Humoral and in vivo cellular immunity against the raw insect-derived recombinant Leishmania infantum antigens KMPII, TRYP, LACK, and papLe22 in dogs from an endemic area

Leishmania infantum causes visceral leishmaniasis, a severe zoonotic and systemic disease that is fatal if left untreated. Identification of the antigens involved in Leishmania-specific protective immune response is a research priority for the development of effective control measures. For this purpose, we evaluated, in 27 dogs from an enzootic zone, specific humoral and cellular immune response by delayed-type hypersensitivity (DTH) skin test both against total L. infantum antigen and the raw Trichoplusia ni insect-derived kinetoplastid membrane protein-11 (rKMPII), tryparedoxin peroxidase (rTRYP), Leishmania homologue of receptors for activated C kinase (rLACK), and 22-kDa potentially aggravating protein of Leishmania (rpapLe22) antigens from this parasite. rTRYP induced the highest number of positive DTH responses (55% of leishmanin skin test [LST]-positive dogs), showing that TRYP antigen is an important T cell immunogen, and it could be a promising vaccine candidate against this disease. When TRYP-DTH and KMPII-DTH tests were evaluated in parallel, 82% of LST-positive dogs were detected, suggesting that both antigens could be considered as components of a standardized DTH immunodiagnostic tool for dogs.     

Analysis of granzyme B activity as a surrogate marker of Leishmania-specific cell-mediated cytotoxicity in zoonotic cutaneous leishmaniasis

The purpose of this study was to analyze Leishmania-specific cell-mediated cytotoxicity in individuals with active or healed zoonotic cutaneous leishmaniasis (ZCL). The (51)Cr-release assay revealed a significant cytotoxicity against L. major-infected autologous macrophages in individuals with active or healed ZCL. As a surrogate marker of cytotoxic response, a test based on the measure of granzyme B activity in parasite-stimulated peripheral blood mononuclear cells was optimized and evaluated in 88 individuals. Increased granzyme B activity was found in 62.5% of patients with active ZCL and in only 30% of individuals with healed ZCL. In both groups, granzyme B activity was significantly higher than in healthy, leishmanin skin test-negative control subjects (P<.05). These data provide additional evidence that Leishmania-specific cell-mediated cytotoxicity is part of the acquired immune response developed against the parasite. Its role in resistance to reinfection should be evaluated.     

甘肃文县婴儿利什曼原虫无症状感染犬的检测

目的评价PCR法、ELISA法和试条法检测我国婴儿利什曼原虫无症状感染犬的潜能。方法用两组PCR引物RV1-RV2和K13A-K13B检测动物源型黑热病疫区健康犬静脉血和骨髓中利什曼原虫特异DNA，以利什曼原虫可溶性抗原为包被抗原的ELISA法和rk39-dipstick试条法分别检测利什曼原虫特异抗体，并比较各种检测方法的敏感性差异。结果PCR法检测抗凝静脉血和骨髓的阳性率分别为50．63％（40／79）和69．62％（55／79），两种样本总检出率为77．21％（61179）；ELISA法检测的阳性率为22．22％（16／72），而rk39-dipstick试条检测的阳性率为33．33％（19／57）。结论我国动物源性黑热病疫区利什曼原虫无症状感染犬的比例相当高，以骨髓为样本的PCR检测法为较精确的犬无症状感染检测方法。     

Leishmania infantum chagasi in northeastern Brazil: asymptomatic infection at the urban perimeter

Visceral leishmaniasis (VL) is endemic in large cities in Brazil, including Natal. We determined the prevalence of asymptomatic human infection with Leishmania infantum chagasi and associated environmental risks around Natal. Infection was detected by Leishmania skin test (LST) and anti-leishmanial antibodies in humans and anti-leishmanial antibodies in dogs. Amongst 345 humans, 24.6% were seropositive, and 38.6% were LST-positive. Prevalence of positive serology was similar in both sexes and across all ages. However, positive LST responses increased with age, suggesting that LST is long-lasting and cumulative. Multinomial logistic analysis showed that LST response varied with location (P = 0.007) and that males were more frequently LST-positive (P = 0.027). Indicators of lower socioeconomic status associated significantly with human infection. Furthermore, there was geographic coincidence of seropositive humans and dogs (r = 0.7926, P = 0.011). These data suggest that dog and human L. i. chagasi infection are intimately interrelated in environmental conditions associated with low income.     

