فهرست مطالب nima khoramabadi

Clostridioides difficile is one of the important causes of hospital infections worldwide. Regarding the importance of Clostridioides difficile infection (CDI) epidemics and limited available prevalence reports of CDI in Iran, the present investigation was done on the incidence of CDI in hospitalized patients from 2017-2018. Our study outlined the requirements for CDI and followed up on the causative agent of antibiotic-associated diarrhea (AAD).

We evaluated the CDI rates by multiplex real-time polymerase chain reaction (PCR) assay directly from inpatient fecal samples with a history of antibiotic therapy (2-8 weeks) and combined with anaerobic culture and toxicity assessments to isolate toxigenic and non-toxigenic types. The results were analyzed through one-way analysis of variance (ANOVA), pairwise two-tailed correlation, and regression using SPSS software version 25.0 (IBM® SPSS® Statistics, USA).

Among 491 fecal samples, 49 (9.9%) toxigenic C. difficile were characterized by real-time PCR, while 40 were isolated by toxigenic culture and cytotoxicity assay. Toxin profiling showed 43 (9.7%) tcdA+/tcdB+ and 6 (1.4%) tcdB+.

A considerable prevalence of CDI among patients with AAD was demonstrated, and causative organisms mostly produce both toxins A and B. Since C. difficile still has problematic treatment and is costly, rapid and early detection may help to curb C. difficile infection more efficaciously.

Brucella spp. are intracellular pathogens, therefore cell-mediated immunity is the main response to inhibit survival and growth of the bacteria in vertebrate host. Many eukaryotic plasmid vectors are being used in setting up DNA vaccines which may show different efficiencies in same conditions. This is important in designing the vaccines and immunization strategies. We looked into the probable differences of immune responses induced by different eukaryotic DNA plasmid vectors (pcDNA3.1 and pVAX1) harboring the same Omp31 gene of B. melitensis. Female BALB/c mice were immunized with pcDNA-omp31 and pVAX-omp31 and further boosted with recombinant Omp31. Subclasses of specific serum IgG against the rOmp31 were measured by ELISA. Cytokines responses to rOmp31 in Splenocyte cultures of the immunized mice were evaluated by measuring the production of IL4, IL-10, IL-12 and IFN-γ. Protective responses of the immunized mice were evaluated by intraperitoneal challenge with pathogenic Brucella melitensis 16M and Brucella ovis PA76250. Both DNA vaccine candidates conferred potent Th1-type responses with higher levels of cytokines and immunoglobulins observed in mice immunized with pVAX-omp31. Although pcDNA-omp31 and pVAX-omp31 both elicited protective immunity, mice immunized with the latter showed a higher protection against both B. melitensis and B. ovis PA76250. The results of this study highlight the significant differences between efficiency of diverse plasmid backbones in DNA vaccines which code for an identical antigen. Comparing various plasmid vectors should be considered as an essential part of the studies aiming construction of DNA vaccines for intracellular pathogens.

Many studies have focused on the potent anti-cancer activity of Arteether (ARE). However, the hydrophobic property of this drug limits its application. To increase the bioavailability of ARE, we formulated a Nanosystem (NS) of Folic Acid (FA), Chitosan (CS), and Fe3O4 for delivery of ARE into breast cancer. The CS-coated Fe3O4 was synthesized by co-precipitation of Fe2+ and Fe3+ in CS gel-like solution. Then, it was conjugated with FA and ARE. The properties of ARE loaded Nanoparticles (NPs) were characterized by Dynamic Light Scattering (DLS), Fourier Transform-Infrared (FTIR) spectra, Scanning Electron Microscopy (SEM), drug loading efficiency, and drug release. The bioactivity of this complex was evaluated in vitro and in vivo settings. Tumor volume was measured, and the cytokines of Interferon-gamma IFN-γ and interleukin 4 (IL-4) were assessed in mice splenocytes. DLS showed an average size of 198nm and the charge of about -7mV. FTIR confirmed the formation of ARE loaded NPs and SEM indicated its solid, dense structure. The drug exhibited a loading capacity of (20%) and significant release in citrate buffer with pH 5.4 compared with phosphate-buffered saline with pH 7.4. The NS showed significant inhibitory effect on the growth of 4T1 cell line and tumor volume. It also augmented IFN-γ and IL-4 production in breast cancer-bearing mice. ARE in FA-CS-Fe3O4 composite NPs may significantly suppress tumor growth. This NS can be utilized in the nano-based drug delivery system for the treatment of breast cancer.

