/c mice by skewing the T_h cell network towards a T_h2 phenotype.Since the difference in B cell-derived IL–10 productionbetween BALB/c and BALB.Xid mice was more prominent in peritonealB cells, the data support the notion that the skewing of theT cell response may be predominantly mediated by the B1 cellsubset.     

Vaccination with the Leishmania infantum acidic ribosomal P0 protein plus CpG oligodeoxynucleotides induces protection against cutaneous leishmaniasis in C57BL/6 mice but does not prevent progressive disease in BALB/c mice

We have examined the efficacy of the administration in mice of a molecularly defined vaccine based on the Leishmania infantum acidic ribosomal protein P0 (rLiP0). Two different challenge models of murine cutaneous leishmaniasis were used: (i) subcutaneous inoculation of L. major parasites in susceptible BALB/c mice (a model widely used for vaccination analysis) and (ii) the intradermal inoculation of a low infective dose in resistant C57BL/6 mice (a model that more accurately reproduces the L. major infection in natural reservoirs and in human hosts). First, we demonstrated that C57BL/6 mice vaccinated with LiP0-DNA or rLiP0 protein plus CpG oligodeoxynucleotides (ODN) were protected against the development of dermal pathology and showed a reduction in the parasite load. This protection was associated with production of gamma interferon (IFN-γ) in the dermal site. Secondly, we showed that immunization with rLiP0 plus CpG ODN is able to induce only partial protection in BALB/c, since these mice finally developed a progressive disease. Further, we demonstrated that LiP0 vaccination induces a Th1 immunological response in both strains of mice. In both cases, the antibodies against LiP0 were predominantly of the immunoglobulin G2a isotype, which was correlated with an rLiP0-stimulated production of IFN-γ in draining lymph nodes. Finally, we demonstrated that LiP0 vaccination does not prevent the Th2 response induced by L. major infection in BALB/c mice. Taken together, these data indicate that the BALB/c model of cutaneous leishmaniasis may undervalue the potential efficacy of some vaccines based on defined proteins, making C57BL/6 a suitable alternative model to test vaccine candidates.     

MURINE CUTANEOUS LEISHMANIASIS: RESISTANCE IN RECONSTITUTED NUDE MICE AND SEVERAL F1 HYBRIDS INFECTED WITH LEISHMANIA TROPICA MAJOR

After cutaneous injection of promastigotes of an isolate of the intramacrophage protozoan parasite, Leishmania tropica major, mouse strains develop chronic cutaneous lesions or show a resolving pattern of disease. On this basis, they can be classified as resistant (e.g. CBA/H and C57BL/6) or susceptible (e.g. BALB/c, BALB/c.H-2^b and BALB/c.H-2^k). Hypothymic nude (nu/nu) mice of either BALB/c, CBA/H or C57BL/6 genotype are susceptible to chronic disease. However, nude mice of these genotypes, including BALB/c, are resistant to chronic cutaneous leishmaniasis when injected at the time of parasite challenge with small numbers of H-2 compatible lymphoid cells from normal mice. Nude mice remain susceptible when injected with fully H-2 incompatible cells. Using cells from H-2 mutant mice for reconstitution of resistance in C57BL/6.nu/nu mice, evidence was obtained that I region compatibility is necessary for cells to mediate host-protective effects. Cells from chronically-diseased BALB/c mice do not have protective effects in BALB/c.nu/nu mice at any cell dose and will abrogate the resistance-promoting effect of lymphoid cell populations from chronically-diseased BALB/c.H-2^k and BALB/c.H-2^b mice can be demonstrated when assayed at certain cell doses in H-2 compatible CBA/H.nu/nu and C57BL/6.nu/nu mice, respectively. The data suggest that chronically-diseased (genetically-susceptible) mice contain a mixture of resistance-promoting and disease-promoting T cells in their peripheral lymphoid organs and that expression of the resistance-promoting subset can occur in nude mice of resistant genotype. Previous data have indicated that Lyl⁺2^? T cells are efficient mediators of both T cell-dependent activities. No evidence for the operation of disease-promoting or resistance-promoting antibodies in perpetuation or resolution of disease has been obtained in extensive serum transfer experiments. Some discrepancies exist in the literature on the question of the dominance of susceptibility or resistance in F₁ hybrid mice. A re-examination of susceptibility/resistance in F₁ hybrids between BALB/c and several other parental strains was undertaken using cloned pathogenic promastigotes derived from a heterogeneous L. t. major isolate in order to reduce effects of parasite heterogeneity in the analysis. Resistance was dominant in some but not all F₁ hybrids, with most showing a delayed healing pattern of disease relative to the resistant parental strain. Despite the use of genetically-homogeneous parasites, the analysis was complicated by variability within groups of F₁ hybrid mice as well as between males and females and between F₁ hybrids of reciprocal crosses. A hypothesis based on antigen and H-2 expression on infected macrophages is advanced to account for the balance between the effects of resistance-promoting and disease-promoting Lyl⁺2^? T cells in mice of various genotypes.     

