DNA methyltransferase 1 and DNA methylation patterning contribute to germinal center B-cell differentiation

The phenotype of germinal center (GC) B cells includes the unique ability to tolerate rapid proliferation and the mutagenic actions of activation induced cytosine deaminase (AICDA). Given the importance of epigenetic patterning in determining cellular phenotypes, we examined DNA methylation and the role of DNA methyltransferases in the formation of GCs. DNA methylation profiling revealed a marked shift in DNA methylation patterning in GC B cells versus resting/naive B cells. This shift included significant differential methylation of 235 genes, with concordant inverse changes in gene expression affecting most notably genes of the NFkB and MAP kinase signaling pathways. GC B cells were predominantly hypomethylated compared with naive B cells and AICDA binding sites were highly overrepresented among hypomethylated loci. GC B cells also exhibited greater DNA methylation heterogeneity than naive B cells. Among DNA methyltransferases (DNMTs), only DNMT1 was significantly up-regulated in GC B cells. Dnmt1 hypomorphic mice displayed deficient GC formation and treatment of mice with the DNA methyltransferase inhibitor decitabine resulted in failure to form GCs after immune stimulation. Notably, the GC B cells of Dnmt1 hypomorphic animals showed evidence of increased DNA damage, suggesting dual roles for DNMT1 in DNA methylation and double strand DNA break repair.     

Fibrinogen β-derived Bβ(15-42) peptide protects against kidney ischemia/reperfusion injury

Ischemia/reperfusion (I/R) injury in the kidney is a major cause of acute kidney injury (AKI) in humans and is associated with significantly high mortality. To identify genes that modulate kidney injury and repair, we conducted genome-wide expression analysis in the rat kidneys after I/R and found that the mRNA levels of fibrinogen (Fg)α, Fgβ, and Fgγ chains significantly increase in the kidney and remain elevated throughout the regeneration process. Cellular characterization of Fgα and Fgγ chain immunoreactive proteins shows a predominant expression in renal tubular cells and the localization of immunoreactive Fgβ chain protein is primarily in the renal interstitium in healthy and regenerating kidney. We also show that urinary excretion of Fg is massively increased after kidney damage and is capable of distinguishing human patients with acute or chronic kidney injury (n = 25) from healthy volunteers (n = 25) with high sensitivity and specificity (area under the receiver operating characteristic of 0.98). Furthermore, we demonstrate that Fgβ-derived Bβ(15-42) peptide administration protects mice from I/R-induced kidney injury by aiding in epithelial cell proliferation and tissue repair. Given that kidney regeneration is a major determinant of outcome for patients with kidney damage, these results provide new opportunities for the use of Fg in diagnosis, prevention, and therapeutic interventions in kidney disease.     

Programmed death-1 expression in liver transplant recipients as a prognostic indicator of cytomegalovirus disease

Immunological parameters that distinguish solid-organ transplant (SOT) recipients at risk for life-threatening cytomegalovirus (CMV) disease are being actively pursued to aid posttransplant management. A candidate marker is programmed death (PD)-1 receptor, whose overexpression has been associated with disease progression during persistent viral infections. To determine whether levels of this negative regulator of T cell activity are altered in SOT recipients with symptoms of CMV disease, a comparative PD-1 expression analysis was done in healthy, CMV-positive individuals and in liver transplant recipients. PD-1 levels were measured among the total population of CD8(+) and CD8(+) T cells binding to CMV-specific major histocompatibility complex class I tetramers. Minimal PD-1 expression was found in the healthy, CMV-positive cohort, and symptomatic SOT recipients had significantly higher PD-1 levels. PD-1 up-regulation was significantly associated with incipient and overt CMV disease and with viremia. Our findings suggest that PD-1 could be developed as a prognostic tool to predict CMV disease and guide therapeutic interventions.     

Lifestyle Modification for Metabolic Syndrome: A Systematic Review

Background

All 5 components of metabolic syndrome have been shown to improve with lifestyle and diet modification. New strategies for achieving adherence to meaningful lifestyle change are needed to optimize atherosclerotic cardiovascular risk reduction. We performed a systematic literature review, based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses framework (PRISMA), investigating optimal methods for achieving lifestyle change in metabolic syndrome.

Methods

We submitted standardized search terms to the PubMed Central, CINAHL, Web of Science, and Ovid databases. Within those results, we selected randomized controlled trials (RCTs) presenting unique methods of achieving lifestyle change in patients with one or more components of the metabolic syndrome. Data extraction using the population, intervention, comparator, outcome, and risk of bias framework (PICO) was used to compare the following endpoints: prevalence of metabolic syndrome, prevalence of individual metabolic syndrome components, mean number of metabolic syndrome components, and amount of weight loss achieved.

