Anticancer potential of fermented milk with autochthonous lactic acid bacteria.

AIMS: The purpose of this study was to investigate the effect of fermented milk supernatants of autochthonous lactic acid bacteria, including Lactobacillus helveticus KMCH1 (ON561781), Lactococcus lactis KMCM3 (ON561782), and Lactiplantibacillus plantarum KMJC4 (ON615217), on human colon cancer (HT-29) and normal mouse fibroblast (L929) cells in vitro. METHODS AND RESULTS: Proteolytic activity, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide test, evaluation of apoptosis induction, and cell cycle arrest by flow cytometry were the assays performed in this study. The measurement of proteolytic activity of three types of fermented milk supernatant using an orthophthalaldehyde reagent showed that the fermented milk supernatant of L. helveticus KMCH1 included the highest proteolysis. Three types of fermented milk supernatant showed anticancer effects on HT-29 cell in a time- and concentration-based manner (at a concentration of 16 mg ml-1 for 72 h of incubation), while the effect of three types of supernatant on inhibition of L929 cell was 3%-10%. Besides, three types of supernatant inhibited HT-29 cell proliferation by inducing apoptosis and cell cycle arrest in the S phase. CONCLUSIONS: Autochthonous lactic acid bacteria strains were able to produce bioactive peptides with anticancer effects in fermented milk. Inhibition of HT-29 cell proliferation was dependent on peptide concentration.

Altered expression of miR-125a and dysregulated cytokines in systemic lupus erythematosus: Unveiling diagnostic and prognostic markers.

BACKGROUND: Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder impacting multiple organs, influenced by genetic factors, especially those related to the immune system. However, there is a need for new biomarkers in SLE. MicroRNA-125a (miR-125a) levels are decreased in T cells, B cells, and dendritic cells of SLE patients. MiR-125a plays a regulatory role in controlling the levels of tumor necrosis factor-alpha (TNF-α) and interleukin 12 (IL-12), which are crucial pro-inflammatory cytokines in SLE pathogenesis. AIM: To assess the levels of miR-125a, IL-12, and TNF-α in SLE patients' plasma, evaluating their diagnostic and prognostic value. METHODS: The study included 100 healthy individuals, 50 newly diagnosed (ND), and 50 SLE patients undergoing treatment. The patients were monitored for a duration of 24 wk to observe and record instances of relapses. MiR-125a expression was measured using real-time reverse transcription polymerase chain reaction, while ELISA kits were used to assess IL-12 and TNF-α production. RESULTS: The results showed significantly reduced miR-125a expression in SLE patients compared to healthy individuals, with the lowest levels in ND patients. TNF-α and IL-12 expression levels were significantly elevated in SLE patients, especially in the early stages of the disease. Receiver operating characteristic curve analyses, and Cox-Mantel Log-rank tests indicated miR-125a, TNF-α, and IL-12 as proper diagnostic biomarkers for SLE. A negative correlation was found between plasma miR-125a expression and IL-12/TNF-α levels in SLE patients. CONCLUSION: Decreased miR-125a levels may be involved in the development of SLE, while elevated levels of IL-12 and TNF-α contribute to immune dysregulation. These findings offer new diagnostic and prognostic markers for SLE. Moreover, the negative correlation observed suggests an interaction between miR-125a, TNF-α, and IL-12. Further research is necessary to uncover the underlying mechanisms that govern these relationships.

Role of ESAT-6 in pathogenicity of Beijing and non-Beijing Mycobacterium tuberculosis isolates.

