Bovine lactoferrin increases the poly(I:C)-induced antiviral response in vitro.

Bovine lactoferrin (bLF) is a naturally occurring glycoprotein with antibacterial and antiviral activities. We evaluated whether bLF can prevent viral infections in the human intestinal epithelial cell line Caco-2. To assess antiviral responses, we measured the levels of interferon (IFN) expression, IFN-stimulated gene expression, and infection with a pseudotyped virus bearing either severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein or vesicular stomatitis virus (VSV)-G protein after treatment of cells with both bLF and polyinosinic-polycytidylic acid, an analog of double-stranded RNA that mimics viral infection. Combination treatment of cells with both bLF and polyinosinic-polycytidylic acid increased mRNA and protein expression of several IFN genes (IFNB, IFNL1, and IFNL2) and IFN-stimulated genes (ISG15, MX1, IFITM1, and IFITM3) in Caco-2 cells. However, treatment with bLF alone did not induce an antiviral response. Furthermore, combination treatment suppressed infection of the SARS-CoV-2 pseudotyped virus more efficiently than did bLF treatment alone, even though combination treatment increased the expression of mRNA encoding ACE2. These results indicate that bLF increases the antiviral response associated with the double-stranded RNA-stimulated signaling pathway. Our results also suggest that bLF and double-stranded RNA analogs can be used to treat viral infections, including those caused by SARS-CoV-2.

Radioactivity and radionuclides in deciduous teeth formed before the Fukushima-Daiichi Nuclear Power Plant accident.

The Fukushima-Daiichi Nuclear Power Plant (FNPP) accident in March of 2011 released substantial amounts of radionuclides into the environment. We collected 4,957 deciduous teeth formed in children before the Fukushima accident to obtain precise control data for teeth formed after the accident. Radioactivity was measured using imaging plates (IP) and epidemiologically assessed using multivariate regression analysis. Additionally, we measured 90Sr, 137Cs, and natural radionuclides which might be present in teeth. Epidemiological studies of IP showed that the amount of radioactivity in teeth from Fukushima prefecture was similar to that from reference prefectures. We found that artificial radionuclides of 90Sr and 137Cs, which were believed to have originated from past nuclear disasters, and natural radionuclides including 40 K and daughter nuclides in the 238U and 232Th series contributed to the generation of radioactivity in teeth. We also found no evidence to suggest that radionuclides originating from the FNPP accident significantly contaminated pre-existing teeth. This is the first large-scale investigation of radioactivity and radionuclides in teeth. The present findings will be indispensable for future studies of teeth formed after the FNPP accident, which will fall out over the next several years and might be more contaminated with radionuclides.

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.

Trehalose inhibits inflammatory cytokine production by protecting IkappaB-alpha reduction in mouse peritoneal macrophages.

OBJECTIVE: The aim of this study was to examine whether trehalose, a disaccharide, could inhibit Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS)-enhanced production of inflammatory cytokines in mouse peritoneal macrophages. DESIGN: Mouse peritoneal macrophages were treated with trehalose and stimulated with P. gingivalis LPS. Interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) levels in the culture supernatant were measured by ELISA. The mRNA levels of the cytokines in macrophages were analysed by semi-quantitative RT-PCR. DNA and protein synthesis were measured by incorporation of [(3)H] thymidine or [(14)C] praline into mouse peritoneal macrophages. IkappaB-alpha reductions were assessed by western blot. RESULTS: Treatment with trehalose suppressed LPS-induced IL-1beta and TNF-alpha production and downregulated transcription of these cytokines. Furthermore, trehalose inhibited LPS-induced reduction of IkappaB-alpha. In addition, we also observed expression of the trehalose receptor (T1R3) in mouse peritoneal macrophages. CONCLUSION: These results may suggest that trehalose inhibits LPS-induced production of IL-1beta and TNF-alpha in mouse peritoneal macrophages by inhibiting degradation of IkappaB-alphavia the trehalose receptor T1R3.

Role of alphav integrin in osteoprotegerin-induced endothelial cell migration and proliferation.