Results

Twenty-eight RCTs (6372 patients) were included. Eight RCTs demonstrated improvement in metabolic syndrome risk factors after 1 year. Team-based, interactive approaches with high-frequency contact with patients who are motivated made the largest and most lasting impact. Technology was found to be a useful tool in achieving lifestyle change, but ineffective when compared with personal contact.

Conclusion

Patient motivation leading to improved lifestyle adherence is a key factor in achieving reduction in metabolic syndrome components. These elements can be enhanced via frequent encounters with the health care system. Use of technologies such as mobile and Internet-based communication can increase the effectiveness of lifestyle change in metabolic syndrome, but should not replace personal contact as the cornerstone of therapy. Our ability to derive quantitative conclusions is limited by inconsistent outcome measures across studies, low power and homogeneity of individual studies, largely motivated study populations, short follow-up periods, loss to follow-up, and lack of or incomplete blinding.     

Mechanisms underlying the augmentation of phenylbiguanide and capsaicin induced cardiorespiratory reflexes by Mesobuthus tamulus venom

Phenylbiguanide (PBG) and capsaicin evoke cardiorespiratory reflexes utilizing two separate pathways. It is known that Indian Red Scorpion (Mesobuthus tumulus; MBT) venom augments PBG (5-HT₃) responses but, the effect of MBT venom on capsaicin (TRPV1)-induced response is not known. Therefore, the present study was undertaken to ascertain whether MBT venom also augments the capsaicin-induced reflex responses involving mechanisms similar to PBG. Experiments were performed on anaesthetized adult rats. Blood pressure, respiratory excursions and ECG were recorded. At the end of each experiment pulmonary water content was determined. PBG (10 μg/kg) produced hypotension, bradycardia and apnoea-bradypnoea. Capsaicin (10 μg/kg) also produced hypotension, bradycardia and apnoea-bradypnoea. MBT venom (100 μg/kg) augmented PBG as well as capsaicin-induced responses and produced pulmonary oedema (increased pulmonary water content). Prostaglandin synthase inhibitor (indomethacin; 10 mg/kg) blocked the venom-induced augmentation of PBG and capsaicin reflexes. Kinin synthase inhibitor (aprotinin; 6000 KIU) and guanylate cyclase (GC) inhibitor (methylene blue; 5 mg/kg) blocked the venom-induced augmentation of PBG response but not the capsaicin response. However, pulmonary oedema was blocked by these antagonists. Phosphodiesterase V inhibitor (sildenafil; 100 μg/kg) augmented the PBG response but not the capsaicin response, though pulmonary oedema was seen in both the groups. The present results indicate that MBT venom also augments the capsaicin-induced responses. The augmentation of capsaicin response involves PGs and pulmonary oedema-independent mechanisms whereas, the augmentation of PBG response involves kinin mediated GC-cGMP pathway and pulmonary oedema-dependent mechanisms.     

Contraceptive Considerations for Women with Gastrointestinal Disorders

Gastroenterologists are in a unique position to assist women with chronic gastrointestinal disorders in order to optimize their health prior to pregnancy. Women, whether with chronic conditions or not, and their infants are more likely to be healthy when pregnancies are planned. Achieving a planned pregnancy at the ideal time or preventing pregnancy altogether requires the use of appropriate contraceptives. There is a broad range of contraceptives available to women in the USA, and the majority of women with digestive diseases will be candidates for all effective methods. Guidance from the Centers for Disease Control and Prevention aids clinicians in prescribing appropriate contraceptives to women with medical disorders. This review will focus on contraception for women with inflammatory bowel disease and chronic liver disease, including liver transplant.     

An insight on the neuropharmacological activity of Telescopium telescopium– a mollusc from the Sunderban mangrove

The present study was carried out to evaluate the biological properties of the tissue extract of a marine snail Telescopium telescopium, collected from the coastal regions of West Bengal India. On extensive pharmacological screening, it was found that the biological extract of T. telescopium (TTE) produced significant central nervous system (CNS)‐depressant activity as observed from the reduced spontaneous motility, potentiation of pentobarbitone induced sleeping time, hypothermia and respiratory depression with transient apnoea. The extract significantly decreased both residual curiosity and also muscle coordination. The fraction, obtained following saturation with 60–80% ammonium sulphate (80S), was also found to demonstrate predominant CNS‐depressant activity. It was observed that both TTE and the 80S fraction significantly altered the brain noradrenaline and homovanillic acid levels without affecting the brain gamma amino butyric acid (GABA) concentration. Based on the present observations, it can be suggested that the CNS‐depressant effects produced by TTE and 80S could be attributable to modified catecholamine metabolism in the brain.     

