Single nucleotide polymorphic discrimination by an electronic dot blot assay on semiconductor microchips.

We have developed a rapid assay for single nucleotide polymorphism (SNP) detection that utilizes electronic circuitry on silicon microchips. The method was validated by the accurate discrimination of blinded DNA samples for the complex quadra-allelic SNP of mannose binding protein. The microchip directed the transport, concentration, and attachment of amplified patient DNA to selected electrodes (test sites) creating an array of DNA samples. Through control of the electric field, the microchip enabled accurate genetic identification of these samples using fluorescently labeled DNA reporter probes. The accuracy of this approach was established by internal controls of dual labeled reporters and by using mismatched sequences in addition to the wild-type and variant reporter sequences to validate the SNP-genotype. The ability to customize this assay for multiple genes has advantages over other existing approaches.

Experimental characterization of the effects of acute stresslike doses of hydrocortisone in human neurogenic hyperalgesia models.

Relative hypothalamic-pituitary-adrenal axis dysfunction has been described as a common feature of several dysfunctional pain syndromes, and its end hormone cortisol may thus constitute a protective factor against the development of chronic pain. We investigated the potential influence of experimentally induced stress-like hypercortisolism on the induction of neurogenic hyperalgesia using 2 human surrogate models: secondary hyperalgesia after intradermal capsaicin injection into the volar forearm, and perceptual windup in normal skin. In a double-blind, placebo-controlled, randomized, crossover study, a psychophysical study was performed in 10 healthy subjects (median age 23 years) examining the effects of 40 mg orally administered hydrocortisone. Numeric pain ratings were assessed for punctate pinprick and light touch stimuli applied to the zone of secondary hyperalgesia adjacent to the capsaicin injection and to the contralateral control side. In addition, visual analog ratings were assessed for repetitive pinprick stimulation of the noninjected arm. Hydrocortisone significantly attenuated the late phase of capsaicin-induced pain by nearly 50%, and hyperalgesia to pinprick stimuli by 33% (both P<.05). Baseline mechanical pain and dynamic mechanical allodynia remained unaltered. Temporal summation (windup) to mechanical pain stimuli and electrically induced windup of second pain (tested in an independent cohort of 10 other subjects) were also unchanged. The selective effects of hydrocortisone on pinprick hyperalgesia but not pinprick pain suggest an antihyperalgesic rather than analgesic effect. The findings suggest that hypothalamic-pituitary-adrenal axis reactivity might be an important mechanism in resilience to dysfunctional pain syndromes.

Structure and expression of the woodchuck herpesvirus genome.

This report describes the genome structure and location from which immediate-early transcription originates in the recently characterized woodchuck herpesvirus (herpesvirus marmota: HVM). Cross-hybridization of restriction fragments indicates that the HVM genome contains a tandem array of 1.5-kb repeat units. Additionally, terminal labeling and exonuclease experiments demonstrate that the repeated sequences lie at the termini of the genome. Hybridization of probes representing immediate-early transcription indicates that only a single predominant species of immediate-early RNA originates from a region near one end of unique sequences in the HVM genome. These results show remarkable similarity with group 2 of the gammaherpesvirinae. However, no homology was detected by conventional Southern blot hybridization between HVM and the gamma-2 prototype, herpesvirus saimiri. Therefore, we propose HVM to be a new member of the gammaherpesvirinae subfamily of herpesviruses.

Replication of macrophage-tropic and T-cell-tropic strains of human immunodeficiency virus type 1 is augmented by macrophage-endothelial cell contact.

Macrophages perform a central role in the pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection and have been implicated as the cell type most prominent in the development of central nervous system impairment. In this study, we evaluated the effect of interaction between macrophages and endothelial cells on HIV-1 replication. Upregulation of HIV-1 replication was consistently observed in monocyte-derived macrophages (hereafter called macrophages) cocultured with either umbilical vein endothelial cells or brain microvascular endothelial cells. HIV-1 p24 antigen production of laboratory-adapted strains and patient-derived isolates was increased 2- to 1,000-fold in macrophage-endothelial cocultures, with little or no detectable replication in cultures containing endothelial cells only. The upregulation of HIV-1 in macrophage-endothelial cocultures was observed not only for viruses with the non-syncytium-inducing, macrophage-tropic phenotype but also for viruses previously characterized as syncytium inducing and T-cell tropic. In contrast, cocultures of macrophages with glioblastoma, astrocytoma, cortical neuronal, fibroblast, and placental cells failed to increase HIV-1 replication. Enhancement of HIV-1 replication in macrophage-endothelial cocultures required cell-to-cell contact; conditioned media from endothelial cells or macrophage-endothelial cocultures failed to augment HIV-1 replication in macrophages. Additionally, antibody to leukocyte function-associated antigen (LFA-1), a macrophage-endothelial cell adhesion molecule, inhibited the enhanced HIV-1 replication in macrophage-endothelial cell cocultures. Thus, these data indicate that macrophage-endothelial cell contact enhances HIV-1 replication in macrophages for both macrophage-tropic and previously characterized T-cell-tropic strains and that antibody against LFA-1 can block the necessary cell-to-cell interaction required for the observed upregulation. These findings may have important implications for understanding the ability of HIV-1 to replicate efficiently in tissue macrophages, including those in the brain and at the blood-brain barrier.

