Computational evaluation of a fusion protein consisted of pertussis toxin and filamentous hemagglutinin from Bordetella pertussis to target Claudin-4 using C-terminal fragment of Clostridium perfringens enterotoxin.

Pertussis, caused by Bordetella pertussis is still one of the controversial diseases worldwide due to its high prevalence in both the developed and the developing countries, especially among young children. As currently approved vaccines are not protective enough and provide Th2-type immune responses, there is an urgent need to develop new vaccines. In the current study, we applied the C-terminal fragment of Clostridium perferingens enterotoxin (C-CPE) as a delivery system and F1S1 fragment (Filamentous hemagglutinin (F1) and subunit 1 of pertussis toxin (S1) of B. pertussis to design a novel chimeric protein in silico, to target Claudin-4 receptors in mice lung cells. To achieve this goal, the primary, secondary and tertiary structures of the fusion protein were evaluated and the interaction of this protein with Claudin-4 receptors was studied. Molecular dynamic (MD) simulation analysis was performed to investigate the physical movement of atoms in a fixed period. According to the results; the full-length fusion protein has consisted of 807 amino acid residues which could be classified as a stable protein. There was a convenient consistency between the 3D predicted structure and the secondary structure prediction. An acceptable percentage of the residues were also detected in the most favored and allowed regions for the model. Based on HADDOCK results, there were no considerable differences between the interactions and MD simulation analysis, indicating that the predicted structures were stable during the simulation. Altogether, the data reported in this study represents the first step toward developing a nasal vaccine candidate against B. pertussis infection. Communicated by Ramaswamy H. Sarma.

Fabrication of chitosan-polyethylene glycol nanocomposite films containing ZIF-8 nanoparticles for application as wound dressing materials.

Biocompatible nanocomposite films based on chitosan (CS) and polyethylene glycol (PEG) polymers containing cephalexin (CFX) antibiotic drug and zeolitic imidazolate framework-8 (ZIF-8) nanoparticles (NPs) were designed and fabricated to develop wound dressing materials capable of controlled drug release. Swelling experiment was performed in three acidic, neutral, and alkaline solutions. The tensile strength test reflected that upon increasing the NPs loading within the films, the tensile strength was enhanced but the elongation at break was diminished. The release of the CFX was intensively increased within approximately 3, 8, and 10 h (burst release) in acidic, neutral, and alkaline media, respectively while after that the CFX was smoothly released over time (sustained release). The antibacterial activities of all films were examined against Gram-positive (S. aureus, B. cereus) and Gram-negative (E. coli, P. aeruginosa, and Acinetobacter) bacteria frequently found in the infected wounds. Moreover, the MTT assay revealed that all films had high cell viabilities towards the L929 fibroblast cells confirming these nanocomposites could be used as favorable wound dressing materials. Finally, the film containing 4% ZIF-8 NPs (film 5) was chosen as the best sample due to it revealed appropriate mechanical properties, swelling, drug release and cell viability among all samples examined.

Review of the current use and evaluation of cell substrates for producing biologicals in selected countries.

In 2010, the WHO guidance document for the evaluation of cell substrates for producing biologicals was replaced with updated recommendations and in May 2013 an implementation workshop on the new recommendations was held in Beijing, China. As part of this workshop, a survey of the use and evaluation of cell substrates for producing biologicals was undertaken and the information obtained was updated in June 2014. The purpose of survey was to capture the status of national requirements related to cell substrates in various countries with particular emphasis on whether or not the updated WHO recommendations had been, or were to be, incorporated into national requirements. This paper reports the outcome of the survey and is based on information provided by regulators in eleven countries. Since the publication of the updated WHO recommendations, several activities such as the implementation workshop and publications have been undertaken by the WHO. The aim of these activities, including the publication of this article, is to contribute to the implementation of WHO recommendations so as to reduce regulatory gaps between national requirements and globally agreed expectations.

Human polymorphonuclear leukocytes produce cytokines in response to Leishmania major promastigotes.

Polymorphonuclear leukocytes (PMN) release cytokines that may influence the development of the subsequent adaptive immune response. Little is known about cytokines produced by human PMN in response to Leishmania (L.). In this study, mRNA expression of Interleukin (IL)-12p40, IL-12p35, Interferon (IFN)-γ, transforming growth factor (TGF)-ß, IL-1, and IL-4 in PMN of volunteers stimulated with L. major promastigotes has been investigated by real-time PCR and the results were confirmed by flow cytometer. The results showed that L. major did not induce mRNA expression of IL12p40, IL12p35, IFN-γ, and TGF-ß in PMN, while IL-1 and IL-4 mRNA were induced. Flow cytometry results confirmed no IFN-γ production by PMN with or without stimulation. IL-12p70 was present in untreated and L. major-treated PMN, and these cells release IL-12 following incubation with L. major. Significant amount of IL-1 even without treatment with promastigotes was detected in PMN. Moreover, the proportion of PMN, which produce IL-1 in response to L. major, was increased compared with the percent of unstimulated IL-1-producing PMN. The results showed the accumulation of small amounts of IL-4 in PMN after stimulation. In conclusion, our results indicate that IL-12 and IL-1 are pre-stored in human PMN, nor L. major induces IL-1 and IL-4, but not IL-12, IFN-γ, nor TGF-ß expression in these cells.

