INTRODUCTION
Cutaneous leishmaniasis is an infectious disease caused by several species of the protozoan parasite Leishmania, including Leishmania braziliensis, L. tropica, L. major, L. aethiopica, and L. mexicana.1,2 Skin lesions are painless but can persist as long-lasting single ulcers or nodes during localized cutaneous leishmaniasis (LL). Lesions result from Leishmania parasites conveyed by the bite of infected sand flies and usually heal spontaneously or after specific treatment. However, under certain circumstances, there are two other forms of cutaneous leishmaniasis that do not spontaneously heal, even after specific treatment. During diffuse cutaneous leishmaniasis (DL), the nodes extend to exposed areas of the skin, such as the arms or face, endowing the patient with a granulomatous appearance. Mucocutaneous leishmaniasis (MCL) commonly devastates the oromucosal tissue to complete loss of cartilage and mucous membrane, leading to patient disfiguration. In these two forms, elimination of the parasite after treatment may be incomplete, which may be an explanation for lesion relapse.3 DL and MCL have been associated with a deficient immune response that is ineffective at clearing the infection; however, a specific immunologic defect has not yet been identified.4
Extensive work with L. major in murine models has revealed that Th1 cells and the Th1-associated cytokines, interferon (IFN)-γ and tumor necrosis factor (TNF)-α, mediate the healing of lesions, whereas Th2 cells and the Th2-associated cytokine, interleukin (IL)-4, enhance the progression of leishmaniasis.5 However, the way cytokine patterns are modulated toward a Th1 or Th2 response is still a matter of discussion. These patterns are being studied in the context of antigen presentation, concentrations and kind of cytokines produced during immune cell priming, and the modulation initiated by the endocrine system.
Dehydroepiandrosterone (DHEA) plays a vital role in regulating the immune responses in some autoimmune and infectious diseases. Low levels of DHEA modify the Th1/Th2 balance, biasing the immune response toward a Th2 response,6,7 influencing the outcome of the disease. For instance, DHEA serum levels decrease during progression of HIV infection8 or tuberculosis,9 shifting Th1-associated cytokine production toward a Th2 pattern. Low DHEA serum levels are common in patients with rheumatoid arthritis (RA), who exhibit high production of antibodies, which could decrease in response to DHEA administration.10 DHEA levels are low in the elderly, and these levels are inversely associated with high IL-6 plasma levels.11–13 Furthermore, DHEA exerts a direct suppression of the human IL-6 gene promoter and inhibits in vitro IL-6 production by human peripheral blood mononuclear cells.14 DHEA also stimulates monocyte-mediated cytotoxicity, natural killer cytotoxicity, and IL-2 secretion on both human15 and murine T cells.16
In many aspects, DHEA has an opposite effect than cortisol. In fact, DHEA has been used to counteract the glucocorticoid side effects incurred during prolonged treatment of autoimmune diseases, such as systemic lupus erythematosus (SLE). Patients experience a diminution of symptoms when they receive DHEA supplementation.17 Glucocorticoids, including cortisol, interfere with the gene expression of some proinflammatory cytokines, through enhancement of inhibitor of nuclear factor (NF)-κB (I-κB) transcription, which prevents signaling through NF-κB.18,19 Glucocorticoids also bias the immune response of T lymphocytes toward a Th2 pattern by inhibiting the expression of signal transducer and activator of transcription (STAT)1 mRNA20 and enhancing the expression of Th2 cytokines through STAT3.21
It is reasonable to assume that deficient production of DHEA in patients with DL may cause them to develop extended disease. In this study, serum DHEA and cortisol were measured in patients with DL and compared with both patients with LL and healthy controls. Production of IL-6, IFN-γ, specific anti-Leishmania antibodies, and Leishmania species of the clinical isolates were also determined to identify their relationship to DHEA and cortisol serum levels under these clinical conditions.
MATERIALS AND METHODS
Patients.
At the time of this study, there were only five live patients with DL in Tabasco, a state in the southeast of Mexico. We studied all 5 patients with DL and15 patients with LL during the course of a project approved by the Research and Ethics Committee of the participating institutions. After informed consent was obtained from patients, their clinical records were complemented by appropriate questions, physical examination, and laboratory tests. All 20 patients agreed to give 20 mL of venous blood. Blood samples were obtained at 8:00 am in the closest health facility to their homes, where they had been diagnosed and received treatment provided by their family physicians. The diagnosis was verified by the presence of Leishmania amastigotes in touch preparation. Patients were treated with eight doses of glucantime, both intramuscularly and intralesion, every third day during the first 15 days. Blood samples were collected from patients with LL after confirmation of diagnosis, as well as before onset of treatment. All five patients with DL underwent multiple previous treatments every 4 months since DL diagnosis had been established; two of five patients with DL had no active lesions when blood samples were taken.
Controls.
Twenty healthy individuals, living in the same neighborhoods as the patients, were invited to participate as controls. After obtaining informed approval, 20 mL of venous blood was drawn on the same day and time as patients, and samples were processed in parallel.
DHEA and cortisol determinations.
DHEA was measured in serum by using a solid-phase 125I radioimmunoassay (Diagnostic Products, Los Angeles, CA). Cortisol was also measured in serum by using a competitive chemoluminescent immunoassay kit (Diagnostic Products). Both determinations of DHEA and cortisol were blinded with respect to their source (patient or control) and tested in triplicate in two separate assays, performed on different days. To avoid influence of sex as a confounding variable, statistical analysis was performed separately. The average of individual determinations was used for statistical analysis.
Analysis of cytokines produced by T cells.
