Mohsen Amin

Staphylococcal enterotoxin B/texosomes as a candidate for breast cancer immunotherapy

A new recombinant construct made up of two components, texosomes (TEX) and staphylococcal enterotoxin B (SEB), showed cytostatic properties against several types of tumor cells in vitro. Here, we aimed to assess the construct’s antitumor immunogenicity in a murine tumor model. SEB was anchored onto purified texosomes and was used for immunization of mice before challenge with 4T1 cells. Tumor size, survival time, necrosis, metastasis rate, and the levels of IL-2, IL-4, IL-17, IL-12, TNF-α, and IFN-γ were measured. Immunization of the mice with TEX-SEB increased the stimulation index of splenocytes significantly compared with the PBS-treated mice (p < 0.01). In addition, there was a significant increase of TNF-α, IL-2, and IFN-γ secreted from isolated splenocytes of the mice immunized by either TEX-SEB, TEX + SEB, TEX, or SEB in comparison with PBS (p < 0.001, p < 0.001, and p < 0.05, respectively), whereas no significant change of IL-4 secretion was observed in any treated groups. Finding from tumor tissue homogenate testing showed that the level of IL-17 and IFN-γ among mice immunized with TEX-SEB was significantly lower than PBS-treated group (p < 0.05). IL-12, IL-4, and TNF-α levels were not significantly different from PBS- and TEX-SEB-immunized groups except in the SEB-immunized mice. Although TEX-SEB immunization relatively prolonged the survival of the mice, it had no inhibitory impact on tumor size. Pathologic manifestations showed the significant rise of necrosis after immunization with TEX-SEB compared to PBS (p < 0.01). Overall, our findings suggest that the presence of SEB rescues tumorigenesis effects of TEX making the construct an appropriate candidate for tumor immunotherapy.

Detection of Class I Integrons in Staphylococcus aureus Isolated From Clinical Samples

Background: Staphylococcus aurous is a major pathogen, causing variety of diseases and death in Iran and in the world. Despite the use of a spectrum of new antibiotics, this organism has caused severe infections in burns as well as in different parts of the body, due to acquired drug resistance. Widespread inappropriate use of antibiotics in treating bacterial infections has led to the selection and circulation of resistant strains and the growing risk of transferring resistant genes to sensitive bacteria. One of the causes of antibiotic resistance in S. aurous strains is the gain of resistance genes including integrase and qac/sul1. Objectives: The purpose of this study was to investigate the presence of class 1 integron in S. aurous strains isolated from clinical samples for the first time in Iran. Materials and Methods: This descriptive study was performed on 200 strains of S. aurous isolated from patients admitted to Baqiyatallah Hospital in Tehran in 2013. These strains were confirmed using biochemical and serological tests and the presence of class 1 integron was determined by polymerase chain reaction (PCR). Results: Among the 200 samples, 1% of the strains (two isolates) contained the class 1 integron gene. The results of this study showed that the highest frequency of the obtained samples belonged to males and the isolates occurred mostly in individuals aged 51-60 years old. The highest number of strains was found in wound samples. The strains were most frequently isolated from the emergency ward and the intensive care unit (ICU). Conclusions: Findings of this study showed that integron can have a limited frequency in S. aurous isolated from clinical sample in Tehran.

Chemical Composition and Antimicrobial Activity of Essential Oils from Different Parts of Daucus littoralis Smith subsp. hyrcanicus Rech. f.

Abstract Daucus littoralis Smith subsp. hyrcanicus Rech. f (Apiaceae) is an endemic species in north of Iran. The chemical composition of the essential oils obtained from different parts (leaves and stems, roots, flowers and fruits) of this plant have been investigated by GC and GC-MS for the first time. The essential oils of all parts were characterized by high amounts of sesquiterpene hydrocarbons (62 to 67 %) especially germaceren D (22 to 36 % in leaves, stems, flowers and roots) and acorenone B (19.7 to 57.5 % in all parts). So this can be considered as a remarkable difference between this species and other Daucus species. The high amounts of phenylpropanoids (8 to 30.3 %) were also found in all parts. Fruits oil was characterized by higher amounts of phenylpropanoids(30.3 %) while other parts were consisted of 8 to 10 % of these compounds. Myristicin was identified in leaves/stems, roots and fruits oil of this plant as one of the major components (8.6 %, 9.5 % and 15.2 % respectively). Elimicin (15.1 %) was identified as a major component in fruits but in lower amount (0.4 to 3.6 %) in other parts. All the essential oils have antimicrobial activity against Staphylococcus aureus, Escherichia coli and Candida albicans with MIC value ranged of 20-40 μl/ml.

Heterologous expression of 3-O-deacylase in Acinetobacter baumannii modulates the endotoxicity of lipopolysaccharide.

