Association of the ESR1 (rs9340799), OLR1 (rs3736234), LIPC (rs2070895), VDR (rs2228570), and CETP (rs708272) Polymorphisms With Risk of Coronary Artery Disease in Iranian Patients.

BACKGROUND: Coronary artery disease (CAD) is a devastating illness and a leading cause of death worldwide, primarily caused by atherosclerosis resulting from a genetic-environmental interaction. This study aimed to investigate the relationship between the ESR1 (rs9340799), OLR1 (rs3736234), LIPC (rs2070895), VDR (rs2228570), and CETP (rs708272) polymorphisms, lipid profile parameters, and CAD risk in a southeast Iranian population. METHODS: A total of 400 subjects (200 CAD patients with hyperlipidemia and 200 healthy controls) were enrolled in this case-control study. Five selected polymorphisms were genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. RESULTS: For all single nucleotide polymorphisms (SNPs), the population under study was in the Hardy-Weinberg equilibrium. The T-risk allele frequency of rs2228570 was associated with an increased risk of CAD. The TT and CT genotypes of rs2228570 had also been associated with the risk of CAD. Additionally, the TT genotype was associated with higher serum low-density lipoprotein cholesterol (LDL-c) and high-density lipoprotein cholesterol (HDL-c) levels. The GG genotype of the rs3736234 was associated with higher body mass index (BMI) and triglyceride (TG) levels, and the AA genotype of the rs708272 was associated with higher HDL-c levels. Based on these findings, we propose that the VDR (rs2228570) polymorphism was associated with serum HDL-c and LDL-c levels and may serve as potential risk factors for CAD within the Iranian population. Moreover, rs3736234 and rs708272 influence the concentrations of TG and HDL-c, respectively. CONCLUSION: These findings provided insights into the complex interplay between genetic variations, cardiovascular risk, and lipid metabolism.

Variations of blood metabolites in single- and multiple-bearing Ossimi ewes during the transition period.

Evaluation of the metabolic profile indices allows early detection and treatment of various metabolic disorders during the transition period in ewes. This study aimed to determine the variations in the blood metabolites around lambing in Ossimi ewes. The blood metabolites were investigated in ewes with single (n = 27) and multiple (n = 9) lambs at 3- and 1-week pre-lambing and 3-week post-lambing. The plasma concentrations of glucose were higher in single-bearing ewes than those in multiple-bearing ewes (p < .05), moreover, its lowest value was measured at 1-week prepartum in both groups. Throughout the study period, the serum concentrations of non-esterified fatty acids (NEFA) were significantly increased in ewes with multiple lambs compared to ewes with single lambs (p < .05), and the highest value was found at 1-week before parturition in both groups. In addition, the serum level of beta-hydroxybutyric acid (BHBA) was higher at 3-week postpartum, and it was significantly increased in multiple-bearing ewes than that in single-bearing ones (p < .05) at 3-week pre-lambing. In both groups, the lowest values of total proteins were determined 1-week before lambing, and its concentrations, at 3- and 1-week prepartum, were higher in ewes with single lambs than those with multiple lambs (p < .05). In contrast, the serum concentrations of albumin were significantly lowered 1-week postpartum (p < .05), and without significant differences between both groups (p > .05). The serum activities of aspartate aminotransferase (AST) and gamma-glutamyltransferase (GGT) were significantly increased at 1-week after parturition in both groups (p < .05). Furthermore, the serum activities of AST were higher in multiple-bearing ewes than those in single-bearing ones at 3-week pre-lambing and 3-week post-lambing (p < .05). Variable positive and negative correlations were determined among the blood metabolites. In conclusion, physiological adaptations are associated with the fluctuation of the blood metabolites around lambing. The higher the number of foetuses the higher the metabolic variations in Ossimi ewes. Therefore, regular metabolic profiling for health monitoring may be necessary to avoid disease development during the transition period.

Design of a chimeric protein composed of FimH, FyuA and CNF-1 virulence factors from uropathogenic Escherichia coli and evaluation its biological activity and immunogenicity in vitro and in vivo.