White blood cells (WBCs) from patients or controls were incubated over 4 hours at 37°C with or without phorbol myristate acetate (PMA) at 25 ng/mL, final concentration; and Ionomycin (Ion) at 1 μg/mL, final concentration. In addition, Brefeldin A (Sigma, St. Louis, MO) was added at 10 μg/mL, final concentration. All samples were incubated for 15 minutes at room temperature with the following antibodies: anti-CD4-FITC or anti-CD8-FITC and anti-CD3-PCY5 (Immunotech Research, Lakewood, NJ). Subsequently, 250 μL of diluted fluorescence-activated cell sorter (FACS) lysis solution (Becton-Dickinson, Franklin Lakes, NJ) was added, and cells were incubated for 10 minutes at room temperature. Cell suspension was washed by centrifugation at 900g for 5 minutes at room temperature, and the pellet was resuspended in 500 μL of 1:10 diluted FACS permeabilizing solution (Becton-Dickinson). All samples were incubated for 10 minutes at room temperature, washed, and resuspended for staining with the following antibodies: anti-hemocyanin IgG2-FITC/anti-hemocyanin IgG1-PE (Pharmingen, San Diego, CA), anti-IFN-γ-PE, or anti-IL-4-PE (Pharmingen). The cell suspension was incubated 30 minutes at room temperature in the dark, washed, and analyzed in an EPICS-ALTRA flow cytometer (Beckman-Coulter, Fullerton, CA). Data were analyzed using the EXPO software (Beckman-Coulter). PMT-5 (PE-CY5) versus PMT1 (granularity) was used to select the population of CD3-positive cells, and 10,000 events were acquired. The difference between stimulated and unstimulated values was used for statistical analysis.
Serum IL-6.
A capture ELISA (Immunotech, Marseille, France) was used to measure IL-6 concentration in serum samples from patients and controls; samples were tested in triplicate, using the means for statistical analysis. Positive and negative controls were assayed at the same time.
Specific anti-Leishmania serum antibodies.
Leishmania mexicana mexicana M379 reference strain parasites were used to determine specific anti-Leishmania serum antibodies titers by dot blot. Parasites growing in Schneider insect medium were lysed by five freeze-thawing cycles in phosphate-buffered saline (PBS; pH 7.2) plus a protease inhibitor cocktail for eukaryotic cells (Sigma); debris was eliminated, and protein concentration was adjusted to 1 mg/mL. Two micro-liters of these preparations was placed on nitrocellulose (NC) membranes, blocked with 2% skim milk, reacted with 1:1,000, 1:2,000, 1:5,000, and 1:10,000 dilutions of serum from patients or controls, incubated overnight at 4°C, reacted with anti-human alkaline phosphatase label secondary antibody (Bio-Rad Laboratories, Hercules, CA), and developed with 5-Bromo-4chloro-3-indolyl phosphate/NitroBlue Tetrazolium (BCIP/NBT) substrate (Sigma).
Leishmania isolates.
Parasites were isolated in Seneckjie media by tissue culture from nodules or ulcers of patients with LL or DL; samples were obtained by needle biopsies, taken before the onset of treatment. When parasites reached 106/mL in culture, samples were prepared on glass slides for indirect immunofluorescent staining (IIF) and incubated with RPMI medium supplemented with 10% fetal calf serum (FCS) and 1:100 penicillin-streptomycin solution (Sigma) for axenic culture.
Identification of Leishmania species.
Strains isolated from patients were typified by IIF, using the monoclonal antibodies (mAb) M2, M7, M9, M11, B2, B4, B5, B16, B19, T1, and T3, which were produced and kindly provided by Diane McMahon-Pratt.22–25 M7 reacts with L. mexicana strains, M2 identifies L. mexicana amazonensis, B4 reacts with L. braziliensis braziliensis, B19 reacts with L. brasiliensis guyanensis; and T1 and T3 react with L. major and L. tropica, and weakly with members of the L. mexicana complex (D. McMahon-Pratt, personal communication, 2003). M9 and M11 react with L. mexicana complex, B2 reacts with the L. braziliensis complex, and B5 reacts with L. brazilienis panamensis and L. braziliensis braziliensis. The reference strains used were MNYC/BZ/62/M379 for L. mexicana, MHOM/VE/74/PMH3 for L. mexicana venezuelensis, MHOM/BR/75/M2903 for L braziliensis, MHOM/PA/71/LS94 for L. panamensis, MHOM/BZ/82/BEL21 for L. mexicana, IFLA/BR/67/PH8 for L. amazonensis, and HOM/BR/74/PP75 for L. donovani chagasi. Briefly, 10 μL of 106 parasites/mL were added per well and air-dried. Slides were fixed with −20°C methanol for 15 minutes and stored at −70°C until tested. Slides were rehydrated in PBS (pH 7.2) and reacted with the WHO anti-Leishmania reference mAb diluted 1:1,000 for 1 hour at 37°C in a humid atmosphere. Slides were washed three times and reacted with a second anti-human FITC-labeled antibody (Sigma) for 1 hour. Results were assessed by observed on an epifluorescence microscope.
Statistical analysis.
DHEA and cortisol data were analyzed by one-way ANOVA, Tukey multiple comparison test. Pearson r was applied for DHEA, cortisol, IFN-γ, or IL-6 correlations. Cox and Steward trend tests were used to compare DHEA values per age ranges. Comparisons between two groups were assessed using Student t test.
RESULTS
Patients.
