Expression and DNA methylation profiles of EZH2-target genes in plasma exosomes and matched primary tumor tissues of the patients with diffuse large B-cell lymphoma.

AIMS: Diffuse large B-cell lymphoma (DLBCL) is the most common type of aggressive lymphoma. This study was designed to compare epigenetic alterations observed in Enhancer of Zeste Homolog 2 (EZH2)-target genes between plasma-derived exosomes and primary tumors in DLBCL patients. MAIN METHODS: Exosomes were isolated from plasma of 21 DLBCL patients and 21 controls. We analyzed the methylation status of the target genes using methylation-specific PCR. We also examined whether the exosomes and the tumor samples contained transcripts of the target genes. KEY FINDINGS: We found that CDKN2A and CDKN2B were methylated in both plasma exosomes and primary tumor tissue samples. None of the transcripts were found in the exosomes except CDKN1B which was expressed in 8 (38%) of the exosome samples. SIGNIFICANCE: This study showed that plasma exosomes might preferably package certain target molecules from primary tumors and the exosomes containing dual methylated DNAs of CDKN2A and CDKN2B, or CDKN1B transcript may contribute to DLBCL pathogenesis.

Increased Expression of Pentraxin 3 in Placental Tissues from Patients with Unexplained Recurrent Pregnancy Loss.

Pentraxin 3 (PTX3), a prototypical member of the long pentraxin subfamily, is a evolutionarily conserved multimeric pattern recognition receptor involved in the humoral component of the innate immune system. Pentraxin 3 is released when tissue is stressed or damaged, and interacts with many different ligands. Pentraxin 3 exerts a pivotal role both as a regulator and as an indicator of inflammatory response in the pathogenesis of many diseases such as sepsis, vasculitis and preeclampsia. Uncontrolled inflammatory response is considered a major cause of unexplained recurrent pregnancy loss (URPL). We determined the PTX3 messenger ribonucleic acid (mRNA) and protein expression levels in placentai tissues from 50 women with URPL, and made comparison with those in 50 age-matched control subjects. In quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry analyses, PTX3 mRNA and protein levels, respectively, were significantly increased in URPL patients compared with their respective controls (p = 0.0001). Although no significant correlations were identified between PTX3 expression levels and clinical parameters such as maternal age, numbers of previous pregnancy losses, and gestational age at miscarriage, PTX3 mRNA expression was significantly higher in patients with no live births than in women with previous live births (p = 0.0001). Our study suggests that tissue-specific expression of PTX3 is associated with URPL. Further larger studies are required to determine whether PTX3 expression can be used as a biomarker to manage URPL in routine clinical practice.

Mutational status of EZH2 and CD79B hot spots in mature B-cell non-Hodgkin's lymphomas: novel CD79B variations have been revealed.

OBJECTIVE: We aimed to determine the hot spot mutational frequencies of Enhancer of Zeste homolog 2 (EZH2) and cluster of differentiation 79B (CD79B) genes in a cohort of mature B-cell non-Hodgkin's lymphomas. PATIENTS AND METHODS: DNA samples from formalin-fixed and paraffin embedded (FFPE) tissues from a total of 37 patients with mature B-cell non-Hodgkin lymphomas were included in the study. Molecular genetic analysis was performed by direct sequencing of the DNA samples. RESULTS: We analyzed formaldehyde fixed-paraffin embedded (FFPE) tumor tissue samples from 17 female and 20 male patients with a median age of 63.7 years at the time of diagnosis. None of the patients had previously reported hot spot mutations in EZH2 and CD79B, but previously unreported single nucleotide variations of CD79B were present in nine patients. rs779833118 was the most frequent variation (7/37 patients, 18.9%). A non-synonymous variation rs757407417, which could have a potentially damaging outcome, was detected in two patients. CONCLUSIONS: None of the patients had well-known hot spot mutations in EZH2 and CD79B. However, we detected novel CD79B variations in mature B-cell non-Hodgkin's lymphoma patients.

Prevalence of Salmonella serovars in chickens in Turkey.

