CD8+ T Cells as a Source of IFN-γ Production in Human Cutaneous Leishmaniasis

Cutaneous leishmaniasis (CL) is usually a self-healing skin lesion caused by different species of Leishmania parasite. Resistance and susceptibility of mice to Leishmania major infection is associated with two types of CD4+ T lymphocytes development: Th1 type response with production of cytokine IFN-γ is associated with resistance, whereas Th2 type response with production of cytokines IL-4 and IL-5 is associated with susceptibility. A clear Th1/Th2 dichotomy similar to murine model is not defined in human leishmaniasis and we need as much information as possible to define marker(s) of protection. We purified CD4+/CD8+ T cells, stimulated them with Leishmania antigens and analysed gene and protein expression of Th1/Th2 cytokines in volunteers with a history of self-healing CL who are presumed to be protected against further Leishmania infection. We have seen significant upregulation of IFN-γ gene expression and high IFN-γ production in the Leishmania stimulated CD4+ T cells and CD8+ T cells. We concluded that both antigen-specific IFN-γ producing CD4+ Th1 cells and IFN-γ producing CD8+ T cells contribute to the long term protection in individuals with a history of CL. This proves the importance of CD8+ T cells as a source of IFN-γ in Th1-like immune responses

Usage of capillary electrophoresis for common hemoglobinopathies screening

Hemoglobinopathies are most common inherited disorders in the world; approximately 7 percent of the worldwide population and 5-6 percent of population of Iran are carriers. The hemoglobin disorders inherit as autosomal recessive and are very common in the Mediterranean area and much of the Asia and Africa. The control of this inherited disorders need to genetic counseling and accurate screening by more advanced and more accurate methods. This study explains features of current Iran hemoglobin disorders, nominates the accessible methods for screening them and introduces the capillary zone electrophoresis as a rapid and more accurate method. The required data were extracted of various articles and then for good explanation, current Iran hemoglobinopathies properties were showed in the tables and electropherograms of important hemoglobin disorders in Iran population were provided for help to interpretation results of blood tests by capillary zone electrophoresis method. Hemoglobin disorders are including thalassemias and hemoglobin variants; Disruption in the production and malfunction of globin chains cause types of hemoglobin disorders. We cannot introduce one of clinical laboratory tests as critical and basic method for screening and distinguishing types of inherited hemoglobin disorders as alone. For distinguishing the types of them must be prepared enough information and data of the hemoglobin disorders and for more accurate analysis must be used simultaneously different methods as gel electrophoresis, high performance liquid chromatography, isoelectric focusing, capillary zone electrophoresis or molecular tests. The capillary electrophoresis is an accurate and rapid method for screening types of the hemoglobin disorders. Other side this method cannot analyze all of them, so must be used biochemical, biophysical and molecular methods for confirmation the results. This review showed we can use the capillary electrophoresis and HPLC as two complementary methods for hemoglobinopathies screening. We can analyze by the methods more hemoglobin disorders and decrease more laboratory errors. Moreover, we must have patient history, hematological indices, information and data of types of hemoglobinopathies. The patient history and complete blood count results as red blood cell count, mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration can be useful and helpful in screening the hemoglobin disorders and then distinguishing all of hemoglobin disorders

Number and subtypes of natural killer cells in patients with allergic rhinitis in comparison to healthy subjects

"n Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi;} Background: Allergic rhinitis is a common disorder with great morbidity. Its prevalence has increased during recent years, therefore attracting attentions to its mechanisms. Type 2 cytokines play a major role in allergies. It has been proposed that Natural killer (NK) cells may be able to produce type 2 cytokines. This study was done to evaluate NK cells number and subtypes in patients with allergic rhinitis, comparing healthy subjects."n"nMethods: In a case control study, patients with allergic rhinitis were compared to healthy non-atopic subjects. Allergic rhinitis was diagnosed according to ARIA guidelines. NK cells quantity was studied by staining of peripheral blood mono nuclear cells with anti-CD16-FITC and anti-CD56-PE and evaluated by two color flowcytometry. Intracellular cytokines were evaluated by tri-color flowcytometry. NK cells were separated by magnetic beads, and cultured for 72 hours. Secretion of IL-4, IL-5, IL-10, IL-13, and IFN-γ was measured by ELISA, in stimulated and unstimulated conditions."n"nResults: Patients had more CD16+ CD56+ NK cells than control group. IL-4+ NK cells were significantly higher in patients (p<0.001), but the number of IFN-γ+ NK cells was not different. Cytokine secretion of NK cells was similar in case and control groups. Although IL-13 level after stimulation seemed higher in patients, the difference was not significant."n"nConclusion: NK cells number is increased in patients with allergic rhinitis and a considerable number of them produce IL-4

Sequence of primer pairs for different amplicons.

<p>Sequence of primer pairs for different amplicons.</p

Relative expression of cytokine genes in SLA stimulated CD4⁺ and CD8⁺ T cells to unstimulated cells (blank wells) of culture.

<p>Total RNA was extracted from stimulated CD4⁺ and CD8⁺ T cells of culture and reverse transcription of mRNA to cDNA was performed using M-MuLV enzyme. Two steps Real-time PCR was set on cDNA samples using SYBR Green I system and specific primer pairs. Threshold cycles (Cts) of each amplicon was used for further analysis. The relative quantities of the target genes were normalized against the relative quantities of the internal standard (GAPDH). Fold-expression changes were calculated using the equation 2^−ΔΔCT. A) relative expression of cytokine genes in CD4⁺ T cells B) relative expression of cytokine genes in CD8⁺ T cells.</p

Basic information of the volunteers.

<p>Age and LST: Mean±S.D.</p><p>Others: Median (Range).</p><p>NA = not applicable.</p

Frequency of purified CD4⁺/CD8⁺ T cells producing intracellular IFN-γ.

<p>A portion of the cells at 72 hrs of SLA stimulation was used for ICS assay. Cells were adjusted at about 5×10⁵/ml and stimulated with PMA + Ionomycin for 5–6 hrs. Monensin was added during the last 4–5 hrs of culture. Cells were permeabilized and stained for intracellular IFN-γ with conjugated mAbs. A) One representative flow cytometry plot showing intracellular IFN-γ positive fractions of gated populations of CD4⁺ and CD8⁺ T cells in HCL volunteers. B) One representative flow cytometry plot showing intracellular IFN-γ positive fractions of gated populations of CD4⁺ and CD8⁺ T cells in healthy controls. C) Flow cytometry data of all volunteers were pooled and are shown as Median (horizontal line) with interquartile ranges (box) and range (whiskers) of intracellular IFN-γ positive cells.</p

The purity of peripheral blood enriched CD4⁺ and CD8⁺ T cell populations after magnetic beads isolation.

<p>CD4⁺ (A) and CD8⁺ (B) lymphocytes were isolated from a cell suspension of PBMC by positive selection using CD4/CD8 cocktail Abs and anti-CD4 or anti-CD8 coated magnetic nanoparticles. The purity of yielded T cell populations was analysed by flow cytometry using conjugated mAbs.</p