The main characteristics of the patients studied are shown in Table 1. Most patients with LL were young men with an age range of 20—50 years old; only three were younger than 20 years of age and two were older than 50 years of age. In the DL group, one patient younger than 20 years of age (19-year-old woman), exhibiting persistent lesions since the age of 6, was unresponsive to multiple glucantime treatment. One man, 39 years old, having inactive DL lesions for the previous 3 years, was diagnosed when he was 27 years old. The other three DL cases were men older than 50 years of age, with active lesions for 4, 12, and 15 years, respectively. While presenting active DL, the five patients received cyclic glucantime treatment. The clinical evolution for patients with DL was chronic (4–15 years; mean, 11 ± 4.18 years), whereas LL was shorter (1–24 months; mean, 7 ± 5.8 months). The patients with LL that were younger than 20 or older than 50 years of age had lesions with longer evolution (12 ± 7.35 months), whereas patients between 20 and 50 years of age had lesions with shorter progression (4.25 ± 2.25 months). Controls were 12–78 years old (mean, 35.82 ± 17.23 years).
DHEA.
Serum concentrations of DHEA were lower in patients with DL (1.68 ± 0.45 ng/mL; range, 0.7–2.66 ng/mL) compared with patients with LL (3.32 ± 0.58 ng/mL; range, 0.31–6.01 ng/mL) or controls (5.28 ± 0.79 ng/mL; range, 1.13–12.96 ng/mL; Figure 1A; ANOVA, P = 0.038). Because DHEA reaches peak values in adults and declines as age advances,16 we considered it necessary to ascertain if DHEA was differentially distributed according to age in the studied patients. In fact, DHEA was lower in patients with LL compared with age-matched controls (P = 0.031, Cox and Steward trend test). DL cases were not included in this analysis because there were no cases for each age interval (Figure 1B). Two patients with LL older than 50 years of age had lesions lasting at least 10 and 24 months, respectively, when they were studied. The youngest patient with LL, an 11-year-old boy, had serum DHEA < 1 ng/mL, with 12-month lesions and no spontaneous resolution. A female patient with DL younger than 20 years old showed low DHEA (2.71 ng/mL) compared with her matched control (4.21 ng/mL). Two other women, 34 and 39 years old, from the LL group also had DHEA values below their matched controls.
Cortisol.
Similar to DHEA, serum cortisol levels were lower in patients with DL compared with controls: 76.0 ± 24.7 versus 140.9 ± 10.65 ng/mL (ANOVA, P = 0.013), whereas patients with LL had intermediate values (101.5 ± 11.82 ng/mL; Figure 2A). There was a positive correlation between DHEA and cortisol serum concentrations (Figure 2B; Pearson r = 0.441; P = 0.013).
Cytokine-producing cells.
Low levels of IFN-γ have been found consistently when DHEA levels are low. IFN-γ was evaluated as the difference between the percentage of CD4+ or CD8+ IFN-γ–positive cells under stimulation and unstimulated cells. The percentage of CD4+ IFN-γ+ cells tended to be lower in patient with DL compared with controls or patients with LL (Table 2; ANOVA, P = 0.42). Although differences were not statistically significant, a positive correlation was found between serum DHEA and IFN-γ–positive cells in patients with DL and LL (Figure 3A; Pearson r = 0.687; P = 0.007). Interestingly, these differences were not observed in controls. There were no differences in the percentage of CD8+ IFN-γ+ cells between groups. Differences between stimulated and unstimulated IL-4–positive cells were close to zero, both in patients and controls. There were no differences in the CD4/CD8 ratio between groups (data not shown).
IL-6.
Serum IL-6 tended to be higher in patients with DL compared with controls or patients with LL (Table 2; ANOVA, P = 0.12). Statistical analysis between the three groups, performed by ANOVA, was not significant, but if only patients with DL were compared with controls by t test with α = 0.05, significant differences were found (P = 0.019). IL-6 correlated inversely to DHEA in both patients with LL and DL (Figure 3B; Pearson r = −0.561; P = 0.1481). No correlation was seen in the control group (data not shown).
Anti-Leishmania antibodies.
Specific anti-Leishmania antibodies could not be found in young patients with LL, even when tested against the Leishmania strain isolated from the same patient. However, high serum titers of specific anti-Leishmania antibodies (up to 1:10,000) were found in all patients with DL when assayed by dot blot. Only one elderly patient with LL, 78 years old, showed a 1:5,000 antibody titer.
Isotyping of isolates.
The Leishmania strains that were isolated from patients with DL or LL were identified as L. mexicana using the reference mAb M7. All the strains were also reactive to the T3 mAb by IIF but not to the T1 mAb. Low reactivity to mAb B5 was observed for some strains, mostly when incubated overnight at 4°C. Serum from patients with DL did not react to L. braziliensis reference strains tested, as they did with L. mexicana reference strain M379 or Bel21.
DISCUSSION
DL is a rare disease, with only 300–500 cases reported by the WHO throughout the world.26 DL is regarded as an extreme of natural evolution of cutaneous leishmaniasis, which grants us an opportunity to study the state of variables in such an advanced state of the disease, where it is extremely difficult to eliminate the parasite. A description of low levels DHEA and cortisol, as presented in this paper, may contribute to our understanding, modify the outcome of the disease, and perhaps, offer new possibilities for novel therapies for its management.
In this study, both DHEA and cortisol were low in patients with DL, who are considered to have chronic inflammation. To understand the results, we may first hypothesize that a common stimulator of these two hormones is altered, as proopiomelanocortin (POMC) or one of its products, such as the adrenocorticotrophic hormone.27 However, there is no explanation for why the number of patients with DL is higher for men than women.4,28,29 A second explanation is given, if independent mechanisms for DHEA or cortisol diminution are considered. For instance, modifications of DHEA could be explained by hormonal changes developed by an organism during normal growth, for example, the young and elderly, considering that DHEA is naturally low at both extremes of life.11–13 Furthermore, low DHEA serum levels have been associated with immunosenescence,11–13 meaning that normal decrease of DHEA in elderly people seems to increase the risk of severity of many diseases and susceptibility to infections.