IgG1 and IgG2a profile of serum antibodies to Leishmania major amastigote in BALB/c and C57BL/6Mice.

It is well accepted that in experimental model of Leishmania major infection, BALB/c mice mount a Th2 response and produce IgG1 predominantly whereas C57BL/6mice enhance Th1 response with the biased production of IgG2a antibodies. Therefore, screening for parasite antigens on the basis of reactivity with sera from infected susceptible or resistant mice might be used for the identification of Th1- or Th2-inducing antigens.In this study, the antigenic profile of Leishmania major amastigote that induce IgG1 or IgG2a isotypes in infected BALB/c or C57BL/6 mice were compared.Western blot analyses revealed that 27, 40, 43, 45 and 114 kDa proteins elicited IgG1 and not IgG2a in both BALB/c and C57BL/6 mice and 55 kDa protein was recognized exclusively by IgG1 of BALB/c mice sera. On the contrary, the bands corresponding to proteins with molecular weights (MW) of 30, 35, 52, 58 and 66 were intensively immunostained with IgG2a from C57BL/6 mice which made them potential candidates for eliciting Th1 response.In conclusion, the results showed that the generation of the immune responses depended on the mouse strain and some leishmanial antigens had an intrinsic potency to elicit Th2 responses.

Toll-like receptor 4 polymorphisms predispose to cutaneous leishmaniasis.

The clinical spectrum of cutaneous leishmaniasis (CL) is extremely variable. Studies in experimental leishmaniasis have revealed a role for TLR4 in control of infection. In the present study the associations between TLR4 mutations (Asp299Gly and Thr399Ile) with outcome of CL have been investigated. Genotyping for Asp299Gly and Thr399Ile was performed in patients with chronic (N = 22) and acute (N = 61) CL, asymptomatic (N = 45) and healthy leishmanin skin test negative individuals (N = 75). The results showed the frequency of the Asp299Gly genotype was increased in patients with chronic disease (OR 25.3, 95% CI 5.2-115.6, P < 0.001) and patients with acute disease (OR 8.03, 95% CI 1.7-37.7, P = 0.006) compared to LST negative subjects. Thr399Ileu genotype was significantly over represented among patients with chronic disease (27.3%, P < 0.001), patients with acute disease (13.1%, P = 0.016), and asymptomatic donors (15.6%, P = 0.008) in comparison with LST negative normal group (1.3%). Both variants were found together more frequently in patients with chronic disease compared to the patients with acute disease (P = 0.045), and asymptomatic donors (P = 0.045). The results provide evidence that polymorphisms of TLR4 gene may lead to the increased susceptibility to and severity of infection by Leishmania major. The concomitant carriage of both mutations increases the susceptibility of individuals to CL.

Lack of association of Toll-Like Receptor 2 Arg753Gln with cutaneous leishmaniasis.

The aim of the present study was to investigate the frequency of Arg753Gln and Arg677Trp polymorphisms of TLR2 in patients with cutaneous leishmaniasis (CL) compared to healthy controls. The polymorphisms were genotyped by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and amplification refractory mutation system assay (ARMS-PCR). The results showed that the frequency of Arg753Gln genotype was 14.3% and 10.1% in CL patients and normal controls, respectively. No one in either group was homozygous for the mutation. There was no significant difference in the genotype frequency. In contrast to the results for Arg753Gln polymorphism, we did not detect any case with Arg677Trp polymorphism in either control or patient group. In conclusion the TLR2 mutations are found equally in CL patients and healthy subjects.

Immune response to Leishmania antigen in anthroponotic cutaneous leishmaniasis.

BACKGROUND: Leishmania (L.) tropica is the causative agent of anthroponotic cutaneous leishmaniasis (ACL) in Iran. The disease often heals within a year; however, the non-healing forms of disease are also known. The immunologic responses to L. major infection have been studied in depth, however little is known about the immune status of L. tropica-infected patients. MATERIALS AND METHODS: This study was conducted to evaluate T-cell responses to Leishmania antigen in non-healing patients, patients with acute lesion, and healthy donors. Peripheral blood mononuclear cells (PBMC) were cultured with antigen and lymphoproliferative responses were determined. Cytokine profile including gamma interferon (IFN-gamma), interleukin (IL)-5, and IL-13 in supernatants of stimulated cells was also determined. RESULTS: The results showed PBMC from both groups of patients proliferated vigorously in response to Leishmania antigens. The levels of IFN-gamma and IL-13 were comparable between patients with acute lesions and non-healing patients. Non-healing patients had significantly higher median levels of IL-5 than patients with acute lesions. The cells from healthy individuals did not respond to Leishmania antigens. CONCLUSIONS: High levels of IFN-gamma, IL-5, and IL-13 in non-healing patients suggest a mixed Th1/Th2 response, whereas patients with acute lesion respond to infection by Th1-type response.