Detection of efflux pump genes in multiresistant Acinetobacter baumannii ST2 in Iran.

Acinetobacter baumannii, as a nosocomial pathogen has become a worldwide concern in recent years. In the current study, the resistance to tetracyclines and colistin were assessed in the isolates from different provinces of Iran.During the timeline of this study, a number of 270 isolates of A. baumannii were collected from tracheal aspirates, wounds, urine and blood cultures. The minimum inhibitory concentration (MIC) for tetracycline, doxycycline, minocycline, tigecycline and colistin were evaluated. Tetracycline resistance genes were assessed by PCR. The mean expression level of adeB, adeJ and adeG were assessed using semi quantitative Real-Time PCR. The clonal relationship of the isolates was evaluated by the repetitive extragenic palindromic PCR (REP-PCR), International Clonal (IC) Lineage Multiplex PCR and multilocus sequence typing (MLST) (Pasteur scheme) methods.The MIC by microdilution method showed that 87.5, 51.4, 28, 0.74 and 0% of the isolates were resistant to tetracycline, doxycycline, minocycline, tigecycline and colistin respectively. The prevalence of tetracycline resistance genes was 99.2, 99.2, 98, 86.7, 10, 3.33, 0.37, 0% for adeB, adeJ, adeG, tetB, tetA(39), tetA, tetM and tetH in tetracycline-resistant isolates. Moreover, the expression level of adeB, adeJ, adeG genes in tigecycline-nonsusceptible A. baumannii (TNAB) strain was higher compared to the tigecycline-susceptible A. baumannii (TSAB). A broad genomic diversity was revealed, but ST2 was the most prevalent ST. Our results indicated that tetracycline resistance in Iran is mediated by resistance-nodulation-cell division (RND) and tetB efflux pumps.

Immunological Assessment of Chitosan or Trimethyl Chitosan-Coated PLGA Nanospheres Containing Fusion Antigen as the Novel Vaccine Candidates Against Tuberculosis.

The crucial challenge in tuberculosis (TB) as a chronic infectious disease is to present a novel vaccine candidate that improves current vaccination and provides efficient protection in individuals. The present study aimed to evaluate the immune efficacy of multi-subunit vaccines containing chitosan (CHT)- or trimethyl chitosan (TMC)-coated PLGA nanospheres to stimulate cell-mediated and mucosal responses against Mycobacterium Tuberculosis (Mtb) in an animal model. The surface-modified PLGA nanoparticles (NPs) containing tri-fusion protein from three Mtb antigens were produced by the double emulsion technique. The subcutaneously or nasally administered PLGA vaccines in the absence or presence of BCG were assessed to compare the levels of mucosal IgA, IgG1, and IgG2a production as well as secretion of IFN-γ, IL-17, IL-4, and TGF-ß cytokines. According to the release profile, the tri-fusion encapsulated in modified PLGA NPs demonstrated a biphasic release profile including initial burst release on the first day and sustained release within 18 days. All designed PLGA vaccines induced a shift of Th1/Th2 balance toward Th1-dominant response. Although immunized mice through subcutaneous injection elicited higher cell-mediated responses relative to the nasal vaccination, the intranasally administered groups stimulated robust mucosal IgA immunity. The modified PLGA NPs using TMC cationic polymer were more efficient to elevate Th1 and mucosal responses in comparison with the CHT-coated PLGA nanospheres. Our findings highlighted that the tri-fusion loaded in TMC-PLGA NPs may represent an efficient prophylactic vaccine and can be considered as a novel candidate against TB.

CD73; a key ectonucleotidase in the development of breast cancer: Recent advances and perspectives.

Tumor cell invasion and metastasis are the definitive cause of mortality in breast cancer (BC). Hypoxia and pro-inflammatory cytokines upregulate the CD73 gene in the tumor microenvironment. Subsequently, CD73 triggers molecular and cellular signaling pathways by both enzymatic and nonenzymatic pathways, which finally leads to breast tumor progression and development. In this paper, we summarize current advances in the understanding of CD73-driven mechanisms that promote BC development and mortality. Furthermore, we evaluate the therapeutic potential of CD73 targeting in BC.

Cloning, Expression, and Refolding of PPE17 Protein of Mycobacterium Tuberculosis as a Promising Vaccine Candidate.