Brucellosis is still an important zoonotic infection and evaluation of immunologic properties of various bacterial antigens along with different vaccination strategies helps in designing efficient vaccines against the disease. The aim of this study is to immunological evaluate the eukaryotic vector pVAX1, carrying the outer membrane protein gene of 31 kDa (Omp31) B.melitensis.
In this study which was carried out in 2014, whole sequence of omp31 of B. melitensis was inserted between BamHI and XhoI of pVAX1 plasmid vector. Female BALB/c mice aged 6-8 weeks (purchased from Pasteur Institute of Iran) were immunized intra-muscularly with 100μg of the construct, followed by either protein or plasmid boosters separately. The level of IL-4, IL-12, IFN-γ, total serum IgG, and specific IgG1 and IgG2a against recombinant Omp31 were evaluated. Finally the protective immune response following exposure to B. melitensis 16M was evaluated.
DNA-vaccine omp31 career with protein reminders Omp31, stimulate higher levels of IFN-γ, IL-12 and IgG2a compared to groups of DNA-vaccine or recombinant protein. Protective immunity was also significantly higher in mice which immunized with DNA vaccine– protein regimen.
Mice which immunized with DNA vaccine–protein regimen showed a significantly higher levels of IL-12 and IFN-γ along with serum IgG2a which together imply augmentation of T cell-mediated immune responses against Omp31. The latter was confirmed by significant protective response to B. melitensis 16M challenge.

Background and Methicillin resistant Staphylococcus aureus (MRSA) is a community-associated pathogen that is so common in hospitals. Antibiotic resistance and poor clinical outcome provide great reasons for using immunization strategies based on antibodies. The aim of this study was to investigate passive immunity using recombinant anti-MRSA ScaF antibody in a mouse model.
In this study, the recombinant protein, ScaF was purified through denaturation method. This protein combined with Freund’s adjuvant was injected into a rabbit to produce antibody. Then, the antibody produced against ScaF was injected to mice infected by S. aureus by two regimens of pre- and after infection treatment. Afterwards, the mortality rate in mice was compared between the control treatment group (vancomycin therapy) and negative control group (non-immune rabbit serum).
Out of 10 mice in two groups of pre- and after infection treatment that received antibody against ScaF antigen 20% and 10% were protected, respectively, that did not show a significant difference compared with non-immune rabbit serum group (negative control).
Passive immunization by antibody cannot play a considerable role in the control of infections caused by S. aureus. Therefore, other mechanisms of immune system for the protection against this bacterium including stimulating of cytokine-dependent cell-mediated immunity may be useful in prevention of infections caused by S. aureus.

Following our interest in reaching for a molded rubber article with possible detergent contact applications, durable silver nanopowder (AgNP) is synthesized by arc discharge, then mixed with varying ratios of ethylene propylene rubber (EPR), affording novel AgNP@EPR nanocomposites. X-ray diffraction (XRD) patterns of AgNP as well as AgNP@EPR show no trace of impurity, while scanning electron microscopy (SEM) indicates an average diameter of 50 nm for the former. Transmission electron microscopy (TEM) images while confirm the SEM results, show quite a few 5 nm AgNP particles lying beside some micro crumbs. Our DC arc discharge technique involves explosion of movable silver anode and static cathode by a current pulse between 5 to 10 A cm-2. A solution blending method is employed for preparation of AgNP@EPR nanocomposites. The AgNP is first dispersed in toluene using an ultrasonic homogenizer, and then thoroughly mixed with EPR in the same solvent whose removal gives nanocomposites of 2, 4, 6 and 8 vol% AgNP in EPR,  showing strong antibacterial activity against both Escherichia coli and Staphylococcus aureus.