Urinary tract infections (UTIs) caused by Uropathogenic Escherichia coli (UPEC) are among the most prevalent bacterial infections in humans. Antibiotic resistance among UPEC isolates is increasing, and designing an effective vaccine can prevent or reduce these infections. FimH adhesin, iron scavenger receptor FyuA, and cytotoxic necrotizing factor -1 (CNF-1) are among the most important virulence factors of UPEC strains. Thus, a novel multi-epitope protein composed of FimH, FyuA, and CNF-1 was designed to evaluate its biological activity and immunogenicity in vitro and in vivo, respectively. The final vaccine design had seven domains, including the N-terminal domain of FimH, four domains of FyuA, and two domains of CNF-1, as determined by immunoinformatics analysis. The results of tertiary structure prediction showed that the chimeric protein had a C-score of -0.25 and Z-score of -1.94. Molecular docking indicated that thirty six ligand residues of the chimeric protein interacted with 53 receptor residues of TLR-4 by hydrogen bonds and hydrophobic interactions. Analysis of protein expression by SDS-PAGE showed an approximately 44 kDa band with different concentrations of IPTG which were confirmed by Western blot. According to ELISA results, the level of IL-8 produced by stimulated Ht29 cells with the chimeric protein was significantly higher than the stimulated Ht29 cells with CNF-1 alone and un-stimulated Ht29 cells. Rabbits subcutaneously immunized with the chimeric protein admixed with Freund adjuvant induced higher level of serum IgG on day 14 after the first vaccination than control rabbits. Furthermore, the booster dose of the chimeric protein significantly enhanced the IgG levels as compared to day 14 and also controls. As, the chimeric protein has suitable B-cell epitopes and MHC-I and MHC-II binding epitopes to stimulate humoral and cellular immunity, it could be a promising vaccine candidate against UTIs caused by UPEC. Evaluating the multi-epitope protein in inducing humoral and cellular immune responses, as well as protection, is ongoing in the mice models.

Stem Cell-Enriched Fat Grafts Versus Autologous Fat Grafts for Facial Reconstruction: A Systematic Review and Meta-analysis.

OBJECTIVE: The aim of the study is to compare the outcomes of stem cell-enrichment fat grafting versus routine fat grafting for facial reconstruction purposes. METHODS: A systematic review and meta-analysis were performed as per the Preferred Reporting Items for Systematic Reviews and Meta-analyses Guidelines and a search of electronic information was conducted to identify all randomized controlled trials, case control studies, and cohort studies comparing the outcomes of stem cell enrichment fat grafting versus routine fat grafting for facial reconstruction purposes. Volume retention and infection rate were primary outcome measures. Secondary outcome measures included patient satisfaction postsurgery, redness and swelling, fat necrosis, cysts, as well as operation time. Fixed and random effects modeling was used for the analysis. RESULTS: Eight studies enrolling 275 subjects were selected. There was a significant difference between the stem cell enrichment fat grafting and routine grafting groups in terms of mean volume retention (standardized mean difference, 2.49; P < 0.00001). However, there was no significant difference between the 2 groups in the rate of infection (odds ratio, 0.36; P = 0.30). For all secondary outcomes, the intervention group had similar results compared with the control group except for the operation time, which was shorter in the latter. CONCLUSIONS: Stem cell-enriched fat grafting is a superior option when compared with the routine fat grafting for facial reconstruction surgery because it improves the mean volume retention and does not worsen patient satisfaction and surgical complications.

Stem Cell Enriched Fat Grafts versus Autologous Fat Grafts in Reconstructive Surgery: Systematic Review and Meta-Analysis.