BACKGROUND: Tuberculosis as a global health problem requires special attention in the contexts of prevention and control. Subunit vaccines are promising strategies to protect burdens of tuberculosis via increasing the BCG protection. The present study aimed to design a vaccine study by means of high-throughput expression and correct refolding of recombinant protein PPE17. METHODS: We aimed to clone, express, and refold PPE17 protein of Mycobacterium tuberculosis in bacterial systems as a promising vaccine candidate. The PPE17 (Rv1168c) gene was PCR amplified and inserted into pET-21b(+) vector, cloned in E. coli TOP10, and finally expressed in E. coli BL21(DE3). RESULTS: The expressed recombinant protein was entirely found in insoluble form (inclusion bodies). The insoluble protein was denatured in 6M guanidine-HCl and refolded by descending denaturant concentration dialysis. Moreover, the recombinant protein was purified by Ni-NTA column chromatography. The changing temperature had no effects on solubilizing protein and the maximum expression was achieved at 0.5 mM concentration of isopropyl-D-thiogalactopyranoside (IPTG) induction. CONCLUSION: Bacterial expression system is a timesaving tool and refolding by descending denaturant concentration dialysis is a rapid and reliable method.

Prevalence of Legionella Species in Water Resources of Iran: A Systematic Review and Meta-Analysis.

BACKGROUND: Legionella species are ubiquitous and naturally found in lakes, rivers, streams and hot springs, and other water resources. The present study aimed to investigate the prevalence of Legionella species in water resources of Iran by a systematic review and meta-analysis. METHODS: In search of papers relevant to the prevalence of Legionella in water resources of Iran, the scientific information database in both English and Persian languages was used. The search was limited to studies between the year 2000 and end of July 2016. Each cohort and cross-sectional study that reported the contamination of water with Legionella was included in the present study. For data analysis, comprehensive meta-analysis software with Cochran's Q and I2 tests were used. P values less than 0.05 were considered statistically significant. RESULTS: The prevalence of Legionella species in water resources of Iran was 27.3% (95% CI: 25.3-29.3). The prevalence of Legionella spp. in hospital water, dental settings water, and other water resources were 28.8% (95% CI: 26.4-31.2), 23.6% (95% CI: 16.1-33.2), and 29.6% (95% CI: 25.6-33.8), respectively. The most common Legionella species was L. pneumophila with a prevalence of 60.5% (95% CI: 53.3-67.2) and the prevalence of all other species was 52.5% (95% CI: 44.7-60.2). The highest prevalence was reported in Isfahan with 55.7% (95% CI: 48.0-63.0). CONCLUSION: Based on the results, the prevalence rate of Legionella species in water resources of Iran was high and the most common Legionella species was L. pneumophila.

Molecular characterization and genetic relatedness of clinically Acinetobacter baumanii isolates conferring increased resistance to the first and second generations of tetracyclines in Iran.

BACKGROUND: The increasing resistance of Acinetobacter baumannii to antibiotics has recently been regarded as a notable therapeutic difficulty. Evaluating resistance rates of some A. baumannii isolates to tetracyclines had an impact on understanding the antibiotic resistance dissemination. By comparing genetic characteristics and relatedness of A. baumannii isolates, we are able to determine the transition dynamics of outbreak isolates. METHODS: A total of 72 non-duplicate isolates of A. baumannii were recovered in 2011 and 2015 and minimum inhibitory concentration (MIC) range distribution of the isolates to tetracyclines was performed by broth micro dilution (BMD) assay, and to determine the lineage relatedness of the outbreak isolates repetitive extragenic palindromic element based on polymerase chain reaction (rep-PCR) and international clonal (ICs) investigations were performed. RESULTS: Resistance rates to tetracycline, doxycycline and minocycline in 2011 were 73, 2 and 0%, while these rates in 2015 increased up to 90, 84 and 52%, respectively. The tetB existed in 100% of all the isolates of both years. tetA was not found in any of the isolates. According to the rep-PCR assays, up to 83% of all isolates clustered distinctly and only 6% of isolates had a common root. The percentage rates of IC1 decreased from 42% in 2011 to 22% in 2015, while those of IC2 increased from 28 to 36%, from 2011 to 2015. CONCLUSIONS: Our data showed that resistance to the first and second generations of tetracyclines is on the rise and the clonal transition dynamics of isolates are in progress in our hospital.