Pathogenic Pseudomonas aeruginosa strains produce a polar flagellum that required for motility, adhesion, invasion and secretion of virulence factors. The aim of this study was to evaluate the effect of prevention and treatment with anti-recombinant type B flagellin antibody in a burned mouse model. One hundred twenty six mice were divided into 16 groups injected with different regimen of anti-recombinant type B flagellin antibody. Infections were caused by sub-dermal injection of P. aeruginosa PAO1 and PAK strains at the burn site. Groups were monitored for mortality for one week. Additionally, functional activity of this antibody was assessed by motility inhibition and opsonophagocytosis assays. Immunotherapy with rabbit antisera substantially increased (85.71%) survival rate of mice challenged with a homologous strain PAO1 compared with the control PBS. The mortality rate in mice infected by the heterologous strain PAK was only 28.57%. This antibody increased phagocytosis killing of the homologous strain but it only had a slight effect on the heterologous strain. Passive immunotherapy protected burned mice challenged with the homologous strain but showed lower efficacy against the heterologous strain.

Designing an effective vaccine against human immunodeficiency virus (HIV)-1 is a global health priority. Multi-epitope vaccines offer several potential advantages that may be promising in case of mutable divergent pathogens such as HIV-1. Herein, a multiepitopic recombinant protein containing various HIV-1 antigens was expressed in E. coli cells and its immunogenicity in combination with different adjuvants was initially evaluated in BALB/c mouse. HIVtop4 sequence spanning the junction of six amino acid fragments (Gag158-186, Pol150-190, ENV296-323, ENV577-610, Tat1-20 and Tat44-61) was designed based on immunoinformatic analysis to reduce the creation of junctional epitopes, improve the cleavage of proteasome and avoid the local accumulation of hydrophobic regions. Synthesized nucleotide sequence corresponding to HIVtop4 was cloned into pET23a plasmid. Expression of pET-HIVtop4 plasmid was induced in BL21 (DE3) E. coli cells by addition of 1 mM IPTG during 3 h culture and the protein was purified by Ni-NTA column chromatography and further confirmed against anti-His antibody in western-blotting. Groups of BALB/c mice (n=6) were immunized three times with 2 weeks interval, subcutaneously with 10 m g of candidate vaccine adjuvanted in Complete Freund’s adjuvant, Montanide ISA70 and Alum with suitable control groups. Two weeks after last immunization lymphocyte proliferation was measured with Brdu, IL-4 and IFN- g cytokines with ELISA, total antibody and IgG1, IgG2a isotypes with indirect ELISA methods. Results showed that Immunization with HIV-1 tat/pol/gag/env led to a significant increase in the proliferative responses of lymphocytes, IL-4 and IFN-γ cytokine production and humoral immune response in comparison with the control groups. In this study we concluded that Tat, Env, Pol, Gag with adjuvants (Montanide, Alum and CFA) has potentials as a candidate vaccine against the HIV-1 virus.

Echinococcosis is a zoonotic parasitic disease of humans and various herbivorous domestic animals transmitted by the contact with domestic and wild carnivores, mainly dogs and foxes. The aim of this study is the production, purifica­tion and evaluation immunogenicity of new construction of EG95 protein. The recombinant plasmid pET32-a+ used for Eg95 expression was con­structed with the EG95 gene of Echinococcus granulosus fused with the thioredoxin tag. This recombinant clone was over expressed in Escherichia coli BL-21 (DE-3). The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in cell lysate. The purification was performed under denaturing conditions in the presence of 8M urea by Ni–NTA column and dialysis. The purified recombinant proteins were confirmed with western blot analysis using polyclonal antiserum. To find out the immunogenicity of the purified protein, the BALB/c mice (10 mice/group) were immunized by injecting 20 µg rEG95 protein formulated in Freund’s and alum adjuvant. Immunization of mice with rEG95 using CFA/IFA and alum adjuvant generated high level of total antibody. In proliferation assay, the lymphocytes were able to mount a strong proliferative response with related production of IFN-ɣ, IL-12 and TNF-α but with low secretion of either IL-4 or IL-10. The humoral and cellu­lar immune responses against rEG95 suggested a mixed Th1/Th2 response with high intensity toward Th1. Our findings suggest that new construct of rEG95 formulated with CFA/IFA and alum adjuvant elicited strong cellular and humoral responses support­ing further development of this vaccine candidate.

