Intestinal Clostridium species lower host susceptibility to enterohemorrhagic Escherichia coli O157:H7 infection.

Susceptibility to enterohemorrhagic Escherichia coli (EHEC) infection varies among humans. The intestinal microbiota seems to play an essential role in host defense against EHEC; thus, we hypothesized that indigenous bacteria, such as Clostridium ramosum and Clostridium perfringens, could influence the susceptibility to EHEC infection. To evaluate the effect of indigenous bacteria on EHEC infection, germ-free mice were precolonized with each indigenous bacterium, and then infected with EHEC O157:H7. Precolonization with C. ramosum or C. perfringens completely prevented death from EHEC infection througout a test period. Precolonization with C. ramosum also reduced the level of secreted Shiga toxin (Stx) 2 and prevented histopathological changes in the kidneys in a similar way to precolonization with Bifidobacterium longum, which is used as a model for preventing EHEC infection. In contrast, the mice precolonized with C. perfringens showed mild renal injuries. When evaluated using an in vitro co-culturing system, again C. ramosum inhibited the growth and Stx production of EHEC more potently than C. perfringens. These results indicate that C. ramosum and C. perfringens suppressed EHEC infection; however, the extent of their preventive effects differed. Therefore, the susceptibility to EHEC infection and its severity can depend on the functional bacteria present in the intestinal microbiota of individuals.

Inhibition of enterohemorrhagic Escherichia coli O157:H7 infection in a gnotobiotic mouse model with pre-colonization by Bacteroides strains.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 has been known to cause outbreaks of hemorrhagic colitis and hemolytic uremic syndrome. We previously demonstrated that intestinal flora contribute to the prevention of EHEC infection in a mouse model. However, it has not yet been determined whether Bacteroides, a predominant genus in the human intestine, contributes to the prevention of EHEC infection. The aim of the present study was to investigate the effect of Bacteroides fragilis (B. fragilis) and Bacteroides vulgatus (B. vulgatus) on EHEC O157:H7 infection in vivo using gnotobiotic mice. These strains were inoculated into germ-free mice to create a gnotobiotic mouse model. EHEC was inoculated into the mice, which were then monitored for 7 days for any change in symptoms. The mice that had been pre-colonized with the Bacteroides strains did not develop lethal EHEC infection, although several inflammatory symptoms were observed in the B. vulgatus pre-colonized group. However, no inflammatory symptoms were identified in the B. fragilis pre-colonized group. Moreover, B. fragilis exerted an inhibitory effect on enterocyte-like cell apoptosis. B. fragilis protected HT29 cells from apoptosis caused by Shiga toxin. In conclusion, the findings of the present study demonstrated that colonization by Bacteroides strains can inhibit EHEC infection.

miR-663a regulates growth of colon cancer cells, after administration of antimicrobial peptides, by targeting CXCR4-p21 pathway.

BACKGROUND: Antimicrobial peptides (AMPs) play important roles in the innate immune system of all life forms and recently have been characterized as multifunctional peptides that have a variety of biological roles such as anticancer agents. However, detailed mechanism of antimicrobial peptides on cancer cells is still largely unknown. METHODS: miRNA array and real-time qPCR were performed to reveal the behavior of miRNA in colon cancer HCT116 cells during the growth suppression induced by the AMPs. Establishment of miR-663a over-expressing HCT116 cells was carried out for the evaluation of growth both in vitro and in vivo. To identify the molecular mechanisms, we used western blotting analysis. RESULTS: miR-663a is upregulated by administration of the human cathelicidin AMP, LL-37, and its analogue peptide, FF/CAP18, in the colon cancer cell line HCT116. Over-expression of miR-663a caused anti-proliferative effects both in vitro and in vivo. We also provide evidence supporting the view that these effects are attributed to suppression of the expression of the chemokine receptor CXCR4, resulting in the abrogation of phosphorylation of Akt and cell cycle arrest in G2/M via p21 activation. CONCLUSIONS: This study contributes to the understanding of the AMPs' mediated anti-cancer mechanisms in colon cancer cells and highlights the possibility of using AMPs and miRNAs towards developing future strategies for cancer therapy.

The Human Cathelicidin Antimicrobial Peptide LL-37 and Mimics are Potential Anticancer Drugs.

