**4. Immune response to** *L. (L.) mexicana*

The skin is the first immune barrier against *Leishmania* promastigotes inoculated by the sand fly vector. The immune response against *Leishmania* spp. is highly complex. Macrophages are the main host cells of *Leishmania* and also responsible for parasite elimination. The activation of the macrophage microbicidal mechanisms depends on the cell-mediated immune response [39]. Thus, the nature and intensity of cellular immune response and their mediators (i.e. cytokines and chemokines) in the lesions are of primary importance for the disease outcome.

Based on this rationale, the characterization of the cytokine expression profile was studied in 13 LCL lesions caused by *L. (L.) mexicana* from the Yucatan Peninsula [40]. Age of the patient ranged from 10 to 29 years and the lesion evolution time from 8 days to 18 months. Lesions were classified as of early (≤2-month duration) and late (≥4-month duration) evolution. Skin biopsies were taken from the border of the lesion to analyse cytokine gene expression by RT-PCR. *Leishmania* amastigotes were present in 8 of the 11 histological sections. Intra-lesional cellular infiltrate was made up of equal proportion of macrophages and plasma cells. Lymphocytes represented 50% of the cellular infiltrate in three of four of the early lesion evolution but only in one of seven of the late-lesion evolution. The analysis of the *in situ* cytokine gene expressions revealed a concomitant presence of Th1 (IL-1α, IFN-γ, TNF-α) and Th2 (IL-6, IL-10, TGF-β) cytokines in all biopsies. The high expression of IFN-γ, cytokine related with macrophage activation, in both early and late evolution suggested that the presence of this cytokine was not sufficient for parasite elimination and control of the disease. A significant increase in IL-1α, TNF-α, Il-10, and TGF-β expressions was observed in late-lesion evolution compared with that in early lesions suggesting the role of these cytokines in the chronicity of LCL caused by *L. (L.) mexicana*. Both IL-10 and TGF-β down-regulate macrophage functions [41, 42]. Thus, the intra-lesional expressions of these cytokines could promote the persistence of the intracellular parasites in the skin. On the other hand, the presence of TNF-α in cutaneous lesions caused by New World *Leishmania* species has been related with lesion formation and loss of integrity of the infected tissue [43]. Further studies to confirm the role of TNF-α in the immunopathogenesis of LCL caused by *L. (L.) mexicana* are needed.

for agriculture, hunting and gum collecting. Although the number of human cases of LCL reported each year has peaked from March to July, if the incubation period is considered, there is a strong correlation with the abundance, rates and timing of infection of both reservoirs and vectors (**Figure 2**). Based on these results, for the first time in the world, a seasonal transmission (from November to March) of LCL caused by *L. (L.) mexicana* in the sylvatic region of the state

**Figure 2.** Monthly percentage of patients and rodents infected by *Leishmania (Leishmania) mexicana*, and relative abun-

The skin is the first immune barrier against *Leishmania* promastigotes inoculated by the sand fly vector. The immune response against *Leishmania* spp. is highly complex. Macrophages are the main host cells of *Leishmania* and also responsible for parasite elimination. The activation of the macrophage microbicidal mechanisms depends on the cell-mediated immune response [39]. Thus, the nature and intensity of cellular immune response and their mediators (i.e. cytokines and chemokines) in the lesions are of primary importance for the disease outcome.

dance of *Lutzomyia cruciata* in a forest at 8 km from the village of La Libertad, Campeche, Mexico.

**4. Immune response to** *L. (L.) mexicana*

of Campeche was determined.

146 The Epidemiology and Ecology of Leishmaniasis

Another study of 20 LCL patients was carried out to analyse the role of IL-12 in the protective immune response to *L. (L.) mexicana* infection. The correlation of IL-12 with its counterregulatory cytokine, IL-10, was also evaluated [44]. The patients were 10–48 years old and their lesions ranged from 10 days to 20 months of evolution. Cytokine expressions were evaluated by RT-PCR. Intra-lesional expression of both IL-10 and IL-12 was present in most of the 20 patients. The more chronic, non-healing lesions had higher levels of IL-12 mRNA indicating that the expression of this cytokine alone was not sufficient to induce healing. The IL-10 expression correlated with both IL-12 and IFN-γ, suggesting that IL-10 promotes disease persistence by a direct inhibition of macrophage activation rather than by suppression of the Th1 response.

Epidemiological studies detected many individuals from the endemic area of LCL without suggestive signs of the disease but with a delayed hypersensitivity skin test (DHT) positive to *Leishmania* antigens. Asymptomatic infection is the most common outcome to the infection in the Yucatan Peninsula [10, 45]. From the immunological point of view, asymptomatic infection is explained as the elicitation of an appropriate immune response capable of controlling parasite replication and maintaining tissue integrity [46]. Therefore, the characterization of this protective immune response in asymptomatic individuals becomes imperative for vaccine designs. Thus, the *in situ* cytokine (IL-4, IL-10, IL-12, IFN-γ) and chemokine (MCP-1, MIP-1α) mRNA expressions were analysed in biopsies of the DHT area of asymptomatic individuals (*n* = 6) and subjects with healed lesions (*n* = 9) and compared with biopsies from active lesions (*n* = 11) [47]. The expression was highly variable. Neither IL-4 nor MIP-1 α was detected in any biopsy. IL-12 was detected in all three groups without significant differences in the median. MCP-1, chemokine that stimulates oxidative burst activity in macrophages thus killing intracellular amastigotes, was expressed in all three groups being significantly higher in active lesions. The most surprising finding was the absence of IFN-γ in both healed lesions and asymptomatic infection. Taken together, these results suggested that IL-12 and MCP-1 in the absence of IFN-γ might be playing a crucial role in the infection outcome at the skin level. Further studies are needed to identify the cytokine and chemokine network and their cell sources in asymptomatic infection. This knowledge is primordial to understand mechanisms involved in immune protection against *L. (L.) mexicana* and to develop better preventive and therapeutic strategies.
