Management of HBV/HIV-coinfected Patients

Coinfection with hepatitis B virus (HBV) and human immunodeficiency virus (HIV) is common as a result of shared routes of transmission, especially in high-risk groups such as injection drug users and persons with hemophilia. HIV is known to influence the natural history of HBV, hastening progression to end-stage liver disease and cirrhosis. Antiretroviral therapy for HIV, and associated immune reconstitution, may result in immune-mediated liver damage as HBV-infected hepatocytes are targeted. This can lead to liver enzyme elevations that may be misattributed to drug-related toxicity. Thus, it is important that HIV-infected patients be tested for HBV and that clinicians be aware of the possibility of atypical serologic markers of HBV in HIV-infected patients. In managing coinfected patients, control of HIV is the priority. In patients with controlled HIV who are candidates for HBV therapy, the goals are the same as in the HBV-monoinfected population: hepatitis B e antigen seroconversion, liver enzyme normalization, and HBV DNA suppression. Treatment options include interferon-based regimens, lamivudine, adefovir, tenofovir, and entecavir. All of these agents have been shown to be relatively effective in HBV-monoinfected patients. However, few randomized, controlled HBV treatment trials have been conducted in coinfected subjects, and thus additional studies are warranted.     

Association of granulocyte-macrophage colony-stimulating factor with the crystalloid granules of human eosinophils

We have previously shown that normal-density human peripheral blood eosinophils transcribe and translate mRNA for granulocyte-macrophage colony-stimulating factor (GM-CSF) and that the intracellular distribution was granular as assessed by light microscopy immunocytochemistry. The present study was conducted to confirm this apparent association between GM-CSF and the crystalloid granule using a subcellular fractionation method for human eosinophils and immunogold electron microscopy (EM). Highly purified (> 99%, by negative selection using anti-CD16 immunomagnetic microbeads) human peripheral blood eosinophils were obtained from four asthmatic subjects (not taking systemic medication), homogenized and density fractionated (5 x 10(7) cells/subject) on linear Nycodenz gradients. Twenty-four fractions were collected from each cell preparation and analyzed for marker enzyme activities as well as total protein. Dot blot analysis with specific monoclonal antibodies (MoAbs) was used to detect the eosinophil granule proteins major basic protein (MBP) and eosinophil cationic protein (ECP). An anti-CD9 MoAb was used as an eosinophil plasma membrane marker. Lactate dehydrogenase (LDH) was used as a cytosolic marker. Immunoreactivity for GM-CSF was detected by a specific enzyme-linked immunosorbent assay using a polyclonal antihuman GM-CSF antibody and confirmed by dot blot. GM-CSF coeluted with the cellular fractions containing granule markers (MBP, ECP, eosinophil peroxidase, hexosaminidase, and arylsulphatase), but not those containing cytoplasm (LDH+) or membrane (CD9+) markers. EM examination of pooled fractions associated with the peak of GM-CSF immunoreactivity confirmed that they contained crystalloid and small granules, but not plasma membrane. In addition, quantification, using immunogold labeling with an anti/GM-CSF MoAb, indicated preferential localization of gold particles over the eosinophil granule cores of intact cells. Thus, our results indicate that GM-CSF resides as a granule-associated, stored mediator in unstimulated human eosinophils.     

An unusual case of vaccine-associated paralytic poliomyelitis

The present article reports a case involving an immunocompetent, previously well child who, despite two previous doses of inactivated poliovirus vaccine, developed severe flaccid paralysis consistent with polio after receiving oral polio vaccine.     