A decrease in serum levels of dehydroepiandrosterone sulfate (DHEAS) and DHEA is typical for noninfectious chronic inflammation where TNF and IL-6 have a predictive role for such changes30,31 as in autoimmune disorders, including inflammatory bowel disease,32 rheumatoid arthritis,33,34 systemic lupus erythematosus,35 and pemphigus.36 These low levels are also seen in infectious diseases with chronic inflammation, such as tuberculosis, where DHEA prevents experimental infection in mice when given within the first few days or before M. tuberculosis inoculum.
We reported here that patients with DL were young or elderly, which was also observed in other studies,37–39 although age groups have not yet been analyzed. Furthermore, LL lesions remained active longer in both young and old patients compared with patients between 20 and 50 years old.
An increase in host resistance to Plasmodium falciparum has been described at the beginning of puberty, a stage that is characterized by increased levels of DHEAS.40 However, because of the fact that populations in endemic areas are highly exposed to this parasite, the immunologic memory may account for the increased host resistance.
DHEA may exert direct and indirect effects over the immune system. The effect may be direct if DHEA is able to regulate the gene expression of several mediators of the immune system, for instance, inhibiting IL-6 and TNF production by monocytes.32,41–45
DHEA is also able to modulate other cellular types, such as rat liver, where it diminishes the expression of transporter associated with antigen processing (TAP)-1 and β2-micro-globulin and enhances gene expression of TAP-2.46 It can affect endothelial cells as well, which respond by increasing their in vitro proliferation47 and genomic and non-genomic regulation of NO synthesis,48 independently of androgen or estrogen receptors. The recent description of a receptor for DHEA signaling thought a G protein49 lends support to understanding direct effects of DHEA.
On the other hand, DHEA may also have indirect actions during its conversion to sexual hormones,50 as is the case of 17β-estradiol, which inhibits TNF51–53 and IL-6 production.54–57 Even if this indirect action could explain greater male susceptibility to Leishmania, additional effects on diverse cells participating in the immune response, as mentioned before, should not be ignored, considering the effect exerted by DHEA over a microorganism. For instance, DHEA seems to extend its actions directly on the protozoa, because a negative effect over the growth of Plasmodium have been recently reported.58
An independent decrease of cortisol levels could be explained if we consider that free cortisol has been reported to be low in critically ill patients with severe infections, such as septic shock, trauma, burns, or surgery.59,60 Low cortisol in our patients may be caused by the chronic inflammation state associated with DL.
Cytokine production during infection by Leishmania has been extensively studied in murine animal models but is poorly studied in human patients. Despite the few papers on cytokine production made in humans with DL,4,28,29,61 they have confirmed low IFN-γ and high IL-4 production in DL in contrast to usually normal production of these cytokines in LL patients or during in vitro stimulation of mononuclear cells in Leishmanianaive humans.62 Low IFN-γ production in patients with DL has been determined by ELISA on supernatants of stimulated cells,28,29,61 by mRNA expression,4 and by percentage of IFN-γ–producing CD4+ cells.29 Our data show that high levels of IL-6 are associated with DL, which is consistent with previous findings on elderly people12 and confirms a direct correlation of low DHEA with low numbers of IFN-γ–producing T cells. Elderly or very young patients with naturally low levels of DHEA who become infected with Leishmania could be at major risk of developing DL. This potential risk may be particularly important in endemic areas.
Murine models have shown important differences in IFN-γ responses depending on the species used to infect the animals.63 However, the contribution of different Leishmania species does not seem to be a major determinant for the development of either LL or DL, because, in this study, only L. mexicana mexicana was found in patients with DL and LL. According to these data, the development of DL could be a consequence of a high initial parasite inoculum64 on susceptible hosts, as well as the amount and types of cytokines induced in response to infection.65 Two other Leishmania species have also been associated with DL, L. braziliensis28 and L. major,29 although none of these Leishmania species were found in this study.
In conclusion, DHEA and cortisol were found to be low in patients with DL, correlating with low IFN-γ and high IL-6 levels. Supplementation with DHEA and cortisol, in addition to specific treatment, could provide additional benefits to patients, biasing the immune response toward a Th1 response, and perhaps, completely eliminating the parasites.
General characteristics of the studied patients with leishmaniasis
| Clinical form | LL | DL | P |
|---|---|---|---|
| * Mean (range). | |||
| † Student t-test. | |||
| ‡ Fisher exact χ2 test. | |||
| Age (years) | 33.87 (11–79)* | 45.4 (19–58)* | 0.213† |
| Sex | 13 Male, 2 Female | 4 Male, 1 Female | 0.6‡ |
| Duration of disease | 7 months (1–24) | 11 years (4–15) | 0.013† |
IFN-γ and IL-6 in the studied patients with Leishmaniasis and controls
| Controls | LL | DL | ANOVA P | |
|---|---|---|---|---|
| † Mean ± SEM. | ||||
| † P = 0.019 for DL against controls (Student t test). | ||||
| IFN-γ (%CD4+ cells) | 18.67 ± 2.7* | 18.52 ± 2.85 | 12.7 ± 3.5 | 0.42 |
| IL-6 (pg/mL) | 90 ± 57 | 129 ± 145 | 259 ± 155† | 0.12 |
Serum DHEA in patients with leishmaniasis. Samples were tested in triplicate by EIA using a standard curve and positive and negative internal controls (A). Histograms show the mean ± SE from 17 controls, 11 LL, and 4 DL. Analysis was performed by ANOVA Tukey test. *DHEA was significantly lower in DL compared with controls, P = 0.038. Even if values were age distributed (B), DHEA remained lower in LL and DL groups compared with controls.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 3; 10.4269/ajtmh.2007.76.566
Serum DHEA in patients with leishmaniasis. Samples were tested in triplicate by EIA using a standard curve and positive and negative internal controls (A). Histograms show the mean ± SE from 17 controls, 11 LL, and 4 DL. Analysis was performed by ANOVA Tukey test. *DHEA was significantly lower in DL compared with controls, P = 0.038. Even if values were age distributed (B), DHEA remained lower in LL and DL groups compared with controls.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 3; 10.4269/ajtmh.2007.76.566
Serum DHEA in patients with leishmaniasis. Samples were tested in triplicate by EIA using a standard curve and positive and negative internal controls (A). Histograms show the mean ± SE from 17 controls, 11 LL, and 4 DL. Analysis was performed by ANOVA Tukey test. *DHEA was significantly lower in DL compared with controls, P = 0.038. Even if values were age distributed (B), DHEA remained lower in LL and DL groups compared with controls.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 3; 10.4269/ajtmh.2007.76.566
Serum cortisol in patients with leishmaniasis. Cortisol was measured at the same time as DHEA in the same sample using a chemoluminescent assay (A). Histograms show the mean ± SE from 18 controls, 11 LL, and 4 DL. Cortisol was lower in DL and LL compared with controls (ANOVA, P = 0.013). Correlation between DHEA and cortisol values was positive under Pearson correlation analysis (r = 0.441, two-tailed P = 0.013; N = 31). Δ, patients with DL; ○, patients with LL; ▪, controls (B).
