Use of the conjugate of disulphated ursodeoxycholic acid with p-aminobenzoic acid for the detection of intestinal bacteria.

The disulphate ester of ursodeoxycholyl-p-aminobenzoic acid (PABA-UCDA) was synthesised and compared with PABA-UDCA for its use in detection of intestinal bacteria. This compound, PABA-UDCA disulphate, had characters in common with PABA-UDCA in that it was deconjugated by cholylglycine hydrolase to release free PABA and bacteria that split glycocholic acid deconjugated PABA-UDCA disulphate. Further, in rat experiments urinary excretions of PABA were measured for six hours after oral administration of 15 mg PABA-UDCA disulphate. Ten control rats excreted (mean (SE) 188.2 (13.6) micrograms of PABA; 10 rats with an intestinal stagnant loop excreted more (530.1 (30.1) micrograms; p < 0.001): whereas 10 rats in each of three groups pretreated by oral administration of various antibiotics excreted less (polymixin B+tinidazole, 4.9 (1.6) micrograms; kanamycin, 31.0 (4.7) micrograms; clindamycin 40.9 (5.5) micrograms; p < 0.001). By contrast with PABA-UDCA, PABA-UDCA disulphate was not actively absorbed from any part of the small intestine in everted gut sac experiments, and showed poor recovery from bile after its intraileal instillation in rats. This indicated that PABA-UDCA disulphate is a single pass type substance in the gut and its oral administration test reflects the sum of the activities of bacteria in the small intestine and colon. The disulphate was easily soluble in water and this allowed its application in an in vitro test involving PABA-UDCA disulphate incubation with intraperitoneal pus (PABA-UDCA disulphate incubation test) from patients with peritonitis. This test was carried out on six patients with peritonitis, and the severity of bacterial peritonitis was expressed quantitatively. From the results obtained PABA-UDCA disulphate was considered a good material to detect intestinal bacteria.     

Heterozygous germline BLM mutations increase susceptibility to asbestos and mesothelioma

Rare biallelic BLM gene mutations cause Bloom syndrome. Whether BLM heterozygous germline mutations (BLM^+/−) cause human cancer remains unclear. We sequenced the germline DNA of 155 mesothelioma patients (33 familial and 122 sporadic). We found 2 deleterious germline BLM^+/− mutations within 2 of 33 families with multiple cases of mesothelioma, one from Turkey (c.569_570del; p.R191Kfs*4) and one from the United States (c.968A>G; p.K323R). Some of the relatives who inherited these mutations developed mesothelioma, while none with nonmutated BLM were affected. Furthermore, among 122 patients with sporadic mesothelioma treated at the US National Cancer Institute, 5 carried pathogenic germline BLM^+/− mutations. Therefore, 7 of 155 apparently unrelated mesothelioma patients carried BLM^+/− mutations, significantly higher (P = 6.7E-10) than the expected frequency in a general, unrelated population from the gnomAD database, and 2 of 7 carried the same missense pathogenic mutation c.968A>G (P = 0.0017 given a 0.00039 allele frequency). Experiments in primary mesothelial cells from Blm^+/− mice and in primary human mesothelial cells in which we silenced BLM revealed that reduced BLM levels promote genomic instability while protecting from cell death and promoted TNF-α release. Blm^+/− mice injected intraperitoneally with asbestos had higher levels of proinflammatory M1 macrophages and of TNF-α, IL-1β, IL-3, IL-10, and IL-12 in the peritoneal lavage, findings linked to asbestos carcinogenesis. Blm^+/− mice exposed to asbestos had a significantly shorter survival and higher incidence of mesothelioma compared to controls. We propose that germline BLM^+/− mutations increase the susceptibility to asbestos carcinogenesis, enhancing the risk of developing mesothelioma.

