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DNA vaccines have emerged as a promising approach for generating antigen-specific immunotherapy. However, due to their low immunogenicity, there is a need to enhance DNA-based vaccine potency. Two main strategies to increase DNA-based vaccine potency are the employment of immuno-adjuvants such as heat shock proteins (HSPs) and a method of improving the delivery of naked plasmid DNA by electroporation. In the current study, we evaluated the effects of linkage of human papillomavirus (HPV) type 16 E7 as a model antigen to N-terminal and C-terminal of glycoprotein 96 (NT-/CT-gp96) on the potency of E7-specific immunity generated by DNA vaccines. We found that subcutaneous DNA injection with E7-CT (gp96) followed by electroporation generates the significant E7-specific IFN-γ immune responses as well as the best protective effects in vaccinated mice as compared to E7 or E7-NT (gp96) DNA vaccines. Therefore, our data indicate that subcutaneous administration of E7 DNA linked to CT (gp96) fragment followed by electroporation can significantly enhance the potency of DNA vaccines. Indeed, the structural domains of immuno-chaperones show the potential of generating effective immune responses against different clinical disorders such as cancer. Altogether, our results show that comparable regions of gp96 (N-/C-terminal fragments of gp96) may have qualitatively different immunological effects in vaccine design., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Listeria monocytogenes has been identified as an important foodborne pathogen in recent years. In humans, it most commonly affects pregnant women, neonates, children, elderly people, and persons with a suppressed immune system. It could contaminate both raw and cooked meat and poultry products. Studies regarding prevalence and risk factors of L. monocytogenes in broilers flocks are limited. Therefore, the present study was conducted to determine the prevalence and risk factors for L. monocytogenes in poultry flocks in Shiraz, southern Iran. During August to September 2009, in total, 100 broiler flocks were selected at slaughter, and 21 specimens were collected from cloacal samples from each flock. Polymerase chain reaction (PCR) was performed on the samples enriched in buffered Listeria enrichment broth (BLEB), using specific primers. Furthermore, enriched samples in BLEB and/or BLEB treated with 5% KOH were subcultured on Palcam medium. Data about farm and flocks were collected using a structured questionnaire. The prevalence of L. monocytogenes was 7% (95% CI, 2-12%) and 1% using PCR and culture, respectively. Results showed that using antibiotics during rearing period was dramatically reduced the rate of isolation (odds ratio [OR]=0.07, p=0.03), whereas house capacity of more than 10,000 birds (OR=24.03, p=0.04) and number of houses (OR=2, p=0.02) significantly increased the prevalence. The correlation between poor management of large poultry flocks and increasing the risk of contamination was more likely due to the recontamination of cooked poultry/undercooking or cross-contamination of other ready-to-eat foods.

The global outbreak of novel A/H1N1 spread in human population worldwide has revealed an emergency need for producing a vaccine against this virus. Current influenza vaccines encounter problems with safety issues and weak response in high-risk population. It has been established that haemagglutinin is the most important viral antigen to which antibody responses are directed, and recombinant subunit vaccines, haemagglutinin of influenza A and B viruses, have been considered in order to facilitate vaccine production. In the present study, we have focused on construction of a recombinant baculovirus encoding the large subunit of novel influenza virus A/H1N1 haemagglutinin. The full genome of haemagglutinin was cloned into pGEM-TEasy vector and sequenced. The large subunit of the haemagglutinin gene was amplified by PCR using specific primers and cloned into pFast- BacHTc donor plasmid, which was then confirmed by restriction enzyme analysis and sequencing and transformed into E. coli DH10Bac competent cells. The bacmid DNA was transfected into insect cells to produce recombinant baculovirus. Expression of recombinant haemagglutinin in insect cells was determined by SDS-PAGE and immunoblotting. It has been shown that the recombinant haemagglutinin (rHA) obtained from the baculovirus insect cell expression system has suitable immunogenicity in human and can be considered as a candidate flu vac- cine. Here we produced large amounts of the HA1 protein of novel influenza A/H1N1 (Iranian isolate) in insect cells. The immunogenicity and efficacy of the recombinant HA1 will be evaluated as a vaccine candidate and compared to the recombinant HA1 produced in a prokaryotic system.