Osteoprotegerin (OPG) is a decoy receptor for the receptor activator of nuclear factor kappaB ligand (RANKL). However, the role of OPG in the endothelium remains unknown. In this study, we demonstrate that OPG stimulates the proliferation and migration of human microvascular endothelial cells (HMVECs). In addition, we show that treatment with integrin alpha(v)beta(3) or integrin alpha(v)beta(5) blocking antibody inhibits endothelial cell migration. In contrast, treatment with anti-alpha(v)beta(3) antibody or anti-alpha(v)beta(5) antibody alone did not inhibit OPG-induced proliferation. However, OPG-induced proliferation was inhibited when these antibodies were applied simultaneously. Furthermore, OPG evoked activation of extracellular signal-regulated kinase (ERK) 1/2, which has been linked to integrin alpha(v) activity. Taken together, these results suggest that integrins alpha(v)beta(3) and/or alpha(v)beta(5) contribute to endothelial cell proliferation and migration induced by OPG.

Osteoprotegerin protects endothelial cells against apoptotic cell death induced by Porphyromonas gingivalis cysteine proteinases.

Osteoprotegerin (OPG) is a key regulator of osteoclastogenesis during the progression of periodontitis. Recent reports suggest that osteoprotegerin may also prevent arterial calcification and contribute to endothelial cell survival. To determine whether the vascular functions of osteoprotegerin are involved in periodontitis, we examined whether osteoprotegerin contributed to the survival of endothelial cells damaged by Porphyromonas gingivalis cysteine proteinases (gingipains). Gingipain proteinases cleave a broad range of host proteins, and are important virulence factors of P. gingivalis, a major causative bacterium of adult periodontitis. Human microvascular endothelial cells (HMVEC) were exposed to activated gingipain extracts from P. gingivalis 381, with and without pretreatment with osteoprotegerin. Cell viability was quantified by the tetrazolium (WST-8) reduction assay, and apoptosis was examined using Hoechst 33342 nuclear staining. After 16 h of treatment with activated gingipain extracts, HMVEC showed near-complete detachment from the tissue culture dish, and apoptosis was evident by 24 h. Pretreatment of HMVEC with osteoprotegerin reduced the extent of both cellular detachment and apoptotic cell death. Our results indicated that osteoprotegerin pretreatment protected HMVEC against detachment and apoptotic cell death induced by gingipain-active bacterial cell extracts. These results also suggest that osteoprotegerin may function as a survival factor for endothelial cells during periodontitis.

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.

NF-kappaB-dependent induction of osteoprotegerin by Porphyromonas gingivalis in endothelial cells.

Porphyromonas gingivalis is a major etiological pathogen of adult periodontitis characterized by alveolar bone resorption. Vascular endothelial cells supply many inflammatory cytokines into periodontal tissue. However, whether the cells contribute to bone metabolism in periodontitis remains unknown. In this study, we investigated the effect of P. gingivalis on osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL) production, both of which are key regulators of bone metabolism, in human microvascular endothelial cells (HMVECs). We showed that P. gingivalis upregulated expression of OPG but not RANKL mRNA in HMVEC. P. gingivalis induced NF-kappaB activation, and the induction of OPG in HMVEC by the pathogen was blocked by the inhibitors of NF-kappaB. In addition, incubation of OPG with P. gingivalis supernatant resulted in loss of the protein. These results indicate that P. gingivalis-stimulated HMVEC secrete OPG via a NF-kappaB-dependent pathway, while the OPG is partly degraded by the bacteria. Thus, microvascular endothelial cells can act as a source of OPG and thereby may play an important role in regulating bone metabolism in periodontitis.

Porphyromonas gingivalis modulates the production of interleukin 8 and monocyte chemotactic protein 1 in human vascular endothelial cells.

Porphyromonas gingivalis seems to perturb the cytokine network to maintain periodontal disease. Although endothelial cells are a leading source of interleukin 8 (IL-8) and monocyte chemotactic protein 1 (MCP-1), the effect of P. gingivalis on the cells remains unclear. We used reverse transcription-PCR (RT-PCR) to assess levels of IL-8 and MCP-1 mRNA and enzyme-linked immunosorbent assays (ELISA) to evaluate their protein production by human umbilical vein endothelial cells (HUVEC) challenged with P. gingivalis. IL-8 and MCP-1 protein levels decreased in response to challenge with P. gingivalis, and N-a- p-tosyl- L-lysine chloromethylketone (TLCK) prevented the degradation of these chemokines. Furthermore, the bacteria upregulated expression of the mRNAs of these chemokines. Our results indicate that P. gingivalis proteases degraded IL-8 and MCP-1. Degradation of chemokines secreted from endothelial cells may decrease the recruitment of leukocytes and their migration through the endothelium, thus contributing to a delay in the host immune defense mechanism.