Characterization of carbapenem-resistant Escherichia coli and Klebsiella: a role for AmpC-producing isolates.

Aim: This study aimed to investigate the role of AmpC enzymes in carbapenem resistance among AmpC/extended-spectrum ß-lactamase (ESBL)-producing clinical isolates of Escherichia coli and Klebsiella spp. Methods: Fifty-six bacterial strains that were AmpC producers were examined. The antibiotic susceptibility test was performed by the disk diffusion and E-test. The prevalence of the plasmid carbapenemase was determined using PCR. Results: The resistance to meropenem in the AmpC+/ESBL+ group was 64%, higher than that reported for the AmpC-/ESBL+ group. Ten isolates of the carbapenem-resistant AmpC producers were negative for carbapenemase-encoding genes. Conclusion: Carbapenem resistance among AmpC-producing isolates with negative results for carbapenemase-encoding genes potentially demonstrates the role of AmpC enzymes among these isolates.

Chitosan nanoparticles containing fusion protein (Hspx-PPE44-EsxV) and resiquimod adjuvant (HPERC) as a novel booster vaccine for Mycobacterium tuberculosis.

This study attempted to explore the immunogenicity of chitosan nanoparticles containing fusion protein (Hspx-PPE44-EsxV; HPE) and resiquimod adjuvant (HPERC) in BALB/c mice. HPE was initially expressed in E. coli BL21 cells. HPE and resiquimod adjuvant were then encapsulated in chitosan nanoparticles (HPERC). One group of mice were subcutaneously vaccinated on days 0, 14, and 28 with HPERC, and the other group was primed with bacilli Calmette-Guérin (BCG) on day 0 and then boosted with HPERC on days 14 and 28. Two weeks after the last injection, IFN-γ, IL-4, and IL-17 in spleen cell culture supernatants, and IgG2a and IgG1 titers in sera were measured. HPERC size was 130.84 ± 12.08 nm (n = 5). Zeta potential of HPERC was 29 ± 4 mv. The highest IFN-γ concentration was detected in BCG-primed mice that were boosted with HPERC. In addition, IL-17 production was significantly increased in all groups compared with that of control, except in those that received nanoparticle (NP), adjuvant (ADJ), NP/ADJ, and fusion protein (Hspx-PPE44-EsxV) (HPE). Comparison of IFN-γ and IL-4 concentration determined that Th1 was activated in BCG-primed and HPERC-boosted group in comparison to the other groups. No significant difference in concentration of IL-4 was observed between groups receiving HPERC and BCG-primed and HPERC-boosted group in comparison to group BCG. Concentrations of IgG2a and IgG1 also increased compared to the control group and the rate of IgG2a was higher compared to IgG1. Chitosan containing HPERC vaccine could induce a high level of specific cytokines in mice. The group of mice which first received BCG and then HPERC as booster vaccine could produce significant amounts of IFN-γ, IL-17, and IgG2a.

T helper type 1 biased immune responses by PPE17 loaded core-shell alginate-chitosan nanoparticles after subcutaneous and intranasal administration.

PURPOSE: Tuberculosis, a cost and life threatening disease, was being subjected for improving vaccine strategies beyond BCG. Thus, a novel particulate delivery system using alginate-coated chitosan nanoparticles including PPE17 protein and CpG were administered through intranasal (IN) and subcutaneous (SC) routes. METHODS: The encapsulated nanoparticles were first characterized for size, surface charge, encapsulation efficiency and in vitro release of PPE17 antigen. The nanoparticles were then administered intranasal and subcutaneously to evaluate the induction of systemic and/or mucosal immune responses in mice. RESULTS: According to our result, the mean size of nanoparticles was measured about 427 nm, and exhibited a negative zeta potential of -37 mV. Following subcutaneous and intranasal administration, the results from cytokines assay showed that an increasing in the level of IFN-γ, and adversely a decrease in the level of IL-4 (presumptive Th1 biased immune response) was happened and also a notable elicitation in IL-17 cytokine was observed. CONCLUSION: In conclusion, our study demonstrated that alginate-coated chitosan nanoparticles showed to be an effective way to improve BCG efficiency as booster strategy for subcutaneous vaccine, and also can induce strong immune responses as prime strategy through intranasal vaccination.