Haplotype Counting by Next-Generation Sequencing for Ultrasensitive Human DNA Detection

Human identity testing is critical to the fields of forensics, paternity, and hematopoietic stem cell transplantation. Most bone marrow (BM) engraftment testing currently uses microsatellites or short tandem repeats that are resolved by capillary electrophoresis. Single-nucleotide polymorphisms (SNPs) are theoretically a better choice among polymorphic DNA; however, ultrasensitive detection of SNPs using next-generation sequencing is currently not possible because of its inherently high error rate. We circumvent this problem by analyzing blocks of closely spaced SNPs, or haplotypes. As proof-of-principle, we chose the HLA-A locus because it is highly polymorphic and is already genotyped to select proper donors for BM transplant recipients. We aligned common HLA-A alleles and identified a region containing 18 closely spaced SNPs, flanked by nonpolymorphic DNA for primer placement. Analysis of cell line mixtures shows that the assay is accurate and precise, and has a lower limit of detection of approximately 0.01%. The BM from a series of hematopoietic stem cell transplantation patients who tested as all donor by short tandem repeat analysis demonstrated 0% to 1.5% patient DNA. Comprehensive analysis of the human genome using the 1000 Genomes database identified many additional loci that could be used for this purpose. This assay may prove useful to identify hematopoietic stem cell transplantation patients destined to relapse, microchimerism associated with solid organ transplantation, forensic applications, and possibly patient identification.CME Accreditation Statement: This activity (“JMD 2014 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“JMD 2014 CME Program in Molecular Diagnostics”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.Myeloablative conditioning and allogeneic stem cell transplantation have historically been limited to the treatment of lethal hematological malignancies in children or young adults. More recently, with the advent of highly immunosuppressive, nonmyeloablative regimens, the clinical use of allogeneic stem cell transplantation has expanded to include older, less fit patients with hematological malignancies and patients with nonmalignant disorders, such as sickle-cell disease.^{1, 2, 3, 4} Nonmyeloablative conditioning regimens offer the additional safeguard of recovery of autologous hematopoiesis in the event of graft rejection and may be a safer option in patients at risk for immune-mediated rejection of the donor graft.Chimerism testing at set intervals is an effective method for detecting graft rejection or recurrence of the original hematopoietic neoplasm after allogeneic hematopoietic stem cell transplantation (HSCT) [with either bone marrow (BM) or peripheral blood stem cells]. Decades ago, BM engraftment (BME) monitoring was performed using Southern blot analysis and minisatellite or variable number of tandem repeats loci.⁵ Today, short tandem repeat (STR) or microsatellite loci are most commonly used for this purpose.^{6, 7, 8} STRs are composed of 10 to 60 tandemly repeated units, in which each unit is 1 to 6 bases in length. They are widely distributed throughout the human genome and highly variable between individuals; therefore, they allow for excellent differentiation between individuals, including patient and donor, even if they are closely related. Most laboratories use multiplex PCR-based kits, originally developed for forensics analysis using Combined DNA Index System loci.^{7, 9, 10} STR analysis most commonly involves PCR amplification using fluorescently labeled primers, followed by amplicon separation by capillary electrophoresis.Other polymorphic DNAs that could be used to monitor BME include single-nucleotide polymorphisms (SNPs).^{11, 12, 13} SNPs are theoretically superior to STR-based analyses because analysis of STR loci by capillary electrophoresis is relatively insensitive [limit of detection (LD), 1% to 5%] and microsatellite alleles of varying length amplify with different efficiencies, thus making them inherently biased. STR amplification can also be difficult in the setting of highly degraded DNA. However, SNPs are less attractive as targets because of their inherently lower informativity (eg, only two possible bases for a bi-allelic SNP versus ≥10 alleles for some microsatellites), requiring many more SNPs to be tested to identify those that distinguish donor from recipient. For example, we previously estimated that one would need to screen >20 to 30 individual SNPs to confidently identify one SNP where the donor is homozygous for one allele and an unrelated recipient is homozygous for the other allele.¹¹ Fewer would need to be included if heterozygotes were included, but more would have to be analyzed for related individuals.Recently emerging next-generation sequencing (NGS) technologies, along with their decreasing costs, are now feasible for clinical testing. However, all NGS technologies currently have high error rates, in the range of 0.04% to 1% at each base,¹⁴ which precludes their use for ultrasensitive detection of one SNP. One solution to this problem is sequencing blocks of closely spaced SNPs (ie, haplotypes). Haplotypes are regions of the genome, where polymorphic areas are sufficiently close that they are inherited together, including either genes (eg, HLA-A and HLA-B) within a locus or multiple SNPs within a region of DNA.Herein, we first used the HLA-A locus as proof-of-principle to demonstrate that this approach permits high sensitivity, precision, and accuracy. We then studied BM samples from a cohort of patients who engrafted after HSCT and tested as all donors by STRs, and found that low-level patient DNA is commonly present. To identify additional loci that could be used for this purpose, we comprehensively analyzed the human genome and identified other regions with highly informative haplotypes. We discuss additional situations where routine haplotyping patient samples could improve patient safety.     

