Optimized simple and affordable procedure for differentiation of monocyte-derived dendritic cells from LRF: An accessible and valid alternative biological source.

Dendritic cells are one of the most popular immune cells, which plays a remarkable role in both immunotherapy and tolerance induction. Due to unwanted side effects of leukocyte presence in donated blood, the policy of blood service is the pre-storage reduction of leukocytes, which today, filtration is the most common method for this purpose. The filtration method has led to diminished access to Buffy coat as a generally used conventional source of biological cells. We developed a simple, affordable, and reproducible method for dendritic cell differentiation from filter-derived monocytes and, the results of the filter study were compared with differentiated DCs from the conventional buffy coat-derived monocytes. The Monocytes were recovered from leukoreduction filter using an optimized protocol with supplemented PBS buffer. Following the adhesion method, CD14+ Monocyte-enriched population with the purity of 94 % was obtained. After cytokine stimulation over a 6-day period and maturation induction by LPS, differentiated DCs were evaluated for morphology, surface markers (CD86, CD40, CD83 and, HLA-DR), antigen uptake potency and IL-12 secretion. Analysis and comparison of the results represented no significant difference between the two groups. Accordingly, we conclude that leukoreduction filters could be introduced as a reliable and research-grade source of monocyte for DC generation in biological research.

Synthesis and characterization of Pseudomonas aeruginosa alginate-tetanus toxoid conjugate.

Chronic infection with Pseudomonas aeruginosa is the main proven perpetrator of lung function decline and ultimate mortality in cystic fibrosis (CF) patients. Mucoid strains of this bacterium elaborate mucoid exopolysaccharide, also referred to as alginate. Alginate-based immunization of naïve animals elicits opsonic antibodies and leads to clearance of mucoid P. aeruginosa from the lungs. Alginate was isolated from mucoid P. aeruginosa strain 8821M by repeated ethanol precipitation, dialysis, proteinase and nuclease digestion, and chromatography. To improve immunogenicity, the purified antigen was coupled to tetanus toxoid (TT) with adipic acid dihydrazide (ADH) as a spacer and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC) as a linker. The reaction mixture was passed through a Sepharose CL-4B column. The resulting conjugate was composed of TT and large-size alginate polymer at a ratio of about 3 : 1; it was non-toxic and non-pyrogenic, and elicited high titres of alginate-specific IgG. Antisera raised against the conjugate had high opsonic activity against the vaccine strain. The alginate conjugate was also able to protect mice against a lethal dose of mucoid P. aeruginosa. These data indicate that an alginate-based vaccine has significant potential to protect against chronic infection with mucoid P. aeruginosa in the CF host.

A retrospective survey of liver fluke disease in livestock based on abattoir data in Shiraz, south of Iran.

Meat-inspection records in an abattoir located in the Fars province (southern part of Iran) from 20 March 1999 to 19 March 2004 were used to determine the prevalence and long-term trend of liver fluke disease in sheep, cattle and goats in the region. A total of 844,039 animals (cattle 131,716; sheep 577,090; goats 135,233) slaughtered in the 5-year period and overall 34,856 (4.1%) livers were condemned. Fasciolosis and dicrocoeliosis were responsible for 54 and 21% of total liver condemnations, respectively. The prevalence of liver condemnations due to fasciolosis was decreased from 3.89, 3.20 and 2.63% in 1999-2000 to 1.07, 0.59 and 0.24% in 2003-2004 for cattle, sheep and goats, respectively. The corresponding features for dicrocoeliosis were similar, declining from 1.47, 1.76 and 2.10% in 1999-2000 to 0.69, 0.34 and 0.25% in 2003-2004, respectively. Drought climatic conditions in conjunction with a greater awareness among farmers could be responsible factors.

Determination of dissociation constants of anti-ALP monoclonal antibodies by an ELISA - based method.

BACKGROUND: Dissociation constant (Kd) is of major significance in immunoassay. Since affinity may be influenced by the immunoassay methodology it is important to determine this parameter under the condition of the assay used. OBJECTIVE: To employ a rapid and simple ELISA- based method for measuring dissociation constants of two Alkaline Phosphatase (ALP) specific MAbs (A1G8F7 and A1G9G3) established in our laboratory. METHODS: A simple and rapid method on enzyme- linked immunosorbent assay (ELISA) was developed for measuring the dissociation constant of antibody ­ antigen reactions. In this method the ability of increasing amounts of antigen to bind to antibody measured in the fluid phase. RESULTS: Based on the data obtained from this study, the dissociation constants of A1G8F7 and A1G9G3 MAbs were 3.8 × 10−9 and 4.3 × 10−9 M, respectively. CONCLUSIONS: A1G8F7 and A1G9G3 MAbs with reasonably high affinity are suggested for used in very immunoassay such as ELISA, immunocytochemistry and immunihistochemistry (APAAP method).

Differential genomics output and susceptibility of Iranian patients with unilocular hydatidose.

BACKGROUND: The objective of this study was to investigate HLA-DRB1*and DQB1* allelic polymorphisms in Iranian patients with hydatidose. This is the first survey dealing with the correlation between HLA-DRB1* and DQB1* alleles and cystic echinococcosis in Iranian patients. METHODS: The study was carried out on 56 patients with confirmed cystic echinococcosis and 30 apparently healthy individuals living in Arak- Markazi Province by HLA-DRB1 and DQB1 typing through PCR-SSP method. The first step was to identify the patients and blood sampling. DNA was prepared from whole blood and PCR-SSP with 31 primer mixes for per sample was used. PCR reaction mixtures were loaded in agarose gels and bands were observed under UV illumination and gel document after electrophoresis. Analysis of results was carried out with specific softwares and frequency and interpretation tables for calculation of P-value in χ(2) test were provided via Fisher's exact test. Significant samples were analyzed by logistic regression and odds-ratios were calculated. RESULTS: A statistically significant positive association was found between HLA-DQB1*03 and the resistance to cystic echinococcosis (P < 0.02) (odds-ratio = 2.87). CONCLUSION: Immunogenetic susceptibility to unilocular hydatidose varies according to the HLA antigens in Arak, Markazi Province, and DQB1*03 molecules are associated with the level of immune response to parasite antigens.

Comparison of three techniques for generation of tolerogenic dendritic cells: siRNA, oligonucleotide antisense, and antibody blocking.

In recent years, a new view of dendritic cells (DCs) as a main regulator of immunity to induce and maintain tolerance has been established. In vitro manipulation of their development and maturation is a topic of DC therapeutic application, which utilizes their inherent tolerogenicity. In this field, the therapeutic potential of antisense, siRNA, and blocking antibody are an interesting goal. In the present study, the efficiency of these three methods--siRNA, antisense, and blocking antibody--against CD40 molecule and its function in DCs and BCL1 cell line are compared. DCs were separated from mouse spleen and then cultured in vitro using Lipofectamine 2000 to deliver both silencers; the efficacy of transfection was estimated by flow cytometry. mRNA expression and protein synthesis were assessed by real time-PCR and flow cytometry, respectively. By Annexin V and propidium iodine staining, we could evaluate the viability of transfected cells. Knocking down the CD40 gene into separate groups of DCs by siRNA, antisense, and blocking antibody treated DCs can cause an increase in IL-4, decrease in IL-12, IFN-γ production, and allostimulation activity. Our results indicated that, in comparison to antisense and blocking antibody, siRNAs appear to be quantitatively more efficient in CD40 downregulation and their differences are significant.