Tulane Medical Center

DNA hypomethylation was the initial epigenetic abnormality recognized in human tumors. However, for several decades after its independent discovery by two laboratories in 1983, it was often ignored as an unwelcome complication, with almost all of the attention on the hypermethylation of promoters of genes that are silenced in cancers (e.g., tumor-suppressor genes). Because it was subsequently shown that global hypomethylation of DNA in cancer was most closely associated with repeated DNA elements, cancer linked-DNA hypomethylation continued to receive rather little attention. DNA hypomethylation in cancer can no longer be considered an oddity, because recent high-resolution genome-wide studies confirm that DNA hypomethylation is the almost constant companion to hypermethylation of the genome in cancer, just usually (but not always) in different sequences. Methylation changes at individual CpG dyads in cancer can have a high degree of dependence not only on the regional context, but also on neighboring sites. DNA demethylation during carcinogenesis may involve hemimethylated dyads as intermediates, followed by spreading of the loss of methylation on both strands. In this review, active demethylation of DNA and the relationship of cancer-associated DNA hypomethylation to cancer stem cells are discussed. Evidence is accumulating for the biological significance and clinical relevance of DNA hypomethylation in cancer, and for cancer-linked demethylation and de novo methylation being highly dynamic processes.

Zebrafish (Danio rerio) are rapidly gaining popularity in translational neuroscience and behavioral research. Physiological similarity to mammals, ease of genetic manipulations, sensitivity to pharmacological and genetic factors, robust behavior, low cost, and potential for high-throughput screening contribute to the growing utility of zebrafish models in this field. Understanding zebrafish behavioral phenotypes provides important insights into neural pathways, physiological biomarkers, and genetic underpinnings of normal and pathological brain function. Novel zebrafish paradigms continue to appear with an encouraging pace, thus necessitating a consistent terminology and improved understanding of the behavioral repertoire. What can zebrafish 'do', and how does their altered brain function translate into behavioral actions? To help address these questions, we have developed a detailed catalog of zebrafish behaviors (Zebrafish Behavior Catalog, ZBC) that covers both larval and adult models. Representing a beginning of creating a more comprehensive ethogram of zebrafish behavior, this effort will improve interpretation of published findings, foster cross-species behavioral modeling, and encourage new groups to apply zebrafish neurobehavioral paradigms in their research. In addition, this glossary creates a framework for developing a zebrafish neurobehavioral ontology, ultimately to become part of a unified animal neurobehavioral ontology, which collectively will contribute to better integration of biological data within and across species.

Analysis of the total base composition of DNA from seven different normal human tissues and eight different types of homogeneous human cell populations revealed considerable tissue-specific and cell-specific differences in the extent of methylation of cytosine residues. The two most highly methylated DNAs were from thymus and brain with 1.00 and 0.98 mole percent 5-methylcytosine (m5C), respectively. The two least methylated DNAs from in vivo sources were placental DNA and sperm DNA, which had 0.76 and 0.84 mole percent m5C, respectively. The differences between these two groups of samples were significant with p less than 0.01. The m5C content of DNA from six human cell lines or strains ranged from 0.57 to 0.85 mole percent. The major and minor base composition of DNA fractionated by reassociation kinetics was also determined. The distribution of m5C among these fractions showed little or no variation with tissue or cell type with the possible exception of sperm DNA. In each case, nonrepetitive DNA sequences were hypomethylated compared to unfractionated DNA.

Rituximab improves outcomes for persons with lymphoproliferative disorders and is increasingly used to treat immune-mediated illnesses. Recent reports describe 2 patients with systemic lupus erythematosus and 1 with rheumatoid arthritis who developed progressive multifocal leukoencephalopathy (PML) after rituximab treatment. We reviewed PML case descriptions among patients treated with rituximab from the Food and Drug Administration, the manufacturer, physicians, and a literature review from 1997 to 2008. Overall, 52 patients with lymphoproliferative disorders, 2 patients with systemic lupus erythematosus, 1 patient with rheumatoid arthritis, 1 patient with an idiopathic autoimmune pancytopenia, and 1 patient with immune thrombocytopenia developed PML after treatment with rituximab and other agents. Other treatments included hematopoietic stem cell transplantation (7 patients), purine analogs (26 patients), or alkylating agents (39 patients). One patient with an autoimmune hemolytic anemia developed PML after treatment with corticosteroids and rituximab, and 1 patient with an autoimmune pancytopenia developed PML after treatment with corticosteroids, azathioprine, and rituximab. Median time from last rituximab dose to PML diagnosis was 5.5 months. Median time to death after PML diagnosis was 2.0 months. The case-fatality rate was 90%. Awareness is needed of the potential for PML among rituximab-treated persons.