Instability of v-src sequences in nonhuman primate tumors cultured in vitro.

We have analyzed the DNA of marmoset tumors induced and marmoset cells transformed by Rous sarcoma virus (RSV) and derivative viruses of various types. Southern blot hybridization was used to determine the presence of v-src gene sequences. We failed to detect v-src DNA in high-passage cells derived from marmoset tumors induced in vivo or from marmoset cell lines transformed in vitro. The inability to detect src sequences was not related to selection of revertants in culture, since all cell lines retained transformed morphology and cells transformed in vitro retained the ability to induce sarcomas after transplantation into adult allogeneic marmosets. By contrast, we detected integrated proviruses in cells analyzed 32 to 60 days after in vitro transformation. The proviral sequences appeared to be identical to the transforming virus but were apparently unstable and continued to transpose.

Tumor necrosis factor alpha negatively regulates hepatitis B virus gene expression in transgenic mice.

It is well known that several inflammatory cytokines can modulate hepatocellular gene expression in a complex physiological process known as the hepatic acute-phase response. Since hepatitis B virus (HBV) characteristically induces a vigorous lymphomononuclear inflammatory response in the liver during acute and chronic hepatitis, it is possible that hepatocellular HBV gene expression may also be modulated by one or more of the cytokines produced by these cells. Using bacterial lipopolysaccharide (LPS) as a surrogate inducer of inflammatory cytokines in vivo, we have tested this hypothesis in a transgenic mouse model system. In experiments with two independent transgenic mouse lineages that express the HBV envelope region under the control of either HBV or cellular promoters, we observed a 50 to 80% reduction in the hepatic steady-state content of a 2.1-kb HBV mRNA following administration of a single intraperitoneal dose of LPS. The regulatory influence of several inflammatory cytokines known to be induced by LPS was also examined in this system. The negative regulatory effect of LPS was consistently reproduced by the administration of a single nontoxic dose of tumor necrosis factor alpha, and it was occasionally observed following the administration of high doses of alpha interferon and interleukin-6, while no effect was detectable in response to high-dose interleukin-1 alpha or to gamma interferon. These observations suggest that tumor necrosis factor alpha and perhaps other cytokines may activate a heretofore unsuspected intracellular pathway that negatively regulates HBV gene expression. The intracellular mechanism(s) responsible for this effect and its pathophysiologic relevance remain to be elucidated.

HBsAg retention sensitizes the hepatocyte to injury by physiological concentrations of interferon-gamma.

The role that inflammatory cytokines may play in the life cycle of the hepatitis B virus and in the pathogenesis of its associated liver disease has not been carefully delineated. In this report, we demonstrate that bacterial lipopolysaccharide, a potent inducer of inflammatory cytokines in vivo, causes a severe acute liver disease in transgenic mice whose hepatocytes produce the hepatitis B virus large envelope polypeptide and retain HBsAg within the endoplasmic reticulum. In contrast, 100-fold higher doses of bacterial lipopolysaccharide do not induce liver cell injury in nontransgenic littermate controls or in transgenic mice whose hepatocytes secrete HBsAg rather than retain it. Coincident with the hepatocellular injury and the influx of inflammatory cells into the liver, a marked reduction occurs in the intrahepatic content of hepatitis B virus steady-state messenger RNA, thereby confirming the selectivity of this process for the HBsAg-positive hepatocyte. Bacterial lipopolysaccharide-induced hepatocellular injury appears to be principally mediated by interferon-gamma because it can be markedly reduced by the prior administration of neutralizing interferon-gamma-specific monoclonal antibodies and because recombinant interferon-gamma is also selectively cytotoxic for the HBsAg-positive transgenic hepatocyte in vivo. Tumor necrosis factor-alpha is also involved in this process because bacterial lipopolysaccharide-induced liver cell injury is significantly reduced by tumor necrosis factor-alpha specific monoclonal antibodies. The role of tumor necrosis factor-alpha in bacterial lipopolysaccharide-induced liver cell injury is less clear than interferon-gamma, however, because unlike interferon-gamma it is also toxic for nontransgenic hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)