OBJECTIVE: To compare the outcomes of stem cell-enrichment fat grafting (SCEFG) versus autologous fat grafting (AFG) for reconstructive purposes. METHODS: A systematic review and meta-analysis was performed as per the preferred reporting items for systematic reviews and meta-analyses. Guidelines and a search of electronic information was conducted to identify all Randomised Controlled Trials (RCTs), case-control studies and cohort studies comparing the outcomes of SCEFG versus AFG. Volume retention, fat necrosis, cancer recurrence, redness and swelling, infection, and cysts were primary outcome measures. Secondary outcome measures included patient satisfaction post-surgery, scar assessment, operation time and number of fat grafting sessions. Fixed and random effects modelling were used for the analysis. RESULTS: 16 studies enrolling 686 subjects were selected. Significant differences between the SCEFG and AFG groups were seen in mean volume retention (standardised mean difference = 3.00, P < 0.0001) and the incidence of redness and swelling (Odds Ratio [OR] = 441, P = 0.003). No significant difference between the two groups in terms of fat necrosis (OR = 2.23, P = 0.26), cancer recurrence (OR = 1.39, P = 0.58), infection (OR = 0.30, P = 0.48) and cysts (OR = 0.88, P = 0.91). For secondary outcomes, both cohorts had similar results in patient satisfaction, scar assessment and number of fat grafting sessions. Operation time was longer for the intervention group. CONCLUSIONS: SCEFG offers better outcomes when compared to AFG for reconstructive surgery as it improves the mean volume retention and does not worsen patient satisfaction and surgical complications except for self-limiting redness and swelling. Further clinical trials are recommended to support this argument and validate the use of SCEFG in clinical practice. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Muller's muscle conjunctival resection versus external levator advancement for ptosis repair: systematic review and meta-analysis.

PURPOSE: To compare the outcome of Muller's muscle conjunctival resection (MMCR) versus external levator advancement (ELA) in patients undergoing ptosis surgery. METHODS: A systematic review and meta-analysis were performed as per the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines, and a search of electronic information was conducted to identify all comparative studies of MMCR versus ELA in ptosis repair. The primary outcome measures were the post-operative marginal reflex distance (MRD1), ptosis under-correction, over-correction, and re-operation rate. Secondary outcome measures included cosmetic appearance, complications, operative time, and learning curve. Fixed-effect modelling was used for the analysis. RESULTS: Seven studies that enrolled 1038 eyelids were identified in the literature. There was no statistically significant difference between the MMCR and ELA groups in post-operative MRD1 (Mean Difference [MD] = 0.13, P = 0.28) and the rate of under-correction odds ratio [OR] = 0.49, P = 0.14). However, ELA had a significantly higher rate of over-correction (OR = 0.17, P = 0.04) and re-operations (OR = 0.26, P = 0.0001) compared to MMCR. For secondary outcomes, MMCR had an improved cosmetic appearance, lower total number of complications and shorter operation time (MD = - 10.96, P < 0.00001). Finally, the two techniques had no significant difference in the learning curves. CONCLUSION: Both MMCR and ELA are effective techniques for the surgical correction of ptosis; however, MMCR surgery is a more predictable and robust technique compared to, ELA with lower rates of over-correction and re-operation.

Preparation and pre-clinical evaluation of flagellin-adjuvanted NOM vaccine candidate formulated with Spike protein against SARS-CoV-2 in mouse model.

From December 2019, the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was started as a cluster of pneumonia cases in Wuhan, Hubei Province, China. The disturbing statistics of SARS-CoV-2 promoted scientists to develop an effective vaccine against this infection. NOM protein is a multi-epitope protein that designed based on Nucleocapsid, ORF3a, and Membrane proteins of SARS-CoV-2. Flagellin is a structural protein that binds to the Toll-like receptor 5 and can enhance the immune response to a particular antigen. In this study, NOM protein as vaccine candidate was linked to the carboxyl and amino terminals of flagellin adjuvant derived from Salmonella enterica subsp. enterica serovar Dublin. Then, informatics evaluations were performed for both NOM protein and NOM protein linked to flagellin (FNOM). The interaction between the NOM and FNOM proteins with the TLR5 were assessed using docking analysis. The FNOM protein, which compared to the NOM protein, had a more suitable 3D structure and a stronger interaction with TLR5, was selected for experimental study. The FNOM and Spike (S) proteins expressed and then purified by Ni-NTA column as vaccine candidates. For analysis of immune response, anti-FNOM and anti-S proteins total IgG and IFN-γ, TNF-α, IL-6, IL-10, IL-22 and IL-17 cytokines were evaluated after vaccination of mice with vaccine candidates. The results indicated that the specific antisera (Total IgG) raised in mice that received FNOM protein formulated with S protein were higher than mice that received FNOM and S proteins alone. Also, IFN-γ and TNF-α levels after the spleen cells stimulation were significantly increased in mice that received the FNOM protein formulated with S protein compared to other groups. Immunogenic evaluations showed that, the FNOM chimeric protein could simultaneously elicit humoral and cell-mediated immune responses. Finally, it could be concluded that the FNOM protein formulated with S protein could be considered as potential vaccine candidate for protection against SARS-CoV-2 in the near future.

