The anticancer effect of potential probiotic L. fermentum and L. plantarum in combination with 5-fluorouracil on colorectal cancer cells.

5-Fluorouracil (5-FU) is an effective chemotherapy drug in the treatment of colorectal cancer (CRC). However, auxiliary or alternative therapies must be sought due to its resistance and potential side effects. Certain probiotic metabolites exhibit anticancer properties. In this study evaluated the anticancer and potential therapeutic activities of cell extracts potential probiotic strains, Limosilactobacillus fermentum and Lactiplantibacillus plantarum isolated from the mule milk and the standard probiotic strain Lacticaseibacillus rhamnosus GG (LGG) against the human colon cancer cell line (HT-29) and the normal cell line (HEK-293) alone or in combination with 5-FU. In this study, L. plantarum and L. fermentum, which were isolated from mule milk, were identified using biochemical and molecular methods. Their probiotic properties were investigated in vitro and compared with the standard probiotic strain of the species L. rhamnosus GG. The MTT assay, acridine orange/ethidium bromide (AO/EB) fluorescent staining, and flow cytometry were employed to measure the viability of cell lines, cell apoptosis, and production rates of Th17 cytokines, respectively. The results demonstrated that the combination of lactobacilli cell extracts and 5-FU decreased cell viability and induced apoptosis in HT-29 cells. Furthermore, this combination protected HEK-293 cells from the cytotoxic effects of 5-FU, enhancing their viability and reducing apoptosis. Moreover, the combination treatment led to an increase in the levels of IL-17A, IFN-γ, and TNF-α, which can enhance anti-tumor immunity. In conclusion, the cell extracts of the lactobacilli strains probably can act as a potential complementary anticancer therapy.

Biofilm stimulating activity of solanidine and Solasodine in Pseudomonas aeruginosa.

BACKGROUND: Biofilm formation has reported as an important virulence associated properties of Pseudomonas aeruginosa that is regulated by quorum-sensing associated genes. Biofilm and quorum-sensing interfering properties of steroidal alkaloids, Solanidine and Solasodine were investigated in the present study. RESULTS: Biofilm formation capacity and relative expression level of five studied genes(lasI, lasR, rhlI, rhlR and algD) were significantly increased dose-dependently after treatment with sub-inhibitory concentrations (32 and 512 µg/ml) of the both Solanidine and Solasodine. Biofilm formation capacity was more stimulated in weak biofilm formers(9 iaolates) in comparison to the strong biofilm producers(11 isolates). The lasI gene was the most induced QS-associated gene among five investigated genes. CONCLUSION: Biofilm inducing properties of the plants alkaloids and probably medicines derived from them has to be considered for revision of therapeutic guidelines. Investigating the biofilm stimulating properties of corticosteroids and other medicines that comes from plant alkaloids also strongly proposed.

Formalin and ferric chloride inactivated Pasteurella multocida type a adjuvanted with bacterial DNA and alum as a new vaccine candidate in sheep pasteurellosis.

The aim of the present study was to investigate humoral and cellular immune responses in sheep inoculated with inactivated P. multocida antigen with alum and bacterial DNA adjuvant by identifying IgG and cytokines from serum and cell culture. Sheep were immunized with iron and formalin-inactivated antigens at an interval of 2 weeks. These immunogens were mixed with alum adjuvant and P. multocida type A DNA (AbDNA). After injection and blood sampling, the serum antibody titer and cellular immune responses (IL-4, IFN-γ, and TNF-α) on serum samples and lymphocyte cell were tested by ELISA. The ELISA results showed a higher antibody titer in the bDNA adjuvant group compared to the alum adjuvant group and the control group. In general, the level of IgG in the serum of immunized animals was significantly increased compared to the control group. The peak antibody titer (1.794) was observed on the 28th day of injection in the IIV-AbDNA group. After immunization, inactivation with iron and bDNA adjuvant increased cytokine production compared to other experimental and control groups. High levels of lymphocyte and serum titers of IL-4, IFN-γ, and TNF-α were also obtained in the IIV-AbDNA group. The findings showed that killed P. multocida type A antigens formulated with bacterial DNA as an adjuvant are candidates for new immunogens against P. multocida infections in sheep. The inactivation of bacteria with iron also enhanced proper immune responses.

Cytotoxic and apoptotic effects of chemically synthesized silver nanoparticles loaded with recombinant Staphylococcus LukS-PV toxin.

