Constructing a hybrid molecule with low capacity of IgE binding from Chenopodium album pollen allergens

Allergen specific immunotherapy is the only remedy to prevent the progression of allergic diseases. Nowadays, using of recombinant allergens with reduced IgE-binding capacity is an ideal tool for allergen immunotherapy. Therefore, in this study we focused on a hybrid molecule (HM) production with reduced IgE reactivity from Chenopodium album pollen allergens. By means of genetic engineering, a head to tail structure of the three allergens of the C. album pollen was designed. The resulting DNA construct coding for a 46kDa HM was inserted into an expression vector and expressed as hexahistidine tagged fusion protein in Escherichia coli. IgE reactivity of the HM was evaluated by western blotting, inhibition ELISA and in vivo skin prick test and its immunogenic property was tested by proliferation assay. The recombinant HM was expressed and purified by nickel-affinity chromatography. Comparison of the recombinant HM with a mixture of three recombinant allergens, as well as natural allergens in the whole C. album pollen extract via immunological experiments revealed that it has a much lower potential of IgE reactivity. Furthermore, in vivo skin prick tests showed that it has a significantly lower potency to induce cutaneous reactions than the mixture of recombinant wild type allergens and whole extract while, it had been preserved immunogenic properties. Our results have demonstrated that assembling three allergens of C. album in a hybrid molecule can reduce its IgE reactivity.     

Evaluating clinical abdominal scoring system in predicting the necessity of laparotomy in blunt abdominal trauma

Objectives

Trauma is among the leading causes of death. Medical management of blunt abdominal trauma (BAT) relies on judging patients for whom laparotomy is mandatory. This study aimed to determine BAT patients' signs, as well as paraclinical data, and to clarify the accuracy, sensitivity, specificity, positive and negative predictive value of clinical abdominal scoring system (CASS), a new scoring system based on clinical signs, in predicting whether a BAT patient needs laparotomy or not.

Methods

Totally 400 patients suspected of BAT that arrived at the emergency department of two university hospitals in Tehran from March 20, 2007 to March 19, 2009 were included in this study. They were evaluated for age, sex, type of trauma, systolic blood pressure, Glasgow coma scale (GCS), pulse rate, time of presentation after trauma, abdominal clinical findings, respiratory rate, temperature, hemoglobin (Hb) concentration, focused abdominal sonography in trauma (FAST) and CASS.

Results

Our measurements showed that CASS had an accuracy of 94%, sensitivity of 100%, specificity of 88%, positive predictive value of 90% and negative predictive value of 100% in determining the necessity of laparotomy in BAT patients. Moreover, in our analysis, systolic blood pressure, GCS, pulse rate, Hb concentration, time of presentation after trauma, abdominal clinical findings and FAST were also shown to be helpful in confirming the need for laparotomy (P<0.05).

Conclusion

CASS is a promising scoring system in rapid detection of the need for laparotomy as well as in minimizing auxiliary expense for further evaluation in BAT patients, thus to promote the cost-benefit ratio and accuracy of diagnosis.     

Comparison of technetium-99m IgG with technetium-99m red blood cells labeling in cardiac blood-pool scintigraphy: a preliminary study

This first clinical prospective study was conducted to use of technetium-99m immunoglobulin G ((99m)Tc-IgG) as compared with autologous (99m)Tc-red blood cells (RBC) in gated blood pool ventriculography. We studied 12 patients who referred to us for a possible diagnosis of liver hemangioma or infection. Six patients underwent gated planar blood pool (GPBP) acquisition using (99m)Tc-RBC and 6 GPBP acquisition using (99m)Tc-IgG. The use of (99m)Tc-IgG in cardiac blood pool studies provided comparable images to (99m)Tc-RBC. In conclusion, (99m)Tc-IgG, which is readily available and needs only a single injection, may be an attractive alternative to (99m)Tc-RBC for the estimation of various cardiac function parameters like left ventricular function.     