A new DNA vaccine expressing HspX-PPE44-EsxV fusion antigens of Mycobacterium tuberculosis induced strong immune responses.

OBJECTIVES: Infection with tuberculosis (TB) is regarded as a major health issue. Due to the emergence of antibiotic resistance during TB treatment, prevention via vaccination is one of the most effective ways of controlling the infection. DNA vaccines are developed at a greater pace due to their ability in generating a long-lasting immune response, higher safety compared to the live vaccines, and relatively lower cost of production. In the present study, we evaluated a new DNA vaccine encoding the fusion HspX-PPE44-EsxV antigens, separately, and in combination with Bacillus Calmette-Guérin (BCG) administration, in a prime-boost method in mice. MATERIALS AND METHODS: A novel DNA vaccine encoding HspX-PPE44-EsxV fusion antigen of Mycobacterium tuberculosis was constructed, and RT-PCR and Western blot analysis were performed to verify the expression of the antigen. Female BALB/c mice were divided into five groups (PBS, BCG, pcDNA3.1 (+) vector, pDNA/HspX-PPE44-EsxV vaccine, and the BCG-prime boost groups). In order to evaluate the immunogenicity of the recombinant vector, BALB/c mice were injected with 100 µg of pDNA at 2-week intervals. Then, cytokine assay was conducted using eBioscience ELISA kits (Ebioscience, AUT) according to manufacturers' instructions to evaluate the concentrations of IL-4, IL-12, TGF-ß, and IFN-γ. RESULTS: The concentrations of INF-γ, IL-12, and TGF-beta were significantly increased compared to the control groups (P<0.001). INF-γ and IL-12 production were increased significantly in pDNA/HspX-PPE44-EsxV+BCG group compared to pDNA/HspX-PPE44-EsxV group (P<0.001). CONCLUSION: This study showed that the present DNA vaccine could induce a high level of specific cytokines in mice. It was also shown that using this DNA vaccine in a BCG prime-boost protocol can produce significant amounts of IFN-γ, IL-12, and TGF-ß.

High prevalence of blaCMY AmpC beta-lactamase in ESBL co-producing Escherichia coli and Klebsiella spp. clinical isolates in the northeast of Iran.

OBJECTIVE: The production of ß-lactamase enzymes such as AmpC ß-lactamases and extended-spectrum ß-lactamases (ESBLs) is among the main mechanisms for resistance to expanded-spectrum cephalosporins. The present study was conducted to investigate the prevalence and molecular epidemiology of plasmid-mediated AmpC beta (ß)-lactamase in ESBL co-producing Escherichia coli (E. coli) and Klebsiella spp. (Klebsiella pneumoniae and Klebsiella oxytoca) clinical isolates in the northeast of Iran. METHODS: A total of 602 E. coli and Klebsiella spp. clinical isolates were collected from three hospitals in Mashhad (northeast of Iran). A combination disk test (CDT) was performed for the phenotypic detection of ESBLs. Screening for the detection of AmpC ß-lactamases was performed by a susceptibility test to a cefoxitin disc among ESBL producing isolates. A confirmatory test for AmpC ß-lactamases was performed using the Mast® D68C test. Identification of plasmid-mediated AmpC cluster genes was done by multiplex polymerase chain reaction (PCR). RESULTS: Among 336 ESBL-producing strains, 230 (68.4%) isolates were resistant to cefoxitin. Results of the Mast® D68C test showed that 30% (69/230) of cefoxitin-resistant isolates simultaneously exhibited ESBL and AmpC activity and 22% (51/230) of isolates probably showed multi-drug resistant (MDR) phenotype. Results of multiplex PCR among ESBL-positive isolates showed that, 16.7% (56/336) of isolates were positive for plasmid-borneampC cluster genes, and CMY (38%) was the most frequent genotype of plasmid mediated AmpC. CONCLUSION: Findings of the study revealed that an increase in the prevalence of ESBL and AmpC co-producer in E. coli and Klebsiella spp. strains may become an important public health issue. Therefore, there is a vital need for surveillance of spread of these clinical isolates.

The therapeutic potential of targeting CD73 and CD73-derived adenosine in melanoma.