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 3; 10.4269/ajtmh.2007.76.566
Serum cortisol in patients with leishmaniasis. Cortisol was measured at the same time as DHEA in the same sample using a chemoluminescent assay (A). Histograms show the mean ± SE from 18 controls, 11 LL, and 4 DL. Cortisol was lower in DL and LL compared with controls (ANOVA, P = 0.013). Correlation between DHEA and cortisol values was positive under Pearson correlation analysis (r = 0.441, two-tailed P = 0.013; N = 31). Δ, patients with DL; ○, patients with LL; ▪, controls (B).
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 3; 10.4269/ajtmh.2007.76.566
Serum cortisol in patients with leishmaniasis. Cortisol was measured at the same time as DHEA in the same sample using a chemoluminescent assay (A). Histograms show the mean ± SE from 18 controls, 11 LL, and 4 DL. Cortisol was lower in DL and LL compared with controls (ANOVA, P = 0.013). Correlation between DHEA and cortisol values was positive under Pearson correlation analysis (r = 0.441, two-tailed P = 0.013; N = 31). Δ, patients with DL; ○, patients with LL; ▪, controls (B).
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 3; 10.4269/ajtmh.2007.76.566
Positive correlation between DHEA and IFN-γ–positive CD4 cells during leishmaniasis (A). Black dots represent DL, and open dots represent LL. Pearson’s correlation: r = 0.687, two-tailed P = 0.007 (N = 14). Negative correlation between IL-6 and DHEA in leishmaniasis (B). Black dots are patients with DL, and open dots are patients with LL. Pearson correlation: r = −0.561, P = 0.1481 (N = 8).
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 3; 10.4269/ajtmh.2007.76.566
Positive correlation between DHEA and IFN-γ–positive CD4 cells during leishmaniasis (A). Black dots represent DL, and open dots represent LL. Pearson’s correlation: r = 0.687, two-tailed P = 0.007 (N = 14). Negative correlation between IL-6 and DHEA in leishmaniasis (B). Black dots are patients with DL, and open dots are patients with LL. Pearson correlation: r = −0.561, P = 0.1481 (N = 8).
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 3; 10.4269/ajtmh.2007.76.566
Positive correlation between DHEA and IFN-γ–positive CD4 cells during leishmaniasis (A). Black dots represent DL, and open dots represent LL. Pearson’s correlation: r = 0.687, two-tailed P = 0.007 (N = 14). Negative correlation between IL-6 and DHEA in leishmaniasis (B). Black dots are patients with DL, and open dots are patients with LL. Pearson correlation: r = −0.561, P = 0.1481 (N = 8).
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 3; 10.4269/ajtmh.2007.76.566
Address correspondence to Norma Galindo-Sevilla, Instituto Nacional de Perinatologia, Subdireccion de Investigacion Clinica, Montes Urales 800, Col. Lomas de Virreyes, Mexico, D. F. 11000. México. E-mail: NGalindoSevilla@hotmail.com
Authors’ addresses: Norma Galindo-Sevilla, Instituto Nacional de Perinatologia, Subdireccion de Investigacion Clinica, Montes Urales 800, Col. Lomas de Virreyes, Mexico, D. F. 11000. México, Telephone: 52-55-5520-9900 ext. 513, Fax: 52-55-5540-6542, E-mail: NGalindoSevilla@hotmail.com. Norma Soto, Universidad Juárez Autónoma de Tabasco, Division Academica de Ciencias de la Salud, Av. Gregorio Méndez 2838-A, Col. Tamulte, Villahermosa, Tabasco, 86100, Mexico, E-mail: sotonlm2003@yahoo.com. Javier Mancilla and Arturo Cerbulo, Instituto Nacional de Perinatologia, Departamento de Infectologia e Inmunologia, Nontes Urales 800, Col. Lomas de Virreyes, Mexico City 11000, Mexico, E-mails: drmancilla@gmail.com and cerbulo@hotmail.com. Elena Zambrano and Roberto Chavira, Institiuto Nacional de Ciencias Médicas y Nutricion, Vasco de Quiroga 15, Col. Section XVI, Tlalpan, Mexico City 14000, Mexico, E-mail: zamgon@laneta.apc.org. Jaime Huerto, Secretaria de Salud de Tabasco, Jurisdicción Comalcalco, Av. Paseo Tabasco 1504, Tabasco 2000, Villahermosa, Tabasco, 86035, Mexico, E-mail: jhuerto@hotmail.com.
Acknowledgments: The authors thank Dr. Charles A. Dinarello and Dr. Leopoldo Flores for critical reading and valuable comments to the manuscript.