Campylobacter species are among the most common bacterial causes of human gastroenteritis in many countries, and poultry meat is considered as a major source of human campylobacteriosis. The present study was conducted to determine the prevalence of infection by Campylobacter jejuni and Campylobacter coli in broiler flocks in Shiraz and to investigate the possible risk factors for the campylobacter infections in this area. For detection of campylobacter, multiplex polymerase chain reaction (mPCR) was used. Between August and September 2009, a total of 100 broiler flocks from 100 commercial broiler farms were selected at slaughter and campylobacter status was determined by mPCR on caecal samples. Data about farms and flocks were collected by questionnaires. Approximately 76% (95% CI: 67-84%) of the flocks were positive for C. jejuni or C. coli. Twenty two percent were positive for C. jejuni, 32% for C. coli and 22% for both species. Results of the statistical analysis using multivariable logistic regression showed that the odds of flock infection decreased when level of owner's education (years) increased (OR=0.86, P=0.04), also odds of infection was nearly five times higher when age at slaughter was ≥45 days compared with <45 days (OR=5.3, P=0.003) and use of antibiotic medications at early stage of production period was negatively associated with the infection status of the flock (OR=0.33, OR=0.059). We found no evidence of the effects of any other factors such as time interval between successive flocks, hygiene measures and number of broiler houses on the farm on the prevalence of campylobacter infection. Getting more attention to the health education issues and planning qualitative studies to reveal the behavioral aspects of the management policy, may be subjects of interest for future researches., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Early detection of myocardial iron overload is crucial for optimal management of patients with beta thalassemia major, which could lead to intensification of iron chelating therapy. In this study, we evaluate the conventional echocardiography and tissue Doppler imaging measurements in patients with beta thalassemia major and further introduce the assessment of atrial ejection force as a feasible price-saving approach for early detection of myocardial iron overload. During a 1-year period, 42 cases of beta thalassemia major aged <21 years and with preserved systolic function were evaluated with magnetic resonance T2* imaging (MRI T2*), conventional echocardiography, and tissue Doppler imaging techniques. Patients were classified into two groups according to their myocardial MRI T2* values, with and without critical iron loading. All patients with echocardiographic evidence of moderate and severe stages of diastolic dysfunction showed critical iron loading in their MRI T2*. After excluding those patients with severe and moderate ventricular diastolic filling abnormality, the atrial ejection force index (P = 0.002) and a number of volume indexes of the left atrium showed a significant difference between the two groups. None of the tissue Doppler imaging measurements showed a statistically significant difference between the two groups. The atrial ejection force index of 7.41, with a sensitivity of 93% and a specificity of 74%, was suggested to detect critical cardiac iron loading. These results imply that combining the atrial ejection force index with the transmitral-derived echocardiographic assessment is a feasible way to detect early stages of myocardial iron overload in patients with beta thalassemia major.

Recent reports have indicated different effects of immunostimulatory sequences containing CpG-Oligodeoxynucleotides (ODN) on various immune cells. However, the exact role of CpG-ODN in the human gut is unclear. In the present study, we assessed potential effects of CpG-ODN on non lymphoid cell (intestinal epithelial cell line HT-29) on a dose-response and time-course basis. Intestinal epithelial cell line HT-29 was treated with CpG-ODN (CpG 2006) and lipopolysaccharide (LPS) at 5, 10, 25, 50 microg/ ml and 1, 5, 10 microg/ ml concentrations, respectively. Following treatments, dose- response and time-course cytotoxicity using a colorimetric method, Metaloproteinase-2 (MMP-2) activity (using gelatin zymography) and apoptosis (using annexin-v flowcytometry method) assays were performed. Chloroquine treatment was also used for its inhibitory effect on endosomal acidification process to verify specific CpG-ODN and Toll Like Receptor 9 (TLR9) interactions. Cytotoxicity analysis of CpG-ODN showed that CpG-ODN increased significantly the proliferation of CpG-ODN treated cells, as compared to untreated cells, at concentrations of 10-25 microg/ml (p < 0.05). Overall MMP-2 activity analysis showed significant differences between treated and untreated cells. However, minimal changes were observed when MMP-2 activity was assessed per cell. Moreover, CpG-ODN treated cells demonstrated an increasing apoptosis rate of 0.8 %, 6.46 % and 14.21% at concentrations of 5, 10, 25 microg/ml, respectively. Collectively, our data indicated that intestinal epithelial cell line HT-29 is highly responsive to CpG effect in vitro and exhibits modified activities. The direct CpG-ODN and TLR-9 interactions in HT-29 cells could provide new approaches in malignant tumor therapeutic strategies.