The overall 5-methylcytosine (m5C) content of DNA from normal tissues varies considerably in a tissue-specific manner. By high-performance liquid chromatography, we have examined the (m5C) contents of enzymatic digests of DNA from 103 human tumors including benign, primary malignant and secondary malignant neoplasms. The diversity and large number of these tumor samples allowed us to compare the range of DNA methylation levels from neoplastic tissues to that of normal tissues from humans. Most of the metastatic neoplasms had significantly lower genomic (m5C) contents than did most of the benign neoplasms or normal tissues. The percentage of primary malignancies with hypomethylated DNA was intermediate between those of metastases and benign neoplasms. These findings might reflect an involvement of extensive demethylation of DNA in tumor progression. Such demethylation could be a source of the continually generated cellular diversity associated with cancer.

One mechanism by which blood-borne cytokines might affect the function of the central nervous system (CNS) is by crossing the blood-brain barrier (BBB) for direct interaction with CNS tissue. Saturable transport systems from blood to the CNS have been described for interleukin (IL)-1 alpha, IL-1 beta, IL-1 receptor antagonist (IL-1ra), IL-6, and tumor necrosis factor-alpha (TNF-alpha). Blood-borne cytokines have been shown to cross the BBB to enter cerebrospinal fluid and interstitial fluid spaces of the brain and spinal cord. IL-2 does not cross the BBB by a saturable transport system. The blood-to-brain uptakes of IL-1 alpha, IL-beta, and IL-1ra are interrelated for most brain sites, but the posterior division of the septum shows selective uptake of blood-borne IL-1 alpha. The saturable transport systems for IL-6 and TNF-alpha are distinguishable from each other and from the IL-1 systems. The amount of blood-borne cytokines entering the brain is modest but comparable to that of other water-soluble compounds, such as morphine, known to cross the BBB in sufficient amounts to affect brain function. CNS to blood efflux of cytokines has also been shown to occur, but the mechanism of passage is unclear. Taken together, the evidence shows that passage of cytokines across the BBB occurs, providing a route by which blood-borne cytokines could potentially affect brain function.

A small portion of the cytosine residues in the DNA of higher eukaryotes as well as in that of many lowe eukaryotes if methylated. The resulting 5-methylcytosine residues occur in specific in the DNA, usually adjacent to guanine residues on the 3' side. This methylation of eukaryotic DNA has been proposed to function in many ways, including control of transcription, maintenance of chromosome structure, repair of DNA, establishment of preferred sites for mutation, oncogenic transformation, and, in certain systems, protection of DNA against enzymatic degradation.

There has been an explosive growth of interest in the multiple interacting paracrine systems that influence renal microvascular function. This review first discusses the membrane activation mechanisms for renal vascular control. Evidence is provided that there are differential activating mechanisms regulating pre- and postglomerular arteriolar vascular smooth muscle cells. The next section deals with the critical role of the endothelium in the control of renal vascular function and covers the recent findings related to the role of nitric oxide and other endothelial-derived factors. This section is followed by an analysis of the roles of vasoactive paracrine systems that have their origin from adjoining tubular structures. The interplay of signals between the epithelial cells and the vascular network to provide feedback regulation of renal hemodynamics is developed. Because of their well-recognized contributions to the regulation of renal microvascular function, three major paracrine systems are discussed in separate sections. Recent findings related to the role of intrarenally formed angiotensin II and the prominence of the AT1 receptors are described. The possible contribution of purinergic compounds is then discussed. Recognition of the emerging role of extracellular ATP operating via P2 receptors as well as the more recognized functions of the P1 receptors provides fertile ground for further studies. In the next section, the family of vasoactive arachidonic acid metabolites is described. Possibilities for a myriad of interacting functions operating both directly on vascular smooth muscle cells and indirectly via influences on endothelial and epithelial cells are discussed. Particular attention is given to the more recent developments related to hemodynamic actions of the cytochrome P-450 metabolites. The final section discusses unique mechanisms that may be responsible for differential regulation of medullary blood flow by locally formed paracrine agents. Several sections provide perspectives on the complex interactions among the multiple mechanisms responsible for paracrine regulation of the renal microcirculation. This plurality of regulatory interactions highlights the need for experimental strategies that include integrative approaches that allow manifestation of indirect as well as direct influences of these paracrine systems on renal microvascular function.

