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Kefir drink is one of the most important probiotic products, which is made using kefir microorganisms in fermenting the milk. Numerous investigation have been accomplished in the field of the therapeutic property of probiotic products. In the present study, we assessed the cytotoxic effect of kefir on the rate of growth and increase of glioblastoma cancer cell as the most severe form of brain tumors. In this experimental study, we used a U87 cancer cell line (glioblastoma). The interaction between cancer cells and different concentrations of kefir drink and supernatants at 24 and 48 hours was considered. The cell cytotoxicity of kefir and sedimentation of cell lysate and extract of kefir was assessed using the MTT test after 24 and 48 hours. The result of the MTT test, treatment of the cells with the 48-hour fermented drink, demonstrated the most cell cytotoxicity in comparison with the control group. Results showed that the toxicity effect in all groups was dose-dependent, and by increasing the concentration, cell survival decreased noticeably. The results indicated that the supernatant of fermented kefir drink as a probiotic product has more toxicity and lethality effect on the glioblastoma cancer cell. This product can be utilized as a replacement or a complementary therapy of cancer.

Introduction. Streptococcus pneumoniae is a significant bacterial pathogen in humans. Currently, there are two types of pneumococcal vaccines, but there are concerns regarding their application. Aim. Since many pneumococcal proteins are serotype-independent, polyhistidine triad protein D (PhtD) has been selected as a vaccine candidate. Methodology. We prepared recombinant PhtD and its C-terminal fragment (PhtD-C) using alum and outer-membrane vesicles (OMVs) as adjuvants. The combinations were injected intraperitoneally into mice, and then total immunoglobulin G (IgG) and specific IgG, IgG1 and IgG2a were measured. A serum bactericidal assay and opsonophagocytosis were also performed as complementary tests. Meningococcal OMVs were used as an adjuvant. Results. The levels of specific IgG and IgG1 against combinations of PhtD and its C-terminal with OMVs and alum as adjuvants increased at the time of the third mouse immunization on day 35. Forty per cent and 60% of S. pneumoniae ATCC 6303 (serotype 3) as a virulent pneumococcal strain, respectively, were killed in the opsonophagocytosis test and these results could also be observed in the serum bactericidal assay. Mice mmunized iwith PhtD and its C-terminal with OMVs and alum as adjuvants survived after 10 days of pneumococcal challenge. Conclusion. The combination of PhtD and PhtD-C with alum produced optimal results, but the combination of PhtD and PhtD-C with OMVs produced minimal results by comparison. The survival rates were also measured, and these corresponded with the results of the immunological assessments. Our findings showed that mice receiving PhtD and PhtD-C plus OMV and alum had higher survival rates than the mice in the other groups.

Protein E (PE) is a conserved entity observed in both nontypeable Haemophilus influenzae (NTHi) and encapsulated H. influenzae . This is a small surface lipoprotein, consisting of only 160 amino acids, involved in the adhesion of H. influenzae to various types of epithelial cells. A 384-bp-long fragment from NTHi PE was cloned into the prokaryotic expression vector pBAD-gIIIA. The recombinant protein was expressed with arabinose and then purified by affinity purification on an Ni - NTA agarose matrix. BALB/c mice were immunized by subcutaneous injection with purified recombinant truncated PE mixed with an alum adjuvant. Serum antibody response and the functional activity of antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA) and serum bactericidal assay (SBA), respectively. Colony PCR, double digestion, and sequencing were used to verify successful cloning of truncated PE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses indicated the presence of a ∼15-kDa recombinant protein. Serum IgG, IgG1, and IgG2a levels were significantly higher in the group immunized by recombinant truncated PE mixed with an alum adjuvant, compared to the non - vaccinated control group. Development of a strong bactericidal effect against NTHi was observed in the serum samples from immunized animals. Our findings suggest that recombinant truncated PE is a potential vaccine candidate for NTHi.

Nonencapsulated, nontypeable Hemophilus influenzae (NTHi) remains an important cause of acute otitis and respiratory diseases in children and adults. NTHi bacteria are one of the major causes of respiratory tract infections, including acute otitis media, cystic fibrosis, and community-acquired pneumonia among children, especially in developing countries. The bacteria can also cause chronic diseases such as chronic bronchitis and chronic obstructive pulmonary disease in the lower respiratory tract of adults. Such bacteria express several outer membrane proteins, some of which have been studied as candidates for vaccine development. Due to the lack of effective vaccines as well as the spread and prevalence of NTHi worldwide, there is an urgent need to design and develop effective vaccine candidates against these strains.