Antimicrobial peptides (AMPs) play a critical role in innate host defense against microbial pathogens in many organisms. The human cathelicidin, LL-37, has a net positive charge and is amphiphilic, and can eliminate pathogenic microbes directly via electrostatic attraction toward negatively charged bacterial membranes. A number of studies have shown that LL-37 participates in various host immune systems, such as inflammatory responses and tissue repair, in addition to its antibacterial properties. Moreover, recent evidence suggests that it is also involved in the regulation of cancer. Indeed, previous studies have suggested that human LL-37 is involved in carcinogenesis via multiple reporters, such as FPR2 (FPRL1), epidermal growth factor receptor, and ERBb2, although LL-37 and its fragments and analogs also show anticancer effects in various cancer cell lines. This discrepancy can be attributed to peptide-based factors, host membrane-based factors, and signal regulation. Here, we describe the association between AMPs and cancer with a focus on anticancer peptide functions and selectivity in an effort to understand potential therapeutic implications.

Antimicrobial peptide FF/CAP18 induces apoptotic cell death in HCT116 colon cancer cells via changes in the metabolic profile.

Metabolic reprogramming is one of the hallmarks of cancer and can be targeted by therapeutic agents. We previously reported that cathelicidin-related or modified antimicrobial peptides, such as FF/CAP18, have antiproliferative effects on the squamous cell carcinoma cell line SAS-H1, and the colon carcinoma cell line HCT116. Although antimicrobial peptides have potential use in the development of new therapeutic strategies, their effects on the metabolism of cancer cells are poorly understood. Here, we investigated changes in the levels of metabolites in HCT116 cells caused by FF/CAP18, via capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Analysis of the 177 intracellular metabolites and 113 metabolites in conditioned medium that were detected by CE-TOFMS, revealed dramatic changes in the metabolic profile of HCT116 cells after treatment with FF/CAP18. The metabolic profile showed that the levels of most metabolites in the major metabolic pathways supported the rapid proliferation of cancer cells. Purine metabolism, glycolysis, and the TCA cycle, were altered in FF/CAP18-treated cells in a dose-dependent manner. Our present study provides mechanistic insights into the anticancer effects of antimicrobial peptides that show great potential as new therapies for colon cancer.

Establishment of cell lines derived from the genus Macaca through controlled expression of cell cycle regulators.

Nonhuman primates are useful animal models for the study of human diseases. However, the number of established cell lines from nonhuman primates is quite limited compared with the number established from other experimental animals. The establishment of nonhuman primate cell lines would allow drug testing on those cell lines before moving experiments into primates. In this study, we established nonhuman primate primary cell lines by introducing the genes for CDK4R24C, cyclin D1, and hTERT. These cell lines proliferated more rapidly than primary cells and bypassed cellular senescence. Karyotype analysis showed that the chromosome patterns were intact in the immortalized cell lines. Furthermore, we showed that the expression of introduced genes could be precisely controlled through the Tet-Off system with the addition of doxycycline. The present study shows that introduction of the CDK4R24C, cyclin D1, and hTERT genes are effective methods of establishing nonhuman primate cell lines.

Susceptibility difference between methicillin-susceptible and methicillin-resistant Staphylococcus aureus to a bovine myeloid antimicrobial peptide (BMAP-28).

A bovine myeloid antimicrobial peptide antimicrobial peptide (BMAP-28) is a member of the cathelicidin family and acts as a component of innate immunity. There are few reports of susceptibility difference of methicillin-resistant Staphylococcus aureus (MRSA) and susceptible strains (MSSA) against BMAP-28. This study aims to clarify how a few amino acid substitutions of BMAP-28 are related to its antimicrobial activity using four analog peptides of BMAP-28. We also compared cellular fatty acid components of MSSA and MRSA using gas chromatography. We found that a few amino acid substitutions of BMAP-28 do not change antimicrobial activity. It was also revealed that the percentage of cis-11-eicosenoic acid in total detected fatty acids of MRSA was significantly higher than that of MSSA. In addition, the percentage of palmitic acid in total detected fatty acids of MRSA tended to be lower than that of MSSA. Our results will provide new information to deal with the question of differences in bacterial susceptibility against BMAP-28.

Ceragenin CSA-13 induces cell cycle arrest and antiproliferative effects in wild-type and p53 null mutant HCT116 colon cancer cells.