Recent advances in leishmaniasis

There has been a rapid increase in the understanding of the mechanisms whereby Leishmania infects mammalian hosts and evades the immune response. This, in turn, is driving the search for vaccines against leishmaniasis. Leishmaniasis is an infection in which the dichotomy between cellular (T-helper cell type 1) and humoral (T-helper cell type 2) responses is clearly characterized in murine models, but is unclear in humans. The diagnosis of infection may be improved by use of the polymerase chain reaction and by serology using a recombinant antigen, K39. The therapeutic choice in visceral leishmaniasis is aided by recent studies of the lipid-associated amphotericin B drugs and aminosidine (paromomycin). Unfortunately, interferon-gamma, allopurinol, and topical aminosidine are all less effective treatments than was originally thought.     

Assessment of interferon-γ levels and leishmanin skin test results in persons recovered for leishmaniasis

Patients who recover from leishmaniasis usually show development of strong immunity and induction of interferon-γ (IFN-γ) and delayed type hypersensitivity. In a randomized trial, we analyzed the IFN-γ response by using a Quantiferon-Leishmania assay against three Leishmania peptide antigens and compared it with results of the leishmanin skin test (LST) in persons residing in areas in Iran to which zoonotic cutaneous leishmaniasis (ZCL, 181 persons), anthroponotic cutaneous leishmaniasis (ACL, 104 persons), and zoonotic visceral leishmaniasis (ZVL, 67 persons) are endemic. The percentage of persons with an IFN-γ-positive response (> 0.2 IU/mL) to three antigens and the mean concentration of IFN-γ induced by the antigens were higher for persons from areas endemic for ZVL than for persons from areas endemic for ZCL and ACL. The percentage of persons with LST-positive results (≥ 5 mm indurations) was 99%, 94%, and 70% for areas with ZCL, ACL, and ZVL, respectively. Our data indicate that the LST is significantly more sensitive than IFN-γ levels in persons who have been cured of cutaneous leishmaniasis than in persons who have been cured of ZVL.     

Diurnal variation in the cellular and humoral immune responses of Japanese quail: role of melatonin

Experiments were conducted to determine if diurnal variations occur in the cellular and humoral immune responses of sexually mature, male Japanese quail and if this diurnal variation is mediated by the daily rhythm of melatonin. In Experiment 1, quail were exposed to LD 12:12 light-dark cycles and immune responses were measured in response to a single antigenic challenge given to different groups every 4 h over a 24 h period. Diurnal changes occurred in both the cellular and humoral immune responses. The cellular response was higher during the light phase than during the dark phase whereas the opposite was true for the humoral immune response. Experiment 2 was designed to determine if melatonin mediated these diurnal immune responses. Quail were maintained in continuous light (LL) to suppress endogenous melatonin production and half of them were given melatonin in the drinking water for 12 h each day for 2 weeks. Contrary to control quail, significant daily variations occurred in both the humoral and cellular immune responses of birds given melatonin. As in Experiment 1, the cellular and humoral immune responses were out of phase with one another, with the humoral response being maximal when melatonin was present. We may conclude that there exists a melatonin dependent diurnal variation in both cellular and humoral immune responses of quail. The responses were inverse to one another during the daily light-dark cycle with the cellular response being maximal during the daily light period and the humoral response being maximal during the daily dark period.     

Detection of antileishmanial IgG antibodies and Leishmania-specific antigens in sera from Yemeni patients with visceral leishmaniasis

As part of an attempt to develop an effective method for the serodiagnosis of visceral leishmaniasis (VL), the sera from 43 Yemeni cases of the disease were screened both for antileishmanial IgG antibodies and for Leishmania-specific antigens. Indirect ELISA and capture ELISA were used to test each serum for the antibodies and antigens, respectively. Sera from patients with diseases other than leishmaniasis (29 for the antibody-detection assays and 42 for the antigen-detection) and from apparently healthy volunteers were also tested. For each type of assay, the threshold for seropositivity was set three standard deviations above the mean absorbance value for the sera from the healthy volunteers. Thirty-seven of the 43 VL sera were found positive for Leishmania-specific IgG antibodies and 37 were found positive for Leishmania-specific antigens. The sensitivities of the antibody- and antigen-detection assays were both therefore 86%. The overall specificity of the antibody-detection ELISA (67%) was, however, slightly higher than that of the capture-ELISA (64%).     

