Rapid detection of human and canine visceral leishmaniasis: assessment of a latex agglutination test based on the A2 antigen from amastigote forms of Leishmania infantum.

The diagnosis of visceral leishmaniasis (VL) in humans and animal reservoir hosts is difficult, particularly in rural areas where the disease is endemic and laboratory facilities are limited. This study aimed to develop a latex agglutination test (LAT) for the rapid detection of anti-Leishmania antibodies against the A2 antigen derived from the amastigote form as well as those against crude antigens derived from the promastigote form of an Iranian strain of Leishmania (Leishmania) infantum. The A2 antigen (42-100 kDa) was prepared from the amastigote form of L. infantum, purified with electroelution and compared with the crude antigen from the promastigote form of L. infantum. Both antigens showed appropriate intensity reactions, were selected using dot blotting of positive and negative pooled sera and used to sensitize 0.9-µm latex beads. The tests were carried out on sera from 43 symptomatic, human patients with VL confirmed by parasitological examination and direct agglutination test (DAT), 30 healthy controls and 32 patients with other infections but without VL. Canine sera were collected from 63 domestic dogs with VL confirmed using parasitological examinations and DAT and 31 healthy dogs from areas non-endemic for VL. Compared with the controls, human sera from DAT-confirmed patients yielded a sensitivity of 88.4% (95% CI, 82.1-94.5%) and specificity of 93.5% (95% CI, 87.0-99.7%) on A2-LAT (amastigote) when 1:3200 was used as the cut-off titre. A good degree of agreement was found between A2-LAT and DAT (0.914). LAT required 3-5 min to complete, versus the 12-18 h needed for DAT. Compared with the controls, A2-LAT of canine sera from DAT-confirmed cases yielded a sensitivity of 95.2% (95% CI, 95.0-95.4%) and specificity of 100% (95% CI 100%) when 1:320 was used as the cut-off titre. A good degree of agreement was found between A2-LAT and DAT (0.968). Similarly, the sensitivity and specificity of Pro.-LAT (promastigote) was calculated to be 88.4% and 91.9%, respectively for human sera and 96.8% and 90.3%, respectively for canine sera. No statistically significant differences were observed between A2-LAT and Pro.-LAT for the detection of human and canine L. infantum infections. In conclusion, A2-LAT and Pro.-LAT could be used in parallel to screen for L. infantum infections in humans and dogs in areas endemic for VL in Iran.

20 Years from the Establishment of Iranian Journal of Allergy, Asthma and Immunology.

No abstract.

T cell immune responses in psoriasis.

A central role for T cells and their cytokines in the pathogenesis of psoriasis has been proposed; however, there are controversies over the details of this issue. The goal of this study is to summarise currently available data on the importance of T cells in psoriasis pathogenesis. A systematic review of the English medical literature was conducted by searching PubMed, Embase, ISI Web of Knowledge, and Iranian databases including Iranmedex, and SID for studies on associations between the involvement of T cell subsets and psoriasis. The results of the present study indicate that alterations in the number and function of different subsets of T-cells are associated with psoriasis. It appears that studies on T cell subsets contributed to understanding the immunopathogenesis of psoriasis. In addition, it may have provided novel therapeutic opportunities in ameliorating immunopathologies.

Preparation and evaluation of a glycerol-preserved direct agglutination antigen for long-term preservation: a comparative study of the detection of anti-Leishmania infantum antibodies in human and dog.

OBJECTIVE: To prepare and evaluate a glycerol-preserved antigen from an Iranian strain of Leishmania infantum (L. infantum) for use in glycerol-preserved direct agglutination tests (GP-DAT) as an alternative to freeze dried direct agglutination tests (FD-DAT) that use freeze-dried antigen. METHODS: Glycerol-preserved DAT antigen was prepared and stored at different temperatures. We tested antigen stored at 4 °C, 22-37 °C and 50 °C over a period of 365 days. Seven hundred twenty-nine serum samples were collected from different geographical zones of Iran from 2007-2009, and 80 of these samples were pooled to produce sera. Each pooled serum contained 10 sera. All positive and negative pooled sera were separately tested for anti-L. infantum antibodies with GP-DAT, FD-DAT and formaldehyde-fixed direct agglutination test (FF-DAT) antigens; tests were performed on both human and dog sera over a period of 12 months. RESULTS: There was strong agreement between the results obtained using GP-DAT and FD-DAT antigens stored at 22-37 °C for 12 months for both human (100%) and dog (100%) pooled sera. The direct agglutination test results were highly reproducible (weighted kappa: GP=0.833, FD=0.979 and FF=0.917). CONCLUSIONS: Because GP-DAT antigen is highly stable over a range of temperatures and is easy to transport in the field, this type of antigen may be particularly useful in areas with endemic visceral leishmaniasis.

Normal range determination of lymphocytes subsets in normal adults in Iran.