Introduction: As the causative agent of meningitis, Neisseria meningitidis has different serogroups. The purpose of this study was to investigate the molecular properties of N. meningitidis strains among Iranian cases. Methods: 450 samples were collected from children under 5 years of age. Detection of Neisseria genus was done by phenotypic and genotypic methods. Multiplex PCR was used to identify the serogroups of N. meningitides. The sequencing of variable regions of porA gene was performed for detection of the subserogroups. Results: From 137 (30.44%) Neisseria isolates, 4 isolates (0.88%) belonged to N. meningitidis and 133 isolates (29.55%) belonged to other species. Multiplex PCR results showed that one isolate belonged to serogroup A while 3 belonged to serogroup B. The analysis of amplified VR1 and VR2 variable regions of porA showed 100% identity of the serogroup A strain with strain BZ83N and the serogroup B strains with strain 528 of N. meningitidis . In accordance with other findings in Asia, serogroups A and B were the most prevalent serogroups of N. meningitidis. Sequencing of variable regions of porA could identify the subserogroups of the isolates. Conclusion: sequencing of porA could be a valuable method for identification of N. meningitidis strains to be used in epidemiological studies as well as improved vaccine designs.

Introduction: Neisseria meningitidis is a major causative agent of bacterial septicemia and meningitis in human. PorA is a major component of the outer membrane of N. meningitidis and functions as a cationic Porin. This study aimed to clone and determine the expression of PorA as the first step for producing a proper antigen in a vaccine study against N. meningitidis. Methods: An approximately 1200-bp fragment of porA gene was amplified by PCR using N. meningitidis serogroup A genomic DNA and then cloned into prokaryotic expression vector pET-28a. The resulting construct (pET28a-porA plasmid) was transformed into competent E.coli BL21 cells for expression of recombinant protein. The proper overexpression of the recombinant protein was verified by SDS-PAGE and Western Blotting. Results: Cloning of porA was confirmed by colony-PCR and enzymatic digestion. The nucleotide sequence homology of the cloned porA gene was 97% , compared to the reference gene (NCBI GenBank accession number AL157959.1). The prokaryotic expression system (pET28a-porA- BL21) could produce our 45-kDa target recombinant protein, efficiently. Conclusion: The prokaryotic expression system and conditions used in this study provides an applicable method for producing recombinant PorA and possibly many other bacterial outer membrane proteins for future vaccine studies.

Background: Nonencapsulated, nontypeable Haemophilus influenzae (NTHi) is considered an important cause of acute otitis in children and respiratory diseases among adults. The bacteria express several outer membrane proteins, some of which have been studied as vaccine candidates. Protein D is a highly conserved surface lipoprotein, which has been found in strains of both encapsulated and nonencapsulated H. influenzae. Objectives: Opsonin and protective antibodies against protein D play major roles in the prevention of infections caused by NTHi. Since NTHi can be also an intracellular organism, it needs to be removed through immune-mediated mechanisms. Methods: In this study, groups of BALB/c mice were subcutaneously immunized with recombinanat truncated D protein and an aluminum hydroxide (alum) adjuvant or protein D combined with an outer membrane vesicle (OMV) adjuvant from Neisseria meningitidis bacteria. Then, opsonophagocytic activities of antibodies were measured in serum samples collected on days 14, 28, and 42. The levels of interleukin-4 (IL-4), IL-10, and interferon-gamma were measured in splenocyte cultures of immunized mice. Results: The opsonophagocytic activity of antibodies significantly increased in the immunized mice, compared to the control group, particularly on day 42. In addition, the secretion level of cytokines significantly increased in both groups of immunized mice, compared to the control group. Conclusions: The results of this study demonstrated that recombinant truncated D protein can induce specific immune responses against nonencapsulated H. influenzae. It was suggested that the recombinant truncated D protein, as a vaccine candidate against the nonencapsulated strains of H. influenza, is essential in protection and development of sustainable immunity against infections caused by this bacterium.