Financial support: This work was supported by CONACyT-SIGOLFO Grant 00-02-002-T.
REFERENCES
- 1↑
Grimaldi G Jr, Tesh RB, McMahon-Pratt D, 1989. A review of the geographic distribution and epidemiology of leishmaniasis in the new world. Am J Trop Med Hyg 41 :687–725.
- 2↑
Handman E, 2001. Leishmaniasis: Current status of vaccine development. Clin Microbiol Rev 14 :229–243.
- 3↑
Costa JML, Saldanha ACR, Melo e Silva AC, Neto AS, Galvao CES, Pedroso e Silva CMO, Silva AR, 1992. Estado actual da leishmaniose cutánea difusa (LCD) no Estado do Maranhao. II. Aspedctos epidemiológicos, clinicoevolutivos. Rev Soc Bras Med Trop 25 :115–123.
- 4↑
Bomfim G, Nascimento C, Costa J, Carvalho EM, Barral-Netto M, Barral A, 1996. Variation of cytokine patterns related to therapeutic response in diffuse cutaneous leishmaniasis. Exp Parasitol 84 :188–194.
- 5↑
Boom WH, Liebster L, Abbas AK, Titus RG, 1990. Patterns of cytokine secretion in murine leishmaniasis: Correlation with disease progression or resolution. Infect Immun 58 :3863–3870.
- 6↑
Cuttolo M, 1997. Do sex hormones modulate the synovial macrophages in rheumatoid arthritis? Ann Rheum Dis 56 :281–283.
- 7↑
Giltay EJ, Fonk JCM, von Blomberg BME, Drexhage HA, Schalkwijk C, Gooren LJG, 2000. In vivo effects of sex steroids on Lymphocyte responsiveness and Immunoglobulin levels in humans. J Clin Endocrin Metabol 85 :1648–1657.
- 8↑
Clerici M, Galli M, Bosis S, Gervasoni C, Moroni M, Norbiato G, 2000. Immunoendocrinologic abnormalities in human immunodeficiency virus infection. Ann N Y Acad Sci 917 :956–961.
- 9↑
Hernandez-Pando R, Streber ML, Orozco H, Arriaga K, Pavon L, Marti O, Lightman SL, Rook GAW, 1998. Emergent immunoregulatory properties of combined glucocorticoid and anti-glucocorticoid steroids in a model of tuberculosis. Q J Med 91 :755–766.
- 10↑
Imrich R, 2002. The role of neuroendocrine system in the pathogenesis of rheumatic diseases (minireview). Endocr Regul 36 :95–106.
- 11↑
Young DG, Skibinski G, Mason JI, James K, 1999. The influence of age and gender on serum dehydroepiandrosterone sulphate (DHEA-S), IL-6, IL-6 soluble receptor (IL-6sR) and transforming growth factor beta 1 (TGF-β1) levels in normal healthy blood donors. Clin Exp Immunol 117 :476–481.
- 12↑
Straub RH, Konecna L, Hrach S, Rothe G, Kreutz M, Schölmerich J, Falk W, Lang B, 1998. Serum dehydroepiandrosterone (DHEA) and DHEA sulfate are negatively correlated with serum inteleukin-6 (IL-6), and DHEA inhibits IL-6 secretion from mononuclear cells in man in vitro: Possible link between endocrinosenescence and immunosenescence. J Clin Endocrin Metabol 83 :2012–2017.
- 13↑
Morley JE, Kaiser F, Raum WJ, Perry HM III, Flood JF, Jensen J, Silver AJ, Roberts E, 1997. Potentially predictive and manipulable blood serum correlates of aging in the healthy human male: progressive decreases in bioavailable testosterone, dehydroepiandrosterone sulfate, and the ratio of insulin-like growth factor 1 to growth hormone. Proc Natl Acad Sci USA 94 :7537–7542.
- 14↑
Keller ET, Chang C, Ershler WB, 1996. Inhibition of NF kappa B activity through maintenance of IkappaB alpha levels contributes to dihydrotestosterone-mediated repressin of the interleu-kin-6 promoter. J Biol Chem 271 :26267–26275.
- 15↑
McLachlan JA, Serkin CD, Bakouche O, 1996. Dehydroepiandrosterone modulation of lipopolysaccharide-stimulated monocyte cytotoxicity. J Immunol 156 :328–335.
- 16↑
Daynes RA, Araneo BA, Dowell TA, Huang K, Dudley D, 1990. Regulation of murine lymphokine production in vivo. III. The lymphoid tissue microenvironment exerts regulatory influences over T helper cell function. J Exp Med 171 :979–996.
- 17↑
Chang DM, Lan JL, Lin H, Luo SF, 2002. Dehydroepiandrosterone treatment of women with mild-to-moderate systemic lupus erythematosus: A multicenter randomized, double-blind, placebo-controlled trial. Arthritis Rheum 46 :2924–2927.
- 18↑
Auphan N, DiDonato JA, Rosette C, Helmberg A, Karin M, 1995. Immunosuppression by glucocorticoids: Inhibition of NF-κB activity through induction of IκB synthesis. Science 270 :286–290.
- 19↑
Scheinman RI, Cogswell PC, Lofquist AL, Baldwin AS Jr, 1995. Role of transcriptional activation of IκBα in mediation of immunosuppression by glucocorticoids. Science 270 :283–286.
- 20↑
Hu X, Li WP, Meng C, Ivashkiv LB, 2003. Inhibition of IFN-γ signaling by glucocorticoids. J Immunol 170 :4833–4839.
- 21↑
Zhang Z, Jones S, Hagood JS, Fuentes NL, Fuller GM, 1997. STAT3 acts as a co-activator of glucocorticoid receptor signaling. J Biol Chem 272 :30607–30610.