Antimicrobial peptides of the cathelicidin family play a central role in the host defense system. Our group has reported previously that cathelicidin-related or cathelicidin-modified antimicrobial peptides, such as FF/CAP-18, have antiproliferative effects on the squamous cell carcinoma cell line SAS-H1 and colon cancer-derived cell line HCT116. Ceragenin CSA-13, which mimics the hydrophobic and cationic morphology of cathelicidin-related peptides, was developed to reduce synthetic costs and resolve stability issues in the presence of proteases. In this study, we evaluated the antiproliferative effect of CSA-13 on HCT116 cells. We evaluated the effects of CSA-13 in HCT116 cells by measuring cell growth, detecting apoptosis, analyzing the cell cycle, and examining mitochondrial membrane depolarization. Treatment with CSA-13 suppressed HCT116 cell proliferation in a dose-dependent manner, increasing the incidence of apoptosis detected by the binding of Annexin V. Furthermore, cell cycle analysis showed that the cell cycle of CSA-13-treated wild-type and p53 null mutant HCT116 cells was arrested at the G1/S phase, indicating that CSA-13 affects the cell cycle by a p53-independent pathway. Our study showed that CSA-13 exerts an antiproliferative effect in cancer cells similar to that of FF/CAP-18, suggesting that membrane-permeabilizing capability is the common underlying mechanism for anticancer and antimicrobial effects of CSA-13 and anitimicrobial peptides.

Anti-proliferative effect of an analogue of the LL-37 peptide in the colon cancer derived cell line HCT116 p53+/+ and p53-/-.

Antimicrobial peptides of the cathelicidin family are found in many mammalian species, and are focused on various effects other than antimicrobial action. In this study, we evaluated the anti-proliferative effect of an analogue peptide, FF/CAP18, derived from an endogenous cathelicidin family member against the colon cancer cell line HCT116. FF/CAP18 significantly decreased the proliferation of HCT116 cells in a dose-dependent fashion. Furthermore, the treatment of HCT116 with FF/CAP18 caused loss of mitochondrial membrane potential, and resulted in the immunoreactivity to the single-strand DNA antibody, suggesting the early stage of apoptosis. Interestingly, the anti-proliferative effect of FF/CAP18 was constant regardless of the genotype of p53 (wild-type and p53 mutant type HCT116 cells). Therefore, the signaling pathway of p53 is not involved in the growth suppression effect of the cathelicidin analogue peptide. These results indicate that the treatment of certain types of cancer cells with FF/CAP18 may increase the sensitivity of the chemotherapeutic reagents, which might relate to the reduction of the side effects.

Antimicrobial activity of a bovine myeloid antimicrobial peptide (BMAP-28) against methicillin-susceptible and methicillin-resistant Staphylococcus aureus.

A bovine myeloid antimicrobial peptide (BMAP-28) is a member of the cathelicidin family which is included in the innate immune system of mammals. Recently, there have been many studies about antimicrobial peptides. This study aims to clarify whether BMAP-28 has bactericidal activity against methicillin-resistant Staphylococcus aureus (MRSA) and compares its activity against methicillin-susceptible S. aureus (MSSA) and MRSA. We found that the peptide was effective in killing MRSA (minimal inhibitory concentration (MIC) range; 5-20 µg/mL). It was also revealed that MSSA (MIC range; 1.25-20 µg/mL) had two levels of susceptibility to BMAP-28. We also examined the effect of BMAP-28 on bacterial shape to visually show its activity. After exposure to the peptide, both MSSA and MRSA cells showed the morphological changes on their surfaces. Our results indicate that BMAP-28 is a promising candidate for medicine against drug-resistant bacteria.

Anti-cell-associated glucosyltransferase immunoglobulin Y suppression of salivary mutans streptococci in healthy young adults.

BACKGROUND: The authors evaluated the suppressive effects of lozenges containing egg yolk antibodies (that is, immunoglobulin Y [IgY]) against Streptococcus mutans cell-associated glucosyltransferase (CA-gtf) on oral colonization by mutans streptococci (MS) in healthy young adults. METHODS: In a five-day double-masked placebo-controlled trial, young adult participants self-administered lozenges containing anti-CA-gtf IgY (Ovalgen DC, GHEN, Gifu-City, Japan) or a placebo at prescribed times each day. On the basis of bacterial colony counts of saliva cultures, the authors analyzed the pretrial and posttrial differences in levels of MS and total anaerobic bacteria among participants in the treatment (anti-CA-gtf IgY) and placebo groups and a control group. RESULTS: Salivary MS scores in participants in the treatment group decreased significantly (P < .001), and the mean anaerobic bacterial count in the treatment group was not statistically different before and after the trial. In the placebo and control groups, posttrial changes in median MS scores and total salivary anaerobic bacterial counts were not statistically significant. CONCLUSIONS: The results of the study show that lozenges containing anti-CA-gtf IgY can suppress oral colonization by MS in healthy young adults. CLINICAL IMPLICATIONS: Lozenges containing anti-CA-gtf IgY may help reduce dental caries risk in humans.

