Casey D. Morrow

Altered microbiota associated with abnormal humoral immune responses to commensal organisms in enthesitis-related arthritis.

IntroductionPrior studies have established altered microbiota and immunologic reactivity to enteric commensal organisms in inflammatory bowel disease (IBD). Since intestinal inflammation is present in a subset of patients with both pediatric and adult spondyloarthritis (SpA), we hypothesized that SpA patients may also have altered microbiota and immune responsiveness to enteric organisms.MethodsStool and blood specimens were collected from children with enthesitis-related arthritis (ERA) and non-inflammatory controls. DNA purified from stool was subject to PCR amplification and sequencing of the variable IV region from the 16S rDNA gene. IgA and IgG Enzyme-linked Immunosorbent Assays (ELISAs) were performed on select species of bacteria in most subjects.ResultsTwenty-five children with ERA and 13 controls were included. The ERA patients had less Faecalibacterium prausnitzii (3.8% versus 10%, P = 0.008) and lachnospiraceae family (12 versus 7.0%, P = 0.020), a statistically significant increase in bifidobacterium (1.8% versus 0%, P = 0.032) and a non-statistically significant increase in Bacteroides (21% versus 11%, P = 0.150). Akkermansia muciniphila was abundant (>2%) in 7/27 ERA patients but none of the controls (P = 0.072.) Cluster analysis revealed two clusters of ERA patients: Cluster one (n = 8) was characterized by high levels of Bacteroides genus, while a second (n = 15) cluster had similar levels as the controls. Seven of 17 (41%) of the ERA subjects in Cluster 2 compared to 0/8 of the subjects in Cluster 1 had abundant Akkermansia muciniphila (P = 0.057). Serum IgA and IgG antibody levels against F. prausnitzii and B. fragilis were similar between patients and controls, whereas the two groups showed divergent responses when the fecal relative abundances of F. prausnitzii and Bacteroides were compared individually against IgA antibody levels recognizing F. prausnitzii and B. fragilis, respectively.ConclusionThe abundance of F. prausnitzii in the stool among patients with ERA is reduced compared to controls, and Bacteroides and A. muciniphila are identified as associative agents in subsets of ERA patients. Differences in the humoral responses to these bacteria may contribute to disease.

Human Immunodeficiency Virus Type-1 Reverse Transcriptase CONTRIBUTION OF MET-184 TO BINDING OF NUCLEOSIDE 5′-TRIPHOSPHATE

Mutations were made in recombinant human immunodeficiency virus type-1 reverse transcriptase (RT) by substituting methionine 184 with alanine (M184A) or valine (M184V), and steady-state and pre-steady-state kinetic constants were determined. The Km values of M184A RT for dNTPs were larger than those of wt RT for RNA-directed synthesis; the kcat values of M184A RT for processive or distributive synthesis were similar. In contrast to M184A RT, the Km and kcat values of M184V RT for dNTP substrates were similar to those of wt RT. The Ki values of M184V RT for 1-β-L-nucleoside analogs were increased 30-500-fold relative to wt RT for both RNA- and DNA-directed synthesis. The Kd and kp values of wt RT and M184V RT for dCTP and cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine 5′-triphosphate (1-β-L-FTCTP) were estimated from pre-steady-state kinetics for single nucleotide incorporation. The Kd value of M184V RT for 1-β-L-FTCTP was 19-fold greater than that of wt RT; the kp values of the two enzymes were similar. These results support the hypothesis that methionine 184 in the highly conserved YMDD region of wt RT participates in the binding of the nucleoside (analog) 5′-triphosphate.

Influences of diet and the gut microbiome on epigenetic modulation in cancer and other diseases

Epigenetic modulation of gene activity occurs in response to non-genetic factors such as body weight status, physical activity, dietary factors, and environmental toxins. In addition, each of these factors is thought to affect and be affected by the gut microbiome. A primary mechanism that links these various factors together in mediating control of gene expression is the production of metabolites that serve as critical cofactors and allosteric regulators of epigenetic processes. Here, we review the involvement of the gut microbiota and its interactions with dietary factors, many of which have known cellular bioactivity, focusing on particular epigenetic processes affected and the influence they have on human health and disease, particularly cancer and response to treatment. Advances in DNA sequencing have expanded the capacity for studying the microbiome. Combining this with rapidly improving techniques to measure the metabolome provides opportunities to understand complex relationships that may underlie the development and progression of cancer as well as treatment-related sequelae. Given broad reaching and fundamental biology, both at the cellular and organismal levels, we propose that interactive research programs, which utilize a wide range of mutually informative experimental model systems—each one optimally suited for answering particular questions—provide the best path forward for breaking ground on new knowledge and ultimately understanding the epigenetic significance of the gut microbiome and its response to dietary factors in cancer prevention and therapy.