A new automated headspace solid-phase microextraction (HS-SPME) sampling device was developed, with the capability of heating the sample matrix and simultaneously cooling the fiber coating. The device was evaluated for the quantitative extraction of polycyclic aromatic hydrocarbons (PAHs) from solid matrices. The proposed device improves the efficiency of the release of analytes from the matrix, facilitates the mass transfer into the headspace and significantly increases the partition coefficients of the analytes, by creating a temperature gap between the cold-fiber (CF) coating and the hot headspace. The reliability and applicability of previously reported cold-fiber devices are significantly enhanced by this improvement. In addition, it can be easily adopted for full automation of extraction, enrichment and introduction of different samples using commercially available autosampling devices. Sand samples spiked with PAHs were used as solid matrices and the effect of different experimental parameters were studied, including the extraction temperature, extraction time, moisture content, and the effect of sonication and modifier under optimal experimental conditions, linear calibration curves were obtained in the range of 0.0009-1000 ng/g, with regression coefficients higher than 0.99 and detection limits that ranged from 0.3 to 3 pg/g. Reproducible, precise and high throughput extraction, monitoring and quantification of PAHs were achieved with the automated cold-fiber headspace solid-phase microextraction (CF-HS-SPME) device coupled to GC-flame ionization detection. Determination of PAHs in certified reference sediments using the proposed approach exhibited acceptable agreement with the standard values.

Background: Previous work has shown that co-incubation of human sperm with Chlamydia trachomatis serovars E and LGV leads to premature sperm death and that this is due primarily to chlamydial lipopolysaccharide (LPS). Here, we investigated the possible involvement of apoptosis in this premature sperm death., Methods: Highly motile preparations of sperm from normozoospermic patients were co-incubated for 6 h with extracted LPS from C. trachomatis serovars E and LGV. Three different methods were used to determine if LPS-treated sperm underwent apoptosis, including: (i) flow cytometry; (ii) measurement of ADP:ATP ratios; and (iii) measurement of mono- and oligonucleosomal DNA fragments. Caspase activity was also investigated by fluorimetry and by use of a pan-caspase inhibitor and caspase-3 inhibitor., Results: All three methods used for detection indicated that C. trachomatis LPS induced some apoptosis in sperm after 6 h when compared with a staurosporine (apoptosis-positive) control. Moreover, a greater degree of apoptosis was seen with C. trachomatis serovar E than with serovar LGV. It was also shown that C. trachomatis LPS-induced apoptosis of sperm could be blocked with a pan-caspase inhibitor and a caspase-3 inhibitor. Moreover, by using a fluorogenic substrate, apoptosis was shown to be caspase-mediated., Conclusions: In general it is believed that apoptosis does not occur in C. trachomatis-infected host cells. However, using three different methods, our findings clearly indicate that co-incubation of sperm with C. trachomatis LPS results in cellular death which is in part due to apoptosis and is caspase-mediated. These findings provide an explanation as to how C. trachomatis can mediate premature death in human sperm.

We have shown previously that the in vitro exposure of spermatozoa to elementary bodies (EBs) of Chlamydia trachomatis can lead to sperm death over a number of hours of incubation. As such, we have hypothesized that the ejaculates of men with a chlamydial infection could contain increased numbers of nonmotile (dead) spermatozoa if they are exposed to EBs prior to ejaculation. To test this hypothesis, the ejaculates of 642 men undergoing diagnostic semen analysis as part of ongoing infertility investigations with their partner were examined. All men were without symptoms of genitourinary infections and semen analysis was performed according to World Health Organisation (WHO) 1999 methods after a 3-5 day abstinence period. In addition to semen analysis, nested plasmid polymerase chain reaction (PCR) was undertaken on the ejaculate to detect the presence of C trachomatis DNA. A total of 31 semen specimens (4.9%) were found to be positive, and in 28 of these, the diagnosis was confirmed using the ligase chain reaction (LCR). Men whose ejaculates were PCR positive for chlamydial DNA had a significantly (P <.05) higher mean concentration of leukocytes (1.71 +/- 2.20 x 10(6) per mL) and a higher mean ejaculate volume (3.45 +/- 1.52 mL) than in those whose ejaculates were PCR negative (leukocyte concentration: 0.67 +/- 2.59 x 10(6) per mL; volume 2.93 +/- 1.38 mL). Leukocytospermia was twice as common in men that were PCR positive for chlamydial DNA (P <.05) but it was not always associated with the presence of chlamydial DNA in semen. However, there was no difference in the mean percent motility between the 2 groups and the proportion of asthenozoospermia also did not differ. Because these results do not confirm the hypothesis proposed from our in vitro experiments, further work needs to be undertaken to understand whether human spermatozoa are actually exposed to elementary bodies of C trachomatis in an infected individual prior to ejaculation.

