Antimicrobial resistance in Campylobacter coli and Campylobacter jejuni in cynomolgus monkeys (Macaca fascicularis) and eradication regimens.

BACKGROUND: Campylobacter spp. are zoonotic pathogens, however, knowledge about their presence and antimicrobial resistance in nonhuman primates is limited. Our animal facility purchased cynomolgus monkeys (Macaca fascicularis) from various Asian countries: China, Cambodia, Indonesia, the Philippines, and Vietnam. METHODS: Colonization by Campylobacter spp. was investigated in 238 of the monkeys from 2009 to 2012 and antimicrobial susceptibility testing was carried out for these isolates. Furthermore, we eradicated these pathogens from these monkeys. RESULTS: Campylobacter spp. were isolated from 47 monkeys from three specific countries: China, Cambodia, and Indonesia, with respective isolation rates of 15%, 36%, and 67%. Two monkeys, which were each infected with Campylobacter jejuni and Campylobacter coli, showed clinical symptoms of diarrhea and bloody feces. In total, 41 isolates of C. coli and 17 isolates of C. jejuni were detected. Antimicrobial susceptibility varied: in the monkeys from China, erythromycin (ERY)-, tetracycline (TET)-, and ciprofloxacin-resistant C. coli, in the monkeys from Cambodia, amoxicillin-intermediate, TET- and ciprofloxacin-resistant C. coli and amoxicillin- and ciprofloxacin-resistant C. jejuni, and in the monkeys from Indonesia, ciprofloxacin-resistant C. coli and TET- and ciprofloxacin-resistant C. jejuni were common (>75%). Multiresistant isolates of C. coli were found in monkeys from all countries and multiresistant isolates of C. jejuni were found in monkeys from Indonesia. The eradication rate with azithromycin was comparable to that with gentamicin (GEN) by oral administration, and was higher than those with amoxicillin-clavulanic acid (AMC) and chloramphenicol (CHL). CONCLUSION: From the perspective of zoonosis, we should acknowledge multiresistant Campylobacter spp. isolated from the monkeys as a serious warning.

Does aberrant DNA methylation occur in human uterine leiomyomas? An attempt of genome-wide screening by MS-RDA.

OBJECTIVE: Uterine leiomyoma are very common benign tumors in women of reproductive age. However, the molecular mechanisms of cause and development of these tumors are poorly understood. This study attempts to examine whether or not aberrant DNA methylation occurred in these tumors. METHODS: We carried out a genome-wide screen for aberrant DNA methylation, adopting methylation-sensitive-representational difference analysis (MS-RDA) using normal adjacent myometria as tester and myoma tissue driver. CONCLUSION: A total of 192 clones identified by MS-RDA were sequenced, 27 DNA fragments derived from CpG islands (CGIs) were isolated, and seven of them were from CGI in the 5' regions of known genes, which include CHARC1, FAM44B, FLJ33655, HSUP, MLLT3, SLC16A1, and ZNF96. Then, methylation statuses of those CGIs were analyzed by methylation-specific polymerase chain reaction using 5 primary samples of human uterine leiomyoma. Aberrant DNA methylation did not observed in 7 genes in 5 human uterine leiomyoma eventually. This study is insufficient to identify aberrant DNA methylation occurring in the human uterine leiomyoma, a large population of primary samples and more attempts, such as the use of cell lines or primary monolayer cultures established from tissue samples, are warranted to clarify this issue.

Expression quantitative trait loci analysis of 13 genes in the rat prostate.

Differential expression of mRNA among animal strains is one of the mechanisms for their diversity. cDNA microarray analysis of the prostates of BUF/Nac (BUF) and ACI/N (ACI) rats, which show different susceptibility to prostate cancers, found 195 differentially expressed genes. To identify loci that control differential expression of 13 genes with diverse expression levels, their expression levels were measured by quantitative RT-PCR in 89 backcross rats, and expression quantitative trait locus (eQTL) analysis was performed. Nine genes [Aldh1a1, Aldr1, Bmp6, Cdkn1a (p21), Cntn6, Ghr, Jund, Nupr1, and RT1-M3] were controlled by cis-acting loci. Cdkn1a, a cell cycle regulator and a candidate for a prostate cancer susceptibility gene, was mapped to its own locus and had polymorphisms, including a 119-bp insertion in the 5' upstream region in BUF rats. Four genes (Kclr, Pbsn, Psat1, and Ptn) were controlled by trans-acting loci. Pbsn, a prostate-specific gene on chromosome X, was controlled by a QTL on chromosome 8. Depending upon which gene that we selected from the genes widely used for normalization (Actb, Gapd, or Ppia), different QTL were mapped for Kclr, Psat1, and Ptn. Normalization using Actb most appropriately explained the expression levels in a congenic strain for chromosome 3. eQTL analysis with precise measurement of expression levels and appropriate normalization was shown to be effective for mapping loci that control gene expression in vivo.