Detection of ESBL and AmpC producing Klebsiella pneumoniae ST11 and ST147 from urinary tract infections in Iran.

In the present study a total of 200 Klebsiella pneumoniae isolates were collected from patients with urinary tract infections (UTIs) in Tehran, Iran. Antibiotic resistance was determined by disk diffusion and broth dilution methods. Detection of extended-spectrum ß-lactamases (ESBLs) and AmpCs was performed using phenotypic tests. Polymerase chain reaction (PCR) was applied to detect the ESBL, AmpC, and integron genes. Analysis of AmpC and cassette arrays of integron genes was performed using DNA sequencing. Plasmids were analyzed by PCR-based replicon typing and conjugation. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were applied to explore the genomic relatedness among the isolates. The highest levels of resistance were observed against ampicillin (100%), followed by piperacillin (57.5%), ceftazidime (46%), trimethoprim/sulfamethoxazole (44%), ciprofloxacin (32.5%), and imipenem (19%). Approximately, 66.5% of isolates harbored at least one of the beta-lactamase genes (blaTEM, blaSHV, blaCTX-M, and blaOXA-1). In addition, 22.5% of isolates carried at least one of the AmpC genes including blaDHA and blaCIT. Integron class I was the most prevalent integron among resistant isolates. According to the results of replicon typing, IncFII, IncL/M, and IncA/C were the most frequent replicons, respectively. All selected isolates were able to transfer blaCTX-M, also two isolates transferred the blaDHA-1 gene to Escherichia coli K12 through conjugation. Finally, 21 isolates were categorized into 4 pulsotypes and 11 unique clusters in PFGE. MLST identified ST147 and ST11 sequence types but ST147 was the most prevalent in the current study.

Investigation of the effects of antimicrobial and anti-biofilm peptide IDR1018 and chitosan nanoparticles on ciprofloxacin-resistant Escherichia coli.

Peptide IDR1018 and chitosan nanoparticles (CNs) showed antimicrobial and anti-biofilm activity against bacteria. In this study, the antimicrobial effects of peptide IDR1018 and CNs were evaluated on 50 clinical isolates of uropathogenic Escherichia coli (UPEC) resistant to ciprofloxacin. Ion gelation method was applied for CNs synthesis. Scanning electron microscope (SEM) and dynamic light scattering (DLS) were utilized to evaluate the nanoparticles. Antimicrobial and synergistic activity of peptide IDR1018 and CNs with ciprofloxacin were evaluated by microtiter broth dilution method. The checkerboard test was used to investigate the antimicrobial effects of IDR1018 and CNS in combination with ciprofloxacin. Anti-biofilm effect of ciprofloxacin, peptide IDR1018, and CNs was evaluated using crystal violet method. Fourteen (28%), 21 (42%), and 15 (30%) of clinical isolates produced strong, moderate, and weak biofilm, respectively. The CNs were spherical and uniform under electron microscopy with an average diameter of 246 nm. The minimum inhibitory concentration (MIC) values were 16-128, 20-40, and 375-750 (µg/ml) for ciprofloxacin, peptide IDR1018, and CNs, respectively. Fractional inhibitory concentration (FIC) analysis indicated a synergistic effect of ciprofloxacin in combination with peptide IDR1018, but in combination with CNs, this antibiotic showed an additive effect. Our results revealed that peptide IDR1018 and CNs have antimicrobial properties on UPEC isolates. Biofilm inhibition and biofilm eradication of clinical isolate were shown by peptide IDR1018 and CNs in a concentration-dependent manner. The antimicrobial agents alone and in combination decreased the number of viable bacteria in the biofilms. Therefore, these components seem to be a treating approach against biofilm-forming UPEC isolates.

Effect of curve location on the severity index for adolescent idiopathic scoliosis: a longitudinal cohort study.