- 22↑
Jaffe CL, McMahon-Pratt D, 1983. Monoclonal antibodies specific for Leishmania tropica. I. Characterization of antigens associated with stage- and species-specific determinants. J Immunol 131 :1987–1993.
- 23
McMahon-Pratt D, Bennett E, Grimaldi G, Jaffe CL, 1985. Subspecies- and species-specific antigens of Leishmania mexicana characterized by monoclonal antibodies. J Immunol 134 :1935–1940.
- 24
McMahon-Pratt D, Bennett E, David JR, 1982. Monoclonal antibodies that distinguish subspecies of Leishmania braziliensis. J Immunol 129 :926–927.
- 25↑
McMahon-Pratt D, David JR, 1981. Monoclonal antibodies that distinguish between New World species of Leishmania. Nature 291 :581–583.
- 26↑
Skeiky YAW, Benson DR, Jackson LM, Costa JLM, Badaro R, Reed SG, 1997. Association of Leishmania heat shock protein 83 antigen and immunoglobulin G4 antibody titers in Brazilian patients with diffuse cutaneous leishmaniasis. Infect Immun 65 :5368–5370.
- 27↑
Robinzon B, Cutolo M, 1999. Should dehydroepiandrosterone replacement therapy be provided with glucocorticoids? Rheumatol 38 :488–495.
- 28↑
Turetz ML, Machado PR, Ko AI, Alves F, Bittencourt A, Almeida RP, Mobashery N, Johnson WD Jr, Carvalho EM, 2002. Disseminated Leishmaniasis: A new and emerging form of leishmaniasis observed in Northeastern Brazil. J Infect Dis 186 :1829–1834.
- 29↑
Ajdary S, Alimohammadian MH, Eslami MB, Kemp K, Kharazmi A, 2000. Comparison of the immune profile of non-healing cutaneous leishmaniasis patients with those with active lesions and those who have recovered from infection. Infect Immun 68 :1760–1764.
- 30↑
Straub RH, Lehle K, Herfarth H, Weber M, Falk W, Preuner J, Schölmerich J, 2002. Dehydroepiandrosterone in relation to other adrenal hormones during an acute inflammatory stressful disease state compared with chronic inflammatory disease: Role of interleukin-6 and tumor necrosis factor. Eur J Endocrinol 146 :365–374.
- 31↑
Schuld A, Mullington J, Friess E, Hermann DM, Galanos C, Holsboer F, Pollmächer T, 2000. Changes in dehydroepiandrosterone (DHEA) and DHEA-sulfate plasma levels during experimental endotoxinemia in healthy volunteers. J Clin Endocrin Metabol 85 :4624–4629.
- 32↑
Straub RH, Vogl D, Gross V, Lang B, Schölmerich J, Andus T, 1998. Association of humoral markers of inflammation and dehydroepiandrostorone sulfate or cortisol serum levels in patients with chronic inflammatory bowel disease. Am J Gastroenterol 93 :2197–2202.
- 33↑
Sambrook PN, Eisman JA, Champion GD, Pocock NA, 1988. Sex hormone status and osteoporosis in postmenopausal women with rheumatoid arthritis. Arthritis Rheum 31 :937–978.
- 34↑
Deighton CM, Watson MJ, Walker DJ, 1992. Sex hormones in postmenopausal HLA-identical rheumatoid arthritis discordant sibling pairs. J Rheumatol 19 :1663–1667.
- 35↑
Lahita RG, Bradlolw HL, Ginzler E, Pang S, New M, 1987. Low plasma androgens in women with systemic lupus erythematosus. Arthritis Rheum 30 :241–248.
- 36↑
De la Torre B, Fransson J, Scheynius A, 1995. Blood dehydroepiandrosterone susphate (DHEA) levels in pemphigoid/Pemphigous and psoriasis. Clin Exp Rheumatol 13 :345–348.
- 37↑
Velasco-Castrejon O, Walton BC, Rivas-Sánchez B, Garcia ME, Lazaro GJ, Hobart O, Roldan S, Floriani-Verdugo J, Munguia-Saldana A, Berzaluce R, 1997. Treatment of cutaneous Leishmaniasis with localized current field (radio frequency) in Tabasco, México. Am J Trop Med Hyg 57 :309–312.
- 38
Velasco O, Savarino SJ, Walton BC, Gam AA, Neva FA, 1989. Diffuse cutaneous leishmaniasis in Mexico. Am J Trop Med Hyg 41 :280–288.
- 39↑
Mandujano-Vera G, 1988. Leishmaniasis en Tabasco, México. Informe de 92 casos, uno de espundia. Patología (México) 26 :87–92.
- 40↑
Kurtis JD, Mtalib R, Onyango FK, Duffy PE, 2001. Human resistance to Plasmodium falciparum increases during puberty and is predicted by dehydroepiandrosterone sulfate levels. Infect Immun 69 :123–128.
- 41↑
Danenberg HD, Alpert G, Lustig S, Ben-Nathan D, 1992. Dehydroepiandrosterone protects mice from endotoxin toxicity and reduces tumor necrosis factor production. Antimicrob Agents Chemother 36 :2275–2279.
- 42
Di Santo E, Foddi MC, Ricciardi-Castagnoli P, Mennini T, Ghezzi P, 1996. DHEA inhibits TNF production in monocytes, astrocytes and microglial cells. Neuroimmunomodulation 3 :285–288.
- 43
Kimura M, Tanaka S, Yamada Y, Kiuchi Y, Yamakawa R, Sekihara H, 1998. Dehydroepiandrosterone decreases serum tumor necrosis factor-alpha and restores insulin sensitivity-independent effect from secondary weight reduction in genetically obese Zucker fatty rats. Endocrinology 139 :3249–3253.