BACKGROUND: Mycobacterium tuberculosis Beijing genotype was associated with tuberculosis outbreaks and increased transmissibility. To understand the variation in virulence between Beijing and non-Beijing clinical isolates of M.tuberculosis genotypes, the esat-6 gene sequencing, and its expression was compared in the macrophage environment. MATERIALS & METHODS: Among 64 nonrepetitive, culture-positive M.tuberculosis, DNA extraction of 24 and 40 pure confirmed Beijing and non-Beijing isolates was accompanied by the boiling method. esat-6 gene PCR amplification and their sequencing were carried out by specific primers and its expression was performed on human macrophage cell line U937 after 6, 12, and 18 h of exposure to bacilli. The esat-6 mRNA transcription and expression in M. tuberculosis treated macrophage by Real-Time PCR and Western blot method. RESULTS: Data analysis based on sequencing of the east-6 gene PCR product showed that this gene exists in all isolates and there are no changes or single nucleotide variation between the Beijing and non-Beijing isolates. In Beijing strains, the esat-6 expression was increased during the study times, but it was constant in non-Beijing isolates. esat-6 gene expression in Beijing isolates reached to about 44.9 times more than non-Beijing isolates after 18 h incubation on the macrophages cell line. CONCLUSION: esat-6 is a conserved gene both in Beijing and non-Beijing isolates of M.tuberculosis. More expression of the east-6 gene in the macrophage model may indicate that this gene is likely to play a more important role in increasing the pathogenicity of Beijing strains.

Anticancer Activity of Ethnopharmacological Plants of Golestan Province/Iran against AGS, HT-29 and KYSE-30 Cell Lines through Promoting the Apoptosis and Immunomodulatory Effects.

The anticancer and immunomodulatory effects of medicinal plants from Golestan province, as a promising source of cancer therapy against gastrointestinal cancer cell lines, were investigated in this study. The ethanolic root/aerial part extracts of 9 medicinal plants were screened for their cytotoxicity against normal mouse fibroblast cells (L-929) and three human cancer cell lines including gastric adenocarcinoma (AGS), colorectal adenocarcinoma (HT-29), and esophagus adenocarcinoma (KYSE-30) by performing MTT assay to determine the IC50 of the extracts. The in-vitro antioxidant activity, total phenolic (TPC), and total flavonoid content (TFC) of extracts was evaluated. Flow cytometry and Real-Time PCR were used for apoptosis assay and evaluation of expression of some genes involved in cell signaling; TLR-4, AKT, ERK1/2, and NFκB. Out of the 9 plant extracts screened, Arctiumlappa root (ALR), showed the most potent cytotoxicity against AGS, KYSE-30, and HT-29 cells with IC50 values of 10, 200, and 2030 µg/mL, respectively. In addition, ALR exerts high TPC (215.8 ± 0.3 mg GAE/g), TFC (69.03 ± 0.7 mg QUE/g) and high radical scavenging activity with IC50 (1250 ± 0.1 µg/mL) in DPPH method. Also, ALR stimulates TLR-4 signaling, increased apoptosis, and decreased cancer cell attachment to the surface compared to the untreated cells. This plant, with a strong cytotoxic effect on cancer cells as well as increased apoptosis and its effect on molecules involved in TLR4 signaling as the immunomodulatory effect can be a suitable candidate for in-vivo studies in the future for cancer therapy.

Utility of Urine Interleukines in Children with Vesicoureteral Reflux and Renal Parenchymal Damage.

PURPOSE: Vesicoureteral reflux (VUR) is the most common risk factor of urinary tract infection in children. Currently, diagnosis of VUR depends on invasive imaging studies, with a high radiologic burden. Therefore, different biomarkers have been introduced for the evaluation of these patients. The objective of this study was to identify alteration of urinary interleukins (ILs) excretion in children with primary VUR and renal parenchymal damage, for further clinical application. MATERIALS AND METHODS: Urinary concentrations of IL-1α, IL-1ß, IL-6, and IL-8 were evaluated in 34 children with VUR (cases) and 36 without VUR (control), during 2018-2019. Urinary concentrations of IL-1, IL-1, IL-6 and IL-8 were measured, using polyclonal antibody ELISA kit, and standardized to urine creatinine (Cr). Patients with infectious or inflammatory disorders, urolithiasis, immune deficiency, acute or chronic kidney disease, and secondary VUR were excluded from the study. RESULTS: Mean age of cases (36.00 ± 27.66) had no significant difference with the control (32.86±29.31) group (p=0.44). The majority of patients had moderate VUR (58.8%), followed by severe (35.3%) and mild (5.9%) grades. Urinary concentration of all ILs/Cr were significantly higher in patients with VUR, compared with those without VUR. There was no significant correlation between urine ILs/Cr with age, gender, serum electrolytes, urine specific gravity, renal ultrasound, laterality or severity of VUR, and DMSA renal scan. All urine ILs/Cr had acceptable sensitivity and accuracy for workup of children with primary VUR. CONCLUSION: Urine IL-1α, IL-1ß, IL-6 and IL-8/Cr were sensitive and accurate additionary screening biomarkers in children with primary VUR.