The hypoxic environment of melanoma results in CD73 upregulation on the surface of various tumor microenvironment (TME) cells including tumor cells, stromal cells and infiltrated immune cells. Consequently, CD73 through both enzymatic and none enzymatic functions affect melanoma progression. Overaccumulation of CD73-derived adenosine through interaction with its four G coupled receptors (A1AR, A2AAR, A2BAR, and A3AR) mediate tumor growth, immune suppression, angiogenesis, and metastasis. This paper aims to comprehensively review the therapeutic potential of CD73 ectonucleotidase targeting in melanoma. To reach this goal, firstly, we summarize the structure, function, regulation, and clinical outcome of CD73 ectonucleotidase. Then, we depict the metabolism and signaling of CD73-derived adenosine along with its progressive role in development of melanoma. Furthermore, the therapeutic potentials of CD73 -adenosine axis targeting is assessed in both preclinical and clinical studies. Targeting CD73-derived adenosine via small molecule inhibitor or monoclonal antibodies studies especially in combination with immune checkpoint blockers including PD-1 and CTLA-4 have shown desirable results for management of melanoma in preclinical studies and several clinical trials have recently been started to evaluate the therapeutic potential of CD73-derived adenosine targeting in solid tumors. Indeed, targeting of CD73-derived adenosine signaling could be considered as a new therapeutic target in melanoma.

Increasing Cellular Immune Response in Liposomal Formulations of DOTAP Encapsulated by Fusion Protein Hspx, PPE44, And Esxv, as a Potential Tuberculosis Vaccine Candidate.

BACKGROUND: Due to the ineffectiveness of the BCG vaccine, especially in adult pulmonary tuberculosis (TB), and variable efficacies against childhood forms of TB, developing an effective TB vaccine is a major priority in controlling this disease. The aim of this study was to evaluate the immunogenicity of a DOTAP liposome formulation containing a fusion protein (FP) containing Mycobacterium tuberculosis HspX, PPE44, and EsxV. METHODS: The FP was expressed in E. coli BL21 cells and confirmed by SDS-PAGE and Western blots. The FP was then encapsulated in various liposomal formulations. Afterwards, liposomal size, zeta potential, and encapsulation efficiency were evaluated. Mice were subcutaneously vaccinated on days 0, 14, and 28 with liposomes containing the FP. Two weeks after the last injection, IFN-γ, IL-4, IL-17, and IL-12 in spleen cell culture supernatants, and IgG2a, IgG1, and IgG2b titers in sera were measured. RESULTS: The FP concentration was 1mg/ml. The encapsulation efficiency of the liposomes varied from 69% in Lip (DOTAP/TDB/CHOL/FP) to 80% in Lip (DOTAP/CHOL/FP). The greatest IFN-γ and IL-12 levels were observed in BCG-primed mice that were boosted with Lip (DOTAP/CHOL/FP). In addition, IL-17 production was significantly greater in all groups than controls except in those that received histidine buffer and FP. The IgG2a/IgG1 ratios were greater in the Lip (DOTAP/TDB/CHOL/FP), Lip (DOTAP/CHOL/FP), Lip (DOTAP/CHOL), and BCG-primed and Lip (DOTAP/CHOL/FP)-boosted groups than in the other groups, indicating a cellular immune response. CONCLUSION: The liposomes containing DOTAP combined with the fusion protein induced a Th1 response. The mice that first received BCG and then Lip (DOTAP/CHOL/FP), produced the most IFN-γ and IL-12, indicating a strong Th1 response.

Enhancement of the effect of BCG vaccine against tuberculosis using DDA/TDB liposomes containing a fusion protein of HspX, PPE44, and EsxV.

Tuberculosis has been a major health problem worldwide for years; therefore, it is important to develop and produce an effective vaccine against this disease. In this study, the immunogenicity of Mycobacterium tuberculosis fusion protein (FP) encapsulated in liposomes containing DDA/TDB was evaluated. The FP was expressed in E. coli BL21 and encapsulated in liposomal formulations. Three weeks after the last subcutaneous immunization, IFN-γ, IL-4, IL-17, and IL-12 in spleen cell culture supernatants, and IgG2a, IgG1, and IgG2b titres in sera were measured. The greatest IFN-γ and IL-12 interleukin concentrations were observed in the DDA/TDB/CHOL liposomes containing the FP. Initial injection with BCG improved the efficacy of the DDA/TDB/CHOL/FP vaccine. The IgG2a/IgG1 ratio was also high in the DDA/TDB/CHOL/FP group; furthermore, the IgG2a/IgG1 ratio was increased in the BCG-primed, DDA/TDB/CHOL/FP-boosted group, indicating induction of a cellular immune response. Our study showed that the FP-containing DDA/TDB/CHOL liposomes induced a Th1 response. However, the groups that first received BCG and then DDA/TDB/CHOL/FP had the greatest Th1 response in terms of IFN-γ and IL-12 production of all the groups. This suggests that these formulations enhance the BCG vaccine's effectiveness.