In this study, 151 (18.6%) of 814 ceca obtained during in-line processing of 28 broiler (Hybro G, Avian, Arbor acres, and Cobb breeds) and 5 layer (Ross, Tetra SL, Isa Brown, and Brown Nick breeds) flocks in Turkey were found to be contaminated with four different Salmonella serovars. Only Salmonella enterica subsp. enterica Serovar Enteritidis (Salmonella Enteritidis) was recovered from layer birds, whereas Salmonella Enteritidis (81.5%). Salmonella Agona (7.6%), Salmonella Thompson (10.1%), and Salmonella Sarajane (0.8%) were isolated from broiler birds. Isolations of Salmonella Agona and Salmonella Thompson from poultry are reported for the first time in Turkey. The isolation of Salmonella Sarajane from chickens is the first report in the world. The standard method of National Poultry Improvement Plan, U.S. Department of Agriculture, was used to detect Salmonella from chicken cecal samples. Primary and delayed secondary enrichments (PE and DSE) were done in tetrathionate-Hajna broth (TTHB). Two different agar media, xylose lysine tergitol 4 (XLT4) and brilliant green with novobiocin (BGN) were used to observe, and compared for their isolation and selective differentiation of, Salmonella-suspected colonies. Isolated salmonellae were then biotyped and serotyped. Ninety-one and 151 salmonellae were isolated with XLT4 agar after PE and DSE, respectively. From the same samples, BGN agar was able to detect only 50 and 131 Salmonella after PE and DSE, respectively. The isolation rate with XLT4 was 11.2% (P < 0.01) with PE, and this rate increased to 18.6% after DSE. Also, the PE isolation rate (11.2%) with XLT4 agar was significantly higher (P < 0.01) than PE with BGN agar (6.1%). Salmonella was isolated from 39.3% (11 of 28) of the broiler flocks and from 60.0% (3 of 5) of the layers. The detection sensitivity of the isolation method was determined as 1 CFU g(-1) experimentally. These data demonstrate the presence of Salmonella Enteritidis, Salmonella Thompson, Salmonella Agona, and Salmonella Sarajane in chicken flocks in Turkey.

Detection of salmonellae in chicken feces by a combination of tetrathionate broth enrichment, capillary PCR, and capillary gel electrophoresis.

This report describes a rapid detection procedure for salmonellae from chicken feces by the combination of tetrathionate primary enrichment (preenrichment [PE])-bacterial lysis-capillary PCR and capillary gel electrophoresis. Pure Salmonella enterica serovar Enteritidis 64K was reisolated and detected by capillary PCR after buffered peptone water and nutrient broth, tetrathionate broth base Hajna (TTBH), and tetrathionate broth (TTB) preenrichments. When the same culture was mixed with intestinal homogenate, bacteriological reisolation and capillary PCR detection was achieved only by TTBH and TTB preenrichments. Capillary gel electrophoresis revealed that a Salmonella genus-specific 281-bp PCR product was detected when Salmonella strains but not non-Salmonella strains were tested. The detection limit of capillary PCR with whole-cell DNA extracted from pure Salmonella enterica serovars Enteritidis 64K, Typhimurium LT2-CIP60-62, and Gallinarum 64K was 3, 3, and 9 CFU ml(-1), respectively. The detection limit of capillary PCR from whole-cell DNA extracted from intestinal homogenate artificially contaminated with the same three strains was 3, 3, and 7 CFU ml(-1), respectively. We compared the results of the capillary PCR and bacteriological examination from the natural samples. Thirty-five of 53 naturally contaminated samples produced a specific PCR product. In 9 of the 35 PCR-positive samples, Salmonella could not be detected bacteriologically either by PE or a primary and delayed secondary enrichment (DSE) combination. In the 18 PCR-negative samples, 4 samples were found to harbor Salmonella by both PE and DSE and 14 samples were positive after DSE. Fifty-three additional intestinal homogenate samples, which were negative by their PE and DSE in bacteriological examination, were found to be also negative by their PCRs. The total time required to detect Salmonella with the capillary PCR method we used was approximately 20 h. If samples are from clinically diseased birds, the total time for PCR and detection is reduced to 2 h since the 18-h PE is not required. These results indicate that TTB enrichment, bacterial lysis, and genus-specific capillary PCR combined with capillary gel electrophoresis constitute a sensitive and selective procedure which has the potential to rapidly identify Salmonella-infected flocks.