Immune responses induced by administration of encapsidated poliovirus replicons which express HIV-1 gag and envelope proteins

Several viruses have been exploited for the development of recombinant vaccine vectors in which to express foreign proteins. Recently, we have described a system utilizing the RNA virus, poliovirus. We have constructed poliovirus genomes in which regions of the capsid have been substituted with gene fragments of the HIV gag and env genes. A complementation system has been designed to encapsidate defective genomes by providing the capsid protein in trans from a recombinant vaccinia virus (VV-P1). Serial passage in the presence of VV-P1 resulted in the generation of stocks of these encapsidated replicons. Infection of cells with these encapsidated replicons resulted in the expression of the recombinant protein as a fusion protein with the poliovirus capsid proteins VP4 and VP1. In this study, we have utilized encapsidated replicons which express the HIV-1-gag capsid protein (p24) as well as 1.5 kb of the HIV-1 env gene. Stocks of these encapsidated replicons were obtained by 20 serial passages in the presence of VV-P1. In addition, passage of the encapsidated replicons in the presence of poliovirus type 2 Lansing resulted in the encapsidation of the replicons by the capsid proteins provided by poliovirus. The administration of the type 2 Lansing/encapsidated replicons expressing HIV-1 gag in BALB/c mice by intramuscular, intrarectal, or intragastric routes resulted in the generation of antibodies in the serum and secretions against both poliovirus and HIV-1 gag. To prove that the replicons alone are immunogenic, we administered replicons expressing either HIV-1 gag or env to transgenic mice which expressed the receptor for poliovirus type 1. Immunization of these mice by the intramuscular route resulted in the generation of serum antibodies specific for poliovirus as well as for HIV-1 antigens. The results obtained led us to the conclusion that the replicons are immunogenic when given alone or in the presence of poliovirus. These results are important for the use of the poliovirus replicons as a recombinant vaccine vector.

Cytokine production in motor neurons by poliovirus replicon vector gene delivery.

Poliovirus replicon vectors transiently express foreign proteins selectively in motor neurons of the anterior horn of the spinal cord. Here we intraspinally inoculated mice transgenic for the poliovirus receptor (PVR) with replicons encoding murine tumor necrosis factor alpha (mTNF-α). We detected high-level expression of mTNF-α in the spinal cords of these animals at 8–12 h post inoculation; this returned to background by 72 h. The mice exhibited ataxia and tail atony, whereas animals given a replicon encoding green fluorescent protein (GFP) exhibited no neurological symptoms. Histology of spinal cords from mice given the replicon encoding mTNF-α revealed neuronal chromatolysis, reactive astrogliosis, decreased expression of myelin basic protein, and demyelination. These animals recovered with only slight residual damage. This study shows that replicon vectors have potential for targeted delivery of therapeutic proteins to the central nervous system and provide a new approach for treatment of spinal cord trauma and neurological disease.

Getting Started with Microbiome Analysis: Sample Acquisition to Bioinformatics

Historically, in order to study microbes, it was necessary to grow them in the laboratory. It was clear though that many microbe communities were refractory to study because none of the members could be grown outside of their native habitat. The development of culture‐independent methods to study microbiota using high‐throughput sequencing of the 16S ribosomal RNA gene variable regions present in all prokaryotic organisms has provided new opportunities to investigate complex microbial communities. In this unit, the process for a microbiome analysis is described. Many of the components required for this process may already exist. A pipeline is described for acquisition of samples from different sites on the human body, isolation of microbial DNA, and DNA sequencing using the Illumina MiSeq sequencing platform. Finally, a new analytical workflow for basic bioinformatics data analysis, QWRAP, is described, which can be used by clinical and basic science investigators. Curr. Protoc. Hum. Genet. 82:18.8.1‐18.8.29.