Immunophenotyping of lymphocytes is very essential for evaluation of immune system. Due to the effect of environmental factors and ethical diversity on immune system, establishment of an internal normal range of lymphocyte subsets is a necessity for each population. The aim of this study was to determine the normal range of T and B lymphocytes, and NK cells in normal Iranian adults. Two hundred and thirty three Iranian normal adult volunteers took part in this study. Complete Blood Count (CBC) was performed for them with Sysmex (KX21) and cells with CD3, CD4, CD8, CD19 and CD16/56 surface markers were simultaneously detected by flow cytometry method with FACstar system. Their percentile and absolute count were determined.The volunteers were 150 male and 83 female. Mean percentages of lymphocyte subpopulation were: CD3 (67.66 ±7.76), CD19 (14.41±5.09), CD4 (39.22±6.7), CD8 (25.42 ±5.4) and CD16/56 (10.14±6.42). Also, their mean absolute count of lymphocyte bearing CD3, CD19, CD4 and CD8 were 1,504±505/µl, 332±186/µl, 827±313/µl and 522±185/µl, respectively.Our results are comparable with similar Asian results from other Asian population, but are different from European population, we therefore conclude that it is necessary for each laboratory to establish an internal normal range for the lymphocytes bearing above- mentioned markers.

Rapid detection of human Leishmania infantum infection: a comparative field study using the fast agglutination screening test and the direct agglutination test.

This study aimed to evaluate the performance of a fast agglutination screening test (FAST) for serodiagnosis of human Leishmania infantum infection in Iran. FAST is based on the direct agglutination test (DAT) but combines with a higher parasite concentration and is performed with only one serum dilution. The validity of FAST for the detection of L. infantum infection in the field was compared with the direct agglutination test on 110 confirmed or patients suspected of infection with leishmaniasis, 177 healthy individuals and 41 patients with other infectious diseases who were from northwestern and southern parts of Iran. In this study, we found a 1:1600 cut-off point empirically by seeking the best correlation (90.8) between sera confirmed with visceral leishmaniasis and healthy control sera. A sensitivity of 95.4% (95% CI, 91.4-99.4) and specificity of 88.5% (95% CI, 84.2-92.8) were found with 1:1600 as a cut-off titer when DAT-confirmed cases were compared with the control groups. A good degree of agreement was found between FAST and DAT (90.8%) by Kappa analysis. FAST requires 2 h for reading the results versus the 12-18 h needed for DAT. As FAST is simple, rapid, sensitive and non-invasive and does not require a higher volume of antigens or much expertise, it can be used for screening and serodiagnosis of human L. infantum infection.

Therapeutic effect of sodium alginate in experimental chronic ulcerative colitis.

The aim of this study was to test the therapeutic efficacy of sodium alginate in a rat model of trinitrobenzene sulfonic acid (TNBS)-induced inflammatory bowel disease. This experiment was carried out using 77 Sprague-Dawley rats which were divided into six groups; normal, control, prophylactic, therapeutic and two experimental groups. Rats were sacrificed 1, 2, 3 and 6 weeks after colitis induction. Severity of colitis was graded macroscopically and assessed using serum and colonic mucosal cytokines and eicosanoids. Intrarectal TNBS (30 mg) produced a significant chronic ulcerative colitis. The lesions were most severe on day seven after TNBS instillation, and then declined, but lesions were still observed after six weeks. TNBS administration also significantly enhanced the serum and colonic mucosal cytokines (TNF-alpha and IL-6) and eicosanoids (LTB4 and PGE2) levels, which paralleled with the severity of colitis. Low viscosity sodium alginate (LVA) solution as therapeutic agent was administered orally as drinking water at concentration of 0.5% (W/V) for six weeks. Results showed that pre-treatment (in prophylactic group) and treatment with LVA were significantly able to reduce colonic damage score, serum level and colonic mucosal production of TNF-alpha, IL-6, LTB4 and PGE2 in pre-treated and treated animals compared with non-treated controls. LVA therapy is able to suppress chronic ulcerative colitis in experimental model.

The short-term effect of mustard gas on the serum immunoglobulin levels.

Mustard gas (MG), as a chemical warfare agent was used by the Iraqi army in Iran-Iraq conflict against military men in the battlefield in 1985.The serum levels of IgG, IgA and IgM of patients exposed to MG in the battlefield were measured by single radial immunodiffusion from day 3 up to one month after exposure to MG. The serum levels of IgG in patients showed significant decrease on day 3 after exposure to MG. However, the levels of IgG in the serum samples collected from the patients during 4-18 days after exposure to MG were found to increase. The increase in serum IgG levels in the sera of patients which were collected during 19-31 days after exposure to MG was found to be highly significant, surpassing those from the controls. The levels of serum IgA in patients during one month after exposure to MG showed alterations similar to those of serum IgG, however the serum alterations of the patients IgA, comparing to those of the normal controls were not significant. The serum levels of IgM in patients did not show marked alterations during one month after exposure to MG comparing to those of the normal controls. The initial decrease in serum levels of IgG in patients is discussed in terms of a possible leakage of IgG into the skin blisters and into other severely affected parts of the body such as respiratory system, whereas the subsequent increase in serum IgG is interpreted as due to (auto) antigenic stimulation of the patients' immune systems.