False Positives in Multiplex PCR-Based Next-Generation Sequencing Have Unique Signatures

Next-generation sequencing shows great promise by allowing rapid mutational analysis of multiple genes in human cancers. Recently, we implemented the multiplex PCR-based Ion AmpliSeq Cancer Hotspot Panel (>200 amplicons in 50 genes) to evaluate EGFR, KRAS, and BRAF in lung and colorectal adenocarcinomas. In 10% of samples, automated analysis identified a novel G873R substitution mutation in EGFR. By examining reads individually, we found this mutation in >5% of reads in 50 of 291 samples and also found similar events in 18 additional amplicons. These apparent mutations are present only in short reads and within 10 bases of either end of the read. We therefore hypothesized that these were from panel primers promiscuously binding to nearly complementary sequences of nontargeted amplicons. Sequences around the mutations matched primer binding sites in the panel in 18 of 19 cases, thus likely corresponding to panel primers. Furthermore, because most primers did not show this effect, we demonstrated that next-generation sequencing may be used to better design multiplex PCR primers through iterative elimination of offending primers to minimize mispriming. Our results indicate the need for careful sequence analysis to avoid false-positive mutations that can arise in multiplex PCR panels. The AmpliSeq Cancer panel is a valuable tool for clinical diagnostics, provided awareness of potential artifacts.Detecting driver mutations in cancer genomes is of increasing importance for patient care, both for prognostic significance and for allowing better utilization of targeted therapies. Determining the mutational status of specific genes, such as KRAS, BRAF, and EGFR in lung adenocarcinoma and KRAS and BRAF in colorectal adenocarcinoma, has become the standard of care in clinical oncology to direct epidermal growth factor receptor (EGFR) inhibitor therapy.^1,2 To achieve this, targeted sequencing of these genes with the use of Sanger sequencing and pyrosequencing is widely available. However, Sanger sequencing is labor intensive and has a relatively poor analytic sensitivity (approximately 20% mutant alleles), requiring specimens with a significant percentage of tumor nuclei (>40%) to detect heterozygous mutations.^3,4 Pyrosequencing, although less labor intensive with a better limit of detection (analytic sensitivity approximately 5%), is typically limited to short regions of DNA, requiring the clustering of mutations (eg, KRAS codons 12 and 13). With each of these approaches, a single amplicon from a single patient is analyzed in a single well or capillary.Massively parallel, next-generation sequencing (NGS) platforms, such as the Ion Torrent Personal Genome Machine (PGM) and the Illumina MiSeq, provide limits of detection superior to pyrosequencing combined with even broader genomic coverage.^5–7 Although NGS platforms have the capability to perform cancer whole genome/exome sequencing, targeted sequencing of panels of amplicons with actionable and hotspot mutations is currently more practical in a clinical laboratory setting.^8–11 One panel, the Ion Torrent AmpliSeq Cancer Hotspot Panel, uses multiplex-PCR to cover >200 amplicons in 50 genes known to be involved in carcinogenesis. We recently transitioned to this platform to determine KRAS, BRAF, and EGFR mutation status in formalin-fixed, paraffin-embedded (FFPE) lung and colon cancer specimens.A major issue with FFPE specimens is significant variability in both the quality and quantity of DNA that can be isolated. Sources of this variability include the amount of tumor in the biopsy, time from biopsy/resection to fixation, and time in formalin before processing.^12,13 As a result, we often are left with relatively low DNA concentrations, which may require use of less DNA than the recommended 10- to 30-ng amount for the AmpliSeq panel.Here, we report that off-target amplification is common in multiplex-PCR–based NGS, yielding 19 mispriming events in 208 amplicons (9%) in our study. We define the signature features to identify mispriming events and show that false-positive mutations can be avoided by using multiple bioinformatic analysis tools in the pipeline. We also show that these events are more common with lower input DNA amounts. We demonstrate that the phenomenon is due to multiplex PCR and is not seen when primers are used in monoplex reactions. Finally, because the vast majority of primers do not show significant mispriming, we hypothesize that NGS may be the ultimate multiplex PCR primer design tool by allowing for sensitive detection of off-target amplification and consequent iterative primer design.