Cystinosis, a rare autosomal recessive lysosomal storage disease, is due to impaired transport of cystine from lysosomes. The disease results in deposition of crystals throughout the body; if untreated, it leads to failure to thrive, profound metabolic imbalance, early end-stage renal disease, thyroid failure, and multiorgan dysfunction. As this review describes, substantial progress has been made in our understanding and treatment of this disorder. The administration of cysteamine, each molecule of which can combine with a half-molecule of cystine (cysteine) to facilitate the exit of cystine from the lysosome, has greatly improved the course of the disease. In addition, the gene for cystinosis, CTNS, which encodes a protein called cystinosin, was isolated in 1998, opening new avenues for understanding this condition.

His-DTrp-Ala-Trp-DPhe-LysNH2, [His1,Lys6] GHRP, is a new synthetic hexapeptide which specifically elicits a dosage-related release of GH in vitro and in vivo without a concomitant release of LH, FSH, TSH, or PRL and, in limited in vivo studies, insulin or glucagon. Our results indicate that this small peptide has the attributes of a hypophysiotropic hormone. In vitro the minimum and maximum active dosages ranged from 1-10 ng/ml in the pituitary incubate assay. It was active in rats, monkeys, lambs, calves, and under special experimental conditions chicks, indicating its lack of species dependency. It was active when administered iv, sc, or ip to rats. After iv injection, GH levels rose within 2 min, peaked at +10-20 min, and by 2 h usually had returned to normal. It was not possible to directly compare the potencies of [His1,Lys6]GHRP, and the GH-releasing factors GHRF-44 and GHRF-40 after a single sc injection in rats because the time course of the GH response of these peptides was different. The GH response of [His1,Lys6]GHRP was longer in duration than either of these larger peptides. Both SRIF-14 and SRIF-28 inhibited the GH response of the hexapeptide; however, SRIF-28 was about four times more active than SRIF-14 in vitro and 7.5 times more active in vivo. When this small peptide was administered sc once or twice daily to immature rats for 9 or 25 days, the BW gain increased above the control. At the end of the weight gain studies the pituitary remained fully responsive to the peptide. Thus, [His1,Lys6] GHRP may be a valuable peptide for investigating the function of the pituitary somatotrophs and, in addition, it has the potential for increasing BW gain of a variety of normal animals by inducing GH release via a direct pituitary site of action.

BACKGROUND: In advanced prostate cancer (APC), successful drug development as well as advances in imaging and molecular characterisation have resulted in multiple areas where there is lack of evidence or low level of evidence. The Advanced Prostate Cancer Consensus Conference (APCCC) 2017 addressed some of these topics. OBJECTIVE: To present the report of APCCC 2017. DESIGN, SETTING, AND PARTICIPANTS: Ten important areas of controversy in APC management were identified: high-risk localised and locally advanced prostate cancer; "oligometastatic" prostate cancer; castration-naïve and castration-resistant prostate cancer; the role of imaging in APC; osteoclast-targeted therapy; molecular characterisation of blood and tissue; genetic counselling/testing; side effects of systemic treatment(s); global access to prostate cancer drugs. A panel of 60 international prostate cancer experts developed the program and the consensus questions. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The panel voted publicly but anonymously on 150 predefined questions, which have been developed following a modified Delphi process. RESULTS AND LIMITATIONS: Voting is based on panellist opinion, and thus is not based on a standard literature review or meta-analysis. The outcomes of the voting had varying degrees of support, as reflected in the wording of this article, as well as in the detailed voting results recorded in Supplementary data. CONCLUSIONS: The presented expert voting results can be used for support in areas of management of men with APC where there is no high-level evidence, but individualised treatment decisions should as always be based on all of the data available, including disease extent and location, prior therapies regardless of type, host factors including comorbidities, as well as patient preferences, current and emerging evidence, and logistical and economic constraints. Inclusion of men with APC in clinical trials should be strongly encouraged. Importantly, APCCC 2017 again identified important areas in need of trials specifically designed to address them. PATIENT SUMMARY: The second Advanced Prostate Cancer Consensus Conference APCCC 2017 did provide a forum for discussion and debates on current treatment options for men with advanced prostate cancer. The aim of the conference is to bring the expertise of world experts to care givers around the world who see less patients with prostate cancer. The conference concluded with a discussion and voting of the expert panel on predefined consensus questions, targeting areas of primary clinical relevance. The results of these expert opinion votes are embedded in the clinical context of current treatment of men with advanced prostate cancer and provide a practical guide to clinicians to assist in the discussions with men with prostate cancer as part of a shared and multidisciplinary decision-making process.