Neuropeptide Y Attenuates Stress‐Induced Bone Loss Through Suppression of Noradrenaline Circuits

Chronic stress and depression have adverse consequences on many organ systems, including the skeleton, but the mechanisms underlying stress‐induced bone loss remain unclear. Here we demonstrate that neuropeptide Y (NPY), centrally and peripherally, plays a critical role in protecting against stress‐induced bone loss. Mice lacking the anxiolytic factor NPY exhibit more anxious behavior and elevated corticosterone levels. Additionally, following a 6‐week restraint, or cold‐stress protocol, Npy‐null mice exhibit three‐fold greater bone loss compared to wild‐type mice, owing to suppression of osteoblast activity. This stress‐protective NPY pathway acts specifically through Y2 receptors. Centrally, Y2 receptors suppress corticotropin‐releasing factor expression and inhibit activation of noradrenergic neurons in the paraventricular nucleus. In the periphery, they act to control noradrenaline release from sympathetic neurons. Specific deletion of arcuate Y2 receptors recapitulates the Npy‐null stress response, coincident with elevated serum noradrenaline. Importantly, specific reintroduction of NPY solely in noradrenergic neurons of otherwise Npy‐null mice blocks the increase in circulating noradrenaline and the stress‐induced bone loss. Thus, NPY protects against excessive stress‐induced bone loss, through Y2 receptor‐mediated modulation of central and peripheral noradrenergic neurons. © 2014 American Society for Bone and Mineral Research.     

The use of 7-amino actinomycin D in identifying apoptosis: simplicity of use and broad spectrum of application compared with other techniques

The detection and quantitation of apoptotic cells is becoming increasingly important in the investigation of the role of apoptosis in cellular proliferation and differentiation. The pathogenesis of hematologic disorders such as aplastic anemia and the development of neoplasia are believed to involve dysregulation of apoptosis. To quantitate accurately the proportion of apoptosis cells within different cell types of a heterogeneous cell population such as blood or bone marrow, a method is required that combines the analysis of large numbers of cells with concurrent immunophenotyping of cell surface antigens. In this study, we have evaluated such a method using the fluorescent DNA binding agent, 7-amino actinomycin D (7AAD), to stain three diverse human cell lines, induced to undergo apoptosis by three different stimuli. Flow cytometric analysis defines three populations on the basis of 7AAD fluorescence and forward light scatter. We have shown by cell sorting and subsequent morphological assessment and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling that the populations defined by 7AAD represent live, apoptotic, and late-apoptotic/dead cells. This method is quick, simple, reproducible, and cheap and will be a valuable tool in the investigation of the role of apoptosis in normal physiology and in disease states.     

Highly methylated genes in colorectal neoplasia: implications for screening.

Discriminant markers are required for accurate cancer screening. We evaluated genes frequently methylated in colorectal neoplasia to identify the most discriminant ones. Four genes specifically methylated in colorectal cancer [bone morphogenetic protein 3 (BMP3), EYA2, aristaless-like homeobox-4 (ALX4), and vimentin] were selected from 41 candidate genes and evaluated on 74 cancers, 62 adenomas, and 70 normal epithelia. Methylation status was analyzed qualitatively and quantitatively and confirmed by bisulfite genomic sequencing. Effect of methylation on gene expression was evaluated in five colon cancer cell lines. K-ras and BRAF mutations were detected by sequencing. Methylation of BMP3, EYA2, ALX4, or vimentin was detected respectively in 66%, 66%, 68%, and 72% of cancers; 74%, 48%, 89%, and 84% of adenomas; and 7%, 5%, 11%, and 11% of normal epithelia (P < 0.01, cancer or adenoma versus normal). Based on area under the curve analyses, discrimination was not significantly improved by combining markers. Comethylation was frequent (two genes or more in 72% of cancers and 84% of adenomas), associated with proximal neoplasm site (P < 0.001), and linked with both BRAF and K-ras mutations (P < 0.01). Cell line experiments supported silencing of expression by methylation in all four study genes. This study shows BMP3, EYA2, ALX4, and vimentin genes are methylated in most colorectal neoplasms but rarely in normal epithelia. Comethylation of these genes is common, and pursuit of complementary markers for methylation-negative neoplasms is a rational strategy to optimize screening sensitivity.