Comparison of two molecular diagnostic methods for identifying Beijing genotype of Mycobacterium tuberculosis.

BACKGROUND AND OBJECTIVES: The Beijing family of Mycobacterium tuberculosis has been identified as a severe pathogen among this species and found in many clinical isolates during the last decade. Early identification of such genotype is important for better prevention and treatment of tuberculosis. The present study performed to compare the efficiency of Real-Time PCR and IS6110-Based Inverse PCR methods to identify the Beijing family. MATERIALS AND METHODS: This study was carried out on 173 clinical isolates of Mycobacterium tuberculosis complex in Golestan Province, northern Iran. DNA extraction performed by boiling and determining the Beijing and non-Beijing strains carried out using Real-Time PCR and IS6110-Based Inverse PCR. RESULTS: In both Real-Time PCR and IS6110-Based Inverse PCR method, 24 specimens (13.9%) of the Beijing family were identified and the result of the IS6110-Based Inverse PCR method showed that all the Beijing strains in this region belonged to the Ancient Beijing sub-lineage. CONCLUSION: Although the efficacy of the two methods in the diagnosis of the Beijing family is similar, the IS6110-Based Inverse PCR is more applicable to the ability to detect new and old Beijing family.

Combination of 5-fluorouracil and Lipopolysaccharide Synergistically Induces Cytotoxicity and Apoptosis in MCF-7 Human Breast Cancer Cells.

Several studies have been conducted to find suitable combinations of drugs to increase the efficacy of chemotherapy and reduce the resistance of tumor cells to treatment. Lipopolysaccharide (LPS), as a ligand for Toll-like receptor 4 (TLR-4), can modify immune responses in different cancers. Although multiple studies have been performed in this area, the effect of LPS on tumor cells remains controversial. In the present study, the cytotoxic effects of 5-fluorouracil (5-FU), with or without LPS, were evaluated in human breast cancer cell line (MCF-7) on apoptosis and gene expression in downstream signaling pathways. MCF-7 was obtained from the Pasteur Institute of Iran. The effects of LPS and 5-FU on cytotoxicity, apoptosis, and gene expression in NF-κB, ERK, and AKT signaling pathways were evaluated by MTT assay, Annexin V/propidium iodide (PI) apoptosis assay, and qRT-PCR, respectively. Our findings showed that LPS alone did not significantly affect cytotoxicity or apoptosis, compared to the control cells (untreated cells), while combined with 5-FU, it caused a significant increase in the apoptosis of cancer cells and decreased cell viability. It was also concluded that LPS in combination with 5-FU increased TLR-4 expression and down-regulated gene expression in NF-κB, ERK, and AKT pathways (p=0.001). Although the role of LPS in tumor inhibition or progression remains controversial, our findings suggest that LPS can be considered a novel complementary approach intranslational oncology research of breast cancer therapy.

Correlation Between HMGB1 and TLR4 Expression in Sinonasal Mucosa in Patients With Chronic Rhinosinusitis.