In the United States, the incidence rate of mesothelioma varies between fewer than one case per 100,000 persons in states with no asbestos industry to two to three cases per 100,000 persons in states with an asbestos industry (1, 2). Asbestos causes DNA damage and apoptosis (3) and promotes a chronic inflammatory reaction that supports the emergence of malignant cells (4). Fortunately, only a small fraction of exposed individuals develop mesothelioma; for example, 4.6% of deaths in miners who worked in asbestos mines for over 10 y were caused by mesothelioma (1). Therefore, multiple cases of mesothelioma in the same family are rare and suggest genetic predisposition (5). In 2001, we discovered that susceptibility to mesothelioma was transmitted in a Mendelian fashion across multiple generations in some Turkish families exposed to the carcinogenic fiber erionite, pointing to gene × environment interaction (G×E) as the cause (6). In 2011, we discovered that carriers of heterozygous germline BRCA1-associated protein–1 (BAP1) mutations (BAP1^+/−) developed mesothelioma and uveal melanoma (5), findings expanded and confirmed by us and by multiple research teams (reviewed in refs. 1, 7, 8). Moreover, heterozygous germline Bap1 mutations (Bap1^+/−) significantly increased susceptibility to asbestos-induced mesothelioma in mice (9, 10), evidence of G×E. Reduced BAP1 levels impair DNA repair (11) as well as different forms of cell death (3, 12) and induce metabolic alterations (13–15) that together favor cancer development and growth.Recent studies revealed that mesothelioma may also develop among carriers of germline mutations of additional tumor-suppressor genes that cause well-defined cancer syndromes, including MLH1 and MLH3 (Lynch syndrome), TP53 (Li–Fraumeni syndrome), and BRCA1-2 (Breast and Ovarian Cancer syndrome) (16, 17). When all germline mutations are combined, it has been estimated that about 12% of mesotheliomas occur in carriers of heterozygous germline mutations of BAP1, the most frequent mutation among patients with mesothelioma, or of other tumor suppressors. Some of these mutations may sensitize the host to asbestos carcinogenesis, according to a G×E scenario (17). Thus, presently, mesothelioma is considered an ideal model to study G×E in cancer (17). As part of the Healthy Nevada Project (HNP), we are studying G×E in northern Nevada, a region with an unusually high risk of exposure to carcinogenic minerals and arsenic, which may be related to the high cancer rates in this region (18). We are investigating genetic variants that may increase cancer risk upon exposure to carcinogens to implement preventive strategies.Biallelic mutations of the Bloom syndrome gene (BLM) cause Bloom syndrome, an autosomal-recessive tumor predisposition syndrome characterized by pre- and postnatal growth deficiency, photosensitivity, type 2 diabetes, and greatly increased risk of developing various types of cancers. BLM is a RecQ helicase enzyme that modulates DNA replication and repair of DNA damage by homologous recombination (19). In patients affected by Bloom syndrome, the absence of the BLM protein causes chromosomal instability, increased number of sister chromatid exchanges, and increased numbers of micronuclei (20–22). In addition, BLM is required for p53-mediated apoptosis (23), a process critical to eliminate cells that have accumulated DNA damage. Impaired DNA repair together with altered apoptosis resulted in increased cancer incidence (17, 24). Of course, inactivating germline BLM heterozygous (BLM^+/−) mutations are much more common than biallelic BLM (BLM^−/−) mutations, with an estimated frequency in the general population of 1 in 900 based on data from the Exome Aggregation Consortium (25). BLM^+/− mutation carriers do not show an obvious phenotype; however, some studies have suggested that carriers of these mutations may have an increased cancer risk (17, 24). Mice carrying Blm^+/− mutations are prone to develop a higher rate of malignancies in the presence of contributing factors, such as concurrent heterozygous mutations of the adenomatous polyposis coli (Apc) gene, or upon infection with murine leukemia virus (26). However, in studies in which Blm^+/− mice were crossed with tuberous sclerosis 1-deficient (Tsc1^+/−) mice that are predisposed to renal cystadenomas and carcinomas, Wilson et al. found that Tsc1^+/− Blm^+/− mice did not show significantly more renal cell carcinomas compared with Tsc1^+/− Blm^WT mice (27). In humans, a large study involving 1,244 patients with colon cancer and 1,839 controls of Ashkenazi Jewish ancestry, in which BLM^+/− frequency is as high as 1 in 100 individuals (28), suggested that carriers of germline BLM^+/− mutations might have a twofold increase in colorectal cancer (CRC) (29). A smaller study did not confirm these results, but reported a trend of increasing incidence of adenomas—premalignant lesions—among BLM^+/− mutation carriers (30). In addition, BLM^+/− mutations were found overrepresented among early-onset (<45 y old) CRC patients (25). Other studies associated BLM^+/− mutations to an increased risk of breast (31, 32) and prostate cancer (33), but the low power of these studies hampered definite conclusions. In summary, it appears possible that BLM^+/− mutations may increase cancer risk in the presence of contributing factors.     

The Interaction Between β‐3 Adrenergic Receptor and Peroxisome Proliferator‐Activated Receptor Gamma Gene Polymorphism to Periodontal Disease in Community‐Dwelling Elderly Japanese

Background: It has been hypothesized that β‐3 adrenergic receptor and peroxisome proliferator‐activated receptor gamma (PPARγ) might have gene–environmental and gene–gene interactions in periodontal disease. The purpose of this study is to elucidate the interaction between β‐3 adrenergic receptor and PPARγ gene polymorphism with periodontal disease. Methods: Three hundred thirty‐two postmenopausal females were enrolled, and their serum high‐sensitivity C‐reactive protein (hsCRP) and hemoglobin A1c (HbA1c) were examined. β‐3 adrenergic receptor and PPARγ genotypes were then determined. An oral examination was performed. The number of remaining teeth was counted, and the probing depth (PD) and clinical attachment level (CAL) were measured. Prevalence‐rate ratios (PRRs) were calculated by multiple Poisson regression analyses to evaluate the relationship among periodontal disease markers, such as the number of sites with CAL 4 to 5 or ≥6 mm or PD 4 to 5 or ≥6 mm, and β‐3 adrenergic receptor polymorphisms, PPARγ polymorphisms, and the interaction term adjusted by age, hsCRP, and HbA1c, after converting the number of remaining teeth (n) to an offset variable. Results: In the participants with body mass index (BMI) ≥25, PRRs of β‐3 adrenergic receptor genotype (Trp/Arg and Arg/Arg) for periodontal disease markers were 0.13 to 0.70 (P <0.0001 to 0.74), those of PPARγ genotype (Pro/Pro) were 0.66 to 3.14 (P = 0.01 to 0.68), and those of the interaction term for the two genotypes were 1.69 to 12.61 (P <0.0001 to 0.33). However, in the participants with BMI <25, a constant tendency was not observed. Conclusion: The results confirmed a positive relationship between the interaction term for β‐3 adrenergic receptor genotype and PPARγ genotype and various periodontal markers in obese elderly females.     