Dendritic cells bearing HLA-G inhibit T-Cell activation in type 1 diabetes.

HLA-G is normally expressed on human trophoblast cells. It is a non-classical MHC molecule class I b. The role of HLA-G in diabetic type 1 is not known. We investigated the role of IFN-beta in induction HLA-G expression on the monocyte derived dendritic cells (DC) in diabetes type 1. Treatment of dendritic cell with IFN-beta in vitro from diabetic patients (n=20) and normal subjects (n=20) resulted to the production and expression of HLA-G on these cells from both groups. However, comparison of DC from the diabetic patients with DC from the controls revealed lower levels of HLA-G molecules in DC from diabetic patients. Using mixed lymphocyte reaction (MLR), it was found that DC expressing HLA-G mediated the inhibition of autologous T cell activation. It is concluded that IFN-beta can increase HLA-G in DC from diabetic patients; subsequently it may prevent the immune regularly pathway in the diabetic pathogenesis.

Serum Antibodies against Hepatitis C Virus in Iranian Patients with Graves' Disease.

Hepatitis C virus (HCV) infection has been associated with a plethora of immune and autoimmune perturbations. A variety of conditions ranging from endocrinopathies to different skin diseases has been described in HCV infection. The aim of this study was to investigate the prevalence and clinical significance of HCV infection in patients with graves' disease (GD). A total of 55 patients with GD (30 women, 25 men, mean age: 35.24 +/- 12.27 years) and 50 control subjects (28 women, 22 men, mean age: 33.34 +/- 11.99 years) were examined. Third generation ELISA test was used for detection of antibodies to HCV in human sera, and anti-HCV seropositivity was confirmed by recombinant immunoblot assay (RIBA).All normal controls were anti-HCV negative whereas anti-HCV antibody was present in 1 patient with GD and confirmed by Western blotting. These results indicate that there was no significant difference of anti-HCV antibodies between patients and controls.In this study no relationship was found between GD and HCV infection, which imply that hepatitis C virus has not a direct causal role in the pathogenesis of GD, however, this does not rule out a "hit and run" virus induced disease.

Identification of hitherto undefined B-cell epitopes by antibodies in the sera of vitiligo patients using phage-display Peptide library.

A random 12 mers phage library was used to screen a pool of immunoglobulin fractions obtained from vitiligo patients. Subsequent to panning experiments, a panel of affinity selected phage from vitiligo patients were obtained. This panel was tested using an ELISA for their reactivity with pooled sera from patients and normal controls. Among the 16 randomly selected clones, two of clones showed distinct positive reactivity with the patient's sera compared with controls. The peptides displayed by these phages expressed the following amino acid sequences: SHMPLANQYQWA and NHVQAWEQFWDS. Thus, screening with phagedisplayed random peptide library of vitiligo sera can reveal peptide sequences that mimic vitiligo-related self-antigen.

The Detection of Dopamine Gene Receptors (DRD1-DRD5) Expression on Human Peripheral Blood Lymphocytes by Real Time PCR.

There is interrelationship between the immune and nervous systems that is accomplished by the molecular mediators. Dopamine is one of the most important neurotransmitters. Five different dopamine receptor genes (DRD1, DRD2, DRD3, DRD4, and DRD5) have been recognized and cloned. The expression of the dopamine receptors is well characterized in the brain but little work has been done to examine their expression in other organ tissues. In certain diseases of the immune and nervous systems, alterations in dopamine receptors gene expression in different cells have been reported. This suggests that dopamine and its receptors have important role in pathophysiology of above-mentioned diseases.In the present study, using Real Time Polymerase Chain Reaction (PCR) technique, we investigated dopamine receptors genes expression in PBMC of normal individuals. The PBMC was separated from normal whole blood by Ficoll-hypaque; the total cellular RNA was then extracted and the cDNA was synthesized. This process followed by real time-PCR using primer pairs specific for five dopamine receptors mRNAs and beta-actin as internal control. The results showed the presence of all types of dopamine receptors in lymphocytes of normal individuals. The specificities of the obtained PCR products for the respective dopamine receptors fragments were confirmed by sequenced analysis capillary system. In conclusion, the present study has shown that human lymphocytes express five dopamine receptors DR1-DR5. However, the conclusive evidence on the possible function of these receptors in lymphocytes remains unknown. Because lymphocytes express all of the five neuronal dopamine receptors, it is quite reasonable to consider them as a model of dopaminergic neuron.