OBJECTIVES: Chronic rhinosinusitis (CRS) is one of the most common inflammations in the upper airway. Despite the wide prevalence of CRS, the pathogenesis of this disease is poorly understood. Several components of the innate immune system may play a significant role in CRS, including Toll-like receptor 4 (TLR4), TLR9, and high-mobility group box 1 protein (HMGB1). This study was conducted to determine the expression of TLR4, TLR9, HMGB1, and pNFκ-B p65 in paraffin-embedded blocks of patients with CRS with nasal polyps compared with those of the control group. METHODS: Twenty-six formalin-fixed, paraffin-embedded samples from patients with confirmed CRS and 26 patients undergoing septoplasty due to anatomic variations and no other inflammatory nasal diseases as the control group were assessed. Expression patterns of HMGB1, TLR9, TLR4, and pNFκ-B p65 genes were examined using real-time quantitative reverse transcription polymerase chain reaction (Real-Time qRT-PCR). Statistical analyses were performed with SPSS and analyzed using unpaired 2-tailed t tests or 1-way analysis of variance. RESULTS: Real-time PCR showed that the expression level of HMGB1 messenger RNA was significantly increased in the tissues of patients with CRS compared with controls (P < .05). The other 3 genes were also upregulated in the patients, but were not significant compared with control. Analysis of the Pearson correlation coefficient (r) revealed a significant positive correlation between HMGB1 and TLR4 (r = 0.79, P < .05) in patients and negative correlation between TLR4 and NfκB in the control group (r = 0.94; P < .05). CONCLUSIONS: Both HMGB1 and TLR4 are increased in the paranasal sinus mucosa of patients with CRS. These results suggest a possible contribution of HMGB1 and its internal receptor (TLR4) in the pathophysiology of CRS.

5-Fluorouracil Induce the Expression of TLR4 on HCT116 Colorectal Cancer Cell Line Expressing Different Variants of TLR4.

Two common single nucleotide polymorphisms (SNPs) of the human TLR4 gene, namely Asp299Gly (D299G) and Thr399Ile (T399I), have been shown to impair the ability of certain individuals to respond properly to TLR4 ligands. 5-Fluorouracil (5-FU) is widely used for the treatment of patients with advanced colon cancers. The present study examined the impact of two common polymorphisms of the TLR4 genes on the response of the HCT116 colorectal cancer cells to 5-FU. HCT116 was transfected with Flag-CMV1-TLR4 wild-type (WT) and D299G, T399I expression plasmids. The cytotoxic effect of 5-FU on transfected cells was assessed by MTT assay. FACS analysis was performed to show the effect of 5-FU and LPS on the expression of different variants of TLR4. The lowest IC50-value was measured in cells expressing the WT TLR4 and non-transfected cells were more resistance to the drug compared to the other cells. 5-FU significantly induced the expression of TLR4 protein in the presence and absence of LPS. 5-FU also induced HMGB1 secretion, Cas3 and PARP activity and these effects were stronger in cells expressing WT TLR4 than the other cells. In conclusion, 5-FU-induced TLR4 expression and LPS had synergistic effect with 5-FU to induced apoptosis in colorectal cancer cells.

Increased NFk-B activity in HCT116 colorectal cancer cell line harboring TLR4 Asp299Gly variant.

Toll-Like Receptor 4 (TLR4), considered one of the most important TLR, recognizes lipopolysaccharide of Gram-negative bacteria. Recognition of ligands by TLRs induces signaling pathways resulting in activation of transcriptional factors such as NF-κB which are involved in the expression of inflammatory cytokines and chemokines. To prevent an inappropriate immune response, a complex network of molecules negatively regulates TLRs and their associated signaling pathways. Two cosegregating single nucleotide polymorphisms of the human TLR4 gene, namely Asp299Gly and Thr399Ile, have been associated with hyporesponsiveness to inhaled LPS. The purpose of this study was to determine the impact of TLR4 gene variant on NF-κB activity in colorectal cancer cell line. HCT116 cells were transfected with wild-type and mutants Flag-CMV1-TLR4 expression vectors. Western blot analysis was performed to evaluate selected molecules involved in TLR4 signaling. NF-κB activity was assessed by dual-luciferase reporter assay and cytokine profiles were evaluated byELISA and Cytometric Bead Array method. Results showed that the activity of pNF-κB was higher in cells harboring TLR4 D299G compared to the other cells. However, the activity of pAKT, pERK1 and pIRAK was higher in wild-type. The results of cytokine measurements showed about four fold higher level of IL-8 in cells with wild-type TLR4. This study suggest that TLR4 Asp299Gly gene variant has an impact on TLR4 signaling and potentially on intestinal homeostasis due to impaired control signals at the epithelial cell level which may lead to chronic intestinal inflammation and interrupted intestinal homeostasis and may eventually lead to colorectal cancer.