The lipopolysaccharide (LPS) of Acinetobacter baumannii is a potent stimulator of proinflammatory cytokines, such as interleukin-6 (IL-6). The 3-O-deacylase (PagL)-modifying enzyme that removes the 3-O-linked acyl chain from the disaccharide backbone of lipid A provides the opportunity to develop a new therapeutic compound that could reduce detrimental inflammatory responses. The plasmid pMMB66EH-PagL obtained by recombinant DNA technology was electroporated into A. baumannii ATCC 19606. Compared with wild-type LPS, outer membrane vesicles and inactivated whole cells of engineered bacteria had a statistically significant decreased ability to produce IL-6. Structural analysis of lipid A by negative-ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry revealed that the profile of lipid A fractions under PagL expression was changed. Taken together, our data showed that recombinant penta-acylated lipid A had less immunoreactivity and that the tetra-acylated version of lipid A with TLR4 antagonist activity decreased the induction of IL-6 production in the murine macrophage cell line J774 A.1.

Cellular immunity survey against urinary tract infection using pVAX/fimH cassette with mammalian and wild type codon usage as a DNA vaccine.

Purpose FimH (the adhesion fragment of type 1 fimbriae) is implicated in uropathogenic Escherichia coli (UPEC) attachment to epithelial cells through interaction with mannose. Recently, some studies have found that UPEC can thrive intracellularly causing recurrent urinary tract infection (UTI). Almost all vaccines have been designed to induce antibodies against UPEC. Yet, the humoral immune response is not potent enough to overcome neither the primary UTI nor recurrent infections. However, DNA vaccines offer the possibility of inducing cell mediated immune responses and may be a promising preventive tool. Materials and Methods In this study, we employed two different open reading frames within mammalian (mam) and wild type (wt) codons of fimH gene. Optimized fragments were cloned in pVAX-1. Expression of the protein in COS-7 was confirmed by western blot analysis after assessing pVAX/fimH(mam) and pVAX/fimH(wt). The constructs were injected to BALB/c mice at plantar surface of feet followed by electroporation. Results The mice immunized with both constructs following booster injection with recombinant FimH showed increased interferon-γ and interleukin-12 responses significantly higher than non-immunized ones (p<0.05). The immunized mice were challenged with UPEC and then the number of bacteria recovered from the immunized mice was compared with the non-immunized ones. Decreased colony count in immunized mice along with cytokine responses confirmed the promising immune response by the DNA vaccines developed in this study. Conclusion In conclusion, DNA vaccines of UPEC proteins may confer some levels of protection which can be improved by multiple constructs or boosters.

Isolation of a new ssDNA aptamer against staphylococcal enterotoxin B based on CNBr‐activated sepharose‐4B affinity chromatography

Staphylococcus aureus are potent human pathogens possessing arsenal of virulence factors. Staphylococcal food poisoning (SFP) and respiratory infections mediated by staphylococcal enterotoxin B (SEB) are common clinical manifestations. Many diagnostic techniques are based on serological detection and quantification of SEB in different food and clinical samples. Aptamers are known as new therapeutic and detection tools which are available in different ssDNA, dsDNA and protein structures. In this study, we used a new set of ssDNA aptamers against SEB. The methods used included preparation of a dsDNA library using standard SEB protein as the target analyte, affinity chromatography matrix in microfuge tubes, SELEX procedures to isolate specific ssDNA‐aptamer as an affinity ligand, aptamer purification using ethanol precipitation method, affinity binding assay using ELISA, aptamer cloning and specificity test. Among 12 readable sequences, three of them were selected as the most appropriate aptamer because of their affinity and specificity to SEB. This study presents a new set of ssDNA aptamer with favorable selectivity to SEB through 12 rounds of SELEX. Selected aptamers were used to detect SEB in infected serum samples. Results showed that SEB c1 aptamer (2 µg SEB/100 nM aptamer) had favorable specificity to SEB (kd = 2.3 × 10−11). In conclusion, aptamers can be considered as useful tools for detecting and evaluating SEB. The results showed that affinity chromatography was an affordable assay with acceptable accuracy to isolate sensitive and selective novel aptamers. Copyright

Texosome-based drug delivery system for cancer therapy： from past to present

Rising worldwide cancer incidence and resistance to current anti-cancer drugs necessitate the need for new pharmaceutical compounds and drug delivery system. Malfunction of the immune system, particularly in the tumor microenvironment, causes tumor growth and enhances tumor progression. Thus, cancer immunotherapy can be an appropriate approach to provoke the systemic immune system to combat tumor expansion. Texosomes, which are endogenous nanovesicles released by all tumor cells, contribute to cell-cell communication and modify the phenotypic features of recipient cells due to the texosomes’ ability to transport biological components. For this reason, texosome-based delivery system can be a valuable strategy for therapeutic purposes. To improve the pharmaceutical behavior of this system and to facilitate its use in medical applications, biotechnology approaches and mimetic techniques have been utilized. In this review, we present the development history of texosome-based delivery systems and discuss the advantages and disadvantages of each system.