Whole-genome analyses of loss of heterozygosity and methylation analysis of four tumor-suppressor genes in N-methyl-N'-nitro-N-nitrosoguanidine-induced rat stomach carcinomas.

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced rat stomach carcinomas are considered to be a good model for differentiated-type human stomach carcinomas. However, as for their molecular basis, only infrequent mutations of Catnb (beta-catenin) and Trp53 (p53) have been observed. Here, we carried out a whole-genome analysis of loss of heterozygosity (LOH) using 21 stomach carcinomas induced by MNNG in F(1) hybrids of ACI and BUF rats, and also analyzed promoter methylation of four tumor-suppressor genes. LOH analysis was performed using 130 polymorphic markers covering rat chromosomes 1-20 with an average interval of 20 Mbp. Despite adapting conditions so that LOH could be detected with up to a 50% contamination of stromal cells, no LOH was detected at any loci. CpG islands in putative promoter regions of four tumor-suppressor genes, Cdh1 (E-cadherin), Cdkn2a (p16), Mlh1, and Rassf1a, were analyzed by methylation-specific polymerase chain reaction (PCR). However, no methylation was detected. In contrast, the promoter region of Pgc (pepsinogen C), which lacks a CpG island, was methylated in all 21-cancer samples. These results indicated that LOH spanning a chromosomal region larger than 30-40 Mbp or silencing of Cdh1, Cdkn2a, Mlh1, and Rassf1a, was not involved in MNNG-induced rat stomach carcinomas. The search for other genes involved in these carcinomas needs to be continued.

Effect of cytogenin, a novel immunomodulator, on streptozotocin-induced diabetes in mice.

The anti-diabetic effect of cytogenin was examined using streptozotocin-induced diabetes in mice. Cytogenin suppressed not only the increase of plasma glucose level but also the body weight reduction in diabetic mice. Histological examination of the pancreas taken from diabetic mice given cytogenin showed that cytogenin decreased the number of macrophages infiltrated into islet of pancreas. Further, cytogenin suppressed the nitric oxide generation by macrophages treated with lipopolysaccharide through decreasing of inducible nitric oxide synthase expression. Cytogenin suppressed interleukin-6 expression by macrophage treated with LPS, suggesting that the anti-diabetic activity of cytogenin might be partly attributed to the suppressive activity against nitric oxide generation.

Linkage and microarray analyses of susceptibility genes in ACI/Seg rats: a model for prostate cancers in the aged.

ACI/Seg (ACI) rats develop prostate cancers spontaneously with aging, similar to humans. Here, to identify genes involved in prostate cancer susceptibility, we did linkage analysis and oligonucleotide microarray analysis. Linkage analysis was done using 118 effective rats, and prostate cancer susceptibility 1 (Pcs1), whose ACI allele dominantly induced prostate cancers, was mapped on chromosome 19 [logarithm of odds (LOD) score of 5.0]. PC resistance 1 (Pcr1), whose ACI allele dominantly and paradoxically suppressed the size of prostate cancers, was mapped on chromosome 2 (LOD score of 5.0). When linkage analysis was done in 51 rats with single or no macroscopic testicular tumors, which had larger prostates and higher testosterone levels than those with bilateral testicular tumors, Pcs2 and Pcr2 were mapped on chromosomes 20 and 1, respectively. By oligonucleotide microarray analysis with 8,800 probe sets and confirmation by quantitative reverse transcription-PCR, only two genes within these four loci were found to be differentially expressed >1.8-fold. Membrane metalloendopeptidase (Mme), known to inhibit androgen-independent growth of prostate cancers, on Pcr1 was expressed 2.0- to 5.5-fold higher in the ACI prostate, in accordance with its paradoxical effect. Cdkn1a on Pcs2 was expressed 1.5- to 4.5-fold lower in the ACI prostate. Additionally, genes responsible for testicular tumors and unilateral renal agenesis were mapped on chromosomes 11 and 14, respectively. These results showed that prostate cancer susceptibility of ACI rats involves at least four loci, and suggested Mme and Cdkn1a as candidates for Pcr1 and Pcs2.

Identification of 27 5' CpG islands aberrantly methylated and 13 genes silenced in human pancreatic cancers.