Pineal melatonin secretion,but not ocular melatonin secretion,is sufficient to maintain normal immune responses in Japanese quail (Coturnix coturnix japonica)

Reports that plasma melatonin is an important immune regulator in avian species have been rather sparse and contradictory. Also, the primary source of immune-modulating melatonin has yet to be determined in birds. In Japanese quail (Coturnix coturnix japonica), the pineal gland and eyes contribute roughly two thirds and one third of the melatonin found in the blood, respectively. Two experiments were conducted to evaluate melatonin as an immune modulator in Japanese quail and to determine the primary source of immune-modulating melatonin in this species. Experiment 1 was designed to evaluate the involvement of the pineal gland and the eyes in immunocompetence. Each of three groups of quail was assigned a surgical treatment and the cellular and humoral immune responses were determined 8 weeks following surgery. The surgical treatments were pinealectomy (Px), sham pinealectomy (SH-Px), and ocular enucleation (eye removal (Ex)). Experiment 2 utilized exogenous melatonin as a replacement to reconstitute immune responses in surgically immunocompromised birds. In this experiment, 50.0 microg/ml of melatonin, or diluent only, was provided to Px and SH-Px birds in the drinking water ad libitum. The cellular and humoral immune responses were determined after 8 weeks of melatonin treatment. In both experiments, a cutaneous basophil hypersensitivity reaction to phytohemagglutinin was measured to evaluate the cellular immune response. To evaluate the humoral immune response, primary antibody titers were determined 7 days postintravenous injection with a Chukar red blood cell suspension. Flow cytometric analysis of peripheral blood lymphocytes was performed to determine the relative percentage of CD4(+) and CD8(+) T- and B-lymphocytes in all treatments of Experiment 2. In Experiment 1, both the SH-Px and Ex surgical treatments produced similar cellular and humoral immune responses, and these responses were significantly greater than those in Px-treated birds. Pinealectomy significantly reduced the cellular and humoral immune responses from SH-Px by 25.8% and 41.3%, respectively. In Experiment 2, Px again resulted in depressed cellular and humoral immune responses. In addition, Px significantly reduced CD8(+) T-lymphocyte numbers compared to SH-Px, while B-lymphocytes remained unchanged. Melatonin administration to Px birds increased the cellular (32.9%) and humoral (30.6%) immune responses to the level of control (SH-Px) birds, although this reconstitution was not due to increased CD8(+) T- or B-lymphocytes. From these data, it was clear that removal of the pineal gland, but not the eyes, reduced cellular and humoral immune responses, which were reconstituted to normal levels by exogenous melatonin. These data suggest that immunodepression is only observed in birds with two thirds of the plasma melatonin removed by pinealectomy. Removal of one third of the plasma melatonin (by ocular enucleation) is not sufficient to reduce cellular and humoral responses in the Japanese quail.     

Seroconversion against Lutzomyia longipalpis saliva concurrent with the development of anti-Leishmania chagasi delayed-type hypersensitivity

Antibody responses to salivary gland sonicate (SGS) from Lutzomyia longipalpis were investigated using serum samples from individuals living in an area where visceral leishmaniasis is endemic. Individuals were classified into 2 groups, according to the alteration of their responses to Leishmania chagasi antigen over the course of 6 months. Group 1 included children who experienced anti-L. chagasi seroconversion from negative to positive; group 2 included children who experienced delayed-type hypersensitivity (DTH) response to L. chagasi antigen conversion from negative to positive. Individuals who experienced seroconversion against L. chagasi antigens did not have increased anti-saliva antibody response, whereas those who developed a positive anti-L. chagasi DTH response had increased immunoglobulin (Ig) G, IgG1 and IgE anti-SGS antibody levels. Despite wide variation, serum samples from individuals in group 2 recognized more bands in SGS than did those from individuals in group 1. This simultaneous appearance of anti-saliva humoral response and anti-L. chagasi cell-mediated immunity supports the hypothesis that induction of immune response against SGS can facilitate induction of a protective response against leishmaniasis.     