The Airway Microbiome at Birth

Alterations of pulmonary microbiome have been recognized in multiple respiratory disorders. It is critically important to ascertain if an airway microbiome exists at birth and if so, whether it is associated with subsequent lung disease. We found an established diverse and similar airway microbiome at birth in both preterm and term infants, which was more diverse and different from that of older preterm infants with established chronic lung disease (bronchopulmonary dysplasia). Consistent temporal dysbiotic changes in the airway microbiome were seen from birth to the development of bronchopulmonary dysplasia in extremely preterm infants. Genus Lactobacillus was decreased at birth in infants with chorioamnionitis and in preterm infants who subsequently went on to develop lung disease. Our results, taken together with previous literature indicating a placental and amniotic fluid microbiome, suggest fetal acquisition of an airway microbiome. We speculate that the early airway microbiome may prime the developing pulmonary immune system, and dysbiosis in its development may set the stage for subsequent lung disease.

Gastric microbiome and gastric cancer.

AbstractCancer of the stomach is the fourth most common cancer worldwide. The single strongest risk factor for gastric cancer is Helicobacter pylori–associated chronic gastric inflammation. Among persons with H. pylori infection, strain-specific components, host immune responses, and environmental factors influence the risk for gastric disease, including adenocarcinoma of the stomach, although only a small proportion of infected persons develop the malignancy. Recent advances in DNA sequencing technology have uncovered a complex community of noncultivatable inhabitants of the human stomach. The interaction between these inhabitants, collectively referred to as the gastric microbiota, and H. pylori likely affects gastric immunobiology and possibly the sequelae of H. pylori infection. Thus, characterization of the gastric microbiota in subjects with and without H. pylori infection could provide new insight into gastric homeostasis and the pathogenesis of H. pylori–associated disease, including gastric cancer.

Selection of Retroviral Reverse Transcription Primer Is Coordinated with tRNA Biogenesis

ABSTRACT Initiation of retrovirus reverse transcription requires the selection of a tRNA primer from the intracellular milieu. To investigate the features of primer selection, a human immunodeficiency virus type 1 (HIV-1) and a murine leukemia virus (MuLV) were created that require yeast tRNAPhe to be supplied in trans for infectivity. Wild-type yeast tRNAPhe expressed in mammalian cells was transported to the cytoplasm and aminoacylated. In contrast, tRNAPhe without the D loop (tRNAPheD−) was retained within the nucleus and did not complement infectivity of either HIV-1 or MuLV; however, infectivity was restored when tRNAPheD− was directly transfected into the cytoplasm of cells. A tRNAPhe mutant (tRNAPheUUA) that did not have the capacity to be aminoacylated was transported to the cytoplasm and did complement infectivity of both HIV-1 and MuLV, albeit at a level less than that with wild-type tRNAPhe. Collectively, our results demonstrate that the tRNA primer captured by HIV-1 and MuLV occurs after nuclear export of tRNA and supports a model in which primer selection for retroviruses is coordinated with tRNA biogenesis at the intracellular site of protein synthesis.

SCH 38057: a picornavirus capsid-binding molecule with antiviral activity after the initial stage of viral uncoating.

The activity of a new water-soluble molecule, SCH 38057, against picornaviruses is described. SCH 38057 inhibited plaque formation of selected entero- and rhinoviruses in a range of 10.2 to 29.1 microM (50% endpoint) and had a therapeutic index of 10 against poliovirus type 2 (polio 2) in HeLa cells. When administered orally or subcutaneously, SCH 38057 protected mice infected with either coxsackievirus B3 (CVB3) or echovirus-9 from mortality. The molecule provided a low level of protection against thermal inactivation of virus, indicating that SCH 38057 interacts with the picornavirus capsid. Binding studies with [3H]SCH 38057 revealed that the molecule binds to CVB3 and human rhinovirus 14 (HRV14) in a ratio of 29 and 19 molecules per viral particle, respectively. The affinity constant for SCH 38057 binding to CVB3 was 7.0 x 10(-4) M. When added to cultures of infected cells at 3 h after infection, SCH 38057 markedly inhibited viral RNA synthesis. This finding with lack of inhibition of attachment and loss of infectious virus after attachment were interpreted to indicate that, although SCH 38057 binds to the viral capsid, the molecule exerts its antiviral effect after the initial stage of picornavirus uncoating, i.e., after conversion of the 156S infectious viral particle to smaller subviral species.