Aberrantly methylated DNA fragments were searched for in human pancreatic cancers, using the genome scanning technique: methylation-sensitive-representational difference analysis (MS-RDA). MS-RDA isolated 111 DNA fragments derived from CpG islands (CGIs), and 35 of them were from CGIs in the 5' regions of known genes. Methylation-specific PCR (MSP) of the CGIs in seven pancreatic cancer cell lines and two pancreatic ductal epithelial cell lines showed that 27 CGIs in the 5' regions were aberrantly methylated in at least one of the cancer cell lines. Quantitative reverse-transcription-PCR analysis showed that downstream genes of all the CGIs were either not expressed or only very weakly expressed in cancer cell lines with the aberrant methylation. In the pancreatic ductal epithelial cell lines, 18 genes were expressed at various levels, and nine genes were not expressed at all. Treatment of a cancer cell line with a demethylating agent, 5-aza-2'-deoxycytidine, restored the expression of 13 genes, RASGRF2, ADAM23, NEF3, NKX2-8, HAND1, EGR4, PRG2, FBN2, CDH2, TLL1, NPTX1, NTSR1 and THBD, showing their silencing by methylation of their 5' CGIs. MSP of 24 primary pancreatic cancers showed that all these genes, except for THBD, were methylated in at least one cancer. Some of those were suggested to be potentially involved in pancreatic cancer development and progression.

Lysyl oxidase is a tumor suppressor gene inactivated by methylation and loss of heterozygosity in human gastric cancers.

Lysyl oxidase (LOX) and HRAS-like suppressor (HRASLS) are silenced in human gastric cancers and are reported to have growth-suppressive activities in ras-transformed mouse/rat fibroblasts. Here, we analyzed whether or not LOX and HRASLS are tumor suppressor genes in human gastric cancers. Loss of heterozygosity and promoter methylation of LOX were detected in 33% (9 of 27) and 27% (26 of 96) of gastric cancers, respectively. Biallelic methylation and loss of heterozygosity with promoter methylation were also demonstrated in gastric cancers. Silencing of LOX was also observed in colon, lung, and ovarian cancer cell lines. As for mutations, only one possible somatic mutation was found by analysis of 96 gastric cancer samples and 58 gastric and other cancer cell lines. When LOX was introduced into a gastric cancer cell line, MKN28, in which LOX and HRASLS were silenced, it reduced the number of anchorage-dependent colonies to 57 to 61%, and the number of anchorage-independent colonies to 11 to 23%. Sizes of tumors formed in nude mice were reduced to 19 to 26%. Growth suppression in soft agar assay was also observed in another gastric cancer cell line, KATOIII. On the other hand, neither loss of heterozygosity nor a somatic mutation was detected in HRASLS, and its introduction into MKN28 did not suppress the growth in vitro or in vivo. These data showed that LOX is a tumor suppressor gene inactivated by methylation and loss of heterozygosity in gastric cancers, and possibly also in other cancers.

Reduced expression of GNA11 and silencing of MCT1 in human breast cancers.

Alteration in the methylation status of a gene is often associated with its altered expression. Based on a genome scanning technique for differences in CpG methylations, methylation-sensitive representational difference analysis, DNA fragments hypermethylated in a human breast cancer were isolated. A DNA fragment was isolated from intron 1 of guanine-nucleotide-binding protein alpha-11 (GNA11). mRNA expression of GNA11 was shown to be decreased in 10 of 16 breast cancers by RT-PCR analysis, and the immunoreactivity of the GNA11 product, Galpha11 subunit of heterotrimeric G-protein, was observed to be reduced in 14 of the 16 cancers by immunohistochemistry. Methylation of a CpG island (CGI) in the 5' region of GNA11 or that of intron 1 did not show a clear correlation with its decreased expression. Another DNA fragment was isolated from a CGI in the 5' upstream region of monocarboxylate transporter 1 (MCT1), and was methylated in 4 of 20 breast cancers. The CGI was also methylated in a human breast cancer cell line, MDA-MB-231, and quantitative RT-PCR showed that its expression was almost lost in the cell line. By treatment of the cells with a demethylating agent, 5-aza-2'-deoxycytidine, the methylation was removed and the expression was restored. GNA11 is involved in signalling of gonadotropin-releasing hormone receptor, which negatively regulates cell growth. MCT1 is involved in cellular transportation of butyrate, which induces cellular differentiation. Downregulation of these two genes was suggested to be involved in human breast cancers.

Reduced Brca1 protein expression in 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced rat mammary carcinomas.