Kinetics of an experimental inflammatory reaction induced by Leishmania major during the implantation of paraffin tablets in mice

In leishmaniasis, macrophages play important but potentially divergent roles. They act as the host cell in which the parasite may reside and replicate, and, at the same time, they act as an effector cell with the potential to eliminate the parasite. In this work, we experimentally induced an inflammatory model that provokes a continued recruitment of the monocytes to the site of inflammation. This model was carried out by means of implanting paraffin tablets under the skin of Balb/c or C57BL/6 mice. Mice were then infected with Leishmania major to determine how the monocyte inflammatory response to paraffin could influence the course of infection with L. major. Mice were sacrificed 15, 21, 30, and 45 days after infection, and skin and inflammatory capsule were collected for histopathology. At 15 days and 21 days, the lesions induced by L. major in combination with paraffin contained markedly increased numbers of parasites relative to lesions in parallel control animals infected with L. major (without paraffin). Both Balb/c and C57BL/6 mice exhibited high parasite numbers in their lesions. The intense parasite burden observed following paraffin implantation would suggest that the monocytes–macrophages that are recruited to the lesion are acting more as a host cell permitting parasite growth than as an effector cell capable of eliminating L. major. At later times, the two strains of mice stratified according to their genetic susceptibility/resistance profiles. Susceptible Balb/c mice continue to have large parasite burdens, whereas the resistant C56BL/6 mice begin to control parasite numbers. This later observation indicates that the genetic difference between susceptible and resistant strains is not due to differences in monocyte recruitment and cannot be reversed through the altering of monocyte inflammation. Received: 20 September 1999 / Accepted: 28 February 2000     

<Emphasis Type="Italic">Leishmania major</Emphasis> infection in susceptible and resistant mice elicit a differential humoral response against a total soluble fraction and defined recombinant antigens of the parasite

In the present work, we analyzed the humoral response of Leishmania major experimentally infected BALB/c and C57BL/6 mice against three Leishmania antigens: total soluble antigen (soluble leishmania antigen(SLA)), a chimerical recombinant protein formed by the genetic fusion of four cytoplasmic proteins (PQ), and a kinetoplastic membrane protein (Kmp-11). We determined the correlation between the immune response against these proteins and the histopathological changes induced in the susceptible and resistant mice after infection. The data showed the existence of wide differences in the recognition of SLA, PQ, and Kmp-11 by the sera from both strains. The anti-SLA titer of BALB/c was 100 times higher than that of C57BL/6 mice. Antibodies against the recombinant Kmp-11 were detected only in infected BALB/c during the first stage of the infection. In contrast, the PQ protein was recognized by the sera from infected BALB/c mice but exclusively when they were in a late-lesion period. The data suggest that the response against the membrane Kmp-11 protein is transient and correlates with early developmental stages of the infection, whereas the response against cytoplasmic proteins as those present in PQ is sustained and could be considered as a marker of an advanced stage of the infection and disease.     