Chronic myeloid leukemia (CML) accounts for approximately 15% of leukemias. LukS-PV, a Panton-Valentine leucocidin (PVL) component, is secreted by Staphylococcus aureus. Silver nanoparticles have increasingly been used for different purposes, most notably for drug delivery and anticancer agents. In this work, the cytotoxicity effect of recombinant LukS-PV protein, chemically synthesized AgNPs, and recombinant LukS-PV protein-loaded silver nanoparticles was investigated on human Chronic myeloid leukemia K562 cells and human normal embryonic kidney HEK293 cells. Cell apoptosis was investigated by staining with Annexin V/propidium iodide. The recombinant LukS-PV protein-loaded silver nanoparticles exhibited dose-dependent cytotoxicity and induced apoptosis in the K562 cells but had little effect on normal HEK293 cells. After 24 h of exposure to recombinant LukS-PV protein-loaded silver nanoparticles (IC50 concentration), flow cytometry showed that 31.17% of K562 cells were apoptotic. These results indicate that recombinant LukS-PV protein-loaded silver nanoparticles maybe are a potential chemotherapeutic agent candidate against K562 cells. Hence, silver nanoparticles could be used as drug carriers for toxin release to cancer cells.

The anticancer effect of recombinant LukS-PV protein and silver nanoparticles loaded with this protein.

LukS-PV is a component of Panton-Valentine leucocidin (PVL) and is secreted by Staphylococcus aureus. Silver nanoparticles exhibit considerable potential as anticancer agents and drug delivery systems. Drug delivery is a way to deliver medicinal combinations to achieve a beneficial therapeutic effect. In the current study, recombinant LukS-PV protein-loaded silver nanoparticles were prepared and their cytotoxicity effect was analyzed on human breast cancer cells and human normal embryonic kidneys cells by MTT assay. Apoptosis was investigated by staining with Annexin V/propidium iodide. The recombinant LukS-PV protein-loaded silver nanoparticles showed dose-dependent cytotoxicity and induced apoptosis in the MCF7 cells and had a lesser effect on HEK293 cells. After 24 h exposure to the recombinant LukS-PV protein-loaded silver nanoparticles (IC50), Annexin V-FITC/PI FCM revealed that 33.2% of MCF7 cells were apoptotic. In conclusion, recombinant LukS-PV protein-loaded silver nanoparticles probably cannot be a better alternative for the targeted healing approaches to cancer therapies. Hence, it is suggested that silver nanoparticles could be utilized as a delivery system for releasing toxins into cancer cells.

Evaluation of the protective immune response of an attenuated strain of Toxoplasma gondii with long-term passages on the Gecko cell line.

Toxoplasma gondii is an obligate intracellular parasite that causes the zoonoses disease, named toxoplasmosis, with global prevalence. Until now, no cost-effective treatment method has been found to deal with toxoplasma, and vaccination is the best way to deal with the infection. In the case of pathogenic protozoa, mainly live vaccines have had successful results compared to other vaccine platforms. This study evaluated the efficacy of a live experimental vaccine through long-term passages on the Gecko cell line (Z1) in inducing a protective immune response in BALB/c mice. Thirty mice were divided into three equal groups; G1: the immunized/challenged group (injection of attenuated strain), G2: the immunized/unchallenged group (injection of attenuated strain), and G3: the control group (injection of culture medium).One month after immunization, the studied mice were challenged with 1ₓ103 live tachyzoites of Toxoplasma acute RH strain. We performed Serological investigations, including evaluating antibodies, interferon-gamma (IFN-γ), and interleukins 2, 4, 10, and 12 (IL-2,4,10,12). At the study's end, a molecular test was performed on brain and liver tissues in the immunized groups to check the presence of parasites. The results from the serological tests for the evaluation of antibodies, interferon-gamma (IFN-γ), and interleukins 10 and 12 (IL-10, 12) show a significant difference (p < 0.05) between the vaccinated group and the control group, which are essential indicators of protective immunity against toxoplasma infection. Thus, in the vaccinated group, the survival rate of mice against the challenge was 70%. Also, in group two (G2), the attenuated strain of Toxoplasma gondii had no pathogenicity, and all mice survived until the end of the study period. Molecular results also showed the absence of parasites in the brain and liver tissues in this immunized group and the parasite was found in only one case of liver tissue in G1. Therefore, the attenuated strain has caused significant and protective humoral and cellular immune responses in vaccinated groups. This study showed that with the long-term passage of the acute strain on the Gecko cell line, it is possible to quickly obtain a non-diseased attenuated strain with the ability to induce protective immunity. This successful finding can introduce further research to achieve a promising vaccine in the target animals.

Parasporin-4, a novel apoptosis inducer of breast cancer cells produced by Bacillus thuringiensis.