OBJECTIVES: Adolescent idiopathic scoliosis (AIS) is the most common spinal disorder in children. A severity index was recently proposed to identify the stable from the progressive scoliosis at the first standardized biplanar radiographic exam. The aim of this work was to extend the validation of the severity index and to determine if curve location influences its predictive capabilities. METHODS: AIS patients with Cobb angle between 10° and 25°, Risser 0-2, and no previous treatment were included. They underwent standing biplanar radiography and 3D reconstruction of the spine and pelvis, which allowed to calculate their severity index. Patients were grouped by curve location (thoracic, thoracolumbar, lumbar). Patients were followed up until skeletal maturity (Risser ≥ 3) or brace prescription. Their outcome was compared to the prediction made by the severity index. RESULTS: In total, 205 AIS patients were included; 82% of them (155/189, 95% confidence interval [74-90%]) were correctly classified by the index, while 16 patients were unclassified. Positive predictive ratio was 78% and negative predictive ratio was 86%. Specificity (78%) was not significantly affected by curve location, while patients with thoracic and lumbar curves showed higher sensitivity (≥ 89%) than those with thoracolumbar curves (74%). CONCLUSIONS: In this multicentric cohort of 205 patients, the severity index was used to predict the risk of progression from mild to moderate scoliosis, with similar results of typical major curve types. This index represents a novel tool to aid the clinician and the patient in the modulation of the follow-up and, for progressive patients, their decision for brace treatment. KEY POINTS: • The severity index of adolescent idiopathic scoliosis has the potential to detect patients with progressive scoliosis as early as the first exam. • Out of 205 patients, 82% were correctly classified as either stable or progressive by the severity index. • The location of the main curve had small effect on the predictive capability of the index.

Surface display of uropathogenic Escherichia coli FimH in Lactococcus lactis: In vitro characterization of recombinant bacteria and its protectivity in animal model.

Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) are very common, leading to high patient morbidity and substantial medical costs. The development of non-antibiotic strategies such as food-grade lactic acid bacterium can be recognized as an attractive and safe alternative way against UTI. Here, we report the construction of Lactococcus lactis (L. lactis) strain genetically modified to produce FimH virulence factor of UPEC on the cell surface. We showed the FimH inserted into the pT1NX vector is actively synthesized on L. lactis. The L. lactis-pT1NX-FimH exhibited an auto-aggregation phenotype in liquid cultures and formed robust biofilm on abiotic surface compared to vector-only bacteria. Then, we developed protective biofilms with L. lactis strains and examined their inhibitory effect for exclusion of uropathogenic biofilm formation. In the natural protective biofilm assays, L. lactis-pT1NX-FimH resulted in significant reduction in the pathogen load when compared to the L. lactis-pT1NX. Evaluation of the colonization ability in the bladder showed that L. lactis expressing FimH survived better in the mice bladder than L. lactis harboring vector. Protection assay against UPEC infection was investigated using a UTI mouse model. L. lactis-pT1NX-FimH displayed high effectiveness in the protection of the bladder as compared to the control group after UPEC challenge. The results suggest that genetically engineered L. lactis-pT1NX-FimH can be used as a safe alternative way for control of biofilm formation in UPEC. Furthermore, the possibility of using L. lactis-pT1NX-FimH as a new promising strategy against UTIs caused by UPEC strains is proposed.

Spine slenderness and wedging in adolescent idiopathic scoliosis and in asymptomatic population: an observational retrospective study.