Reduced expression of BRCA1 protein, caused by the hypermethylation of its gene promoter and by other mechanisms, is observed in most sporadic human breast cancers, whereas its somatic mutations are rare. In the present study, we demonstrate that immunoreactivity of Brca1 was reduced in almost all rat mammary carcinomas induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), whereas hypermethylation of its promoter, mutations in its entire coding region, and decreased mRNA expression were absent. Using two kinds of polyclonal antibodies against human BRCA1 that recognized the N and C termini, respectively, immunoreactivity of Brca1 was found to be reduced in all 17 carcinomas examined and, especially, to be almost completely lost in 8 or 10 carcinomas. The reduction was confirmed by immunoblot analysis of five mammary carcinomas and normal mammary epithelial cells. Sequencing of the Brca1 promoter region after bisulfite modification in 10 PhIP-induced mammary carcinomas showed that the region was completely unmethylated in all of them. No mutations were detected in the entire coding region by direct sequencing of the Brca1 cDNA. Decrease in mRNA levels was not detected by quantitative real-time reverse transcription-polymerase chain reaction. These data suggested that PhIP-induced mammary carcinomas could model human breast cancers that show reduced BRCA1 immunoreactivity without promoter hypermethylation and with normal mRNA expression. As underlying mechanisms, alterations in posttranscriptional regulation or stability of Brca1 protein were suggested.

Profiling and selection of genes differentially expressed in the pylorus of rat strains with different proliferative responses and stomach cancer susceptibility.

Rat stomach cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are widely used as a model of differentiated-type human stomach cancers. ACI/NJcl (ACI) rats show persistent and strong cell proliferation in response to gastric mucosal damage by MNNG while BUF/NacJcl (BUF) rats show transient and limited cell proliferation. This difference is considered as one of the mechanisms for the high susceptibility of ACI rats to MNNG-induced stomach carcinogenesis. To identify genes involved in the differential induction of cell proliferation, cDNA subtraction was performed using RNA isolated from the pylorus of ACI and BUF rats treated with MNNG. By the temporal patterns of their expressions, the isolated 16 genes were overviewed and clustered into groups. Expression of the genes in group 1 (such as MHC class I and class II genes and interferon-inducible genes Iigp, Mx2 and Ubd) was induced by MNNG treatment, and the genes in group 2 (such as cellular retinoic acid-binding protein II (CrabpII)) were constantly expressed regardless of MNNG treatment. Then, expression profiles among multiple rat strains were compared with the extents of induction of cell proliferation. Iigp, CrabpII and EST222005 were found to show relatively good accordance, and these three genes were considered as candidates for genes that control differential induction of cell proliferation. Presence of polymorphisms at the genomic DNA level was indicated for CrabpII and EST222005, and these two genes were considered to be better candidates than IIGP: It was shown that the temporal profiles and profiles among strains, taking advantage of animal models, are useful to select candidate genes from a collection of genes isolated by various genome-wide scanning methods.

Promoter hypermethylation and post-transcriptional mechanisms for reduced BRCA1 immunoreactivity in sporadic human breast cancers.

BACKGROUND: Germline mutation of BRCA1 is well known to cause familial breast cancer. Although somatic mutations of the BRCA1 gene are rare in sporadic breast cancers, a high incidence of reduced BRCA1 immunoreactivity has been demonstrated. As one of the mechanisms for this, gene silencing by hypermethylation of the BRCA1 promoter region has been reported. Here, we show the presence of a post-transcriptional mechanism by examining promoter hypermethylation, mRNA expression levels and immunoreactivity of BRCA1 in sporadic human breast cancers METHODS: Paired samples of 20 invasive ductal carcinomas and one invasive lobular carcinoma were obtained from sporadic breast cancer cases. The BRCA1 protein expression levels were determined by immunohistochemistry using a well-characterized antibody. The methylation status of the BRCA1 promoter region was determined by sequencing after bisulfite modification. The mRNA expression levels were determined by semi-quantitative reverse transcription polymerase chain reaction (PCR). Mutations in the entire BRCA1 coding region were analyzed by PCR single-strand conformation polymorphism analysis. RESULTS: Reduced immunoreactivity was observed in 13 of the 21 cancers. Hypermethylation was observed in five of the 13 cancers with reduced immunoreactivity and mRNA expression was almost absent in these five cancers. In the remaining eight cancers, mRNA expression was not decreased. None of the 21 cancers examined harbored BRCA1 mutations. CONCLUSION: These findings showed that post-transcriptional mechanisms, such as low efficiency of translation or reduced stability of BRCA1 protein, are also involved in reduced BRCA1 immunoreactivity in sporadic breast cancers.