Eosinophilic inflammation in lymph nodes of dogs with visceral leishmaniasis

Eosinophils are traditionally associated with the immune response against helminth parasites. However, several studies have demonstrated that these cells have a role regarding protective immunity in leishmaniasis. Here, we examined the relationship between the presence of eosinophils and parasite load in biopsy samples from dogs, obtained through fine needle puncture and aspiration of lymph nodes. Histological slides containing biopsy material from lymph nodes of dogs with canine visceral leishmaniasis and healthy dogs were used to obtain baseline eosinophil counts. Subsequently, scrapings were taken from slides for DNA extraction and determination of parasite load, using real‐time PCR (qRT‐PCR). Additionally, production of nitric oxide (NO) and reactive oxygen species (ROS) levels by eosinophils in the peripheral blood of dogs with canine visceral leishmaniasis and healthy dogs was measured. The eosinophil percentage were higher in lymph nodes of infected group, and the parasite load showed a significant negative correlation with the eosinophil count. The production of NO and ROS by eosinophils in the peripheral blood was higher in the dogs with canine visceral leishmaniasis. All the results together suggest that eosinophils may participate in antileishmanial immunity in canine visceral leishmaniasis.     

Leucine rich repeats are the main epitopes in Leishmania infantum PSA during canine and human visceral leishmaniasis

The PSA protein is one of the major antigens of the surface of the Leishmania infantum parasite membrane. We describe the immune humoral response against the PSA in dogs and human patients with visceral leishmaniasis caused by L. infantum. The immunodominant region of the PSA was determined by subcloning, expression and purification of three fragments covering the complete protein. The analysis revealed that the antibodies are mostly directed against the central region, which is formed exclusively by leucine rich repeats. This region is recognized by 100% of the sera from the infected dogs and 40% of the human sera. These percentages are significantly higher than those observed when the complete protein was used as antigen. The analysis of the isotype of the G immunoglobulins raised against the immunodominant determinants of the PSA indicates that both IgG1 and IgG2 classes are produced during natural infections but that the IgG2 predominates over that of the IgG1.     

Variations in the serum levels of soluble CD23, nitric oxide and IgE across the spectrum of American cutaneous leishmaniasis

Serum IgE levels and the expression of low affinity receptor for IgE (Fc epsilon RII/CD23) are increased in cutaneous leishmaniasis. The ligation of CD23 receptor by IgE-anti-IgE immune complexes results in nitric oxide (NO) production, which is a critical leishmanicidal factor. Human monocytes/macrophages express Fc epsilon RII/CD23 after activation with IFN-gamma and IL-4. In the present study, we assessed the relationship between NO, total and Leishmania-specific IgE and soluble CD23 across the clinical spectrum of American cutaneous leishmaniasis (ACL). Sixty-nine ACL patients and 22 endemic controls were studied. NO concentration was estimated using the Greiss method. Soluble CD23, total IgE and anti-Leishmania IgE levels were measured using ELISA. Soluble CD23 was increased in the four patient groups (0.001相似文献   

Assessment of humoral immune response in patients with chronic hepatitis C virus infection