BACKGROUND: Parasporin (PS) proteins have cytocidal activity preferential for various human malignant cells. The purpose of this investigation was to see if the PS separated from B. thuringiensis strain E8 isolate had any particular cytotoxicity against breast cancer. METHODS AND RESULTS: The extracted spores-crystal proteins were solubilized and digested with proteinase K. Cytotoxicity effects were analysed by MTT assay. Caspases activities were measured using ELISA. SDS-PAGE analysis was performed for determination of molecular weight of Cry protein. Identification of extracted proteins function was evaluated by MALDI-TOF MS analysis. Breast cancer cells line (MCF-7) was highly susceptible to 1 mg/mL PS and showed apoptosis characteristics, but it has no effects on the normal cells (HEK293). Apoptosis evaluation showed that caspases 1, 3, 9 and BAX were remarkably up-regulated in cancer cells, indicating the intrinsic pathway activation in these cells. PS Size was determined using SDS-PAGE in E8 isolate as 34 kDa and a 25 kDa digested peptide was identified as PS4. The function of PS4 was reported as an ABC-transporter by spectrometry. CONCLUSION: The data of the present study show that PS4 is a selective cytotoxic protein against breast cancer and a molecule with a lot of potentials for next researches.

Is the Presence of Helicobacter Pylori in the Colonic Mucosa, Provocative of Activity in Ulcerative Colitis?

Background: Epidemiologic studies have shown world-wide increasing incidence of ulcerative colitis (UC) as an autoimmune disease of intestine. In the meantime, gastrointestinal H. Pylori infection is being decreased. Objectives: There are very few studies about comparing the presence of H. Pylori in the colon and the disease activity of UC. There is no study form Iran. In this study, we tried to investigate the presence of H. Pylori in the mucosa of colon by molecular and microbiological as well as pathological methods to find any association between the presence of this organism in the colon and the presence and activity of UC. Patients and Methods: In 100 patients who referred to colonoscopy clinic, colonoscopy was performed. Fifty-seven patients with the new diagnosis of UC were considered as cases and 43 patients with normal screening colonoscopy for polyps were considered as controls. Colon biopsies were evaluated according to histopathology, clinical findings, and laboratory results to confirm the diagnosis and the degree of activity in the cases of UC. Molecular studies were also performed to evaluate the presence of H. Pylori genome in the colon biopsies. A sample of colon was also cultured for H. Pylori. ELISA test was performed in a sample of blood to evaluate the level of IL-10 and IL-17 as regulatory cytokines of inflammation. Results: Cases with the diagnosis of UC showed significantly higher number of positive colonic H. Pylori comparing to normal colonic mucosa. Also, the presence of H. Pylori genome in the colon was associated with higher activity in the cases with UC and higher levels of inflammatory mediators especially IL17 and lower levels of inhibitory mediators such as IL-10. Conclusion: Colonic colonization of H. Pylori was higher in the patients with UC and higher activity of this disease comparing with normal control colonic mucosa.

Antibiotic resistance and phylogenetic comparison of human, pet animals and raw milk Staphylococcus aureus isolates.

The present study was conducted to compare the S. aureus isolates from different sources in the basis of resistance phenotypic and genotypic features and phylogenetic differences. Total of 70 S. aureus isolates (including 25 human, 25 raw milk and 20 pet animal isolates) were subjected to the antimicrobial susceptibility testing, polymerase chain reaction (PCR) detection of the resistance genes and DNA fingerprinting using random amplification of polymorphic DNA-PCR (RAPD-PCR) to survey the variability of the isolates. Among 70 S. aureus, 55 (78.5%) isolates were MRSA. The isolates showed the highest antibiotic resistance to methicillin, ampicillin and penicillin (78.5%) and showed the lowest resistance to ciprofloxacin (12.8%). ErmB and tetM resistance genes were present in all isolates and the vanA gene was not detected in any of the isolates. Thirteen distinct clusters were identified in RAPD-PCR fingerprinting. Statistical analysis showed that the isolates without resistance to antibiotics were significantly in associated with raw milk origin (P < 0.05). According to the results of the study, S. aureus strains with pets and raw milk origin are significant sources of antibiotic-resistant isolates such as MRSA. They are also carriers of resistance genes that can be transmit to human isolates and cause drug resistance in human infections. Identifying the source of these infections is possible with a reliable genotyping method such as RAPD-PCR.

Antimicrobial resistance pattern, genetic distribution of ESBL genes, biofilm-forming potential, and virulence potential of Pseudomonas aeruginosa isolated from the burn patients in Tehran Hospitals, Iran.