PURPOSE: The origin of the deformity due to adolescent idiopathic scoliosis (AIS) is not known, but mechanical instability of the spine could be involved in its progression. Spine slenderness (the ratio of vertebral height to transversal size) could facilitate this instability, thus playing a role in scoliosis progression. The purpose of this work was to investigate slenderness and wedging of vertebrae and intervertebral discs in AIS patients, relative to their curve topology and to the morphology of control subjects. METHODS: A total of 321 AIS patients (272 girls, 14 ± 2 years old, median Risser sign 3, Cobb angle 35° ± 18°) and 83 controls were retrospectively included (56 girls, median Risser 2, 14 ± 3 years). Standing biplanar radiography and 3D reconstruction of the spine were performed. Geometrical features were computed: spinal length, vertebral and disc sizes, slenderness ratio, frontal and sagittal wedging angles. Measurement reproducibility was evaluated. RESULTS: AIS girls before 11 years of age had slightly longer spines than controls (p = 0.04, Mann-Whitney test). AIS vertebrae were significantly more slender than controls at almost all levels, almost independently of topology. Frontal wedging of apical vertebrae was higher in AIS, as expected, but also lower junctional discs showed higher wedging than controls. CONCLUSION: AIS patients showed more slender spines than the asymptomatic population. Analysis of wedging suggests that lower junctional discs and apex vertebra could be locations of mechanical instability. Numerical simulation and longitudinal clinical follow-up of patients could clarify the impact of wedging, slenderness and growth on the biomechanics of scoliosis progression. These slides can be retrieved under Electronic Supplementary Material.

Alterations of 3D acetabular and lower limb parameters in adolescent idiopathic scoliosis.

PURPOSE: To evaluate the 3D deformity of the acetabula and lower limbs in subjects with adolescent idiopathic scoliosis (AIS) and their relationship with spino-pelvic alignment. METHODS: Two hundred and seventy-four subjects with AIS (frontal Cobb: 33.5° ± 18° [10°-110°]) and 84 controls were enrolled. All subjects underwent full-body biplanar X-rays with subsequent 3D reconstructions. Classic spino-pelvic and lower limb parameters were collected as well as acetabular parameters: acetabular orientation in the 3 planes (tilt, anteversion and abduction), center-edge angle (CEA) and anterior and posterior sector angles. Subjects with AIS were represented by both lower limb sides and classified by elevated (ES) or lowered (LS), depending on the frontal pelvic obliquity. Parameters were then compared between groups. Determinants of acetabular and lower limb alterations were investigated among spino-pelvic parameters. RESULTS: Acetabular abduction was higher on the ES in AIS (59.2° ± 6°) when compared to both LS (55.6° ± 6°) and controls (57.5° ± 3.9°, p < 0.001). CEA and acetabular anteversion were higher on the LS in AIS (32° ± 6.1°, 20.5° ± 5.7°) when compared to both ES (28.7° ± 5.1°, 19.8° ± 5.1°) and controls (29.8° ± 4.8°, 19.1° ± 4°, respectively, p < 0.001). Anterior sector angle was lower on both ES and LS in AIS when compared to controls. CEA, acetabular abduction and acetabular anteversion were found to be mostly determined (adjusted R2: 0.08-0.32) by pelvic tilt and less by frontal pelvic obliquity, frontal Cobb and T1T12. CONCLUSIONS: Subjects with AIS had a more abducted acetabulum at the lowered side, more anteverted acetabulum and a lack of anterior coverage of both acetabula. These alterations were strongly related to pelvic tilt.

Human Amniotic Membrane: A New Option for Graft Donor Sites - Systematic Review and Meta-analysis.

There are currently no standardised guidelines on the optimum dressing used for graft donor sites. The aim was to compare the outcomes of human amniotic membrane (HAM) vs routine dressings in split-thickness skin graft (STSG) donor site healing. A systematic review and meta-analysis was performed and a search of electronic information was conducted to identify all randomised controlled trials comparing the outcomes of HAM vs routine dressings in STSG donor sites. Wound healing and infection rate were primary outcome measures. Secondary outcome measures included severity of pain, discharge from donor site, the number of dressing changes, pruritus, and comfort. Fixed effect modelling was used for the analysis. Four studies enrolling 157 patients were identified. There was a significant difference between HAM and routine groups with wound healing time (P < .0001) and proportion of wounds healed by day 12 (P = .01). There was no significant difference between the two groups in infection rates (P = .27). For all secondary outcomes, HAM had improved results. HAM dressings are a superior option when compared with routine dressings used in current clinical practice for STSG donor sites as they improve wound healing and do not increase the infection rate.

Antibiotic resistance, virulence and genetic diversity of Klebsiella pneumoniae in community- and hospital-acquired urinary tract infections in Iran.