Microclimate and development of pressure ulcers and superficial skin changes

This study aims to evaluate the microclimate and development of pressure ulcers and superficial skin changes. A prospective cohort study was conducted in an acute care ward in Indonesia. Risk factors for pressure ulcers and superficial skin changes were identified based on the Bergstrom Braden conceptual model. Microclimate data were collected every 3 days for 15 days while the development of pressure ulcers and superficial skin changes was observed every day. Pressure ulcers and superficial skin changes were developed in 20 of the 71 participants. Total mean difference in skin temperature was higher for patients with pressure ulcers and superficial skin changes (0·9 ± 0·6°C) compared with controls (0·6 ± 0·8°C) (P = 0·071). Binary logistic regression predictor values for pressure ulcers and superficial skin changes were 0·111 for type of sheet and 0·347 for Braden Scale results. In conclusion, difference in skin temperature seems to be a predictor for pressure ulcer development and superficial skin changes, while synthetic fibre sheets are able to maintain a beneficial microclimate.     

Enhanced Gastric Ghrelin Production and Secretion in Rats with Gastric Outlet Obstruction

Background and Aim  

Ghrelin has distinct effects on gastrointestinal motility through the vagus nerve and gastric excitatory neural plexus. The objectives of this study were to investigate the dynamics of ghrelin and expression of neuromuscular markers in a newly established surgically manipulated rat model of gastric outlet obstruction (GOO), akin to the pyloric stricture associated with duodenal ulcer, advanced gastric cancer, and other conditions, in the clinical setting.     

Massive bone reconstruction with heat‐treated bone graft loaded autologous bone marrow‐derived stromal cells and β‐tricalcium phosphate composites in canine models

Bone marrow‐derived stromal cells (BMSCs) contain mesenchymal stem cells that are capable of forming various mesenchymal tissues. We hypothesized that BMSCs and β‐tricalcium phosphate (β‐TCP) composites would promote the remodeling of large‐sized autologous devitalized bone grafts; therefore, the aim of this study was to evaluate the effects of the composites on the remodeling of autologous devitalized bone grafts. Autologous BMSCs cultured in culture medium containing dexamethasone (10^?7 M) were loaded into porous β‐TCP granules under low‐pressure. Theses BMSC/TCP composites were put into the bone marrow cavity of autologous heat‐treated bone (femoral diaphysis, 65‐mm long, 100°C, 30 min) and put back to the harvest site. In the contralateral side, β‐TCP without BMSC were used in the same manner as the opposite side as the control. Treatment with the BMSC/TCP composites resulted in a significant increase in thickness, bone mineral density, and matured bone volume of the cortical bone at the center of the graft compared to the control. Histological analysis showed matured regenerated bone in the BMSC loaded group. These results indicate that BMSC/TCP composites facilitated bone regeneration and maturation at the graft site of large‐sized devitalized bone. This method could potentially be applied for clinical use in the reconstruction of large bone defects such as those associated with bone tumors. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1308–1316, 2013

     

Interleukin-6 gene promoter methylation in rheumatoid arthritis and chronic periodontitis

Background: Methylation status of the cytokine genes may play a role in the pathogenesis of inflammatory diseases, such as rheumatoid arthritis (RA) and chronic periodontitis (CP). This study was undertaken to evaluate whether the DNA methylation profile of the interleukin‐6 (IL‐6) gene promoter was unique to individuals with RA and CP. Methods: The study participants consisted of 30 patients with RA, 30 patients with CP, and 30 age‐, sex‐, and smoking status–balanced healthy controls. Genomic DNA isolated from peripheral blood was modified by sodium bisulfite and analyzed for DNA methylation levels of IL‐6 gene with direct sequencing. Levels of IL‐6 were determined by an enzyme‐linked immunosorbent assay. Results: The region of IL‐6 gene promoter from ?1200 to +27 bp was shown to contain 19 CpG motifs. The methylation levels of the CpG motif at ?74 bp were significantly lower in patients with RA and CP than those in controls (P = 0.0001). Both levels of serum IL‐6 and IL‐6 production by mononuclear cells were significantly different between individuals with and without the methylation at ?74 bp (P = 0.03). The +19 bp motif exhibited differential levels of the methylation among the groups, which was not associated with serum levels of IL‐6. The other 17 CpG motifs exhibited comparable levels of the methylation between the groups. Conclusion: These results suggest that hypomethylated status of a single CpG in the IL‐6 promoter region may lead to increased levels of serum IL‐6, implicating a role in the pathogenesis of RA and CP.