NUDT2 Disruption Elevates Diadenosine Tetraphosphate (Ap4A) and Down-Regulates Immune Response and Cancer Promotion Genes.

Regulation of gene expression is one of several roles proposed for the stress-induced nucleotide diadenosine tetraphosphate (Ap4A). We have examined this directly by a comparative RNA-Seq analysis of KBM-7 chronic myelogenous leukemia cells and KBM-7 cells in which the NUDT2 Ap4A hydrolase gene had been disrupted (NuKO cells), causing a 175-fold increase in intracellular Ap4A. 6,288 differentially expressed genes were identified with P < 0.05. Of these, 980 were up-regulated and 705 down-regulated in NuKO cells with a fold-change ≥ 2. Ingenuity® Pathway Analysis (IPA®) was used to assign these genes to known canonical pathways and functional networks. Pathways associated with interferon responses, pattern recognition receptors and inflammation scored highly in the down-regulated set of genes while functions associated with MHC class II antigens were prominent among the up-regulated genes, which otherwise showed little organization into major functional gene sets. Tryptophan catabolism was also strongly down-regulated as were numerous genes known to be involved in tumor promotion in other systems, with roles in the epithelial-mesenchymal transition, proliferation, invasion and metastasis. Conversely, some pro-apoptotic genes were up-regulated. Major upstream factors predicted by IPA® for gene down-regulation included NFκB, STAT1/2, IRF3/4 and SP1 but no major factors controlling gene up-regulation were identified. Potential mechanisms for gene regulation mediated by Ap4A and/or NUDT2 disruption include binding of Ap4A to the HINT1 co-repressor, autocrine activation of purinoceptors by Ap4A, chromatin remodeling, effects of NUDT2 loss on transcript stability, and inhibition of ATP-dependent regulatory factors such as protein kinases by Ap4A. Existing evidence favors the last of these as the most probable mechanism. Regardless, our results suggest that the NUDT2 protein could be a novel cancer chemotherapeutic target, with its inhibition potentially exerting strong anti-tumor effects via multiple pathways involving metastasis, invasion, immunosuppression and apoptosis

A RAPID IN VITRO METHOD FOR THE EVALUTION OF CANDIDATE REPELLENTS AGAINST LEPTOTROMBIDIUM CHIGGERS

Scrub typhus is an acute febrile zoonotic disease resulting from infection with the Gram-negative intracellular bacteria Orientia (formerly Rickettsia) tsutsugamushi (Hyachi) (Seong et al. 2001). The disease is endemic in much of south and central Asia, with approximately one million cases each year and more than a billion people at risk worldwide (Rosenberg 1997). Scrub typhus is transmitted by several species of larval trombiculid mites which are commonly known as chiggers (Tanskul et al. 1998). Repellents provide an effective method of protecting individuals from arthropods (Gupta and Rutledge 1994). In this study 6 essential oils were tested to evaluate their repellent activity against the chigger, Leptotrombidium imphalum Vercammen-Grandjean and Langston. A rapid and economic in vitro procedure which requires only 5 min and a small number of chiggers was used to determine the median effective doses. The results showed that clove oil was significantly more effective than others with ED50 of 53.2 µg followed by vetiver oil

A RAPID IN VITRO METHOD FOR THE EVALUATION OF CANDIDATE REPELLENTS AGAINST LEPTOTROMBIDIUM CHIGGERS

Scrub typhus is an acute febrile zoonotic disease resulting from infection with the gram-negative intracellular bacteria Orientia (formerly Rickettsia) tsutsugamushi (Hyachi) (Seong et al. 2001). The disease is endemic in much of south and central Asia, with approximately one million cases each year and more than a billion people at risk worldwide (Rosenberg 1997). Scrub typhus is transmitted by several species of larval trombiculid mites which are commonly known as chiggers (Tanskul et al. 1998). Repellents provide an effective method of protecting individuals from arthropods (Gupta and Rutledge 1994). In this study 6 essential oils were tested to evaluate their repellent activity against the chigger, Leptotrombidium imphalum Vercammen-Grandjean and Langston. A rapid and economic in vitro procedure which requires only 5 min and a small number of chiggers was used to determine the median effective doses. The results showed that clove oil was significantly more effective than others with ED50 of 53.2 µg followed by vetiver oil