Hepatitis C infection is a major public health problem worldwide. Hepatitis C virus (HCV) infection has been identified as a major causative agent of post-transfusion hepatitis. The host immune response to HCV infection is composed of both non- specific immune response, including interferon (IFN) production and natural killer (NK) cell activity and a virus-specific immune response, including humoral and cellular components. Susceptibility to infection has been related to immunological disturbances. Several studies have provided experimental evidence of disorders of both cellular and humoral immunity. Humoral Immunity is dependent mainly on immunoglobulins and little data are available about serum immunoglobulin values in chronic hepatitis C. The present study aimed to evaluate humoral immune response by measuring the concentration of serum immunoglobulin isotypes (IgG, IgM, IgA) and IgG-subclasses level (IgG1-4) in chronic hepatitis C patients and healthy controls. This study included 50 patients with chronic hepatitis C. All of them had positive serum anti-HCV antibodies, positive serum HCV-RNA by PCR, and histologically-proven chronic hepatitis. The results were compared with 25 healthy controls. Total IgG, IgA and IgM were assayed by nephelometry. IgG subclasses were assayed using human IgG subclasses enzyme immunoassay. Serum protein electrophoresis was performed in agarose gel. The results showed that no significant difference in serum immunoglobulin levels were found among patients with chronic hepatitis C of minimal liver damage( Knodell index < or =3) and patients with mild liver disease (Knodell index > 3). A significant increase in total serum IgG, IgG1 and IgG2 levels were found in patients with chronic hepatitis than in healthy controls but no difference was found in IgG3 and IgG4 in both patients and controls. Mean serum IgM was increased in patients with HCV infection compared with healthy controls. No significant difference was found in IgA level in both the patients and healthy controls. Our data revealed an increase of humoral immune response in chronic hepatitis C infection. This is evidenced by an elevation in serum immunoglobulin isotypes; IgG and its subclasses IgG1 and IgG2 and IgM. These findings may provide some new insights into the antibody response to HCV.     

Immunology of canine leishmaniasis

The role of dogs as the main reservoir of visceral leishmaniasis has led to an increased interest in the immune responses and in Leishmania antigens implicated in protective cellular immunity in canine visceral leishmaniasis. The primary goal is to control the prevalence of human disease. Immune responses in canine visceral leishmaniasis are reviewed. Cellular immune responses toward a Th1 subset mediated by IFN-gamma and TNF-alpha predominate in asymptomatic dogs exhibiting apparent resistance to visceral leishmaniasis. On the other hand, while the role of Th2 cytokines, such as IL-4 and IL-10, in symptomatic animals is still controversial, there is increasing evidence for a correlation of these cytokines with progressive disease. CD8+ cytotoxic T cells seem also likely to be involved in resistance to visceral leishmaniasis. Several Leishmania antigens implicated in protective immune responses are described and some pivotal points for development of an effective vaccine against canine visceral leishmaniasis are discussed.     

The relationship between severity of ulcerated lesions and immune responses in the early stage of cutaneous leishmaniasis in Ecuador

The relationship was examined between the severity of ulcerated lesions and immune responses in 19 Ecuadorian patients in the early stages of New World cutaneous leishmaniasis. As an immunological assay, the humoral immune response was assessed by enzyme-linked immunosorbent assay (ELISA) and the cell-mediated response by delayed type skin test for leishmanial antigen (leishmanin test). There was a statistically significant correlation (r = 0.61, P less than 0.01) between the total area of ulcerated lesions and the reciprocal titre of ELISA in identical subjects. However, no significant difference was observed in the ELISA titre between patients with a single lesion and those with multiple lesions (chi 2 = 7.06, df = 5, P greater than 0.2). A correlation (r = 0.48, P less than 0.05) was observed between the area of lesions and the intensity of leishmanin test; further, the ELISA titre also correlated to the intensity of leishmanin test in the identical subjects (r = 0.71, P less than 0.01). These results suggest that the severity of ulcerated lesions relates to the activation of both the humoral and cell-mediated immune systems in the early stage of New World cutaneous leishmaniasis.     

Persistence of cellular and humoral response to synthetic peptides from defined Plasmodium falciparum antigens.

The cellular and humoral immune responses to synthetic peptides reproducing the repeat sequences of two major vaccine candidates (circumsporozoite protein and Pfl55/RESA) were investigated in two groups of African subjects according to the length of their stay outside endemic areas. The relation between the lymphoproliferative response and the antibody levels to these antigens was studied. The results confirm the existence of T-cell epitopes within the repeat sequences of the CS protein and the Pfl55/RESA capable of inducing lymphocyte proliferation. Cellular response to all studied peptides was more frequently observed in individuals living in France for less than one year than in individuals living in France for a longer time. T-cell proliferation in the presence of the tetrapeptide and of the octapeptide from the C-terminus repeat of Pfl55/RESA was related, with an immunodominance of the tetrapeptide over the octapeptide. Cellular responses to the CS protein repeat and to the 11-amino-acid peptide from the Pfl55/RESA N-terminus were the longest lasting after termination of exposure. In a given individual, cellular and humoral responses were not related for any peptide studied.     