Heterologous Expression, Purification, and Characterization of the HspX, Ppe44, and EsxV Proteins of Mycobacterium tuberculosis.

BACKGROUND: Subunit vaccines are appropriate vaccine candidates for the prevention of some infections. In this study, three immunogenic proteins of Mycobacterium tuberculosis, including HspX, Ppe44, and EsxV as a new construction, were expressed alone and as a fusion protein to develop a new vaccine candidate against tuberculosis infection. METHODS: To make the fusion protein, the three genes were linked together by AEAAAKEAAAKA linkers and inserted into pET21b and pET32b vectors. Escherichia coli (E. coli) Top10 cells were transformed with the plasmid, and the purified plasmid was used to transform E. coli BL21 cells. Protein expression was induced with IPTG. After optimizing protein expression, the recombinant proteins were purified by Ni-NTA chromatography. Protein purification was confirmed by SDS-PAGE and Western blotting with an anti-poly histidine-peroxidase monoclonal antibody against the 6His-tags at the proteins' C termini. RESULTS: Directional cloning was confirmed by polymerase chain reaction (PCR), restriction enzyme digestion, and sequencing. The highest expression of the tri-fusion protein and HspX were obtained by the addition of 0.2 mM of IPTG to E. coli BL-21 cells at 37 °C and 18 h of incubation. For Ppe44 and EsxV, the optimum expression conditions were 18 °C and 16 h of incubation. SDS-PAGE and Western blots confirmed that the desired proteins were produced. CONCLUSION: The three desired proteins and the fusion protein were successfully expressed and the conditions for optimum expression determined. These recombinant proteins will be evaluated as vaccine candidates against tuberculosis. Further studies are needed to evaluate the abilities of these proteins to induce strong immunological responses.

Diversity of aminoglycoside modifying enzymes and 16S rRNA methylases in Acinetobacter baumannii and Acinetobacter nosocomialis species in Iran; wide distribution of aadA1 and armA.

PURPOSE: Acinetobacter baumannii-calcoaceticus complex (ABC) make a great burden on health-care systems due to hospital-acquired infections and antibacterial resistance. Aminoglycoside in combination with other antibacterials used as treatment options. However, ABC species overcome this class of antibacterials in different ways. This study provides a comprehensive report on the distribution of aminoglycoside modifying enzymes (AMEs) and 16S rRNA methylase in Acinetobacter baumannii and Acinetobacter nosocomialis isolated from various provinces in Iran. METHODS: During six month of study, from eight referral centers in seven provinces across the country, Iran, 178 A. baumannii and 43 A. nosocomialis isolates were collected. The minimum inhibitory concentration of amikacin, gentamicin, netilmicin, kanamycin and tobramycin were measured by microbroth dilution method. AMEs and 16S rRNA methylase variants were sought by PCR. RESULTS: High rates of resistance were seen in all centers. MIC50 and MIC90 for all A. baumannii and A. nosocomialis isolates from different centers were > 512 mg/L. The most frequent AME was ant(3″)-Ia (aadA1) in both of A. baumannii (74.1%) and A. nosocomialis (86%). armA was detected in A. baumannii and A. nosocomialis at the frequency of 41.6% and 67.4%, respectively. rmtA, B, C, D, aac(3)-Ia (aacC1) and aac(6')-Im were not detected, neither in A. baumannii nor A. nosocomialis. Moreover, aac(6')-Ih was only found in A. baumannii isolates. The distribution of some of the ARGs was limited to a definite center. CONCLUSION: The overall high-level carriage of ARGs in Acinetobacter species may limited usage of this class of antibacterials as a treatment option.

Aluminum hydroxide nanoparticles show strong activity to stimulate Th-1 immune response against tuberculosis.