The reliability of rifampicin resistance as a proxy for multidrug-resistant tuberculosis: a systematic review of studies from Iran

In Iran, patients showing rifampicin (RIF) resistance detected by the Xpert® MTB/RIF assay are considered as candidates for multidrug-resistant tuberculosis (MDR-TB) treatment. Despite the fact that RIF resistance has been used as a proxy for MDR-TB, little is known about the proportion of isoniazid (INH) resistance patterns in RIF-resistant TB. We systematically searched MEDLINE, Embase, and other databases up to March 2017 for studies addressing the proportion of INH resistance patterns in RIF-resistant TB in Iran. The data were pooled using a random effects model. Heterogeneity was assessed using Cochran’s Q and I2 statistics. A total of 11 articles met the eligibility criteria. Data analysis demonstrated that 33.3% of RIF-resistant isolates from new TB cases and 14.8% of RIF-resistant isolates from previously treated cases did not display resistance to INH. The relatively high proportion of INH susceptibility among isolates with RIF resistance indicated that RIF resistance may no longer predict MDR-TB in Iran. Therefore, the detection of RIF resistance by the Xpert MTB/RIF assay will require complementary detection of INH resistance by other drug susceptibility testing (DST) methods in order to establish the diagnosis of MDR-TB.

Staggered Target SELEX, a novel approach to isolate non-cross-reactive aptamer for detection of SEA by apta-qPCR

BACKGROUND AND OBJECTIVE Aptamers or chemical antibodies are oligonucleotides (DNA or RNA) that are able to bind to various targets with high specificity and affinity such as toxins which are isolated by an in vitro method known as SELEX. To date, there are many SELEX procedures for the isolation of novel aptamers against proteins. However not all modified SELEX are suitable for similar protein based on sequence homology such as staphylococcal enterotoxins. Staphylococcal enterotoxin type A (SEA) is the most prevalent toxin involved in staphylococcal food poisoning (SFP) worldwide. SEA is homologous to Staphylococcal enterotoxin type D (SED) and Staphylococcal enterotoxin type E (SEE) about 50% and 83%, respectively. Here, we have developed Staggered Target SELEX (ST-SELEX) as a novel designed SELEX procedure to acquire specific non-cross-reactive aptamers against SEA as a model protein. METHODS In this study, isolated ssDNA aptamers by ST-SELEX were used for detection of SEA via apta-Real time PCR (apta-qPCR). After in silico analysis of SEA protein with SEE and finding the specific region on the surface of protein, ST-SELEX was carried out in two steps (classical SELEX and Second SELEX). Finally, after isolating high specific aptamers, the apta-qPCR was used for the detection of the SEA. In this technique, poly-clonal antibody against SEA was immobilized on protein G sepharose beads (Ab-PGs). Then, the SEA protein was captured by poly clonal antibody as the target that immobilized on sepharose beads. The isolated aptamers were bound on the surface of SEA protein that captured by Ab-PGs. Finally, the heat-released aptamers were amplified by qPCR. RESULT Our investigation showed that the aptamers were generated in vitro by a ten-round selection process based on ST-SELEX procedure with dissociation constant (KD) value 7.44± 0.6 nM and limit of detection (LOD) of 146.67 fM. DISCUSSION AND CONCLUSION The advantage of ST-SELEX compared to other SELEX methods was to select a specific non cross-reactive aptamer against two or more proteins with high sequence homology. These aptamers can be used in sensitive detection methods such as apta-qPCR.

Prokaryotic toxins provoke different types of cell deaths in the eukaryotic cells

Abstract Bacterial toxins play major roles in developing infections and can cause diverse morphological and physiological changes via certain biochemical mechanisms leading to cell death. Most of the bacterial toxins induce apoptosis in host cell by intrinsic and extrinsic apoptosis pathways that are caused by intracellular organelle dysfunction and caspase cascade activation. Some of bacterial toxins encourage the programmed necrosis (necroptosis) in mature RBCs. In addition, pore-forming toxins can induce autophagy. Another kind of cell death, the pyroptosis, is induced by Salmonella typhimurium and Pseudomonas aeruginosa and Shigella flexneri and the lethal toxin of Bacillus anthracis. Therefore, recently scientists utilize cytotoxic properties of bacterial toxins for designing novel anti-tumor drugs.