Variant Toll-like receptor4 (Asp299Gly and Thr399Ile alleles) and Toll-like receptor2 (Arg753Gln and Arg677Trp alleles) in colorectal cancer.

The innate immune system recognizes the presence of bacterial products through the expression of a family of membrane receptors known as Toll-like receptors (TLRs). Polymorphisms in TLRs have been shown to be associated with increased susceptibility to diseases such as inflammatory bowel disease. The aim of this study was to determine whether there was a correlation between polymorphisms of TLR4 (Asp299Gly; Thr399Ile) and TLR2 (Arg677Trp; Arg753Gln) genes and risk of colorectal cancer. DNA from 60 colorectal carcinoma patients from 3 major races in Malaysia (22 Malays, 20 Chinese and 18 Indians) and blood from 50 apparently healthy individuals were evaluated. Control group were matched to study group by race and age. The polymorphisms were determined by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP). Genotyping results showed two out of sixty tumour specimens (3.3%) harbored both variant TLR4 Asp299Gly and Thr399Ile alleles. In contrast, DNA isolated from blood cells of 50 apparently healthy individuals harbored wild type TLR4. In the case of TLR2 Arg753Gln genotyping, all of the fifty normal and 60 tumours were of the wild type genotype. TLR2 Arg677Trp genotyping showed a heterozygous pattern in all samples. However, this may not be a true polymorphism of the TLR2 gene as it is likely due to a variation of a duplicated ( pseudogene) region. There was only a low incidence (2/60; 3.3%) of TLR4 polymorphism at the Asp299Gly and Thr399Ile alleles in colorectal cancer patients. All normal and tumour samples harbored the wild type TLR2 Arg753 allele. Our study suggests that variant TLR4 (Asp299Gly and Thr399Ile alleles) as well as TLR2 (Arg753Gln allele) are not associated with risk of colorectal cancer.

Herbal plants and their derivatives as growth and health promoters in animal nutrition.

The purpose of this review is to summarize the effectiveness, modes of action and commercial application of herbal plants and their derivatives as growth promoters for animal. Feed supplements are a group of feed ingredients that can cause a desired animal response in a non-nutrient role such as pH shift, growth, or metabolic modifier (Hutjens, 1991). Common feed additives used in animal diets include immunostimulators, antimicrobials, antioxidants, pH control agents and enzymes. Herbal plants, are a new class of growth promoters and in recent years this feed additives have gained extensive attention in the feed industry. They are a wide variety of herbs, spices, and products derived thereof, and are mainly essential oils. Although numerous reports have demonstrated antioxidative and antimicrobial and immune stimulation efficacy in vitro, respective experimental in vivo evidence is still quite limited. A limited number of experimental comparisons of herbal plants feed additives with antibiotics or organic acid have suggested similar effects on the animal gut microflora. Gut microflora has significant effects on host nutrition, health, and growth performance by interacting with nutrient utilization and the development of gut system of the host. In addition, some phytogenic compounds seem to promote intestinal mucus production. However, the future of using herbs in animal feeding will in great measure depend on the knowledge of chemical structure, their value and characteristics of practical herbs or their extract physiological needs and well-being of animal, and, above all on consumer's preferences and expectations.