Many materials such as aluminum hydroxide have been tried as adjuvants to compensate low inherent immunogenicity of subunit vaccines. The aim of this study was to evaluate the specific immune response following the administration of aluminum hydroxide nanoparticles with EsxV antigen. The physiochemical properties of the nanoparticle were characterized in vitro. After subcutaneous immunization, cytokine secretion patterns including IFN-gama,IL-4, and TGF-beta levels were measured by indirect enzyme linked immunosorbent assay (ELISA). Aluminum hydroxide-NPs were demonstrated excellent effects to raise of IFN-γ secretion in compare to EsxV alone. Administration of aluminum hydroxide nanoparticles stimulates strong cellular immune response and could be considered as appropriate adjuvant against TB infection.

Different methods to determine the encapsulation efficiency of protein in PLGA nanoparticles.

BACKGROUND: Effective encapsulation of drugs into the delivery systems could increase the efficiency of nanoparticles in prevention and treatment of diseases. OBJECTIVE: The purpose of this study was to compare the different methods for determination of encapsulation efficiency of a model protein in the PLGA nanoparticles. METHODS: The various direct methods include dichloromethane, acetonitrile, modified acetonitrile and NaOH based extraction and radioactive methods were used to directly calculate the encapsulation efficiency of the loaded protein in the PLGA nanoparticles. Furthermore, indirect methods include BCA, Fluorescent and radioactive methods were compared. RESULTS: The encapsulation efficiencies determined by indirect methods include dichloromethane, acetonitrile, modified acetonitrile, NaOH based extraction and radioactive methods were 12.62% ± 1.97, 17.43% ± 2.51, 64.69% ± 4.31, 86.36% ± 2.25 and 90.15% ± 1.78, respectively. Moreover, the encapsulation efficiencies determined by indirect methods include BCA, fluorescent and radioactive methods were 81.46% ± 1.92, 88.23% ± 1.15 and 89.6% ± 1.9, respectively. CONCLUSIONS: Among the results obtained by indirect methods, radioactive and fluorescent methods showed more reliable. Moreover, NaOH and radioactive methods were the most reliable methods among the direct methods.

Development of an effective delivery system for intranasal immunization against Mycobacterium tuberculosis ESAT-6 antigen.

Introduction The early secreted antigenic target 6-kDa protein (ESAT-6) plays an important role in immune protection against Tuberculosis. Owing to its great potential to increase immune response, chitosan can be considered as a suitable biodegradable polymer for intranasal administration. Methods The physiochemical properties of the nanoparticle were measured in vitro. Two weeks after the last intranasal administration, blood samples were collected and specific IgG, IFN-gama, and IL-4 levels were measured by ELISA. Results Chitosan nanoparticles containing ESAT-6 demonstrated stronger ability to induce IFN-gama, IL-4, and IgG antibody level than the control groups. Conclusion Administration of chitosan nanoparticles can be a suitable method to induce more appropriate immune responses against low inherent immunogenic tuberculosis proteins through intranasal routs.

Construction of a Novel DNA Vaccine Candidate Encoding an HspX-PPE44-EsxV Fusion Antigen of Mycobacterium tuberculosis.

BACKGROUND: Mycobacterium tuberculosis is the causative agent of tuberculosis (TB). Bacille Calmette-Guerin (BCG) vaccine, is not effective in adults, therefore, many efforts have been made to produce an effective adult TB vaccine. The aim of this study was to develop a new tuberculosis DNA vaccine candidate encoding a recombinant HspX-PPE44-EsxV fusion antigen of M. tuberculosis. METHODS: A fusion DNA segment consisting of HspX, linker, PPE44, linker, and EsxV, after codon optimization, was designed. The fusion DNA was cloned and its sequence confirmed. Then, expression of a recombinant pcDNA3.1 (+)/HspX-PPE44-EsxV plasmid in Chinese hamster ovary (CHO) cells was verified by RT-PCR and Western-blot analysis. RESULTS: A 1968 bp band in RT-PCR and a 68 kDa band on Western-blot analysis confirmed transcription and expression of recombinant hspX-ppe44-esxV in eukaryotic cells. CONCLUSION: A recombinant DNA segment encoding the HspX-PPE44-EsxV fusion antigen of M. tuberculosis was constructed and considered to be tested as a new TB DNA vaccine candidate.