One of the most common diseases between zoonosis - especially in developing countries – is brucellosis. Identification of Brucella cell antigen combinations in terms of the amount and type of immune response in infected hosts, are important in vaccine design. 31kDa Brucella cell surface protein (BCSP31) is shared among all Brucellae. We aimed to define specific immune responses to BCSP31 which are elicited during infection of murine host with B. abortus. Surface protein of 31 kDa (BCSP31) is present in all strains, and here, the host immune response during murine infection with Brucella abortus which are formed in proportion to the proteins are studied. Recombinant BCSP31 (rBCSP31) of B. abortus was produced and purified. One group of BALB/c mice were infected with pathogenic B. abortus 544 and maintained for 60 days; a no-infected group of mice also was considered. Specific serum antibodies directed to rBCSP31 was evaluated through ELISA. Splenocytes of mice were cultured and stimulated with rBCSP31; thereafter, IL-4, IL-12 and IFN-γ cytokine responses of spleen lymphocytes were assessed. We detected a remarkable IgG titer along with IgG1 and IgG2a specific to recombinant BCSP31 in serum samples from infected mice. Significant titers of IgG, IgG1 and IgG2a antibodies than BCSP31 in the the serum of mice infected with the virulent strain were observed. The Evaluation of Splenocytes responses to rBCSP31 also showed a significant IL-12 and IFN-γ production along with remarkable IL-4 production in infected mice. All responses were significantly different from that of non-infected mice (p<0.05). These findings suggest that specific humoral and cell-mediated responses to BCSP31 is formed during murine host infection with B. abortus. Based on these findings, rBCSP31 can be used in further design of immunogenic strategies for vaccination against brucellosis.

Antigen 85 complex of Mycobacterium tuberculosis includes three immunogenic proteins which are important TB vaccine candidates. The use of these protein as a component of subunit vaccines, as a part of DNA vaccines or recombinant BCG boosters, enhances their recombinant production. Recombinant production of these mycobacterial proteins located in the cell wall somehow differs from the other proteins, as they are partially apolar. Therefore, this study aims to produce recombinant Ag85C as a vaccine candidate. Ag85C gene was cloned in pJET1.2 and subsequently in pET32a(+). Both recombinant plasmids were sequenced. Expression of the recombinant protein was induced with 1mM IPTG. Recombinant Ag85C was purified through dissolving the inclusions in 8M urea buffer, absorbed to Ni-NTA resins, washed by buffers with decreasing urea concentrations, and finally eluted in aqueous solution. Western blot analysis was performed using anti-6His tag antibody, rabbit anti-M. tuberculosis polyclonal antibody, and the serum from hospitalized TB patients. Ag85C successfully cloned in both plasmid vectors. The recombinant Ag85C expressed in the E. coli host and was purified with significant yield. Western blot results along with that of sequencing ensure accurate production of recombinant Ag85C, retaining its partial epitopes.

Haemophilus influenzae type b (Hib) is a gram negative bacterium and one of the most common causative agents of acute meningitis in infants and less than 5 years old children worldwide. The production of Hib capsular polysaccharide; polyribosyl ribitolphosphate (PRP) is important for the production of conjugate vaccines against Hib infections. The aim of this study is the improvement of Large-scale PRP production by Hib. Haemophilus influenzae type b standard strain ATCC10211 was cultivated in 2L fermentors contain 1.5L CY (casaminoacid yeast extract) medium with normal or modified concentrations of glucose, yeast extract, hemin and NAD (nicotinamide adenine dinucleotide). Seed culture of two fermentors was inoculated to 50 L fermentor, separately and range of PRP production and Dry cell weight (DCW) were studied. Cultivation of Hib in 50L fermentor contained modified CY medium with 6gl-1 Glucose, 2.5 gl-1 Yeast extract, 0.03 gl-1 Hemin and 0.015 gl-1 NAD, with controlled pH at 7.3 and 30% Dissolved oxygen tension (DOT) resulted to about 5.1 gl-1 DCW and 1.16 gl-1 PRP, that was significantly higher than normal CY medium. In conclusion, by modification in some medium components of CY medium, control of Dissolved oxygen tension and pH, the Large-scale production of PRP is improved. Improvement of PRP production leads to reduce the final cost of Hib conjugate vaccines.