In susceptible mice,Leishmania major induce very rapid interleukin-4 production by CD4+ T cells which are NK1.1−

Susceptibility of BALB/c mice to infection with Leishmania major is associated with a T helper type 2 (Th2) response. Since interleukin-4 (IL-4) is critically required early for Th2 cell development, the kinetics of IL-4 mRNA expression was compared in susceptible and resistant mice during the first days of infection. In contrast to resistant mice, susceptible mice exhibited a peak of IL-4 mRNA in their spleens 90 min after i.v. injection of parasites and in lymph nodes 16 h after s.c. injection. IL-12 and interferon-γ (IFN-γ) down-regulated this early peak of IL-4 mRNA; the effect of IL-12 was IFN-γ dependent. Treatment of resistant C57BL/6 mice with anti-IFN-γ allowed the expression of this early IL-4 response to L. major. The increased IL-4 mRNA expression occurred in Vβ8, 7, 2^? CD4⁺ cells in BALB/c mice and NK1.1^? CD4⁺ cells in anti-IFN-γ treated C57BL/6 mice. These results show that the NK1.1⁺ CD4⁺ cells, responsible for the rapid burst of IL-4 production after i.v. injection of anti-CD3, do not contribute to the early IL-4 response to L. major.     

Standardization and performance evaluation of mononuclear cell cytokine secretion assays in a multicenter study

Background

Burkholderia pseudomalleiis the causative agent for melioidosis. For many bacterial infections, cytokine dysregulation is one of the contributing factors to the severe clinical outcomes in the susceptible hosts. The C57BL/6 and BALB/c mice have been established as a differential model of susceptibility in murine melioidosis. In this study, we compared the innate IFN-γ response toB. pseudomalleibetween the C57BL/6 and BALB/c splenocytes and characterized the hyperproduction of IFN-γ in the relatively susceptible BALB/c micein vitro.

Results

Naïve BALB/c splenocytes were found to produce more IFN-γ in response to live bacterial infection compared to C57BL/6 splenocytes. Natural killer cells were found to be the major producers of IFN-γ, while T cells and Gr-1^intermediatecells also contributed to the IFN-γ response. Although anti-Gr-1 depletion substantially reduced the IFN-γ response, this was not due to the contribution of Gr-1^high, Ly-6G expressing neutrophils. We found no differences in the cell types making IFN-γ between BALB/c and C57BL/6 splenocytes. Although IL-12 is essential for the IFN-γ response, BALB/c and C57BL/6 splenocytes made similar amounts of IL-12 after infection. However, BALB/c splenocytes produced higher proinflammatory cytokines such as IL-1β, TNF-α, IL-6, IL-18 than C57BL/6 splenocytes after infection withB. pseudomallei.

Conclusion

Higher percentages of Gr-1 expressing NK and T cells, poorer ability in controlling bacteria growth, and higher IL-18 could be the factors contributing to IFN-γ hyperproduction in BALB/c mice.     

Generation and characterization of transgenic mice expressing cobra venom factor

Cobra venom factor (CVF), the anticomplementary protein in cobra venom, activates the alternative complement pathway, eventually leading to complement consumption. Here, we describe the development of a transgenic mouse model for CVF. We generated a DNA construct containing the full-length cDNA for single-chain pre-pro-CVF. Expression of CVF was controlled by the alpha(1)-antitrypsin promoter to achieve liver-specific expression. Linearized DNA was microinjected into murine ovary cells (strain CD(2)F(1) (BALB/cxDBA/2J)) and the newborn mice were analyzed for stable integration of CVF DNA. After establishing the transgene, mice were propagated in a BALB/c background. The CVF mRNA was detected in the liver and, in some animals, in the kidney. CVF protein was detected in small amounts in the serum. Serum complement hemolytic activity in CVF-transgenic mice was virtually absent. The concentration of plasma C3 was significantly reduced. The CVF-transgenic animals show no unusual phenotype. They provide an animal model to study the effect of long-term complement depletion by continued activation, as well as the role of complement in host immune response and pathogenesis of disease.     