Interaction of W-substituted analogs of cyclo-RRRWFW with bacterial lipopolysaccharides: the role of the aromatic cluster in antimicrobial activity

The activity of cyclo-RRRWFW (c-WFW) against Escherichia coli has been shown to be modulated by the aromatic motif and the lipopolysaccharides (LPS) in the bacterial outer membrane. To identify interaction sites and to elucidate the mode of c-WFW action, peptides were synthesized by the replacement of tryptophan (W) with analogs having altered hydrophobicity, dipole and quadrupole moments, hydrogen-bonding ability, amphipathicity, and ring size. The peptide activity against Bacillus subtilis and erythrocytes increased with increasing hydrophobicity, whereas the effect on E. coli revealed a more complex pattern. Although they had no effect on the E. coli inner membrane even at concentrations higher than the MIC, peptides permeabilized the outer membrane according to their antimicrobial activity pattern, suggesting a major role of LPS in peptide transport across the wall. For isothermal titration calorimetry (ITC) studies of peptide-lipid bilayer interaction, we used POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline), either alone or in mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG), to mimic the charge properties of eukaryotic and bacterial membranes, respectively, as well as in mixtures with lipid A, rough LPS, and smooth LPS as models of the outer membrane of E. coli. Peptide accumulation was determined by both electrostatic and hydrophobic interactions. The susceptibility of the lipid systems followed the order of POPC-smooth LPS > POPC-rough LPS > POPC-lipid A = POPC-POPG > POPC. Low peptide hydrophobicity and enhanced flexibility reduced binding. The influence of the other properties on the free energy of partitioning was low, but an enhanced hydrogen-bonding ability and dipole moment resulted in remarkable variations in the contribution of enthalpy and entropy. In the presence of rough and smooth LPS, the binding-modulating role of these parameters decreased. The highly differentiated activity pattern against E. coli was poorly reflected in peptide binding to LPS-containing membranes. However, stronger partitioning into POPC-smooth LPS than into POPC-rough LPS uncovered a significant role of O-antigen and outer core oligosaccharides in peptide transport and the permeabilization of the outer membrane and the anti-E. coli activity of the cyclic peptides.     

Cloning and Expression of the Allergen Cro s 2 Profilin from Saffron (Crocus sativus)

BackgroundProfilin is a panallergen that is recognized by IgE in allergic patients. Allergy to saffron (Crocus sativus) pollen has been described in people exposed to its pollen. Saffron contains a profilin that may cause allergic reactions in atopic subjects. The aim of this study was to describe the cloning, expression and purification of saffron profilin from pollen.MethodsCloning of saffron profilin was performed by polymerase chain reaction using specific primers from saffron pollen RNA. Expression was carried out in Escherichia coli BL21 (DE3) using a vector pET-102- TOPO. A recombinant fusion protein was expressed and the recombinant profilin was purified by metal precipitation. Immunological characterization was performed by immunoblotting experiments.ResultsThe 34 kDa- recombinant saffron profilin, Cro s 2, as a fusion protein was purified. Immunoblotting tested with the sera of allergic patients showed a specific reaction with the recombinant Cro s 2 band.ConclusionsThe sequence of Cro s 2 showed a high degree of identity and similarity to other plant profilins and the recombinant saffron profilin, Cro s 2, may be used for target-specific diagnosis and structural analyses and investigation of cross reactivity of Cro s 2 with other plant profilins.     

Production, quality control and pharmacokinetic studies of Ho-EDTMP for therapeutic applications

¹⁶⁶Ho-EDTMP is a major therapeutic agent which is widely used in bone palliation therapy. In this study, a ¹⁶⁶Ho-EDTMP complex was prepared successfully using an in-house synthesized EDTMP ligand and ¹⁶⁶HoCl₃. Ho-166 chloride was obtained by thermal neutron irradiation (1 × 10¹³ ncm⁻²s⁻¹) of natural Ho(NO₃)₃ samples (specific activity = 3–5 GBq/mg), dissolved in acidic media. The radiochemical purity of ¹⁶⁶Ho-EDTMP was checked by ITLC (>99%) and stability studies in presence of human serum and final preparation were performed. The biodistribution of ¹⁶⁶Ho-EDTMP and ¹⁶⁶HoCl₃ in wild-type rats was checked by scarification. SPECT imaging of ¹⁶⁶Ho-EDTMP was also performed in wild-type rats. A comparative accumulation study for ¹⁶⁶Ho-EDTMP and ¹⁶⁶HoCl₃ was performed for vital organs up to 48h. Significant bone accumulation (>70%) of the tracer in 48h was observed.