Tertiary structure-related activity of tick defensin (persulcatusin) in the taiga tick, Ixodes persulcatus.

Defensins are small cysteine-rich cationic proteins found in both vertebrates and invertebrates constituting the front line of host innate immunity. To examine the importance of the tertiary structure of tick defensin in its antimicrobial activity, we synthesized two types of the peptides with tertiary structure or primary one on basis of the information of the sequence in the defensin originated from the taiga tick, Ixodes persulcatus. Chemically synthesized peptides were used to investigate the activity spectrum against Staphylococcus aureus, Borrelia garinii and flora-associated bacteria. Both synthetic peptides showed antimicrobial activity against S. aureus in short-time killing within 1 h, but they do not show the activity against B. garinii, Stenotrophomonas maltophila and Bacillus spp., which were frequently isolated from the midgut of I. persulcatus. The teriary structure brought more potent activity to S. aureus than primary one in short-time killing. We also examined its antimicrobial activity by evaluation of growth inhibition in the presence of the synthetic peptides. Minimum inhibitory concentration (MIC) was ranged from 1.2 to 5.0 µg/ml in tertiary peptide and from 10 to 40 µg/ml in primary peptide, when 10 strains of S. aureus were used. From the curve of cumulative inhibition rates, MIC50 (MIC which half of the strains showed) to S. aureus is about 1.2 µg/ml in the peptide with tertiary structure and about 10 µg/ml in the linear one. Corynebacterium renale is 10 times or more sensitive to tertiary peptide than primary one. In conclusion, the presence of 3 disulfide bridges, which stabilize the molecule and maintain the tertiary structure, is considered to have an effect on their antimicrobial activities against Gram-positive bacteria such as S. aureus.

High expression of MeCP2 in JC virus-infected cells of progressive multifocal leukoencephalopathy brains.

Mutations of the methyl CpG binding protein 2 (MeCP2) gene are a major cause of Rett syndrome. To investigate whether the expression of this gene was related to JC virus (JCV) infection, we examined brains of four progressive multifocal leukoencephalopathy (PML) patients. JCV infection was confirmed by immunohistochemical labeling with antibodies against JCV VP1, agnoprotein and large T antigen. MeCP2 expression was examined by immunohistochemistry using a specific polyclonal antibody against MeCP2. In normal brains and uninfected cortices of PML brains, MeCP2 expression was observed in the nuclei of neurons, but not observed in glial and endothelial cell nuclei. However, in PML brains intense immunolabeling was observed in abnormally enlarged glial nuclei of JCV-infected cells. Double immunolabeling using antibodies against large T antigen (visualized as blue) and MeCP2 (visualised as red) revealed dark red JCV-infected nuclei, which confirmed that the JCV infected nuclei expressed MeCP2. We conclude that MeCP2 is highly expressed in the JCV-infected nuclei of PML brain and these results may provide a new insight into the mechanism which regulates the MeCP2 expression in glial cells by the infection of JCV.

Antimicrobial activity of three tick defensins and four mammalian cathelicidin-derived synthetic peptides against Lyme disease spirochetes and bacteria isolated from the midgut.

In this study, chemically synthesized tick defensins and cathelicidin-derived mammalian peptides were used to investigate the activity spectrum against Borrelia garinii and symbiotic Stenotrophomonas maltophila. Synthetic tick defensins showed antimicrobial activity against Staphylococcus aureus but not B. garinii and S. maltophila. Mammalian peptides which have cationic property similar to tick defensins, showed antimicrobial activity similar to tick defensins. The antimicrobial peptides in ticks and mammalian hosts have common characteristics against microbial invasion in the innate immune system.

Detection of Salmonella invA by isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) in Zambia.

The isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) is a new isothermal DNA amplification method composed of exo Bca DNA polymerase, RNaseH and DNA-RNA chimeric primers. We detected invA of Salmonella from chicken carcasses, egg yolk and cattle fecal samples. Fifty-three of 59 isolates were invA-positive in ICAN-chromatostrip detection. The result was consistent with those obtained by standard PCR. Salmonella invA was detected in 12 of 14 carcass rinses by ICAN, while in 7 of 14 rinses by standard PCR. These results indicate that ICAN is an efficient, sensitive and simple system to detect invA of Salmonella species in developing countries such as Zambia.

Neutrophil and lymphocyte responses to oral Streptococcus in Adamantiades-Behçet's disease.

Immune reactions against microorganisms play an important pathogenic role in Adamantiades-Behçet's disease. We had previously obtained Streptococcus sanguinis (strain BD113-20), isolated from the oral cavity of patients with Adamantiades-Behçet's syndrome. To investigate the pathogenesis of this isolate, we examined neutrophil reactions and levels of cytokine production by lymphocytes after stimulation with the strain. The reactions of neutrophils were examined by chemiluminescence assay using whole blood. The amounts of interferon gamma (IFN-gamma) and interleukin (IL)-4, IL-8, IL-10 and IL-12, produced by peripheral blood mononuclear cells, were measured by ELISA. Strain BD113-20 activated neutrophils from Adamantiades-Behçet's patients and healthy volunteers, and, in addition it increased the IFN-gamma production by lymphocytes. Lymphocytes from Adamantiades-Behçet's patients showed a dominant T helper-1 immune response. Results indicated that both bacterial stimulation and host hypersensitivity might be involved in the symptoms and pathogenesis of Adamantiades-Behçet's disease.

C-terminal domain of human CAP18 antimicrobial peptide induces apoptosis in oral squamous cell carcinoma SAS-H1 cells.

Mammalian myeloid and epithelial cells express many antimicrobiotic peptides that contribute to innate host defense against invading microbes. In the present study, a 27-mer peptide of the C-terminal domain (hCAP18(109-135)) and analogs of the antimicrobial peptide human cathelicidin LL-37/human cationic antimicrobial protein 18 (hCAP18) were examined for tumoricidal activity. In vitro results showed that hCAP18(109-135) induced apoptosis in human oral squamous cell carcinoma (OSCC), SAS-H1 cells. The hCAP18(109-135) induced mitochondrial depolarization and apoptosis in SAS-H1 cells, but not in healthy human gingival fibroblasts (HGF) and human keratinocyte line HaCaT cells. Caspases were not activated during hCAP18(109-135)-induced apoptosis in SAS-H1 cells. The results indicate that hCAP18(109-135) may induce caspase-independent apoptosis in OSCC but not in normal cells. hCAP18(109-135) can therefore be a useful anti-tumor therapeutic agent in the treatment of OSCC.

SS1 Helicobacter pylori disrupts the paracellular barrier of the gastric mucosa and leads to neutrophilic gastritis in mice.

Helicobacter pylori induces severe neutrophilic infiltration in the lamina propria of the stomach, which leads to gastritis in humans. The possible involvement of a paracellular route for bacterial nutrients and etiologic agents that may play an important role in colonization of the bacteria and cause gastritis has been suggested. To study the functions of the paracellular barrier of gastric surface epithelium, SS1, a strain of H. pylori adapted to the murine stomach, was inoculated into the stomachs of C57BL/6 mice. At 4 months after inoculation, SS1 had achieved a high level of colonization (10(6)-10(7) colony-forming units/g tissue) associated with neutrophilic infiltration in the lamina propria of the junctional zone. Disruption of the paracellular barrier was observed in the SS1-infected stomachs, as revealed by the invasion of a lanthanum tracer into the paracellular space of the surface epithelium. Only 2% of junctions were permeable in control stomachs, whereas 72% of the paracellular barrier was disrupted in the SS1-infected gastric epithelia. Furthermore, distribution of tight junction-related molecules such as 7H6 antigen, occludin, and cortical actin was affected in the surface epithelium by SS1 infection. The linear expression pattern of occludin was disrupted and became irregular or punctuated. The 7H6 antigen accumulated as aggregates in the apical portion of the surface epithelium and cortical actin became irregular and punctuated. Taken together, these results indicate that infection by SS1 directly or indirectly caused an increase in paracellular permeability and altered the localization of tight junction-related molecules of the gastric surface epithelium. This observation suggests that the paracellular pathway may play a significant role in establishing H. pylori-induced gastritis in the clinical setting.