The binding subunit of Escherichia coli heat-labile enterotoxin (LT-B) is a highly active oral immunogen. Transgenic tobacco and potato plants were made with the use of genes encoding LT-B or an LT-B fusion protein with a microsomal retention sequence. The plants expressed the foreign peptides, both of which formed oligomers that bound the natural ligand. Mice immunized by gavage produced serum and gut mucosal anti-LT-B immunoglobulins that neutralized the enterotoxin in cell protection assays. Feeding mice fresh transgenic potato tubers also caused oral immunization.

Are newer types of antihypertensive agents, which are currently more costly to purchase on average, as good or better than diuretics in reducing coronary heart disease incidence and progression? Will lowering LDL cholesterol in moderately hypercholesterolemic older individuals reduce the incidence of cardiovascular disease and total mortality? These important medical practice and public health questions are to be addressed by the Antihypertensive and Lipid Lowering Treatment to Prevent Heart Attack Trial (ALLHAT), a randomized, double-blind trial in 40,000 high-risk hypertensive patients. ALLHAT is designed to determine whether the combined incidence of fatal coronary heart disease (CHD) and nonfatal myocardial infarction differs between persons randomized to diuretic (chlorthalidone) treatment and each of three alternative treatments--a calcium antagonist (amlodipine), an angiotensin converting enzyme inhibitor (lisinopril), and an alpha-adrenergic blocker (doxazosin). ALLHAT also contains a randomized, open-label, lipid-lowering trial designed to determine whether lowering LDL cholesterol in 20,000 moderately hypercholesterolemic patients (a subset of the 40,000) with a 3-hydroxymethylglutaryl coenzyme A (HMG CoA) reductase inhibitor, pravastatin, will reduce all-cause mortality compared to a control group receiving "usual care." ALLHAT's main eligibility criteria are: 1) age 55 or older; 2) systolic or diastolic hypertension; and 3) one or more additional risk factors for heart attack (eg, evidence of atherosclerotic disease or type II diabetes). For the lipid-lowering trial, participants must have an LDL cholesterol of 120 to 189 mg/dL (100 to 129 mg/dL for those with known CHD) and a triglyceride level below 350 mg/dL. The mean duration of treatment and follow-up is planned to be 6 years. Further features of the rationale, design, objectives, treatment program, and study organization of ALLHAT are described in this article.

A sensitive and specific radioimmunoassay for somatostatin (SRIF) has been used to determine the regional distribution of SRIF in rat brain. The hypothalamus contained the highest concentration of SRIF. Lower, but significant amounts of SRIF were present outside of the hypothalalmus. Within the hypothalamus, the concentration of SRIF was highest in the median eminence and arcuate nucleus although all of the hypothalmic nuclei contained some fo this material. The implications of this distribution are discussed.