Elementary bodies (EBs) of Chlamydia trachomatis serovar E are more toxic to sperm than those from serovar LGV. In this study, lipopolysaccharide (LPS) was prepared from the EBs of both serovars and incubated with human spermatozoa at concentrations that matched the LPS concentration of EBs. The effects of EBs and LPS on sperm motility, viability and acrosomal status were then determined. Sperm motility was measured by computer-assisted sperm analysis and the hypo-osmotic swelling test was used to determine the proportion of dead cells. Acrosomal status was examined using a standard mAb assay. Over a 6 h incubation, LPS from both serovars resulted in a marked reduction in sperm motility (and a concomitant increase in the proportion of dead spermatozoa) in a manner similar to that seen in response to EBs of serovar E. In addition, when sperm were incubated with a range of doses of EBs and LPS, probit analysis revealed that the greater spermicidal effects of EBs from serovar E (when compared with serovar LGV) were not observed when sperm were incubated with LPS from the two serovars. This suggests that the more potent effect of EBs of serovar E cannot be explained entirely by differences in the composition of LPS. Interestingly, Escherichia coli LPS was required in doses 500 times more concentrated than chlamydial LPS in order to kill a similar proportion of sperm, suggesting that bacterial LPSs may differ in their spermicidal properties. However, that chlamydial LPS was spermicidal was demonstrated by the use of polymyxin B (a polycationic antibiotic known to neutralize LPS effects), confirming that the effects observed were primarily a result of LPS activity.

The aim of this work was to investigate the effect of elementary bodies (EB) of Chlamydia trachomatis serovars E and LGV on sperm motility, viability and acrosomal status. Highly motile preparations of spermatozoa from normozoospermic patients were co-incubated for 6 h with 0.54x10(6) EB per ml. At 1, 3 and 6 h of incubation, sperm motility was determined by computer-assisted semen analysis (CASA) and the proportion of dead cells determined by the hypo-osmotic swelling (HOS) test. Acrosomal status was also examined using a standard monoclonal antibody assay. In the absence of EB, the percentage of motile spermatozoa remained >69% over the 6h incubation and the proportion of dead spermatozoa at <12%. However, during the incubation with EB of serovar E there was a significant decline in the percentage of motile spermatozoa (P < 0.05), and a corresponding increase in the proportion of dead spermatozoa (P < 0.05) at all time-points. However, following incubation with serovar LGV, only the percentage of dead spermatozoa after 6 h incubation was significantly different from the control (P < 0.05). The amount of acrosome-reacted spermatozoa remained unchanged (<16%) in all incubations at all time-points. Dose-response experiments indicated that increasing the concentration of EB to 2.5x10(6) per ml did not significantly alter the results. Furthermore, co-incubation of spermatozoa with dead EB (killed by heat treatment) abolished the chlamydia-mediated response, indicating that the effect is a result of the live organism and not soluble components or membrane elements. These data suggest that a detrimental effect on sperm function by some serovars may be an as yet unrecognized component of infertility problems.

Elementary bodies (EBs) of the obligate intracellular bacterium Chlamydia trachomatis are responsible for the first step of attachment to host cells. We have studied the effects of EBs on human sperm protein tyrosine phosphorylation, which is important to sperm function. Indirect immunofluorescence using antiphosphotyrosine antibodies showed that serovar E, but not LGV, caused increased tyrosine phosphorylation which was localized to the sperm tail region. Immunoblotting revealed that serovar E caused a marked increase in tyrosine phosphorylation of 80- and 95-kDa sperm proteins, whereas serovar LGV caused increased phosphorylation of only the 80-kDa moiety. Considering the importance of tyrosine phosphorylation for sperm capacitation and other aspects of sperm function, we conclude that EBs may affect these events.