Antigen 85 complex of Mycobacterium tuberculosis includes three immunogenic proteins which are TB vaccine candidates of great importance. As they are very hard to be achieved in natural form, recombinant production of them fuels immunological experiments. Production of such apolar mycobacterial proteins located in the cell wall faces substantial challenges mainly regarding their solubility. This study reports the production of soluble recombinant Ag85B with an efficient yield. Ag85B gene was cloned in pJET1.2 and subsequently in pET32a (+). Both recombinant plasmids were sequenced. Expression of the recombinant protein was induced with 1mM IPTG. Recombinant Ag85B was purified through dissolving inclusions in 8M urea buffer, absorbing to Ni-NTA resins, washing by buffers with decreasing urea concentrations and finally eluted in imidazole. Western blot analysis was performed using anti-6His tag antibody, rabbit anti- M. tuberculosis polyclonal antibody and serum of hospitalized TB patients. Ag85B gene was successfully cloned in both plasmid vectors. The recombinant Ag85B was expressed in E. coli host and purified with significant yield. Western blot results along with those of sequencing ensured accurate production of recombinant Ag85B and retaining of its antigenic structure.

A quantitative TaqMn Real-Time PCR assay was developed and its diagnostic value on human serum and livestock samples were evaluated. Brucella species could be distributed through communities as a biological agent. Rapid detection of biological threat agents is critical for timely therapeutic administration. Quantitative real-time PCR pro vides a rapid, sensitive and specific tool for molecular identification of this agent. We evaluated a new real-time PCR set by numerous human and livestock samples. For primers and probe designation BCSP31 of B. melitensis 16 M was used. Different sero-positive human samples and tissue samples livestock with suspected brucellosis were then assayed after primary adaptation of Q-PCR in the laboratory. The detection limit of the assay was 10 fg (equivalent to 2 genome). Totally 50 samples (25 sero-positive, 25 sero-negative) of patients and 75 samples from slaughtered livestock (due to brucellosis) were collected and assayed by Q-PCR, along with control samples. Brucella genome were detected by this real-time set from nearly 42% (5 out of 12) of negative samples by conventional PCR. This system also detected brucella contamination in 46 out of 75 animal samples.

Molecular detection techniques are believed to be key tools for both prevention and treatment follow up of brucellosis within live stock and human beings. Consequently rapid، reliable، easy to perform and automated systems for Brucella detection are urgently needed to allow early diagnosis and adequate antibiotic therapy in time. Brucellosis is a worldwide re-emerging zoonosis causing high economic losses and severe human disease. In attempt to improve current molecular detection of Brucella melitensis، we compared a conventional PCR with PCR- ELISA، to detect brucella genome within standard strains and clinical isolates. Primer sets based on “omp-31” sequence of B. melitensis 16M were designed. The primer specificity was checked with appropriate online bioinformatics softwares. The primer specificity was also confirmed by testing the reaction with non-Brucella strains. A biotinylated probe complementary to an internal sequence of the PCR products was designed. The labeled non-specific fragments bound to streptavidin-coated wells، saturating the solid phase streptavidin by biotin-streptavidin interaction. Compared with conventional PCR، the PCR- ELISA proved to have more sensitivity for Brucella genome after appropriate optimization. Few human serum، whole blood and also different affected tissue samples from slaughtered livestock with brucellosis were used for protocol evaluation. Further samples should be tested before final conclusion about the results.