BACKGROUND: Erectile dysfunction in men is common. We evaluated a system by which alprostadil (prostaglandin E1) is delivered transurethrally to treat this disorder. METHODS: Alprostadil was delivered transurethrally in a double-blind, placebo-controlled study of 1511 men, 27 to 88 years of age, who had chronic erectile dysfunction from various organic causes. The men were first tested in the clinic with up to four doses of the drug (125, 250, 500, and 1000 microg); those who had sufficient responses were randomly assigned to treatment with either the effective dose of alprostadil or placebo for three months at home. RESULTS: During in-clinic testing, 996 men (65.9 percent) had erections sufficient for intercourse. Of these men, 961 reported the results of at least one home treatment; 299 of the 461 treated with alprostadil (64.9 percent) had intercourse successfully at least once, as compared with 93 of the 500 who received placebo (18.6 percent, P<0.001). On average, 7 of 10 alprostadil administrations were followed by intercourse in men responsive to treatment. The efficacy of alprostadil was similar regardless of age or the cause of erectile dysfunction, including vascular disease, diabetes, surgery, and trauma (P<0.001 for all comparisons with placebo). The most common side effect was mild penile pain, which occurred after 10.8 percent of alprostadil treatments, but the pain rarely resulted in refusal to continue in the study. Hypotension occurred in the clinic in 3.3 percent of men receiving alprostadil. Hypotension-related symptoms were uncommon at home. No men had priapism or penile fibrosis. CONCLUSIONS: In men with erectile dysfunction, transurethral alprostadil therapy resulted in erections in the clinic and in intercourse at home.

VITAMIN D is a seco-steroid in which the B-ring of the cyclopentanoperhydrophenanthrene structure is cleaved. The vitamin is either obtained from the diet or is synthesized in the skin from 7-dehydrocholesterol (Fig. 1) and is itself biologically inert (for reviews see Refs. 1–8). Vitamin D and its metabolites circulate in the blood primarily bound to and in equilibrium association with the vitamin D-binding globulin [DBP]. In the liver, the enzyme 25-hydroxylase converts the vitamin to the inactive hormonal precursor 25-hydroxyvitamin D [25(OH)D]. Subsequently, the renal lα-hydroxylase enzyme converts 25(OH)D to the biologically active form 1,25-dihydroxyvitamin D [1,25(OH)2D]. Both hydroxylase enzymes are cytochrome P450-containing enzymes. la-Hydroxylase activity is tightly controlled by a number of ionic and endocrine factors including stimulation by PTH and reduced plasma Ca2+ or inhibition by 1,25(OH)2D in a classic negative feedback loop. Although not illustrated in Fig. 1, under conditions of adequate plasma Ca2+ levels, 25(OH)D is alternatively converted to 24,25(OH)2D by the reciprocally regulated renal P450- dependent 24-hydroxylase enzyme. Whether this metabolite of vitamin D is biologically effective or whether its production simply initiates a catabolic pathway has been a topic of active debate (2, 4, 5, 9). The seco-steroid hormone 1,25(OH)2D exerts its principal biological activities through specific intracellular receptors (Fig. 1), which are nuclear transcription factors of the steroidthyroid receptor gene superfamily (7, 10), a relationship previously postulated on the basis of the biochemical similarities between all these receptor species (1, 11). As now considered likely for many of these ligand-dependent transcription factors, the 1,25(OH)2D receptor (VDR) (12, 13), seems to be predominately a nuclear protein (1), a fact that will be of importance in later discussions of its signal transduction mechanisms. The mechanism of action of the VDR is similar to that of the other steroid hormone receptors (Fig. 2), involving specific interactions of the receptor zinc-finger regions with specific nucleotide sequences (hormone response elements) usually in the 5′-regulatory region of the affected genes (1, 7, 10, 15). Similarly, 1,25(OH)2D regulation of cell function by altering gene transcription and mRNA and protein synthesis is similar to that of the other members of this gene family (1).

Interventions added to androgen-deprivation therapy in men with metastatic prostate cancer have recently been noted to increase survival. Decisions about which treatments to use and in what order to maximize the benefit should be made on the basis of high-quality clinical trials.