Klebsiella pneumoniae is among the most important causes of urinary tract infection (UTI). The aim of this study was to investigate the prevalence and correlation of antibiotic resistance with virulence characteristics and genetic diversity in K. pneumoniae isolated from UTIs in Iran. Phenotypic tests and antibiotic susceptibility were carried out on the isolates. Detection of the virulence and extended-spectrum ß-lactamase (ESBL) genes was performed by polymerase chain reaction. Pulsed-field gel electrophoresis (PFGE) was used for exploring the genomic relatedness. Hemolysin, biofilm, and hypermucoviscosity formation were observed in 87.1%, 86.4%, and 12.1% of isolates, respectively. The antibiotic resistance rate of K. pneumoniae isolates ranged from 12.1% for meropenem to 100% for amoxicillin. The prevalence of virulence genes ranged from 1.4% for cnf-1 to 100% for mrkD, fimH, kpn, and entB genes. In this study, 91.7%, 33.3%, and 4.2% of phenotypically ESBL-producers were positive for blaCTX-M, blaTEM, and blaSHV genes, respectively. An association was observed between the presence of traT, fyuA, or cnf-1 genes with antibiotic resistance. Two clone types were obtained by PFGE that indicate different K. pneumoniae clones in community- and hospital-acquired UTIs. The findings of this study are valuable in development of treatment strategies against UTIs in Iran.

Construction and evaluation of the immune protection of a recombinant divalent protein composed of the MrpA from MR/P fimbriae and flagellin of Proteus mirabilis strain against urinary tract infection.

Urinary tract infections (UTI) caused by Proteus mirabilis are prevalent among the catheterized patients. There is no effective vaccine to reduce the frequency of UTIs caused by P. mirabilis. In the present study, the immune responses and effectiveness of different combinations of MrpA and flagellin (FliC) of P. mirabilis were assessed intranasally in the mice model. The addition of FliC as adjuvant to MrpA in fusion form significantly raised the mucosal IgA and cellular (IFN-γ and IL-17) responses and maintained the serum IgG responses for 180 days after the first vaccination. Furthermore, MrpA in fusion form with FliC significantly increased the systemic, mucosal and IFN-γ responses of the FliC alone. In a bladder challenge assay with P. mirabilis, the fusion MrpA.FliC and the mixture of MrpA and FliC significantly decreased the colony count of the bacteria in the bladder and kidneys of mice in comparison to the control mice. It suggests a complex of the systemic, mucosal and cellular responses are needed for protection of the bladder and kidneys against P. mirabilis UTI. In our knowledge, the adjuvant property of the recombinant P. mirabilis flagellin was evaluated for the first time in a vaccine combination administered by an intranasal route. Our results suggest the recombinant flagellin of P. mirabilis could be used as an intranasal adjuvant in combination with other potential antigens against UTIs.

Determination immunogenic property of truncated MrpH.FliC as a vaccine candidate against urinary tract infections caused by Proteus mirabilis.

Proteus mirabilis is common cause of urinary tract infections (UTIs) especially in complicated UTIs which are resistant to antibiotic therapy, Consequently, an ideal vaccine is inevitably required. The N-terminal domain of MrpH (Truncated form of MrpH) lies between the most critical antigens of P. mirabilis to consider as vaccine candidate. FliC of Salmonella typhimurium induces several pathways of immunity system, which leads to produce antibody and cytokines. In this study, adjuvant properties of FliC and efficacy of truncated MrpH as important antigen, in tMrpH.FliC were determined in in vitro and in vivo circumstances. Three proteins including: FliC, MrpH and tMrpH.FliC were injected to mice and subsequently sera and supernatant of cell culture were collected to evaluate different immune responses. According to our findings, tMrpH.FliC could stimulate both humoral and cellular immune responses, so that serum IgG, urine IgA, IL.4, IFN-γ and IL.17 were increased significantly in comparison to MrpH and FliC alone, this augmentation was considerable. Results showed significant decrease of bacterial load in all of the challenged groups compared to the control group, although this protective effect was the highest in mice vaccinated with tMrpH.FliC. Our results showed truncated MrpH, without an unwanted domain is an ideal vaccine target and FliC, as adjuvant, increases its immunogenic property. Thus, fusion protein tMrpH.FliC can be considered as promising vaccine against P. mirabilis.