Murine immune response induced by Leishmania major during the implantation of paraffin tablets

We carried out a model of chronic inflammation using a subcutaneous paraffin tablet in mice experimentally infected with Leishmania major. It was previously reported that the parasite load following paraffin implantation occurred at a peak of 21 days in both BALB/c and C57BL/6 mice. At the present study, we have investigated what cytokines and chemokines are directly related to the parasite load in C57BL/6 mice. All mice were divided in four groups: mice implanted with paraffin tablets; mice experimentally infected with L. major; mice implanted with paraffin tablets and experimentally infected with L. major; and mice submitted only to the surgery were used for the Real-Time Polymerase Chain Reaction (RT-PCR) controls. Fragments of skin tissue and the tissue surrounding the paraffin tablets (inflammatory capsule) were collected for histopathology and RT-PCR studies. By 21 days, a diffuse chronic inflammatory reaction was mainly observed in the deep dermis where macrophages parasitized with Leishmania amastigotes were also found. RT-PCR analysis has shown that BALB/c mice showed strong IL-4 and IL-10 mRNA expression than controls with very little expression of IFN-γ. In contrast, both IFN-γ and IL-10 mRNA was found in higher levels in C57BL/6 animals. Moreover, in C57BL/6 mice the expression of chemokines mRNA of CCL3/MIP-1α was more highly expressed than CCL2/MCP-1. We conclude that the Th1 immune response C57BL/6 did not change to a Th2 response, even though C57BL/6 animals presented higher parasitism than BALB/c mice 21 days after infection and paraffin implantation.     

F1 Cross‐Breed Between Susceptible BALB/c and Resistant Swiss mice Infected with Leishmania major Exhibit an Intermediate Phenotype for Lesion Sizes and Type 1 Cytokines but Show Low Level of Total IgG Antibodies

Our current understanding of the host immune response during leishmaniases largely derives from studies performed in mice due to the intrusive techniques required to study infected human patients. Swiss mice are highly resistant to Leishmania infections in concordance with observed response in humans, while BALB/c mice indicate a high‐susceptibility phenotype. Developing a cross‐breed between BALB/c and Swiss mice may have important consequences on disease development, immune responses and parasite killing, as yet, response of the cross‐breed to Leishmania infection is superficial. The aim of the present study was to determine disease course and immune responses in F₁ cross‐breed between BALB/c and Swiss albino mice infected with L. major. Three mice groups were infected intradermally with stationary‐phase L. major parasites with parental strains (BALB/c and Swiss albino) as controls. Lesion development was monitored weekly for 8 weeks and monocyte chemotactic protein (MCP‐1), macrophage inflammatory protein (MIP‐1α), interferon‐gamma (IFN‐γ) and IgG antibody quantified by enzyme‐linked immunosorbent assay. The data were analysed using one‐way analysis of variance and Tukey–Kramer test. Results indicated F₁ mice having intermediate lesion sizes, type 1 cytokine levels and footpad parasite loads as compared to the parental strains. However, the F₁ mice had low levels of IgG antibodies and parasite burden in the spleen. (P < 0.05). This study concludes that the F₁ cross‐breed between resistant and susceptible mice may be used as a requisite model to study the role of genetics in leishmaniases and perhaps other intracellular parasites.     

Treatment of mice with the anticoccidial drug Toltrazuril does not interfere with the development of a specific cellular intestinal immune response to Eimeria falciformis

Immunity against Eimeria-infections is highly specific and it depends on cell-mediated effector mechanisms. Infections of BALB/c mice with 1000 sporulated oocysts of Eimeria falciformis led to protection against challenge infections. Treatment with the anticoccidium Toltrazuril, during primary infection, terminated the ongoing disease and did not interfere with the establishment of protective immunity against challenge infections. Mesenteric lymph node cells of infected, treated as well as non-treated and challenged BALB/c mice, showed a similar proliferation upon stimulation with parasite antigen. In contrast, neither cells of the Peyers patches, intraepithelial lymphocytes, nor spleen cells responded to stimulation with parasite antigens. Cells from all compartments and of all investigated groups proliferated and released the cytokines IFN- and IL-4 in response to the mitogen Concanavalin A. The number of cells releasing IFN- or IL-4 was not dependent on the status of infection or previous treatment with Toltrazuril. The serum IgG response against total sporozoite antigens of individual mice showed that in addition, a systemic humoral response developed in infected mice, independent of a previous drug treatment, although the specific IgG antibody concentration was higher in non-treated mice. Thus, Toltrazuril does not impair the parasite specific intestinal cellular and systemic antibody response and does not prevent the development of protection against challenge infection.