Effects of lighting conditions and melatonin supplementation on the cellular and humoral immune responses in Japanese quail Coturnix coturnix japonica

Two experiments were conducted to determine the effects of lighting conditions and melatonin supplementation on the cellular and humoral immune responses in Japanese quail. The first experiment was designed to evaluate differing light regimes as immune modulators in both adult and juvenile quail. The cellular and humoral immune responses were determined for three lighting conditions; short days (8:16LD), long days (16:8LD), and constant light (LL). In the second experiment, melatonin was administered in varying doses to adult quail placed in LL. The doses used in this experiment were 0.0, 0.5, 5.0, and 50.0 microg/ml melatonin given in the drinking water for 16 h per day for 2 weeks. The cellular and humoral immune responses were evaluated after 1 week of melatonin treatment. In both experiments, a cutaneous basophil hypersensitivity reaction to phytohemagglutinin (PHA-P) was measured to evaluate the cellular immune response. To evaluate the humoral immune response, primary antibody titers were calculated 7 days postintravenous injection with a Chukar red blood cell suspension. In the adult birds of experiment 1, both the 8:16LD and 16:8LD treatments produced similar cellular and humoral immune responses but these responses were significantly greater than those observed in LL. The juvenile birds held under 8:16LD also had significantly greater cellular and humoral immune responses as compared to juvenile birds held in LL. In experiment 2, there was a clear melatonin dose response on immune function in LL. The humoral immune response increased to a peak at the 5.0 microg/ml dose while the cellular immune response increased across all dose levels. From the present study it was clear that quail placed in daily light-dark cycles (LD), possessing a diurnal rhythm of melatonin, had significantly elevated immune responses as compared to those birds in LL. Furthermore, melatonin supplemented to birds exposed to LL was immuno-enhancing. This suggests that melatonin may be a mediator of the differences seen between LD and LL lighting conditions and may have important immune modulating properties.     

Correlation between histopathology, immune response, clinical presentation, and evolution in Leishmania braziliensis infection

Skin biopsies from 221 parasitologically confirmed cases of tegumentary leishmaniasis caused by Leishmania braziliensis spp. were evaluated with respect to histopathology, the qualitative and quantitative nature of the cellular infiltrate, and the presence of Leishmania amastigotes. These variables were cross correlated with the Leishmania-specific immune response, clinical presentation, and response to treatment. Physical evidence of prior leishmanial lesions was associated with the absence of amastigotes (P less than or equal to 0.001) and the presence of giant (P = 0.03) and epitheloid cells (P = 0.03) in the biopsy of the active lesion. The presence of amastigotes was inversely related to the duration of the lesion (P less than or equal to 0.001) and the presence of eosinophils (P less than or equal to 0.01), whereas the presence of adenopathy (P = 0.01), necrosis (P = 0.001), histiocytes (P = 0.001), and increased serum antibody titer (P = 0.02) were directly associated with the presence of amastigotes. The lymphocyte transformation response was correlated with the presence of granulomas (P = 0.001), but showed no correlation with cutaneous delayed type hypersensitivity. The presence of epithelioid (P = 0.04) and giant cells (P = 0.03) was associated with less drug being required to achieve healing. In contrast, necrosis was associated with a greater amount of drug to achieve healing (P = 0.05). The observed correlations between tissue responses and immune and clinical parameters provide further evidence for the role of antibody and other soluble mediators of the cellular immune response in the evolution of disease.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Transfer Factor Therapy on Mixed Lymphocyte Culture Reactivity

We have studied the effect of dialyzable transfer factor therapy on three patients with immunodeficiency disease and in one patient who demonstrated no evidence of deficiency of either humoral or cellular immunity. We found evidence for nonspecificity in the effect of transfer factor on mixed lymphocyte culture reactivity. The data suggest that in patients with immunodeficiency disease a maturation of lymphocytes may lead to a generalized increased immune responsiveness. More profoundly, our data show that transfer factor may induce changes in the expression of histocompatibility determinants. We observed changes in the expression of determinants capable of stimulating in the mixed lymphocyte culture reaction as well as an increase in the capacity of lymphocytes to respond.