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 ethnical 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 l,504 +/- 505/microl, 332 +/- 186/microl, 827 +/- 313/microl and 522 +/- 185/microl, 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

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

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

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

detection of dopamine gene receptors [DRD[1]-DRD[5]] 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

Identification of hitherto undefined B-cell epitopes by antibodies in the sera of vitillgo 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