Evaluation of prevalence, immunogenicity and efficacy of FyuA iron receptor in uropathogenic Escherichia coli isolates as a vaccine target against urinary tract infection.

Uropathogenic Escherichia coli (UPEC) are among the most prevalent agents of urinary tract infections (UTIs). Antibiotic resistance reaches the need for alternative treatment approaches such as vaccination against UTIs. There is no ideal vaccine against UTIs, thus there is a need to evaluate different targets of uropathogens against UTIs. Ferric scavenger receptor FyuA in UPEC has the properties of an ideal vaccine candidate against UTIs. In the present study, the prevalence of FyuA among UPEC isolates, its immunogenicity with and without alum adjuvant, and its efficacy against experimental UTI were assessed. Totally, fyuA gene was present in 77% of the UPEC isolates tested. Alignments of FyuA exhibited a high degree of conservation among different submitted UPEC isolates in GenBank. The bioinformatics studies showed the high confidence value and stability of the FyuA structure. SDS-PAGE and Western blot confirmed the purification of FyuA with high yield by nickel resins. Mice vaccinated subcutaneously with the FyuA induced a significantly higher humoral response (total IgG, IgG1 and IgG2a) than control mice that alum enhanced these responses. The FuyA alone showed the ability to reduce the colonization of UPEC in bladder and kidney of mice as compared to the control group. But the addition of alum to FyuA increased the protection level against UPEC in these organs. Since, FyuA induced significant IgG1 (Th2) and IgG2a (Th1) responses and protected the mice against experimental UTI, it could be a promising target against UPEC infections.

Evaluation of immunological responses to recombinant Porin A protein (rPoA) from native strains of Neisseria meningitidis serogroups A and B using OMV as an adjuvant in BALB/c mice.

Neisseria meningitidis is one of the main causes of sepsis and meningitis, which are two serious life-threatening diseases in both children and adolescents. Porin A (porA) from both serogroup A and B were cloned into the pET28a plasmid and expressed in E. coli BL21 (DE3). The protein was expressed in Escherichia coli BL21 (DE3) and confirmed by SDS-PAGE and Western blot analysis. BALB/c mice were subcutaneously injected three times with 25 µg of the recombinant PorA. Specific total IgG antibodies and isotypes were evaluated using ELISA assay. Opsonophagocytic assay (OPA) and Serum Bactericidal assay (SBA) were performed. Results showed that vaccinated mice exhibited higher levels of anti-Porin A (p < 0.05) with a predominant IgG1 response compared to the control group. Results from in vitro experiments indicated that N. meningitidis was opsonized with immunized-mice sera, and compared to non-immunized mice, immunized mice displayed significantly increased phagocytic uptake and effective intracellular killing. In this study, serogroup B N. meningitidis OMV of strain CSBPI G-245 and complete and incomplete Freund's adjuvant were used. Results demonstrated that Porin A could be a valuable target for the development of immunotherapeutic strategies against N. meningitidis.

Use of flagellin and cholera toxin as adjuvants in intranasal vaccination of mice to enhance protective immune responses against uropathogenic Escherichia coli antigens.

Urinary tract infections (UTIs) caused by Uropathogenic Escherichia coli (UPEC) are among the most common infections in human. Antibiotics are common therapy for UTIs, but increase in antibiotic resistance will complicate future treatment of the infections, making the development of an efficacious UTI vaccine more urgent. In this study, we have evaluated intranasally the efficacy of FliC and FimH antigens of UPEC in different vaccine formulations with and without cholera toxin (CT) adjuvant. Immunization of mice with FliC in fusion form or admixed with FimH elicited higher levels of serum, mucosal and cell-mediated responses than FimH alone. Furthermore, the use of CT in synergism with FliC resulted in the stimulation of a mixed Th1 and Th2 responses against FimH and FliC as antigen and maintained the antibody responses for at least 24 weeks following the last vaccine dose. Of the vaccine preparations, Fusion, Fusion + CT, and FimH admixed with FliC and CT showed the best protection against UPEC. These data indicated that intranasal administration of a FliC and CT adjuvant-based vaccine has the potential to provide protective responses against UPEC strains.