- 44
Padgett DA, Loria M, 1998. Endocrine regulation of murine macrophage function: effects of dehydroepiandrosterone, androstenediol, and androstenetriol. J Neuroimmunol 84 :61–68.
- 45↑
Daynes RA, Araneo BA, Ershler WB, Maloney C, Li G, Ryu S, 1993. Altered regulation of IL-6 production with normal aging. Possible linkage to the age-associated decline in dehydroepiandrosterone and its sulfate derivative. J Immunol 150 :5219–5230.
- 46↑
Gu S, Ripp SL, Prough RA, Geoghegan TE, 2003. Dehydroepiandrosterone affects the expression of multiple genes in rat liver including 11b-hydroxysteroid dehydrogenase type 1: A cDNA Array analysis. Mol Pharmacol 63 :722–731.
- 47↑
Williams MRI, Dawood T, Ling S, Dai A, Lew R, Myles K, Funder JW, Sudhir K, Komesaroff PA, 2004. Dehydroepiandrosterone increases endothelial cell proliferation in vitro and improves endothelial function in vivo by mechanisms independent of androgen and estrogen receptors. J Clin Endocrinol Metab 89 :4708–4715.
- 48↑
Simoncini T, Mannella P, Fornari L, Gaetano V, Caruso A, Genazzani AR, 2003. Dehydroepiandrosterone modulates endothelial nitric oxide synthesis via direct genomic and nongenomic mechanisms. Endocrinology 144 :3449–3455.
- 49↑
Liu D, Dillon JS, 2002. Dehydroepiandrosterone activates endothelial cell nitricoxide synthase by a specific plasma membrane receptor coupled to Gα (i2,3). J Biol Chem 277 :21379–21388.
- 50↑
Beck SG, Handa RJ, 2004. Dehydroepiandrosterone (DHEA): A misunderstood adrenal hormone and spine-tingling neurosteroid? Endocrinology 145 :1039–1041.
- 51↑
Deshpande R, Khalili H, Pergolizzi RG, Michael SD, Chang MD, 1997. Estradiol down-regulates LPS-induced cytokine production and NFkB activation in murine macrophages. Am J Reprod Immunol 38 :46–54.
- 52
Ralston SH, Russell RG, Gowen M, 1990. Estrogen inhibits release of tumor necrosis factor from peripheral blood mononuclear cells in postmenopausal women. J Bone Miner Res 5 :983–988.
- 53↑
Shanker G, Sorci-Thomas M, Adams MR, 1994. Estrogens modulates the expression of tumor necrosis factor alpha mRNA in phorbol ester-stimulated human monocytic THP-1 cells. Lymphokine Cytokine Res 13 :377–382.
- 54↑
Pottratz ST, Bellido T, Mocharla H, Crabb D, Manolagas SC, 1994. 17 Beta-estradiol inhibitis expression of human interleu-kin-6 promoter-reporter constructs by a receptor-dependent mechanism. J Clin Invest 93 :944–950.
- 55
Ray A, Prefontaine KE, Ray P, 1994. Down-modulation of in-teleuki-6 gene expression by 17 beta-estradiol in the absence of high affinity DNA binding by the estrogen receptor. J Biol Chem 269 :12940–12946.
- 56
Kassem M, Harris SA, Spelsberg TC, Riggs BL, 1996. Estrogen inhibits interleukin-6 production and gene expression in a human osteoblastic cell line with high levels of estrogens receptors. J Bone Miner Res 11 :193–199.
- 57↑
Sukovich DA, Kauser K, Shirley FD, DelVecchio V, Halks-Miller M, Rubanyi GM, 1998. Expression of interleukin-6 in atherosclerotic lesion of male ApoE-knockout mice: Inhibition by 17beta-estradiol. Arterio Thromb Vasc Biol 18 :1498–1505.
- 58↑
Freilich D, Ferris S, Wallace M, Leach L, Kallen A, Frincke J, Ahlem C, Hacker M, Nelson D, Hebert J, 2000. 16α-Bromoepiandrosterone, a dehydroepiandrosterone (DHEA) analogue, inhibits Plasmodium falciparum and Plasmodium berghei growth. Am J Trop Med Hyg 63 :280–283.
- 59↑
Cooper MS, Stewart PM, 2003. Current concepts: Corticosteroid insufficiency in acutely ill patients. N Engl J Med 348 :727–734.
- 60↑
Hamrahian AH, Osen TS, Arafah BA, 2004. Measurements of serum free cortisol in critically ill patients. N Engl J Med 350 :1629–1638.
- 61↑
Diaz NL, Zerpa O, Ponce LV, Convit J, Rondon AJ, Tapia FJ, 2002. Intermediate or chronic cutaneous leishmaniasis: Leukocyte immunophenotypes and cytokine characterisation of the lesion. Exp Dermatol 11 :34–41.
- 62↑
Rogers KA, Titus RG, 2004. Characterization of the early cellular immune response to Leishmania major using peripheral blood mononuclear cells from Leishmania-naive humans. Am J Trop Med Hyg 71 :568–576.
- 63↑
Colmenares M, Sujata K, Goldsmith-Pestana K, McMahon-Pratt D, 2002. Mechanisms of pathogenesis: Differences amongst Leishmania species. Trans R Soc Trop Med Hyg 96 :S3–S17.
- 64↑
Sacks D, Noben-Trauth N, 2002. The immunology of susceptibility and resistance to Leishmania major in mice. Nat Rev Immunol 2 :845–858.
- 65↑
Lang T, Courret N, Colle J, Milon G, Antoine J, 2003. The levels and patterns of cytokines produced by CD4 T lymphocytes of BALB/c mice infected with Leishmania major by inoculation into the ear dermis depend on the infectiousness and size of the inoculum. Infect Immun 71 :2674–2683.