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

Interleukin-28B (IL28B) single-nucleotide polymorphisms (SNPs) constitute important host-related factors influencing the response rate to Hepatitis C virus (HCV) standard antiviral therapy. In the last few years, several new technologies for SNP detection have been developed. However, the sensitivity and specificity of various methods are different and needs evaluation. Five different methods (resolution melting curve [RMC], polymerase chain reaction-restriction fragment length polymorphism [PCR-RFLP], PCR-sequencing analysis, amplification refractory mutation system [ARMS], and zip nucleic acid probe-based real-time PCR [ZNA]) were developed for genotyping rs12979860 associated with IL28B. In this study, limit of detection (LD), costs and turnaround time of these methods were compared in 350 subjects. As for IL28B rs12979860 polymorphisms, 348/350 (99.4%) samples were consistent among the five methods, while results for 2/350 (0.57%) samples were concordant by ZNAs and PCR-sequencing, and discordant by other methods. Without considering the cost of DNA extraction, the price of each reaction for ARMS-PCR, RMC, PCR-RFLP, ZNA and PCR-sequencing were respectively: US$3.10, US$5.0, US$5.50, US$8.50 and US$17.0. RMC was the fastest method, while the ZNA method was easy to use, reliable and effective. Lower LD was determined to be 50-60 copies/μL for the PCR-RFLP, RMC and ARMS-PCR assays; whilst ZNA assay was able to detect 2-3 copies/μL. In conclusion, in the current study, all four methods are suitable for IL28B rs12979860 genotyping, but the ZNA assay can be a reliable tool. Due to its lower LD for SNP identification, this method is better than others for detecting this type of polymorphism.

Manitol fermentation, and DNase tests). S. aureus isolates were investigated for the variability of HVR of mecA gene by PCR method. Results: Among 261 collected nasal swabs, 70 (27%) were S. aureus. Of these, 29(41%) isolates were resistant to Oxacillin and 32 (46%) of those had mec-HVR gene. The polymerase chain reaction (PCR) products showed five different patterns of HVR. Also among 109 clinical samples, 52 (48%) of them were S. aureus. Of these, 40 (77%) were resistant to Oxacillin and 45 (87%) of them carried the mec-HVR gene. The PCR products showed 11 different patterns of HVR. Conclusions: Molecular typing of MRSA isolates by HVR amplification has shown a high diversity among the strains and can be used as a basis for tracking the contaminations and the source of hospital infections from staff to patients and vice versa.

Background: Staphylococcus aureus is one of the important human pathogens which are mainly isolated from wound, skin and contaminated respiratory excretions. Because many of hospital staff and patients carry this pathogen in their nose or skin, close contacts and touching have special role in spreading the infection in hospitals. Also, antibiotic resistant S. aureus, especially Methicillin Resistant S. aureus (MRSA) have been seen among subjects. Thus, there should be an investigation for Bacteria colonization in nose of hospital staff and patients. Furthermore, investigation of antibiotic resistance pattern and examination of genotyping properties of resistant strains have a high efficacy in control and recognition of infection origin. Objective: The current study aimed to determine the characteristics of S. aureus isolated from patients and staff in hospitals and compare them based on coa and spa typing methods. Materials and Methods: In the current study, 157 clinical specimens were collected from patients who were treated at the Ahvaz medical university hospitals including 79 specimens (50.3) from Sina hospital, 34 specimens (21.7) from Imam Khomeini hospital, and 44 specimens (28) from Golestan hospital and 157 nose swab specimens from the staff of these hospitals were collected during 2010. coa, spa genes of isolated Bacteria were amplified using PCR. Result: PCR results showed seven different patterns for staff and five different patterns for patients based on spa gene, and for coa gene five and six different patterns respectively. In addition, the prevalence of MRSA was 52.5 in staff and 83.7 in patients' specimens. Comparison of genetic diversity of spa, and coa genes in Ahvaz university hospitals doesn't show significant difference (Chi-square and fisher's exact test). Concloutions: The outcome of this study show that spa and coa typing are suitable methods for MRSA isolates typing because it is easy to use and interpret them, and that these methods can be useful in infection source detection and its control especially in epidemic situations. © 2013, Ahvaz Jundishapur University of Medical Sciences.