The vascular insulin-like growth factor (IGF)-1 system includes the IGFs, the IGF-1 receptor (IGF-1R), and multiple binding proteins. This growth factor system exerts multiple physiologic effects on the vasculature through both endocrine and autocrine/paracrine mechanisms. The effects of IGF-1 are mediated principally through the IGF-1R but are modulated by complex interactions with multiple IGF binding proteins that themselves are regulated by phosphorylation, proteolysis, polymerization, and cell or matrix association. During the last decade, a significant body of evidence has accumulated, indicating that expression of the components of the IGF system are regulated by multiple factors, including growth factors, cytokines, lipoproteins, reactive oxygen species, and hemodynamic forces. In addition, cross-talk between the IGF system and other growth factors and integrin receptors has been demonstrated. There is accumulating evidence of a role for IGF-1 in multiple vascular pathologies, including atherosclerosis, hypertension, restenosis, angiogenesis, and diabetic vascular disease. This review will discuss the regulation of expression of IGF-1, IGF-1R, and IGF binding proteins in the vasculature and summarize evidence implicating involvement of this system in vascular diseases.

BACKGROUND: The outcomes of kidney transplantation and immunosuppression in people infected with human immunodeficiency virus (HIV) are incompletely understood. METHODS: We undertook a prospective, nonrandomized trial of kidney transplantation in HIV-infected candidates who had CD4+ T-cell counts of at least 200 per cubic millimeter and undetectable plasma HIV type 1 (HIV-1) RNA levels while being treated with a stable antiretroviral regimen. Post-transplantation management was provided in accordance with study protocols that defined prophylaxis against opportunistic infection, indications for biopsy, and acceptable approaches to immunosuppression, management of rejection, and antiretroviral therapy. RESULTS: Between November 2003 and June 2009, a total of 150 patients underwent kidney transplantation; survivors were followed for a median period of 1.7 years. Patient survival rates (±SD) at 1 year and 3 years were 94.6±2.0% and 88.2±3.8%, respectively, and the corresponding mean graft-survival rates were 90.4% and 73.7%. In general, these rates fall somewhere between those reported in the national database for older kidney-transplant recipients (≥65 years) and those reported for all kidney-transplant recipients. A multivariate proportional-hazards analysis showed that the risk of graft loss was increased among patients treated for rejection (hazard ratio, 2.8; 95% confidence interval [CI], 1.2 to 6.6; P=0.02) and those receiving antithymocyte globulin induction therapy (hazard ratio, 2.5; 95% CI, 1.1 to 5.6; P=0.03); living-donor transplants were protective (hazard ratio, 0.2; 95% CI, 0.04 to 0.8; P=0.02). A higher-than-expected rejection rate was observed, with 1-year and 3-year estimates of 31% (95% CI, 24 to 40) and 41% (95% CI, 32 to 52), respectively. HIV infection remained well controlled, with stable CD4+ T-cell counts and few HIV-associated complications. CONCLUSIONS: In this cohort of carefully selected HIV-infected patients, both patient- and graft-survival rates were high at 1 and 3 years, with no increases in complications associated with HIV infection. The unexpectedly high rejection rates are of serious concern and indicate the need for better immunotherapy. (Funded by the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT00074386.).

The term monoclonal gammopathy of renal significance (MGRS) was introduced by the International Kidney and Monoclonal Gammopathy Research Group (IKMG) in 2012. The IKMG met in April 2017 to refine the definition of MGRS and to update the diagnostic criteria for MGRS-related diseases. Accordingly, in this Expert Consensus Document, the IKMG redefines MGRS as a clonal proliferative disorder that produces a nephrotoxic monoclonal immunoglobulin and does not meet previously defined haematological criteria for treatment of a specific malignancy. The diagnosis of MGRS-related disease is established by kidney biopsy and immunofluorescence studies to identify the monotypic immunoglobulin deposits (although these deposits are minimal in patients with either C3 glomerulopathy or thrombotic microangiopathy). Accordingly, the IKMG recommends a kidney biopsy in patients suspected of having MGRS to maximize the chance of correct diagnosis. Serum and urine protein electrophoresis and immunofixation, as well as analyses of serum free light chains, should also be performed to identify the monoclonal immunoglobulin, which helps to establish the diagnosis of MGRS and might also be useful for assessing responses to treatment. Finally, bone marrow aspiration and biopsy should be conducted to identify the lymphoproliferative clone. Flow cytometry can be helpful in identifying small clones. Additional genetic tests and fluorescent in situ hybridization studies are helpful for clonal identification and for generating treatment recommendations. Treatment of MGRS was not addressed at the 2017 IKMG meeting; consequently, this Expert Consensus Document does not include any recommendations for the treatment of patients with MGRS.