INTRODUCTION: according to the studies performed, researchers considered Pseudomonas aeruginosa (P. aeruginosa) as the major cause of infectious diseases like burn and wound infection that makes it one of the most threatening opportunistic pathogens. The present research aimed at investigating antimicrobial resistance, biofilm-forming abilities, and frequency of the genes contributed to blaVEB-1, blaPER-1, and blaPSE-1 genes and virulence of P. aeruginosa separated from the burn infections in Tehran, Iran. METHODS: we evaluated the resistance of 156 P. aeruginosa isolates to fifteen antimicrobial agents and generation of the ESBL and MBL enzymes phenotypically based on the CLSI instructions. Moreover, the biofilm forming potential has been assayed in a microtitre plate. In addition, PCR has been used to examine the frequency of virulence-and biofilm-related genes. Furthermore, the PCR of blaVEB-1, blaPSE-1, and blaPER-1 genes has been amplified. RESULTS: according to the results, 72.2% of P. aeruginosa isolates have been MDR and 35.6% and 55.5% have been positive for producing MBL and ESBL, respectively. Moreover, 67.8% have been positive for forming biofilms. It has been found that 15.3% isolates are ESBL-positive; from among them 60% belong to the females and 40% belong to the males. In addition, one and two isolates respectively harbored the blaVEB-1and blaPER-1genes. CONCLUSION: the present research outputs indicated the higher frequency of the multi drug resistance and higher percent of the virulence-related genes in the clinical P. aeruginosa isolates in Iran.

Anti-colon cancer activity of Bifidobacterium metabolites on colon cancer cell line SW742.

BACKGROUND/AIMS: Bacteria species, which are used as probiotics, are lactic acid bacteria. The majority of them are under the genera Bifidobacterium and Lactobacillus. The aim of the present study was to isolate and identify Bifidobacterium and to evaluate the effects of their 24 h and 120 h cell-free supernatants (CFS) from both cultures on colon cancer cell line. MATERIALS AND METHODS: In the present study, 84 samples of dairy products, infant feces, and probiotic capsule were collected, and Bifidobacterium was isolated. Gram stain, biochemical tests, and molecular identification were done for the isolation and identification of Bifidobacterium. Cytotoxicity effects of CFS derived from both cultures of isolated Bifidobacterium were assessed on colon cancer cell lines. RESULTS: In the present study, 17 isolates of Bifidobacterium were identified. The results show that Bifidobacterium was most frequently associated with infant feces and dairy products, whereas the lowest rate was associated with local milk. After the effects of CFS on colon cancer cell line, two isolates were identified from infant feces and probiotic capsule; they had the highest ability in inhibiting the growth of cancer cells. Bifidobacterium bifidum was effective in combating cancer cells and was associated with a substantial improvement in gastrointestinal cancer. CONCLUSION: The study has shown that the regular ingested probiotics could prevent the development of colorectal cancer. During the present study, the produced CFS could inhibit the growth of colon cancer cells. In conclusion, probiotics have good potential to be introduced as a new approach to colon cancer treatment.

Anti-cancer Parasporin Toxins of New Bacillus thuringiensis Against Human Colon (HCT-116) and Blood (CCRF-CEM) Cancer Cell Lines.

Bacillus thuringiensis is one of the most important microorganisms used against cancer cell lines in latest studies all over the world. This study aims to perform the isolation, molecular identification, and to identify novel B. thuringiensis strains that specifically targeting human cancer cell-killing activities in Iran. A total of 88 B. thuringiensis isolates were recovered from Iran. Upon the treatment of the non-hemolytic crystal proteins by proteinase K, five isolates belonging to three biotypes, thuringiensis, kurstaki and sotto of B. thuringiensis are found to have different cytotoxicity toward HCT-116 and CCRF-CEM cell lines. Digested inclusions of the isolates consisted of one major poly peptide of 34-kDa, as estimated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis. The structure, molecular identification, and functionality of five isolates inclusion proteins have shown to be closely like to parasporin-2 but their size of activated protein is not similar to this parasporin. It is unclear that discovered damaging proteins are parasporin-2. This 34-kD protein exhibited varying degrees of cytocidal activity toward human colon and blood cancer cells and caused cell swelling and the formation of blebs in the surface of the cells or alteration in cytoskeleton. The soil in the humid and temperate climates of Iran is a good reservoir for parasporin producing B. thuringiensis. The isolated B. thuringiensis strains exhibit specific and different cytocidal activities against human colon and blood cancer cells. Parasporin is a novel cytotoxic protein to human cancer cells produced by B. thuringiensis and these toxins appeared to attack an identical target on human cancer cells.