A RAPID IN VITRO METHOD FOR THE EVALUTION OF CANDIDATE REPELLENTS AGAINST LEPTOTROMBIDIUM CHIGGERS

Scrub typhus is an acute febrile zoonotic disease resulting from infection with the Gram-negative intracellular bacteria Orientia (formerly Rickettsia) tsutsugamushi (Hyachi) (Seong et al. 2001). The disease is endemic in much of south and central Asia, with approximately one million cases each year and more than a billion people at risk worldwide (Rosenberg 1997). Scrub typhus is transmitted by several species of larval trombiculid mites which are commonly known as chiggers (Tanskul et al. 1998). Repellents provide an effective method of protecting individuals from arthropods (Gupta and Rutledge 1994). In this study 6 essential oils were tested to evaluate their repellent activity against the chigger, Leptotrombidium imphalum Vercammen-Grandjean and Langston. A rapid and economic in vitro procedure which requires only 5 min and a small number of chiggers was used to determine the median effective doses. The results showed that clove oil was significantly more effective than others with ED50 of 53.2 µg followed by vetiver oil

Rickettsia-Macrophage Interactions: Host Cell Responses to Rickettsia akari and Rickettsia typhi

The existence of intracellular rickettsiae requires entry, survival, and replication in the eukaryotic host cells and exit to initiate new infection. While endothelial cells are the preferred target cells for most pathogenic rickettsiae, infection of monocytes/macrophages may also contribute to the establishment of rickettsial infection and resulting pathogenesis. We initiated studies to characterize macrophage-Rickettsia akari and -Rickettsia typhi interactions and to determine how rickettsiae survive within phagocytic cells. Flow cytometry, microscopic analysis, and LDH release demonstrated that R. akari and R. typhi caused negligible cytotoxicity in mouse peritoneal macrophages as well as in macrophage-like cell line, P388D1. Host cells responded to rickettsial infection with increased secretion of proinflammatory cytokines such as interleukin-1β (IL-1β) and IL-6. Furthermore, macrophage infection with R. akari and R. typhi resulted in differential synthesis and expression of IL-β and IL-6, which may correlate with the existence of biological differences among these two closely related bacteria. In contrast, levels of gamma interferon (IFN-γ), IL-10, and IL-12 in supernatants of infected P388D1 cells and mouse peritoneal macrophages did not change significantly during the course of infection and remained below the enzyme-linked immunosorbent assay cytokine detection limits. In addition, differential expression of cytokines was observed between R. akari- and R. typhi-infected macrophages, which may correlate with the biological differences among these closely related bacteria

Isolation and Propagation of the Ap-Variant 1 Strain of Anaplasma phagocytophilum in a Tick Cell Line▿

The first tissue culture isolates of the unique Anaplasma phagocytophilum strain, Ap-Variant 1, were obtained in the Ixodes scapularis tick-derived cell line ISE6. Two isolates were from goat blood samples: one from a goat infected with I. scapularis ticks from Rhode Island and a second from a goat infected by serial passage of blood from the first infected goat. Eight isolates were made directly from I. scapularis ticks collected from white-tailed deer in Minnesota and represent the first isolations of an Anaplasma species directly from ticks. Each of the 10 isolates had a 16S rRNA gene sequence identical to that previously described for Ap-Variant 1, but differences within the ank gene were found that suggest natural variation. Prevalence of Anaplasma in the Minnesota ticks was 63.9%; 23 of 36 ticks tested by PCR were positive. Six of the tick-derived isolates were obtained from a set of 18 PCR-positive ticks, for a 33.3% isolation success rate. The conservation of host tropism among the Rhode Island and Minnesota isolates of Ap-Variant 1 was examined by use of experimental infections of mice and a goat. A Minnesota tick-derived isolate (MN-61-2) was used to inoculate naïve animals, and this isolate was able to infect a goat but unable to infect each of five mice, confirming that the Minnesota isolates have the same host tropism as Ap-Variant 1 from the northeastern United States. Light and electron microscopy of the Ap-Variant 1 isolate MN-61-2 in ISE6 cells showed cytoplasmic inclusions characteristic of A. phagocytophilum with pleomorphic bacteria in membrane-bound vacuoles and both electron-dense and electron-lucent forms