Western Blot Analysis of Leishmania infantum Antigens in Se-ra of Patients with Visceral Leishmaniasis

Background: Visceral leishmaniasis (VL) is endemic in the northwest and south of Iran. Untreated cases of VL could cause death. The aim of the present study was to evaluate the diagnostic performance of western blotting to detect a specific immunodominant proteins pattern for Leishmania infantum infection using human sera infected with VL. Methods: We studied a panel of 122 cryopreserved human serum samples from the leishmaniasis Research Laboratory, Tehran University of Medical Sciences, Tehran, Iran from 2010 to 2017. Serum samples were collected from visceral (Group I, n: 43) and cutaneous leishmaniasis (CL) (Group II, n: 8) patients, healthy individuals from endemic (Group III, n: 13) and non-endemic (Group IV, n: 16) areas for VL, and patients with other infectious diseases (Group V, n: 42). Total antigens were prepared from the Iranian strain of L. infantum promastigote form. Results: In western blotting method, 34 protein bands of 14 to 163 kDa were recognized using the sera of VL pa­tients. The polypeptide fractions with the highest frequency including 29, 51, and 62 kDa fractions were detected using 81.4%, 79%, and 81.4% of the sera, respectively. These bands were not detected using the sera of the negative control. Moreover, 19-23, 27, 31-35, 143-163, and 109 kDa fractions were detected specifically using the sera of the patients with VL. Conclusion: This technique could be a primary step for further exploration of VL immunodominant antigens for cloning (or any technique) further investigations for future planning

BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING EGFP reporter protein: its immunogenicity in Leishmania-infected BALB/c mice

Abstract Optical reporter genes such as green fluorescent protein (GFP) and luciferase are efficiently and widely used in monitoring and studying the protective/therapeutic potential of candidate agents in leishmaniasis. But several observations and controversial reports have generated a main concern, whether enhanced GFP (EGFP) affects immune response. To address this issue, we studied the immunogenicity of EGFP in vivo by two lines of stably transfected parasites (Leishmania major EGFP or L. major EGFP-LUC ) in BALB/c model and/or as a recombinant protein (rEGFP) produced in vitro by bacteria in parallel. Disease progression was followed by footpad swelling measurements and parasite burden in draining lymph nodes using microtitration assay and real-time PCR, and immune responses were also evaluated in spleen. EGFP-expressing parasites generated larger swellings in comparison with wild-type (L. major) while mice immunized with rEGFP and challenged with wild-type parasite were quite comparable in footpad swelling with control group without significant difference. However, both conventional and molecular approaches revealed no significant difference in parasite load between different groups. More importantly, no significant inflammatory responses were detected in groups with higher swelling size measured by interferon-γ (IFN-γ), interleukin (IL)-10, IL-5, and nitric oxide against frozen and thawed lysate of parasite as stimulator. Altogether, these results clearly revealed that EGFP protein expressed in prokaryotic and eukaryotic hosts is not an immunological reactive molecule and acts as a neutral protein without any side effects in mice. So, EGFP expressing Leishmania could be a safe and reliable substitution for wildtypes that simplifies in situ follow-up and eliminates the animal scarification wherever needed during the study

A Novel Spot-Enhancement Anisotropic Diffusion Method for the Improvement of Segmentation in Two-dimensional Gel Electrophoresis Images, Based on the Watershed Transform Algorithm

Introduction Two-dimensional gel electrophoresis (2DGE) is a powerful technique in proteomics for protein separation. In this technique, spot segmentation is an essential stage, which can be challenging due to problems such as overlapping spots, streaks, artifacts and noise. Watershed transform is one of the common methods for image segmentation. Nevertheless, in 2DGE image segmentation, the noise and artifacts of images cause over-segmentation in the watershed algorithm. Materials and Methods In this study, we proposed a novel spot-enhancement anisotropic diffusion (SEAD) method, based on multi-scale second-order derivatives and eigensystemto enhance the spots and remove noise and artifacts. The proposed SEAD algorithm was plugged to a watershed transform in order to improve the performance of watershed segmentation algorithm. Results The performance of the proposed SEAD method was evaluated on synthetic and real 2DGE images. The proposed algorithm was compared with other segmentation methodsin terms of different criteria including efficiency, precision and true positive rate. The performance of the methods were evaluated in the presence of noise and the results were evaluated by t-test. According to the count of detected spots, precision and efficiency of the proposed method were 0.82 and 0.67 respectively.  The precision and efficiency values of the comparative methods were as follows: 0.65 and 0.42 for MCW algorithm, 0.40 and 0.37 for BWT method, 0.74 and 0.53 for the method proposed by Kostopoulou and 0.76 and 0.55 for the method proposed by Mylona. Conclusion The comparison of the proposed method with four other conventional methods revealed the superiority and effectiveness of the proposed SEAD method